BMSCs来源成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架构建血管化组织工程骨研究

目的探讨大鼠BMSCs来源的成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架植入大鼠桡骨缺损处的成骨作用和成血管作用。方法取分离培养至第3代的SD大鼠BMSCs行成骨和成内皮细胞诱导并鉴定。分别将内皮细胞(A组)、成骨细胞(B组)、混合细胞(成骨细胞和内皮细胞比例为1∶1,C组)均匀滴加于壳聚糖-羟基磷灰石多孔支架上制备3组细胞-支架复合物,MTT检测支架内细胞增殖活性。取2月龄雄性SD大鼠30只,制作大鼠桡骨5 mm长缺损模型并分别植入3组细胞-支架复合物(n=10)。术后4、8、12周分别取移植物行HE染色观察,CD34免疫组织化学染色计数微血管密度,RT-PCR法检测骨桥蛋白(osteopontin,OPN)和骨保护素(osteoprotegrin,OPG)mRNA表达。结果 BMSCs成骨诱导7 d后ALP染色可见细胞质内蓝染颗粒,细胞核呈红染;内皮细胞诱导14 d后,CD34免疫细胞化学染色可见细胞内棕色颗粒。MTT检测示3组细胞活性随时间延长逐渐升高。HE染色示,术后12周A组未见明显类骨质形成,而有较密集的微血管结构及较多纤维组织形成;B、C组可见均质的类骨质,呈条索状和岛状分布,可见大量成骨样细胞存在。术后各时间点A、C组微血管密度均显著高于B组(P<0.05);A组术后12周微血管密度高于C组(P<0.05),其余2个时间点A、C组间差异无统计学意义(P>0.05)。A组3个时间点OPN和OPG mRNA表达水平均较低,与B、C组比较差异有统计学意义(P<0.05);B、C组分别于术后8、12周OPN mRNA表达达峰值,4周时OPG mRNA表达达峰值。结论 BMSCs来源的成骨细胞和内皮细胞按1∶1比例共培养于壳聚糖-羟基磷灰石多孔支架作为组织工程骨移植物,可以促进大鼠桡骨缺损部位骨的形成和血管化,促进骨缺损愈合。

Study on bone marrow mesenchymal stem cells derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite scaffold to construct vascularized tissue engineered bone

Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells(BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite(CS/HA) scaffold in repairing radial defect in rats.Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells.The endothelial cells,osteoblasts,and mixed osteoblasts and endothelial cells(1∶ 1) were compound with CS/HA scaffold in groups A,B,and C respectively to prepare the cell-scaffold composites.The cell proliferation was detected by MTT.The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted,respectively.At 4,8,and 12 weeks after transplantation,the graft was harvested to perform HE staining and CD34 immunohistochemistry staining.The mRNA expressions of osteopontin(OPN) and osteoprotegerin(OPG) were detected by RT-PCR.Results Alkaline phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red.CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction.MTT test showed that the proliferation level of the cells in 3 groups increased with the time.HE staining showed that no obvious osteoid formation,denser microvessel,and more fibrous tissue were seen at 12 weeks in group A;homogeneous osteoid which distributed with cord or island,and many osteoblast-like cells were seen in groups B and C.The microvessel density was significantly higher in groups A and C than group B at 3 time points(P < 0.05),and in group A than in group C at 12 weeks(P < 0.05).The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points(P < 0.05).In groups B and C,the OPN mRNA expressions reached peak at 8 and 12 weeks,respectively,and OPG mRNA expressions reached peak at 4 weeks.Conclusion BMSCs derived osteoblasts and endothelial cells(1∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.