Congresso Nazionale Biomateriali - Lecce 18

Transcript

Congresso Nazionale Biomateriali - Lecce 18-20 giugno 2012
3
4
5
PROGRAM
17:30 - 17:45
A. Fiorani, M. Gioffrè, C. Gualandi, M. L. Focarete, S. Panzavolta, B. Bracci, A. Bigi, P. Torricelli
(University of Bologna).
Biomimetic gelatin/poly(L-lactic acid) composite scaffolds by co-electrospinning (pag. 22)
17:45 - 18:00
C. Piconi, P. Fabbri, F. Mazzanti, G. Magnani, E. Burresi, P. Nitti, C. Mingazzini (Ghimas S.p.A., Brindisi).
Experimental determination of low temperature degradation behaviour of a new zirconia-alumina composite - Preliminary results (pag. 23)
18:00 - 18:15
A. Tampieri, T. D’Alessandro, M. Sandri, S. Panseri, C. Cunha (Institute of Science and Technology for Ceramics, CNR, Faenza).
Superparamagnetic bio-mimetic hybrid composites (pag. 24)
18:15 - 19:15
SIB Social Members’ Assembly
TUESDAY JUNE 19 - MORNING SESSIONS
Third session 09:00 - 10:00
Design and characterization of biomaterials and tissues Chairmen: R. De Santis and A. Maffezzoli
09:00 - 09:15
I. E. De Napoli, E. M. Zanetti , A. L. Audenino, R. Quarto, G. Catapano (University of Calabria).
The effects of hollow fiber membrane (HFM) geometry and transport properties on the performance of HFM bioreactors for bone tissue engineering (pag. 25)
09:15 - 09:30
S. Longoni, M. Sartori (S. Apollonia Dental Centre, Lazzate).
Fractal modeling: medical devices designed by tissues (pag. 26)
09:30 - 09:45
C. Giannini, D. Altamura, R. Lassandro, L. De Caro, D. Siliqi, M. Ladisa (Istituto di Cristallografia, CNR, Bari).
X-Ray Microimaging Laboratory (XMI-LAB) (pag. 27)
09:45 - 10:00
G. Ceccarelli, D. Prè, L. Benedetti, M. Imbriani, L. Visai, M.G. Cusella De Angelis (University of Pavia).
High frequency vibration enhances the expression of osteogenic genes and extracellular matrix
deposition in human Bone Marrow Stromal Cells (hBMSCs) (pag. 28)
10:00 - 10:30
Coffee break
Fourth session 10:30 - 12:15
Cell-biomaterial interactions Chairmen: L. Ambrosio and L. Rimondini
10:30 - 11:00
Lectio Magistralis
Prof. Josep A. Planell (Institute of Bioengineering of Catalonia, Spain)
Functional scaffolds: materials and fabrication (pag. 29)
11:00 - 11:15
S. Martino, I. Armentano, F. D’Angelo, I. Cacciotti, R. Tiribuzi, M. Quattrocelli,
C. Del Gaudio, E. Fortunati, M. Imbriani, A. Caraffa, G. G. Cerulli, L. Visai, J. M. Kenny, M. Sampaolesi, A. Bianco, A. Orlacchio (University of Perugia).
Tuning multi-/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats (pag. 30)
11:15 - 11:30
I. E. Palamà, A. M. L. Coluccia, S. D’Amone, M. Riehle and G. Gigli (Institute of Nanoscience CNR, Lecce).
Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials (pag. 31)
6
PROGRAM
11:30 - 11:45
F. Intranuovo, M. Domingos, A. Gloria, R. Gristina, P. J. Bártolo, P. Favia (University of Bari).
Low pressure plasma processes to control cell colonization inside 3D scaffolds (pag. 32)
11:45 - 12:00
A. Cochis, A Carletta, L. Altomare, S. Farè, S. Merlin, A. Follenzi, M. Prat and L. Rimondini (Università del Piemonte Orientale, Novara).
Engineered cell sheets using thermo-reversible hydrogel (pag. 33)
12:00 - 12:15
L. Lungaro, V. Goranov, A. Riminucci, C. Dionigi, T. Shelyakova, A. Russo, M. Sandri, A. Tampieri, R. De Santis,
L. Ambrosio, Haranava Y, V. A. Dediu (Istituto per lo Studio dei Materiali Nanostrutturati, CNR, Bologna).
Interaction of magnetized cells with magnetic scaffolds (pag. 34)
Poster session 12:15 - 13:30
12:15 - 13:30
Poster session
The authors should stand in front of the poster for discussion
13:30 - 14:30
Lunch: Buffet
TUESDAY JUNE 19 - AFTERNOON SESSIONS
Fifth session 15:00 - 16:30
Drug and gene delivery Chairmen: G. Catapano e A. Sannino
15:00 - 15:15
F. Ravanetti, E. Scarpa, M. Negri, M. Campanini, G. Salviati, A. Cacchioli (University of Parma).
Interactions between Cells and SiC/SiO2 Nanowires: Scanning and Transmission Electron Microscopy investigations (pag. 35)
15:15 - 15:30
I. E. Palamà, A. M. L. Coluccia, S. D’Amone, G. Gigli (Institute of Nanoscience CNR, Lecce).
Polyelectrolyte complexes (PECs) as intelligent and safe nanocarriers for gene/drug delivery (pag. 36)
15:30 - 15:45
S. Fedi, G. Giani and R. Barbucci (University of Siena).
Hybrid magnetic hydrogel: a potential system for controlled drug delivery by means of alternating magnetic fields (pag. 37)
15:45 - 16:00
C. Malloggi, D. Pezzoli, F. Olimpieri, S. Bertini, R. Chiesa, A. Volonterio, G. Candiani (Pol. di MI).
Development of copolymers for gene delivery: influence of degree of grafting on transfection behavior (pag. 38)
16:00 - 16:15
E. Nicolì, M. Bosetti, F. Boccafoschi, A. Calarco, L. Fusaro, G. Peluso, M. Cannas (Università del Piemonte Orientale, Novara).
Nanotoxicity of PEI-based nanoparticles is reduced by acetylation of polyethylenimine amines in human primary cells (pag. 39)
16:15 - 16.30
D. Vergara, C. Bellomo, X. Zhang, V. Vergaro, A. Tinelli, V. Lorusso, R. Rinaldi, Y. M. Lvov, S. Leporatti, M. Maffia (University of Salento).
Lapatinib/Paclitaxel polyelectrolyte nanocapsules for overcoming multidrug resistance in ovarian cancer (pag. 40)
16:30 - 17:00
Coffee break
7
PROGRAM
Sixth session 17:00 - 18:30
Biomaterials and scaffolds for Tissue Engineering Chairmen: A. Cigada e M.C. Tanzi
17:00 - 17:15
A. La Gatta, A. Papa, C. Schiraldi, A. D’Agostino, M. De Rosa (Second University of Naples).
Novel cross-linked hyaluronic acid hydrogels for biomedical applications (pag. 41)
17:15 - 17:30
A. D’Agostino, A. La Gatta, T. Busico, A. Papa, M. De Rosa, C. Schiraldi (Second University of Naples).
GAGs (Hyaluronan and chondroitin sulphate) in combination with synthetic polymers: potential applications (pag. 42)
17:30 - 17:45
F. C. Pavia, V. La Carrubba and V. Brucato (Università di Palermo).
Preparation and hydrolytic degradation of poly lactic acid based scaffolds (pag. 43)
17:45 - 18:00
E. De Giglio, G. M. Farinola, D. Cafagna, R. Rizzi, V. Fino, S. Cometa, C. Ferretti, M. Mattioli-Belmonte (University of Bari).
Innovative glycerol-based biocompatible copolymer as material for potential biomedical applications (pag. 44)
18:00 - 18:15
V. Guarino, A. Borriello, M. A. Alvarez-Perez, L. Ambrosio (Institute of Composite and Biomedical Materials, CNR, Naples).
Polyaniline based scaffolds for the regeneration of electro-active tissues (pag. 45)
18:15 - 18:30
N. Rescignano, I. Armentano, S. Montesano, C. Elvira, S. Martino, A. Orlacchio, J. M. Kenny (University of Perugia).
PCL scaffolds produced by scCO2 technique and nanoparticle incorporation (pag. 46)
20:30 - 22:30
Gala Dinner
WEDNESDAY JUNE 20 - MORNING SESSIONS
Seventh session 09:00 - 10:30
Surface modification of biomaterials Chairmen: N. Cioffi and M. Mattioli-Belmonte
09:00 - 09:15
C. Cristallini, N. Barbani, E. Bellotti, F. Manetti, E. Rosellini, M. Gagliardi, E. Del Gaudio, F. Tricoli, S. Mantero (Institute of Composite and Biomedical Materials, CNR, Pisa).
Modulation of MMP-9/TIMP activity in preventing cardiac disfunction through a combination of molecularly imprinting technology and biodegradable microfabricated systems (pag. 47)
09:15 - 09:30
E. Sardella, R. A. Salama, R. Gristina, A. N. Habib, G. H. Waly, P. Favia (Institute of Inorganic Methodologies and Plasmas, CNR, Bari).
An innovative method to induce cell ingrowth through 3D porous scaffolds by poor cell adhesive coatings (pag. 48)
09:30 - 09:45
F. Boccafoschi, L. Fusaro, C. Mosca, M. Bosetti, M. Cannas (Università del Piemonte Orientale, Novara).
Grafted-Modified PLLA: New Approaches for Phenotype Guiding in Cardiovascular Tissue Engineering (pag. 49)
09:45 - 10:00
F. Taraballi, C. Lupo, L. Russo, S. Zanini, C. Riccardi, S. Panseri, C. Cunha, M. Campione, M. Marcacci, L. Cipolla and F. Nicotra (University of Milano-Bicocca).
In vitro assessment of collagen films functionalized with NH2 and COOH groups by plasma treatment (pag. 50)
10:00 - 10:15
C. Della Valle, S. Panzuto, E. Saino, L. Visai, M. Santin, R. Chiesa (Politecnico di Milano).
Antibacterial anodic spark deposition treatments for titanium (pag. 51)
8
PROGRAM
10:15 - 10:30 V. Nassisi, P. Alifano, D. Delle Side, A. Talà, S. M. Tredici and L. Velardi (University of Salento).
Enhancing antibacterial properties of UHMWPE via ion implantation (pag. 52)
10:30 - 11:00
Coffee break
Eighth session 11:00 - 12:00
Bone Tissue Engineering 2 Chairmen: V. Guarino and C. Piconi
11:00 - 11:15
A. Licciulli, G. Casarano, S. Franza, D. Diso, A. Chiechi, G. Vantaggiato (University of Salento).
In vivo studies and prototyping of Zirconia dental implants (Y-PSZ) (pag. 53)
11:15 - 11:30
M. Carrozzo, K.P. Sanosh, F. Gervaso, A. Sannino, A. Licciulli (University of Salento).
Mechanical performance and in vitro studies of wollastonite/hydroxyapatite composite scaffold for bone tissue engineering (pag. 54)
11:30 - 11:45
F. Mazzanti, G. Magnani, L. Beaulardi (ENEA, Faenza).
High performance zirconia-alumina biomaterial (pag. 55)
11:45 - 12:00
D. Pasqui, M. De Cagna, M. Fini, P. Torricelli and R. Barbucci (University of Siena).
Carboxymethylcellulose hydrogel/hydroxyapatite composite biomaterial for bone tissue engineering applications (pag. 56)
12:00 - 12:30
Award for the best oral communication and poster
12:30 - 12:45
End of the Congress
9
PROGRAM
POSTER SESSION
Tuesday morning, June 19: 12:15 - 13:30
P1: P. Nitti, A. Gallo, A. Casillo, B. Palazzo, L. Ambrosio, C. Piconi (Ghimas S.p.A., Brindisi).
A new composite chitosan-nano-hydroxyapatite scaffold for bone regeneration: fabrication, characterization and cell response (pag. 57)
P2: C. Lupo, F. Taraballi, L. Russo, M. Sandri, A. Tampieri, A. Paleari, J. Jiménez-Barbero, L. Cipolla, F. Nicotra (University of Milano-Bicocca).
Covalent functionalization of hydroxyapatite: comparing different methodologies (pag. 58)
P3: G. Camporeale, D. Pignatelli, F. Intranuovo, E. Sardella, R. Gristina, P. Favia (University of Bari).
Dielectric barrier discharges on 2D/3D polymeric scaffolds for applications in Tissue Engineering (pag. 59)
P4: L. Russo, E. Valliant, L. Gabrielli, L. Cipolla, J. Jiménez-Barbero, F. Nicotra, J. R. Jones (University of Milano-Bicocca).
Preparation and characterization of new Silica/PEG hybrid materials (pag. 60)
P5: M. Madaghiele, F. I. Errico, M. G. Raucci, L. Ambrosio, A. Sannino and A. Maffezzoli (University of Salento).
Synthesis and characterization of novel photoreactive cellulose derivatives for use as soft tissue fillers (pag. 61)
P6: M. Pollini, F. Paladini, L. Salvatore, A. Licciulli, A. Maffezzoli and A. Sannino (University of Salento).
Silver-treated flax fabrics with durable antibacterial properties for biomedical application (pag. 62)
P7: E. De Angelis, F. Ravanetti, A. Cacchioli, A. Corradi, R. Bettini, P. Borghetti (University of Parma).
Differentiation related gene expression of equine articular chondrocytes cultured in chitosan scaffold (pag. 63)
P8: U. D’Amora, T. Russo, A. Gloria, R. De Santis, S. Zeppetelli, M. Sandri, A. Tampieri, L. Ambrosio (Institute of Composite and Biomedical
Materials, CNR, Naples).
Multifunctional PCL/biomimetic hydroxyapatite nanocomposite scaffolds to guide hard tissue engineering (pag. 64)
P9: F. Salamanna, V. Masciale, M. Maglio, A. Ferrari, A. Parrilli, M. Cadossi, M. Fini, R. Giardino (Rizzoli Orthopaedic Institute, Bologna).
Microtomographic analysis of retrieval hip resurfacing arthroplasty failed at different times (pag. 65)
P10: R. De Santis, A. Gloria, U. D’Amora, T. Russo, S. Zeppetelli, T. D’Alessandro, M. Sandri, M. Banobre-Lopez, Y. Pineiro-Redondo, A.
Tampieri, J. Rivas, V. Dediu and L. Ambrosio (Institute of Composite and Biomedical Materials, CNR, Naples).
The effect of iron-doped hydroxyapatite nanoparticle inclusion on mechanical, magnetic and biological performances of innovative
PCL-based substrates for hard tissue engineering (pag. 66)
P11: A. Gloria, T. Russo, U. D’Amora, R. De Santis, V. D’Antò, F. Bollino, M. Catauro, S. Rengo, L. Ambrosio (Institute of Composite
and Biomedical Materials, CNR, Naples).
Advanced composite PCL/organic-inorganic hybrid fillers: from 2D innovative substrates to 3D rapid-prototyped scaffolds for tissue
engineering (pag. 67)
P12: L. Salvatore, F. Paladini, M. Pollini, A. Sannino (University of Salento).
Synthesis and characterization of silver-doped collagen based patch with antimicrobial properties (pag. 68)
P13: T. Russo, A. Gloria, U. D’Amora, F. Causa, E. Battista, R Della Moglie, S. Zeppetelli, R. De Santis, P.A. Netti and L. Ambrosio
(Institute of Composite and Biomedical Materials, CNR, Naples).
3D advanced rapid-prototyped PCL scaffolds: effect of surface modification and peptide conjugation on mechanical and biological
performance. (pag. 69)
10
PROGRAM
P14: S. Montesano, I. Armentano E. Lizundia , F. D’Angelo, E. Fortunati, S. Mattioli, R. Tiribuzi, M. Sampaolesi, J. R. Sarasua, J. M.
Kenny, S. Martino, A. Orlacchio (University of Perugia).
Inclusion of multi walled carbon nanotubes on PLLA supports the myogenic differentiation of murine progenitor cells (pag. 70)
P15: G. M. L. Messina, R. Lettieri, M. Venanzi, F. Formaggio, C. Toniolo, G. Marletta (University of Catania).
Peptide confinement in nanopores for antibacterial surfaces (pag. 71)
P16: M. Falconi, A. Bigi, R. Giardino, M. Fini, A. Mazzotti, G. Teti (University of Bologna).
Morphological evaluation of adesion and proliferation of MG63 cells grown on gelatin/genipin scaffold (pag. 72)
P17: E. Fortunati, I. Armentano, Q. Zhou, A. Iannoni, M. Vercellino, L. Berglund, J. M. Kenny, M. Imbriani, L. Visai (University of Pavia).
Antibacterial properties of PLA composites based on silver nanoparticles and crystalline cellulose (pag. 73)
P18: M. C. Sportelli, M. Valentini, L. Giannossa, M. A. Nitti, N. Ditaranto, A. Valentini, N. Cioffi, L. Sabbatini (University of Bari).
Zinc oxide nanoantimicrobials wet chemical vs ion beam sputtering syntheses of nanoparticles and nano-coatings (pag. 74)
P19: M. A. Nitti, M. C. Sportelli, M. Valentini, N. Cioffi, G. M. Tantillo, A. Valentini, G. Casamassima (University of Bari).
Ion beam deposition and characterization of inorganic nanoparticles-fluoropolymer antimicrobial coatings for textile applications (pag. 75)
P20: F. Carfì Pavia, S. Rigogliuso, V. La Carrubba, G. Ghersi and V. Brucato (Università di Palermo).
Vascular grafts based on PLLA/PLA blends (pag. 76)
P21: D. Pignatelli, G. Dilecce, B. R. Pistillo, S. De Benedictis, P. Favia, R. Gristina (University of Bari).
The influence of air DBDs plasma process on eukaryotic cell lines (pag. 77)
P22: M. Mattioli-Belmonte, C. Ferretti, A. Trapani, R. Iatta, M. Orciani, D. Cafagna, R. Lazzarini, A. Romanelli, E. De Giglio (Università
Politecnica delle Marche).
Effect of a novel in situ antibiotic delivery systems on osteoblasts (pag. 78)
P23: N. Bloise, I. Cislaghi, D. Merli, A. Profumo, M. Fagnoni, P. Mustarelli, M. Imbriani, L. Visai (University of Pavia).
Preparation and characterization of a multiwalled carbon nanotube/mitoxantrone adduct for a targeted drug delivery system (pag. 79)
P24: C. Malloggi, G. Scaparrotti, R. Chiesa, D. Pezzoli, G. Candiani (Politecnico di Milano).
Comparative screening of cationic polymers for gene delivery and optimization of transfection parameters (pag. 80)
P25: A. Ruffini, M. Sandri, A. Tampieri (Institute of Science and Technology for Ceramics, Faenza).
Chitosan-based pH sensitive polymers as drug delivery systems: kinetic release study (pag. 81)
P26: M. Tunesi, E. Prina, A. Cigada, C. Giordano, D. Albani (Politecnico di Milano).
Innovative hydrogels for neuroprotective protein delivery in neurodegeneration (pag. 82)
P27: E. Prina, M. Tunesi, F. Daniele, A. Cigada, C. Giordano, D. Albani (Politecnico di Milano).
Neurodegenerative disorders: novel hydrogels for drug delivery strategies. (pag. 83)
P28: C. Esposito Corcione, F. Scalera, A. Sannino, A. Maffezzoli (University of Salento).
UV curable hydroxyapatite suspensions for bone tissue substitutes prototyping (pag. 84)
P29: F. Paladini, M. Pollini, A. Talà, P. Alifano, A. Sannino (University of Salento).
Long-term antibacterial efficacy of silver treated catheters for haemodialysis in preventing biofilm formation (pag. 85)
11
PROGRAM
P30: A. Sola, D. Bellucci, M. G. Raucci, S. Zeppetelli, L. Ambrosio, V. Cannillo (University of Modena and Reggio Emilia).
Sintering and bioactivity of glasses belonging to the Na2O-CaO-P2O5-SiO2 system (pag. 86)
P31: D. Bellucci, G. Bolelli, R. Gadow, A. Killinger, L. Lusvarghi, A. Sola, N. Stiegler, V. Cannillo (University of Modena and Reggio Emilia)
High-Velocity Suspension Flame Sprayed (HVSFS) bioactive coatings for orthopedic applications. (pag. 87)
P32: D. Bellucci, F. Chiellini, G. Ciardelli, M. Gazzarri, P. Gentile, A. Sola, V. Cannillo (University of Modena and Reggio Emilia).
An innovative processing route to realize scaffolds for bone tissue engineering (pag. 88)
P33: M. Pollini, F. Paladini, L. Salvatore, A. Licciulli, A. Maffezzoli and A. Sannino (University of Salento).
Assessment of the hypoallergenicity of silver functionalized cotton with long-term antimicrobial properties (pag. 89)
P34: M. Dapporto, S. Sprio, T. D’Alessandro, C. Cunha and A. Tampieri (Institute of Science and Technology for Ceramics, CNR, Faenza).
Novel biomimetic bone cements based on Sr-substituted hydroxyapatite for regenerative vertebroplasty (pag. 90)
P35: S. Minardi, E. Tasciotti, M. Sandri, S.M. Khaled, J.O. Martinez, B.S. Brown, M. Ferrari, A. Tampieri (Institute of Science and Technology
for Ceramics, CNR, Faenza).
Intelligent multifunctional scaffold (pag. 91)
12
ABSTRACT BOOK
13
CURRENT SURGICAL OPTIONS FOR ARTICULAR CARTILAGE REPAIR AND FUTURE PERSPECTIVES
Peretti G.M.
Department of Biomedical Sciences for Health, University of Milan
[email protected]
INTRODUCTION
The repair of chondral or osteochondral lesions still represents a big challenge for the orthopaedic surgeon. Many efforts have been
done and are currently part of research and clinical programs to try to find the solution for this issue. Generally, the surgical options
for articular cartilage repair can be divided into three groups: techniques without transplant of cells or tissues; techniques based on
the transplantation of tissues; the tissue engineering techniques.
SURGICAL TECHNIQUES WHICH DO NOT REQUIRE CELL OR TISSUE TRANSPLANT
Several procedures were developed with the attempt of inducing bleeding from the bone marrow and, consequently, the migration
of growth factors and stem cells into the lesion site. These techniques are simple and are performed arthroscopically, but present
the limit of generating fibro-cartilaginous tissue.
SURGICAL TECHNIQUES WHICH REQUIRE TISSUE TRANSPLANT
A different approach for the repair of a chondral lesion consists in the transplantation to the lesion site of a mature osteocartilaginous tissue. In this procedure, healthy osteochondral cylinders are taken from low bearing areas of the joint and transplanted to the
defect site. This procedure has the disadvantage of the creation of one or more iatrogenic lesions having overall the same dimension
of that to be repaired.
TISSUE ENGINEERING TECHNIQUES FOR CHONDRAL REGENERATION
The current tissue engineering techniques for chondral regeneration can be divided into three groups: first, the transplantation of
chondrocytes or other cells in solution; second, the transplantation of cells previously seeded onto a scaffold; third, the implantation
of acellular scaffold capable of the recruitment of nucleated mesenchymal stem cells from the bone marrow and the blood of the
sub-chondral bone. Eventually, these scaffolds should also provide the proper stimuli for the correct differentiation of the reparative
cells. The original technique was proposed by Brittberg in the 1994 and was called A.C.I. (Autologous Chondrocyte Implantation).
This procedure requires two surgical steps; during the first step, a cartilage biopsy is taken from an area of healthy cartilage and
then transferred in a laboratory for cells’ isolation and expansion. Cells are expanded in a monolayer culture until they reach the
sufficient number to fill the defect. During the second step, the patient undergoes a second surgery; at this time, a periosteal flap is
sutured at the boundaries of the lesion and the cells are injected in solution under the flap. In the following years, some authors have
proposed to seed the cells onto biocompatible scaffolds. The materials employed as cell carriers are different. These techniques still
require two surgical steps and several new materials have been developed. Beside these techniques that involve the transplantation
of cells, others approaches are currently used, based on the employment of non-seeded membrane. These products are based on
the natural healing potential of full thickness acute lesions of the articular cartilage, as bone marrow-stimulation techniques do.
FUTURE PERSPECTIVES
The research in the field of cartilage repair is becoming more and more active and involves the use of different biomaterials, growth
factors, isolated or incorporated in their structure, able to induce or maintain the cartilaginous phenotype of the reparative cells.
Our group has being studying the use of fresh or expanded cells for the development of engineered substitute in combinations with
hydrogel and open matrix collagen scaffolds. The coupling of such a materials with bone compatible material brought to interesting
results in the repair of osteocartilaginous lesion in pre-clinical models. Moreover, thanks to the evolution of the research in the
material science and in the imaging, it will be soon possible to create a custom-made scaffold, based on the dimension and size
of the patient’s lesion.
CONCLUSIONS
In the past years, a lot of efforts have been done by studying the lesions’ biology and trying to improve the natural healing potential
of the articular cartilage. All the bone marrow-stimulation techniques are based on this principle, but have the limit of the low quality
of the newly formed tissue. In order to improve the biomechanical properties of the neo-tissue, many different procedures based on
tissue engineering methodologies were proposed that can potentially reproduce a hyaline cartilage by more complex and expensive
procedure. Nowadays, none of the techniques described can completely and systematically restore the injured cartilaginous tissue.
On the other hand, important future perspective are originating from the research activity in a multidisciplinary scenario, involving
cells, biomaterials, gene-based therapies and the use of novel stimulating factors.
14
TISSUE ENGINEERING FOR CARTILAGE REPAIR: IN VITRO DEVELOPMENT OF AN OSTEOCHONDRAL SCAFFOLD
Deponti D., Di Giancamillo A., Gervaso F., Pozzi A., Ballis R., Scalera F., Domenicucci M., Domeneghini C., Sannino A., Peretti G.M.
1 Istituto Scientifico San Raffaele di Milano
2 Dipartimento di Scienze e Biotecnologie Veterinarie per la Salute Alimentare, Università degli Studi di Milano
3 Dipartimento di Ingegneria dell’Innovazione, Università del Salento, Lecce
4 Istituto Ortopedico Galeazzi, Milano
5 Dipartimento di Scienze dello Sport, Nutrizione e Salute, Università degli Studi di Milano
[email protected]
INTRODUCTION
Articular cartilage lesions have poor healing potential. Basic science and clinical investigation have led to different approaches to
address this problem, in particular with the use of biocompatible scaffolds seeded with autologous chondrocytes. We developed
an osteochondral scaffold with a biphasic structure: a chondral phase made of collagen I, designed for hosting the autologous
chondrocytes, and a bony phase made of hydroxyapatite structured for the scaffold integration into the bone beneath the chondral
layer. This scaffold was developed for the repair of a chondral lesion in a swine model. The aim of the present work was the in
vitro optimization of the chondral phase, and, in particular, the analysis of the effect of the addition of the fibrin glue as embedding
scaffold for the seeded chondrocytes.
EXPERIMENTAL METHODS
Fresh chondrocytes were isolated from swine articular cartilage and seeded onto the collagen sponges: some samples
were seeded with cells resuspended in medium, other samples were seeded with cells resuspended in fibrinogen that was
then polymerized into fibrin glue by the addiction of thrombin. The samples were cultured in vitro for three weeks. Chondrocytes were isolated from swine articular cartilage and expanded in the presence of specific growth factors (FGF-2 and
TGFß1) to stimulate proliferation and the maintenance of a chondral phenotype, then they were resuspended in fibrinogen and
seeded onto the chondral composite that was cultured in vitro for 1, 3 and 5 weeks in the presence of TGFß3: the optimal
time for a preliminary maturation of the composite was identified by morphological, phenotypical and biomechanical analyses.
RESULTS AND DISCUSSION
Histological and immunohistochemical data demonstrated that the presence of fibrin glue ameliorated cell distribution and survival
into the chondral composite: the cells were surrounded by a matrix that was positive for GAGs and collagen II and it was more
abundant in the peripheral portion of the scaffold than the inner. The collagen sponges seeded without fibrin showed a reduced
matrix deposition compared to those seeded with fibrin glue; moreover, few cells were observed and they were restricted to some
clusters anchored to the collagen fibers of the sponge.
The second part of this work showed that one week of in vitro culture was not sufficient for the rescue of the chondrocyte phenotype
and for the acquirement of significant biochemical and mechanical properties. The prolongation of the culture to 3 weeks promoted a significant rescue of the cell phenotype resulting in a composite with improved morphological, biochemical and mechanical
properties. In fact, the cells started to rescue their original morphology, characterized by the presence of lacunae around them and
by a peri-cellular matrix that was positive for GAGs production and for collagen II. Moreover, the composites showed higher levels
of cellularity and GAGs production with respect to the other experimental samples: these acquired properties resulted in a higher
stiffness of the seeded scaffolds that mediated a higher resistance to compression. The prolongation of the culture to 5 weeks promoted a rescue in the phenotype similar to that of the scaffolds cultured for 3 weeks; however, the cellularity and GAGs content were
lower and similar to that of the scaffolds cultured for 1 weeks, demonstrating that a further extension of the in vitro culture caused
a reduction in the biochemical properties of the scaffolds. Moreover, the prolongation of the culture affected also the stiffness of the
scaffolds that was reduced leading to a lower resistance to compression.
CONCLUSION
This study developed a collagenic scaffold that, in combination with fibrin glue, was able to support chondrocytes survival and
synthetic activity in vitro in a static culture; in particular, this model was able to turn the engineered samples into a tissue with
chondral-like properties when cultured in vitro for at least 3 weeks.
ACKNOWLEDGMENTS
This work was supported by the Cariplo Fundation.
15
CARTILAGE REPAIR OF OSTEOCONDRAL DEFECTS WITH SCAFFOLD OF CHITOSAN ENRICHED WITH D (+) RAFFINOSE
Edoardo Scarpa¹, Francesca Ravanetti¹, Paolo Borghetti², Fabio Leonardi³, Filippo Maria Martini³, Ruggero Bettini4, Antonio Cacchioli¹
Department of Animal Health: 1 Anatomy Unit; 2 Pathology Unit; 3 Surgery Unit, Faculty of Veterinary Medicine
4 Department of Pharmacy, University of Parma, Italy
[email protected]
INTRODUCTION
The progressive loss of articular cartilage tissue after traumatic events or several pathologies are leading causes of disability
worldwide¹. The poor healing capacity of this tissue, and the subsequent creation of fibrocartilage, are problems that tissue engineering is trying to face. To this propose, a field of research focuses on the identification of suitable scaffolds of biomaterials able to
allow the creation of new functional tissue. Natural polymers represent a promising and widely studied resource for the creation of
these three-dimensional scaffolds; among them, chitosan, due to its structural similarity with glycosaminoglycans (GAGs), its biocompatibility and biodegradability, is considered a very interesting candidate. The aim of our study was to assess whether chitosan
scaffolds, modified by the addition of D (+) raffinose and with a degree of deacetylation (DA) of 92.8%, could be a good substrate
for promoting the repair of osteochondral full-thickness defects.
The tested graft were cylinders scaffolds of chitosan and D (+) raffinose2, with a diameter of 2.6 mm and a length of 5 mm. They
were analysed by scanning electron microscopy (SEM) and morphometric analyses to evaluate the three dimensional structure and
the porosity before and after the grafting. Six male White New Zealand rabbits were used for this study. We created full-thickness
cartilage defects, diameter of 2.7 mm, of knee joint on medial condyle and trochlear groove of the distal femur, which are respectively bearing and not bearing area. After 2 and 4 weeks from the insertion of the scaffolds, we evaluated the reparative response
using as reference a surgical control. For the SEM analysis samples were fixed with 2,5 % glutaraldehyde in 0,1M sodium cacodylate buffer (pH 7,3). They were dehydrated and then critical-point-dried with liquid CO2 (CPD 030 Baltec). Subsequently, they were
sputter-coated with a gold-palladium layer using a SCD 0,40 coating device (Balzer Union) and observed using a Zeiss DSM 950
Scanning Electron Microscope at 10 kV. Samples were fixed (4% formalin) and decalcified (Diapath). For the histological analyses,
samples were paraffin embedded (Bio-Optica), 4-µm thick sections were cut from the paraffin blocks and then they were stained.
All samples were evaluated both in gross morphology and with the O’Driscoll reported method3; moreover we investigated collagen
fibers density and orientation by means of polarized microscopy. The data collected were grouped by descriptive statistics, evaluated by the analysis of variance (ANOVA) with 3 factors and 3 replications and by post hoc test (Tukey, Scheffe).
SEM analyses showed that scaffolds could be divided into two circumferential areas. Specifically, the outer one has a lower average
pore area than the inner one, with a difference that is statistically highly significant both before and after the graft (p <0.01). Also,
there were no changes of porosity of each scaffold due to the graft. The observed knee gross morphologies showed no significant
differences between controls and treatment after 2 weeks, instead they were detectable after 4 weeks and only in the trochlear groove. Histological analyses of the newly formed tissue, in both experimental times, do not highlight the structure and cell morphology
characteristic of hyaline cartilage tissue. The scoring results obtained by histological observations indicate that scaffolds grafted in
medial femoral condyles (bearing area) were not well- performing if compared with surgical controls (p ≤ 0.01) in both experimental times. On the other hand, the trochlear groove showed a significant difference between treated and controls only after 4 weeks (p
≤ 0.01). We also noticed that treated trochlear groove defects had a better score than treated medial condyles, although these data
were no statistically significant. Analysis in polarized light on treated samples resulted in the formation of areolar connective tissue
inside bearing areas and compact connective tissue in not bearing areas. As expected, surgical controls showed the generation of
fibrocartilage.
CONCLUSION
Our study suggest that tested material does not show an optimal tissue regeneration from a quantitative point of view, but the quality
of newly generated tissue is not minor. We assume that structure improvements of chitosan and D (+) raffinose scaffolds would
make this material suitable biomaterial to regenerate cartilage tissue.
REFERENCES
1. Ge Z. et al., J Biomed Mater. Res. A. Epub, 2012
2. Bettini R. et al., Eur J Pharm Biopharm. 68(1):74-81, 2008
3. O’Driscoll S.W. et al., J Bone Joint Surg. Am. 68:1017–1035,1986
16
SILK FIBROIN PROSTHESIS EVALUATION FOR ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION
Masciale Valentina¹, Sartori Maria², Maglio Melania¹, Giavaresi Gianluca¹,², Farè Silvia³, Tanzi Maria Cristina³, Alessandrino Antonio4, Freddi Giuliano4, Fini Milena¹,², Giardino Roberto²
1 Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopaedic Institute, Bologna, Italy
2 Laboratory of Laboratory of Biocompatibility, Innovative Technology and Advanced Therapy, RIT-Rizzoli Orthopaedic Institute, Bologna, Italy
3 Biomaterials Laboratory, Bioengineering Department, Politecnico di Milano, Milano, Italy
4 Innovhub-Stazioni Sperimentali per l’Industria, Div. Stazione Sperimentale per la Seta, Milano, Italy
[email protected]
INTRODUCTION
The anterior cruciate ligament (ACL) is the ligament of knee mostly affected by traumatic lesions which cause mechanical instability, pain
and damage to the medial collateral ligament, chondral and meniscal lesions until developing secondary osteoarthritis¹. The intra-articular
environment and the poor regenerative capabilities of ACL prevent spontaneous healing of the damaged ligament¹. Consequently, the surgical reconstruction based on the autologous tissue transplantation (patellar and semitendinosus tendons) represents the “gold standard”
for the ACL reconstruction surgery1,2. However, the laxity of ligament and the morbidity of sampling sites, can have a negative impact on
the post-operative rehab and on the complete recovery after the ACL reconstruction3,4. Moreover, the prosthesis currently used can be
affected by complications such as poor integration with damaged tissue and/or inflammatory processes caused by a foreign body. The
aim of this study was to assess the biocompatibility, the integration ability of a silk fibroin 3D textile structure with joint, peri-implant bone
tissue and native ACL in large animal model5,6.
The research protocol was approved by the Ethics Committee of Rizzoli Orthopaedic Institute and by public authorities in agreement with
the Law by Decree 116/92. Ten adult sheep underwent to surgical reconstruction of ACL (right knee) through a femoral-tibial tunneling in
which the silk fibroin textile structure was inserted. The controlateral knee was left untreated as control. The animals were divided in two
groups of five animals each and at the end of the experimental times, 3 and 6 months, the joints were explanted and a semiquantitative
score was applied (Joint Damage Score)7,11. After macroscopic evaluation, the joint was analyzed by computed microtomography, µ-CT
(Skyscan 1172) in order to evaluate the tunnel volume and the bone structure around silk fibroin structure. Thus bone samples were
embedded in resin for qualitative histological evaluations.
Macroscopic evaluations of the articular surfaces showed a significative difference
between the operated limb against the healthy controlateral, while no significative
differences were detected between the two experimental times. Microtomographic
evaluations of bone tunnel volume showed an enlargement of tunnel volume with
no differences between 3 and 6 months. Histological evaluation highlighted at three
months the formation of a fibro-vascular tissue between the silk fibroin textile structure and bone tissue, in agreement with other preclinical studies. At six months a
progressive organization and maturation of fibro-vascular structures can be observed (Figure 1), with histological and morphological characteristics which make them
similar to a ligamentous tissue.
a
b
Figure 1
Histological images of
bone tunnel after six
months from implant,
stained with Toluidine
Blue and Fast Green:
(a) bone tunnel (4x);
(b) bone tunnel with
polarized filter, where
“Sharpey like-fibers”
are visible (arrows)
(4x); FC: collagen
fibers; O: bone tissue;
P: fibroin structure.
CONCLUSION
The obtained results highlight the presence of remodeling phenomena and progressive organization of the structures around the silk fibroin
structure, that support its integration ability with surrounding structures. Further studies at longer experimental time are necessary to
improve these findings and to investigate the progression of the regenerative process.
REFERENCES
1. Freeman JW, Afr J Biotechnol. 8:7182-7189, 2009
2. Frank CB. et al., J Orthop Res. 30:384-392, 2012
3. Petrogliano FA. et al, Arthroscopy. 22:441-451, 2006
4. Fan H. et al., Biomaterials. 29:3324-3337, 2008.
5. Altmann GH. et al., Biomaterials. 23:4131-4144, 2002
6. Seo YK. et al., J Orthop Res. 27:495-503, 2009
7. Drez DJ Jr. et al., Am J Sports Med. 19:256-263,1991
8. Jackson D. et al., Am J Sports Med. 20:644-656, 1992
9. Cummings JF. et al., J Orthop Res. 20:338-345, 2002
10. Le Graverand MP. et al., J Orthop Res. 20:535-544, 2002
11. Heard BJ. et al., J Orthop Res. 29:1185-1192, 2011
17
EFFECTIVENESS OF A PURE ORIENTED COLLAGEN I MEMBRANE IN TENDON REPAIR: AN EXPERIMENTAL STUDY.
Monica Mattioli-Belmonte¹, Alberto Busilacchi¹, Alessandra Giuliani², Barbara Lonzi³, Sandra Manzotti¹ and Antonio Gigante
1 Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy.
2 Department of Clinical Sciences and Stomatology, Università Politecnica delle Marche, Ancona, Italy
3 Department of Industrial Engineering and Mathematical Sciences, Università Politecnica delle Marche, Ancona, Italy.
[email protected]
INTRODUCTION
Tendons have poor spontaneous regenerative capabilities, and complete regeneration is never achieved despite intensive remodelling¹.
Finding suitable tendon replacement material has proved difficult; autografts are associated with substantial donor site morbidity, whereas cadaveric allografts may trigger an adverse immune response, besides being limited in supply2. Considerable effort has therefore
been devoted to finding artificial substitutes for tendon lesion repair or filling. Since fiber architecture and alignment confer a number of
functional properties, architecture must be one of the first point when engineering replacement constructs. In this experimental study we
characterized a multilamellar collagen I membrane with oriented collagen fiber deposition and we assessed its safety and tendon regeneration properties in an experimental animal model.
For the in vitro study human dermal fibroblasts and tenocytes were seeded onto
the multilamellar membrane obtained from purified equine Achilles tendon. Evaluation of the effect of fiber orientation on cell viability and cytoskeletal organization
was performed. In vivo study was performed in New Zealand White rabbits. The
central third of the patellar tendon (PT) of 10 animals was sectioned and grafted
with the collagen membrane; the contralateral PT was sectioned longitudinally and
sutured (Figure 1). Animals were euthanized 1 or 6 months from surgery and tendons subjected to histological and Synchrotron Radiation-based Computed Microtomography (SRµCT) examination and 3D structure analysis. ligamentous tissue.
Figure 1. Drawing showing tendon excision and application
of the collagen membrane to the treated tendon; the control
lesion was merely sutured.
In vitro results demonstrate that the multilamellar collagen I membrane with oriented fibers
has good mechanical properties and affords optimum cell proliferation and adhesion. Its
fiber arrangement provides an instructive pattern for cell growth (Figure 2) and may serve
to guide the alignment of cells migrating from the ends of a crushed or frayed tendon to
obtain a strong, correctly structured tendon, thus providing a viable clinical option for tendon repair. Histological and µCT findings of in vivo experimentation showed satisfactory
membrane integration with native tendon. Histological examination also showed ongoing
angiogenesis in treated tendons with no adverse side-effects (inflammation, rejection, calcification). Therefore the multilamellar collagen I membrane can be considered as a safe
and effective tool for tendon repair and augmentation.
Figure 2. SEM micrographs of tenocytes cultured
on the collagen I membrane with cells extending
along the collagen fibers.
REFERENCES
1. Lin TW. et al., J Biomech 37: 865-877, 2004.
2. DeFranco et al., J Am Acad Orthop Surg 12: 298-304, 2004
ACKNOWLEDGMENTS
Authors wish to thank: Dr. B. Parma (Opocrin, Corlo di Formigine, Italy) for the collagen I membranes and Dr. F. Orlando for veterinary
controls. They also acknowledge ELETTRA (Trieste, Italy) and ESRF (Grenoble, France) User Offices for providing beam-time.
18
NERVE REGENERATION INSIDE COLLAGEN-BASED ARTIFICIAL NERVE-GUIDE: A SECOND-LOOK IN HUMANS
Antonio Merolli, Lorenzo Rocchi, Rocco De Vitis
Orthopaedics & Hand Surgery Unit, The Catholic University in Rome
“Columbus” Complex, via Moscati 31, I-00168 Rome, Italy
[email protected]
INTRODUCTION
We implanted nerve-guides in patients with gap-injuires up to 30 millimeters, from elbow to fingers. Most of patients were treated in
emergency and presented associated lesions. We proposed a collagen implant (NeuroMatrix, Stryker; or, Neuragen, Integra) as an option
to autologous grafting or neurectomy, informing the patients that a second operation could be enterprised like, for example, a tenolysis
in multiple tendon lesions. During these successive surgery we had a second-look at nerve-guides and assessed macroscopically what
was inside.
MATERIALS AND METHODS
In the early 50 implants we treated 15 digital; 11 metacarpal; 10 median nerves; 7 ulnar; 6 superficial radial nerves. We performed 21
second-look: 6 in digital; 2 in metacarpal; 5 in median; 4 in ulnar; 4 in superficial radial implants. Of the remaining 29 implants, in 7 there
were no associated lesions requiring a second-look. Protective sensory recovery and motor function was assessed by clinical examination.
RESULTS
Digital implants showed a good sensory recovery and absence of painful neuroma. Guides in proximity of a joint did not add any discomfort and, at second-look, did not show collapse. Second look after 4 months showed a nerve-regenerate inside all guides. In
metacarpal implants there was absence of painful neuroma, however the sensory recovery was obtaind only in four patients. In median
implants there was absence of painful neuroma and sensory recovery in all cases, however motor recovery was not obtained in six cases;
furthermore, electrophysiological studies suggested that a Martin-Gruber anastomosis was performing most of the job in motor recovery.
Second-look after 9 months showed a regenerate of variable entity. In ulnar implants there was absence of painful neuroma but sensory
recovery was present in only two short-gap lesions and motor recovery in only one; second-look after 9 months showed a regenerate only
in the guide with recovery. In all superficial radial implants there was, notably, absence of painful neuroma; sensory recovery was present
in five cases. Second-look after 9 months showed a regenerate in highly degraded guides.
DISCUSSION
Despite the wealth of animal studies on nerve regeneration, macroscopic observations of nerve regeneration in Humans are rare and
mostly limited to case-reports. We pursued a sistematic approach and were able to gather an higher number of patients. Our data are in
accordance with what is known from in-vivo animal study, but the fact that they are collected in humans strengthen their clinical relevance.
CONCLUSIONS
Pain was never recorded in all patients in this serie. An adequate sensory recovery occurred in all patients where a nerve-regenerate was
found inside the guide. The degree of degradation appeard variable and not directly related to the time passed. Motor recovery seemed
to be confined only in gap-lesion shorter than 10 millimeters. All patients which received the second-look operation were satisfied of the
further improvement in their condition.
19
FROM BENCH TO BOARDROOM: CREATING SUCCESSFUL START UP COMPANIES BASED ON ACADEMIC SCIENCE
Eric Elenko
Pure Tech Ventures
Boston, Massachusetts, USA
A key goal of many academicians is seeing their work help patients and a large amount of academic research is driven by a desire to
improve human health. In order for breakthrough discoveries made in the lab to reach large numbers of patients, the discoveries must be
commercialized into actual products. Starting a company focused on translation of a discovery is a way of ensuring that the maximum
potential utility of that discovery is realized. A startup company has the ability to attract funding from the private sector which can enable
it to engage in the required capital intensive activities needed for a product to either gain regulatory approval or become mature enough to
be of interest to larger, more established companies. While starting a life science focused company holds the promise of both scientific
and financial reward, success rests on taking into account a number of critical factors and avoidance of potential pitfalls. This talk will
focus on key considerations in starting a new company focused on translating academic research and the unique venture creation model
being pursued by PureTech Ventures in translating academic research into nascent startups.
20
X-RAY MICRO-CT FOR VISUALIZATION OF 3D DISTRIBUTION OF CELLS SEEDED ONTO A POLYMERIC SCAFFOLD
Maglio Melania¹, Parrilli Annapaola2, Masciale Valentina1, Salerno Aurelio3,4, Netti Paolo Antonio3,5, Rimondini Lia6, Fini Milena1,2,
Giardino Roberto2
2 Biocompatibility, Technological Innovations and Advanced Therapies Laboratory (BITTA)
Rizzoli Orthopaedic Institute, Bologna, Italy
3 Interdisciplinary Research Centre of Biomaterials, University of Naples Federico II, Naples, Italy
4 Institute for Composite and Biomedical Materials, National Research Council, (IMCB-CNR), Naples, Italy
5 Centre for Advanced Biomaterials for Health Care (CRIB-IIT), Italian Institute of Technology, Naples, Italy
6Department of Medical Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy
[email protected]
INTRODUCTION
Skeletal lesions due to injuries and degenerative joint disease are more and more frequently related to lifestyle and longer life expectancy.
The need to restore or regenerate tissues cannot be achieved by autografts because of their limited availability and marked associated
morbidity rate. Regenerative medicine, with the use of biocompatible and bioactive materials, growth factors and cells, can be a new chance to overcome these limits1. Consequently, new techniques have been developed to display the architecture of materials and especially
the cellular distribution inside the constructs2. The aim of the present study is to describe the use of the microtomographic approach (μCT) to observe biomaterial colonization in term of cell localization and make direct comparison with SEM images of biomaterial surface.
Porous PCL scaffolds with a bi-modal pore size distribution and with suitable characteristics for bone tissue engineering were prepared
for the cell adhesion. Osteoblastic-like cells from human osteosarcoma, MG63, were seeded at different densities (100k, 200k and 400k
cells/cm2) onto PCL scaffolds, while PCL alone was used as control. Tridimensional MG63 colonization and proliferation were assessed
at two experimental times, one and two weeks. The specimens and the controls were analyzed by microtomographic system Skyscan
1172 before the gold-coating for SEM analysis. Three scaffolds were also analyzed without any staining to evaluate their porosity and
interconnectivity. The pixel size was 2.5 μm and the scan duration was nearly one hour. DataViewer software was used to identify surface
sections and allow the direct comparison with SEM surface images.
PCL scaffolds showed a fair porosity value (43.04 % ± 1.17%) and perfect interconnectivity (99.74% ± 0.12%) to allow cell colonization inside the scaffolds and the
contrast agent to pass through them. Differences in cell proliferation were observed
as a function of the starting seeding density. In particular both at one week and at
two weeks after seeding, the grey level contrasts were stronger with a cell density of
100k cells/cm2 or 400k cells/cm2. Conversely, the images obtained from the 200k
cells/cm2 seeding were less bright. The 3D results of cell density within the scaffold,
expressed as ratio between the volume of cells detected and the total volume of the
scaffold, show an increase over time for all concentrations of cells seeded (Figure 1).
Figure 1. 3D
models of
PCL scaffolds
seeded with
different
concentration
of MG-63 cells
at one and two
weeks after
seeding
CONCLUSION
SEM and μ-CT might be two complementary approaches for the evaluation of tissue engineering techniques that combine biomaterials and
cells: SEM is useful to investigate the biomaterial surface, whereas μ-CT allows the scaffold to be assessed three-dimensionally in terms
of cell colonization and increasing material degradation rate3. Besides, due to its 3D analysis nature, μ-CT allows the combination of the
results derived from qualitative surface analysis improving the understanding of cell colonization process. Furthermore, the morphological
results of this study suggest that a suitable balance between different factors is necessary for the correct scaffold colonization and for
the subsequent extracellular matrix synthesis. Not only can scaffold properties such as porosity, pore size and pore geometry be tailored
to dictate the mechanism of tissue regeneration and the structure of the resulting tissue, but so can the way in which cells proliferate and
adhere to three-dimensional scaffolds.
REFERENCES
1. Sundelacruz S. et al., Semin. Cell Dev. Biol. 20:646-655, 2009
2. Dorsay S.M. et al., Biomaterials. 30:2967-2974, 2009
3. Chen Y. et al., Biomaterials. 32:5003-5014, 2011
21
POROUS POLYURETHANE COMPOSITES AS SCAFFOLDS FOR BONE REGENERATION:
COMPARISON BETWEEN MICRO- AND NANO- SIZED HA FILLERS
Serena Bertoldi, Silvia Farè, Maria Cristina Tanzi
Biomaterials Laboratory, Bioengineering Department, Politecnico di Milano, Milan, Italy
[email protected]
INTRODUCTION
Composite materials can be used for scaffold production, thus merging the appealing properties of polymers, in terms of chemicophysical properties and ease of fabrication into 3D porous structures, with mechanical strength and osteoconductive properties of phosphate ceramics [1]. Among polymer matrices, polyurethanes (PU) combine versatility in reagents chemistry, mechanical properties and
biological performance with easy processability into porous structures. We have developed biocompatible PU foams via a gas foaming
procedure by use of water as expanding agent during synthesis [2]. Their use as scaffolds able to support cell adhesion [3], and to be
subcutaneously integrated in the rat model [4] was also investigated. This work was aimed at investigating the development of PU-based
composites by use of micro- and nano- sized hydroxyapatite (HA) fillers, and to comparatively investigate their properties and their potential use as scaffold for bone tissue engineering.
Porous PU composites were synthesized with a co-expansion process in the presence of the same weight (25% w/wpolyol) or the same
volume (8.1 cm3) of micro HA (A6021, Plasma Technik, PU-Cmicro) or nano HA (ad hoc sintered, PU-Cnano1 and PU-Cnano2). MDI
prepolymer (Bayer, Germany), polyether-polyol mixture, FeAcetyl Acetonate as catalyst and water (2% w/wpolyol) as expanding agent
were used for PU foam preparation. The name PU-Cnano1 identifies the composite produced with the same weight of HA used to obtain
the PU-Cmicro, whereas PU-Cnano2 is produced with the same volume of HA. A PU-foam was synthesized as control. Crystallinity of
micro- and nano- sized HA powders was investigated by X-ray diffractometry (XRD, Philips PW1710) and granulometry by SEM (Stereoscan Cambridge 360). Morphology of PU-foam and PU-Composites was characterized by SEM, density, average pore size, open pore
percentage and pore interconnection by micro-CT (Skyscan 1172). Mechanical compressive properties were investigated under wet
conditions in distilled water (Instron 4301).
As observed by XRD, micro-sized HA showed higher cristallinity when
compared to the nano-sized one, whose diffraction pattern showed wider
peaks, typical of a nano-sized powder. Physico-morphological properties
of PU-foam and PU-Composites are reported in Table 1. The addition of
both an equal weight (25% w/wpolyol) or an equal volume (8.1 cm3) of
micro- or nano-sized HA increased the material density, in comparison
with the PU matrix (p>0.05), however the expansion process had no influence, as confirmed by the satisfactory values of open porosity and average pore size of the composites.
Table 1: Properties of PU-foam and PU-Composites
The presence of micro-HA improved the compression
stiffness and strength in comparison with the PU-matrix
(p>0.05).
Instead, the addition of a 25% w/wpolyol amount of nanosized HA (PU-Cnano1) resulted excessive, as it weakened
the resulting material (Figure 1). This result can be attributed to the higher surface area of nano-sized HA in respect
to the micro-sized one. In fact, when the same volume,
and therefore the same surface area, of micro or nano
HA was used, no differences in the mechanical properties were observed between PU-Cmicro and PU-Cnano2
(p<0.05), and both types of composites showed higher
stiffness when compared to the PU matrix.
Figure 1: Compressive properties of PU-foam and the different P
Composites testes in wet conditions
CONCLUSIONS
The proposed co-expansion method, not affecting the foams morphological properties, can be considered a valid technique for PU-Composite scaffolds preparation. Preliminary results of in vitro assays indicated good cytocompatibility for all PU-Composites obtained with
micro- and nano-sized HA. Future developments are focused on the production of PU-Composites with a reduced amount of nano-sized
HA, also varying its cristallinity (Progentix Orthobiology BV).
1. Hutmacher D.W. et al., J Tissue Eng Regen Med. 1:245-60, 2007
2. Guelcher S.A et al., Tissue Eng B. 14:3-17, 2008
3. Bertoldi S. et al. J Mater Sci: Mater Med. 21:1005-11, 2010
4. Farè S. et al. 2007 SFB Annual Meeting, p.190.
22
BIOMIMETIC GELATIN/POLY(L-LACTIC ACID) COMPOSITE SCAFFOLDS BY CO-ELECTROSPINNING
Andrea Fiorani¹, Michela Gioffrè², C. Gualandi,a M. L. Focarete,a S. Panzavolta,b B. Bracci,b A. Bigib, P. Torricelli³
1 Polymer Science, Department of Chemistry “G. Ciamician”, University of Bologna, Italy,
2 Biomimetics and Material Chemistry, Department of Chemistry “G. Ciamician”, University of Bologna, Italy
3 Preclinical and Surgical Studies Labor atory, Codivilla Putti Research Institute - Rizzoli Orthopaedic Institute, Bologna, Italy
[email protected]
INTRODUCTION
The basic approach to bone tissue engineering involves the
development of highly porous biodegradable 3D-scaffolds, with
interconnected pore network structure for cellular in-growth,
revascularization, adequate nutrition and oxygen supply.
Electrospinning is a simple and cost-effective technique that enables
to fabricate scaffolds, from both synthetic and natural polymers,
mimicking the three-dimensional nano-scaled features of ECM¹. Synthetic bioresorbable polymers provide structural functionalities to the
scaffold; on the other hand, natural polymers display unique bioactive properties and excellent cellular affinity2. To combine natural and
synthetic polymers, electrospinning of blends has been proposed as
a useful strategy, such as in the case of polylactic acid and gelatin3,5.
In the current work a different strategy named concomitantly electrospinning (co-electrospinning ) is presented. Nanofibrous scaffolds
made up of poly(L)lactic acid (PLLA) fibers and Type A Gelatin (Gel)
fibers were fabricated by co-electrospinning with the aim of combining the bioactivity of gelatin together with the structural stability of
PLLA in a unique scaffold.
PLLA was dissolved at a concentration of 13% (w/v) in DCM/DMF
(65/35, v/v) whereas gelatin solution was 30% (w/v) in acetic acid/
double distilled water (60/40, v/v). Mats containing different amounts
of PLLA and gelatin fibers were produced by changing solutions flow
rates during the process. Gelatin content in the as electrospun mats
was evaluated by detecting the amount of gelatin released in water by
Biuret assay. In order to prevent gelatin fiber dissolution in physiological environment, gelatin was crosslinked through scaffold immersion in 5% (w/v) genipin solution in ethanol for 7 days at 37°C6.
In this study we demonstrated that the co-electrospinning method
allows to obtain scaffolds displaying a homogeneous distribution
of bead-free and regular gelatin and PLLA fibers with diameters in
the range of 500-600 nanometers. Scaffolds with PLLA/Gel ratio of
about 30/70, 50/50 and 70/30 w/w were fabricated. To verify the
distribution of the two types of fibers in the scaffold, PLLA and gelatin solutions were mixed individually with different fluorescence
probes: FITC for gelatin and Rhodamine B for PLLA (Fig. 1). An optimized crosslinking procedure was applied to retain the nanofibrous
morphology after exposure to aqueous
solution and to achieve scaffold stability in physiological environment. Mechanical properties were found to depend on scaffold
composition. In particular, the scaffold elastic modulus decreased as
PLLA content increased. Scaffold were used for in vitro cell culture to
test biocompatibility and cells proliferation.
Fig. 1
SEM micrograps
and laser confocal
fluorescence images
of electrospun mats.
Bar in fluorescence
images: 50 μm.
CONCLUSION
In this work we report the fabrication of electrospun mats
made of mixed PLLA and gelatin fibers by the co-electrospinning technique. We verified the possibility to tune scaffold properties by acting on mat composition.
Furthermore, preliminary in vitro tests indicate that the scaffolds support chondrocytes proliferation and growth.
REFERENCES
1. Agarwal S. et al., Polymer. 49:5603-5621, 2008
2. Sionkowska A., Prog. Polym Sci. 36:1254– 1276, 2011
3. Chong E.J. et al., Acta Biomater. 3:321–330, 2007
4. Kim H.W. et al., J. Biomed. Mater. Res. Part A. 87A:25–32, 2008
5. Shu-Ying G. et al., Mat. Sci. Eng. C. 29:1822–1828, 2009
6. Panzavolta S. et al., Acta Biomater. 7: 1702–1709, 2011
AKNOWLEDGEMENTS
This work was supported by MIUR
(FIRB °RBAP10MLK7).
23
EXPERIMENTAL DETERMINATION OF LOW TEMPERATURE DEGRADATION BEHAVIOUR
OF A NEW ZIRCONIA-ALUMINA COMPOSITE – PRELIMINARY RESULTS
Corrado Piconi¹ , Paride Fabbri2, Francesca Mazzanti2, Giuseppe Magnani2, Emiliano Burresi2, Paola Nitti1, Claudio Mingazzini2
1 GHIMAS SpA, MeLab, Brindisi, Italy,
2 ENEA, Faenza Technical Unit on Material Technologies (UTTMATF), Faenza (RA), Italy
[email protected]
INTRODUCTION
Zirconia Toughened Ceramics (ZTCs), especially Yttria- Tetragonal Zirconia Polycrystal (YTZP), are in clinical use since the second half of
the 1980s [1]. So far the main use of YTZP is in dentistry for fixtures, crowns and bridges [2] while in ball heads for Total Hip Replacements
(THR) it was abandoned after the year 2000 due to the worldwide withdrawal of this kind of ceramic components [3]. Zirconia still fulfils the
role of toughening phase in ceramic composites, and especially Alumina Matrix Composite is a well established material in orthopedics [4].
The metastable nature of the tetragonal phase of the zirconia polymorph is the root of the mechanical behaviour of ZTCs, that are exploiting
the zirconia tetragonal-to-monoclinic transformation in the process zone ahead of a crack tip as dissipative mechanism of the fracture
energy. It is well known that due to its metastable nature the tetragonal phase can switch spontaneously to monoclinic, a behavior known
as Low Temperature Degradation (LTD). The nucleation of monoclinic phase, that starts on the surface, eventually leads to degradation
and loss in strength of the ceramic components [5]. In view of their use as biomaterials, the kinetic of LTD in ZTCs should then be carefully
assessed, because this behaviour – which is enhanced by the humid environment – may result the main limiting factor of the lifetime of
ZTC components.
Disk-shaped samples of zirconia-alumina composite (40/60 wt% ) have been obtained from a mechanically blended batch of commercial
zirconia and alumina powders, that had been cold isostatically pressed before firing, following a route described elsewhere [6].
Hydrothermal treatment of the disks has been performed in saturated steam at 134°C and 120 °C in autoclave and in distilled water at 90 °C.
Monoclinic content has been assessed according to the International Standard ENV 14273 [7].
The relationship between the monoclinic phase and the aging time at different temperatures is well described by a sigmoidal curve,
according to Johnson-Mehl-Avrami equation [8,9]. The amount of monoclinic volumetric fraction observed increases with the aging time
and temperature up to about 90 % of the total zirconia content.
The rate of monoclinic formation obtained at the different aging temperatures is lower then the ones reported by other Authors for YTZP
either for ZTC composites [8,9]. The value of the process activation energy can be calculated, following the Arrhenius kinetic equation, in
about 115 kJ/mol, that indicates the good hydrothermal stability of this new composite. After 10 hours treatment at 134°C in steam, that
are corresponding to about 40 years in vivo [10], only about 12 vol% of the zirconia contained in the material is in the monoclinic state.
CONCLUSION
The aging kinetic of the ZTC developed by ENEA, as well as the mechanical properties and biological safety of the material already reported
[6,11], characterize this ceramic composite as potentially suitable for applications in load bearing components of medical devices.
REFERENCES
1. Piconi C and Maccauro G, Biomaterials 1999; 20:1-25.
2. Piconi C, Rimondini L, Cerroni L. La Zirconia in Odontoiatria. Elsevier Masson, Milano 2008.
3. Piconi C, et al. J Mater Sci Mater Med 2006;17:289-300.
4. Piconi C, Maccauro G, Muratori F. Key Eng Mater 2005; 284-286: 979-82.
5. Piconi C, et al. Hip Intl 2007;17:119-130.
6. Magnani G and Brillante A. J Eur Ceram Soc 2005; 25: 3383-92.
7. UNI ENV 14273 (2002).
8. Schneider J, et al. J Am Ceram Soc 2008; 91:3613-8.
9. Chevalier J, et al. J Am Ceram Soc 1999; 82:2150-4.
10. Pezzotti G. et al. J Am Ceram Soc 2009;92:1817-22.
11. Maccauro G, et al. Int J Immunopathol Pharmacol 2009; 22 : 773-9.
ACKNOWLEDGMENTS
This activity has been performed as part of the ENEA-GHIMAS R&D agreement.
24
SUPERPARAMAGNETIC BIO-MIMETIC HYBRID COMPOSITES
Anna Tampieri¹, Teresa D’Alessandro¹, Monica Sandri¹, Silvia Panseri¹,², Carla Cunha¹,²
1 Institute of Science and Technology for Ceramics, National Research Council, Italy
2 Laboratory of Biomechanics and Technology Innovation, Rizzoli Orthopaedic Institute, Italy
[email protected]
INTRODUCTION
In the last decade bio-inspired design and manufacture of scaffolds for tissue engineering have received increasing interests to provide
the microenvironment similar to the natural extracellular matrix (ECM) for regenerative cells and tissues¹. The reproduction of biomimetic
conditions of bone formation allows to obtain hybrid constructs where the mineral phase is nucleated upon guidance by the chemical
features and physical confinement imposed by the polymeric matrix. The chemic-physical and ultra-structural feature of the nucleated mineral phase resemble the neo-formed natural bone.2,3 With the purpose to increase the osteogenic and angiogenic capacity of biologically
inspired scaffolds for bone and osteochondral regeneration, through magnetically-driven release of specific growth factor, superparamagnetic bio-hybrid composites were performed nucleating on collagen fibres an iron doped hydroxyapatite (Fe-HA) endowed with intrinsic
superparamagnetism.
To perform a bio-inspired mineralization process, an aqueous solution of phosphoric acid (H3PO4) was added to a 1 wt% collagen gel
and dropped in a basic suspension of calcium hydroxide (Ca(OH)2) previously enriched with Fe3+ and Fe2+ iron ions. Suitable amounts
of reactive allow to prepare composite materials with different Fe-HA/Coll wt% ratios (i.e. 70/30 and 40/60). Afterwards, in order to realize
scaffolds resembling the morphological features of the osteochondral region, three different composites with different mineralization and
magnetization grade were prepared, layered and freeze-dried: i) the upper one, mimicking the cartilagineous layer, constituted by pure
Collagen; ii) the intermediate one, mimicking the tidemark, formed by Fe-HA/Coll 40/60 wt%; iii) the lower one, mimicking the subchondral
bone, composed by Fe-HA/Coll 70/30 wt%. XRD, SEM, TEM, chemical and magnetic characterization were performed on the developed
materials. In-vitro studies were conducted to evaluate cell adhesion and proliferation in each single layer. Fe-HA/Coll(70/30)wt% scaffolds
were implanted in rabbit femoral condyle, and in addition 1.2 T cylindrical NdFeB magnets were implanted in direct contact to the scaffolds to achieve a good magnetic fixation. Bone regeneration was investigated paying attention to any effects of magnetic field and to the
magnetic fixation.
Chemic-physic, morpho-structural and magnetic characterization put in evidence that the careful set up of synthesis conditions allowed
the nucleation of Fe-HA nano-particles with defined substitution of Ca2+ by Fe2+/Fe3+ ions, with very limited amount of magnetite as
secondary phase. As demonstrated from the X-ray analysis in flat chamber, where the nano-sized HA crystallites grow inside collagen
fibres with their c axes preferentially oriented parallel to the direction of orientation of the fibres. From the magnetic characterization, the
Fe-HA/Coll composites result endowed with superparamagnetic and hyperthermic properties under the effect of an alternating electromagnetic field. By XRD combined with computer simulations on structural models, the clear indication that both Fe species are in Casubstitutional positions and not in interstitial positions into the HA lattice was obtained. The position Ca(2) with sixfold coordination and
Ca(1) with fourfold coordination are occupied in a reciprocal ratio so that to give magnetic susceptibility to the Fe-HA owing to strictly
defined synthesis parameters5. From in-vitro and in-vivo tests, these new biohybrid material showed optimal cell adhesion and proliferation and capability of bone regeneration.
CONCLUSIONS
Innovative magnetic scaffolds were developed, totally free from iron oxides, based on collagen (Coll) and a biocompatible and bioresorbable superparamagnetic inorganic phase made of iron doped hydroxyapatite (Fe-HA).5 The magnetic properties and the bioactivity of the
new Fe-HA/Coll bio-hybrid composite together with its biocompatibility may open the door of the regenerative medicine to a conceptually
new family of biomimetic scaffolds for tissue regeneration, able to be biologically manipulated or activated in situ by means of an external
magnetic field.
REFERENCES
1. H. J. Chung, T. G Park, Advanced Drug Delivery Reviews 59:249-262, 2007.
2. Mann, S. (ed.) Bio-mineralization: Principles and Concepts in Bioinorganic Materials Chemistry, Oxford University Press 2001
3. A.Tampieriet al.J.Biomed.Mater.Res.67A:618-25, 2003
4. Tampieri A et al. Nanotechnol. 22:015104 (8pp), 2011.
5. A. Tampieri et al. WO2012014172 Intrinsically Magnetic Hydroxyapatite.
AKNOWLEDGEMENTS
European Project Magnetic Scaffolds For In Vivo Tissue Engineering Acronym: “MAGISTER” NMP3-LA-2008-214685 (2008-13)
25
THE EFFECTS OF HOLLOW FIBER MEMBRANE (HFM) GEOMETRY AND TRANSPORT PROPERTIES
ON THE PERFORMANCE OF HFM BIOREACTORS FOR BONE TISSUE ENGINEERING
Ilaria E. De Napoli¹, Elisabetta M. Zanetti², Alberto L. Audenino³, R. Quarto4, G. Catapano¹
1 University of Calabria, Rende, 2 University of Perugia, Perugia, 3 Politecnico di Torino, Torino,
4 CBA University of Genova, Genova. Italy
[email protected]
INTRODUCTION
In most bioreactors currently used for bone tissue engineering, O2 and nutrients starvation causes formation of a necrotic core in osteogenic cell constructs just a few mm´s large. Hollow fibre membrane bioreactors (HFMBs) in which cells are cultured around and among
the membranes and medium flows in the membrane lumen feature a distributed and delocalized O2 and glucose supply similar to naturally
vascularized bone tissue. Recently, they have been shown to be promising to culture osteogenic cells for bone tissue engineering (TE)
[1]. So far, models have been proposed to optimize HFMB design in which the membranes are usually considered a separation barriers,
the geometry, wall structure and transport properties of which are often neglected or accounted for only to the second approximation [2].
In this paper, mathematical models of momentum and mass transport in HFMBs were developed accounting for the actual wall structure
and were used to investigate the effects of membrane geometry and transport properties on HFMB performance for bone TE.
Models are based on a multi-compartment description of HFMBs based on the Krogh cylinder assumption, and on a quasi-steady state
analysis of nutrients profiles for a closed shell HFMB. Membrane wall and the densely cell packed shell is considered an isotropic porous
medium, where momentum is transported according to the Darcy-Brinkmann equation. Momentum continuity applies at the liquid-porous
media interfaces. Relevant non-dimensional parameters were identified, and governing momentum and mass transport equations were
numerically solved for dissolved oxygen and glucose with a finite element commercial code. Membrane geometry and transport properties
typical of ultrafiltration (UF) to microfiltration (MF) membranes were used, so were metabolic parameters for various osteogenic cells..
Dimensional analysis shows that, indeed, membrane wall thickness and radius are present in most dimensionless groups determining
bioreactor performance, hence they directly affect it. Membrane transport properties with respect to the freely permeating species affect
HFMB performance combined with membrane length through the pressure modulus, and the shell-side Thiele modulus.
Model predictions show that membrane wall thickness significantly affects the concentration profiles of dissolved O2 and glucose only
when operated under diffusion control. Under such conditions, membranes with a small wall thickness may ensure culture of cells with
low metabolic requirements up to 106 cells/ml with low incidence of necrotic zones in the bioreactor shell. When the HFMBs are designed
and operated in the presence of high convective flows, increasing membrane wall thicknesses slightly affect nutrients transport towards
the cells.
Consistent with that reported in the course of tracer experiments with HFMBs [3], model predictions show that increasing the membrane
hydraulic permeability (e.g. from that typical of UF to MF membranes) may greatly enhance the effectiveness of nutrient transport towards
the cells. Similarly, decreasing the membrane inner radius enhances nutrients transport to the cells and helps reducing the onset of necrotic zones in the bioreactor. In both cases above, the extent of the enhancement observed depends on the membrane length, as well
as on bioreactor geometry and the conditions under which it is operated. Simultaneous optimization of membrane choice and bioreactor
geometry and operating conditions may permit cell culture above 107 cell /ml, hence close to that in the natural bone, depending on the
actual cell metabolic requirements.
CONCLUSIONS
Albeit often neglected, membrane geometry and transport properties significantly affect HFMB performance. Their choice is crucial to the
design of HFMBs for bone TE at low cell density. At high cell density, the choice of membrane geometry and transport properties should
be optimized simultaneously with the conditions under which the HFMB is operated.
REFERENCES
1. De Napoli I.E. et al., J. Mem. Sci. 379:341-352, 2011
2. Brotherton J.D. & Chao, Biotechnol. Prog. 12:575-590, 1996
3. De Napoli I.E., Catapano G., Int. J. Artif. Organs 33:381-391 2010.
ACKNOWLEDGMENTS
The authors acknowledge the partial financial support (ex 60% MIUR) of the University of Calabria, Rende, Italy.
26
FRACTAL MODELING: MEDICAL DEVICES DESIGNED BY TISSUES
Salvatore Longoni¹, Matteo Sartori¹
1 S. Apollonia Dental Center, R&D Department, Lazzate (MB), Italy
[email protected]
INTRODUCTION
Nowadays many studies show that mechanical distortion of cells
and local geometry drive tissue sculpting during the development of
many different organs. This knowledge should begin to impact the
design and fabrication of medical devices such as dental implants
and biomaterial scaffolds in order to better promote osseointegration and new bone formation.1,2
Starting from Recursive Quadrant Analysis (RQA) of human jawbone microarchitecture3,4, the aim is to present a new reverse engineering process to encode new geometries for medical devices based
on a multi-modular tensegrity fractal model.
.
Human digital jaw bone images were analysed with RQA to identify
the number and structure of their basic fractal components that
were named “islands”. These islands were clusters of quadrants
grouped together on the basis of their size, position and adjacency
relationships. The procedure was performed automatically by a
software conceived by the Authors and it was conducted both for
the colour white (bone trabeculae) and for the colour black (spaces
containing bone marrow). White and black fractal islands were the
starting point for reverse engineering.
To obtain a dental implant thread design each white fractal island
was placed onto Y axis followed by a black fractal island. In order
to enhance bone implant contact, fractal black islands were exposed to bone and fractal white islands were exposed to the implant
core. Biomaterial blocks were designed generating a multi-modular
tensegrity 3d mesh: average fractal white and black islands were
arranged in space in relation to the quadrant number, dimension
and adjacency relationship. They were linked together reproducing
specific morphological dimensions, position and proportion between white trabeculae and black space of human jaw bone.
According to RQA fractal bone quality classification, 3 different implant threads were obtained: high, medium and low quality bone
threads. They were different in terms of shape, pitch and depth
in order to achieve an optimal filling of the intra-trabecular spaces
(Fig 1). Tailored trabecular and cortical artificial bone blocks were
computer designed matching the macro and micro-geometry of the
recipient site. They were exported as *.stl and *.csv files and they
were ready to be produced using bioplotting and/or rapid prototyping technology (Fig 2).
For the first time microarchitecture, size and shape of medical devices are not determined by engineering rules but are directly designed by tissues.
Figure 1: Encoding implants threads
Figure 2: Encoding bone blocks
CONCLUSIONS
This study introduces a new philosophy: bone decoding and with
this data, device encoding. Further research will explore the cellular and clinical response to these devices with a microarchitecture
driven by nature. Moreover, changing the fractal scale, the method
will be also used to analyze tissues and encode specific device
surfaces.
REFERENCES
Ingber D.E., J. Cell. Sci. 116:1397-408, 2003
Ingber D.E., J. Cell. Sci. 116:1157-73, 2003
Longoni S. and Sartori M., Nature Precedings: hdl:10101
npre.2010.4512., 2010
Sánchez I., Uzcátegui G.J. Dent. 39:273-92, 2011
27
X-RAY MICROIMAGING LABORATORY (XMI-LAB)
Cinzia Giannini , Davide Altamura, Rocco Lassandro, Liberato De Caro, Dritan Siliqi, Massimo Ladisa
Istituto di Cristallografia (IC-CNR), via Amendola 122/O, 70126 Bari, Italy
[email protected]
INTRODUCTION
High flux X-ray micro-focus sources have been a prerogative of
synchrotron radiation for many years. Although the best performances can be obtained with synchrotron radiation, brilliances of
4*109 photons/sec/mm2/mR2 can be nowadays obtained for laboratory instrumentation with the new Rigaku Fr-E+ SuperBright
synchrotron-class generators. With these novel rotating anode generators for microfocus X-ray high brilliance sources, detailed material analysis are no more exclusively achieved through synchrotron sources, which are normally overbooked and with an access
conditioned to the submission of a proposal (twice per year) and to
a screening process by a referee committee. The present laboratory
has one of these synchrotron-class microsources, being only 9 the
other Fr-E+ SuperBright microsource present in the world.
REFERENCES:
Giannini, C., Siliqi, D., Bunk, O., Beraudi, A., Ladisa, M., Altamura, D., Stea
S. & Baruffaldi, F. (in preparation).
Altamura, D., De Caro, L., Corricelli, M., Falqui, A., Striccoli, M., Curri M. L.
& Giannini C., (in press 2012) Crystal Growth & Design.
Altamura, D., Corricelli, M., De Caro, L., Guagliardi, A. , Falqui, A., Genovese, A., Nikulin, A. Y., Curri, M. L., Striccoli, M. & Giannini C. (2010) Crystal
Growth & Design 10, 3770-3774
ACKNOWLEDGEMENTS
This laboratory has been entirely financed by the SEED project “Xray synchrotron class rotating anode microsource for the structural
micro imaging of nanomaterials and engineered biotissues (XMILAB)”- IIT Protocol n.21537 of 23/12/2009.
A first-generation-synchrotron-class x-ray laboratory microsource,
coupled to a three pinhole camera, is here presented (Fig.1). It allows i) the small and wide angle x-ray scattering (SWAXS) images
to be simultaneously acquired, ii) SWAXS lens-less scanning microscopy.
As representative applications, the structural complexity of a biological natural material (human bone biopsy), and of metamaterials
(colloidal nanocrystal assembly) are inspected at different length
scales, studying the atomic/molecular ordering by (grazing incidence-) wide angle x-ray scattering and the morphological/structural
conformation by (grazing incidence-) small angle x-ray scattering.
In particular, the grazing incidence measurement geometries are
needed for inspecting materials laying on top of surfaces or buried
underneath surfaces.
CONCLUSIONS
The presented test-cases have been selected in order to provide representative examples on relevant research fields, in line with European Commission Horizon 2020 Framework Programmes for Research and Technical Development objectives. Indeed, the aforesaid
instrumentation will be mainly employed for the study of: Advances
materials, such as nanostructured metamaterials, namely nanocrystal architectures based on self assembled nanoparticles Health
and wellbeing, with particular focus on natural biomaterials studies,
especially bone, teeth.
Fig1. XMI-LAB (a: scheme; b: Fr-E+ SuperBright microsource;
c: SWAXS system)
28
HIGH FREQUENCY VIBRATION ENHANCES THE EXPRESSION OF OSTEOGENIC GENES AND EXTRACELLULAR MATRIX
DEPOSITION IN HUMAN BONE MARROW STROMAL CELLS (HBMSCS).
Gabriele Ceccarelli¹,², Deborah Prè2,3, Laura Benedetti1,2, Marcello Imbriani1,5, Livia Visai2,4,5 and M.G. Cusella De Angelis1,2
1 Department of Public Health, Neuroscience, Experimental Medicine and Forensic, University of Pavia, Pavia, Italy
2 C.I.T, Tissue Engineering Centre, University of Pavia, Pavia, Italy
3 Department of Industrial and Information Sciences , University of Pavia, Pavia, Italy
4 Department of Molecular Medicine, University of Pavia, Pavia, Italy
5 Salvatore Maugeri Foundation IRCCS, Pavia, Italy and International Center for Studies and Research in Biomedicine (I.C.B.), Luxembourg
[email protected]
INTRODUCTION
Human bone marrow stromal cells have the potentiality to differentiate into ligament, tendon, muscle, nerve, endothelium and
bone [1]. Previous studies have demonstrated the efficacy of
high frequency vibration in accelerating the in vitro differentiation
of SAOS-2 cells [2] and human Adipose-Derived Stem Cells (hASCs) toward bone tissue [3]. So, we decided to treat BMSC cells
with high frequency vibration (HFV) in order to determine whether
the promising results obtained with SAOS-2 and hASCs could be
extended to hBMSCs. The cells were stimulated with 30 Hz vibrations for 45 minutes a day, for 21 and 40 days as in previous
experiments with other cell lines.
Fig.1: gene expression results
To stimulate the cells, a previously described custom made “bioreactor” was used [2]. We cultured hBMSCs in osteogenic medium
(15% Osteogenic Stimulatory supplement™, 10-8M Dexamethasone, 50 µg/mL Ascorbic Acid and 3.5mM ß-Glycerophosphate).
hBMSCs were divided into two groups of samples: one subjected
to mechanical treatment (T) and one as control, (C). We measured
the expression of osteogenic genes with (q) Real-Time PCR: OP,
RUNX2, ALP and BOSP. In addition we evaluated the levels of the
more important osteogenic proteins (collagen I, collagen III, osteocalcin, human decorin, osteopontin, alkaline phosphatase, osteonectin and bone sialoprotein), commonly used to test the level of
bone differentiation [4].
The results of the (q)Real-time PCR are presented in Fig.1. At 40
days, the expression of BOSP and OP was higher in treated cells
with respect to control ones (Fig. 1C and 1D, p<0,001). Also
RUNX-2, that is an important transcription factor associated with
osteoblasts differentiation, was higher in treated cells with respect
to controls (Fig.1B). Also the effects at 21 days was evident: all
the osteogenic genes were higher in treated hBMSCs with respect
to control cells (Fig.1). In order to evaluate the amount of the extracellular matrix constituents produced by the cells, an ELISA assay
was performed. In Table 1, the protein content results are presented for treated and control samples, as fg/(cells x dish). At 21 days
and at 40 days the deposition of bone proteins in HFV stimulated
samples was considerably enhanced (p<0.05) in comparison
with the control samples.
Table 1: normalized amount of extracellular matrix proteins
CONCLUSION
Although these encouraging findings indicate that high frequency
vibration treatment accelerates the differentiation of BMSCs toward
bone, other tests should be carried out on BMSCs plated on scaffolds or on specific biomaterials in order to translate this information into clinical applications.
REFERENCES
Derubeis A. et al., Ann Biomed Eng. 2004 Jan;32(1):160-5. Review.
Prè D. et al., Tissue Eng Part C Methods. 2009 Dec;15(4):669-79.
Prè D. et al., Bone. 2011 Aug;49(2):295-303. Epub 2011 Apr 30.
Saino E. et al., Eur Cell Mater. 2011 Jan 14;21:59-72; discussion 72.
29
FUNCTIONAL SCAFFOLDS: MATERIALS AND FABRICATION
Josep A. Planell
Institute for Bioengineering of Catalonia, Technical University of Catalonia, CIBER-BBN, Barcelona, Spain
[email protected]
Different strategies are being developed in regenerative medicine in order to repair diseased tissues or organs. Scaffolds of different
structures and made with different biomaterials are designed for different clinical applications aiming to deliver signals that guide the fate
of cells in the biological environment. Functional scaffolds are meant to perform specific tasks such as delivering biochemical cues and/
or providing topographical, mechanical or other physical stimuli.
Every clinical application will require a specific scaffold with specific properties such as having an appropriate geometry, being easy to
handle and to implant or even being injectable. Moreover, depending on the tissue or organ, specific functionalities adequate to the biological environment will have to be envisaged. This means that the fabrication technique will play a leading role in defining the geometry and
the easiness to handle and to implant the scaffold. The control of processing at the macro/micro/nano level will then be a crucial issue.
The specificity of the biomaterial in bulk and at the surface will control the interaction with cells. Biodegradable polymers or composites
are probably the most adequate substrates and their surface functionalization will tailor their interaction with the biological environment.
Our group of research currently works with well known biodegradable polymers such as PLA and PLGA. Hybrid or composite materials
containing calcium phosphate glasses are an option to modify their bulk properties. These materials can be processed into different
geometries and architectures by using different fabrication techniques such as rapid prototyping, electrospinning and macro/micro/
nano particles production. Their surface functionalization, chemical and/or topographical will make the scaffolds truly functional. Some
examples are provided for the regeneration of different tissues in ophthalmology, nervous, soft tissues or bone.
30
TUNING MULTI-/PLURI-POTENT STEM CELL FATE BY ELECTROSPUN POLY(L-LACTIC ACID)-CALCIUM-DEFICIENT
HYDROXYAPATITE NANOCOMPOSITE MATS
Sabata Martino1, Francesco D’Angelo1, Ilaria Armentano2, Ilaria Cacciotti3, Roberto Tiribuzi1, Mattia Quattrocelli4, Costantino
Del Gaudio3, Elena Fortunati2, M. Imbriani5, Auro Caraffa6, Giuliano Giorgio Cerulli6, Livia Visai5,7, Josè Maria Kenny2,8, Maurilio
Sampaolesi4, Alessandra Bianco3, Aldo Orlacchio1.
1 Department of Experimental Medicine and Biochemical Science, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia Italy
2 Materials Engineering Centre, UdR INSTM, NIPLAB, University of Perugia, Terni, Italy
3 Department of Industrial Engineering, UdR INSTM Roma Tor Vergata, University of Rome “Tor Vergata”, Rome, Italy
4 Translational Cardiomyology Lab, SCIL, Catholic University of Leuven, Leuven, Belgium
5 Department of Molecular Medicine and Center for Tissue Engineering (C.I.T), University of Pavia, Pavia, Italy
6 Department of Orthopedics and Traumatology, University of Perugia, Perugia, Italy
7 Salvatore Maugeri Foundation IRCCS, Pavia, Italy and International Centre For Studies And Research In Biomedicine (I.C.B.), L-2015, Luxembourg
8 Institute of Polymer Science and Technology, CSIC, Madrid, Spain.
INTRODUCTION
In this study we investigated whether multipotent (human bone marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent
stem cells (murine induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous
mats of poly(L-lactic acid) (PLLA) loaded with 1%wt or 8%wt of calcium deficient nanohydroxyapatite (d-HAp).
Materials: Poly(L-lactic acid) (PLLA) loaded with 1%wt or 8%wt of calcium deficient nanohydroxyapatite (d-HAp) was produced as
described by Bianco et al (1).
Osteogenic differentiation of stem cells hBM-MSCs and 5 days-old embryoid bodies seeded on neat PLLA and PLLA/d-HAp
anocomposites mats were incubated with the appropriate basal growth medium (BM) for 21 days in a humidified incubator at 37°C
and 5% CO2. The medium was changed every 3 days. As control of osteogenic differentiation similar experiments were performed
maintaining stem cell-mats cultures in osteogenic medium using MSCs Differentiation Basal Medium-Osteogenic supplemented with the
SingleQuots® containg: Dexamethasone, L-Glutamine, Ascorbate, Pen/Strep, Mesenchymal Cell Growth Supplement, ß-Glycerophosphate.
The osteogenic stem cell differentiation was monitored by:Histological analysis; Alkaline phosphatase staining; Alizarin Red S staining;
Immunofluorescences; measurement of ECM proteins; Real Time RT-PCR;FE-SEM.
We selected adult human bone marrow-mesenchymal stem cells (hBM-MSCs) as representative multipotential stem cells and on the
basis of their capability to generate differentiated cells even if they are cultured on biomaterials.As pluripotent stem cells we chosen
murine induced pluripotent stem cells (iPSCs). These stem cells may be generated in vitro from somatic differentiated cells1 and offer the
advantage to produce patient-specific donor cells for cell replacement and/or tissue engineering applications.Finally we adopted murine
embryonic stem cells (ESCs), as natural stem cell control of iPSCs and based on their pluripotency capability.2
Our results showed that PLLA/d-HAp nanocomposites have an active role in inducing human multipotent (hBM-MSCs) and murine
pluripotent (iPSCs and ESCs) stem cell differentiation towards the osteogenic lineage in the absence of exogeneous soluble differentiating
agents.
The lack of osteogenic differentiation of both murine pluripotent and human multipotent stem cells cultured on neat PLLA under
the above experimental conditions addresses this result to the new properties acquired by the PLLA/d-HAp nanocomposite.
CONCLUSION
Altogether these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was
independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the new mechanical
properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.
REFERENCE
1. Bianco A et al. J Bioactive and Compatible Polymers. 2011, 26, 225-241.
2. D’angelo F et al Biomacromolecule.2012 april 12
ACKNOWLEDGMENTS
“The authors would like to thank This study was supported by the Italian Fondazione Cassa di Risparmio di Perugia (grant no.
2010.011.0445 to A.O.), the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (grant: PRIN no.20084XRSBS_001to
A.O.), as well as the Istituto Nazionale Biostrutture e Biosistemi.
31
MODULATION OF ALIGNMENT AND DIFFERENTIATION OF SKELETAL MYOBLASTS BY BIOMIMETIC MATERIALS
Ilaria E. Palamà1,2, Addolorata M. L. Coluccia³, Stefania D’Amone¹, Mathis Riehle4 and Giuseppe Gigli1,5,6
1 NNL, Institute of Nanoscience CNR, Lecce, ITALY
2 Scuola Superiore ISUFI, University of Salento, Lecce, ITALY
3 Hematology and Clinical Proteomics Unit, “Vito Fazzi” Hospital, University of Salento, ITALY
4 Centre for Cell Engineering, University of Glasgow, Glasgow, UK
5 Dipartimento Ingegneria dell’Innovazione, University of Salento, Lecce, ITALY
6 Center for Biomolecular Nanotechnologies (CNB) of IIT, Arnesano (Le), ITALY
[email protected]
INTRODUCTION
Alignment of cells plays a significant key role in skeletal muscle
tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated
myotubes formed through differentiation and fusion of myoblasts.1
Unfortunately, myoblasts cannot differentiate autonomously into
organized arrays of myotubes when cultured in vitro, which results
in disordered and branched myotubes and may lead to failure in
clinical applications.2 Therefore, it is suggested that physical/chemical topography may determine the spatial organization of myoblasts.
Substrates with lines (size 300x150µm) were fabricated by replicating a patterned SU8 master with PDMS 10:1. A 50:1 PDMS
membrane, was spin coated onto a pre coated Threalose glass.
Next, patterned PDMS and membrane are treated by O2 plasma,
and put in contact. PDMS substrates were immersed in a solution
of Dextrane (DXS), followed by washes with H2O; then immersed
in a solution of Protamine (PRM) followed by washes. This procedure was repeated to assemble 6 layers of polyelectrolytes, with a
positively charged PRM as the last layers. To promote the stability
of the PEMs in solutions, the films were cross-linked by exposure
to glutaraldehyde solutions.
We herein describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation.
The surface consists of a double-sheet PDMS structure coated
with layer-by-layer self-assembled multilayers of biocompatible
polyelectrolytes (PEMs) to provide topographical and biochemical stimuli controlling C2C12 myoblasts seeding and differentiation3. The combination of stiffness modulation of the substrate and
PEM’s coatings allows the myoblasts to organize in tightly aligned
arrays of mature myotubes without switching experimental culture
conditions from standard growth media (GM) to differentiative media (DM) (e.g. by lowering serum concentration) We analyzed the
strength of the cell-substratum adhesion using a parallel plate perfusion chamber, as percentage of elongated or round-shaped cells.
The adhesive strength of C2C12 cells is significantly modulated by
PEM multilayers.
Fig. 1. Fluorescent images of C2C12 cells on PDMS substrate coated with
fibronectin (A,B) or (DXS/PRM)3 multilayers (C,D) after 7 days and stained
with fluorescent phalloidin (red) or α-actinin (green). Nuclei stained with
DAPI (blue). The white arrows indicate the directions of the patterns.
CONCLUSION
We have fabricated cell adhesive substrates in which multilayered
polyelectrolyte films ending in (DXS)/(PRM) enhance cell adhesion
and differentiation of myoblasts by modification of the physicalchemical properties of the surface. The combination of stiffness
modulation of the substrate and PEMs coating allow the myoblasts
to differentiating in myotubes in standard proliferative media. To our
knowledge, this is the first evidence showing the effect of PEMmediated control of PDMS soft and stiff microdomains on myoblasts morphology, spatial organization and skeletal differentiation.
Our findings have relevance to the interpretation of in vitro data as
well as to the study of cellular interactions with biomaterials.
REFERENCES
[1.] P.M. Wingmore, F. Maleki, D.J.R. Evans, M. McErlain. Dev. Dyn,
1996, 207, (2):215-221
[2.] R.G. Dennis, P.E. II Kosnik, In Vitro Cell Dev Biol Anim, 2000, 36,
5:327-335
[3.] I.E. Palamà, S. D’Amone, AML Coluccia, M. Biasiucci and G. Gigli,
Integrative Biology, 2012, 4, 228-236
32
LOW PRESSURE PLASMA PROCESSES TO CONTROL CELL COLONIZATION INSIDE 3D SCAFFOLDS
F. Intranuovo¹, M. Domingos², A. Gloria³, R. Gristina3,4, P. J. Bártolo², P. Favia1,4
1 Department of Chemistry, University of Bari, 70126 Bari, Italy
2 Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, 2430-028 Marinha Grande, Portugal
3 Institute of Composite and Biomedical Materials, National Research Council, 80125 Naples, Italy
4 Institute of Inorganic Methods and Plasmas, IMIP-CNR, 70126 Bari, Italy
[email protected]
INTRODUCTION
Plasma processes are widely used to tailor the surface properties of materials for tissue engineering applications [1]. This is a crucial
aspect, as surface properties influence the cell-material interactions in the tissue engineered constructs. Further, the production of 3D
porous biodegradable scaffolds with proper porosity, pore size, shape, chemical composition and mechanical integrity, able to act as
temporary backbone for the regeneration or repair of a living tissue, represents a paramount challenge for scientists working on tissue
engineering applications [2]. Usually, cell adhesion inside the scaffold’s core regions is hampered by the tortuosity of the poor interconnected polymer structure, leading to limited and heterogeneous cell colonization of scaffolds. With this study, poly(ß-caprolactone)
(PCL) scaffolds, produced by means of conventional and additive manufacturing techniques, were treated using low pressure plasma
depositions and treatments, with the aim of controlling the chemical composition throughout the 3D scaffold thickness, correlating it to a
homogeneous cell adhesion from the top to the bottom of the 3D structure.
PCL scaffolds (10mm diameter, 4mm thickness) were produced with a conventional and simple Solvent Casting/Particulate Leaching
technique, using PCL/CHCl3 (20/80 wt/wt) solutions and NaCl crystals as porogen. Other PCL scaffolds were produced with an innovative Additive Manufacturing system, the BioCell Printing technique (10mm length, 10mm width, 8mm height) [3], with a single lay-down
pattern of 0/90° and a filament distance of 650µm.
Scaffolds were treated in a stainless steel parallel-plate plasma reactor, with low pressure plasma depositions, fed with C2H4/N2 mixtures, followed by H2 post treatment, or plasma treatments with O2/H2 mixtures. Chemical (XPS), Wettability (WCA absorption kinetics),
morphological (SEM) and mechanical (compression tests) characterizations were performed on scaffolds, before and after plasma
modifications. In vitro biological analyses were performed on both plasma treated and untreated scaffolds, using Saos2 osteoblast cells.
MTT assay and fluorescence actin staining were performed.
With the C2H4/N2 plasma process, nitrogen-rich hydrocarbon films were deposited homogeneously along the scaffold thickness, with
nitrogen percentages of ca 10%. Wettability also increased, obtaining WCAs of 27±1°, starting from the hydrophobic PCL material having
a WCA of 76±1°. With the O2/H2 plasma treatments instead, hydroxyl groups were grafted on the PCL scaffolds, as confirmed by XPS
analyses. A great effect on the wettability has been observed, with absorption kinetics very fast when the O2 power was increased. MTT
assay clearly attested for an improved cell proliferation after the plasma modification. This has been confirmed by data of area occupied
by cells, calculated from fluorescence staining images, along the scaffolds thickness.
CONCLUSION
The combination of scaffolding parameters and plasma processes have led to the production of 3D scaffolds with a chemical composition
biocompatible with osteoblast-like cells. Enhanced cell adhesion and proliferation were achieved. In particular, comparing the results
obtained for the differently produced scaffolds, it was clear how the scaffolds produced by means of the Additive Manufacturing technique, were better interconnected, more homogeneously covered by the plasma coatings or grafted species, and gave rise to the best cell
performances, in terms of cell adhesion, proliferation and morphology.
REFERENCES
1. Intranuovo F., Howard D., White L., Johal R.K., Ghaemmaghami A.M., Favia P., Howdle S.M., Shakesheff K.M., Alexander M.R. Acta Biomaterialia 7
(9), 3336-3344, 2011.
2. Hutmacher D.W., Schantz J.T., Xu Fu Lam C., Cheng Tan K., Chye Lim T. J. Tissue Eng. Regen. Med. 1, 245-260, 2007.
3. Bártolo P., Domingos M., Gloria A., Ciurana J., CIRP Annals Manufacturing Technology, 60(1), 271-274, 2011.
AKNOWLEDGEMENTS
Authors would like to thank Mr S. Cosmai for technical support and the PRIN 2008 9CWS4C 002 MIUR project for funding.
33
ENGINEERED CELL SHEETS USING THERMO-REVERSIBLE HYDROGEL
Cochis A.¹, Carletta A.², Altomare L.³, Farè S.², Merlin S.¹, Follenzi A.¹ , Prat M.¹ and Rimondini L.¹
1. Laboratories of Histology and of Biomedical and Dental Materials Laboratory, Department of Health Sciences, Università del Piemonte Orientale,
“A. Avogadro”, Novara, Italy
2. Laboratory of Biomaterials, Department of Bioengineering, Politecnico di Milano, Milan, Italy
3 Department of Chemistry, Materials and Chemical Engineering ‘G. Natta’, Politecnico di Milano, Milan, Italy
[email protected]
INTRODUCTION
Cell sheet (CS) technology allows to obtain sheets of interconnected cells in contact with their extracellular matrix (ECM) with potential
application in many fields of medicine including regeneration of oral tissues [1].
Thermo-reversible behavior of methylcellulose (MC) gels is related to increases in temperature and in salt concentration in the MC aqueous
solutions. This phase-transition make it a promising functional hydrogel for biomedical applications [2]. In the present study we assessed
the fibroblasts CS formation using a thermo-reversible MC hydrogel.
Hydrogel was prepared by dissolving 8% w/v MC in a 0.05 M Na2SO4 solution, at 4°C and then heating it at 37ºC prior to use. Mouse
fibroblast (NIH-3T3), previously transduced with Green Fluorescent Protein (Gfp), were seeded (5x105/cm2) on hydrogel and cultured
until confluent; afterwards, cells sheet was detached by cooling down temperature (30 min, 4°C). Cells sheets were characterized
by immunofluorescence (IF) staining and MTT assay. In-vitro adhesion to a new surface was characterized. Moreover, the CSs were
subcutaneously implanted into SCID mice and histomorphometrically evaluated
CSs were successfully collected by cooling down temperature. IF staining confirmed cells monolayer aggregation and ECM integrity (Fig
1). CS adhesion to a new surface was confirmed and proliferation was observed after 24 h. CSs survived and grew in vivo and were also
successfully vascularized.
Figure 1. CS detached (A) and phalloidin staining of upper (B) and lateral
(C) side of the cell sheet.
CONCLUSIONS
CSs were obtained using MC thermoreversible hydrogel. This technique seems promising in tissue engineering of various tissues including
oral mucosa and periodontal ligament.
REFERENCES
1. Hannachi IE, Yamato M and Okano T. Cell Sheet technology and cell patterning for biofabrication. Biofabrication 2009; 1:022002.
2. Chen CH. et al. Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels. Biomacromolecules 2006; 7:736-743.
ACKNOWLEDGMENTS
The authors would like to thanks Cariplo Foundation grant “Nano-Micro-Structured Polymeric Matrices for Engineered
Cardiac Proto-Tissues”, 2008.
34
INTERACTION OF MAGNETIZED CELLS WITH MAGNETIC SCAFFOLDS
L. Lungaro¹, V. Goranov¹, A. Riminucci¹, C. Dionigi¹, T. Shelyakova², A. Russo², M. Sandri³, A. Tampieri³, R. De Santis4, L. Ambrosio4,
Haranava Y.5, V. A. Dediu¹
1 Istituto per lo Studio dei Materiali Nanostrutturati (ISMN-CNR), Via P. Gobetti 101, Bologna 40129, Italy,
2 Istituti Ortopedici Rizzoli, Laboratorio di Biomeccanica, Via di Barbiano 1/10, Bologna 40136, Italy
3 Istituto di Scienza e Tecnologia dei Materiali Ceramici (ISTEC-CNR), Via Granarolo 64, Faenza 48108, Italy
4 Istituto per i Materiali Compositi e Biomedici (IMCB), Piazzale Vincenzo Tecchio 80, Napoli 80125, Italy
5 Biodevice Systems s.r.o., Bulharska 996/20, Prague 11000, Czech Republic.
[email protected]
INTRODUCTION
Nowadays the interest to the tissue engineering is continuously increasing in both scientific and application fields. In particular, scaffolds
have become fundamental tools in bone graft substitution and the research in this direction has achieved impressive accomplishments. On
the other hand, the need to support the scaffold with cells, growth factors and others evidenced a long-standing problem consisting on the
difficulty to reload after implantation the scaffolds with the bio-agents. Recently proposed magnetic scaffolds [1] represent a conceptually
new solution to this problem. The magnetic scaffold is able, via magnetic driving, to attract and take up growth factors, stem cells or other
bio-agents bound to magnetic particles.
Scaffold preparation
Magnetized and nonmagnetized porous hydroxyapatite scaffolds (ISTEC-CNR Foaming HA(REF), having a cylindrical shape of 8 mm in
diameter and sickness of 2,5 – 4mm. Inner capacity of each scaffold were determined as the ratio between wet and dry weights. The
scaffolds with inner capacity 1,4±0,27mg were choosed for experiment. Scaffolds were placed in 24-well tissue culture plates (with or
without Magnet) and used for cell seeding.
Magnet
Big magnet from ISMN-CNR (ø ≈ 8cm) was placed directly under the bottom of 24-well tissue culture plates for cell loading during 60min.
Magnetic nanoparticles
Magnetite superparamagnetic fluorescent nanoparticles (Chemicell) of 10-20 nm magnetic diameter and PAA coverage (MNPs) where
used.
Cell loading into scaffold
Rabbit bone marrow stem cells (rBMSCs) were obtain from medullar aspirates of rabbit according to the Ethic Committee Rules at Rizzoli
Hospital with a standard methodology with following incubation under usual culture conditions. After 3d passage the cells were labeled
with MNPs overnight and used for scaffold cell loading in concentration 0,5x106 per scaffold.
Quantitative cell analysis (MTT-test).
The standard MTT protocol was used for analysis of cell amount and viability. Shortly 20μl (5mg/ml) of MTT (3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) was added to every experimental sample. After 3,5 hours incubation at 37°C and 5% CO2 the
medium was carefully aspirated and 1ml of DMSO was added to each well for dye liberalization. The absorbance of dye was read at 570
nm using ELISA plate reader (STATFAX 3200).
Statistical Analysis
The data were estimated using EXCELL.
RESULTS
At least 15% increase of cell seeding efficiency in magnetic scaffolds were observed in comparison with nonmagnetic one in case of
magnetically labeled cells were used in vitro. No decrease in cell viability was observed in all the experimental groups.
CONCLUSION
These preliminary results suggest that magnetic scaffold represents a possible solution to attract and take up in vivo, via magnetic driving,
growth factors, stem cells or other bio-agents bound to magnetic particles. The use of magnetized scaffolds permits to avoid the necessity
to reload the scaffolds after implantation with the bio-agents required for the recostituition of the tissue.
REFERENCES
[1] N. Bock, A. Riminucci, C. Dionigi, A. Russo, A. Tampieri, E. Landi, V.A. Goranov, M. Marcacci, V. Dediu “A Novel Route in Bone Tissue Engineering:
Magnetic Biomimetic Scaffolds”, Acta Biomaterialia 6, 2010, 786.
ACKNOWLEDGMENTS
The financial support from EU project ‘‘MAGISTER” NMP3-LA-2008-21468 is acknowledged
35
INTERACTIONS BETWEEN CELLS AND SIC/SIO2 NANOWIRES: SCANNING AND TRANSMISSION ELECTRON
MICROSCOPY INVESTIGATIONS
Francesca Ravanetti¹, Edoardo Scarpa¹, Marco Negri², Marco Campanini², Giancarlo Salviati², Antonio Cacchioli¹
1 Department of Animal Health, Anatomy Unit, University of Parma, Italy,
2 IMEM-CNR Institute, Parco Area delle Scienze 37/A, Parma, Italy
[email protected]
INTRODUCTION
The development of hybrid organic / inorganic multifunctional nanosystems, optimized for clinical treatment of degenerative cellular processes in solid tumours1, is nowadays an issue of great interest nanomedicine. The comprehension of the way of interaction between the
nanomaterial and cells and the procedure of their internalization into the latters are key steps for the application of these nanosystems.
The present study focuses on the understanding of the mechanisms of interaction between the tumour cells (A549) and nanowires of
silicon carbide / silicon dioxide2 (SiC/SiO2). The aim is a subsequent enrichment of these nanowires and the creation of nano systems for
therapeutic targeting. To this purpose, we analyzed in Scanning Electron Microscopy (SEM) the morphology of the cultured tumour cells
at increasing concentrations of nanowires and, by means of Transmission Electron Microscopy (TEM), we studied at ultrastructural level
the internalization of nanowires in cells and their interaction with the plasma membrane and cytoplasmic organelles.
SiC/SiO2 nanowires with uniform and knotted-core structures have been synthesized on Fe-coated Si substrates by chemical vapor deposition (CVD) in an open tube that allows the deposition of a solid to the hot surface of a substrate, through a chemical reaction in the gas
phase2. The nanowires consist of a SiC core wrapped with an amorphous SiO2 shell.
As a cellular model adenocarcinomic human alveolar basal epithelial cells (A549) were used. For SEM analyses: cells treated for 24 hours
with 50 and 100 µg/ml nanowires were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.3). They were dehydrated
and then critical-point-dried with liquid CO2 (CPD 030 Baltec). Subsequently, specimens were then sputter-coated with a gold–palladium
layer using a SCD 040 coating device (Balzer Union) and observed using a Zeiss DSM 950 scanning electron microscope at accelerating
voltage of 10 kV (Zeiss). For TEM analyses, the A549 cells treated for 24 hours with 100 μg/ml nanowires were detached, resuspended,
and finally centrifuged for 2’ at 1800 rpm in order to obtain a cell pellet. The latter was fixed with 2.5% glutaraldehyde in 0.1M sodium
cacodylate buffer (pH 7.3) and stained with eosin 1% (Sigma), dehydrated in acetone and then embedded in Durcupan (Fluka). Semi-thin
sections of 0,5 µm were made and stained with Toluidine Blue 0,5% (Sigma). Ultrathin sections were observed with an EM Philips 300
at 80 kV3.
RESULT AND DISCUSSION
SEM analyses did not show alterations in A549 cells morphology associate to the treatment with increasing concentrations of nanowires.
However, at the concentration of 100 µg/ml, we frequently observed morphologies referable to a cellular suffering and to a programmed
cell death. Most specifically, there was an increasing detachment of cells from the well surface in conjunction with nanowires agglomerates; these cells showed morphological changes related to apoptotic death, such as membrane blebbing and round apoptitic bodies. As
far as concern the interaction between cells and nanowires we noticed cytoplasmic protrusions, as lamellipodia and filopodia, mediating
the contact between the cell membrane and the nanowires. In transmission electron microscopy nanowires showed a thin electron-dense
layer on their surface indicating material /protein deposition. The ultrastructural study, performed using by TEM, proves the ability of cells
to internalize the nanowires. Cells that internalizing nanowires showed pseudopodia typical of phagocytosis, and intercellular bundles of
nanowires; sometimes round vesicular structures were found around them. Regarding the contact with cytoskeleton, actin microfilaments
are involved in the mechanism of internalization.
CONCLUSIONS
Our results demonstrate the effective internalization of nanowires in the cell through active mechanisms. The data seem promising for the
subsequent functionalization of the nanowires and the creation of nanosystems to use for therapeutic targeting.
REFERENCES
1. Singh R. et al., J. Biomed Nanotechnol. 7(4):489-503, 2011
2. Fabbri F. et al., Nanotechnology 21, 345702, 2010
3. Müller KH. et al., ACS Nano 23;4(11):6767-79, 2010
AKNOWLEDGEMENTS
The authors acknowledge financial support by Fondazione Cariparma through project “Nanosistemi ibridi multifunzionali innovativi per
applicazioni biomediche (BioNiMed)”.
36
POLYELECTROLYTE COMPLEXES (PECS) AS INTELLIGENT AND SAFE NANOCARRIERS FOR GENE/ DRUG DELIVERY
Ilaria E. Palamà1,2, Addolorata M. L. Coluccia³, Stefania D’Amone¹ and Giuseppe Gigli1,4,5
1 NNL, Institute of Nanoscience CNR, Lecce, ITALY
2 Scuola Superiore ISUFI, University of Salento, Lecce, ITALY
3 Hematology and Clinical Proteomics Unit, “Vito Fazzi” Hospital, University of Salento, ITALY
4 Dipartimento Ingegneria dell’Innovazione, University of Salento, Lecce, ITALY
5 Center for Biomolecular Nanotechnologies (CNB) of IIT, Arnesano (Le), ITALY
[email protected]
INTRODUCTION
Polymeric particles have sparked major interest as nano-sized delivery systems that increase drug’s retention effects and efficacy at
single cell level through a temporal or spatial control (targeted therapy).1,2 From a biological and therapeutic perspective, the intrinsic
advantage of these supramolecular assemblies lies in the potential of acting as biocompatible and biodegradable carriers under
physiological conditions. Due to their versatility in templating and
surface coating, several polymeric nano-systems such as polyelectrolyte capsules,2,3 polyionic complexes (PICs) or polyelectrolyte
complexes (PECs) are being largely characterized by addressing
their uptake kinetics and processing in living cells.
Polyelectrolyte complexes (PECs) were obtained by mixing in equal
volume two polyelectrolyte solutions: Dextran sulphate (DXS) and
poly(allylamine hydrochloride) (PAH).4 The physio/chemical properties of PECs were evaluated by the measurement of particle
size, ς potential, AFM and SEM, showing that DXS/PAH polyelectrolyte complexes were monodispersed with regular rounded-shape
features and average diameters of 250 nm. Fluorescently labelled
DXS and FITC-DXS were used to follow cell uptake efficiency of
PECs and biodegradability by using confocal laser scanning microscopy (CLSM).
We describe a simple and reproducible procedure to obtain the
spontaneous formation of PECs by mixing oppositely charged
polyions, such as PAH and DXS. These polyelectrolytes present different chemical features: DXS is a biocompatible and biodegradable
polyelectrolyte while the PAH is biocompatible and not biodegradable polyelectrolyte. Strong green fluorescent staining (Fig. 1) can
be observed after 3 hours of incubation, which means PECs have
been successfully delivered into cells. The cell uptake of the PECs
was confirmed by performing scans along the Z axis of cells, thus
showing the PECs localization into the cellular cytoplasm. These
findings can be explained by electrostatic interactions between the
positive surface charge of PECs and the negatively charged cell
plasma membrane followed by spontaneous internalisation of PECs
inside cells through pinocytosis or endocytosis.
Fig. 1. CLSM images of
PECs intracellular uptake
by HeLa cells. In blue DAPI
fluorescence of nuclei. In
green FITC fluorescence of
PECs. Each confocal image
reports the corresponding
Z-stack optical sections (1,
2) to confirm the PECs
internalization.
Quantitative MTT and Trypan Blue assays were employed to
validate PECs as feasible and safe nanoscaled carriers at single-cell
level without adverse effects on metabolism and viability.
CONCLUSION
In this study, novel PECs were prepared through an electrostatic
interaction between DXS and PAH. These PECs were spontaneously
internalised into different cell lines, showing a predominant cytoplasmatic localization. The intracellular degradation of DXS can lead
to the release of therapeutic cargo into cells, while the presence of
biosynthetic polyelectrolyte can function as reserve system. This
implies that PECs can be used as biocompatible and biodegradable
carriers, hence as promising gene/drug delivery vehicles at singlecell level.
REFERENCES
1. M. Jenkins, Biomedical Polymers, Woodhead Publishing, Cambridge,
UK, 2007
2. I.E. Palamà, S. Leporatti, E. De Luca et al., Nanomedicine, 5( 3): 419431, 2010
3. I.E. Palamà, A. M. L. Coluccia, A. D. Torre et al., Science of Advanced
Materials, 2: 1-9, 2010
4. I.E. Palamà, M. Musarò, A. M. L. Coluccia, S. D’Amone and G. Gigli,
Journal of Drug Delivery, 1-7, 2011
37
HYBRID MAGNETIC HYDROGEL: A POTENTIAL SYSTEM FOR CONTROLLED DRUG DELIVERY BY MEANS OF
ALTERNATING MAGNETIC FIELDS
Serena Fedi, Gabriele Giani and Rolando Barbucci
CRISMA ( Centro Ricerca Interuniversitario Sistemi Medici Avanzati), University of Siena, Viale Matteotti 15 53034 Colle Val D’elsa, Siena, Italy.
[email protected]
INTRODUCTION
Novel hybrid magnetic hydrogels demonstrate their influence in
several areas, particularly in biomedical science where these innovative materials are showing interesting applications for controlled
drug delivery. ¹-² This innovative strategy for targeted drug delivery consists of coupling the drug to magnetic nanoparticles (NPs)
that can be guided to the target by means of external magnetic
fields.³-4nce they reach the target, the NPs release the drug under
the influence of an alternate magnetic field. A number of different
approaches for the preparation of these hybrid hydrogels
have been suggested and developed but these strategies share
the limitation that the NPs are only physically embedded within the
hydrogel and this may imply a release of NPs from the hydrogel
matrix towards the external environment. One strategy to overcome
these problems involves the use of NPs as cross-linker agents.5
The aim of this study is to obtain polysaccharide hybrid hydrogels
with magnetic NPs as cross-linker agents and test these as systems for the release of a drug (or a model of drug) by means of
the application of an alternating magnetic fields (AMF).
The organic part of the hydrogels consists of carboxymethylcellulose (CMC) or hyaluronic acid polymers, while the inorganic part is
made up of CoFe2O4 or Fe3O4 magnetic NPs.
In order to create a covalent bond between the polymer and the
inorganic phase, we functionalized NPs with (3-aminopropyl) trimethoxysilane (APTMS) with the aim to introduce -NH2 groups on
the metal oxide surface. Infrared spectroscopy, XPS and cyclic voltammetry measurements confirm the presence of amino groups on
the surface of nanoparticles.
The reaction for the synthesis of the hydrogel involves the formation of amide bonds between the carboxylic groups of CMC or hyaluronic acid and the amine groups of the functionalized NPs in the
presence of EDC and NHS.
The scheme of the hybrid hydrogel is reported in Fig. 1.
The presence of NH2 groups on the NP surface was ascertained
by XPS measurements and for the first time, by cyclic voltammetry
in solution. This technique allowed us to determine the thickness
of silane layer on NP.
Figure 1
Figure 2 Cyclic Voltammograms of (a)
NP saturated solution after 30”sonication, (b) NP-NH2 0.023 g in 0.020 l after
30”sonication in DMSO solution and (c)
APTMS 2,3 x10-3 mol dm-3 in CH2Cl2
solution (unfortunately, the corresponding
process in DMSO is prevented by solvent
discharge).
CONCLUSION
The technique developed in this project suggests a method of
preparing hybrid hydrogel with magnetic nanoparticles covalent
bound in the matrix. This new system can be loaded with a large
amount of drug and positioned near the target site. Recent results
demonstrate the release of drug-like molecules by means of application of magnetic stimulus.
REFERENCES
1. Barbucci R. et al.; Soft Matter, 3524:3532- 6, 2010
2. Peppas N.A. et al.; Hydrogels in Medicine and Pharmacy ; ed. N. A.
Peppas and A. G.Mikos ; CRC 309 press, Boca Raton, USA, vol. 1;1987.
3. Gaihre B. et al.; International Journal of Pharmaceutics, 180:189365, 2009.
4. Drbohlavova J. et al.; Pharmacological Research, 144:149- 62, 2010.
5. Barbucci R. et al.; Soft Matter, , 5558:5565-7, 2011
6. S. Satarkar, Z. and Hilt, Acta Biomateria11:16-4, 2008.
ACKNOWLEDGMENTS
The authors thank the M.I.U.R. for financial support for the F.I.R.B.
project RBAP11ZJFA, 2010
38
POLYELECTROLYTE COMPLEXES (PECS) AS INTELLIGENT AND SAFE NANOCARRIERS FOR GENE/ DRUG DELIVERY
Chiara Malloggi¹,², Daniele Pezzoli², Francesca Olimpieri¹, Sabrina Bertini³, Roberto Chiesa¹, Alessandro Volonterio¹,
Gabriele Candiani1,2
1 CMIC Department, Politecnico di Milano, Milan, Italy
2 INSTM, Unità Politecnico, Milan, Italy
3 Istituto G. Ronzoni, Milan, Italy
[email protected]
INTRODUCTION
Successful non-viral gene delivery currently requires compromises
to achieve useful transfection levels while minimizing cytotoxicity.
A straightforward approach widely exploited to develop effective
transfectants relies on the synthesis of copolymers such as chitosan grafted with low molecular weight branched polyethylenimine
(Chi-g-bPEIx). Unfortunately, despite the vast amount of work that
has been done in developing promising polymeric gene delivery
vectors, the possible influence of the degree of grafting on the overall behavior of copolymers has been largely overlooked.
Seven Chi-g-bPEIx copolymers with different amounts of 2 kDa
bPEI grafted onto medium molecular weight 75-85% deacetylated
chitosan backbone were synthesized as previously described1.
The compositions of the intermediates and of Chi-g-bPEIx copolymers were evaluated by 1H NMR. The amine content and the
degree of grafting of 2 kDa bPEI on chitosan were characterized
by 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay. Polyplexes
were prepared by adding plasmid DNA to chitosan, Chi-g-bPEIx
or bPEI, yielding different N/P ratios. The DNA binding ability of
chitosan, copolymers and bPEI was monitored by a fluorophoreexclusion assay and by gel retardation assay. Size and ς-potential
of polyplexes were determined by Dynamic Light Scattering (DLS)
and Laser Doppler Velocimetry (LDV). For transfections, HeLa,
COS-7 and U87-MG cell lines and primary articular chondrocytes
were seeded at a density of 1.5 x 104 cells/cm2 and the day after,
polyplexes were added to cells. 24 h post-transfection, cytotoxicity
was evaluated by AlamarBlue® viability assay, cells were lysed
and luciferase activity was measured by Luciferase Assay System
and normalized to the total protein content. Statistical analysis was
performed by ANOVA test. Significance was retained when p <
0.05.
We synthesized a series of seven Chi-g-bPEIx copolymers with different degrees of grafting of 2 kDa bPEI onto to MMW chitosan.
As expected, the degree of grafting of copolymers increased by
increasing the amount of bPEI added to chitosan during the synthesis (Fig. 1).
DNA complexation ability experiments showed that all Chi-gbPEIx were able to complex plasmid DNA at nitrogen (N) to
plasmid DNA phosphate (P) (N/P) ratios ≥ 2.75. At N/P 30,
all complexes had almost the same hydrodynamic mean diameter
but, along the Chi-g-bPEIx series, the higher the degree of grafting,
the greater the ζ-potential of the resulting polyplexes.
Moreover, we tested the overall gene delivery behavior of Chi-gbPEIx copolymers at N/P 30 in three cell lines transfected in com-
plete medium (DMEM with 10% FBS). Interestingly, along the Chig-bPEIx series, the higher the degree of grafting, the greater the
cytotoxicity of the resulting polyplexes. It is worth noting that, in all
cell lines tested, the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared
to other Chi-g-bPEIx copolymers (p < 0.05 vs all). Furthermore,
in primary articular chondrocytes, Chi-g-bPEI2.7% showed similar efficiency but lower cytotoxicity than the gold standard 25 kDa
bPEI.
Fig. 1. Degree of grafting of Chi-g-bPEIx copolymers as a function of 2 kDa
bPEI equiv.
CONCLUSION
This work underlines for the first time the pivotal role of the degree
of grafting in modulating the overall transfection effectiveness of
Chi-g-bPEIx copolymers. Crucially, we demonstrated that, along
the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had
a direct effect on cytotoxicity. In this scenario, ongoing studies aim
to shed light on putative mechanisms of DNA complexation with
different copolymers that might explain our findings.
REFERENCES
1. Jiang H. L. et al., J. Control Release 2007; 117(2): 273-80.
2. Pezzoli D. et al., PLoS One 2012; 7(4): e34711.
ACKNOWLEDGMENTS
This work was partly supported by the Politecnico di Milano, 5xmille Junior, “SURGES” Project and by the Italian Ministry for Education, University and Research (MIUR) - FIRB Futuro in Ricerca
2008, Grant RBFR08XH0H.
39
NANOTOXICITY OF PEI-BASED NANOPARTICLES IS REDUCED BY ACETYLATION OF POLYETHYLENIMINE AMINES IN
HUMAN PRIMARY CELLS
Elena Nicolì1, Michela Bosetti1, Francesca Boccafoschi3, Anna Calarco2, Luca Fusaro3, Gianfranco Peluso2, Mario Cannas3
1 Dipartimento di Scienze del Farmaco, University of Eastern Piedmont, Novara, Italy
2 Institute of Protein Biochemistry, CNR, Napoli, Italy
3 Dipartimento di Scienze della Salute, University of Eastern Piedmont, Novara, Italy
[email protected]
INTRODUCTION
Cationic nanoparticles have the advantages of high electrostatic interaction with the cellular surface, quick uptake and high capacity to bind
nucleic acids so they are good non viral vectors candidates1. PEI is a highly positive charged polymer but it is known to have toxic effects
to cells based on its chemical structure (i.e. primary and secondary amines amount). With the aim of reducing its toxicity, nanoparticles
of PEI-PCL (PEI-NPs) have been created and compared to similar nanobits produced using acetylated PEI (AcPEI-NPs)2. Nanotoxicity is
often underestimated, but the ultrasmall size of nanoparticles could interfere with biological systems, with possible interaction at cellular,
sub-cellular and protein levels3. In this study we have evaluated cell uptake, biocompatibility and genotoxicity of the two NPs.
Human primary cells have been used for the experiments: fibroblasts, myoblasts, endothelial cells, lymphocytes and monocytes. We
have evaluated the kinetic of nanoparticle internalization into the cells by flow cytometry and time lapse confocal microscopy using
cumarin 6 loaded NPs (1A,1B). Cytotoxicity of PEI-NPs and AcPEI-NPs have been tested by trypan blue exclusion assay and by LDH
quantification (1C). The genotoxicity has been analysed by sister chromatid exchanges (SCEs) and the micronuclei (MNi) formation (1D).
The immunotoxicity has been evaluated through the quantification in chemiluminescence of intracellular reactive oxygen species (ROS)
and cytokine secretion resulting from monocytes activation after exposure of NPs (1E).
The results showed that both NPs have an equivalent behavior in terms of cellular uptake: faster (2-5 minutes) at higher concentrations
(300μg/ml) and slower (15-30 minutes) at lower concentrations (10μg/ml) with no differences comparing the two nanomaterials. AcPEINPs revealed no cytotoxicity at all the concentrations used and on all the studied cells; PEI-NPs resulted cytotoxic only on primary
myoblasts when used at the highest concentration. PEI-NPs increased SCEs and MNi statistically reduced by PEI acetylation. The
genotoxic effect of PEI-NPs was probably related to reactive oxygen species (ROS) production, since there is a DNA damage without a
nuclear localization. Our data showed an increase of ROS release when cells were treated with PEI-NPs and not in presence of AcPEI-NPs
indicating that ROS increment may affects cell function by directly acting on cell components, including lipids, proteins, and DNA and may
be able to perturbate the cell antioxidant systems by decreasing its natural defense. No cytokine release has been evidenced, in ELISA
test, after exposure of both NPs.
a
b
c
d
e
f
Figure 1. A) flow citometry results; B) confocal microscopy images; C) LDH-assay results; D) genotoxicity results; E) ROS quantification.
CONCLUSION
Our study shows that acetylation reduces the genotoxicity of PEI-NPs, that seems to be dependent by the amine acetylation without
affecting nanoparticles uptake and biocompatibility profile. This results indicated AcPEI-NPs as good non viral vector.
REFERENCES
1. Hover J. et al, Acc Chem Res, 2012.
2. Tamura A. et al, Nanomedicine, 2010.
3. Hillegass J.M. et al, Nanomed Nanobiotech., 2010.
ACKNOWLEDGMENTS
The authors would like to thank the Research Programs of National Interest (Grant no: 1407/Ric/2008) for providing financial support to
this project.
40
LAPATINIB/PACLITAXEL POLYELECTROLYTE NANOCAPSULES FOR OVERCOMING MULTIDRUG RESISTANCE IN
OVARIAN CANCER
Daniele Vergara¹,², Claudia Bellomo³, Xingcai Zhang4, Viviana Vergaro³, Andrea Tinelli5, Vito Lorusso6, Ross Rinaldi7, Yuri M.
Lvov4, Stefano Leporatti³, Michele Maffia¹,²
1 Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy
2 Laboratory of Clinical Proteomic, “Giovanni Paolo II” Hospital, ASL-Lecce, Italy
3 NNL-Istituto Nanoscienze, CNR Via Arnesano 16, 73100, Lecce, Italy
4 Institute for Micromanufacturing, Louisiana Tech University, Ruston, LA, USA; 71270
5 Department of Obstetrics and Gynaecology, Vito Fazzi Hospital, Lecce, Italy
6 Department of Medical Oncology, Vito Fazzi Hospital, Lecce, Italy
7 NNL-Istituto Nanoscienze, CNR University of Salento, Department of Innovation Engineering, Technology District, Lecce, Italy
[email protected]
INTRODUCTION
Paclitaxel (PTX) is a drug of natural origin presently used for the treatment of solid tumors including ovarian and breast cancers. PTX
promotes microtubule assembly and stability, thus leading to the disruption of the normal microtubule network required for mitosis. We
hypothesized that PTX clinical efficacy can be increased through a strategy that combines improvements in PTX delivery and combination
of targeted therapies to inhibit one or more cellular pathways involved in drug resistance. In particular, ATP-binding cassette (ABC)
transporters, such as the multiple drug resistance transporter MDR1 (multidrug resistance 1, or P-glycoprotein or ABCB1), have been
associated with a poorly response to several chemotherapeutics including PTX. These observations set the stage for the development
of efficacious instruments to increase PTX efficacy by limiting adverse side effects and increasing its cytotoxic action. In this regard,
nanotechnology has been recognized as a fundamental tool in cancer research (1) and the potential of nanocarriers to increase drug
efficacy is well described (2).
Stable nanocolloids of PTX and Lapatinib were prepared by the sonication assisted layer-by-layer (SLBL) technology method. To build up the
nanocapsules for a simultaneous controlled release of two drugs, we used a sonication assisted layer-by-layer technique. Biodegradable
chitosan (polycation) and alginic acid (polyanion) were chosen for a biocompatible and biodegradable coating on drug nanoparticles.
By using as model a chemioresistance ovarian cancer cell line, OVCAR-3, we observed that the cytotoxic activity of PTX is increased
when the drug is combined with Lapatinib, an inhibitor of the intracellular tyrosine kinase domains of both the EGFR and Her-2 receptors.
Lapatinib is also active at the ATP-binding site of ABC transporters, key mediators of PTX resistance.
For this reason, a nanoformulation of two drugs in one nanocapsule locating PTX in the core and Lapatinib on the shell periphery was
realized by SLBL technology. With this strategy both drugs can be temporally co-localized in the tumor cell for optimal synergy, limiting
possible differences in the pharmacokinetics and tumor accumulation of the two different agents. Results obtained by MTT test confirmed
the enhanced cytotoxic activity of this nanopreparation compared to free PTX.
CONCLUSION
Here, we describe a SLBL method to efficiently converting PTX into drug nanoparticles. It allows to combine many necessary factors
for an efficient drug delivery system: i) control of nanocolloid size within 100-300 nm, ii) high drug content of ca 70 wt %, iii) shell
biocompatibility and biodegradability, and iv) sustained controlled release. Overall, these characteristics, including the small size and the
net negative charge that can be advantage for their penetration to and within tumors, make nanoparticles attractive candidates for possible
in vivo applications.
REFERENCES
1. Ferrari M., Nat Rev Cancer 5:161-171, 2005.
2. Vergaro V. et al., Advanced Drug Delivery Review 63:847-864, 2011.
ACKNOWLEDGMENTS
This work was funded by the Italian Ministry of University and Research (MIUR) through the FIRB project no. RBLA03ER38, “Con il
contributo del Ministero degli Affari Esteri (MAE), Direzione Generale per la promozione e la Cooperazione Sociale ” (Italy –USA Large
Scale Bilateral Project “Nano-trasportatori per la terapia del cancro”). This work was also supported by the PS105 ARTI strategic project
“Development and realization of bio-chip for molecular diagnostic and typization of human pathogenic viruses (HPV, HCV)” of Apulia
Region and by a contribution of “ANGELA SERRA” Found. For Cancer Research, Parabita (Lecce, Italy).
41
NOVEL CROSS-LINKED HYALURONIC ACID HYDROGELS FOR BIOMEDICAL APPLICATIONS
Annalisa La Gatta, Agata Papa, Chiara Schiraldi, Antonella D’Agostino, Mario De Rosa
Department of Experimental Medicine, Faculty of Medicine, Second University of Naples, Via L. De Crecchio 7, 80138 Naples, Italy
[email protected]; [email protected]
INTRODUCTION
Hyaluronic acid (HA) is recognized as a performing biopolymer for tissue engineering purposes due to a unique combination of properties
among which magnificent biocompatibility, non immunogenicity, capacity to degrade in safe products, high hygroscopicity [1]. Actually HA
has been widely used in the development of matrices conceived as scaffolds. When used for this purpose, HA has to be stabilized (mainly
through crosslinking) to achieve the proper stability (resistance to chemical and enzymatic degradation) and mechanical performance in
the biological environment. Many crosslinking strategies have been employed among which crosslinking processes involving the hydroxyl
groups of the biopolymer. The latter are mainly achieved using a diglycidyl ether as the crosslinker [1].
This report presents an approach for the fabrication of HA based scaffolds using 1,4-butandiol diglycidyl ether as the crosslinker and
their characterization.
A 12,5%w/w aqueous solution of a low molecular weight HA (Mw = 220kDa) was lyophilized in order to obtain a microporous substrate,
which was crosslinked in a non solvent for the biopolymer with increasing 1,4-butandiol diglycidy ether (BDDE) amounts (22,5-112%
equivalents with respect to the hydroxyl groups on HA). Swelling properties of the resulting hydrogels, that proved not soluble in water,
were investigated in water and phosphate buffer saline (PBS, pH 7.4, 0.165M ionic strength) by means of gravimetric measurements.
Additionally, the swelling degree was evaluated as a function of the ionic strength (0-0.165M) and of the pH (3-9) of the swelling medium.
In vitro enzymatic degradation studies were performed by incubating materials with BTH at 100 and 1000U/mL at 37°C; degradation
was monitored by measuring the amount of solubilized HA during incubation by the carbazole assay[2]. The stability of the hydrogels
in cell culture medium (DMEM added with Calf Bovine Serum 10%) at 37°C was evaluating likewise. A biological evaluation of the
materials including cytotoxicity and cytocompatibility tests was performed using NIH 3T3 fibroblasts and performing the MTT test for the
quantitative analyses [3].
The crosslinking process performed led to products insoluble in aqueous environment presenting an interconnected porous 3D
architecture as confirmed by SEM observation. Swelling studies revealed that materials were able to absorb large amounts of water
increasing their dry weight up to one hundred folds. Consistently, the swelling degree was found to decrease at the increasing
of the BDDE amount used. As expected, the amount of absorbed water was higher in water than in PBS. Swelling capability was
found to decrease at the increase of the ionic strength and to increase with the pH of the swelling medium due to the presence of
carboxylic groups (polyelectrolitic behaviour). The hydrogels exhibited high stability in cellular culture medium and proved highly
resistant to hydrolysis catalyzed by bovine testicular hyaluronidase (the hydrogel with the higher crosslinking degree was found
to resist more than two weeks when incubated with BTH 1000U/mL). This result is very interesting when compared to other HA
based products crosslinked via digòycidyl ethers. The biological evaluation of the materials proved their non cytotoxicity and
their appropriateness to promote cell adhesion and proliferation thus supporting their use in tissue engineering applications.
CONCLUSIONS
The physico-chemical and biological characterization performed proved the developed hydrogels as interesting and suitable materials for
tissue engineering applications.
REFERENCES
1. Schiraldi, C.et al., In Biopolymers; Elnashar, M., Ed.; published by Sciyo, Rijeka, Croatia 2010; pp. 387–412.
2. La Gatta A. et al., Anal Biochem. 2010, 404, 21-29.
3. La Gatta A. et al., J Biomed Mater Res. 2009, 90A, 292-302.
42
GAGS (HYALURONAN AND CHONDROITIN SULPHATE) IN COMBINATION WITH SYNTHETIC POLYMERS:
POTENTIAL APPLICATIONS
A.D’Agostino, A. La Gatta, T. Busico, A. Papa, M.De Rosa and C.Schiraldi
Department of Experimental Medicine, Faculty of Medicine, Second University of Naples, Via L. De Crecchio 7, 80138 Naples, Italy
[email protected] ; chiara.schiraldi @unina2.it
INTRODUCTION
A great number of naturally occurring polymers have been studied and proposed for the preparation of materials designed for Tissue
Engineering (TE) applications [1].
Bearing this in mind, the presence of ECM molecules in a scaffold is fundamental for creating an environment mimicking the cellular
surroundings [2,3].
The aim of this research is to combine natural polymers as hyaluronan (HA) and chondroitin sulphate (CS) with two synthetic polymers:
polyvinylalchol(PVA) and a co-polymer of hydroxyethylmethacrylate (HEMA) and methacryloxyethyltrimethylammonium (METAC) in order
to obtain respectively novel physical and chemical networks.
Physical network PVA 9%-CS 1%, PVA 9%-CS 2% PVA 9%- HA 1%, PVA 9%-HA 2% w/v were obtained by four cycles of freeze-thawing.
HEMA and METAC monomers were co-polymerized in the presence of aqueous solutions of each GAG (HA or CS) at 1% and 2% w/v final
concentrations using AIBN as thermal initiator. A curing cycle at different temperature and time on acrylic materials was made in a forcedair circulation oven. Swelling behavior in water and physiological solution were studied. Rheological measurement with a oscillatory
rheometer were performed in order to obtain mechanical information on novel materials. Biological response was studied using vitality
assays (MTT test), SEM analysis and time lapse videomicroscopy (OKOLAB). In particular for PVA materials, experiments in fluorescence
were accomplished to evaluate better cell behaviour and acquire images otherwise not sufficiently clear due to their opacity.
The swelling studies showed, in both the materials containing CS, a lower water uptake respect to the ones containing HA. A different
behavior in the trend of the swelling curves of the acrylic based materials is due to the presence of charges of METAC monomers; the
PVA, on the contrary, is an un-charged polymer. The hydrogels containing CS seemed more compact and semi-interpenetrated systems.
The presence of CS does not affect the mechanical features of the polymers, while HA make them more rigid. All the materials resulted
not toxic and showed a better biological response in presence of GAGs. In particular, HA led to a better citocompatibility in PVA matrices,
while CS elicit a better biological response in acrylic resins.
CONCLUSION
GAGs-based semi-interpenetrating hydrogels obtain through physical or chemical strategies were synthesized in this study. The introduction
of polysaccharides in synthetic polymers (PVA and pHEMA-METAC) enhances the general performances of polymers and supports the
hypothesis of applications of these materials in tissue engineering.
REFERENCES
1. Puppi D, Chiellini F, Piras AM, Chiellini E. Prog Polym Sci. 2010; 35: 403-440.
2. Wang TW, Sun JS, Wu HC, Huang YC, Lin FH. J Biomed Mater Res Part B
3. Tan H, Wu J, Lao L, Gao C. Acta Biomater. 2009; 5: 328-337.
43
PREPARATION AND HYDROLYTIC DEGRADATION OF POLY LACTIC ACID BASED SCAFFOLDS
Francesco Carfì Pavia1, Vincenzo La Carrubba² and Valerio Brucato1
1 DICGIM, Università di Palermo, Italy
2 DICAM, Università di Palermo, Italy
[email protected]
INTRODUCTION
A crucial aspect of the tissue engineering regards the possibility to design an effective method to tune the degradation rate of the scaffold.
Poly-Lactic-Acid (PLA) is biodegradable thermoplastic polyester. Since lactic acid is a chiral molecule, it exists in two forms, Poly-L-Lactic
Acid (PLLA) and Poly-D-Lactic-Acid (PDLA). The involvement of D- and L-units in the sequences of PLLA and PDLA, respectively, exerts
a profound effect on their thermal and mechanical properties1. For the aforementioned reasons, blending PLLA with PDLLA is an effective
method for controlling the polymer crystallization, morphology, and hydrolysis nature2. In the present work Poly-L-lactic acid and two
Poly lactic acid based blends (PLLA/PLA 90/10 and 70/30) were utilized to produce porous foams for tissue engineering applications via
Thermally Induced Phase Separation (TIPS). The foams obtained were characterized via Differential Scanning Calorimetry (DSC) in order
to verify if PLA influences theirs final thermal properties. Moreover, a hydrolytic degradation was carried out on the scaffolds in order to
observe theirs differences in terms of the weight loss.
A homogeneous ternary solution composed by PLLA or PLLA/PLA blends (90/10, 80/20, and 70/30), dioxane and water was prepared,
with a constant dioxane to water volumetric ratio of 84.5/15.5. The concentration of PLLA was 6% wt/wt. The solution was initially kept at
84 °C. The temperature was then suddenly lowered to a value within the unstable region for 30 minutes. Later a quench by direct immersion
in an ethyl alcohol bath at a temperature of -25 °C was performed. The foams obtained were subjected to washing in deionised water and
drying at 20 °C under vacuum, in order to completely remove any remaining solvent trace. Enthalpies of melting and crystallization were
determined with a DSC Jade Perkin Elmer calorimeter by recording heating and cooling scans at 10 °C/min. The so-obtained disk-shaped
scaffolds (diameter 17 mm, height 2 mm) were placed into wells in containing PBS 1X (pH 7,4) and stored at 37 °C. The medium was
changed every weeks and its pH was measured. After 2, 4, 7 and 10 weeks samples were extracted from the well, washed in distilled
water to remove the PBS and dried at 40 °C under vacuum for three days. The percentage weight loss of the samples was calculated using
the sample weight before and after the degradation (Wbefore and Wafter respectively) Wloss (%) =100(Wbefore - Wafter)/ Wbefore
Figure 1: Enthalpies of melting (A) and crystallization (B) for PLLA/PLA
scaffolds prepared at different PLLA/PLA ratios.
Figure 2: weight loss of the scaffolds after 10 weeks.
In figure 1A and 1 B the latent heats of melting and crystallization of the PLLA/PLA foams at the different PLLA/PLA ratios are shown.
It is easy to notice that the ΔHm decreases by increasing the PLA/PLLA ratio. This behavior was also observed for the enthalpy of
crystallization. In both cases it has been noticed a lowering of crystallinity level with respect to the level predicted by ideal mixing rules
(dotted line). It is reasonable to suppose that crystallization kinetics of the scaffolds is hindered by blending.The percentage of weight loss
of the scaffolds is shown in figure 2. It is easy to notice that the higher the amount of PLA in the blend, the higher the weight loss. As a
matter of fact, after 10 weeks, pure PLLA scaffolds have lost less than 2% of their original weight; PLLA/PLA 90/10 scaffolds lost about
the 3% and 70/30 lost more than 6%. The results confirm that when adding PLA, a faster degradation occurs.
CONCLUSION
Porous PLLA/PLA foams were produced via TIPS. A Differential scanning calorimetry analysis revealed a lowering of enthalpy of melting
and crystallization when increasing the PLA content. Crystallinity level lower than predicted by ideal mixing rule were found, showing that
crystallization kinetics is hindered by blending. Hydrolytic degradation test has confirmed an increase in weight loss when increasing the
PLA content. The results have confirmed that, blending PLLA with PLA is an effective method for controlling the scaffold degradation rate
REFERENCES
1. Yamane H. et al., Polymer 44:2569-2575, 2003
2. Tsuji H. et al., Biomaterials 25:5449-5455, 2004
44
INNOVATIVE GLYCEROL-BASED BIOCOMPATIBLE COPOLYMER AS MATERIAL FOR POTENTIAL BIOMEDICAL
APPLICATIONS
Elvira De Giglio¹, Gianluca M. Farinola1, Damiana Cafagna1, Ruggiero Rizzi1, Vincenzo Fino2, Stefania Cometa3,
Concetta Ferretti4 and Monica Mattioli-Belmonte4
1 Dept. of Chemistry, University of Bari Aldo Moro, Italy,
2 Synchimia srl, Spin-off of the University of Bari Aldo Moro, Italy
3 Dept. of Chemistry & Industrial Chemistry, University of Pisa, Italy
4 Dept. of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
[email protected]
INTRODUCTION
Polyglycerol that can be produced from abundant renewable sources, has excellent hydrophilic characteristics thanks to pendant hydroxyl
groups and ether linkages. Polyglycerol-based materials were successfully synthesized. Biocompatibility and biodegradability make them
good starting materials in the biomedical and biotechnological fields as drug delivery, tissue engineering, and hydrogel adhesives for
wound dressing1, 2. Their commercial relevance has increased considerably in the last few years because of the rising inevitable formation
as a by-product of biodiesel production. In this study, we present the synthesis of a new copolymer based on glycerol and tartaric acid.
The copolymer was thermally characterized by TGA and DSC, while its chemical composition was assessed by XPS, FT-IR and NMR
analyses. A preliminary biological investigation with fibroblast and epithelial cell lines was performed.
The polycondensation of the activated tartaric acid with glycerol (ratio 1:1) was carried out at 100°C for 1 hour under stirring. The
thermal degradation pattern of the synthesized polymer was evaluated by TGA; while DSC was used to study its thermal characteristics.
The obtained copolymer was characterized by XPS, FT-IR and 1H-NMR. For biocompatibility tests, the copolymer was dissolved in
methanol and deposed by spinning onto flat slides. Samples were placed into 24-well polystyrene culture plates (TCPs), seeded with
4x104 fibroblasts (L6 - CRL-2648™ ATCC, USA) or 2.7x104 HaCaT epithelial cells (5% CO2, T=37°C) for 48 h and 7 days. In the control
cultures, cells were placed directly into TCPs at the same density of investigated samples. MTT viability test, SEM morphological analysis
and cytoskeletal actin evaluation were performed. For cytoskeletal analysis cells were incubated with falloidin for actin detection and with
DAPI to evaluate nuclear morphology.
XPS, FT-IR and NMR investigations confirmed the formation of ramified and/or cross-linked structures in the synthesized glycerol-based
copolymer. TGA analysis showed the copolymer thermal stability under 100°C and the DSC investigation denoted an high Tg; while at
room temperature the copolymer appeared as a solid yellow resinous form. Both cytotypes displayed a good biocompatibility onto the
glycerol-based copolymer.
After 7 days the viability was equal to 100±.6.% and 86±.5.% for HaCaT
epithelial and fibroblasts cells, respectively. SEM morphological observations
were in agreement with MTT data. Immunofluorescence staining (see Figure a
and b) and SEM morphological analysis of HaCaT (Figure c and d) displayed
the extent of cell adhesion on the polymeric surface, as well as the presence of
cell junctions (arrows: zonula adherens) between epithelial cells that were more
evident after 7 days of culture (Figure b and d).
CONCLUSION
A new glycerol-based copolymer was synthesized and characterized in order to obtain a biodegradable wound dressing system. Preliminary
biological investigation of fibroblast and epithelial cell lines seeded on the copolymer demonstrated a good biocompatibility. Further cell
and drug release studies are currently in progress.
REFERENCES
1. Steinhilber D. et al., Int. J. Artif. Organs 34:118-122, 2011
2. Salehpour S. et al., Eur. J. Lipid Sci. Technol. 114:92-99, 2012
ACKNOWLEDGMENTS
This study was supported by grants to Dr De Giglio (National grant Progetto Strategico cod. CIP PS_046 “Studio e Sviluppo di Materiali
Innovativi per Applicazioni in Chirurgia Laser della Cornea” founded by the Apulia Region).
45
POLYANILINE BASED SCAFFOLDS FOR THE REGENERATION OF ELECTRO-ACTIVE TISSUES
Vincenzo Guarino, Anna Borriello, Marco A. Alvarez-Perez, Luigi Ambrosio
Institute of Composite and Biomedical Materials, National Research Council of Italy, Naples
[email protected]; [email protected]
INTRODUCTION
Currently, the use of conducting polymers (CPs) are progressively exploring in scaffold design to electrically stimulate cells, thus regulating
specific cellular activities and, ultimately, the process of regeneration of damaged tissues. In particular, many studies of electroactive
tissues (i.e, nerve, muscle, myocardium) responding to electrical impulses, demonstrate the success of variously doped CPs to entrap/
release biological molecules as therapeutic proteins and molecular signals including NGF or IGF. However, several limitations of using CPs
concern their reduced ability to naturally degrade and toxicity response1. In this context, the combination of CPs with synthetic polymers
(i.e, polylactide, polycaprolactone, etc) may offers an interesting strategy to adapt conductive signals to cell environment2, preserving the
surrounding tissues from indesired toxic response, without limiting their electrical conductive potential. Here, we propose two different
solutions for the development of scaffolds for nerve and myocardiium: 1) hybrid macroporous scaffolds via photopolymerization of
polyethilenglicole with diacrilate groups (PEGDA) for nerve regeneration; 2) Electrospun PCL membranes as conductive patches for
cardiac tissue diseases. In the first case, polyaniline (PANi) will be integrated to the polymer matrix by in situ reaction of aniline in PEGDA/
water solution. In the latter one, PANi short fibres have been previously synthesized, and then mixed to the PCL solution before the fibre
formation via electrospinning process.
PEGDA and PEGDA/PANi scaffolds were obtained by protopolymerization and salt leaching technique. In detail, sodium chloride crystals
(90% wt) with predefined sizes, from 150 to 300 µm, were added to a PEGDA/water solution (50/50wt/wt). PEGDA/water solution with
camphorsulfonic acid (CSA)(0.5g/ml) was used to dissolve the aniline. Different PANi/PEGDA ratios, from 1 to 10% wt/wt, were used.
DAROCUR1173 has been also selected as precursor (0.5%wt). After photo-polymerization (10 min) (λ= 365 nm, 8W), scaffolds were
immersed in bi-distilled water (4 days) to start the ionic dissolution of salt crystals, and then, in ethanol/water solutions to completely
remove the retained water. As for the electrospun membranes, ultrafine CSA-PANi short fibres were obtained by aniline polymerization as
follows: Ammonium peroxydisulfate initiator and 3.2 mmol amount of aniline monomer were separately dissolved in a 1M CSA-doped acid
solution and rapidly mixed together all at once. The synthesis was left to proceed for 2 h at room temperature. Then, PANi short fibres (1%
wt) were integrated to polycaprolactone (PCL - Mw 65kDa – Sigma Aldrich) in chloroform solution. The mixture was electrospun to obtain
micrometric fibrous membranes. Both scaffolds were morphologically characterized by SEM/FESEM and TEM microscopy while basic
conductive properties were investigated in terms of proton and/or electrical conductivity of substrates by home-ade equipments. Finally,
In vitro response was assessed by culture of PC-12 cell and human mesenchymal stem cells (hMSC)
As for hybrid macroprous hydrogels, the photo-crosslinking reaction was preliminary optimized in terms of precursors, UV exposure time,
and source/sample distance. This allowed to develop porous scaffolds to obtain an homogeneous porous structure - 80% of porosity
- and pore size of ca 140 μm. The addition of PANi did not evidently affect the pore architecture but drastically influenced the scaffold
conductivity. Dry and wet mesurements showed that the electrical conductivity significantly increased with the increase of the PANI
content from (7.6 ± 0.3)x10-8 to (1.1 ± 0.5)x10-3 mS/cm, switching from an insulating to conductive behaviour. We proved that different
PANi content also affects the cell behaviour showing a more significant neurites sprouting from cell bodies as the PANi content increases.
Similarly, the integration of PANi short nanofibres, up to 1% wt, into PCL electrospun membranes did not provoke any remarkable alteration
of the fibre architecture as confirmed by SEM/image analysis investigation which showed a broader distribution of fibres with only a slight
reduction of the average fibre diameter from 7.1 to 6.4 μm. Meanwhile, biological assays to evaluate of cell survival rate (MTT assay) and
immunostaining of sarcomeric a-actinin of cardiomyocites-like cells clearly indicated the ability of PANi short nanofibres to promote the
cardiogenic differentiation of hMSC into cardiomyocite-like cells.
CONCLUSION
These preliminary results corroborate the idea that conductive polymers such as PANi can efficiently contribute to stimulate the basic cell
mechanisms, so predicting the coming of a new generation of smart devices able to guide the regeneration of electroactive tissues such
as nerve and myocardium.
REFERENCES
1. Li et al Biomaterials 27 (2006) 2705–15
2. Borriello et al J Mater Sci:Mat Med (2011)22:1053-62
ACKNOWLEDGMENTS
TISSUENET (n.RBPR05RSM2) and MERIT n. RBNE08HM7T for financial support.
46
PCL SCAFFOLDS PRODUCED BY SCCO2 TECHNIQUE AND NANOPARTICLE INCORPORATION
Nicoletta Rescignano1,2, Ilaria Armentano², Simona Montesano³, Carlos Elvira¹, Sabata Martino³, Aldo Orlacchio³ and Josè M.
Kenny1,2
1 ICTP-CSIC, Juan de la Cierva 3, Madrid, Spain,
2 Materials Engineering Center, University of Perugia, Str. Pentima 4, 05100 Terni, Italy,
3 Department of Experimental Medicine and Biochemical Science, University of Perugia, Perugia, Italy,
[email protected]
INTRODUCTION
The most often utilized biodegradable synthetic polymers for 3D scaffolds in tissue engineering are saturated poly-α-hydroxy esters, including
poly(lactic acid) (PLA) and poly(glycolic acid) (PGA), and their copolymer poly(lactic-co-glycolide) (PLGA) [1-3]. Polycaprolactone (PCL)
is also an important member of the aliphatic polyester family. It has been used to effectively entrap antibiotic drugs and thus a construct
made with PCL can be considered as a drug-delivery system, being used to enhance bone ingrowth and regeneration in the treatment of
bone defects [4]. Several techniques were employed for PCL scaffold production. Solvent casting/particulate leaching [5] results in thin
scaffolds with a good controlled interconnected porosity and pore size. Emulsion freeze-drying or thermally induced phase separation
leads to pore size between 20 and 100μm, 3D-printing [6] requires the use of a complex and specific equipment. The supercritical ﬂuid
(SCF) processing has been developed as an alternative to conventional manufacturing methods. This technique combines liquid like
densities (resulting in high solvent power) with gas like viscosities (resulting in high diffusion rates) [7]. Another important beneﬁt of SCF
based production methods is the reduction in problems associated with residual solvents.
Materials
Polycaprolactone (PCL), used for scaffold preparation, was purchased by Sigma Aldrich. The NPs were synthesized from Poly(L-lactide)
PLLA (the double emulsion method) and the chloroform was used as solvent.
Methods
Supercritical carbon dioxide foaming gas (scCO2) was used for the scaffold formation. Before scaffold production, the PCL films were
prepared by two techniques:
- solvent casting
- microextrusion and moulding press.
Field Emission Scanning Electron Microscopy (FESEM) was used to analyze the morphology of the PCL scaffolds. The differential scanning
calorimeter was employed in this study to detect the change in thermal behaviour after the scCO2 treatment. To improve the wettability and
the roughness of scaffold surface in this work we performed an oxygen plasma treatment.
The morphological characterization in figure 1 shows that the PCL scaffolds can be successfully fabricated by using supercritical gas
foaming technique (scCO2). The results demonstrated that scCO2 PCL have the typical skin-core structure, the cell sizes are more than
30μm for all samples. The microstructures of PCL scaffolds appeared to be macroporous, and an interconnected open pore microstructure
was achieved. The pores distribution and dimension are influenced by several parameters: pressure, depressurization rate and the
saturation time.
a
b
The change of surface topography was observed on the pores after the plasma treatment.
c
d
Figure 1 FESEM images of PCL scaffolds.
CONCLUSION
It is demonstrated in the present work that porous PCL scaffolds can be successfully prepared by scCO2 methods, this technique possess
advantages over standard processing methods for the production of scaffolds for use in tissue engineering.
REFERENCES
1. Mano J.F. et al., Compos. Sci. Technol. 64:789-817, 2004., 2. Seal B.L. et al., Mater. Sci. Eng: R: Rep., 2001, 3. Jagur-Grodzinski J. Reactive Funct.
Polym. 39:99-138, 1999. 4. Pitt C.G. et al., Biomaterials; 2:215-220, 1981. 5. Devin J.E. et al., J Biomater. Sci. Polymer. 7:661-669, 1996 6. Hutmacher
D.W. Biomaterials 2000; 21: 2529-2543. 7. Kazarian S.G., Martirosyan G.G. J Pharmaceutics 2002; 232: 81-90.
47
MODULATION OF MMP-9/TIMP ACTIVITY IN PREVENTING CARDIAC DISFUNCTION THROUGH A COMBINATION OF
MOLECULARLY IMPRINTING TECHNOLOGY AND BIODEGRADABLE MICROFABRICATED SYSTEMS
Caterina Cristallini¹ , Niccoletta Barbani², Elena Bellotti², Francesca Manetti², Elisabetta Rosellini², Mariacristina Gagliardi²,
Elisa Del Gaudio², Fabio Tricoli¹, Sara Mantero³
1 CNR Institute for Composite and Biomedical Materials, University of Pisa, Italy
2 Department of Chemical Engineering, University of Pisa, Italy
3 Department of Bioengineering, Politecnico di Milano, Italy
[email protected]
INTRODUCTION
The optimal scaffold to engineer cardiac muscle tissue has to satisfy a large repertory of requirements such as biodegradability, great
compliance towards the natural tissue, specific microarchitecture to stimulate cell growth and differentiation. Recently the authors
prepared and characterised three-dimensional microfabricated and bioartificial scaffolds through soft lithography able to mimic the
cardiac extracellular matrix architecture. The feasibility of manufacturing new multi-functional systems with a specific function at meso,
micro- and nano-scale level for cardiac tissue engineering was demonstrated. In this work we have investigated more extensively for each
level the properties of multi-functional systems obtained.
A meso-scale feature can be addressed by developing scaffolds based on blends of biodegradable synthetic polymers and natural
polymers. To optimize degradation rate and mechanical properties of scaffold biomaterial, a ternary bioartificial material was prepared
using a newly-synthesised tri-block copolymer, poly(ε-caprolactone) and gelatin. To modulate alignment and differentiation of stem cells
the three-component biomaterial was processed by soft lithography in an anisotropic microfabricated structure. The behavior of constructs
was examined by physico-chemical, morphological, mechanical and degradation analyses. To evaluate the materials potential for in vitro
cardiac engineering bioreactor tests were also performed.
Finally, molecularly imprinted nanoparticles (MIPs) able to recognize a specific metalloproteinase (MMP-9) were prepared to modify
microfabricated systems with the aim of reducing or preventing left ventricle remodelling. After deposition, evaluation of rebinding
specificity and selectivity of MIPs towards MMP-9 in respect to its specific inhibitor was carried out.
To obtain ternary bioartificial material, a mixture of poly(e-caprolactone) (PCL), three-block copolymer (PVL-POE-PVL), and gelatin (GEL)
was prepared starting from organic solvent and aqueous solutions of the components in a selected composition. Previously, a threeblock copolymer was synthesised in our lab by ring opening polymerisation of δ-valerolactone (VL) in the presence of poly(ethylene
glycol) (PEG, 35kDa) and VL/PEG percentage weight ratio 85/15. The ternary bioartificial mixture PCL/PVL-POE-PVL/GEL (72/18/10 w/w)
was homogeneously distributed over the PDMS mould having a rectangular micropatterning. Bioartificial systems were characterized by
infrared analysis (FT-IR Chemical Imaging), scanning electron microscopy (SEM), chromatography (HPLC, GPC), mechanical analysis
(DMA) and bioreactor tests. For nanoparticle synthesis, methacrylic acid (MAA) and poly(ethylene glycol) ethyl ether methacrylate ((PEGMA) as monomers and TRIM as cross-linker were used. Human MMP-9 (92 kDa) metalloproteinase enzyme was used to obtain MIPs.
HPLC and light scattering analyses were performed to characterize nanoparticles. Rebinding and selectivity tests were carried out in PBS
solution at various enzyme concentrations also after deposition on microfabricated structure.
FTIR analysis confirmed the ternary composition of microfabricated materials and their homogeneous chemical distribution. DMA
analysis showed improved mechanical properties in terms of elasticity in comparison with results previously obtained for two component
bioartificial systems. The reliability of microfabricated material to bioreactor working conditions was also verified. Dynamic light scattering
tests allowed to measure the size and size distribution of imprinted nanoparticles. Deposition procedure of nanoparticles onto systems
was optimized by varying a wide range of particle dispersion parameters.
Figure shows SEM analysis of microfabricated systems after deposition of MMP-9 imprinted
nanoparticles. Rebinding tests verified the molecular recognition ability of the systems towards
the specific enzyme. Stem cell response to the developed constructs, functionalized or not, is
currently being analysed. In this work, it was optimized the production and characterization of
nano-modified materials to control enzyme activity at early remodeling phase after infarction and
benefit cardiac tissue engineering.
ACKNOWLEDGMENTS
This work is supported by PRIN-2008
48
AN INNOVATIVE METHOD TO INDUCE CELL INGROWTH THROUGH 3D POROUS SCAFFOLDS BY POOR CELL
ADHESIVE COATINGS
Eloisa Sardella¹, R. A. Salama4, R. Gristina¹, A. N. Habib4, G. H. Waly4, P. Favia1,2,3
1 CNR-Institute of Inorganic Methodologies and Plasmas, IMIP-CNR
2 Department of Chemistry, University of Bari “A. Moro”
3 Plasma Solution Srl, Spin Off dell’Università di Bari “A. Moro”
4 Department of Biomaterials, Faculty of Oral and Dental Medicine, Cairo University
[email protected]
INTRODUCTION
Every year a large part of western countries’ medical charges is assigned to medical care of patients suffering from tissues and/or
organs alterations. Health centers spend for this purposes more than 10 million euro/year and this expense grow continuously due to the
population mean age’s increase. Tissue engineering (TE) represents a valid alternative to organ/tissue transplantation. A fundamental step
for TE is the cell seeding directly on porous tridimensional artificial structures called scaffolds. However on artificial polymer scaffolds
made of PCL or PLA as an example often higher cell colonization at the periphery and inadequate colonization at the center of the scaffold
were noted due to the hydrophobic character of the materials [1,2]. In this work a single and multistep innovative approaches based
on plasma technology are proposed as an interesting alternative. A plasma deposition of Polyethylene oxide like (PEO-like) coatings is
proposed alone and in combination with O2 plasma treatment to stimulate cell ingrowth.
Poly ε-caprolactone (PCL) scaffolds, having a pore size ranging from 150μm to 300μm, fabricated by solvent casting/particulate leaching,
were treated in a low pressure plasma reactor. A mixture of diethyl glycol dimethyl ether vapor and argon (Ar5sccm, DEGDME 0.4sccm),
at input power values of either 5 watt (PEO5W) or 10 watt (PEO10W) at 400mTorr was used to produce PEO-like coatings wich are
cell-repulsive at different extent. Oxygen plasma treatment (OXY, 100sccm Ar, 20sccm O2, 200mTorr, 200W, 15min) was performed to
hydrophilize the inherently hydrophobic PCL scaffold. This aimed at investigating the outcome of deposition of PEO-like coating on cell
distribution within the scaffold core when applied on inherently hydrophilic polymers. Physico-chemical assessment of both the treated
and untreated surfaces was carried out. Scaffolds were then seeded with Saos-2 osteoblast cell line and cell viability test was performed.
To assess cell distribution within the scaffold, the cells were fixed and viewed with a fluorescence microscope.
XPS C1s spectra of PEO5W and PEO10W coatings deposited on porous
PCL revealed the presence of different functionalities centered at: 285.0
eV (hydrocarbon C-C/C-H), 286.5 eV (ether COR, alcohol COH) and 287.9
eV (carbonyl C=O or ketal O-C-O) as binding energy. For PEO5W coatings
deposited on PCL scaffolds, the ether (alcohol) groups (i.e. PEO-character
[3]), reflecting the non-fouling character, were approximately equal to 54%
while that one acquired on PEO10W coatings are 31%. The PEO-character
was higher in PEO5W and O2 + PEO5W (coatings PEO5W deposited
after a pre-treatment with O2) coatings, compared to PEO10W and O2 +
PEO10W samples.
Figure 1: The % area covered by Saos-2 osteoblast cells within
the scaffold core after 1 and 5 days post-culture
Cell viabilty results showed that plasma treatment was an effective approach to enhance cell adhesion. Comparing both the top and the
core of the scaffolds, 5 days after cell culture, fewer cells were present in the scaffold core with respect to the surface. However, the %
of area covered by cells in case of top surface of PCL scaffolds coated with PEO5W and PEO10W was twice that within the core. For
native PCL, O2, O2 + PEO 1 and O2+PEO10W, the % of area covered by cells within the scaffold core was at least two-third that on the
top surface of the scaffold (figure 1).
REFERENCES
1. Barry J. J. A., et al. Adv. Funct. Mater. 15, 1134–1140, 2005;
2. F. Intranuovo, E. Sardella, et al.; Surface and Coatings Technology; 205, 2, S548–S551, 2011;
3. E. Sardella, et al.; Plasma Processes and Polym., 1, 63-72, 2004
ACKNOWLEDGMENTS
S. Cosmai is greatly acknowledged for his assistance in the lab. The project PRIN 2008 - 20089CWS4C is gratefully acknowledged for
the financial support.
49
GRAFTED-MODIFIED PLLA: NEW APPROACHES FOR PHENOTYPE GUIDING IN CARDIOVASCULAR TISSUE
ENGINEERING
Francesca Boccafoschi¹, Luca Fusaro1, Cecilia Mosca1, Michela Bosetti2, Mario Cannas1
1 Health Sciences Department, University of Piemonte Orientale “A. Avogadro”, 28100 Novara, Italy
2 Dipartimento di Scienze del Farmaco, University of Piemonte Orientale “A. Avogadro”, 28100 Novara, Italy
[email protected]
INTRODUCTION
Vessels can be prone to many pathologies, both traumatic, such as thrombosis and aneurysm, and chronic, such as arteriosclerosis.
Various methods are used to treat these pathologies, such as prosthetic implants for repairing or substitution of damaged vessel. Polymeric
materials are primarily used, because of the high variability in mechanical, physical and chemical properties. Biodegradable polymers
are object of the majority of studies, because of their ability to be degraded by the host organism, avoiding late stent thrombosis unlike
permanent grafts. [1]
Because of its particular mechanical properties, Poly-L-lactide acid (PLLA), both alone [2] or as a copolymer [3], is one of the most used
polymers in vascular substitutes research.
A key issue of constructing a functional scaffold for vascular grafting is to control the cell phenotype during the regenerative
process. [4] One approach to control cell phenotype is surface grafting, able to bind a bioactive peptide to polymeric surface.
Most used peptides are RGD, a fibronectin derived adhesion motif [5], and SIKVAV, a laminin derived angiogenetic motif. [6] Aim of this
work is to evaluate adhesion, proliferation and differentiation of C2C12 myoblast murine cells and H9C2 cardiomyocytes rat cells, when
seeded on differently grafted PLLA surfaces, in order to show eventual variance of cells behavior for every type of grafted surface.
PLLA films were obtained by casting technique, solubilizing solid PLLA in a 5% solution in chloroform, and pouring this solution
in a mould. Films obtained were hydrolysed for 30’ in a solution of 0,5 M NaOH in 50% ethanol/water solution. Grafting procedure
was performed pouring over the PLLA film a solution of 14mM EDC, 5,5mM NHS and 50mM MES, for 1 h. This solution was
then replaced with 0,5 mg/ml RGD, SIKVAV or 3:1 molar ratio mix of the two peptides, for 24 h at 4° C. Grafted surface were then
characterized by XPS spectroscopy, to verify the chemical composition of PLLA surfaces, and the accomplishment of the reaction.
To verify cells adhesion, proliferation and differentiation, several assays were performed. SEM microscopy was performed on cells
cultured on grafted surfaces for 96 h, to evaluate cell morphology. To verify cells proliferation, MTT assay was performed on cells cultured
on control and grafted PLLA for 24, 48, 72 and 96 h.
Finally, Western blot technique was used to investigate the biological markers of proliferation (PCNA), and differentiation (Myf5, Myogenin
and Myosin Heavy Chain), in cell cultured on control and grafted surfaces for 24, 48, 72 and 96 h.
Surface characterization, through contact angle measurement, showed that water contact angle on grafted and hydrolysed
surfaces is lower than control surface contact angle, meaning that modified surfaces are more hydrophilic than
control PLLA. XPS showed, as expected, nitrogen presence on grafted PLLA, due to the presence of grafted peptides.
SEM images showed that cells cultured on SIKVAV and mix grafted PLLA had characteristic spindle shape, while cells cultured on control
and grafted PLLA exhibited a spread shape.
MTT assay showed no significant differences of proliferation between cells cultured on differently grafted surfaces. Western blot assay
showed for PCNA similar results to MTT assay, while, concerning cell differentiation, cells cultured on SIKVAV and peptide mix grafted
surfaces increased the expression of a contractile phenotype with respect to cells cultured on control and RGD grafted surfaces.
CONCLUSION
In this work an evaluation of proliferation and differentiation of myoblasts and cardiac cells on grafted PLLA surfaces was performed. An
overall view of the results suggested that SIKVAV peptide significantly promotes differentiation mainly on cardiac cells. These results could
lead to the use of SIKVAV peptide on grafted scaffolds for cardiovascular applications.
REFERENCES
1. Pfisterer ME. Circulation. 118: 1117-1119, 2008.
2. Chu CF. et al. Biochim Biophys Acta. 1472: 479-485, 1999.
3. Cohn D. et al., Biomaterials; 26: 2297-2305, 2008.
4. Moussallem MD. et al., Biomacromolecules.10: 3062-3068, 2009.
5. Hersel U. et al.,. Biomaterials. 24: 4385-4415, 2003.
6. Kibbey MC. et al., NCI Journal. 21: 1633-1638, 1992.
50
IN VITRO ASSESSMENT OF COLLAGEN FILMS FUNCTIONALIZED WITH NH2 AND COOH GROUPS BY PLASMA TREATMENT
Francesca Taraballi¹, Cristina Lupo¹, Laura Russo1, Stefano Zanini2, Claudia Riccardi2, Silvia Panseri3,4, Carla Cunha3,4, Marcello
Campione5, Maurilio Marcacci3, Laura Cipolla1 and Francesco Nicotra1
1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy
2 Department of Physics “G. Occhialini”, University of Milano-Bicocca,University of Milano-Bicocca, Milano-Italy
3 Laboratory of Biomechanics and Technology Innovation, Rizzoli Orthopaedic Institute, Bologna, Italy
4 Laboratory of Bioceramics and Bio-hybrid Composites, Institute of Science and Technology for Ceramics, National Research Council, Faenza, Italy
5 Department of Geology and Geotechnology, University of Milano-Bicocca, Milano, Italy
[email protected]
The promising trends in biotechnology and tissue engineering are based on the development of advanced materials with biomimetic
features in order to recreate the native environment promoting the appropriate cell behavior for tissue regeneration1,2.
Articular cartilage exhibits a well-ordered organization with an extracellular matrix arranged as a network of collagen fibers and proteoglycans
that allow for cell adhesion, mechanical support, transduction of chemical and mechanical signals from the surrounding tissue to the cells.
Among natural polymers, collagen is universally applied as biomaterial in regenerative medicine because of its unique biocompatibility,
and structural property.
Robust techniques for surface “biodecoration” are currently required and the appropriate surface functionalization still remains a critical
variable for the optimal performance of a wide range of biomaterials. Covalent bonding of bioactive molecules to material surface is a valid
strategy in order to allow a sufficiently strong and specific affinity of biomolecules with the surface itself; in addition covalent bonding may
permit site-directed immobilization and preservation of specific conformation and exposition to control biological responses.
Plasma processes allow to tune surface properties of materials with negligible effect on their bulk3,4. The need of improving cell/surface
interaction has decisively introduced plasma techniques in the field of biomaterials.
In the present work, we investigated the use of a plasma modification strategy enabling the introduction of carboxylic and amino groups
on the surface of collagen films and electrospun nanofibrous collagen scaffolds (Scheme 1). Novel functional groups, were subsequently
reacted with a fluorescent probe via a covalent bond, in order to better understand the chemical reactivity of the carboxylic and amino
groups introduced by deposition.
Here, we have prepared type I collagen films functionalized by plasma
technology with the carboxyl group -COOH and the amino group
-NH2. Non-functionalized films were used as control.
MG-63 human osteoblast-like cells were seeded onto the collagen
film surfaces and cell proliferation, viability and morphology were
assessed at 1, 3 and 7 days.
Surface modifications of collagen films and fibrous scaffolds were
evaluated by means of contact angle measurements and Atomic force
microscope measurment.
Contact Angle: A) Untreated and Plasma treated collagen films.
B) Untreated.
RESULTS AND CONCLUSIONS
High cell proliferation and high viability was found for all films. Good biocompatibility was also demonstrated by morphological cell
analysis, with both functionalized and control films equally cell colonized. These results show that the in vitro behaviour of collagen films
functionalized with -COOH and -NH2 groups is identical, with no difference to control, indicating that the introduction of functionalization
groups onto collagen surface does not impair cellular performance, opening a range of possibilities for collagen surface modification with
different bioactive molecules for articular cartilage regenerative applications.
REFERENCES
1. Lutolf MP et al. Nature Biotechnol. 23:47-55, 2005.
2. Ratner BD et al. Ann. Rev. Biomed. Eng. 6:41-75, 2004.
3. Daw et al, Plasmas Polym. 4:113-132, 1999.
4. Zanini S et al., Eur. Phys. J. 2:159-164, 2009
5. Riccardi C. et al. New J. Phys. 073008, 2010.
ACKNOWLEDGMENTS
We gratefully acknowledge Fondazione Cariplo grant n° 2010-0378
51
ANTIBACTERIAL ANODIC SPARK DEPOSITION TREATMENTS FOR TITANIUM
C.DellaValle¹, S.Panzuto¹, E.Saino², L.Visai², M.Santin³, R.Chiesa¹
1 Politecnico di Milano, Dipartimento di Chimica, Materiali e Ingegneria Chimica “G. Natta”, IT
2 Università degli Studi di Pavia, IT 3School of Pharmacy and Biomolecular Sciences, University of Brighton, UK
INTRODUCTION
The direct osteointegration between titanium surfaces and bone
tissue represents one of the primary goal for many dental and orthopaedic application. This can be obtained throgh the modification
of titanium oxide layer by mimicking hydroxyapatite mineralization
and osteoconductive properties.
Actually, an additional complications related to the use of implanted materials is represented by the infection of clinical devices. To
reach this challenging goal the biomaterials research is nowadays
focused on new surface treatments development for titanium, performed to prevent bacterial
adhesion and to reduce the risk of infection. The aim of this
study was to provide antibacterial effects on titanium surface using the Anodic Spark Deposition (ASD) technique, which
allows to obtain homogeneous and stable modified oxidized
surface. The starting-point was an ASD silicon-based osteointegrative treatment previously developed [1] and now in
clinical use (dental field). Therefore the goal of this work was to develop a new biomimetic treatments on titanium with high osteintegrative potential characterized by effective antibacterial properties.
MATERIALS AND METHODS
Four surfaces treatments were performed on commercially pure
grade 2 titanium samples (10 mm diam., 0.5 mm thick). ASD treatments were performed in electrolytic solution containing silicon,
calcium, phosphate and sodium ions. This basic solution was enriched with silver salts, or silver nanoparticles or gallium salts mixed
with chelating agents, these necessary to avoid the salts precipitation. The obtained surfaces were anodized in galvano-static condition with a current density of 10mA/cm2 reaching 295V.
i) SiBNa: Ca-P-Si-Na coated titanium grade II by ASD treatment
used ad control followed by acid etching in 5M NaOH for 2h at
60°C;ii) AgNPs: Antibacterial treatment obtained adding silver nanoparticles in the basic electrolytic solution;
iii) NitAgC: Antibacterial treatment obtained adding silver nitrate and
L-cysteine in the basic electrolytic solution;
iv) NitGalC: Antibacterial treatment obtained adding gallium nitrate
and L-cysteine in the basic electrolytic solution.
v) NitGalO: Antibacterial treatment obtained adding gallium nitrate
and Oxalic Acid in the basic electrolytic solution.
Surface properties were evaluated with scanning electron microscopy (SEM), electron dispersion spectroscopy (EDS), thin film
x-ray diffraction (TF- XRD), laser profilometry (LP) and Glow Discharge Optical Emission Spectroscopy (GDOES). Optical contact
angle measurements (OCA), in static conditions, were performed
to investigate the surfaces wettability and the ICP-OES was used to
study the silver release in PBS up to 7 days. For the biological in vitro characterization, a suspension of 3T3 murine fibroblast cultured
in DMEM and Saos2 osteoblast like-cell from human
osteosarcoma cultured in MCM were seeded on the specimens,
respectively of 3×104 cells and 1.5×104 cells. Cells viability and
proliferation were studied with HPI 24h, 48h and 72h after cell
seeding. Viability was also confirmed by images obtained after
DAPI staining. Phalloidin staining was used to investigate the cells
spreading and morphology after 48h and 72h. A preliminary antibacterial study was performed after 3h and 24h of incubation with
Streptococcus mutans (CCUG35176), Staphylococcus epidermidis (RP62A) and Escherichia coli (RB) to investigate the bacteria
viability on treated surfaces.
The SEM and EDS results showed that the biomimetic antibacterial
treatments on titanium are characterized by a stable and homogeneous microporous surface (Fig. 1) enriched with silicon, calcium,
phosphorus, sodium, and showing the presence of silver or gallium
depending on the antibacterial agent in the electrolytic solution.
Also the GD-OES results confirm the stable presence of gallium or
silver in all of the antibacterial surfaces.
Fig. 1. SEM pictures of AgNPs (A), NitAgC (B), NitGalC (C) and NitGalO (D)
Fig. 2. S.mutans CCUG 35176 viability after 3h and 24h of incubation
on treated surfaces. Data were expressed as the percentage of bacteria
growth onto disks and bacteria growth on plastic (positive control)
XRD analysis showed the presence of TiO2 with anatase structure
(2ϑ=25°) for all ASD treated materials. The antibacterial titanium
surfaces, in particular AgNPs, was found to possess an excellent
hydrophilicity showing a water contact angle as low as 18°, very
close to the control SiBNa. The ICP-OES highlights a constant silver
release on the AgNPs and on NitAgC up to 7 days. No cytotoxic
effects were observed on all investigated surfaces which showed similar levels of cellular activity than the control for each time
point for both 3T3 and Saos2 cells line. The images obtained after
staining of nuclei and F-actin filaments confirmed the presence of
health cells strongly adherent to the treated surfaces. A significant
reduction of bacteria adhesion and proliferation was observed for
the treatments containing gallium and silver for all of the tested
gram + and gram - bacteria strains (Fig 2).
CONCLUSIONS
Considering the results obtained in the present study the developed
antibacterial ASD treatments on titanium can be considered promising treatments capable to combine osteointegrative properties to
antibacterial activity.
REFERENCES
[1] WO 2010/013120 A1 Silicon-based biomimetic treatment for
the osteointegration of metal substrates.
52
ENHANCING ANTIBACTERIAL PROPERTIES OF UHMWPE VIA ION IMPLANTATION
Vincenzo Nassisi¹, Pietro Alifano2, Domenico Delle Side1, Adelfia Talà2, Salvatore Maurizio Tredici2 and Luciano Velardi1
1 Department of Mathematic and Physics, INFN, LEAS Laboratory, University of Salento, Italy,
2 Di.S.Te.B.A., Microbiology Laboratory, University of Salento, Italy.
[email protected]
INTRODUCTION
In the last decade, the demand for biomaterials of antimicrobial quality sensibly increased. The essential properties of these materials
must be the biocompatibility, wettability, durability and its antibacterial characteristics. One of the most important biomaterial for medical
applications is the ultra high molecular weight polyethylene (UHMWPE) that it is used to make components of prosthetic knee, hip and
shoulder1.
It is well known that the presence in UHMWPE of Ag atoms increase its antibacterial properties2 while Cu and its alloys are known as
natural antimicrobial materials3. We propose the use of ion implantation on the surface of this material4 in order to modify its antibacterial
properties. The proposed technique consists in the application of a dose of the above ions inside the first layer of the sample to be treated.
The goal can be achieved if the ions are preventively accelerated4.
The implantation of UHMWPE was performed by means of the accelerator Platone, located at the LEAS Laboratory of the University of
Salento. It is a laser ion source with a double stages accelerating gaps5. It consists of an excimer KrF laser and an implantation chamber.
The accelerating voltage applied was of 60 kV, while the implantation dose was of the order of 1016 ions/cm2.
After this treatment the implanted samples were challenged with Staphylococcus aureus, together with the untreated ones. After a week
of incubation, the biofilm maturated on the UHMWPE sample surfaces was stained using green-fluorescent nucleic acid stain (SYTO9;
Molecular Probes, USA). After 15 min dark incubation, biofilm was viewed with a Nikon Optiphot-2 microscope with an episcopicfluorescence attachment (EFD-3, Nikon).
The analysis of the UHMWPE samples with fluorescence microscopy revealed a strong reduction of bacterial adhesion on Cu-treated
UHMWPE and even more with Ag-treated UHMWPE (Fig. 1) as compared to untreated (control) samples (Fig. 2). Quantitative analysis
revealed that the mean values of adherence were reduced of 20% and 7% with Ag and Cu ions, respectively.
Figure 1: Photo of the untreated sample that
showing the adherence of Staphylococcus aureus.
Figure 2: Photo of the Ag-treated sample (a) and Cu-treated sample (b) that
showing a redaction of adherence of Staphylococcus aureus.
CONCLUSION
In this work we have used, for the first time, the Platone accelerator to improve the antibacterial proprieties of the biocompatible
UHMWPE by means of ion implantation. This technique seems to be interesting, since it can open the way to an easier realization
of antibacterial materials using Cu and Ag ions. Higher ion doses and accelerating voltages could improve these good results.
It is also in progress the use of Ti ions to improve the above qualities.
REFERENCES
1. Lee H.B., Application of synthetic polymers in implants. Editors: T. Seagusa, T. Higashimura and A. Abe. Frontiers of Macromolecular Science. Oxford,
Blackwell Scientific Publications, 1989
2. Roe D. et al., J. Antimicrob. Chemother. 61, 869–876, 2008
3. Kubacka A. et al., Applied Catalysis B: Environmental 104, 346–352, 2011
4. Velardi L. et al., Rad. Eff. Def. Solids 165, 637, 2010
5. Lorusso A. et al., Nucl. Instrum. Methods B. 266, 2008
53
IN VIVO STUDIES AND PROTOTYPING OF ZIRCONIA DENTAL IMPLANTS (Y-PSZ)
Antonio Licciulli1, Giuseppe Casarano1, Sergio Franza2, Daniela Diso2, Antonio Chiechi2, Giovanni Vantaggiato3
1 Department of Engineering for Innovation, University of Salento, Via Monteroni, Campus Ecotekne, 73100 Lecce (Italy)
2 Salentec srl, Spin Off of Salento University,Via dell’Esercito 8, 73020 Cavallino (Lecce), (Italy)
3 “City of Lecce Hospital” , Via Prov. per Arnesano km 4 - 73100 , Lecce, (Italy)
[email protected]
INTRODUCTION
Strong and tough yttria-stabilized Zirconia (Y-PSZ) have widened the application spectra of ceramics in dentistry, enabling the use of
all-ceramic restorations also in posterior regions where high-strength structures are required permitting a substantial reduction in core
thickness. Zirconia ceramics, obtained by slip casting forming have been developed by Salento University in collaboration with Salentec.
Long test campaign has showed Zirconia meets all the requirements of ISO 13356:2008 “Implants for surgery – Ceramic materials
based on Y-TZP“. Indeed it obtain CE mark for sale in January 2010. This material has been used in this study both to produce dental
implant prototype by computer numerical controlled lathe and to test the osseointegration by in vivo studies conducted in Chieti University
Laboratories.
Microstructure. Microstructural Analysis of pre-sintered and sintered Zirconia were carried out using SEM Zeiss model EVO-40.
Accelerated Aging Test. Five bars 45x4x3 mm were exposed to water steam at 134°C, 2 bars for a period of 5 hours to evaluate resistance
to ageing. Fraction of monoclinic phase was evaluated before and after ageing by X ray diffractometry analysis
Mechanical properties. Four point bending testes were performed on sintered bars 45 x 4 x 3 mm. Toughness was measured by SEVNB
method according to prEN 14425-5. Cyclic limit fatigue in physiological solution was evaluated by 1.000.000 cycles at peak stress of 320
with a frequency of 10 Hz under a sinusoidal stress wave form [MPa].
Prototyping of implant. Preliminary studies for prototyping titanium/zirconia and all zirconia dental implants have been conducted by using
2 axes - CNC lathe and pre-sintered Salentec zirconia blocks. In the first case Z-look® System has been used as starting geometry. In
second case De Bortoli® Titanium Implant has been modified by substituting a part of it with zirconia element.
In vivo test of osseointegration. Three Zirconia/Titanium dental implant prototype have been implanted in oral cave of sheeps. They will be
extracted to evaluate % of osseointegration by SEM analyses.
Four-point bending strength is 940 ± 130 [MPa], all 5 samples survive to 106 fatigue cycles at peak stress of 320 [MPa] in physiological
solution, fracture toughness measured by SEVNB method is 9,4 ± 0,6 [MPa√m], four-point bending strength after ageing in water steam
at 134°C, 2 bar for 5 hours is 840 ± 135 [MPa].
In fig. 1-a) we can see a 3Y-TZP cylinder (size: 3,7 mm x 11 mm) prepared for in vivo test. In fig. 1-b) we can see a prototype of zirconia
implant obtained by CNC turning with Schaublin Machines SA 225 TM-CNC FANUC (fig.3.43) with two axes (X and Z). It’s possible to
appreciate the shiny surface without cracks. Fig. 1-c) shows a comparison between zirconia/titanium implant obtained in this study and
a commercial titanium implant.
Fig. 1 a) Zirconia cylinder for in-vivo test; b) Zirconia Dental Implant
prototype based on Z-Look® system and obtained by CNC turning; c)
Zirconia/Titanium dental implant prototype obtained by CNC turning and
the starting commercial system (De Bortoli®) by which was obtained.
a)
b)
c)
CONCLUSION
Slip cast Tetragonal stabilized Zirconia Y-PSZ has been obtained with superior mechanical properties. Due to the high packing factor in the
green, a low shrinkage has been achieved which limit non isotropic deformations.
The presintered zirconia shows excellent compatibility with lathe machining. Several prototypal dental implants were successfully obtained
so that an extensive campaign has been started. The proposed method is very interesting for the production of custom dental implants
such us installations of the existing root replication, immediate load implants. A significant aesthetic improvement is offered by both the
system zirconia/titanium and all ceramic.
54
MECHANICAL PERFORMANCE AND IN VITRO STUDIES OF WOLLOSTONITE/HYDROXYAPATITE COMPOSITE SCAFFOLD
FOR BONE TISSUE ENGINEERING
Marina Carrozzo, K.P. Sanosh, F. Gervaso, A. Sannino and A. Licciulli
Department of Engineering for Innovation, University of Salento, Via Monteroni, Campus Ecotekne, 73100 Lecce (Italy)
[email protected]
INTRODUCTION
Porous bioceramics are gaining more prominence in bone repair
surgery. The polymeric sponge replication method has received particular attention because it can provide very high porosity with good
interconnections between pores. Calcium phosphate scaffolds for
bone graft applications has some limitations due to their low mechanical strength and bioresorption rate. In the present study wollostonite/hydroxyaptite (WS/HA) scaffolds were produced using polymer
replica method and their mechanical, bioactivity, biodegradation properties were analysed and compared with pure HA scaffolds.
Moreover, in order to asses the applicability of these novel 3-D macroporous foam systems as scaffolds for bone tissue regeneration.
an in vitro study to investigate the osteoblast-like cell (MG63) response was performed.
Sub micron sized HA synthesized in laboratory and commercial calcium silicate powders (50:50 wt %) were used for making the slurry (70 wt%). PVA and Dolapix were used as binder and dispersant
respectively. Polyurethane sponges were used as the template for
slurry impregnation. After impregnation, the samples were dried in
air and sintered at 1300°C for 3 hr to get the scaffolds. The phase
purity and morphology of the scaffold were analysed using XRD and
SEM. The porosity, mechanical property, bioactivity in SBF and biodegradibility in Tris-HCl of WS/HA scaffold were also evaluated and
compared with pure HA scaffold made with same method.
Cell viability and proliferation on scaffold were assessed by the MTT
test. Osteogenic differentiation was evaluated by alkaline phosphatase activity assay for 19 days. 3-D confocal microscopy studies
have been carried out, scanning from 0 to 250 μm in each foam, in
order to determine the cell morphology after internalization into the
scaffolds. For this purpose DAPI and phalloidin were used as fluorescence probes.
XRD revealed the scaffolds were composed of pure wollostonite and
hydroxyapatite. The scaffolds show a high and highly interconnected
porosity (>90%) (Fig 1.a). Compression tests revealed a two fold
increase in strength with respect to pure HA scaffold (0.5 to 1 MPa).
Formation of apatite layer on the scaffold by soaking in SBF solution shows the material is bioactive. Tris-HCl experiment proved the
WS/HA scaffolds were highly biodegradable with respect to pure HA
scaffold.
The MTT tests showed that MG63 cells proliferation increases
increasing the culture time, confirming that the WS/HA scaffolds promoted cell proliferation without cytotoxic effects and
it is highly biocompatible to cells. The morphological features
of cells were used to determine the biocompatibility of the
scaffold (Fig 1.b). The MG63 could attach, spread and proliferate on the scaffold, suggesting a positive cellular behaviour.
The presence of cell groups interconnected at different depths
of the scaffold was also observed evidencing a good intercellular communication.
Fig. 1
SEM micrograps and laser confocal fluorescence images of
electrospun mats. Bar in fluorescence images: 50 μm.
CONCLUSION
In the present work, high resistance interconnected macrochanneled porous WS/HA scaffolds were obtained by polymer
replica method. The incorporation of wollostonite in HA (50:50
wt %) shows an increase in mechanical, bioactive and biodegradable properties with respect to pure HA scaffold.
Moreover, adhesion, proliferation and osteogenic responses
of MG63 cell in the developed 3D highly porous WS/HA scaffold were investigated. The developed interconnected macroporous foams show excellent 3-D cellular internalization as
well as a good cell response in terms of adhesion, proliferation and differentiation.
55
HIGH PERFORMANCE ZIRCONIA-ALUMINA BIOMATERIAL
Francesca Mazzanti, Giuseppe Magnani, Leandro Beaulardi
ENEA, Faenza Technical Unit on Material Technologies, Faenza (RA), Italy,
[email protected]
INTRODUCTION
Yttria stabilized Tetragonal Zirconia Polycrystals (Y-TZP) were introduced in the biomaterials world as structural material several years ago
with the aim to overcome the problems connected to the utilization of monolithic alumina. Y-TZP materials, containing approximately 2–3%
mol Y2O3, are completely constituted by tetragonal grains and, due to the transformation toughening mechanism present high bending
strength and high fracture toughness. However, the hardness (and so the wear resistance) of Y-TZP is not high enough for applications
where high wear resistance is required. A solution could be the usage of ZTA (Zirconia Toughened Alumina), which combines the benefits
of alumina (high hardness, high stiffness) and those of zirconia (high strength and high toughness).
In this work the properties of a new ZTA material were presented in order to evaluate it as biomaterial for orthopaedic prosthesis and dental
devices.
Sintered samples of ZTA composite are prepared following
the method reported in [1]. Densities of sintered samples
were determined by Archimede’s method. Hardness (HV)
and fracture toughness (KIc) were determined by means of
Vickers indentation. Flexural strength (MOR) was measured
by 4-point bend tests at room temperature. Microstructural
characterization was performed by SEM-EDS. XRD and Raman
spectroscopy were used to evaluate the transformability of the
zirconia tetragonal phase.
1 µm
Figure 1: SEM image of ZTA material
Fine grains of alumina (black grains) and zirconia (white grains) were put in evidence by SEM (Figure 1).
XRD revealed an amount of tetragonal phase higher than 95% vol [1]. Typical mechanical properties of the new ZTA are summarized in Table 1. Here,
properties of some commercial materials are also reported. The new ZTA material developed by ENEA shows better mechanical properties than
commercial materials used in the dental restoration.
Toughness
(MPa m1/2)
4 pt flexural
strength
(MPa)
14-16
6-9
1000-1400
12
9-10
900
12
10
Composition
Hardness
(GPa)
ENEA
ZTA
CERCON
ZrO2
LAVA
ZrO2
KAVO
ZrO2
PROCERA
ALLCERAM
Al2O3
16
3.5-4.5
500
PROCERA
ALLZIRCON
ZrO2
11
10
900
Product
800
1000
IN-CERAM YZ
ZrO2
11
6
1000
IN-CERAM Al
Al2O3
11.5
4
500
Table 1: Typical mechanical properties of ENEA and commercial materials
Figure 2: Distribution of monoclinic phase on the fracture surface
The main reason is the high transformability of the tetragonal phase. Raman spectroscopy was used to map the monoclinic phase on the fracture surface
(Figure 2). The in-vivo e in-vitro tests confirmed the biocompatibility of the ZTA material [2-4].
CONCLUSION
A new ZTA biomaterial was developed. It shows high hardness, toughness and flexural strength and can be proposed as material for ortophaedic
prosthesis and dental devices.
REFERENCES
1. G.Magnani et al., J. Eur.Ceram.Soc., 2005;25: 3383-3392.
2. G. Maccauro et al., Immunopathol Pharmacol., 2009; 22:773-779.
3. G. Maccauro et al., Immunopathol Pharmacol., 2010; 23: 841-846.
4. M.S. Spinelli et al., Immunopathol Pharmacol., 2011;24:153-156.
56
CARBOXYMETHYLCELLULOSE HYDROGEL/ HYDROXYAPATITE COMPOSITE BIOMATERIAL FOR BONE TISSUE
ENGINEERING APPLICATIONS
Daniela Pasqui1, Milena De Cagna1, Milena Fini2, Paola Torricelli2 and Rolando Barbucci1
1 CRISMA Interuniversity Research Centre for Advanced Medical System, University of Siena, Italy
2 Experimental Surgery Department, Rizzoli Orthopedic Institute, Italy
[email protected]
INTRODUCTION
Natural bone is a complex inorganic-organic nanocomposite material, in which hydroxyapatite (HA) nanocrystals and collagen fibrils are
well organized into hierarchical architecture over several length scales.1 Previous works reported the preparation of composites containing
HA in conjugation with some bioactive polymers or proteins, such as collagen, gelatin, chitosan, silk fibroin and chondroitin sulfate.2 In this
work, we reported a new hybrid material (CMC-HA) containing HA in combination with a carboxymethylcellulose (CMC) based hydrogel.
The novelty consists of inserting HA nanocrystals within the hydrogel matrix, rather than mixing it to a polymer. The combination of the
characteristics of both organic and inorganic components inside a single material leads to the formation of a promising biomaterial for
bone tissue regenerative medicine.
The CMC hydrogel, previously synthesized3, is made to swell in an aqueaus solution of hydroxiapatite using suspensions containing
different increasing amounts of HA in order to determne the maximum amount that can be up-taken by the hydrogel. The mechanical
properties of CMC-HA hybrid material were evaluated by rheological analyses and comapred to those of bare CMC hydrogel. FT-IR
and ToF-SIMS analyses provided information about the presence, the interaction of HA with CMC polymer chains and distribution of
HA crystals inside the hydrogel. The hydrogel surface was viewed by AFM measurements. Finally, the possible release of HA from the
hydrogel matrix was evaluated.
By increasing the concentration of HA suspension a hydrogel containing different amount of HA can be prepared. The maximum amount of
HA that can be loaded is about 0.5g/g hydrogel (Fig 1). The presence of HA affects the charcteristics of CMC hydrogel. CMC-HA shows
a lower swelling degree than that of CMC hydrogel. The storage modulus (G’) and the loss modulus (G”) of CMC-HA are higher that those
of bare CMC hydrogel due to the presence of the inorganic phase. ToF-SIMS analysis demonstrates that HA is homogeneusly distributed
both inside and on the external surface of the hydrogel (see chemical maps in Fig 2). AFM measurements indicates the presence of HA
clusters raging between 400 and 500 nm which make the hydrogel surface rougher than that of bare CMC hydrogel (few nm). A small
release (1.25%) of HA from the hydrogel matrix was observed within the first 24h. After that period a no additional release of HA form the
composite was revealed.
Fig 1: Uptake of HA in the CMC hydrogel. The plot reports the amount of
HA contained inside the hydrogels vs the amount used for the loading
Fig 2: ToF-SIMS images showing the distribution of Ca and P signal
referred to HA and the fragment at m/z 129 belonging to CMC polymer.
CONCLUSION
A novel hydrogel containing HA without releasing it was prepared. The presence of HA affects the characteristics of the CMC hydrogel
especially in terms of mechanical properties. This novel biomaterial seems to show suitable properties to be considered as a good
candidate for bone tissue engineering.
REFERENCES
1. Du C. et al., J. Biomed. Mater. Res. 50:518-527, 2000
2. Wang L. et al., Carbohyd. Polym. 68:740-745, 2007
3. Barbucci et al., Soft Matter 6:3524-3532, 2010
ACKNOWLEDGMENTS
The authors would like to thank Prof. Bigi (University of Bologna) for providing HA powders and the FIRB project: “Scaffold per la
rigenerazione dei tessuti scheletrici: valutazione preclinica della loro compatibilità ed efficienza” n° RBAP10MLK7 for providing financial
support.
57
A NEW COMPOSITE CHITOSAN-NANO-HYDROXYAPATITE SCAFFOLD FOR BONE REGENERATION:
FABRICATION, CHARACTERIZATION AND CELL RESPONSE
Paola Nitti1, Anna Gallo1, Antonella Casillo2, Barbara Palazzo1, Luigi Ambrosio 2, Corrado Piconi1
1 GHIMAS SpA, MeLab, Brindisi, Italy
2 CNR-IMCB, Napoli, Italy
[email protected]
INTRODUCTION
In bone tissue engineering, the biodegradable scaffold is a temporary template introduced at the defective site to support
tissue regeneration, while it gradually degrades and is replaced by newly formed bone1. An ideal scaffold is characterized by
excellent biocompatibility, controllable biodegradability, cytocompatibility, suitable microstructure and adequate mechanical
properties. Additionally, it must be capable of promoting cell adhesion and retaining the metabolic functions of attached cells. As
a scaffold, the natural biopolymer chitosan is currently a subject of interest in tissue engineering2. Its ability to support cell
attachment and proliferation is attributed to its chemical properties e.g. its structural similarity to the extracellular matrix of bone
and cartilage3. Aim of the study was to investigate a method to nucleate HAp nano-crystals into the chitosan scaffold structure.
Chitosan sponges with interconnected porous structure were obtained by freeze drying. A nano-HAp coating was grown onto the sponge
surface. The structural morphology of scaffolds was examined in a scanning electron microscope (SEM). Moreover the scaffolds have
been characterized by X-ray diffraction (XRD), Fourier Transform and Attenuated Total Reflectance Infrared Spectroscopy (FTIR-ATR) and
thermo-gravimetric (TGA) analysis.
Cytocompatibility in vitro assay have been conducted by MG63 osteoblasts to evaluate their vitality and proliferation on the
scaffold. Cell differentiation has been assessed by co-culture of mesenchimal cells (hMSC), cell morphology has been
established by SEM. The cytotoxicity of the residues of the enzymatic degradation has been investigated by L929 cell line.
Calcium phosphate nano-crystals can be nucleated into the scaffolds structure trough an in situ crystal growth. The presence of mineral
nano-coating joined to the chitosan has been highlighted trough SEM images. The XRD patterns of HAp nano-crystals nucleated onto
chitosan scaffolds are very similar to the ones of a biological apatite obtained from deproteinated bone. The above observations makes us
believe that the chitosan network not only serves as matrix to the nHAp particles but also provided an anchoring site for nHAp particles
in the structure.
Pure chitosan and nano-composite scaffolds showed a good osteoblasts proliferation, actually in nano-composite scaffolds vitality is
reduced respect to pure chitosan, to advantage of an alkaline phosphatase activity (ALP), indicating that cells are addressed towards a
osteogenic differentiation. Additionally another protein expressed in the late phases of hMSC osteogenic differentiation has been observed
by ospeopontin marker (OPN).
Mesenchimal cells spread onto the scaffold extending their filopodia in and around the interconnected porous structure.
Cytocompatibility tests by L929 fibroblasts put in evidence the absence of adverse reactions to the degradation products of chitosan and
chitosan-nHAp nano-composite scaffolds.
CONCLUSION
The process developed has demonstrated its suitability in the production of chitosan scaffolds able to nucleate in situ biomimetic HAp
nano-crystals on their pore surfaces, as well the positive role of the nHAp coating in enhancing the cellularization of cell-nanocomposite
construct. Osteoblasts exhibited superior initial cell attachment, spreading and proliferation on the nano-composite scaffold in comparison
to chitosan scaffolds. The enhanced in vitro performance induced by nHA coating was demonstrated by the good proliferation and
osteogenic differentiation of mesenchimal cells, indicative of improved poroperties of nano-composites with respect to pure chitosan
scaffolds.
REFERENCES
1. Langer R. et al., Science 260:920-6, 1993
2. Suh JKF. et al., Biomaterials 21:2589-98, 2000
3. Khor E. et al., Biomaterials 24:232339-49,2003
58
COVALENT FUNCTIONALIZATION OF HYDROXYAPATITE: COMPARING DIFFERENT METHODOLOGIES
Cristina Lupo1, Francesca Taraballi1, Laura. Russo1, Monica Sandri2, Anna Tampieri2, Alberto Paleari3, Jesús. Jiménez Barbero4,
Laura Cipolla1, and Francesco Nicotra1
1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy
2 Institute of Science and Technology for Ceramics-CNR, Faenza (RA), Italy
3 Department of Materials Science, University of Milano-Bicocca, Milano, Italy
4 Centro de Investigaciones Biológicas, CSIC, Madrid 28040, Spain
[email protected]
INTRODUCTION
Among various biocompatible materials, synthetic hydroxyapatite (HAp) and carbonate hydroxyapatite (CHA) are widely used in many
biomedical applications. HAp is the natural mineral ingredient of bones, tooth and calcified tissues in vertebrate. Synthetic HAp and CHA
are used for human implant coatings possessing beneficial biocompatibility and osteoconductivity [1], [2].
The combination of inorganic materials, such as CHA, and organic signaling molecules is very promising in tissue engineering. HA primarily
facilitates cell adhesion and provides a suitable microenvironment for bone progenitor cells. Indeed stoichiometric HA displays a proven
osteoconductivity and its geometry may deeply influence specific tissue responses, as it happens for vascular in-growth. Nonetheless,
researchers try to upgrade these inorganic materials by the addition of biological cues, activating peculiar specific cell-biomaterial
interactions, thereby triggering additional, and possibly more specific biological responses. Unfortunately hydroxyapatite possess a paucity
of reactive functional groups on its surface; therefore covalent linkage of biomolecules on this material is still challenging. In this report we
wish to compare different methodologies for covalent functionalisation of the hydroxyl groups naturally present in hydroxyapatite.
CHA granules (size 400–600 µm) were functionalized by three different synthetic methodologies: i) direct condensation of hydroxyl groups
with sulfonyl chloride derivatives, as activated chemical species. As chemical probe, dansyl chloride was used, due to its fluorescence
features (Fig.1); ii) two steps functionalization with a) tetraalkoxysilanes (TEOS) and b) 3-(aminopropyl)-triethoxysilane (APTES) and iii)
functionalization via sol-gel methods.
The resulting materials were characterized by ATR/FT-IR and NMR solid state in order to establish the efficiency of the methods.
Fig 1.Dansyl functionalization of HA
CONCLUSIONS
NMR experiments revealed that CHA functionalisation occurs effective in any of the condition tested, however the functionalisation degree
is dependent on the applied methodology. FTIR confirm the correct exposure of functional groups/molecules on hydroxyapatite surface.
These results open the way to the decoration of hydroxyapatite with organic bio-molecules able to elicit the desired cellular response, a
relevant issue for tissue engineering applications.
REFERENCES
1. Hersel, U et al Biomaterials 24:4385-4415, 2003.
2. Garcia, AJ et al. Dent. Res. 84: 951-957, 2005.
3. Lis, H. et al, Chem. Rev. 98:637-674, 1998.
ACKNOWLEDGMENTS
We gratefully acknowledge MIUR, under project FIRB RBPO68JL9, and Fondazione Cariplo, grant n° 2008/3175 for financial support.
59
DIELECTRIC BARRIER DISCHARGES ON 2D/3D POLYMERIC SCAFFOLDS FOR APPLICATIONS IN TISSUE ENGINEERING
Giuseppe Camporeale¹, Daniela Pignatelli1, Francesca Intranuovo1, Eloisa Sardella2, Roberto Gristina2, Pietro Favia1,2
1 Department of Chemistry, University of Bari, 70126 Bari, Italy
2 Institute of Inorganic Methods and Plasmas, IMIP-CNR, 70126 Bari, Italy INSTM unit, University of Bari
[email protected]
INTRODUCTION
Cold plasma (deposition, etching, treatments) are widely utilized in research and applications to modify at will surface properties
(hydrophobic/philic character, roughness, distribution of chemical groups, charge, etc.) of biomedical materials [1]. Low (LP) are utilized
more often than atmospheric pressure (AP) processes in this field, but the latter are gaining popularity, especially in the field of Plasma
Medicine where AP plasmas are applied directly on living tissues for blood coagulation, wound healing, cancer treatments and other
therapeutic purposes [2]. Among other AP plasma sources Dielectric Barrier Discharges (DBD) are mostly utilized in this field; it has been
found, for example, that different mammalian cells lines show dose-dependent responses to air plasma. In particular, low doses of air
plasma applied to tumor-like cells leads to an inhibition of their growth or have deadly effects on them [3]. DBD are still poorly utilized
to improve surface properties of 3D scaffolds for Tissue Engineering, LP plasmas are more utilized. Safinia et al. demonstrated that AP
plasma treatments can graft O- and N-containing chemical groups at the surface of 3D scaffolds to improve their wettability and affinity
with eukaryotic cells [4]. The aim of our work is to test how air plasma DBD treatments of different kinds of human cells seeded on 2D
and 3D substrates influence their biological behavior. Moreover, we want to assess to which extent such treatments modify the surface of
the materials used, and how these modifications influence cell behavior.
EXPERIMENTAL
3D polycaprolactone (PCL, Mn 70000-90000) scaffolds (20mm dia, 2mm thick) were prepared with the Solvent Casting/Particulate
Leaching technique from PCL/CHCl3 (20/80 wt/wt) solutions and NaCl porogen crystals. Control PCL flat substrates were prepared by
spin-coating a PCL/THF (10% wt/V) solution on PET disks (10 mm dia). Three types of Petri dishes were also plasma-treated: Poly-Styrene
(PS) for bacteriological culture, “Non-Treated” Iwaki Cell Culture Poly-Styrene (ntCCPS) and treated Iwaki Tissue Culture (CCPS) dishes.
PCL (scaffolds and flat substrates) and Petri dishes were processed in air in a homemade parallel-plate AP DBD. The device is equipped
with two electrodes made of copper (upper, connected to the electric field, covered with quartz) and steel (lower, grounded), respectively.
Petri dishes with/without PCL substrates were treated on the lower electrode, always with the same 2 mm gap. Treatments was performed
at fixed voltage (25 kV), frequency (27.37 kHz), power per pulse (120W), period (2s) and duty cycle (5%). The number of plasma pulses
N was varied from 0 to 27, each delivers 12 J to the substrate. Water Contact Angle measurements were performed on processed samples
before, immediately and 3h after (to check ageing effects) plasma modifications, as well as X-ray Photoelectron Spectroscopy analysis.
Cell culture of Saos2 immortalized osteoblasts and NHDF fibroblasts were performed on the samples, followed by MTT and fluorescence
analyses.
WCA and XPS analysis clearly show how chemical composition and wettability of material surfaces are influenced by plasma
pulses. The distribution of plasma-grafted oxygen and nitrogen containing is a function of the number of plasma pulses.
Changes produced by the pulses depend heavily on the treated substrate; an increase of the wettability is always recorded with
increasing the number of pulses. Ageing effects due to the hydrophobic recovery of the treated surfaces are measured too.
Cells seeded on each substrate and directly treated in air plasmas show specific proliferation trends as a function of the kind of treated
material and of the number of pulses. The exposure to plasma pulses has effects on both cells and material surfaces; disentangling the two
effects is not easy, since cell growth could be affected in opposite way by the number of pulses. For example, at low pulse number cells
may be activated and their growth favored by the slight increase of hydrophilicity imparted to the substrate. High pulse number, instead,
have negative effects on cells, while they are more effective in increasing hydrophilicity and cell-compatibility of substrates.
REFERENCES
[1] Favia P. et al., S. Guceri & A. Fridman ed., NATO Science for Peace & Security, 2008
[2] Fridman G. et al., Plasma Process. Polym. 5:503-533, 2008ù
[3] Kalghatgi S. et al., PLoS ONE 6(1):1-11, 2011.[4] Safinia L. et al., Macromol. Biosci. 7:315-327, 2007.
ACKNOWLEDGMENTS
Authors would like to thanks Mr S. Cosmai and Mr D. Benedetti for technical support and the RINOVATIS Project of Dhitech (Distretto
Tecnologico Pugliese High Tech) for funding.
60
PREPARATION AND CHARACTERIZATION OF NEW SILICA/PEG HYBRID MATERIALS
Laura Russo1,3, Esther Valliant3, Luca Gabrielli1, Laura Cipolla1, Jesús Jiménez-Barbero2, Francesco Nicotra1, Julian R. Jones3
1 Department of Biotechnology and Biosciences, University of Milan – Bicocca, Piazza della Scienza 2 Milan, Italy
2 Centro de Investigaciones Biológicas, CSIC, c/ Ramiro de Maeztu 9, Madrid 28040 (Spain)
3 Department of Materials, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
[email protected]
INTRODUCTION
Bioactive materials are being designed to stimulate specific cellular responses at the molecular level1. Bioactive glass scaffolds are amorphous,
silicate-based resorbable materials that bond to bone and stimulate new bone growth, making them good candidate materials for tissue engineering;
however, they are brittle. To overcome this drawback, hybrid inorganic-organic nanocomposites have been designed and synthesised, in order
to improve toughness2.
The aim of the present work is to fabricate tailored scaffolds with defined mechanical properties and congruent linear degradation for bone regeneration.
Previous work has shown that obtaining covalent coupling between the organic and the inorganic is key for obtaining these properties.2 The objective was
to synthesise and characterize a polyoxyethylene bis(amine)/ silica hybrid material with composition 35 wt% organic and 65 wt% inorganic with covalent
coupling between the PEG and the silica. PEG contains two amino terminal groups available for the covalent functionalisation with the epoxy group of a
coupling agent, glycidopropyltrimethoxysilane (GPTMS).
Two different stoichiometric ratios of functionalisation (GPTMS:NH2-PEG-NH2 2:1 and 4:1) were employed for the fabrication of the hybrid by
the sol-gel process. The inorganic sol was prepared by the hydrolysis of the silica precursor tetraethyl orthosilicate (TEOS) in acidic conditions
in a separate beaker. As the functionalised polymer is added to the silica sol, the methoxysilane groups hydrolyse and then O-Si-O bonds form,
creating covalent bonds between the polymer and the silica (a class II hybrid). The two solutions were added together to create the hybrid sol
(Fig. 1). The hybrid sol was poured into teflon moulds and were sealed and aged at 40° C and dried at 60°C.
Figure 1
Figure 2. SEM photomicrographs of
hybrids A) 1:2 B) 1:4 hybrids
PEG-based hybrid networks with alkoxysilane groups were prepared by epoxy-amino reaction and subsequently addition of hydrolyzed TEOS, to obtain
two different hybrid materials.
New hybrid material was developed for bone tissue engineering via the sol-gel process and proven to have an excellent balance between strain to failure
(10%) and compressive strength (30MPa). Changing the covalent coupling had no significant effect on the mechanical properties for these materials.
This could be due to the low molecular weight of the dPEG (1500 mw). The fracture surfaces of 1:2 and 1:4 hybrid materials were compared using
SEM (Figure 5). The 1:4 hybrids exhibited a fracture surface indicative of brittle fracture (Figure 5b), very similar to a glass. However the 1.2 hybrids
has a more ductile mode of failure (Figure 5a), although the fracture surface would still be considered brittle compared to plastic failure in polymers.
The novel hybrid materials were then characterized in terms of morphological, mechanical and biological properties. Solution NMR studies was performed
during functionalisation reaction, to characterize chemical reactivity of organic and inorganic moieties in the different steps of material preparation.
REFERENCES
1. Hench LL, Polak JM.(2002).Science, 295:1014
2. 4 Mahony, O, Tsigkou, O, Ionescu, C, Minelli, C, Hanly, R, Ling, L, Smith, ME, Stevens, MM, Jones, JR “Adv.Funct. Mater., 2010, 20: 3835
ACKNOWLEDGMENTS
We gratefully acknowledge FONDAZIONE CARIPLO, project 2008/3175, C.I.B. the EPSRC and Imperial College London for financial support.
61
SYNTHESIS AND CHARACTERIZATION OF NOVEL PHOTOREACTIVE CELLULOSE DERIVATIVES FOR USE AS SOFT TISSUE FILLERS
Marta Madaghiele¹, Francesco Ivo Errico1, Maria Grazia Raucci2, Luigi Ambrosio2, Alessandro Sannino1 and Alfonso Maffezzoli1
1 Department of Engineering for Innovation, University of Salento, Via per Monteroni, 73100 Lecce, Italy
2 Institute for Biomedical and Composite Materials, National Research Council, Piazzale Tecchio 80, 80125 Naples, Italy
[email protected]
INTRODUCTION
Photocrosslinkable hydrogels are being investigated for a number of biomedical applications, since they can be delivered in a minimally
invasive manner (e.g. by injection) and then crosslinked in situ. In this work, photoreactive hydroxyethylcellulose (HEC) derivatives
were synthesized by functionalization with cinnamate moieties, which are known to form dimers when UV exposed [1]. The aim was to
evaluate whether aqueous solutions of these HEC derivatives could photo-crosslink upon UV exposure. The resulting hydrogels might be
extremely advantageous compared to non-crosslinked fillers currently used (e.g. hyaluronic acid) because of their higher stability in the
physiological environment, as well as because of the absence of any additional chemicals (e.g. photoinitiator), which would be detrimental
for biocompatibility.
Cinnamated HEC (cHEC) was synthesized in toluene/water/ethanol mixtures of cinnamoyl chloride (CC) and HEC (Sigma-Aldrich), adapting
a protocol described in the literature [1]. Several parameters were varied in order to assess the extent and kinetic of chemical functionalization
(Table 1). Fourier Transform Raman spectroscopy (FT-Raman), UV/Visible spectrophotometry and solubility tests in water (1% w/v) were
used to evaluate the efficacy of the functionalizations, using unmodified HEC as a control. The cytotoxicity of the novel cellulose derivatives
was assessed by evaluating the viability and adhesion of human HeLa cells when cultured either in direct or indirect contact with the
derivatives, also comparing the results with those obtained for methacrylated HEC (mHEC). The latter, which requires the use of a photoinitiator
to be crosslinked, was synthesized for the sake of comparison, according to a well established protocol for similar polysaccharides [2].
The synthesis of hydrogels from aqueous solutions of cHEC (2% and 4% w/v) was then attempted by means of exposure to UV light (UV
lamp with peak of emission at 365 nm), and compared with the synthesis of hydrogels made of mHEC, with the same polymer content.
As confirmed by FT-Raman spectra, the synthesis of cHEC
was performed successfully when using triethylamine (TEA) as
a catalyst (Table 1). In addition to the peaks characteristic of
cinnamate moieties, the spectra highlighted the presence of the
ester linkage at 1700 cm-1, which is representative of the chemical
bond between cellulose and cynnamate groups. In some cases,
a signal due to cinnamic acid dimerization was detectable. The
UV/Visible spectra of cHEC displayed a strong UV absorption at
about 290-300 nm. As expected, grafting of CC to HEC reduced
or inhibited the water solubility of the cellulose derivative; however,
one formulation, among those prepared, was found to be water
soluble and thus of interest for the synthesis of hydrogels
(table 1 bold row). The cytotoxicity tests carried out on this
formulation and on mHEC (both of which freeze dried, from a 2%
w/v solution) revealed that both derivatives are not cytotoxic, as
found for unmodified HEC. Whereas hydrogels based on mHEC
could be formed by exposure of solutions to UV light for 5 min
(using Irgacure 1173 as a photoiniator), solutions with the same
percentages of cHEC, in the absence of any photoinitiator, did
not crosslink upon UV exposure, for times of exposure up to 90
minutes. Since the reactivity of cinnamate groups was detected
in UV/Visible spectra as well as in some FT-Raman spectra (i.e.
cinnamic acid dimers), the lack of crosslinking in water solution
could be ascribed to the use of either low cellulose concentrations
or unsuitable UV wavelengths.
HEC/CC
molar ratio
TEA/CC
molar ratio
Time
Hours
Ester Bond
(Yes/No)
Water
solubility 1%
w/v
1/3
0
Overnight
No
1/3
1/1
Overnight
Yes
No
1/3
1/1
1
Yes
No
Yes
1/3
1/1
3
Yes
No
1/4.5
1/1
Overnight
Yes
No
1/1.5
1/1
3
Yes
Yes
1/0.5
1/1
3
Yes
Partial
CONCLUSION
The results showed that photoreactive and cytocompatible cellulose
derivatives can be synthesized by either cinnamate or methacrylate
functionalization. Water-soluble cinnamated hydroxyethylcellulose
(cHEC) shows potential for the synthesis of hydrogels upon UV
exposure, starting from a photoinitiator-free aqueous solution.
Although photocrosslinking was not achieved using the working
parameters adopted in this work, the synthesis of cHEC hydrogels
appears feasible, if properly tuning the cellulose concentration in
solution and the emission band of the UV lamp.
REFERENCES
1. Kim YT, Yun SH, Oh SS, Lee ES. Applied Chemistry 8, 1: 187-190. 2.
Leach JB, Schmidt CE. Biomaterials 26: 125-135, 2005.
62
SILVER-TREATED FLAX WITH DURABLE ANTIBACTERIAL PROPERTIES FOR BIOMEDICAL APPLICATION
Mauro Pollini1, Federica Paladini1, Luca Salvatore1, Antonio Licciulli1, Alfonso Maffezzoli1 and Alessandro Sannino1
1 Department of Engineering for Innovation, University of Salento, 73100 Lecce, Italy
[email protected]
INTRODUCTION
Colonization of several types of bacteria, especially of Staphylococcus aureus, represents the most common complication in patients
affected by atopic dermatitis (AD) [1], a chronic disease of pruritus and eczematous lesions. In the treatment of this disease, some
solutions have been tried, among which antibacterial drugs or topical steroids. The antimicrobial activity of silver is well known since
ancient time. In this work, silver was deposited on flax yarns adopting a patented silver deposition technique [2] described in previous
works on different substrates [3], in order to produce antibacterial textile for biomedical applications.
White flax yarns were coated with silver according to an innovative technology based on the photo-reduction in situ of a silver salt. The
flax yarns were impregnated with a silver solution containing silver nitrate and methanol. Then, the wet substrates were exposed to a
UV source to induce the photoreduction. SEM-EDX analysis was performed to study the size and the distribution of the silver particles.
The antibacterial capability was checked against E.coli and S. aureus through agar diffusion tests. Thermogravimetric analysis TGA was
adopted to verify the durability of the coating after several washing cycles.
Flax textile with durable antibacterial properties was obtained through an innovative silver deposition technology. High coverage and
uniform distribution of silver particles somewhere aggregated in bigger clusters are evident on the fibers. The size of the particles ranges
from about 100 nm to bigger submicron clusters (Fig.1). The bactericidal capability of the coating against Gram positive and Gram
negative bacteria was confirmed through agar diffusion tests even after several washing cycles. Thermogravimetric analysis performed
on silver-treated flax before laundries and after 1, 5, 10 and 20 industrial washing cycles confirmed the excellent adhesion of the coating
to the substrates. The initial amount of silver was 1.65 wt%, after 1 cycle it is 1.53 wt% and then, up to 20 cycles, it remains always about
1.48 wt%, confirming the good adhesion and stability of silver clusters on the substrate. Very small amount of silver is released just after
the first washing cycle due to very small amount of silver not completely reacted during the deposition treatment.
Figure 1. SEM
micrography of
untreated and silver
treated sample
showing the size
and the distribution
of silver particles
Figure 2.
Antibacterial
capability of silver
treated samples,
before and after
twenty washing
cycles against
Escherichia coli and
CONCLUSION
In this work antimicrobial flax was obtained by the in situ photo-reduction of a silver salt. The strong adhesion of the coating to the
substrate and the antibacterial efficacy were demonstrated even after several washing cycles. The developed textile could represent a
strong instrument to contain skin diseases and infections.
REFERENCES
1. T. Biederman, Acta Derm Venereol. 86 (2006) 99
2. M.Pollini, A.Sannino, A.Maffezzoli, A.Licciulli. European Patent No. EP1986499, 2008.
3. M.Pollini et al., Nano-Antimicrobials Progress and Prospects, 1st ed. Springer, pp 313-336.
63
DIFFERENTIATION RELATED GENE EXPRESSION OF EQUINE ARTICULAR CHONDROCYTES CULTURED
IN CHITOSAN SCAFFOLD
Elena De Angelis1, Francesca Ravanetti², Antonio Cacchioli 2, Attilio Corradi1, Ruggero Bettini3, Paolo Borghetti1
1 Patology Unity,
2 Anatomy Unity, Department of Animal Health, University of Parma, 43126 Parma, Italy
3 Department of Pharmacy, University of Parma, Campus Univeritario, 43124 Parma, Italy.
[email protected]
INTRODUCTION
Cartilage injury remains a major challenge in orthopaedic surgery due to the fact that articular cartilage has only limited capacity for
intrinsic healing and tissue engineering represents a promising approach for cartilage reconstruction. Tissue engineering involves the
development of new biomaterials wich should be the ones that most closely mimic the naturally occurring environment in the tissue1.
The present study investigated on the capacity of the innovative biomaterial chitosan to promote the maintenance of differentiated equine
articular chondrocytes in vitro. Chitosan is a linear polysaccharide with a structure similar to glycosaminoglycans present in cartilaginous
extracellular matrix, it represented an interesting natural biomaterial and shows many advantages: it is biocompatible, biodegradable, nontoxic and not expensive2. In the present study modified chitosan scaffold (Patent, WO 2008/077949), prepared according to the method
described by Bettini et al.3, were seeded with isolated primary chondrocytes and their differentiation markers expression were compared
to the same chondrocytes cultured into other three-dimensional systems of culture: alginate beads and micro-masses.
Chondrocytes were obtained from articular cartilage of fetlock joint of properly slaughtered horses through a protocol previously
described4. Cartilage was digested by enzymatic treatment and chondrocytes obtained were seeded on the scaffolds at the
density of 2 x 106 cells/scaffold, 4 x 105 chondrocytes were pelleted into conical vials for micromasses culture5 and 4 x 106 cells/
ml were re-suspended in 1,2% sodium alginate solution for alginate beads preparation6; all cultures were cultured in complete
D-MEM+10%FCS and incubated at 37°C in a humidified atmosphere for 2 and 4 weeks before gene expression analysis.
Total RNA of each sample was extracted using TRIreagent (Ambion, Inc., Austin, TX), 1 μg of total RNA was reverse transcripted using
an High Capacity RNA-to-cDNA Master Mix kit (Applied Biosystems). cDNA obtained was used as a template for subsequent polymerase
chain reaction using DreamTaq Green DNA polymerase (Fermentas life science, Canada) and 5µl of both forward and reverse primers (2.5
µM) (MWG-Biotech AG) for each specific primers: Coll type I, Coll typeII, aggrecan and versican; QuantumRNA 18S was used (Ambion,
Inc.) as an internal positive control. The PCR products were separated by electrophoresis on 2% agarose gel in SybrSafe (Invitrogen), and
visualized under UV light.
The chondrocytes seeded in micro-masses, after 2 weeks expressed collagen type-II, aggrecan and very low versican whereas collagen
type-I was absent; the situation changed at 4 weeks of culture when versican increased and the expression of collagen type-I was
appeared, markers usually up-regulated during de-differentiation; this indicated that this culture system could induce the production of a
matrix more similar to fibrocartilagineous tissue overtime. The chondrocytes seeded in alginate beads highly expressed collagen type-II
and aggrecan, whereas versican, which was initially absent, appeared weakly at 4 weeks with an increase of the expression of collagen
type-II; no expression of collagen type-I was detected. The results obtained by PCR analysis of the chondrocytes seeded on chitosan
scaffolds showed the presence of the mRNA expression of collagen type-II and aggrecan while collagen type-I and versican were totally
absent. Altogether, these results highlight how the traditional three-dimensional cell culture systems, such as beads but not micromasses
maintain the differentiation of the isolated chondrocytes. Also the modified chitosan used in our experiments seems to offer a good support
for the maintenance of differentiated state of chondrocytes. This behavior could be trace back both to the similarity of the chitosan with
the GAGs structure and to the characteristics of the physiological environment in which chondrocytes live in vivo.
CONCLUSION
These scaffolds prepared with the modified chitosan, could be therefore used as a temporary substrate for viable and differentiated cells,
providing the necessary physical support for the tissue reconstruction.
REFERENCES
1.Suh JK et al., Biomaterials 21: 2589-2598, 2000.
2.Shahidi F et al., Adv Food Nutr Res 49: 93-135, 2005.
3.Bettini R et al., Eur J Pharm Biopharm. 68:74-81, 2008.
4.Borghetti P et al., Tissue & Cell 27(2): 173-183, 1995.
5. Zang Z et al., J Anat 205: 229-237,2004.
6.Guo J et al., Connect Tissue Res 19:227-297, 1989.
ACKNOWLEDGMENTS
The authors would like to thank the “Fondazione Cariparma” for providing financial support to this project”.
64
MULTIFUNCTIONAL PCL/BIOMIMETIC HYDROXYAPATITE NANOCOMPOSITE SCAFFOLDS TO GUIDE HARD TISSUE
ENGINEERING
U. D’Amora1, T. Russo1, A. Gloria1, R. De Santis1, S. Zeppetelli1, M. Sandri2, A. Tampieri2 and L. Ambrosio1
1 Institute of Composite and Biomedical Materials, National Research Council, Naples, Italy
2 Institute of Science and Technology for Ceramics - National Research Council, Faenza, Italy
[email protected]
INTRODUCTION
Among rapid prototyping techniques, 3D fiber deposition showed great potential to manufacture well-defined and custom-made scaffolds
with specific architectures, precise pore shape and size, and suitable mechanical performances. In the field of bone tissue engineering,
much attention has been driven toward the synthesis of new biomimetic hydroxyapatite. As emerged in literature, if compared to stoichiometric hydroxyapatite (HA), Mg- and Mg,CO3-substituted HA enhanced the behaviour of MSC and MG-63 cells in term of adhesion, proliferation and metabolic activation [1]. The aim of this study was to develop 3D fiber-deposited poly(ε-caprolactone)/Mg,CO3-substituted
HA nanocomposite scaffolds, and to study the effect of the biomimetic HA nanoparticles on the biological and mechanical performances.
Poly(ε-caprolactone) (PCL, Mw=65000–Aldrich) pellets were dissolved in tetrahydrofuran through stirring at room temperature. Mg,CO3substituted HA (MCHA) nanoparticles and ethanol were added to the polymer/solvent solution during stirring. A PCL/MCHA weight ratio
(wt/wt) of 80/20 was used. Accordingly, a homogenous paste was obtained and the solvent was suitably removed. Nanocomposite
scaffolds were built layer by layer using a Bioplotter dispensing machine (Envisiontec GmbH, Germany), also taking into account the possibility to design different custom made cylindrical scaffolds for in vivo tests, (6 mm in diameter, D, and 10 mm in height, H), characterized
by a lay-down pattern of 0°5/90°5 and 1500 µm x 1500 µm x 1500 µm pore size (Figure 1).
a
b
Figure 1. Two different views of 3D cylindrical scaffolds obtained through 3D Fiber Deposition technique and characterized by a lay-down pattern of 0°5/90°5 and 1500 µm x 1500 µm
x 1500 µm pore size.
Micro-computed tomography (Micro-CT) was performed through a SkyScan 1072 (Aartselaar, Belgium) system to analyze the architecture, pore shape and size of the nanocomposite scaffolds. Compression tests were carried out on PCL/MCHA nanocomposite scaffolds
to evaluate their mechanical properties by using a 5566 INSTRON dynamometer.
Human mesenchymal stem cells (hMSCs) were seeded onto the 3D nanocomposite scaffolds in order to qualitatively study cell adhesion
and spreading through confocal laser scanning microscopy (Zeiss LSM 510/ConfoCor 2).
As shown through compression tests, mechanical behaviour of 3D PCL/MCHA nanocomposite scaffolds is qualitatively similar to that
of PCL ones. The stress-strain curve is characterized by an initial linear region at low values of strain, suggesting an initial stiff mechanical response. This zone is followed by a region with lower stiffness, and finally it can be noticed another stiff portion of the stress-strain
curve. However, PCL/MCHA nanocomposite scaffolds have provided values of compressive modulus that are higher than those obtained
from the PCL structures. Micro-CT analysis has evidenced the morphological and architectural features of the structures, confirming that
well-organized nanocomposite scaffolds with a repeatable micro-structure, precise pore size and shape, have been obtained. Confocal
analyses have evidenced that the inclusion of MCHA nanoparticles seems to enhance cell adhesion and spreading.
CONCLUSION
In this study the possibility to make appropriate modifications into hydroxyapatite reticular structure for guiding specific cell responses
together with the ability to tailor the morphology, hence the mechanical and transport properties of nanocomposite scaffolds through a
suitable topological optimization process were evidenced.
REFERENCES
1. Landi E. et al. J. Europ. Ceram. Soc. 26:2593–2601, 2006
ACKNOWLEDGMENTS
The authors woul like to thank the project “Materiali Innovativi per lo Sviluppo di BioProtesi Articolari” FIRB RBIPO68JL9 for providing
financial support.
65
MICROTOMOGRAPHIC ANALYSIS OF RETRIEVAL HIP RESURFACING ARTHROPLASTY FAILED AT DIFFERENT TIMES
Salamanna Francesca¹, Masciale Valentina2, Maglio Melania2, Ferrari Andrea2, Parrilli Annapaola1, Cadossi Matteo3, Fini Milena1,2,
Giardino Roberto1,2
1 Laboratory of Biocompatibility, Innovative Technologies and Advanced Therapies, Rizzoli Research Innovation Technology, Rizzoli Orthopaedic Institute, Bologna, Italy
3 II Orthopaedic and Traumatology Clinic, Rizzoli Orthopaedic Institute, Bologna, Italy
[email protected]
INTRODUCTION
Joint replacement is continuously evolving to reduce the invasiveness of surgery, prolong the implant life, decrease complications and
improve the patient’s life quality. Hip Resurfacing Arthroplasty (HR) is emerging as an alternative to conventional total hip arthroplasty
and has been proposed as an option for the treatment of degenerative hip disease in young, active individuals1. The retrieval of failed
prostheses and the analysis of human implanted devices is one of the most valuable tools to provide information about prostheses that
have been submitted to clinical loading and biological and chemical microenvironment during their stay in the body 2-6.
The aim of the study was to analyze by a new and specific quantitative microtomographic (µCT) method the characteristics of bone quality
and its microarchitecture in retrieved metal-on-metal Birmingthan Hip Arthroplasty (BHR).
A series of 948 BHR were performed between 2001 and 2009. Among these 9 retrieved specimens of failed BHR were divided according
to the time to fracture: 3 specimens failed at less than 6 months (Group 1), 3 failed between 6 months and 3 years (Group 2) and 3 failed
later than 3 years (Group 3). µCT assessment was carried out on embedded section of the samples using the Skyscan 1172 computed
microtomographic system (Kontich, Belgium). Microtomographic three-dimensional (3D) analyses were performed considering 3 Volumes
of Interest (VOI) in each compartment: at the top, within 0.8 cm from the HR dome, in the centre, from 0.8 to 1.6 cm from the HR dome,
and at the bottom, 1.6 to 2.4 cm from the HR dome (Figure 1). The morphometric parameters considered were: Bone volume density (BV/
TV); Trabecular thickness (TbTh, μm); Trabecular separation (TbSp, μm); Trabecular number (TbN,mm-1).
Figure 1: Hip resurfacing arthoplasty
prosthesis. The red dots highlight the 6
VOIs considered for each specimen.
Figure2: Histograms of bone parameters
The results of µCT parameters, reported in Figure 2, showed that bone rarefaction progressively change over time. Statistically significant
differences between Groups in the top ROI were found for BV/TV (p = 0.002), Tb.N (p = 0.003) and Tb.Sp (p = 0.048). In the top ROI,
the multiple comparison test showed that Group 2 and Group 3 presented statistical significantly lower BV/TV (Group 2: 61%, p = 0.002;
Group 3: 41%, p = 0.011 ) and Tb.N (Group 2: 53%, p = 0.013; Group 3: 40%, p = 0.03), and higher Tb.Sp (Group 3: 71%, p = 0.040)
when compared to those of Group 1.
CONCLUSION
The main goal of this study was to evaluate the characteristics of bone quality and its microarchitecture in a series of femoral heads that
failed at different times for different reasons by adopting a novel and specific quantitative µCT. 3D measurement showed that bone density
decreases over time especially in group 3 in compared with group 1 and 2. Moreover, 3D information carried out on different ROIs (top,
central and bottom) showed a significant decrease in bone quality over time in the top ROI near cupola. The present study showed that the
morphometric parameters considered are crucial for the understanding of mechanical properties and the causes of the reported HR failure.
REFERENCES
1. McMinn D et al., Clin Orthop. 329:89-98, 1996.
2. Daniel J et al., J Bone Joint Surg. 92:20-7, 2010.
3. van Gerwen M et al., Acta Orthop. 81:680-3, 2010.
4. McMinn DJ et al., Int Orthop. 35:231-7, 2011.
5. Spierings PT. Acta Orthop. 79:727-30, 2008.
6.Shimmin AJ et al., J Bone joint Surg. 87-B:463-74, 2005
66
THE EFFECT OF IRON-DOPED HYDROXYAPATITE NANOPARTICLE INCLUSION ON MECHANICAL, MAGNETIC AND
BIOLOGICAL PERFORMANCES OF INNOVATIVE PCL-BASED SUBSTRATES FOR HARD TISSUE ENGINEERING
R.De Santis1, A. Gloria1, U. D’Amora1, T. Russo1, S. Zeppetelli1, T. D’Alessandro2, M. Sandri2, M. Banobre-Lopez3,
Y. Pineiro-Redondo3, A. Tampieri2, J. Rivas3, V. Dediu4 and L. Ambrosio1
1 Institute of Composite and Biomedical Materials - National Research Council, Naples Italy,
2 Institute of Science and Technology for Ceramics - National Research Council, Faenza, Italy
3 Applied Physics Department, University of Santiago de Compostela, Santiago de Compostela, Spain
4 Institute for Nanostructured Materials - National Research Council, Bologna, Italy
[email protected]
INTRODUCTION
The aim of this research was to develop fully biodegradable and magnetic nanocomposite substrates for hard tissue engineering made of
a poly(ε-caprolactone) (PCL) matrix reinforced with iron-doped hydroxyapatite (FeHA) nanoparticles. The effect of nanoparticle inclusion
on the mechanical, magnetic and biological performances was investigated.
Poly(ε-caprolactone) (PCL, Mw=65000 – Aldrich) pellets were dissolved in tetrahydrofuran (THF) with stirring at room temperature. FeHA
(Fe/Ca=0.20 mol and a Fe(III)/Fe(II) ratio of 1:1, prepared through neutralization method [1] in presence of Fe(III) and Fe(II) doping ions
[2]) nanoparticles and, then, ethanol were added to the PCL/THF solution during stirring. PCL/FeHA nanoparticles 90/10 weight ratio (wt/
wt) was used. Moulding & solvent casting approach [3] was used to make cylindrical specimens with a diameter of 6.4 mm and a height of
0.5 mm. Specimens were placed under hood to remove the solvent. Small punch tests (ASTM F 2183) were performed on PCL/FeHA disk
specimens at a rate of 0.5 mm/min, using a 5566 INSTRON dynamometer. Magnetic measurements were performed in a Vibrant Magnetic Measurement magnetometer operating at room temperature under a maximum magnetic field of 1 Tesla [4]. Magnetic hyperthermia
measurements were performed applying a radio-frequency magnetic field to the samples. Both frequency and amplitude of the oscillating
magnetic field (f=260 kHz and 27 mT) were generated with a home-made alternating current source. Confocal laser scanning microscope
(Zeiss LSM 510/Confocor 2) was used to study human mesenchymal stem cell (hMSC) adhesion and spreading on the substrates at 7,
14 and 21 days after cell seeding. Cell viability and proliferation were assessed by using Alamar BlueTM assay, whilst cell differentiation
was evaluated by measurement of ALP activity.
Results from small punch tests have shown load-displacement curves characterized by an initial linear trend, followed by a decrease of the
curve slope until a maximum load is reached. The inclusion of FeHA nanoparticles seems to improve mechanical behavior (Table 1). Magnetic characterization has shown superparamagnetic behaviour of the doped nanocomposite, with a measurable saturation magnetization,
stressing its ability to be magnetized by applying a magnetic field without remanence once the field is turned off. Magnetic hyperthermia
measurements have evidenced an effective temperature increase of the nanocomposite (table 1).
Materials
PCL
Maximum load Displacement Magnetic Satu(N)
at maximum load
ration
(mm)
emu /g
15.30 ± 1.30
1.60 ± 0.20
-
PCL/FeHA 90/10 22.51± 0.60
wt/wt
2.40 ± 0.45
0.26±0.1
Temperature
increase
°C
3.0±0.1
Table 1: Results from small punch tests and magnetic characterization
performed on PCL and PCL/FeHA nanocomposite substrates reported
as mean value ± standard deviation.
Confocal analyses have shown interesting results in terms of cell adhesion and spreading of hMSCs, at least in vitro. The Alamar BlueTM assay has provided informations on cell proliferation and viability over the culture time. The results obtained in terms of percentage of Alamar
BlueTM reduction were higher for PCL/FeHA nanocomposite substrates than for neat PCL. The osteogenic differentiation of the hMSCs cells
has been assessed by normalized ALP activity. The significant increase for nanocomposite substrates, from day 7 to 14 clearly suggests
that cells were induced to differentiate during this period.
CONCLUSION
The present study may be considered as a first approach toward the design of 3D magnetic nanocomposite scaffolds for hard tissue
engineering, benefiting from the eventual effect of the magnetic field on the tissue regeneration process.
REFERENCES
1. A. Tampieri et al. Acta Biomater., 8: 843-851, 2012. 2. A. Tampieri et al. Italian Patent No. MI2010A001420, 2010
3. T. Russo et al. J. Appl. Biomater. Biomech., 8: 146-152, 2010. 4. M. Banobre-Lopez et al. J. Appl. Phys., 109 : 07B313, 2011
ACKNOWLEDGMENTS
The research leading to these results has received funding from the European Community’s 7th Framework Programme under grant agreement no. NMP3-LA-2008-214685 project MAGISTER, www.magister-project.eu.
67
ADVANCED COMPOSITE PCL/ORGANIC-INORGANIC HYBRID FILLERS:
FROM 2D INNOVATIVE SUBSTRATES TO 3D RAPID-PROTOTYPED SCAFFOLDS FOR TISSUE ENGINEERING
A. Gloria1, T. Russo1, U. D’Amora1, R. De Santis1,V. D’Antò2, F. Bollino3, M. Catauro3, S. Rengo2 and L. Ambrosio1
1 Institute of Composite and Biomedical Materials, National Research Council, Naples, Italy,
2 Department of Oral and Maxillofacial Sciences, University of Naples “Federico II”, Naples, Italy
3 Department of Mechanical and Aerospace Engineering, Second University of Naples, Aversa, Italy
[email protected]
INTRODUCTION
The bioactivity of sol-gel synthesized poly(ε-caprolactone)/TiO2 or poly(ε-caprolactone)/ZrO2 organic-inorganic hybrid materials has been
already shown by the formation of a hydroxyapatite layer on the surfaces of samples soaked in a fluid simulating the composition of human blood plasma [1, 2]. In designing innovative 2D composite substrates for hard tissue engineering, Russo et al. (2010) proposed to
embed PCL/TiO2 or PCL/ZrO2 hybrid fillers into a PCL matrix [3]. This strategy allowed to enhance the mechanical performance of the neat
PCL preserving the bioactive features of the hybrid particles. Benefiting from basic and further analyses performed on the 2D composite
substrates, the aim of the present work was to design and study 3D rapid-prototyped scaffolds consisting of a PCL matrix reinforced with
sol-gel hybrid fillers.
PCL pellets (Aldrich; molecular weight [Mw] = 65,000) were dissolved in tetrahydrofuran (THF) with stirring at room temperature. Organic–inorganic particles (diameter lower than 38 μm) and, subsequently, ethanol were added to the PCL/THF solution during stirring. A
PCL/filler weight ratio (wt/wt) of 80/20 was used. To optimize the particle dispersion in the polymer solution, an ultrasonic bath (Branson 1510 MT) was also employed. Composite pellets consisting of PCL loaded with sol-gel synthesized organic-inorganic hybrid fillers
were processed through 3D fiber deposition technique in order to develop cylindrical scaffolds (6 mm in diameter, 3 mm in height) with
a 0°/0°/90°/90° lay-down pattern. In particular, 3D scaffolds were built by extruding and depositing the fibers along specific directions
according to the selected lay-down pattern. PCL/filler pellets were initially placed in a stainless steel syringe, then heated to a temperature
of about 140°C using a heated cartridge unit placed on the mobile arm of a 3D plotter dispensing machine (Envisiontec GmbH, Germany).
An appropriate nitrogen pressure was successively applied to the syringe through a cap. The nozzle used to extrude PCL/filler fibers had an
inner diameter of 500 µm. Scaffolds were characterized by the fiber diameter, the fiber spacing (1200 µm) and the layer thickness, which
influence the overall pore size and porosity. A deposition speed of about 30 mm/min was employed. In order to evaluate the effect of the
inclusion of the hybrid fillers on the mechanical performances of the scaffolds, compression tests were carried out at a rate of 1 mm/min
up to a strain value of 0.6 mm/mm by using a 5566 Instron dynamometer. On the other hand, human bone marrow–derived mesenchymal
stem cells (BMSCs) and dental pulp stem cells (DPSCs) with a density of 5x104cells/sample were seeded onto 3D scaffolds in order to
evaluate cell proliferation by using Alamar BlueTM assay.
Compression tests have highlighted that the mechanical behavior of the 3D
fiber-deposited composite scaffolds is improved if compared to that of 3D neat
PCL scaffolds. The stress-strain curve is characterized by an initial linear region,
suggesting an initial stiff mechanical response of the structure, followed by a
zone with lower stiffness and a final stiff portion of the stress–strain curve (Figure 1). Furthermore, biological analyses have shown the role of the inclusion of
the hybrid fillers in improving cell proliferation in terms of higher values of the
percentage of Alamar BlueTM reduction.
CONCLUSION
In the present study, the potential of 3D fiber deposition technique to design morphologically controlled scaffolds consisting of poly(εcaprolactone) reinforced with PCL/TiO2 or PCL/ZrO2 hybrid fillers has been demonstrated.
REFERENCES
1. Catauro M. et al. J. Biomed. Mater. Res. A 77: 340-350, 2006 .
2. Catauro M. et al. J. Mater. Sci. Mater. Med. 19: 531-540, 2008
3. Russo T. et al. J. Appl. Biomater. Biomech. 8: 146-152, 2010
ACKNOWLEDGMENTS
The authors thank PRIN 2007 “Sol-gel synthesis and characterization of organic–inorganic hybrid materials to process scaffolds for craniofacial tissue engineering” (grant number 20072L2KRX) for financial support.
68
SYNTHESIS AND CHARACTERIZATION OF SILVER-DOPED COLLAGEN BASED PATCH WITH ANTIMICROBIAL
PROPERTIES
Luca Salvatore¹, Federica Paladini1, Mauro Pollini1, Alessandro Sannino1
[email protected]
INTRODUCTION
Collagen is the major structural protein of the natural extracellular matrix. During the past 50 years collagen-based materials have been
successfully used in a wide variety of medical and cosmetic applications due its low antigenicity, reasonable mechanical strength,
requisite haemostatic properties, biostability, capacity to promote cell and tissue attachment/growth, ease of handling [1,2]. Additionally,
collagen films, gels and sponges can be produced thanks to its good malleability [3].
Nowadays, infections are one of the most important causes of morbidity and mortality in clinical practice. The use of collagen-based
products with suitable antimicrobial properties could represent a strong instrument to minimize the risk of transmission in clinical
procedures.
In this work an innovative method to produce silver-doped collagen based patches (AgCPs) with antimicrobial properties is
proposed. AgCPs were obtained through the in situ synthesis and incorporation of silver nanoparticles into the collagen matrix.
Thermal and physical analyses were carried out to analyse the influence of silver treatment on the properties of collagen and to evaluate the
biological performance of the constructs. Antibacterial capabilities of the matrices were also assessed by Agar diffusion test on S.aureus.
Micro-fibrillar type I bovine dermis collagen (2%wt) was blended with a 0.05 M acetic acid solution for 360 min at 10°C. Glycerine (5%wt)
was added to this mixture and blended for an additional 120 min at 10°C. A white slurry characterized by a quite high viscosity was
obtained by this procedure. A silver solution was prepared dissolving a 0.1%wt of silver nitrate in a mixture of methanol (10%wt) and
deionized water. 3%wt of silver solution was added to the collagen slurry and kept under gentle stirring over night to obtain a homogeneous
distribution of the silver salt. The suspension was degassed by centrifugation to remove bubbles. Then, it was poured into aluminium
pans (12.4 x 12.4 cm) and exposed to a UV source (500 W/cm2) for 1 h in order induce the photo-reduction of AgNO3 and the synthesis/
adhesion of nanoparticles on collagen fibers. The silver treated collagen slurry (AgCS) was then frozen from 20°C to -40°C over a period of
65 min and held constant at -40°C for 60 min. The frozen slurry was sublimated for 17 h (100 mTorr, 0°C) to produce the porous collagen
structure. The matrices were then cross-linked and sterilized by heating through dehydrothermal standard treatment (DHT; T = 121°C, p
< 100mTorr, t = 48 h) and standard dry-heat sterilization (DHS; T = 160°C, p < 100 mTorr, t = 2 h), respectively.
Thermal stability of silver-doped collagen-based matrices (AgCPs) was investigated in comparison with not-treated collagen-based
matrices (CPs). Shrinkage temperature, Ts (or denaturation temperature) of the sponge-like samples was determined through Differential
Scanning Calorimetry (DSC). Results from DSC analysis showed no significant differences between AgCPs and CPs samples, as Ts was
211.71 ± 0.53°C and 212.04 ± 1.18°C for CPs and AgCPs respectively.
The evaluation of the cross-linking density ρx of the materials was calculated adopting the theory of the rubber elasticity. No considerable
difference can be observed between Ag-treated and not-treated samples, being the calculated values almost the same (~ 10.2 ± 2.3
mol/m3). Chemical stability in terms of resistance towards enzymatic proteolysis were also evaluated. In vitro degradation tests were
performed using bacterial collagenase. A slightly higher degradation can be observed in AgCPs only after the first steps of the experiment.
After 4 hours, no significant difference occurs in the degradation profile between silver-treated and untreated samples. The antibacterial
capability of silver treated samples was demonstrated by agar diffusion tests on S. aureus.
CONCLUSION
In this work silver-doped collagen based patches with antimicrobial properties have been developed through an innovative technique
for the synthesis and adhesion of silver particles to collagen fibers in situ. Thermal and physical-chemical analyses demonstrated that
no significant differences occur between untreated and silver treated samples. Antibacterial capability was demonstrated on S. aureus,
suggesting that the developed constructs could represent a strong instrument to contain the risk of infection in clinical practice.
REFERENCES
1. Yannas IV. Tissue and organ regeneration in adults. New York: Springer; 2001.
2. O’Brien FJ et al. Biomaterials 2005; 26(4):433–41.
3. M. Mckegney et al. J. Mat. Sc.: Mat. In Med. 12 (2001) 833-844.
69
3D ADVANCED RAPID-PROTOTYPED PCL SCAFFOLDS: EFFECT OF SURFACE MODIFICATION AND PEPTIDE CONJUGATION ON MECHANICAL AND BIOLOGICAL PERFORMANCES
T. Russo1, A. Gloria1, U. D’Amora1, F. Causa2, E. Battista2, R Della Moglie2, S. Zeppetelli1, R. De Santis1, P. A. Netti2 and L. Ambrosio1
1 Institute of Composite and Biomedical Materials, National Research Council, Naples, Italy,
2 Interdisciplinary Research Center on Biomaterials (CRIB) at Italian Institute of Technology (IIT@CRIB), Naples, Italy
[email protected]
INTRODUCTION
3D fiber deposition technique represents a powerful tool to design morphologically-controlled scaffolds with specific architecture, precise
pore shape and size, suitable mechanical properties. However cell adhesion to scaffold surface results a critical factor, since it is strongly
related to the surface properties of biomaterials. Surface modifications with bioactive molecules such as RGD peptides seem to enhance
cell recognition, but these approaches may often undermine mechanical properties. Many surface modification techniques have been
developed to alter the surface properties of biomaterials without alteration of the bulk properties [1]. In this study, 3D rapid-prototyped
poly-ε-caprolactone (PCL) scaffolds were surface functionalized by aminolysis and then bioactivated by RGD-peptide covalent immobilization. The aim of this study was to evaluate the effect of surface modification and peptide conjugation on mechanical and biological
performances of the 3D fiber-deposited scaffolds.
3D cylindrical scaffolds (10 mm in diameter, 10.2 mm in height) were obtained through 3D Fiber Deposition technique, using a Bioplotter
dispensing machine (Envisiontec GmbH, Germany). PCL pellets were initially placed in a stainless steel syringe and, then, heated at a
temperature of 120°C through a cartridge unit placed on the mobile arm of the XYZ plotter. A nitrogen pressure of 8-8.5 bar was applied
to the syringe through a cap. 3D scaffolds were obtained by alternatively extruding and depositing the polymer fibers with different angle
steps between two successive layers, making two different patterns: 0°/90° and 0°/45°/90°/135°.
3D morphologically-controlled scaffolds were aminolyzed at different time through immersion in 0.08 g/ml 1,6-hexanediamine/2-propanol
solution at 37°C in batch mixer processing conditions. Successively, the GRGDY (Inbios, Italy) peptides were covalently grafted onto
the surface in a two step way by using di-ethylene glycol diglycidyl ether in mild aqueous condition. A peptide with reverse sequence
(GYGDR) was also used as control. The amounts of NH2 groups on the PCL fiber surface was assessed through Kaiser test, which is a
ninhidryn-based procedure, whilst micro-BCA assay (Sigma-Aldrich) was used to quantify the peptide density directly onto the bioactivated surfaces above melting temperature of PCL samples. Moreover, the effect of aminolysis on the surface properties were highlighted
through nanoindentation tests performed on the aminolyzed PCL fibers of the scaffold in a load range 1-5 mN, using a Nanotest Platform
(Micromaterials, U.K.). To evaluate the effect of surface modification on the macromechanical and bulk properties, 3D cylindrical scaffolds
were compressed at a strain rate of 1 mm/min, whilst tensile tests were performed on PCL fibers. Finally, biological tests on RGD-activated
scaffolds seeded with NIH 3T3 cells were carried out to investigate cell adhesion through scanning electron microscopy and confocal
analysis using phalloidin.
Results from nanoindentation measurements have evidenced that the surface modification via aminolysis provides an alteration of the
surface properties since hardness values of aminolyzed fibers (0.1 - 0.03 GPa) result lower than those obtained for not-aminolyzed ones
(0.50-0.27 GPa). Although aminolysis provides an alteration of the surface properties, compression tests (Table I) have highlighted that
it does not alter the material bulk properties and the macromechanical behavior of the 3D rapid-prototyped scaffolds, as also confirmed
by tensile tests.
Lay-Down Pattern
0°/90°
Compressive Modulus
E (MPa)
Maximum Stress
σ (MPa)
PCL
PCL-NH2
PCL
PCL-NH2
89.1 ± 6.9
87.9 ± 8.1
13.5 ± 1.3
13.2 ± 1.5
Table I: Effect of surface modification via aminolysis on the mechanical properties of 3D rapid-prototyped scaffolds. Compressive modulus and maximum stress
reported as mean value ± standard deviation, for PCL scaffolds characterized by a
0°/90° lay-down pattern before (PCL) and after aminolysis (PCL-NH2).
SEM and confocal analyses performed on cell constructs have shown a better interaction with RGD-modified scaffolds: an increase in
adhered number and a more evident spreading of NIH 3T3 cells were observed.
CONCLUSION
Aminolysis, as shown in this work, may be considered an easy-to-perform chemical technique to engraft amino groups along polyesters
chains, providing active sites through which other biomolecules, such as RGD peptides, can be immobilized, obtaining cytocompatible
surface and decreasing the surface hydrophobicity.
REFERENCES
1. Zhu Y. et al. 3: 1312–1319, 2002
70
INCLUSION OF MULTI WALLED CARBON NANOTUBES ON PLLA SUPPORTS THE MYOGENIC DIFFERENTIATION
OF MURINE PROGENITOR CELLS.
S. Montesano¹, I. Armentano², E. Lizundia3 , F. D’Angelo1, E. Fortunati2, S. Mattioli2, R. Tiribuzi1, M. Sampaolesi4, J. R. Sarasua3, J.
M. Kenny2,3, S. Martino1 and A. Orlacchio1
1 Department of Experimental Medicine and Biochemical Science, Section of Biochemistry and Molecular Biology, University of Perugia, Via del
Giochetto, Perugia, Italy
2 Materials Engineering Centre, UdR INSTM, NIPLAB, University of Perugia, Terni, Italy
3 Institute of Polymer Science and Technology, CSIC, Madrid
4 Translational Cardiomyology Lab, SCIL, Catholic University of Leuven, Leuven, Belgium
5 Department of Molecular Medicine and Center for Tissue Engineering (C.I.T), University of Pavia, Pavia, Italy
INTRODUCTION
In this study, is presented the use of a new class of biomaterials comprised of conductive PLLA/MWCNTs (poly(L-lactic acid) containing
various contents of Multi Walled Carbon NanoTubes) as suitable support for the differentiation of murine progenitor muscle cells, C2C12,
for skeletal muscle tissue engineering applications.
Materials: PLLA/MWCNT samples containing 0.1, 0.5, 1, and 3 wt.% as respect to PLLA initial weight and designed as 0.1MWCNTs,
0.5MWCNTs, 1MWCNTs, 3MWCNTs respectively, were prepared by solvent casting method (1). Neat PLLA film was also prepared for
comparison. Cells: Murine myoblasts were C2C12 cells were seeded on each materials at a density of 2x103 cells/mL in control medium
consisting of DMEM medium containing 10% heat-inactivated fetal bovine serum, 2mM of L-glutamine, and 100U/mL of penicillin–
streptomycin in a humidified atmosphere and 5% carbon dioxide (CO2) at 37°C. The medium was changed every 3 days.
For C2C12 differentiation experiments the serum content in the growth media was decreased to 2% after 24 hours post seeding. At each
time point (3, 7, 14 days) cells were harvested and further analysed.
Conductive nanocomposite films based on PLLA and different amount of MWCNTs were successfully developed and characterized,
showing a 3 dimensional nanotube network in the percolated formulations.
C2C12 cultured on PLLA and PLLA/MWCNTs (0.1MWCNTs, 0.5MWCNTs, 1MWCNTs, 3MWCNTs), showed comparable levels of
mitochondrial dehydrogenase activity and absence of signs of toxicity such as appearance of cellular debris in the culture medium.
C2C12 cells were cultured on the smoothest (bottom side) and the roughness surfaces (upper side), of neat PLLA, and nanocomposites
and the cell-mats interaction evaluated. Results shown the interaction of C2C12 with neat PLLA, 0.1MWCNTs, 0.5MWCNTs, 1MWCNTs
and 3MWCNTs appeared earliest after 1 day of culture with the typical myoblasts-“tapered” morphology. Morphological images were
similar for C2C12 seeded on both smoothest and roughness surface in each substrates.
Finally C2C12 cells were induced to differentiate in presence of medium containing 2% FBS for 17 days. The muscle differentiation was
monitored evaluating the expression of the MyoD transcription factor as early marker and the expression of MHC as late differentiation
marker. After 3 days of culture in differentiation medium the percentages of MyoD positive cells were 31%, 25%, 39%, 36%, 21% for
PLLA, 0.1MWCNTs, 0.5MWCNTs, 1MWCNTs and 3MWCNTs, respectively. After 17 days in differentiation medium, myotubes were found
on neat PLLA, 0.1MWCNTs, 0.5MWCNTs, 1MWCNTs and 3MWCNTs. No differences in terms of myotubes number were observed on
each biomaterials .
CONCLUSION
Together these data demonstrated that PLLA and PLLA/MWCNTs supported the differentiation of murine progenitor cells C2C12 toward
muscle cells and indicated that even high percentage of MWCNTs have any toxic effect thereby are suitable for regenerative medicine
application.
REFERENCES
1. Lizundia E., R. Sarasua, F. D’angelo, A. Orlacchio, S. Martino, J. M Kenny, I. Armentano. Macromolar Bioscence,2012, 10.1002/mabi. 201200008
Accepted.
ACKNOWLEDGMENTS
The authors would like to thank the Italian Fondazione Cassa di Risparmio di Perugia (grant no. 2010.011.0445 to A.O.), the Italian
Ministero dell’Istruzione, dell’Università e della Ricerca (grant: PRIN no.20084XRSBS_001to A.O.), as well as the Istituto Nazionale
Biostrutture e Biosistemi.
71
PEPTIDE CONFINEMENT IN NANOPORES FOR ANTIBACTERIAL SURFACES
Grazia M.L. Messina1, Raffaella Lettieri2, Mariano Venanzi2, Fernando Formaggio3, Claudio Toniolo3 and Giovanni Marletta1
1 Laboratory for Molecular Surface and Nanotechnology (LAMSUN) – Dept. of Chemical Sciences – Univ. of Catania and CSGI – CT - Italy
2 Dept. of Chemical Sciences and Technologies- University of Rome Tor Vergata – Rome - Italy
3 Dept. of Chemistry – University of Padova – Padova – Italy
[email protected]
INTRODUCTION
Nanotechnology deals with design, characterization, building and application of structures and devices keeping a check on shape and
dimension on nanometer scale. This is why, basic research on nanopatterned surfaces become increasingly importantIn view of the
relevance of nanostructured surfaces for biomedical applications, including sensing, spatially resolved surface-cell interactions, drug
delivery, etc.., the idea of confining biofunctional compounds in nanosized structures is gaining more and more interest. Accordingly, in
this communication we present a versatile approach to nanostructurate polymeric films, in order to achieve the selective confinement
of a multipurpose biological platform, consisting in suitable liposome formulation, driven by an antibacterial peptide anchored within the
surface nanostructures.
We present a versatile and simple approach for rapidly fabricating nanopatterned surfaces on micrometer scale. Monodisperse inorganic
nanospheres were spin-coated onto gold surfaces. Subsequent annealing process leads to the formation of a monolayer of ordered
colloidal crystals on substrates. A polymer layer is then deposited on samples, embedding the nanoparticle distribution. Finally, the
selective removal of the nanospheres leads to the formation of nanostructured micron-sized area. The nanopatterned surfaces consisted
of 2D nanopore arrays, having internal area of gold surrounded by polymeric matrix. The nanopores depth can be modulated.
Trichogin GA IV, an antimicrobial peptide functionalized at the N-terminus by a thiol-group, was shown to be selectively chemisorbed within
the obtained nanopores, due to the interaction of its thiol termination with the nanopore-paving gold . The Trichogin GA IV strong affinity for
the liposome structures is then exploited to drive the selective confinement of phospholipid bilayers within the functionalized nanopores.
It is found that liposomes are efficiently included within the trichogin-functionalised pores (fig.1a and b), and that only the liposomes in
direct contact with trichogin remain firmly attached within the pores (fig.1c).
The formation of a peptide self-assembled monolayer covalently linked to the gold surface inside the pores, as well as the immobilization
of liposome bilayers was demonstrated by using cyclic voltammetry (CV), Atomic Force Microscopy (AFM), Quartz Crystal Microbalance
with Dissipation monitoring (QCM-D) and X-ray Photoelectron Spectroscopy (XPS), obtaining information on the peptide/liposome
interaction as well as on the best suited conditions of liposome immobilization.
Fig. 1: AFM height images and corresponding 3D
and section analysis for nanostructured surface.
CONCLUSION
The immobilization of Trichogin GA IV, an antimicrobial peptide functionalized at the N-terminus by a thiol-group inside nanopores was
studied by using different techniques. Then the interaction between peptide and liposome was investigated to demonstrate the selective
confinement of phopholipid bilayers within functionalized nanopores.
REFERENCES
1. Moon J.H. et al., Chem. Commun. 4107-4109, 2005
2. McCarty L.S. et al., Angew. Chem. Int. Ed. 46:206-209, 2007
3. Chen X. et al., Adv. Mater. 15:1413-1417, 2003
4. Wang L. et al., J. Am. Chem. Soc. 130:2142-2143, 2008
5. Yuan J. et al., Chem. Commun. 994-995, 2004
72
MORPHOLOGICAL EVALUATION OF ADESION AND PROLIFERATION OF MG63 CELLS GROWN ON GELATIN/GENIPIN
SCAFFOLD
Mirella Falconi¹, Adriana Bigi2, Roberto Giardino, Milena Fini, Antonio Mazzotti1,4, Gabriella Teti4
1 Department of Human Anatomy, University of Bologna, Italy
2 Department of Chemistry ‘‘G. Ciamician’’, University of Bologna, Italy
3 Laboratory of Preclinical Surgical Studies, Rizzoli Orthopaedic Institute, Bologna, Italy
4 Clinic of Orthopaedics and Traumatology, Rizzoli Orthopaedic Institute, Bologna, Italy
[email protected]
INTRODUCTION
Composite scaffolds prepared from natural polymers are expected to have enhanced differentiation properties and as a result they gained
much attention in recent years for use in tissue-engineering applications. Although there are various natural polymers available for this
purpose, gelatin is commonly studied because of their inherent properties1.
Gelatin is a denatured collagen and commercially available as biodegradable polymer. It has been exstensively utilized for pharmaceutical
and medical purposes, and its biosafety has been proven through long clinical applications 2. The main disadvantage of gelatin is due to its
poor mechanical properties, which limit its possible applications as a biomaterial 3. Gelatin materials for long-term biomedical applications
must be submitted to crosslinking, which improves both the thermal and the mechanical stability of the material.
Genipin is a naturally occurring crosslinking agent, which seems to display promising characteristics 4.
The objectives of this study was to evaluate the effects and the influence of a gelatin /genipin porous composite as scaffold for human
osteoblast like cells testing cell viability, adhesion, proliferation and differentiation processes.
MG63 cells were seeded on gelatin/genepin scaffolds for 7, 14, and 21 days. Cell proliferation assay, light microscopy, transmission
electron microscopy (TEM) and scanning electron microscopy (SEM) were carried out to evaluate cell viability, morphological and
ultramorphological changes induced by cell/scaffold interaction.
Cell proliferation assay showed an increase of cells on gelatin scaffold during the experimental time, suggesting the lack of genipin release
which in high concentrations could have toxic effects.
SEM showed a round shape of MG63 cells on the surface of gelatin scaffold even after 3 days from the seeding, suggesting a decrease
on growing rate compared to control samples.
The inner structure of the gelatin /genipin sponge is gradually colonized by MG63 cells that appear localized on the big pores of the
structure, covering its entire surface. TEM analysis showed that after 20 days of cell/scaffold incubation, cells have changed their shape
from a round to a fibroblast like ones, and their cell bodies were strictly connected with the scaffold material.
CONCLUSION
Our results showed that the porous gelatine/genipin scaffold presented here is an appropriate substrate for cell adhesion, proliferation
and differentiation for tissue engineering.
REFERENCES
1. Isikli C. et al., J Tissue Eng Regen Med. 2012 6:135-43.
2. Zekorn D. Bibl. Haematol. 1969. 33:131 – 140.
3. Panzavolta S. et al., Acta Biomat. 5: 636 - 643, 2009
4. Bigi A. et al. Biomaterias 23: 4827 – 4832.
ACKNOWLEDGMENTS
The authors would like to thank the Italian Ministry of Research and Technology (MURST) (FIRB grant (RBAP10MLK7_005 and a PRIN
2009 grant) for providing financial support to this project.
73
ANTIBACTERIAL PROPERTIES OF PLA COMPOSITES BASED ON SILVER NANOPARTICLES AND CRYSTALLINE
CELLULOSE
Elena Fortunati1, Ilaria Armentano1, Qi Zhou3, Antonio Iannoni1, Marco Vercellino5, Lars Berglund4, Josè M. Kenny1,2, Marcello
Imbriani6,7 Livia Visai5,7
1 Materials Engineering Center, UdR INSTM, University of Perugia, Terni, Italy
2 Institute of Polymer Science and Technology, CSIC, Madrid , Spain
3 School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden
4 Wallenberg Wood Science Center, Royal Institute of Technology, Stockholm, Sweden
5 Department of Molecular Medicine, and Center for Tissue Engineering (C.I.T), University of Pavia, Pavia, Italy
6 Department of Public Health, Neuroscience, Experimental Medicine and Forensic, University of Pavia, Pavia, Italy
7 Fondazione S.Maugeri, IRCCS, Pavia, Italy and International Centre For Studies And Research In Biomedicine (ICB), Luxembourg
[email protected]
INTRODUCTION
Poly-Lactide Acid (PLA) is becoming popular as a biodegradable engineering plastic owing to its high mechanical strength, easy
processability compared to other biopolymers. The preparation of micro- and nano-composites represents a promising method to
improve the physical properties of biopolymers. The incorporation of antimicrobial substances in food-packaging materials to control
undesirable growth of microorganisms on the surface of foods, represents a great challenge.1 Microcrystalline cellulose (MCC) has
been used as additive to improve the properties of polymer, while nanocrystalline cellulose (CNC), produced from the acid hydrolysis
of MCC and properly modified, presents increased crystalline phase. CNC shows high mechanical properties, great biodegradability
and biocompatibility, high stiffness, and low density.2 These new multifunctional materials were investigated in terms of morphological,
mechanical, thermal and antibacterial response.
Material preparation and characterization. PLA 3051D, supplied by Nature Works®, was used as polymer matrix and commercial silver
(Ag) nanopowder were added in order to provide antibacterial properties to the system. Microcrystalline cellulose (MCC, dimensions of
10-15 μm) was by Sigma Aldrich and cellulose nanocrystal (CNC) suspension was prepared from MCC by sulphuric acid hydrolysis.3
Cellulose nanocrystals were modified with an acid phosphate ester of ethoxylated nonylphenol (s-CNC). Binary and ternary films with
5%wt of MCC, 5%wt of pristine CNC or surfactant modified s-CNC and with 1%wt of Ag were prepared by a melt extrusion followed by
a filmature process. New multifunctional materials were characterized in terms of morphology, mechanical, thermal properties. In vitro
bacterial assays on PLA nanocomposite Bacteria growth inhibition were tested on Escherichia coli RB and S. aureus 8325-4 strains using
the CFU method as previously reported.4 The bacterial strains were incubated with each sample at different temperatures (37 °C, 24 °C
and 4 °C) for 3 h and 24 h, respectively.
Cellulose nanocrystals produced by acid hydrolysis appear individualized,
with the typical dimensions ranging from 100 to 200 nm in length and
15 nm in width. Morphology studies of PLA composite films prepared
combining MCC and Ag nanoparticles revealed a well dispersion of silver
and the presence of microcrystalline cellulose aggregates, while the
mechanical properties demonstrated the MCC reinforcing effect. Moreover,
a bactericidal effect of all the analyzed PLA composites on S.aureus and
E.coli was detected suggesting possible applications which requires an
antibacterial effect constant over time (Fig. 1 and 2).
Fig. 1. Antibacterial properties
of new multifunctional PLA
composites in micro dimension.
Fig.2. Antibacterial properties
of new multifunctional PLA
composites in nano dimension.
CONCLUSION
PLA composites reinforced with micro- and nano-crystalline cellulose combined with silver nanoparticles offer a good perspective for
food packaging applications.
REFERENCES
1. Vermeiren L. et al., Trends Food Sci Tech. 1999:77-86.
2. Fortunati E.et al., Polym Degrad Stab 2010, 95,2200-6.
3. Zhou Q. et al., Macromolecules, 2009,42,5430.
4. Petrini P. et al., IJAO. 2006;29(4):434-42.
ACKNOWLEDGMENTS
The authors would like to acknowledge financial support by Regione Lombardia (2010) and by FONDAZIONE ALMA MATER TICINENSIS
(2010) and by (ICB).
74
ZINC OXIDE NANOANTIMICROBIALS. WET CHEMICAL VS ION BEAM SPUTTERING SYNTHESES OF NANOPARTICLES
AND NANO-COATINGS.
Maria Chiara Sportelli1, Marco Valentini2, Lorena Giannossa1, Maria Angela Nitti2, Nicoletta Ditaranto1, Antonio Valentini2 Nicola
Cioffi1, Luigia Sabbatini1
1 Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro” Bari-Italy,
2 Dipartimento Interateneo di Fisica, Università degli Studi di Bari “Aldo Moro” Bari-Italy
[email protected]
INTRODUCTION
The development of antimicrobial agents and surface coatings is being attracting increased interest, due to the continuous spread of
multiple antibiotic resistant strains of various infectious organisms. Inorganic nanoparticles NPs with antibacterial activity can be used
under different forms (e.g. powders, coatings, and as a part of organic/inorganic nanocomposites) in numerous industrial sectors1-2. The
main advantages of using inorganic oxides when compared with organic/molecular antimicrobial agents are their thermal stability, and
their robustness and long shelf life2-3. ZnO can form various nanostructures suitable for a wide variety of applications in materials science
and antimicrobial treatments. The main advantages of using ZnO-NPs compared with its bulk oxide is its improved efficiency and reactivity
in several fields4.
Hydrolytic (Sol-Gel) synthesis of ZnO nano-colloids was carried out by warming up an aqueous solution of ZnCl2 0.1M at 70°C and pH=2.
After 1h, aqueous ammonia solution (0.1M) was added drop-wise, until a dense white gel was formed, at pH 9. The gel was then dried
and –if needed, the as-prepared ZnO-NPs were subjected to further thermal annealing at 550°C.
Ion Beam Sputtering deposition of ZnO-NP thin films was carried out by sputtering a single ZnO target with a Kaufman source under vacuum
conditions and sample rotation.5 Surfactant-stabilized ZnO-NPs were electrosynthesized by means of the so-called Electrochemical
Deposition under Oxidizing Conditions (EDOC)6, using a two- or three-electrode electrolysis cell, equipped with an Ag/AgBr (0.1M in
isopropanol) reference electrode, a zinc anode and a platinum cathode. The electrolytic solution was composed of a 0.1M tetra-alkylammonium salts dissolved in isopropanol. The current density was kept at about 3 mA/cm2.
TEM microscopy was performed by a FEI Tecnai 12 instrument operated at 120 kV. The surface analysis of the nanomaterials was carried
out by X-ray Photoelectron Spectroscopy (XPS). XP spectra were acquired by Theta Probe VG Scientific spectrometer equipped with a
monochromatized AlKα source (spot = 300µm).
Different chemical and electrochemical routes to ZnO nanoparticles (ZnONPs) were comparatively investigated in the present study.
Processes were chosen in order to produce complimentary nanomaterials that can be used as colloidal additives (wet-chemical processes)
or as direct coatings (IBS method) for the controlled modification of . Moreover, wet processes were carried out in the presence or without
making use of surfactants, thus leading to core-shell –e.g. stabilized- particles or to bare –e.g. non-stabilized- NPs. The resulting materials
have been used to proficiently modify different substrates and manufactured goods such as textiles, air-filters, etc.
Nanoantimicrobials morphology was systematically assessed by electron microscopy while the surface chemical status of ZnONPs and
ZnONP-modified goods was investigated by XPS. In particular, surface atomic percentages were collected for all the samples, and were
used to drive the syntheses optimization. Zinc Chemical speciation was assessed combining the results recorded for high resolution XP
regions, as well as those relevant to the main Auger Zn L3M45M45 signal. The latter ones showing more pronounced shifts, as a function of
changes in the zinc chemical environment. In general, hydrolytic (Sol-Gel) routes provided less regular morphologies but implied greener
experimental conditions. EDOC procedure showed higher potentialities for the size-controlled preparation of core-shell nanocolloids
stabilized by quaternary ammonium species possessing their own disinfecting characteristics. Finally, physical methods such as IBS
proved to be successful in the controlled deposition of thin nano-coatings in all those contexts in which wet-impregnation of manufactured
goods with aggressive solvents was not feasible.
CONCLUSION
Comparative approach to the size-selective development of ZnO nanoantimicrobials is presented, aimed at conferring antibacterial
properties to textiles and other manufactured goods.
REFERENCES
1. N. L. Rosi and C. A. Mirkin, Chem. Rev. 105: 1547-1562 (2005) - 2. Z. L.Wang, J. Phys. Condens. Matter 16: R829 (2004).
3. P. K. Stoimerov et al., Langmuir 18:6679-6686 (2002) - 4. L. Zhang et al., J. Nanopart. Res. 9: 479-489 (2007).
5. A. Valentini et al., J. Appl.Phys. 73: 1143-1145 (1993) - 6. H. Natter et al., Scripta Mater. 44: 2209-2212 (2001).
ACKNOWLEDGMENTS
Financial support from Italian MIUR Project PON01_02210 is gratefully acknowledged.
Financial support from Regione Puglia, in the scientific Research Project “Laboratorio di tecnologie di modificazione superficiale di fibre
naturali per il rilancio del settore tessile in Puglia” - Avviso pubblico n. 16/2009 POR PUGLIA 2007 – 2013 “Reti di Laboratori Pubblici di
Ricerca,” is acknowledged for the TEM facility.
75
ION BEAM DEPOSITION AND CHARACTERIZATION OF INORGANIC NANOPARTICLES-FLUOROPOLYMER ANTIMICROBIAL
COATINGS FOR TEXTILE APPLICATIONS
Maria Angela Nitti¹, Maria Chiara Sportelli2, Marco Valentini3, Nicola Cioffi2 , Giuseppina Marilia Tantillo4, Antonio Valentini1,
Giuseppe Casamassima1
1 Department of Physics, University of Bari “Aldo Moro” Bari - Italy,
2 Department of Chemistry, University of Bari “Aldo Moro” Bari - Italy
3 INFN- Sezione di Bari - Italy
4 Department of Animal Health and Welfare, University of Bari “Aldo Moro” Bari - Italy
[email protected]
INTRODUCTION
In the field of textile, innovative technologies are expected to allow surface modification treatments of natural or synthetic fibers in order
to obtain stain resistant, wear resistant and antibacterial fabrics. Ion Beam co-Sputtering (IBS) of an inorganic material target made of
metal or metal oxide and a polytetrafluoroethylene one has been used for the production of new nanocoatings composed of nanoparticles
(NPs) embedded in a polymer matrix, thus combining the antimicrobial properties of NPs1 with the anti-stain ones of the CF2 matrix. IBS
allows to produce antibacterial/antistain products with tunable chemical-physical properties, enabling to change the NPs loading and, as
a consequence, the release of bioactive ions, thus modulating the final coating properties. As a result, a control on the proliferation of
microorganisms is achieved in an operating regime which prevents toxicity for humans.
NPs of Copper or Zinc Oxide have been dispersed in CF2 to obtain antimicrobial coatings, 150 nm thick, on 5x5 cm2 pieces of textile
substrates, by sputtering a Cu or a ZnO target simultaneously to a CF2 one. All the samples were fully characterized by means of
X-ray Photoelectron Spectroscopy (XPS) and Transmission Electron Microscopy (TEM), in order to obtain surface chemical composition
and morphology information, respectively. XP spectra were acquired by Theta Probe VG Scientific spectrometer equipped with a
monochromatized AlKα source (spot = 300 μm). TEM was performed by a FEI Tecnai 12 instrument operated at 120 kV. Biological tests
were performed in order to study the coatings’ bioactivity.
The main conditions of the IBS deposition technique of nanoantimicrobial
coatings are reported in the following table, where fv2 represents the
volume fraction of Cu or ZnO incorporated in the composite, while ECu/
EZnO and ICu /IZnO represent, respectively, the energy and the current
intensity of the Ar+ ion beam bombarding the inorganic target. An
analogous notation has been introduced to indicate the energy and current
of the Ar+ beam used to bombard the target of Teflon. XPS analytical
technique has allowed to obtain information on the concentration of
dispersed inorganic NPs, on increasing their loading in the composite.
TEM images have revealed the inorganic nanoclusters size and their
homogeneous in-plane distribution in the composite coatings. Biological
tests have shown the marked bioactive action of Teflon-Cu/ZnO coatings
on the tested microoganisms. These results proved the IBS to be a
successful technique for the production of thin nanoantimicrobial coatings
alternative to the photo-wet impregnation3.
CFxCu (5%)
0,05
ETef
(eV)
750
CFxCu (10%)
0,10
750
50
700
55
2.78
CFxCu (15%)
0,15
Sample
fV
50
ITef
(mA)
50
1100
EZnO
(eV)
800
65
IZnO
(mA)
35
3.12
ρcomp
(g/cm3)
2.27
Sample
fV
ITef
(mA)
50
ECu
(eV)
450
ICu
(mA)
40
ρcomp
(g/cm3)
2.44
CFxZnO(5%)
0,05
750
ETef
(eV)
1000
CFxZnO(10%)
0,10
900
40
1000
40
2.45
CFxZnO(15%)
0,15
900
50
1250
45
2.63
CONCLUSION
An IBS synthesis protocol, that ensures optimal performance
in different innovative fields of the textile substrates modified
with inorganic NPs-fluoropolymer antimicrobial coatings,
has been determined.
Coatings’ bioactivity is demonstrated to depend on the
inorganic NPs composition and concentration in the
coating, since it affects the pronounced ion release from
the nanocoatings.
REFERENCES
1. N. Cioffi et al., Anal. Bioanal. Chem. (2005) 381: 607-616
2. N. Cioffi et al., Chem. Mater. (2002) 14: 804-811.
3. M. Pollini et al., J. Mater. Sci.: Mater. Med. (June 13, 2009)
ACKNOWLEDGMENTS
Financial support from Italian MIUR Project PON01_02210 is gratefully acknowledged.
Financial support from Regione Puglia, in the scientific Research Project “Laboratorio di tecnologie di modificazione superficiale di fibre
naturali per il rilancio del settore tessile in Puglia” - Avviso pubblico n. 16/2009 POR PUGLIA 2007 – 2013 “Reti di Laboratori Pubblici di
Ricerca,” is acknowledged for the TEM facility.
76
VASCULAR GRAFTS BASED ON PLLA/PLA BLENDS
Francesco Carfì Pavia1, Salvatrice Rigogliuso2, Vincenzo La Carrubba³, Giulio Ghersi2 and Valerio Brucato1
1 DICGIM, Università di Palermo, Italy
2 STEMBIO, Università di Palermo, Italy
3 DICAM, Università di Palermo, Italy
[email protected]
Tubular scaffolds for vascular tissue engineering (VTE) were produced and characterized by utilizing two PLLA/PLA blends in order to tune their
biodegradability. Cell cultures into the scaffold were carried out and the non-cytotoxicity of scaffolds, adhesion and cell proliferation were evaluated.
The scaffolds do not induce cell toxicity and cells are able to cover scaffold internal surface, making the devices suitable for the designed application.
INTRODUCTION
Cardiovascular diseases remain the leading cause of mortality in western nations, with an estimated prevalence of almost 80 million in
the USA alone. In particular, coronary artery disease is the leading cause of death, accounting for 53% of the total mortality related to
cardiovascular disease1. Currently, occluded vessels with diameters below 6mm are bypassed with autologous native blood vessels, such
as the saphenous vein. However, those surgical techniques have generally proved inadequate2. Regenerative medicine and, in particular,
tissue engineering approaches are being investigated as potential solutions to these problems. To this end, autologous vascular cells are
seeded in biodegradable (tubular) scaffolds which are subsequently cultured in vitro or immediately implanted. The goal of this study was
to produce PLLA/PLA blends based tubular scaffolds in order to tune the initial level of the crystallinity, thus controlling the biodegradation
rate. In order to verify the suitability of these products for tissue engineering applications cell cultures were performed to evaluate the noncytotoxicity, cell adhesion and proliferation.
The tubular scaffolds were obtained by performing a Diffusion Induced Phase Separation (DIPS) process, after a dip coating, around a
nylon fiber with a diameter of ~700 μm. Briefly, the fiber was first immersed into a PLA/dioxane or PLA/PLLA blend/dioxane solution
at a constant temperature (35°C). Then the fiber was slowly pulled-out at different constant rates (10-40 cm/min) from the solution and
immersed into a second bath (DIPS bath), containing pure water at the same temperature, for 10 min. Finally, the fiber was extracted from
the bath, rinsed in distilled water and dried at 40°C for 48 h. The scaffolds were analyzed by scanning electron microscopy (SEM) with a
Philips 505 Microscope on sample cross section. ECV304 continuous human endothelial cells from ECACC were seeded inside scaffolds
2,5 cm long, at 15 104 cell/cm. After 14 days scaffolds were extracted from culture medium, fixed in 4% formaldehyde solution for 10 min
at RT and washed in PBS. Tubes were sectioned across the transverse direction in smaller pieces and longitudinally opened. To evaluate
the possible effects of cytotoxicity, cells were stained with a solution of Acridine Orange- Ethidium Bromide [100 µg/ml] in PBS for 10 sec.
Evaluations were performed by confocal microscopy (Olympus 1X70-Melles Griot laser system).
Thickness of the porous walls for 90/10, 75/25 PLLA/PLA scaffolds is shown in figure 1a, where it is possible to notice an increase in the
thickness by raising the fiber extraction rate. However, in order to obtain easy-to-handle samples (>100 μm) when utilizing a 75/25 PLLA/
PLA solution, the fiber extraction rate must be increased with respect to 90/10 samples. This could be explained by recalling that viscosity
of the polymer/dioxane solution significantly decreases when passing from 10 to 25% PLA content.
a
b
Figure 1: (a) thickness of the tubular scaffolds
in function of fiber extraction rate; (b) confocal
microscope image of the ECV grown inside the
scaffold for 14 days.
A cell culture inside the vessel-like scaffolds was carried out with ECV304 endothelial cells, the first to form during embryonic
development. Figure 1b illustrates an image of a longitudinal section of a scaffold with cells grown for 14 days. Cells show a good level
of adhesion to the polymeric substrate. Moreover, the typical apoptotic cell morphology was not detected, confirming the biocompatibility.
CONCLUSION
Tubular scaffolds for tissue engineering applications based on PLLA/PLA blends were produced to tune the biodegradation rate. They
present an open structure with interconnected pores along the wall. It was possible to change the thickness of the wall by varying the
solution pull-out rate. Biological cultures in the scaffolds with ECV304 show a good level of adhesion and proliferation with a development
of an homogeneous monolayer, confirming the non-cytotoxicity of the materials. These results indicate these products as promising
devices for vascular tissue engineering.
REFERENCES
1.Soletti L. et al., Acta Biomat. 6:110–122, 2011 - 2. Conte M.S., Faseb Journal 12:43-45, 1998
77
THE INFLUENCE OF AIR DBDS PLASMA PROCESS ON EUKARYOTIC CELL LINES
Daniela Pignatelli1, Giorgio Dilecce2, Bianca Rita Pistillo1, Santolo De Benedictis2, Pietro Favia1,2,3, Roberto Gristina2
1 Department of Chemistry, University of Bari, Italy
2 Institute of Inorganic Methodologies and Plasma (IMIP) CNR, Italy
3 Plasma Solution Srl, Spin off of the University of Bari
[email protected]
INTRODUCTION
In the last ten years the use of atmospheric pressure plasma processes to directly treat living tissues was successful in different therapeutic
fields such as sterilization and decontamination of wounds, wound healing and treatment of cancer, etc [1,2]. These interactions can
involve lethal or positive effects on living cell. Such effects can be attributed to nature and distribution of active species generated in
the plasma such as Reactive Oxygen (ROS, e.g., ozone and others) and Nitrogen (RNS, e.g. nitrous oxide and others) species, ions, UV
radiations and heat, that are known to play a role in activation/deactivation paths of eukaryotic cells, bacteria and living tissues.
In order to understand the mechanisms underlying the different interaction between plasma and eukaryotic cells, in vitro experiments with
cell lines represent a very powerful tool.
In this study the effects of different doses of DBD (Dieletric Barrier Discharge) air plasma on different cell lines, an immortal one, SAOS-2
osteoblastoma, and a primary one, NHDF(Normal Human Dermal Fibroblasts), were investigated.
In the home made plasma source discharges were operated in air and in pulse, with cells positioned on the bottom of a a Petri dish
which works as dieletric of the ground DBD electrode (Figure 1). Discharges were operated with a sinusoidal voltage at 27.37 kHz, fully
modulated with a rectangular pulse characterized by a tON = 0.1 s and a tOFF = 1.9 s and by a finite number N of tON events.
Cells were grown on 60mm diameter Petri dishes for 24 hrs and after 24 and 72 hours after plasma exposure cells behavior was studied
by means of MTT test, Coomassie blue and Actin staining by Phalloidin, and compared with the one observed on cells not processed by
plasma.
Saos-2 and NHDF were both exposed to different number of pulse, 1,3,9 and 27 respectively. They have clearly shown a different behavior,
in term of cell proliferation and morphology.
At 24 and 72 hours after plasma treatment, MTT results on SAOS-2 shown a strong decrease in cell proliferation when exposed at 9 and
27 pulses. Furthermore, after 27 pulse, an evident cell morphological change occurred. This result was also observed when the actin
cytoskeleton morphology was studied. Differently for the control cells and for the ones processed with a smaller dose of plasma, most of
the cells exposed to 9 and 27 pulses did not show the presence of stress fibers but mainly spots of actin proteins. To confirm the effect of
plasma on actin proteins, the actin gene expression has been studied on SAOS-2. The obtained results confirmed a very low expression
of actin on cells grown for 72 hours after have being exposed to 9 and 27 pulses of plasma.
NHDF cells shown a different behaviour. In fact, MTT results revealed an increase in cell proliferation after 3 and 9 pulses while cells
exposed to 27 pulses showed a good growth and no cell morphological change was observed.
CONCLUSION
Atmospheric plasma discharges applied on the two selected cell type have shown negative and positive effects, strongly dependent on cell
type. The negative effect on cell growth observed in SAOS-2 could be open important perspectives for treatment of cancer cells, while the
results obtained on the fibroblast cell line can give interesting hints on the fields of wound healing and tissue regeneration. Future study will
focus on the analysis of expression of other genes that can be involved in the cellular mechanism of response to plasma. Furthermore it
will be necessary to investigate which reactive species produced by plasma are responsible to cell behaviour.
REFERENCES
1. Plasma Processes & Polymers 3(6/7), 2006; Special Issue on Plasma Processes for Biomedical Applications, organized by P. Favia.
2. Plasma Processes & Polymers 5(7), 2008; Special Issue on Plasma Medicine, organized by A. Fridman and M. Laroussi.
ACKNOWLEDGMENTS
Mr S. Cosmai and Mr D. Benedetti are gratefully acknowledged for their technical assistance in lab.
78
EFFECT OF A NOVEL IN SITU ANTIBIOTIC DELIVERY SYSTEMS ON OSTEOBLASTS
Monica Mattioli-Belmonte1, Concetta Ferretti1, A. Trapani2, R. Iatta3, M. Orciani1, D. Cafagna4, R. Lazzarini1,A. Romanelli4 and Elvira
De Giglio4
1 Dept. of Clinical and Molecular sciences, Università Politecnica delle Marche, Ancona, Italy.
2 Dept. of Pharmaceutical Chemistry, University of Bari Aldo Moro, Italy
3 Dept. of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Italy
4 Dept. of Chemistry, University of Bari Aldo Moro, Italy.
[email protected]
INTRODUCTION
Orthopedic infections represent one of the major causes of implant failure1. The initial bacterial adhesion onto biomaterial surfaces is
believed to be a critical event2 in the pathogenesis of implant-associated infections, Therefore an important strategy for their prevention is
the modification of the material surface in order to avoid bacterial adhesion. In this respect a local antimicrobial prophylaxis could consist
of antibiotic incorporation onto titanium coatings surfaces3. One essential requirement of this strategy is that titanium coating should
not hamper tissue-integration. Ciprofloxacin (CIP) loaded chitosan nanoparticles (CSNPs) coatings onto titanium surface efficacy in the
inhibition of Gram-positive bacterial growth. However few data are available on their effects on osteoblasts.
CSNPs loading CIP were prepared according to a modified ionic gelation method3. Subsequently, CIP loaded CSNPs were set by casting
onto titanium sheets. X-ray Photoelectron Spectroscopy (XPS) analysis was performed on pure materials and on CIP loaded CSNPs.
Antibacterial activity of the investigated nanoparticles-based coatings was performed on S. aureus and P. aeruginosa cultures. Evaluation
of biocompatibility was performed by MTT test and SEM morphological analysis using MG63 osteoblast-like cells. Moreover, gene
expression of runx2, bmp2, cdh11 and Collagen Type I were performed by quantitative Real Time PCR (q-PCR). In order to compare MG63
gene expression onto NPsCPX loaded samples toward untreated titanium surfaces, ΔΔCq method was employed.
The tested drug delivery system was able to inhibit S. aureus and P. aeruginosa growths. Good cell viability was evidenced when CIPloaded nanoparticulate coatings were exposed to MG63 osteoblasts-like cells (Fig. 1).
No changes were detected in the q-PCR expression of early genes of osteoblast differentiation (i. e. runx2, cdh11), while a significant
increase in bmp2 expression was observed. Collagen Type I production confirmed the good viability of MG63 on CSNPs coating (Fig. 2)
6,00
Figure 1
4,50
3,00
Figure 2. Relative expression of collagen Type I, runx2,
cdh11 and bmp2 mRNAs in MG-63 human osteoblastlike cells observed on CIP loaded CSNPs coating.
1,50
0
CONCLUSION
The obtained data are interesting for a potential application of this new nanoparticle-based coating as drug delivery system to prevent
serious bacterial infections frequently related to orthopedic surgery without affecting osteoblast functions pertinent to new bone formation.
REFERENCES
1. Qiu Y, et al. Int J Artif Organs 30: 828–41, 2007.
2. Busscher HJ, et al., FEMS Microbiol Lett 128:, 229-34, 1995.
3. De Giglio E, et al. Acta Biomaterialia 7: 882-91, 2011.
4. Trapani A, et al. Nanotechnology 19:185101-10, 2008
ACKNOWLEDGMENTS
The authors would like to thank the FIRB Research Programme (Grant no: RBAP10MLK7) for providing a partial financial support to this
project.
79
PREPARATION AND CHARACTERIZATION OF A MULTIWALLED CARBON NANOTUBE/MITOXANTRONE
ADDUCT FOR A TARGETED DRUG DELIVERY SYSTEM
Nora Bloise¹, Irina Cislaghi2, Daniele Merli2, Antonella Profumo2, Maurizio Fagnoni2, Piercarlo Mustarelli2, M. Imbriani3,4, L.Visai1,4
1 Dep. of Molecular Medicine and Center for Tissue Engineering (C.I.T.), University of Pavia, Italy
2 Dep. of Chemistry, University of Pavia, Italy
3 Dep.of Public Health, Neuroscience, Experimental Medicine and Forensic, University of Pavia, Italy
4 Salvatore Maugeri Foundation, IRCCS, Pavia, Italy and International Center for Studies and Research in Biomedicine (I.C.B.), Luxembourg
[email protected]
INTRODUCTION
Drug delivery system is considered a valuable strategy of administering a pharmaceutical compound to achieve a therapeutic effect. The
aim is to target the drug in particular areas of the body where it is active (targeted delivery) and to permit a sustained release for a given
period of time in a controlled manner. Mitoxantrone (MTX) is an anthracenedione antineoplastic agent, very effective but scarcely used due
to its high toxicity 1,2. The idea to use MTX adsorbed on multiwalled carbon nanotubes (MWCNTs) has a dual purpose: a targeted delivery
in cancer tissue and a drug gradual release after reaching the specific site.
MWCNT-MTX preparation: MWCNT were purified and oxidized through a well known method [2]. MTX was adsorbed on oxidized MWCNTs
by interactions with CNT carboxylic group and drug amine groups. The adduct has been characterized through Raman Spectroscopy,
Thermo Gravimetric Analysis (TGA), and TEM. Citotoxicity Test: SAOS-2 cells were cultured in McCoy’s 5A medium, supplemented with
15% fetal bovine serum, 1% L-glutamine, 0.4 % antibiotic, 2% sodium pyruvate, and 0.2% fungizone, at 37°C with 5% CO2. A suspension
of 1x105 cells were seeded in 96-well tissue culture dishes and incubated with increasing concentrations of MWNCTs at different time.
Trypan blue dye exclusion assay was used to investigate the effect of MWCNTs on cell viability. All the results were compared with those
obtained with a comparable amount of MTX in solution.
Maximum loading of 100 mg MTX/g CNTs was obtained; MTX adsorption was confirmed by HPLC and Raman Spectroscopy. The release
of the drug from MWCNTs-MTX suspended in the cultural medium followed a first order kinetics.
Mitoxantrone loading on carbon nanotubes produced a reduction in SAOS-2 cell viability slightly lower if compared to MTX solution. Cell
viability in the presence of MTX loaded carbon nanotubes was dose- and time-dependent (Fig.1).
Fig.1 Cell viability of SAOS-2 incubated with
MTX and MTX adsorbed on CNTs.
CONCLUSION
These preliminary results showed that MWCNTs-MTX are as much effective as the free drug in killing tumor cell line; its physico-chemical
and pharmacokinetics properties may support its use as an in-situ neo-adjuvant and/or adjuvant cyotoxic device.
REFERENCES
1. Brunton L.L et al., Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 11th Edition, McGraw-Hill, N.Y., 2006
2. Merli D. et al., Journal of Nanoscience and Nanotechnology, 11: 1-7, 2011
ACKNOWLEDGMENTS
The authors would like to acknowledge financial support from “Project SAL-45” financed by the Regione Lombardia (2010) and by a
project financed by the Fondazione Alma Mater Ticinensis (2010) and by I.C.B.
80
COMPARATIVE SCREENING OF CATIONIC POLYMERS FOR GENE DELIVERY AND OPTIMIZATION OF TRANSFECTION
PARAMETERS
Chiara Malloggi¹,², Gloria Scaparrotti1, Roberto Chiesa1, Daniele Pezzoli2, Gabriele Candiani1,2
1 CMIC Department, Politecnico di Milano, Milan, Italy;
INSTM, Unità Politecnico, Milan, Italy
2
[email protected]
INTRODUCTION
Successful non-viral gene delivery currently requires compromises to formulate non-cytotoxic and highly efficient transfectants. However,
transgene expression levels depend not only on the reagent formulation but also on the experimental conditions and on various biological
parameters1,2. With the aim of overcoming the limitations of currently available non-viral gene delivery vectors, we provide a comparative
screening among commercially sourced cationic polymers in order to find out the best candidate which combines high transfection
efficiency with low cytotoxicity. Most important, we have evaluated the influence of different biological parameters on the transfection
behavior of different polymers to in order to obtain a model that allows a simple and quick selection of the best transfection condition for
a given polymeric vector.
Polyplexes were prepared at r. t. by adding an aqueous solution of plasmid DNA (pGL3) to aqueous solution of each polymer, in the
opportune buffer, at the desired polymer concentration, yielding different N/P ratios. For transfections, HeLa cell lines were plated in 96well cell culture plates at a density of 2.0 x 104 cells/cm2. The day after, 102 ng/well of pGL3 was complexed and then added to cells in a
final volumes of 100 µl/well of complete DMEM (DMEM supplemented with 10% FBS). 24 h post-transfection, cytotoxicity was evaluated
by AlamarBlue® viability assay, cells were lysed and luciferase activity was measured by Luciferase Assay System and normalized to the
total protein content. Statistical analysis was performed by ANOVA test. Significance was retained when p < 0.05.
We have compared the overall transfection behavior of the following polymers: 25 kDa linear polyethylenimine (lPEI), 50100 kDa branched polyethylenimine (bPEI), 15-30 kDa poly-L-lysine (PLL) and polyamidoamine generation 7 (PAMAM G7).
We have determined the optimal transfection conditions in terms of N/P ratio and complexation buffer, which correspond to the point
that conferred low cytotoxicity and higher transfection efficiency that correspond to N/P 40 in 150 mM NaCl for 25 kDa lPEI, N/P 30 in
10 mM Hepes for 50-100 kDa bPEI, N/P 3 in 10 mM Hepes for 15-30 kDa for PLL and N/P 3 in 10 mM Hepes for PAMAM G7 (Fig. 1).
Most important, 25 kDa lPEI was by far the most effective commercially sourced polymer for gene delivery.With the aim of evaluating
the influence of three different biological parameters, such as the volume of cell culture medium during transfection, the density of cells
transfected and the dose of polyplexes administered to cells, on the transfection efficiency and the cytotoxicity of the resulting polyplexes,
we have performed transfection experiments complexing each polymer in his optimal transfection conditions. Interestingly, the volume of
cell culture medium directly affected both transfection efficiency and cytotoxicity, and is polymer-specific.
Fig. 1. Transfection efficiency and cytotoxicity on HeLa cell
line of lPEI, bPEI, PLL and PAMAM complexed with pGL3 in
their optimal transfection conditions.
CONCLUSION
We have performed a comparative analysis in terms of transfection effectiveness among commercially available cationic polymers. For all
polyplexes, the transfection efficiency and the cytotoxicity were dependent on the N/P ratio and on the complexation buffer.Moreover, we
have evaluated the influence of different biological parameters on transfection efficiency of different polyplexes. We have demonstrated
that the volume of the cell culture medium during transfection is the most prominent parameter influencing the overall behavior of polymerbased gene delivery vectors.
REFERENCES
1. Gebhart C. L. and Kabanov A. V., J of Control Release 73:401-416, 2001
2. Kunath K. et al., J. of Control Release 89:113-125, 2003§
ACKNOWLEDGMENTS
This work was partly supported by the Politecnico di Milano, 5xmille Junior, “SURGES” Project and by the Italian Ministry for Education,
University and Research (MIUR) - FIRB Futuro in Ricerca 2008, Grant RBFR08XH0H.
81
CHITOSAN-BASED PH SENSITIVE POLYMERS AS DRUG DELIVERY SYSTEMS: KINETIC RELEASE STUDY
Andrea Ruffini1, Monica Sandri1 and Anna Tampieri1
1 CNR-ISTEC - Institute of Science and Technology for Ceramics - Via Granarolo, 64 - 48018 Faenza (RA) - ITALY
[email protected], [email protected], [email protected]
INTRODUCTION
Chitosan-based hydrogel polymers have been extensively studied as carriers in the pharmaceutical and medical fields, where they
have shown promise for drug delivery as a result of their controlled and sustained release properties, as well as biodegradability and
biocompatibility with tissue and cells1-3. pH sensitive polymers are materials which swell or collapse depending on the pH of their surrounding
environment. This behavior is exhibited due to the presence of certain functional groups in the polymer chain. In the present work, some
semi-interpenetrating polymer network structures (semi-IPN) based on chitosan were studied. The compositional and structural effects
of these materials on swelling and drug releasing properties are being explored, in response to changes in physiological pH conditions.
Bi-component semi-IPN formed by chitosan and natural (Na-alginate or gelatin) or synthetic (PEG or PVP) polymers were prepared.
Chitosan was cross-linked by ionic or covalent agents. Commercial chitosan was dissolved in acetic acid under stirring at room
temperature. To this solution, secondary polymer and cross-linked agent were added and again stirred. The mixtures were put into
cylindrical cases to obtain the final cylindrical shapes after freeze-drying. Drug was loaded by mixing or immersing methods.
Various conditions were changed: kind of cross-linking species and secondary polymer, chitosan/polymer weight ratio, cross-linking
concentration. The swelling behavior was studied by immersing the cylinder hydrogels in media of different pH at 37°C (es. fig. 1). The
kinetic releases were evaluated by UV-Vis spectroscopy, analyzing the drug concentration in solution at different times. Methylene blue
was used as drug molecule and observed at 614nm.
Swelling and delivery behaviour of drug were found to be dependent on pH of the
medium, on the amount of secondary polymer in the gel, on the weight ratio of
chitosan/polymer and on the amount of cross-linking agent.
The effect of pH on the swelling of the chitosan hydrogels is explained on the
basis of protonation of the amino groups of chitosan. In the acidic medium, the
protonation of the amino groups leads to repulsion in the polymer chains, thus
allowing more water in the hydrogel network. At higher pH, deprotonation of the
animo groups takes place and repulsion in polymer chains is reduced. This results
in the shrinking of the gels and therefore, the amount of water in the gel decreases.
The kinetic rate diagrams suggest that the quick release occurred at low pH, while
the release was slower at pH near neutrality. The release profile was characterized
by an initial quick effect, followed by a continuous and controlled release phase.
All the hydrogels swelled rapidly and reached equilibrium within two days. The
pH sensitive containing natural polymers showed higher release under the same
conditions of the material containing artificial polymers.
Figure 1
Swelling behaviour of chitosan-based pH sensitive hydrogel (containing gelatin) at 37°C after 2
days: dried, pH=7, pH=5.5, pH=4
“Table 1 show that men in the father-watching
condition cried significantly more...”
CONCLUSION
Chitosan-based pH sensitive hydrogels containing varying amounts of different polymer were prepared by cross-linking. Swelling
properties and drug release were studied at different pH. At lower pH, more swelling of the hydrogels was observed compared to high pH
because of protonation of amino acids in the acidic medium.
REFERENCES
1. Rani M. et al., BioResources, 5(4), 2010
2. Khurma J.R & Nand A.V., Polymer Bullettin, 59, 2008
3. Muzzarelli R.A.A., Charbohydrate Polymers, 77, 2009
ACKNOWLEDGMENTS
The Authors wish to acknowledge the financial support of European Commission under the contract number “NMP-2009-2.3-1” – OPHIS
Project
82
INNOVATIVE HYDROGELS FOR NEUROPROTECTIVE PROTEIN DELIVERY IN NEURODEGENERATION
Marta Tunesi1, Elisabetta Prina1, Alberto Cigada1, Carmen Giordano1, Diego Albani2
1 Chemistry, Materials and Chemical Engineering Department “G. Natta”, Politecnico di Milano, Milan, ltaly
2 Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy
[email protected]
INTRODUCTION
The growth of knowledge about pathogenetic mechanisms related to neurodegenerative disorders such as Parkinson’s (PD)
and Alzheimer’s disease (AD) has led to the discovery of new molecular targets of therapeutic interest that might reduce
neurodegeneration. Novel therapies based on neuroprotective protein delivery have recently been proposed to counteract
oxidative stress, pathologic protein misfolding and aggregation in both PD and AD or to improve the cognitive impairment1.
In this scenario, hydrogels are appealing materials to design suitable protein delivery systems, thanks to their tunable chemical,
physical and mechanical properties and well known biocompatibility; in fact, embedding therapeutic proteins within polymer
matrices offers the advantage to promote their controlled release while minimizing both their stability and activity loss occurring in
an unprotect environment2,3. The present study has focused on the preparation and characterization of novel agar/Carbomer/
poly(ethylene glycol)-based hydrogels patented by Politecnico di Milano4, that might be subcutaneously injected and exploited
as reservoirs for the controlled, continuous and more efficient release of therapeutic proteins against neurodegeneration5.
Four hydrogels containing agarose (A), Carbomer (C) and poly(ethylene glycol) (PEG) 600 were tested and compared to their PEGfree counterparts, that were considered as controls. More in detail, starting from two selected A/C-based formulations differing from A
m/v ratio with respect to C solution (1:2 and 1:4, labelled as High A and Low A, respectively), A/C/PEG-based matrices were obtained
by adding 10% or 20% (v/v) of PEG 600 and referred to as (a) High A+10% PEG, (b) High A+20% PEG, (c) Low A+10% PEG
and (d) Low A+20% PEG. All the matrices were preliminary characterized from a chemical-physical point of view by evaluating their
initial water content and swelling behaviour with time in phosphate buffered saline solution (PBS) and distilled water at pH=2, pH=7
and pH=12. In order to assess the absence of toxic leachables after hydrogel preparation with a cell model suitable to mimic the
cited subcutaneous application, L929 immortalized fibroblasts were exposed for 24h to culture medium previously incubated for 1, 3
and 7 days with proposed materials and their viability was evaluated by Alamar Blue® assay. In vitro hydrogel biological performance
was also preliminary tested with neural cells; in particular, U-87MG glioblastoma astrocytoma cells were embedded within matrices
and their viability was evaluated 3 days later after extraction from hydrogels and cell counting following trypan blue staining.
Initial water content is higher for PEG-free matrices than for A/C/PEG-based ones; in particular, for a fixed A/C ratio it decreases by
increasing PEG content. Swelling behaviour depends on pH, A/C ratio and PEG concentration. In particular, in water it is lower at pH=2
than at pH=7 or pH=12, while in PBS the increase of A/C ratio, as well as PEG content, leads to a swelling ratio decrease.
In vitro biological assessments have evidenced that at the selected time points L929 cell viability shows values comparable
with controls for all the materials (Fig.1), indicating that eventually coming out leachables have negligible effects on cell
viability. Concerning U-87MG cells, data have indicated that cells survive the period of latency within proposed matrices;
in particular, their viability is higher for A/C/PEG-based gels than for PEG-free ones, confirming PEG enhances cell viability.
Fig.1: L929 cell viability 24h after exposure to culture medium
incubated for 1,3 and 7 days with proposed hydrogels: results are
expressed as a percentage with respect to controls
CONCLUSION
All collected data suggest that proposed A/C/PEG-based hydrogels are potentially interesting candidates to design suitable protein delivery
systems for the treatment of neurodegenerative disorders.
REFERENCES
1. Giordano C. et al., Int J Artif Organs 32:836-50, 2009
2. Soderquist R.G. et al., Expert Opin Drug Deliv 7:285-93, 2010
3. Drury J.L. et al., Biomaterials 24:4337–51, 2003
4. Daniele F. et al.,Politecnico di Milano. Patent WO2009/144569. 2008
5. Lin C.C. et al., Pharm Res 26:631-43, 2009
83
NEURODEGENERATIVE DISORDERS: NOVEL HYDROGELS FOR DRUG DELIVERY STRATEGIES
Elisabetta Prina¹, Marta Tunesi1, Francesco Daniele1, Alberto Cigada1, Carmen Giordano1, Diego Albani2
1 Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Milan, Italy
2 Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy
[email protected]
INTRODUCTION
The global market for central nervous system (CNS) pathologies is now one of the largest therapeutic sectors; in particular, neurodegenerative
diseases include more spread disorders such as Alzheimer’s and Parkinson’s diseases and rare disorders such as Huntington’s disease,
amyotrophic lateral sclerosis and multiple sclerosis. CNS disorders constitute a bottleneck in medical practice: clinical symptoms are
different, but they share common features and for most of them etiopathogenetic mechanisms are not fully understood. Because of this,
for both chronic and neurodegenerative disorders therapies are symptomatic rather than disease-modifying: drugs may give temporary
improvements, but treatments able to stop or reverse the pathological processes are not currently available. In addition, the majority of
applied drugs is systemically administered, thus large doses are usually required for a desired local effect. This is not only costly, but it
may also result in serious side effects for the patients1. In this context, hydrogels are an interesting tool to be exploited for the development
of drug delivery systems, thanks to their tunable chemical and physical properties, high water content and biocompatibility2. In this
work, we aim to preliminarily investigate the performance of novel agar/Carbomer/collagen-based hydrogels patented by Politecnico
di Milano3, to be subcutaneously injected and used as reservoirs for the controlled, continuous and efficient delivery of neurotrophic or
neuroprotective drugs for the treatment of severe neurological disorders affecting the brain, such as Parkinson’s and Alzheimer’s diseases.
Starting from two agar/Carbomer-based formulations differing from agarose (A) content (0.125% or 0.25% (m/v)) with respect to
Carbomer (C) solution, two agar/Carbomer/collagen-based hydrogels were obtained by adding 0.011% (m/v) of collagen (COLL) solution
to each formulation. The matrices were labelled as Low A+COLL (A/C 0.125% + 0.011% COLL) and High A+COLL (A/C 0.25% +
0.011% COLL), while the selected starting A/C formulations were referred to as Low A (A/C 0.125%) and High A (A/C 0.25%) and
were considered as controls in the experimental assessments. In particular, the hydrogels were characterized from a chemical-physical
point of view by evaluating their initial water content and swelling behaviour with time in phosphate buffered saline solution (PBS). In
order to model the previously introduced subcutaneous application, hydrogel in vitro biological response was investigated with L929
fibroblast-like cells; furthermore SH-SY5Y neuronal-like cells were also used to investigate matrix possible effects on neural cells.
More in detail, with both selected cell models the absence of any toxic leachables coming out from the hydrogels was assessed by
evaluating cell viability by Alamar Blue® assay at different time points after culture medium incubation with matrices for 1, 3 and 7 days.
Collected results have indicated that agar-Carbomer-collagen hydrogels have a higher swelling ratio with time than their counterparts with
the same A content; furthermore High A+COLL matrices have shown a lower swelling ratio than Low A+COLL hydrogels. Data have also
suggested that COLL adding does not remarkably modify the A/C matrix initial water content.
From the biological point of view, with both investigated models after culturing for 24h with culture medium collected at day 1, 3 and
7 following hydrogel incubation L929 and SH-SY5Y viability is comparable with controls (Fig.1), suggesting the absence of remarkable
negative effects on cell viability at the selected time points.
Fig.1: Results from cell viability assay for SH-SY5Y cells grown for
24h with culture medium incubated for 1,3 and 7 days with proposed
hydrogels: data are reported as a percentage with respect to controls
CONCLUSION
The present preliminary investigations have highlighted that the selected novel agar/Carbomer/collagen-based systems might be potentially
exploited to design innovative drug delivery devices to release neurotrophic or neuroprotective molecules with interesting properties for
CNS applications.
REFERENCES
1. Giordano C. et al., Int J Artif Organs 32:836-50, 2009
2. Hoare T.R. et al., Polymer 49:1993-2007, 2008
3. Daniele F. et al.,Politecnico di Milano. Patent WO2009/144569. 2008
84
UV CURABLE HYDROXYAPATITE SUSPENSIONS FOR BONE TISSUE SUBSTITUTES PROTOTYPING
Carola Esposito Corcione, Francesca Scalera, Alessandro Sannino, Alfonso Maffezzoli
1 Department of Engineering for Innovation, University of Salento, Italy
[email protected]
INTRODUCTION
The need of reconstructing complex bone defects in the maxillo-facial region as a result of trauma, tumor surgery or congenital
malformation has become a hot topic in the field of tissue engineering. Digital tools such as 3D CAD systems and rapid prototyping
(RP) machines are a useful tool to realize custom made bone scaffolds. RP techniques allow the construction of complex physical
models based on 3D clinical images elaborated by suitable software and CAD systems. Hydroxyapatite (HA) is one of the most
used material for bone restoring because of its composition very closed to human bones and teeth. Producing a custom-made
scaffold in a ceramic material directly by RP is therefore an exciting challenge. The final purpose of the present study is to fabricate
a green part in HA powder dispersed in UV curable media by stereolithography, without using any mould. To this aim, the influence
of different solid loads of the ceramic suspension on the kinetic mechanism of the photochemical reaction was investigated.
A bifunctional epoxy monomer was obtained from Dow Chemicals (former Union Carbide) under the trade name of Cyracure®–
UVR-6105 (3,4-epoxycyclohexylmethyl-3,epoxycyclohexane carboxylate, CE). The photoinitiator (PI) used is Cyracure® UVI6976. Both CE and PI were selected since dental composites containing them were found biocompatible in vitro studies.1
A commercial HA powder (Plasma Biotal PB260R, North Derbyshire, UK) was used. Three reactive ceramic suspensions
with different solid load (10, 20, 30%wt) were prepared and characterized. The curing of the suspensions was carried out
in a Photo Differential Scanning Calorimeter (Mettler Toledo DSC1 StareSystem), able to allow the irradiation of the sample
by means of a UV/visible lamp at 370 nm. Isothermal scans were run at 25°C in nitrogen and air atmosphere with light radiations
intensity of 3.7 µW/mm2. After each isothermal p-DSC test, a second dynamic scan was always performed on the same
specimen using the same DSC without the UV light source equipment. These thermal dynamic tests were always performed
in nitrogen atmosphere, at a heating rate of 10°C/min, from -10°C to 250°C, in order to monitor the residual overall reactivity.
The maximum heat of reaction Hmax and time to reach the peak value (tpeak) obtained from the p-DSC isothermal scan in inert atmosphere
and the respective residual heat of reaction, calculated starting from the thermal dynamic scan were reported in Tab
Figure 1: extent of reaction versus time
Table 1: kinetic parameters by p-DSC and DSC scans
The reactivity, expressed in terms of both heat developed and rate of reaction, was generally found to decrease by increasing the content
of HA, remained almost unvaried the reaction time. This effect could be attributed to the light scattering of the ceramic powders in the UV
curable resin3. On the other hand Hresidual was found to increase with HA content.
The extent of reaction, α, defined as: where H(t) is the heat of reaction developed at time t was calculated for all
the mixtures produced. In Figure 1 an evident reduction of the value of α for the mixtures with 0% and 30% of HA is shown.
CONCLUSION
The kinetic characterization of UV curable HA suspensions suggested that the rate of reaction decreasing, by increasing the weight
percentage of the ceramic powder, remained nevertheless suitable for the construction in the stereolithography apparatus. A further
investigation related to the viscosity of the suspensions will be performed in order to select the mixture to realize a green part in HA powder
dispersed in UV curable media by stereolithography.
REFERENCES
1. J.D. Eick et al., Dent. Mater. 18:413–421, 2002.
2. C. E.Corcione et al., J. Mater.Sci. 40:1-6, 2005.
85
LONG-TERM ANTIBACTERIAL EFFICACY OF SILVER TREATED CATHETERS FOR HAEMODIALYSIS IN PREVENTING BIOFILM FORMATION
Federica Paladini¹, Mauro Pollini1, Adelfia Talà2, Pietro Alifano2, Alessandro Sannino1
1 Department Engineering for Innovation, University of Salento, Italy
2 Di.S.Te.B.A., Microbiology Laboratory, University of Salento, Italy
[email protected]
INTRODUCTION
Nanotechnology applied to medicine represents a huge instrument to solve some important biomedical problems [1]. The most prominent
nano-product in medicine is nano-silver because it exhibits unusual physicochemical properties and biological activities due to its
nanometric size [2]. An innovative and patented technique was used to synthesize nanometric silver particles by photo-reduction of a silver
salt in an alcohol [3-4] on temporary polyurethane catheters for haemodialysis. The efficacy of the coating in preventing bacterial adhesion
during its working life was verified through fluorescence microscopy. Physiological conditions of the human body were reproduced for 30
days. ICP-AES was adopted to study silver release in water and in simulated body fluid SBF. Thermo-gravimetric analysis TGA was used
to analyse any loss of weight in treated samples.
“Carbothane” catheters for haemodialysis were treated by dipping in an alcoholic silver solution and then exposing them to UV irradiation
in order to induce the photo-reduction of silver and the formation of silver nanoparticles on the surface of the devices. A washing system
was developed to reproduce human body condition for 30 days. Samples of washing water and SBF were collected at defined times
of 1, 7, 14, 21 and 30 days to analyse silver release through ICP-AES. The biological characterization was conducted adopting the
washing system using as flowing solution M9 minimal medium inoculated with Staphylococcus aureus (6*106 CFU/ml). The bacterial
film maturated on the catheter surfaces was stained using green-fluorescent nucleic acid stain and it was analysed through fluorescence
microscopy. Thermo-gravimetric analysis of silver treated catheters after soaking in deionized water and SBF was performed to show the
stability of the coating for the whole working life of the device.
ICP-AES graph in Figure 1 shows a decreasing release of silver ions from the first day to the 30th day for both water for SBF. The maximum
amounts of silver release in deionized water was 0.24±0.007073 ppm and 0.14±0.002762 ppm in SBF. The fluorescence microscopy
analysis of the untreated catheter and the silver treated catheter revealed a strong reduction of bacterial adhesion on catheter treated with
silver ions with respect to untreated catheter (Figure 2) demonstrating the efficacy of the silver coating in preventing S. aureus adhesion
and biofilm formation. The amount of silver is stable for all the samples also after 30 days soaking confirming the good adhesion and
stability of silver cluster on the polymeric substrate both in water and in SBF.
CONCLUSION
Antibacterial catheters for haemodialysis were obtained by an innovative silver deposition technology. The impressive efficacy of the
coating in preventing adhesion of Staphylococcus aureus on the surface was demonstrated through fluorescence microscopy. The
assessment of silver release by ICP-AES demonstrated that silver coating was not affected by flowing aqueous solution and biological
fluid. Furthermore, the values of silver release were well below the limit of toxicity reported in literature.
REFERENCES
1. M. Singh, S. Singh, S. Prasad and I.S. Gambhir. Dig J Nanomater Bios. 2008, 3, 115.
2. X. Chen and H.J. Schluesener. Toxicol Lett. 2008, 176, 1.
3. M. Pollini, A. Sannino, A. Maffezzoli, A. Licciulli. European Patent No. EP1986499, 2008.
4. M. Pollini, F. Paladini, M. Catalano, A. Taurino, A. Licciulli, A. Maffezzoli and A. Sannino. Mater Sci: Mater Med. 2011, 22, 2005.
86
HIGH-VELOCITY SUSPENSION FLAME SPRAYED (HVSFS) BIOACTIVE COATINGS FOR ORTHOPEDIC APPLICATIONS
Devis Bellucci1, Giovanni Bolelli1, Rainer Gadow2, Andreas Killinger2, Luca Lusvarghi1,Antonella Sola1, Nico Stiegler2 and
Valeria Cannillo¹
1 Department of Materials and Environmental Engineering, University of Modena and Reggio Emilia, Modena (MO), Italy
2 Institute for Manufacturing Technologies of Ceramic Components and Composites (IMTCCC), Universität Stuttgart, Germany
[email protected]
INTRODUCTION
Load-bearing orthopedic prostheses are commonly made of a metallic substrate, usually Ti and its alloys, with a proper bioactive coating
to promote osseointegration. The coatings mainly consist of hydroxyapatite1, HA (Ca5(PO4)3OH), which is chemically similar to the mineral
component of bone, or bioactive glasses2, which are special glasses possessing a high bone-bonding ability thanks to their composition.
These coatings are generally deposited by thermal spraying processes, which benefit from their relatively low cost and their ability to
treat quite complex surfaces3. In particular, the present contribution was focused on an emerging technique, namely the High-Velocity
Suspension Flame Spraying (HVSFS) method, which operates on suspensions instead of dry powders and hence offers the opportunity
of spraying sub-micrometer sized particles, which are expected to create denser and more reliable coatings4. In particular, the HVSFS
technique was tested to deposit HA coatings as well as bioactive glass ones, using both an experimental glass belonging to the K2O-CaOP2O5-SiO2 system, Bio-K5, and a glass having a composition analogous to the 45S5 Bioglass®, BG2.
The substrates were planar 50 mm x 50 mm x 3 mm Ti (grade 2) plates, cleaned with acetone and sand-blasted. As regards the HA
coatings, two different suspensions (Ce.Ri.Col., , Italy) were employed, since the nano-sized particles were dispersed either in water
(solid: 13 wt%) or diethylene glycol, DEG (solid: 10 wt%). Various spraying conditions were considered and compared. For both glasses,
the same procedure was followed. In fact the micro-sized glass powders were dispersed in a water-isopropanol solution (solid: 20 wt%;
deflocculating agent: 0.5 wt%). The spraying parameters were systematically changed in order to evaluate the relation between processing
conditions and final microstructure. The coatings were characterized by means of SEM, XRD and in-vitro tests in a Simulated Body Fluid,
SBF6.
When HA powders were processed, water-based suspensions resulted in relatively low deposition efficiencies (<40%) and produced
poorly crystallized coatings, which tended to dissolve very rapidly in SBF. Instead the DEG-based suspensions, if appropriately sprayed,
reached higher deposition efficiencies (45-55%) and resulted in compositionally graded coatings, since the degree of crystallinity increased
from the bottom to the top layer. The high crystallinity of the working surface made these coatings much more stable in SBF7. As regards
the BG samples, the coating thickness and porosity were very sensible to the spraying parameters. However all the BG coatings resulted to
be completely glassy. Also in this case, a through-thickness micro-structural gradient was observed, as the deposition mechanisms of the
glass droplets changed at every torch cycle because of the progressive increase in the system temperature. When immersed in SBF, all the
coatings were promptly covered by a HA layer of re-precipitation8.The Bio-K samples were peculiar, since the coatings remained entirely
glassy, but the glass underwent some structural alterations during processing (as revealed by Raman micro-spectroscopy). Nevertheless,
if soaked in SBF, the Bio-K coatings were able to develop a uniform HA layer of re-precipitation, thus suggesting that the bioactivity of the
original glass was not compromised9.
CONCLUSION
The HVSFS method proved to be a reliable technique to produce HA and bioactive glass coatings, with a fine and dense microstructure
and a controlled bioactivity in SBF.
REFERENCES
1. Sun L. et al., J Biomed Mater Res 58:570-92, 2001
2. Hench L.L., J Am Ceram Soc 74:1487-510, 1991
3. Heimann R.B., Surf Coat Technol 201:2012-9, 2006
4. Killinger A. et al., Surf Coat Technol 201:1922-9, 2006
5. Cannillo V. and Sola A., Ceram Int 35:3389-93, 2009
6. Kokubo T. et al., Biomaterials 27:2907-15, 2006
7. Stiegler N. et al., J Therm Spray Technol 21:275-87, 2012
8. Altomare L. et al., J Mater Sci: Mater Med 22:1303-19, 2011
9. Bolelli G. et al., Surf Coat Technol 205:1145-9, 2010
ACKNOWLEDGEMENTS
The Vigoni project (Ateneo Italo-Tedesco and Deutscher Akademischer Austausch Dienst, DAAD) support is kindly acknowledged.
87
SINTERING AND BIOACTIVITY OF GLASSES BELONGING TO THE NA2O-CAO-P2O5-SIO2 SYSTEM
Antonella Sola1, Devis Bellucci1, Maria Grazia Raucci2, Stefania Zeppetelli2, Luigi Ambrosio2 and Valeria Cannillo1
1 Department of Materials and Environmental Engineering – University of Modena and Reggio Emilia - Via Vignolese 905 – 41125 Modena, Italy
2 Institute of Composite and Biomedical Materials IMCB – National Research Council of Italy Naples, Italy
[email protected]
INTRODUCTION
Bioactive glasses are ideal candidates to fabricate biomedical devices for bone prostheses, due to their excellent bone-bonding ability1.
Unfortunately several processing routes include a thermal treatment which is likely to promote crystallization of the glass, thus altering its
properties and, in particular, its bioactivity2.
The present contribution investigated the effect of the Na2O-to-CaO ratio on the thermal behavior and bioactivity of glasses belonging to
the Na2O-CaO-P2O5-SiO2 system3.
Two glasses3,4 were examined in the so-called “bioactive bonding boundary” of the Na2O-CaO-P2O5-SiO2 system, with a fixed 2.6 mol%
amount of P2O5: the BG_Na glass, which was relatively rich in Na2O (33 mol%), and the BG_Ca glass, which was relatively rich in CaO
(46 mol%). As a term of comparison, a glass with a composition analogous to the standard 45S5 Bioglass®, hereafter BG glass, was
also considered5. The thermal behavior was investigated by means of an integrated approach, including Differential Thermal Analysis
(rate: 10°C/min), heating microscopy (same rate) and optical thermal dilatometry (same rate). On account of the thermal results, sintered
samples were produced by heat-treating pressed powder specimens (particle size < 70 μm) at an optimal temperature (3 h). The eventual
crystallization of the sintered samples was assesed via X-ray diffraction (10-70° 2θ range, 0.017° 2θ step size). The bioactivity was
preliminary studied with standard tests in a Simulated Body Fluid6 and then further analysed with cell culture tests (human osteoblast-like
MG-63 cells). The cell morphology and colonization of MG-63 onto bioglasses were evaluated by scanning electron microscopy. Alamar
Blue test was used to evaluate the biocompatibility of materials.
According to the thermal characterization, the (first) glass transition temperature increased with decreasing Na2O-to-CaO ratio in the
series BG_Na (490°C) BG (530°C)
BG_Ca (700°C). The same trend was observed for the (first) crystallization temperature, which
increased in the series BG_Na (590°C)
BG (620°C) BG_Ca (850°C), and for the (first) sintering temperature, which also increased
in the series BG_Na (530°C)
BG (570°C)
BG_Ca (800°C). Interestingly, for both the BG glass and the BG_Na glass, the (first)
crystallization hindered the sintering process, which was completed in a second step, at a higher temperature (around 1050°C for BG and
880°C for BG_Na). Instead the BG_Ca glass completely sintered before crystallizing. On account of these considerations, the sintered
specimens were obtained at 1050°C for the GB glass, 880°C for the BG_Na glass and 800°C for the BG_Ca glass. The former two samples
were widely crystallized, with a prevalent presence of sodium calcium silicates. Instead the latter sample was marginally crystallized, with
traces of wollastonite, which is typical of silicate glasses with a relatively low Na2O-to-CaO ratio7. The amorphous phase still present in
the BG_Ca sintered sample, as well as the development of wollastonite which is a biomaterial itself, greatly favored the reactions in SBF.
Also the cell culture and cytotoxicity tests performed on the sintered glasses confirmed the high bioactivity of the CaO-rich composition,
even after a heat treatment.
CONCLUSION
Decreasing the Na2O-to-CaO ratio in the composition of bioactive glasses belonging to the Na2O-CaO-P2O5-SiO2 system may help to sinter
the glass without inducing its crystallization, thus preserving the bioactivity of the original glass even after a heat treatment.
REFERENCES
1. Rahaman M.N. et al., Acta Biomater 7:2355-2373, 2011
2. Boccaccini A.R. et al., Faraday Discuss 136:27-44, 2007
3. Sola A. et al., J Biomed Mater Res Part A 100A:305-322, 2012
4. Lockyer M.G.W. et al., J Non-Crys Sol 188:207-219, 1995
5. Hench L.L., J Mater Sci: Mater M 17:967-978, 2006
6. Kokubo T. and Takadama H., Biomaterials 27:2907-2915, 2006
7. Arstila H. et al., J Eur Ceram Soc 27:1543-1546, 2007.
ACKNOWLEDGMENTS
Eng. Silvia Tessarini is acknowledged for her contribution in the experimental activity.
88
AN INNOVATIVE PROCESSING ROUTE TO REALIZE SCAFFOLDS FOR BONE TISSUE ENGINEERING
Devis Bellucci1, Federica Chiellini2, Gianluca Ciardelli3, Matteo Gazzarri2, Piergiorgio Gentile3, Antonella Sola1 and Valeria Cannillo1
1 Department of Materials and Environmental Engineering – University of Modena and Reggio Emilia, Modena, Italy.
2 Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab) & UdR INSTM, Department of Chemistry &
Industrial Chemistry, University of Pisa, Via Vecchia Livornese 1291, 56122 S. Piero a Grado, Pisa, Italy.
3 Department of Mechanics and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Turin, Italy.
[email protected]
INTRODUCTION
The production of bioceramic scaffolds to foster cell adhesion and proliferation in a 3D architecture is an important issue in bone tissue
engineering [1]. Among biomaterials suitable for bone tissue engineering, the 45S5 Bioglass® plays an important role for its excellent
bioactivity degree [2]. Unfortunately, the production of bioceramic scaffolds by means of the widely used replication method, where highly
porous ceramic foams are obtained by coating a polymeric sponge, usually lead to samples with poor mechanical properties and fragile
surfaces [3]. In this work a new protocol, based on a modified replication method, is proposed to obtain bioactive glass scaffolds [4].
These samples, named “shell scaffolds”, are reinforced by an external resistant surface that, like a porous shell, provides both mechanical
support and high permeability [5]. The mechanical behavior of the new scaffolds has been investigated through compressive tests;
moreover, a biological evaluation of the realized samples to sustain cell viability and proliferation has been carried out.
Shell scaffolds were obtained combining a modified replication technique [3] with the usual polymer burning-out method [6]. A polyurethane
sponge was used as template for the scaffolds. A 45S5 Bioglass® slurry was prepared by dispersing glass powders into distilled water
together with a polyvinylic binder and polyethylene powders (PE), acting as further pore generating agents. The sponges were immersed
in the slurry and then retrieved from the suspension; unlike the traditional replication technique, they were not squeezed but kept fully
loaded with the slurry. The samples were then rapidly dried under a multidirectional vigorous air flux at 100o C for 15 minutes, taking care
to keep them slurry-loaded. Subsequently, the samples were heat-treated at 1050o C for three hours to burn out the organic phase and
sinter the 45S5 Bioglass® structure. The bioactivity of the shell scaffolds was preliminary tested in a simulated body fluid solution (SBF)
and then further analyzed with cell culture tests (mouse calvaria-derived pre-osteoblastic cell line MC3T3-E1), while their microstructure
was investigated in a scanning electron microscope. Finally, the scaffolds’ mechanical behaviour, before and after immersion in SBF, was
assessed by means of compression tests.
The microstructural analysis of the scaffolds reveals the sensible difference between the external shell, which shows large pores but
also thick walls, thus guaranteeing permeability and manageability at the same time, and the internal pores network, resembling the
original sponge structure. The pore sizes fall within the 200–500 μm range, while the average porosity of the samples, resulting from
density measurements, is about 80% vol. The shell scaffolds were then soaked in a simulated body fluid solution to evaluate their in
vitro bioactivity. Already after three days in SBF, many hydroxyapatite agglomerates were observed on the sample surface. Preliminary
biological evaluations suggest a promising role of the obtained samples for applications in bone tissue regeneration. Finally, shell scaffolds
result satisfactory in terms of their mechanical strength (compressive stress: 0.3–0.4 and 0.7–0.8 MPa, depending on sample porosity),
which falls in the range reported for spongy bone.
CONCLUSION
A new approach to realize highly porous and manageable bioceramic scaffolds for bone tissue engineering was developed. The resulting
samples are characterized by a compact and at the same time permeable shell which surrounds the porous internal network. The obtained
samples look rather promising in terms of biological performance, while the positive effect of the external shell on the samples’ mechanical
strength has been confirmed.
REFERENCES
[1] Hollister SJ., Nat. Mater. 5:518-524, 2005.
[2] Hench LL, J. Am. Ceram. Soc. 74:1487-1510, 1991.
[3] Chen QZ et al., Biomaterials 27:2414-2425, 2006.
[4] Bellucci D et al., Mater Lett 64:203-206, 2010.
[5] Bellucci D et al., J Mater Sci: Mater Med, in press (2012) DOI 10.1007/s10856-012-4622-6.
[6] Vitale-Brovarone et al., J Mater Sci: Mater Med 20: 809-820 (2009).
89
ASSESSMENT OF THE HYPOALLERGENICITY OF SILVER FUNCTIONALIZED COTTON WITH LONG-TERM ANTIMICROBIAL
PROPERTIES
Mauro Pollini1,2, Federica Paladini1, Luca Salvatore1, Antonio Licciulli1,2, Alfonso Maffezzoli1 and Alessandro Sannino1,2
2 Silvertech Ltd, Department of Engineering for Innovation, 73100 Lecce, Italy
[email protected]
INTRODUCTION
Textile is a fertile ground for a multitude of microorganisms and an excellent substrate for the bacterial proliferation under appropriate
moisture and temperature conditions [1]. Colonization of several types of bacteria, especially Staphylococcus aureus, represents the
most common complication in patients affected by atopic dermatitis (AD), a chronic disease of pruritus and eczematous lesions [2].
In this work, silver was deposited on cotton fibers applying a patented silver deposition technique [3] described in previous works on
different substrates [4]. The focus of the work was the evaluation of any possible interaction of silver treated cotton with human skin,
in terms of skin irritation and hypoallergenicity. The presence of silver and the antibacterial capability against Gram positive and Gram
negative bacteria were verified even after several washing cycles in order to develop a product with long-term antibacterial capability and
no dangerous effect on human health.
Cotton yarns kindly provided by Silvertech Ltd were treated with silver according to a technology developed and patented at the University
of Salento [3,4]. 20 subjects were recruited for the skin irritation test and 20 for the hypoallergenicity test (average age 40, not pregnant
and not breast-feeding women). The skin irritation and hypoallergenicity tests consist in an occlusive application of the samples by means
of Finn chambers to the selected subjects. “Immediate skin irritation potential” (ISIP value), and the “skin irritation potential” (SIP value)
were evaluated 30 minutes (T1) after occlusive application to evaluate (ISIP), 48 hours (T2) after occlusive application, 24 hours after
removal of the occlusion (T3) to evaluate (SIP). Allergic reactions were evaluated 48 hours (T1) after occlusive application of the product,
24 hours (T2), 48 hours (T3) after occlusion removal. The antimicrobial activity of silver coated materials was verified by the procedure
described in Standard SNV 195920-1992. SEM-EDX analysis was performed to analyse the distribution of the silver particles on the
substrate.
The occlusive application of silver treated cotton for 30 minutes (T1) did not induce any reaction related to ‘Immediate skin irritation
potential’. The occlusive application of the tested product for 48 hours on 20 healthy subjects did not induce any reaction related to a ‘Skin
irritation potential’, as assessed at the occlusion removal (T2) and 24 hours after the product removal (T3).
No allergic reaction was observed. The skin reactions were checked at 24 and 48 hours after the occlusion removal and the test yielded
0% of allergic reactions as result. The antibacterial capability was verified on E.coli and S.aureus even after several washing cycles. The
strong adhesion of the silver clusters to the substrate was demonstrated through the EDX analysis by mapping the presence of silver on
cotton substrate (Figure 1).
Figure 1. Distribution of the silver
particles on cellulosic fibers
CONCLUSION
Silver treated cotton obtained through the photochemical deposition of silver nanoparticles have
demonstrated a strong antibacterial capability on E. coli and S.aureus even after several washing
cycles, thus indicating the long-term effectiveness of the material. No effects of skin irritation and
hypoallergenicity associated to the tested materials occurred in the recruited subjects. The excellent
adhesion of the silver particles to the substrate analysis ensured the stability of the coating and
its resistance to laundries. This study suggested that the developed product can be proposed for
application in biomedical and daily comfort fields with no dangerous effects in terms skin irritation
REFERENCES
1. Borkow G, Gabbay J. Med Hypotheses. 2008; 70:990-4
2. Biederman T. Acta Derm Venereol. 2006; 86:99-109
3. Pollini M. et al. Antibacterial surface treatments based on Silver clusters deposition 2008 EP20050850988
4. Pollini M et al. Engineering nanostructered silver coatings for antimicrobial applications. In Cioffi N, Rai M editors. Nano-Antimicrobials - Progress
and Prospects. Springer 2012. pp 313-36
90
NOVEL BIOMIMETIC BONE CEMENTS BASED ON SR-SUBSTITUTED HYDROXYAPATITE FOR REGENERATIVE
VERTEBROPLASTY
Simone Sprio¹, Teresa D’Alessandro¹, Carla Cunha¹,² and Anna Tampieri¹
1 Institute of Science and Technology for Ceramics, National Research Council, Italy
2 Rizzoli Orthopaedic Institute, Laboratory of Biomechanics and Technology Innovation, Bologna, ITALY,
[email protected]
INTRODUCTION Osteoporosis is a metabolic bone disease that results in bone fragility and increased susceptibility to fracture. As populations
age globally, the incidence of osteoporosis-related fractures will rise substantially over the coming decades1. Vertebroplasty and balloon
kyphoplasty are widely used in spinal surgery2. They consist in the injection of acrylic bone cement based on polymethylmetaacrylate
(PMMA) into damaged vertebrae. However PMMA exhibit no bioactivity and its use is accompanied by several drawbacks, such as the
strong exothermic effect developed during polymerization and the excessively high stiffness of the set cement, that can easily induce
fractures in adjacent vertebrae3. The present work focuses on the development of injectable pastes based on α-TCP partially substituted
with defined amounts of Strontium ions (2-20 mol%) as anti-osteoporotic agent4 that, upon hardening at physiological conditions,
hydrolyses and transforms in Sr-substituted hydroxyapatite (Sr-HA). Polymeric phases including natural polymers were incorporated
in the ceramic paste to enhance injectability and cohesion as well as to promote the formation of high open and interconnected macroporosity upon hardening, for proper cell conductivity and enhancement of osteointegration. This approach is a promising way towards
local regenerative therapies for vertebral bodies affected by ostoporosis-derived osteopenia, also providing mechanical sustain soon after
injection in vivo.
α-TCP was prepared by high temperature reaction (i.e. >1300 °C) of CaCO3 and CaHPO4 and successive quenching in air at room
temperature. Strontium ions were incorporated in the αTCP structure by addition of SrCO3. The particle size of the final product was
adjusted by powder milling in ethanol. The pastes were prepared by mixing the ceramic powders with sodium phosphate solutions.
Powder-to-liquid ratio was adjusted to optimise the paste injectability, setting time and viscosity. Polymeric phases were also added.
Polyethylene glycole (PEG) in different molecular weights was added to improve cohesion and injectability in liquid media. Sodium alginate
powder was also added in different amounts. The initial and final setting time were evaluated by Gilmore needles. X-ray diffraction (XRD),
Rietveld analysis, Fourier-Transform Infrared Spectroscopy (FTIR), thermal (TG) and chemical analysis (ICP) were used to assess phase
composition and degree of ion substitution. Mercury porosimetry and SEM image analysis were used to define the micro-macro porosity
and the interconnectivity. The particle size distribution was determined by X-ray sedimentography. Compressive strength and Young´s
modulus were evaluated vs. quenching temperature and amount of strontium, using a standard mechanical testing device. Cytotoxicity
tests were carried out on hardened cements.
XRD analysis confirmed that in all the investigated compositions Sr-substituted α-TCP completely converted in Sr-substituted HA upon
cement setting, following specific transformation kinetics. In some cases small amounts of α-TCP formed as secondary phase, as a result
of the set conditions of the thermal synthesis route. No other crystalline compounds were detected. It was found that the degree of powder
milling and the amount of strontium ions influenced the setting time and the kinetic transformation of Sr-α-TCP, so that a final setting time
of 5-10 minutes can be achieved. The addition of PEG gave positive effect on paste cohesion and injectability in simulated body fluid. The
addition of bio-resorbable polymeric phases allowed the formation of inteconnected pores with size in the range 200-500 µm, that results
feasible for cell conduction. The presence of a bio-resorbable phase such as α-TCP provoked a slight reduction of mechanical strength;
however compressive strength was found in the range 20-35 MPa and elastic modulus resulted ~ 1 GPa. All the obtained cements
resulted not cytotoxic, irrespective of the amount of strontium introduced.
CONCLUSION
On the basis of the obtained results the developed composite pastes promise to function as biomimetic, minimally invasive bone
cements potentially providing a jump-start for bone regeneration and eliminating the need for more radical surgical interventions for
spinal regions weakened by osteoporosis.
REFERENCES
1. Brewer L et al., Eur. J. Clin. Pharmacol. 67:321-331, 2011
2. Lewis G, J. Biomed. Mater. Res. Part B: Appl. Biomater. 76B :456-468, 2006
3. Baroud G et al., Joint Bone Spine, 73:144-150, 2006
4. Landi E et al., Acta Biomater. 3(6): 961-969, 2007
ACKNOWLEDGMENTS
The Authors gratefully acknowledge the European Commission for financial support under the FP7 contract number n° NMP3-SL-2010SMALL-3-246373 (OPHIS).
91
INTELLIGENT MULTIFUNCTIONAL SCAFFOLD
Silvia Minardi1,2, Ennio Tasciotti1,2, Monica Sandri1, SM Khaled2, Jonathan O. Martinez2,3, Brandon S. Brown2,3
Mauro Ferrari1,2, Anna Tampieri1,2
1 Department of Bioceramics and Biohybrid materials, Institute for Science and Technology for Ceramics – CNR, Italy
2 Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX, US
3 The University of Texas-Graduate School Of Biomedical Sciences At Houston, Houston, TX
[email protected]
INTRODUCTION
The broad medical impact of osteoarthritis and the limits in the current therapies have led to the rapid expansion in the field of biomaterials
for osteochondral repair[1]. The main challenge of osteochondral tissue engineering is represented by the need to regenerate two different
and adjacent tissues (cartilage and bone) and their interface (mineralized cartilage)[2]. Thus, we developed a biomimetic tri-layered
scaffold integrated with mesoporous silicon-PLGA composite microspheres (PLGA-pSi). The scaffold has been designed to mimic the
chemical, physical and morphological cues of the natural osteochondral region, combined with a concurrent delivery of biochemical
information to direct the concerted regeneration of bone and cartilage and their interface. The inclusion of the delivery system, into the
boundaries of the collagen based scaffold, constitutes a coating which can prevent disruption of the biochemical information within the
scaffold by phagocytic cells occurring in the post-operative.
To fabricate the layers of the biomimetic scaffold, three biomaterials were synthesized: (i) the cartilagineous-like layer, made of collagen
and hyaluronic acid; (ii) the mineralized cartilage-like layer, made of a hydroxyapatite/collagen 40/60 wt% composite; (iii) the subchondral
bone-like layer, composed of hydroxyapatite/collagen 70/30 wt% composite. A biologically inspired mineralization process was used
to nucleate biomimetic Magnesium-doped hydroxyapatite crystals on the type I collagen fibers during their self-assembling. PLGA-pSi,
with an average diameter of 15 µm, was synthesized through a modified S/O/W emulsion method as published elsewhere. PLGA-pSi
microspheres were blended and integrated into the three biomaterials, and a monolithic scaffold was generated through a freeze drying
process. The scaffold was characterized by XRD, FTIR, TGA, ICP-AES and SEM. Both 2D and 3D internalization studies of PLGA-pSi
microspheres of different dimensions were performed with macrophages, and studied through SEM imaging, confocal and time-lapse
microscopy.
During the biologically inspired mineralization process, an almost amorphous mineral phase was directly nucleated on the organic
template (type I collagen), as it occurs in the natural process. The PLGA-pSi microspheres were integrated in the collagen-based and the
mineralized collagen material: the microspheres were evidently integrated inside the collagen boundaries and mineralized collagen fibers.
The coating of the PLGA-pSi by the collagen boundaries enable to define it as a triple controlled release model. The integration of the
PLGA microspheres into the scaffold can also prevent the phagocytosis by macrophages, retarding the microsphere to be individuated,
preserving the loaded biochemical information.
Fig. 1 SEM images shows the microspheres integrated into
the (a) collagen and (b) mineralized collagen-based layers of
the scaffold. (c) A detail of the microspheres.
a)
(b)
(c)
CONCLUSIONS
We generated a chemically and structurally graded biomimetic scaffold, able to support osteochondral repair, integrated with PLGA-pSi
composite microspheres for the release of growth factors. The ultimate goal of our investigation is to precisely regulate the temporal and
spatial in vivo formation of chemical and biochemical gradients, as the crucial step to enhance the signaling capability of scaffolds for
tissue regeneration.
REFERENCES
Panseri et al., Knee Surg Sports Traumatol Arthrosc,. 20(6): p. 1182-91, 2012.
Tampieri et al., Biomaterials, 29(26): p. 3539-46, 2012.
92
goodbye
93

Congresso Nazionale Biomateriali - Lecce 18

Transcript

Documenti analoghi

NAZIONALE PILOTI vs TURKEY ALL STARS Wednesday 4th May

One of the fountains of Bellagio Hotel in Las Vegas (USA), made of

Call for Papers

talent without show

IANVA – La mano di Gloria

Alimini Lakes - Dipartimento di Bioscienze

the technical data sheet

Gamma-rays from Dark Matter Mini

Brochure 2015 - SEGOVIA

XII Congress of Mediterranean Society of Myology

FIRST INTERNATIONAL CONFERENCE ON THE FUTURE OF

here - ArtMoorHouse

Technicians and Master Builders for the Dome of St. Peter`s in

June 2005 - After the tsunami - Qui

Liceo Scientifico “A. Labriola - Programma di Lingua e Letteratura

Eötvös-Kürschák Competitions