Preparation And Characterisation Of Collagen And Complex Scaffolds For Cartilage Tissue Engineering

Current articular cartilage tissue engineering endeavors, using synthetic poly-mers as scaffolds, have been somewhat successful. However, for the poorbiocompatibility, using of these materials has not yielded a satisfactory, functionalreplacement for articular cartilage. Therefore, this project focuses on analternative to these materials, collagen, and 3-hydroxybutyrate and3-hydroxyhexanoate (PHBHHx). and complex materials.1. Isolation, Identifying and culture of seed Chondrocytes.The articular cartilage of 48-hours old infant rabbit was isolated and slicedundergoed sequential digestions with pronase and collagenase for different time,chondrocytes were cultured and subcultured in vitro from P₀ to P₈. Theattachment, proliferation and the variation of biological characteristices calculatedby the HE staining,toluidine blue staining The resoults showed that theattachment and proliferation of chondrocytic undergoed 4 hours digestion withcollagenase were the best, The phenotypic of the fourth passage chondrocytes isstability and are the best seed-cells for cartilage tissue engineering.2. Preparation and characterization of collagen scaffolds.A series of porous collagen scaffolds were fabricated with different collagenconcentration, pre-freezing temperature and cross-linking agents by freeze-drying.The average pore diameter, mechanical, and degradation. of the porous scaffoldswere analysised and compared each other. Chondrocytes of rabbit were separatedand cultured on these scaffolds to evaluate their biocompatibility。The resoultsshowed that the collagen concentration, pre-freezing temperature cross-linkingagents have a crucial influence on the structure and properties of collagenscaffolds. The suitable collagen scaffolds were obtained by adjusting the collagenconcentration, pre-freezing temperature and the cross-linking agents; The biggerof the pore size was the faster cell proliferation was achieved in this research. 3. Constructing the complex of Chondrocytes and collagen scaffolds invitroThe sixth passage chondrocytes were cultured in the 0.6ï¼…collagenscaffolds(pre-temperature:-50,cross-lingking agent:EDC).The scaffolds wereevaluated in terms of their ability to support the chongdroncytes attachment,viability, cell morphology by SEM and HE stain and the phenotype ofchondrocyte was analyzed by the expression of collagen typeâ…¡using the methodof RT-PCR. Results indicated that the phenotype of the sixth passagechondrocytes is stability and strong in the collagen scaffolds in vitro and are thebest seed-cells for cartilage tissue engineering. This kind of collagen scaffolds canbe Useed for cartilage tissue engineering.4. Preparation and ccharacterization of PHBHHx/collagen-heparinscaffoldsHighly porous PHBHHx scaffolds were prepared by a solventcasting/particulate leaching method.heparinized collange was immobilized ontothe surface of PHBHHx scaffolds. The physical and chemical properties of thesynthetic scaffolds were characterized by (SEM), (EDS),,contact angle, heparincontent analysis and so on. The cell proliferation, morphology were evaluated bymicroscopy. The results indicated that the heparinized-collagen could improve theaffinity of PHBHHx to water. The triple composite of PHBHHx/collagen-heparinhad a ability to control release the bFGF and excelled in the chondroncytesproliferation compared to unmodified PHBHHx scaffolds. The modifiedPHBHHx is a kind of ideal biodegradable scaffolds for cartilage tissueengineering. this novel scaffold could make a new development in the field ofcartilage tissue engineering.