自体松质骨和骨髓间充质干细胞联合移植治疗家兔股骨头缺血性坏死的实验研究

Mechanism of action of spongy bone autografting combined with BMSCs transplantation on avascular necrosis of femoral head in rabbits

目的:探讨自体松质骨和骨髓间充质干细胞联合移植治疗股骨头缺血性坏死的可能机制,为该方法应用于临床提供依据。方法:兔龄4～5个月的新西兰大耳白兔24只,雌雄不限,随机分为2组,每组12只。采用液氮冷冻股骨头负重区的方法造成股骨头缺血坏死模型后,A组进行自体松质骨移植,B组进行自体松质骨和骨髓间充质干细胞联合移植。每组术后2、4、6、8周分别处死3只动物,制作股骨头标本,进行X线检查和组织学观察,并对术后8周的股骨头标本进行扫描电镜观察。结果:①X线观察结果。A组术后2周时,有2个标本钻孔区密度不均匀,钻孔边缘清晰可见;4周时,有3个标本钻孔区密度增高不显著,钻孔边缘出现硬化线;6周时有4个标本钻孔区显示有骨吸收现象,出现股骨头关节面塌陷;8周时有3个标本骨吸收现象明显,而骨小梁结构不清晰。B组术后2周,股骨头形态均正常,钻孔区有密度增高影,质地均匀,钻孔边缘尚可分辨;4周时股骨头形态均正常,钻孔区密度进一步升高,质地均匀,钻孔边缘变得模糊;6周时股骨头形态仍正常,钻孔区密度高而均匀,钻孔边缘出现放射状骨小梁接合现象;8周时钻孔区密度接近周围骨质,股骨头内出现张力性和压力性骨小梁,钻孔边缘交界区骨小梁接合良好。②组织学观察结果。A组术后2周时,移植骨发生坏死,4个标本出现再血管化,移植区出现破骨和成骨细胞,成骨细胞的数目较少,移植骨周围附加新生骨较薄,有少量连接现象;另2个标本局部新生血管较少,成骨细胞募集不良,新生骨组织的形成较少,无连接现象。术后4周时,3个标本新生骨增加,连接现象增多,出现骨塑形;另3个标本移植骨无明显修复现象。术后6周时,有2个标本新生骨量增加,有骨塑形现象;另4个标本移植骨无明显修复表现,出现骨吸收现象。术后8周时,3个标本可见到塑形的板层骨小梁和成熟的骨髓组织;另3个标本出现较明显的骨吸收现象,局部仍有坏死骨组织存在,无明显的骨组织修复现象。B组术后2周时,植入的松质骨发生坏死,出现局部再血管化,破骨细胞和大量的成骨细胞出现在移植区,植入的松质骨被边缘生成的1层较厚的新生骨连接在一起;4周时骨小梁增粗变大,连接进一步加强,并出现骨髓组织;6周时骨塑形现象活跃;8周时骨塑形现象仍然存在,可以见到塑形的板层骨小梁和成熟的骨髓组织。③扫描电镜观察结果。术后8周时,A组有3个标本在扫描电镜下显示移植骨保持原有骨小梁结构,部分骨小梁出现虫蚀样破坏。B组标本在扫描电镜下显示,骨小梁密集,骨髓腔间隙较小,力学结构欠完善,骨胶原纤维排列不均匀、不整齐。结论:自体松质骨和骨髓间充质干细胞联合移植治疗股骨头缺血性坏死,细胞移植为股骨头的骨质修复提供了充足的骨髓间充质干细胞,骨修复现象活跃,可提高骨移植的成功率,促进股骨头骨组织修复。

Objective：To investigate the mechanism of action of spongy bone autografting combined with bone marrow mesenchymal stem cells（BMSCs） transplantation on the experimental avascular necrosis of femoral head（ANFH） for the purpose of application in clinic. Methods：Twenty-four New Zealand white rabbits ,4 to 5 months old, were divided into 2 groups randomly, 12 cases in each group. After the rabbit model of ANFH were built by freezing the weight loading region of femoral head with liquid nitrogen, rabbits in group A were adminis- trated with spongy bone autografting, while rabbits in group B were administrated with spongy bone autografting combined with BMSCs transplantation. Three rabbits selected from each group were sacrificed 2,4,6,8 weeks after the transplantation for making specimens of femoral head, which were observed with radioscopy, histological examination. Moreover, those specimens obtained 8 weeks after the transplantation were observed with scanning electron microscope. Results： Radiological observations：In group A, two specimens presented uneven density in drilled area and clear images of drilled holes 2 weeks after operation, sclerosis line was showed at the margin of drilled area in 3 specimens and increase in density were not significant in drilled area 4 weeks after operation, bone resorption of drilled area and femoral head collapse appeared in 4 specimens 6 weeks after operation, three specimens presented obvious bone resorption and disordered structure of trabeculae of bone 8 weeks after operation. In group B, the shape of femoral head was normal and the bone density increased uniformly in the drilled area while the margin of drilled area could be distinguished 2 weeks after operation ,the shape of femoral head was normal and the bone density further increased uniformly in the drilled area and the margin of drilled area became obscured 4 weeks after operation, the shape of femoral head was still normal and the bone density was high and uniform in the drilled area and radial conjunction of the trabeculae was presen- ted at the margin of drilled area 6 weeks after operation, the bone density in the drilled area approached that of the surrounding substantia ossea and tensile trabeculae and compression trabeculae formed in the femoral head and good conjunctions of the trabeculae were presented in the junctional zone of the margin of drilled area 8 weeks after operation. Histological observations ： In group A, two weeks after operation, osteonecrosis of bone graft appeared, revaseularization appeared in 4 specimens, osteoclasts and fewer osteoblasts appeared in transplantation zone, the new bone around bone graft was slight and a few conjunctions were presented, there were no conjunction and few neovascularization were presented in the other 2 specimens, osteoblasts recruitment was poor. Four weeks after operation, new bone and conjunction increased and bone modeling appeared in 3 specimens, while no remodeling of bone graft was presented in the other 3 specimens. Six weeks after operation, new bone mass increased and bone remodeling occured in 2 specimens, there were no obvious repair of bone graft phenomenon and bone resorption occurred in the other 4 specimens. Eight weeks after operation, moulded lamellar bone trabeculae and mature myeloid tissue could be observed in 3 specimens. In the other 3 specimens obvious bone resorption phenomenon were observed while no obvious bone repair phenomenon appeared, and bone necrosis could be seen in partial area. In group B, two weeks after operation, necrosis of bone graft and partial revaseularization appeared, osteoblasts and osteoclasts appeared in the transplant area, grafted spongy bone were connected by new bone. Bone trabecula augmented and conjunction enhanced and myeloid tissue appeared 4 weeks after operation. Bone remodeling was active 6 weeks after operation and it still existed 8 weeks after operation, meanwhile,the lamellar bone trabeeula and matured myeloid tissue could be observed. Scanning electron microscope observations ： Eight weeks after operation, trabecular structure was maintained and moth-eaten bony destruction was found in part bone trabecula in 3 specimens of group A. Grafted bone trabeculae in group B were different from normal biomechanics structure, they arranged closely and had a narrow cavitas medullaris, whlie, osteocollagenous fibers arranged unevenly and ir- regularly. Conclusion： In the treatment of ANFH, cellular transplant provides sufficient BMSCs and the result is active repairing of bone tissue of the femoral head. Spongy bone autografting combined with BMSCs transplantation can obviously promote the repairing of bone tissues of the femoral head and raise the achievement rate of bone transplantation.