SD大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损的实验研究

A study on repairing of tibial defect of the SD rat with bone marrow mesenchymal stem cells combined with bone matrix gelatin

目的:探讨大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损的可行性。方法:采用第5代(P5)SD大鼠骨髓间充质干细胞(MSCs),经Hoeschst 33344(sigma)荧光杂料标记后,调配制成的1×106个／ml细胞浓度后与骨基质明胶(BMG)共同培养6 h,然后植入SD大鼠双侧胫骨的实验性骨缺损中(A组),同时作胫骨骨缺损单纯BMG植入(B组),无植入物(C组)两组对照。术后8周处死,取骨缺损区组织进行组织学观察,其中A组行荧光染料标记测定以确认骨缺损区的骨痂是否来源于MSCS。结果:①A组:胫骨骨缺损区可见大量新生不规则骨纤维组织、软骨及纤维骨痂填充,可见骨细胞、骨组织和骨小梁,已形成骨髓腔。②B组:胫骨骨缺损区可见大量纤维组织、少量新生不规则骨纤维组织及骨骼肌组织,伴有多核巨细胞和少量炎性细胞,缺损区边缘带有骨痂组织。③C组:胫骨骨缺损区可见大量纤维组织及骨骼肌组织填充生长,伴有多核巨细胞和少量炎性细胞,缺损区边缘带有少量骨痂组织。荧光染色鉴定确认胫骨骨缺损区的骨痂来源于MSCs。结论:大鼠骨基质明胶吸附骨髓间充质干细胞修复骨缺损具有安全性、可行性及有效性。

Objectives: To examme thee teasibility ot repairing the tibial detect of the SD rat with bone marrow mesenchymal stem cells combined with bone matrix gelatin. Methods: Bone marrow mesenchymal stem cells (MSCs)of the SD rat were cultured and transferred in vitro to the fifth passage. After fluorescence labeling with Hoechst 33344(sigma), MSCs were harvested, and adjusted cell density to 1×106/ml and incubated with bone matrix gelatin(BMG)for 6 hours. Then the BMG involving or uninvolving MSCs was implanted into the experimental sugical defect in the tibia of SD rats (Group A). Control groups included Group B and Group C (without implantation). Rats were sacrificed and histological analysis of the defects was performed 8 weeks after implantation. The origin of the new bone formed in the defects in Group A was also studied. Results: Group A: the defect was filled with large amount of osteofibrous tissue, chondrocytic tissue and fibrous new bone. Osteocytes could be observed and bone marrow cavity was formed. Fluorescent positive cells confirmed the derivation of new bone from the MSCs. Group B: The defect was filled with large amount of fibrous tissue, small amount of osteofibrous tissue and muscle tissue. Infiltration of multiple giant cells and small number of inflammatory cells and capillary hyperplasia could be observed. Group C: The defect was largely filled with fibrous and muscle tissue associating with infiltration of multiple giant cells and inflammatory cells. Capillary hyperplasia could be also observed. Only small amount of new bone formed in the marginal zone of the defect. No abnormal tumor cells were found in the defect. Conclusions: Repairing the tibial defects of the SD rats with bone marrow mesenchymal stem cells combined with bone matrix gelatin is safe, feasible and effective.