摘要
目的:以经体外扩增及细胞因子诱导后已进入软骨细胞分化谱系的兔自体滑膜间充质干细胞(SMSCs)为种子,借助猪小肠粘膜下层(SIS)为支架,以SMSCs-SIS复合物的形式回植入兔体内半月板缺损处,探讨以此修复半月板损伤的可行性。方法:通过有限稀释单克隆培养法将兔SMSCs由滑膜组织中分离出来并加以纯化,并进一步在体外培养条件下研究其形态学、超微结构、分子表型、增殖动力学、核型以及致瘤性等基本的生物学特性;在体外用BMP-2、TGF-β3和DEX协同刺激SMSCs后,以RT-PCR检测I、II型胶原及软骨特异性聚集蛋白聚糖(Aggrecan)的mRNA表达,细胞免疫荧光化学染色检测细胞分化过程中I、II型胶原的表达,碱性甲苯胺蓝细胞化学染色检测软骨特异性糖胺聚糖(GAG)的表达,借此判断BMP-2、TGF-β3和DEX是否能够诱导SMSCs进入软骨细胞分化谱系;采用Abraham方法处理SIS,并检测其生物安全性和组织相容性,评价其作为种子细胞支架的可能性;最后将经体外扩增及细胞因子诱导已进入软骨细胞分化谱系的SMSCs种植在SIS支架上,回植入体内,6周、12周后取修复组织块行HE染色,碱性甲苯胺蓝染色,I型、II型胶原与S100蛋白的免疫化学组织染色,探讨以此修复损伤半月板结构和功能的可行性。结果:在体外培养条件下兔SMSCs的增生分裂能力十分活跃,可以通过离体培养使其实现数目的扩增;DNA含量检测、染色体核型分析、荷瘤实验结果表明,SMSCs是正常的二倍体细胞,无致瘤性,可作为半月板组织工程的种子细胞。在体外,SMSCs经BMP-2、TGF-β3和DEX协同诱导后14天,RT-PCR检测到I型、II型胶原及软骨特异性聚集蛋白聚糖(aggrecan)mRNA的表达,细胞免疫荧光化学染色也证实有I型、II型胶原的表达,同时碱性甲苯胺蓝细胞化学染色也证实有软骨细胞特异性胞外基质GAG成分,以上即证明SMSCs已进入软骨细胞分化谱系;本实验制成的SIS经组织切片和扫描电镜的观察表明是一种海绵样结构,其表面及内部均含有丰富的微孔结构,并与SMCSs有良好的组织相容性,可作为SMCSs的支架材料。SMSCs-SIS复合物植入半月板缺损区12周后大体观察原半月板缺损区有半月板样组织生成,HE染色可见纤维软骨样结构,周围胶原纤维结构明显,排列规则;碱性甲苯胺蓝染色可见蓝色异染基质-GAG;I型胶原、II型胶原与S100蛋白的免疫化学组织染色呈强阳性。而胶原对照及空白对照组则仅见纤维组织样的修复组织。结论:利用自体SMSCs经体外扩增及细胞因子刺激后,以SIS为支架,按一定的密度复合预培养后,回植入半月板缺损区的方法,是一种较为可行的修复半月板损伤的方法。为进一步提高这一方法的有效性,还需要在生物材料科学、生物力学和生物化学等方面进行更为深入的研究。
The feasibility study of rabbit meniscus repair was evaluated with the use of rabbit autologous synovial-derived mesenchymal stem cells(SMSCs) and pig’s small intestinal submucosa(SIS). Synovium cell populations were enzymatically released from the synovial tissue and were expanded in monolayer with serial passages at confluence. Cell clones were obtained by limiting dilution. After the synergic stimulation of such cytokines as BMP-2、TGF-β3 and DEX, the SMSCs were detected by RT-PCR and by histo- and immunohisto-chemistry for the expression of chondrocyte-related maker to confirm the pluropotential cell’s entering into the chondrogenic lineage. Subsequently, SIS scaffold was synthesized and SMSCs that had entered into the chondrogenic lineage were added into the porous SIS scaffold. Then, the SMSCs-SIS were implanted in vivo to the menisci-injured rabbit knees as the substitute for the injured meniscus.
After 6, 12 weeks postoperation, the implants were evaluated by gross observations and by histo- and immunohisto-chemistry. As a result, the SMSCs-enriched SIS implants underwent inflammation, degradation, SMSCs differentiation and remodeling stages in vivo, and consequently formed a meniscus-like fibrocartilage tissue. The collage I, collage II and S-100 protein were expressed at area of the repaired meniscal tissue by immunohistochemistry. The glycosaminoglycan(GAG) was detected by toluidine blue staining. All above proved the repaired meniscal tissue were chondrocyte-like fibrochondrocytes. On the contrast, the results of control group showed that both SIS implants without SMSCs and no substitute had limited regenerating tissue, further evaluations by histological observations showed no evidence of fibrochondrocytes, and hence these regenerated tissue were fibrous rather than fibrocartilagous.
In conclusion, the inducing of rabbit meniscus repair by autologous SMSCs and porous SIS scaffold is proved to be feasible in this study. However, further studies such as improving biomaterial design, evaluating the biomechanical properties of the regenerated tissue and ensuring clinical safety et al. are necessary to enhance the efficiency of the idea.
引文
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1.吴海山,徐青镭主编.膝半月板外科与组织工程学重建.上海:第二军医大学出版社, 1999年.
2. Allman AJ, McPherson TB, Badylak SF, el a1. Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response. Transplantation, 2001, 71(11): 1631-1640.
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4. Grimes M, Pembroke JT, McGloughlin T. The effect of choice of sterilisation method on the biocompatibility and biodegradability of SIS (small intestinal submucosa). Biomed Mater Eng, 2005, 15(1-2): 65-71.
5. Roeder R, Wolfe J, Lianakis N, et al. Compliance, elastic modulus, and burst pressure of small-intestine submucosa (SIS) small-diameter vascular grafts. Boimed Mater Res, 1999, 47: 65-70.
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1.吴海山,徐青蕾.膝半月板外科与组织工程学重建.上海:第二军医大学出版社. 1999. 7-9.
2. Andersson-Molina H, Karlsson H, Rockborn P. Arthroscopic partial and total meniscectomy: a long-term follow-up study with matched controls. Arthroscopy. 2002, 18: 183-189.
3. Bonneux I, Vandekerckhove B. Arthroscopic partial lateral meniscectomy: long-term results in athletes. Acta Orthop Belg, 2002, 68: 356-361.
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