脱钙松质骨体外构建组织工程软骨的实验研究
Allogeneic demineralized cancellous bone for cartilage tissue engineering

摘要：目的 软骨细胞复合同种异体脱钙松质骨(demineralized cancellous bone, DCB)体外构建组织工程软骨，评价DCB作为组织工程软骨支架的可行性。方法 取新西兰兔长骨干骺端松质骨制备DCB，扫描电镜观察；分离幼兔关节软骨细胞，原代培养后种植在同种异体DCB体外培养，分别于7、14、21、28、35、42d取材进行苏木素-伊红(HE)、甲苯胺蓝(TB)、Ⅰ型和Ⅱ型胶原染色。结果 制备的DCB成三维立体多孔结构，孔隙大小100～500μm，有良好的可塑性和一定的力学强度；软骨细胞在DCB表面和空隙内生长、分化良好，形成软骨陷窝，分泌基质蛋白聚糖和Ⅱ型胶原。培养42d软骨细胞在DCB表面及孔隙内形成软骨样组织。结论 DCB复合同种异体软骨细胞在体外成功构建了组织工程软骨，是一种较理想的软骨组织工程支架材料。
ABSTRACT: Objective Chondrocytes were seeded on allogeneic demineralized cancellous bone (DCB) constructing tissue-engineered cartilage to evaluate the feasibility of DCB as scaffolds for tissue engineering cartilage. Methods Allogeneic DCB was prepared by successive defatting and decalcification and then observed under scanning electronic microscope. One-month old rabbit articular chondrocytes were isolated and proliferated by monolayer culture. Passage 1 chondrocytes were seeded on DCB to construct tissue-engineered cartilage in vitro for 6 weeks. Specimens were taken after 7, 14, 21, 28, 35 and 42 days’ culture and evaluated by hematoxylin and eosin (HE), toluidine blue (TB), and immunohistochemistry for collagen type Ⅰ and Ⅱ. Results Prepared DCB showed three-dimensional porous structure of 100-500μm with good plasticity and certain mechanical strength. Chondrocytes grew well on and inside the DCB, and were located in lacunae and expressed matrix proteoglycan and type collagen Ⅱ, which formed cartiageous tissues on DCB up to 42 days’ culture. Conclusion DCB combined with allogeneic chondrocytes successfully constructed tissue-engineered cartilage in vitro, which is an ideal scaffold for tissue engineering cartilage