经碱性成纤维细胞生长因子修饰骨髓基质干细胞注射式修复全层软骨缺损

Injectable repair of full-thickness cartilage defects with basic fibroblast growth factor-modified bone marrow mesenchymal cells

目的 探讨经碱性成纤维细胞生长因子（bFGF）修饰的骨髓基质干细胞（BMSCs）注射式修复全层软骨缺损的可行性.方法 体外培养家兔自体骨髓基质干细胞,并以bFGF处理修饰细胞,免疫组织化学Ⅱ型胶原蛋白表达、逆转录-聚合酶链反应（RT-PCR）检测蛋白聚糖表达.将其与藻酸钙凝胶支架复合注射式植入兔股骨髁全层软骨缺损处,同时设立凝胶支架对照组和空白对照组.术后8周取材观察修复效果,并行苏木素-伊红（HE）、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色检测;透射电镜观察修复组织的微观结构.结果 BMSCs的细胞群体倍增时间（PDT）为33.8 h,经bFGF处理的BMSCs可检测到Ⅱ型胶原和蛋白聚糖的表达.至术后8周可见质硬的类白色修复组织完全充填软骨缺损处,组织学检查可见大量软骨样细胞分布于深染的细胞外基质中,检测到Ⅱ型胶原的表达.透射电镜可见丰富的细胞器和胞外基质.凝胶支架对照组和空白对照组仅有部分质软组织充填缺损处,未检测到Ⅱ型胶原的表达.结论 经hFGF修饰的自体骨髓基质干细胞藻酸钙凝胶复合物可用于修复全层软骨缺损.

Objective To testify the feasibility of the basic fibroblast growth factor （bFGF）-modified bone marrow mesenchymal stem cells to repair full-thickness cartilage defects by injection.Methods The bone marrow stromal cells （BMSCs） were derived from bone nla.rYow of the rabbits and cultured in vitro,and modified by bFGF.The Expression of the type Ⅱ collagen and aggrecan Was detected by immunohistochemistry and RT-PCR.Twenty-one New Zealand white rabbits were divided into three groups randomly.The cultured BMSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the femoral condyle,and the de^cts repaired with gel or without treatment served as control groups.After 8 weeks,the reconstructed tissue Was evaluated macroscopically and microscopically.Histologic analysis and electron microscopic detection were also performed to observe the structure of repair tissue.Results The population doubling time（PDrr）of BMSCs was about 33.8 h.The exprezsion of the type Ⅱ collagen and aggrecan was detected after bFGF application.The reconstructed tissue with BMSCs was similar with the microstructure of hyaline cartilage,and contained ulore ceils and thick extracellular matrix than other groups.Type Ⅱ collagen was detected within the matrix by immunohistochemical staining.Plenty of collagen fibers around cells could be seen under the transmission electron microscopy.Only partial soft connective tissue filled the bottom of the defects in other two control groups.Conclusion The compound of bFGF-modified BMSCs with injectable calcium alginate gel is applicable for full-thickness cartilage defects.