Zhou Guang-Dong, Wang Xiao-Yun, Miao Chun-Lei, Liu Tian-Yi, Zhu Lian, Liu De-Li, Cui Lei, Liu Wei, Cao Yi-Lin
Department of Plastic Surgery of the 9th People's Hospital, Shanghai Second Medical University, Shanghai Tissue Engineering Research Center, Shanghai 200011, China.
Zhonghua Yi Xue Za Zhi. 2004 Jun 2;84(11):925-31.
To test the possibility of using bone marrow stromal cells (BMSC) and biodegradable polymers to repair articular osteochondral defects at non-weight bearing area of porcine knee joints.
Bone marrows were harvested from 18 hybrid pigs. BMSC were cultured and in vitro expanded and induced with dexamethasone (group A) or with dexamethasone and transforming growth factor-beta1 (TGF-beta1) (group B) respectively. Immunohistochemistry and RT-PCR were used to evaluate chondrogenic differentiation of induced cells. Part of BMSC of 2 animals were retrovirally-labeled with green fluorescent protein (GFP). After induction and label, cells were seeded on a construct of polyglycolic acid (PGA) and polylactic acid (PLA) and co-cultured for 1 week before implantation. Total 4 osteochondral defects (8 mm in diameter, 5 mm in depth) in each animal were created at the non-weight bearing areas of knee joints on both sides. The defects were repaired with dexamethasone induced BMSC-PGA/PLA construct in group A, with dexamethasone and TGF-beta1 induced BMSC-PGA/PLA construct in group B, with PGA/PLA construct alone (group C) or left untreated (group D) as controls. Animals were sacrificed at 3 months (n = 6) or 6 months (n = 10) post-repair. Gross observation, histology, glycosaminoglycan (GAG) quantification and biomechanical test were applied to analyze the results. The two animals with GFP-labeled cells were sacrificed at 7 months post-repair to observe with confocal microscope the distribution of GFP-labeled cells in repaired tissue.
Stronger expression of type II collagen and aggrecan were observed in BMSCs induced with both dexamethasone and TGF-beta1. At both time points, Gross observation and histology showed that the defects in most of group A were repaired by engineered fibrocartilage and cancellous bone with an irregular surface, minority defects were repaired by engineered hyaline cartilage and cancellous bone. However, in most of group B, the defects were completely repaired by engineered hyaline cartilage and cancellous bone. No repair or only fibrous tissue were observed in groups C and D. Besides, the compressive moduli of repaired cartilage in groups A and B reached 30.37% and 43.82% of normal amount at 3 months and 62.69% and 80.27% at 6 months respectively, which was further supported by the high levels of GAG contents in engineered cartilage of group A (78.03% of normal contents) and group B (no statistical difference from normal contents). More importantly, confocal microscope revealed the presence of GFP-labeled cells in engineered cartilage lacuna and repaired underlying cancellous bone.
The results demonstrated that implanted BMSC can differentiate into either chondrocytes or osteoblasts at different local environments and repair a complex articular defect with both engineered cartilage and bone. TGF-beta1 and dexamethasone in vitro induction can promote chondrogenic differentiation of BMSC and thus improve the results of repairing articular defects.
测试使用骨髓基质细胞（BMSC）和可生物降解聚合物修复猪膝关节非负重区关节软骨下骨缺损的可能性。
从18只杂交猪中采集骨髓。BMSC分别用地塞米松（A组）或地塞米松和转化生长因子-β1（TGF-β1）（B组）进行培养、体外扩增和诱导。采用免疫组织化学和逆转录-聚合酶链反应（RT-PCR）评估诱导细胞的软骨形成分化。将2只动物的部分BMSC用绿色荧光蛋白（GFP）进行逆转录病毒标记。诱导和标记后，将细胞接种在聚乙醇酸（PGA）和聚乳酸（PLA）构建体上，共培养1周后植入。每只动物在双侧膝关节非负重区制造4个软骨下骨缺损（直径8 mm，深度5 mm）。A组用经地塞米松诱导的BMSC-PGA/PLA构建体修复缺损，B组用经地塞米松和TGF-β1诱导的BMSC-PGA/PLA构建体修复缺损，C组用单独的PGA/PLA构建体修复缺损，D组不进行处理作为对照。在修复后3个月（n = 6）或6个月（n = 10）处死动物。应用大体观察、组织学、糖胺聚糖（GAG）定量和生物力学测试分析结果。在修复后7个月处死2只带有GFP标记细胞的动物，用共聚焦显微镜观察GFP标记细胞在修复组织中的分布。
在地塞米松和TGF-β1诱导的BMSC中观察到Ⅱ型胶原和聚集蛋白聚糖的表达更强。在两个时间点，大体观察和组织学显示，A组大多数缺损由工程化纤维软骨和松质骨修复，表面不规则，少数缺损由工程化透明软骨和松质骨修复。然而，B组大多数缺损由工程化透明软骨和松质骨完全修复。C组和D组未观察到修复或仅有纤维组织。此外，A组和B组修复软骨的压缩模量在3个月时分别达到正常量的30.37%和43.82%，在6个月时分别达到62.69%和80.27%，A组工程化软骨中高水平的GAG含量（正常含量的78.03%）和B组（与正常含量无统计学差异）进一步支持了这一结果。更重要的是，共聚焦显微镜显示在工程化软骨陷窝和修复的下方松质骨中存在GFP标记的细胞。
结果表明，植入的BMSC可在不同局部环境中分化为软骨细胞或成骨细胞，并通过工程化软骨和骨修复复杂的关节缺损。TGF-β1和地塞米松体外诱导可促进BMSC的软骨形成分化，从而改善关节缺损的修复效果。
