Regeneration of Articular Cartilage Tissue from Cultured Chondrocytes using a Photo-polymerizing Gelatin as a Cell Carrier

基于以下三点概念，我们开发了一种苯乙烯化明胶作为软骨软骨细胞移植的支架：1)支架可以在手术部位原位交联（通过可见光照射）以填补关节软骨组织的任何间隙；2)被其包裹的移植细胞能够增殖并产生软骨基质；3)支架具有适当的硬度和弹性，交联后可生物降解。在这项研究中，我们研究了苯乙烯明胶作为软骨细胞移植支架的潜力和问题。将苯乙烯基与赖氨酸明胶残渣连接，在含20%胎牛血清和相当于明胶重量0.1%的樟脑醌的培养基中与培养的兔软骨细胞混合，合成苯乙烯化明胶。然后用牙科卤素灯用无害可见光（400-520 nm）照射2分钟，交联。结果表明：1)在聚苯乙烯基取代率低于45%（基团/分子），聚苯乙烯基明胶与其溶剂（介质）的混合质量比为30%的条件下，交联后的包埋软骨细胞能够存活、增殖并生成软骨基质；2)RT-PCR结果显示明胶水凝胶培养的关节软骨细胞表达II型胶原和聚集蛋白核心蛋白mRNA；细胞移植三周后，在明胶中观察到软骨基质的组织学变化。然而，作为软骨基质的代表性成分，硫酸化糖胺聚糖的产量（40-50%）不如使用胶原凝胶代替苯乙烯化明胶作为支架时高；在本例中，柱化基团的取代比约为45%组/分子，柱化明胶与其溶剂介质的混合物质量比为30%（光聚合后的柱化明胶水凝胶弹性约为13-15 KPa（约为活体实际软骨组织的1/40））。这些结果表明，尽管在程化明胶水凝胶中嵌入的软骨细胞增殖和软骨基质生成不足，其交联体在体内的弹性和生物降解性仍不理想，证明我们的软骨细胞移植支架概念是可以实现的和有前景的。少

We have developed a styrenated gelatin as a scaffold for cartilage chondrocyte trans-plantation based on the following three-point concept : 1)The scaffold can be cross-linked in situ (by visible light irradiation) at the surgery site to fill any gaps in the articular cartilage tissue ; 2)Transplanted cells enclosed with in it can proliferate and produce cartilage matrices, and ; 3)The scaffold has proper hardness and elasticity and yet will biodegrade after it is cross-linked. In this study, we examined the potential and the problems of styrenated gelatin as a scaffold for chondrocyte transplantation.Styrenated gelatin was synthesized by connecting styrene groups with lysine gelatin residue and mixed with cultured rabbit chondrocytes in a medium containing 20% fetal bovine serum with camphorquinone equivalent to 0.1% of the gelatin weight. Then the mixed solution was irradiated with harmless visible light (400-520 nm) for 2 minutes using a dental halogen lamp and cross-linked.The resu … More lts showed that : 1)Embedded chondrocytes can survive, proliferate, and produce cartilage matrices after it is cross-linked under the condition that the substitu-tion ratio of the stylenated group was below ca. 45% (group/molecule), and the mixture weight ratio between the stylenated gelatin and its dissolvent (medium) was 30% ; 2)RT-PCR showed that the articular chondrocytes cultured in the gelatin hydrogel expressed the mRNA of type II collagen and aggrecan core protein ; these cartilaginous matrices were observed histologically throughout the gelatin three weeks after the cell trans-plantation. However, sulfated glycosaminoglycan production, a representative component of the cartilage matrix, was not as high (40-50%) as when collagen gel instead of styrenated gelatin was used as the scaffold ; in this case, the substitution ratio of the stylenated group was ca. 45% group/molecule, and the weight ratio of the mixture between the stylenated gelatin and its dissolvent medium was 30% (the elasticity of the stylenated gelatin hydrogel was around 13-15 KPa (ca. 1/40 of the actual cartilage tissue of the living body) just after it was photo-polymerized).These results suggest that, even though the embedded chondrocyte proliferation and cartilage matrix production in the stylenated gelatin hydrogel are insufficient and the elasticity and biodegradability of its cross-linked body in-vivo are still unsatisfactory, proving that our chondrocyte transplantation scaffold concept is realizable and promising. Less