仿生活性人工软骨的体外构建及其异位成软骨分化实验研究

应用先进快速成形技术(RP)制备32枚粒度均匀(尺寸均为4mm×4mm×4mm)的聚乳酸-聚羟乙酸(PLGA)人工载体,该载体经I型胶原表面修饰后均分为A、B两组。A组载体复合人骨形态发生蛋白-2基因转染(rAAV-hBM P-2)的兔骨髓基质细胞(M SC s,2×104个细胞/枚);B组每枚载体复合等量、同代次、未基因转染M SC s。体外培养第5 d,从两组各取12枚细胞-载体复合物植入裸鼠皮下,术后30 d取材观察。结果发现rAAV-hBM P-2转染的M SC s成功表达目的基因。RP制备的PLGA载体具有良好的空间结构,大孔及材料表面微孔孔径分别为300μm和3~5μm。体外培养3~5 d,两组载体均复合生长着大量种子细胞。皮下埋植30 d,A组植入物形成较为典型的软骨细胞及基质,II型胶原蛋白表达阳性;同期B组植入物无软骨组织形成。A组聚酯材料面积百分率显著低于B组(P<0.01)。结果表明RP结合载体材料表面修饰,能制备出兼具理想孔隙结构和良好生物相容性的组织工程支架载体,该载体高效复合rAAV-hBM P-2转染的M SC s为组织工程软骨构建创造有利条件。

The Experimental Study of Biomimetic Artificial Cartilage Fabrication In Vitro and Ectopic Chondrogenesis In Vivo

Tri-dimensional poly (DL-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated using a rapid prototyping (RP) technique and the gene of human bone morphogenetic protein 2 (hBMP-2) was transferred into rabbit bone marrow stromal cells (MSCs) via recombinant adeno-associated virus vectors (rAAV-hBMP-2). Thirty-two PLGA scaffolds, size (4 mm X 4 mm X 4 mm), were coated with collagen type I and equally divided into 2 groups. In group A, each scaffold was loaded with 2 X 10(4) hBMP-2 (+) MSCs to establish a hBMP-2 (+) MSCs/PLGA composite. In group B, each scaffold was loaded with 2 X 10(4) hBMP-2 (-) MSCs to establish a hBMP-2 (-) MSCs/PLGA composite. The composites in both groups were cultured for subcutaneous implantation in nude mice. All animals were killed 30 days after implantation and the differentiation of composites was evaluated. As a result, MSCs infected with rAAV-hBMP-2 efficiently expressed hBMP-2 protein. RP-based PLGA scaffolds had ideal microarchitecture. The diameters of macropore and micropore of the scaffolds were 300 microm and 3-5 microm, respectively. At 3-5 days after culture, a number of seeding cells well grew on the scaffolds of both groups. The composites in group A had chondrogenesis ability in vivo and the expression of collagen type II was positive. In group B, however, only polymers and fiber tissues were predominantly found. The percentage of polymer remnant area was significantly lower in group A than in group B (P<0.01). Our results therefore indicate that RP-based PLGA scaffolds efficiently coated with collagen type I have good biocompatibility with hBMP-2 (+) MSCs and the techniques developed in this study may favor cartilage tissue engineering.