以纤维蛋白胶为载体的注射型骨修复材料对兔骨髓基质细胞增殖和分化的影响

Effects of the injectable osteoinductive material with fibrin glue as carrier on the proliferation and differentiation of marrow matrix cell of rabbit

目的观察以纤维蛋白胶(FS)为载体的注射型骨修复材料对体外培养的兔骨髓基质细胞(MSCs)增殖和分化的影响。方法取3天龄新西兰兔MSCs进行培养,实验分为5组,A组(实验组)培养液中加入含1μg/ml重组人骨形态发生蛋白2(rhBMP-2)和1μg/ml重组人碱性成纤维细胞生长因子(bFGF)的纤维蛋白胶,B组(对照1组)培养液中加入单纯的纤维蛋白胶,C组(对照2组)培养液中加入含1μg/mlbFGF的纤维蛋白胶,D组(对照3组)培养液中加入含1μg/mlrhBMP-2的纤维蛋白胶,E组为单纯对照组(培养液中不加入任何材料)。采用细胞培养、组织化学及电镜观察等方法对各组细胞增殖情况、贴壁率、碱性磷酸酶(ALP)活性、I型胶原表达、超微结构变化等进行研究。结果各组材料的促增殖作用和对细胞贴壁率的影响由强到弱均依次为C组、A组、B组、D组、E组,差异有显著性(P<0.05)。各组细胞ALP活性及I型胶原表达水平由强到弱均依次为A组、D组、B组、C组、E组,差异有显著性(P<0.05)。扫描电镜观察发现,以纤维蛋白胶为载体的注射型骨修复材料表面粗糙,有微孔存在,各组细胞与其融合生长。透射电镜观察发现:A组的绝大部分细胞呈成骨细胞表型,细胞增殖旺盛,分化好,胞质内有丰富的粗面内质网和线粒体,并明显扩张,内有蛋白样物质,细胞外基质丰富,细胞周围可见大量的胶原纤维;而各对照组或是细胞增殖活性高,但向成骨细胞表型分化差,或是向成骨细胞表型分化好,但细胞增殖活性较差,细胞外基质及胶原纤维少。结论以纤维蛋白胶为载体的注射型骨修复材料(FS+bFGF+rhBMP-2)可显著促进MSCs的增殖及其向成骨细胞的分化。

Objective To investigate the effect of the injectable osteoinductive material with fibrin glue （FG） as carrier compounded with rhBMP-2 and bFGF on the proliferation and differentiation of marrow matrix cells （MMCs） of rabbit, in order to lay an experimental foundation for clinical application in the future. Method MMCs were isolated and cultured from hone marrow of a 3-day-old rabbit. The effects of the material were investigated in experiment group （group A： FG＋bFGF＋rhBMP-2）, with control groups consisting of control group 1 （group B： FG）, control group 2 （group C： FG＋bFGF）, control group 3 （group D： FG＋rhBMP-2） and single control group （group E： free of material）. The observation objects included proliferation rate, adhesive rates, expression of type I collagen and alkaline phosphatase, cell growth condition in the material and the ultrastructure of rabbit MMCs with the aid of electron microscopy, histochemistry and cell culturing. The concentration of rhBMP-2 and bFGF in all groups was uniform （1μg/ml）. Results The proliferation rates and the adhesive rates of rabbit MMCs in group A was significantly higher than that in group B, group D and group E, but was lower than in group C （ P〈0. 05）. The expression level of type I collagen and alkaline phosphatase in experiment group A was significantly higher than in all control groups （P〈0. 05）. Scanning electron microscopy showed that many pores were present on the rough surface of the material, with confluent growth of both the cells and the material. Transmission electron microscope showed that MMCs in group A were mostly of the phenotype of osteoblast with high proliferation activity and high differentiation degree toward osteoblast, and there was a rich extracellular matrix and collagen fibers, while the MMCs in control groups had either low differentiation degree toward osteoblast with poor extracellular matrix and collagen fibers or low proliferation activity. Conclusion The injectable nsteoinductive material with fibrin glue as carrier compounded with rhBMP-2 and bFGF could significantly accelerate the proliferation and differentiation of marrow matrix cells towards osteoblasts in rabbit.