rhBMP_2／TGF-β对β-磷酸三钙与骨髓基质细胞自体皮下成骨能力的影响

Effects of rhBMP_2/ TGF-β on heterotopic osteogenesis of autogenous marrow stromal cells and β-tricalcium phosphate scaffold

目的:观察rhBMP2／TGF-β对β-磷酸三钙与骨髓基质细胞在自体皮下成骨能力的影响。方法:应用矿化条件培养液体外培养成年新西兰兔的骨髓基质细胞,收集细胞并以5×106·mL-1浓度分成4组,A组加入 rhBMP2(15μg)／TGF-β(30 ng),B组加入rhBMP2(15μg),C组不加因子作为对照,然后将细胞接种在β-磷酸三钙上,体外培养3 d,D组细胞不接种材料上,继续体外培养。组织化学方法检测碱性磷酸酶。将复合物埋植于新西兰兔自体皮下。5周后常规HE染色、免疫组织化学染色并进行图像分析,观察I型胶原、骨形成蛋白合成情况。结果:A、B组的碱性磷酸酶平均A值显著高于C、D组(P<0．01);复合物植入皮下5周时 HE染色见各组均有条索状骨基质形成,A、B组I型胶原平均灰度值明显低于C组(P<0．01),各组免疫组织化学检测及图像分析显示骨形成蛋白的表达差异无显著性(P>0．05)。结论:β-磷酸三钙与骨髓基质细胞在自体皮下能够形成骨组织,rhBMP2／TGF-β较rhBMP2有更强的促进骨形成作用。

Abstract： Objective To observe heterotopic osteogenesis of autogenous marrow stromal cells on β-tricalcium phosphate （β-TCP） scaffold, and the effects of recombinant human bone morphogenetic protein 2 （rhBMP-2） and transforming growth factor-beta （TGF-β） on osteogenesis. Methods The Newzealand rabbits autogenous marrow stromal cells were cultured by mineralized condition medium. The cell suspension was collected. All the cells with concentration of 5×10^6 · mL^-1 were divided into four groups. Group A： rhBMP-2 （15/tg） /TGF-β （30 ng） were added; group B： TGF-β 30 ng was administered; group C： without growth factor as control; group D： continual cultured and no loading on β-TCP. MSCs in group A, B, C were planted on β-TCP. Alkaline phosphatase （ALP） activity was examined by histochemistry. Then MSCs loading on β-TCP were embedded in autogenous subcutaneous. Four weeks later, the osteogenesis was observed by HE staining, type Ⅰ collagen and bone morphogenetic protein synthesis were examined by immunohistochemistry stains. Results The ALP activities in groups A and B were significantly higher than those in group C and D （P〈0.01）. Five weeks later, some strip new bone formations were observed by HE staining in all groups, the bone matrix area in group A was significantly bigger than that in group B （P〈0.01）. The average gray degree of type Ⅰ collagen in groups A and B were lower than that in group C. There was not obviously difference of bone morphogenetic protein expression between group A, B and C. Conclusion β-TCP and MSCs can promote heterotopic osteogenesis, rhBMP-2 and TGF-β have the stronger effect to enhance osteogenesis than rhBMP 2.