He Dawei, Jin Yan, Luo Kai, Li Shibao
The Fourth Military Medical University, Xi'an Shaanxi 710032, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Feb;20(2):116-20.
To observe the heterotopic osteogenesis of the autogenous marrow stromal cells (MSCs) on the ceramic bovine bone (CBB)/hydrogel scaffold (HG) and the effects of the recombinant human bone morphogenetic protein-2 (rhBMP-2) and the transforming growth factor beta (TGF-beta) on osteogenesis.
The autogenous marrow stromal cells were cultured by the mineralized condition medium (10% FBS, dexamethasone 10 nmol, L-vitamin C 50 mg/L, beta-sodium glycerophosphate DMEM culture medium 10 mmol). At 5 days, the MSCs differentiation was observed by Type I collagen, the Mend calcium-cobalt staining, and the Von-Kossa staining. The cell suspension of 5 x 10(6)/ml was obtained. There were three groups: Group A: added in rhBMP-2 (10 microg)-TGF-beta (0.05 microg); Group B: added in TGF-beta (0.05 microg); and Group C (the control group): without the growth factor. Then, the MSCs loading on CBB/HG were embedded in the autogenous subcutaneous area at 4 and 8 weeks, and the osteogenesis was observed by the HE staining and the modified Mallory's trichrome staining, with an image analysis. Type I collagen and the bone morphogenetic synthesis were examined by the immunohistochemistry stains.
Most MSCs induced by the mineralized condition medium at 5 days became smaller and polygon-shaped, and the cytodendrite became shorter. The MSCs were observed by the Mend calcium-cobalt staining. Some brown and black grains were found in the cytochylema. The MSCs were positive for the Type I collagen immunohistochemistry stains. At 20 days, the mineralized nubs were found by the Von Kossas stains. At 4 weeks, some strips of the new bone were observed by the HE staining and the modified Mallory's trichrome staining in all the groups. The bone matrix area was significantly larger in Group A than in Group B(P < 0.01). The average gray degrees of Type I collagen were lower in Groups A and B than in Group C. However, there was no significant difference in the bone morphogenesis among the three groups. At 8 weeks, there were significantly more snatch strips and macula mature bone formation in Groups A and B than in Group C. The Type I collagen and the bone morphogenesis were not significantly different among the three groups.
The autogenous marrow stromal cells on the ceramic bovine bone /hydrogel scaffold can promote the heterotopic osteogenesis, and the combined use of rhBMP-2 and TGF-beta is better than the only use of rhBMP-2 or TGF-beta in promoting osteogenesis.
观察自体骨髓基质细胞（MSCs）在陶瓷牛骨（CBB）/水凝胶支架（HG）上的异位成骨情况以及重组人骨形态发生蛋白-2（rhBMP-2）和转化生长因子β（TGF-β）对成骨的影响。
采用矿化条件培养基（10%胎牛血清、地塞米松10 nmol、L-维生素C 50 mg/L、β-甘油磷酸钠10 mmol的DMEM培养基）培养自体骨髓基质细胞。第5天，通过Ⅰ型胶原、门氏钙钴染色和冯科萨染色观察MSCs的分化情况。获得浓度为5×10⁶/ml的细胞悬液。分为三组：A组：加入rhBMP-2（10 μg）-TGF-β（0.05 μg）；B组：加入TGF-β（0.05 μg）；C组（对照组）：不添加生长因子。然后，将负载MSCs的CBB/HG在4周和8周时植入自体皮下区域，通过苏木精-伊红（HE）染色和改良马洛里三色染色观察成骨情况，并进行图像分析。通过免疫组织化学染色检测Ⅰ型胶原和骨形态发生合成情况。
矿化条件培养基诱导的大多数MSCs在第5天变得更小且呈多边形，细胞突起变短。通过门氏钙钴染色观察MSCs。在细胞浆中发现一些棕色和黑色颗粒。MSCs的Ⅰ型胶原免疫组织化学染色呈阳性。第20天，通过冯科萨染色发现矿化小结。第4周，所有组通过HE染色和改良马洛里三色染色均观察到一些新骨条带。A组的骨基质面积显著大于B组（P<0.01）。A组和B组的Ⅰ型胶原平均灰度低于C组。然而，三组之间的骨形态发生没有显著差异。第8周，A组和B组的成熟骨形成的条带和斑片明显多于C组。三组之间的Ⅰ型胶原和骨形态发生没有显著差异。
陶瓷牛骨/水凝胶支架上的自体骨髓基质细胞可促进异位成骨，rhBMP-2与TGF-β联合使用在促进成骨方面优于单独使用rhBMP-2或TGF-β。
