陶瓷化骨-水凝胶与骨髓基质细胞自体皮下成骨实验

目的观察重组人骨形成蛋白(recombinant human bone morphogenetic protem 2,rhBMP-2)-转化生长因子β(transforming growth factor β,TGF-β)与陶瓷化骨-水凝胶和骨髓基质细胞复合后,植入自体皮下成骨能力的影响.方法用矿化液诱导第1代成年SD大鼠骨髓基质细胞(marrow stromal cells,MSCs)5 d后,应用改良钙钴法染色、Von Kossa染色、免疫组织化学染色观察诱导后的MSCs向成骨细胞分化情况.将收集的细胞(5×106/ml)分成3组,A组加入rhBMP-2(5μg)-TGF-β(0.05μg),B组加入rhBMP-25μg,C组不加生长因子作为空白对照;然后将细胞接种在陶瓷化骨-水凝胶上,植入自体12只SD大鼠皮下,每组6只,每只大鼠3种材料各1块.术后4、8周取材,行组织学观察骨基质形成情况,图像分析测量单位面积骨形成量,免疫组织化学染色观察Ⅰ型胶原、BMP合成情况.结果 MSCs经过5 d矿化诱导后,大部分细胞变小,胞浆突起变短,呈多边形.改良钙钴法染色见胞浆含有棕黑色颗粒的阳性细胞,Ⅰ型胶原免疫织组化学染色见大部分细胞胞核未着色,胞浆呈棕黄色;Von Kossa染色见20 d已经形成矿化结节.动物体内植入后4周,各组均有形状不规则的条索状骨基质形成,图像分析示A组的骨基质面积明显多于B组(P＜0.01);A、B组Ⅰ型胶原平均灰度值明显低于C组(P＜0.01),各组BMP的表达无明显差异(P＞0.05).8周时各组均有形状不规则的条索状、斑块状砖红色骨基质形成,A、B两组成熟骨面积明显多于C组(P＜0.01);Ⅰ型胶原、BMP均有阳性表达,但各组间平均灰度值无明显差异(P＞0.05).结论陶瓷化骨-水凝胶与MSCs复合物植入自体皮下后能形成骨组织,rhBMP 2-TGF-β复合生长因子较单纯rhBMP-2有更强的促进骨形成作用.

HETEROTOPIC OSTEOGENESIS OF AUTOGENOUS MARROW STROMAL CELLS ON CERAMIC BOVINE BONE/HYDROGEL SCAFFOLD

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 snatchy 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. CONCLUSION: 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.