成骨细胞及血管内皮细胞复合同种异体颗粒骨治疗大鼠股骨头坏死

Osteoblasts and vascular endothelial cells combined with allogeneic morselized bone graft for treating rat osteonecrosis of the femoral head

目的探讨骨髓基质干细胞(bone marrow stromal stem cells,BMSCs)来源的大鼠同种异体成骨细胞(osteo-blast,OB)及血管内皮细胞(vascular endothelial cells,VEC)与同种异体颗粒骨复合后治疗大鼠创伤性股骨头坏死(osteo-necrosis of the femoral head,ONFH)的可能性。方法 36只6月龄SD大鼠,分为模型组、同种骨组及OB+VEC+同种骨组(n=12)。同时取4周龄SD大鼠BMSCs体外分离培养,取第3代细胞复合同种颗粒骨;制备大鼠创伤性股骨头坏死动物模型(圆韧带离断法)并分别在术后1、2、4、6周处死动物并取材。扫描电镜观察两种细胞在支架上的生长情况;免疫细胞化学技术检测诱导后血管内皮细胞CD34、CD31的表达;碱性磷酸酶(ALP)染色及Ⅰ型胶原免疫细胞化学染色鉴定成骨细胞;5-溴脱氧尿嘧啶(5-bromo-2-deoxy Uridine,BrdU)体外标记成骨细胞及内皮细胞,动物体内示踪两种细胞动力学。光镜观察病理形态改变;通过骨形态计量学参数计算新骨生成量,包括骨小梁面积百分率(percent trabecular area,%Tb.Ar)、骨小梁厚度(trabecular bone thickness,Tb.Th)、骨小梁数量(trabecular bone number,Tb.N)、骨小梁分离度(trabecular bone separation degree,Tb.Sp)。结果 BMSCs诱导来源的成骨细胞其ALP染色及Ⅰ型胶原免疫组化染色均呈阳性反应;血管内皮细胞表达了其标志物CD31、CD34。两种细胞与同种骨有良好的组织细胞相容性,细胞在其表面及壁内平铺、伸展、生长状态良好。OB+VEC+同种骨组新骨生成量均多于模型组及同种骨组,各项新骨形成参数均高于模型组及同种骨组(P<0.01)。结论 OB+VEC+同种骨支架材料在治疗实验性大鼠ONFH中其促成骨能力优于单纯同种异体颗粒骨。

Objective To study the possibility of bone marrow stromal stem cells（BMSCs）-derived allogeneic osteoblasts（OBs）/vascular endothelial cells（VECs） combined with allogeneic morselized bone grafts for treating rat traumatic osteonecrosis of the femoral head（ONFH）.Methods Thirty-six six-month-old SD rats were divided into three groups： model group,allogeneic bone group,and OB＋VEC＋allogeneic bone group,each including 12 rats.BMSCs,extracted from 4-week-old SD rats,were cultured in vitro,and the obtained third-generation cells were combined with allogeneic morselized bone.An animal model for rat traumatic ONFH was built through removing round ligaments of femoral heads,and the animals were sacrificed in the first,second,fourth,and sixth weeks after implanting OB＋VEC＋allogeneic morselized bone or allogeneic morselized bone to collect the femoral heads.The growth of OBs and VECs on allogeneic morselized bone was observed with a scanning electronic microscope.The expression of CD34 and CD31 in VECs was detected through an immunocytochemical method.OBs were identified through alkaline phosphatase（ALP） staining and collagen type-Ⅰ immunocytochemical staining.OBs and VECs were labeled with 5-bromo-2-deoxy uridine（BrdU） in vitro,so as to trace the kinetics of OBs and VECs in vivo.Pathological morphology changes were observed with an optical microscope.New bone formation was calculated according to bone histomorphometry parameters,including percent trabecular area（%Tb.Ar）,trabecular bone thickness（Tb.Th）,trabecular bone number（Tb.N）,and trabecular bone separation degree（Tb.Sp）.Results ALP staining and collagen type-Ⅰ immunohistochemical staining of the BMSC-derived OBs gave positive results.The VECs expressed CD31 and CD34.The OBs and VECs exhibited excellent biocompatibility with the allogeneic bone,and were in good spreading,extension,and growth conditions on its surfaces.The OB＋VEC＋allogeneic bone group accomplished more new bone formation than the model group and allogeneic bone group,and each new bone formation parameter of the OB＋VEC＋allogeneic bone group was higher than those in the model group and allogeneic bone group（P0.01）.Conclusion The OB＋VEC＋allogeneic bone composite has higher ability to promote new bone formation in treatment of rat traumatic ONFH than allogeneic morselized bone alone.