同种异体骨髓间充质干细胞在比格犬体内异位构建组织工程骨

Ectopic osteogenesis of tissue-engineered bone using allogenic bone marrow mesenchymal stem cells in beagle dogs

背景:选用同种异体骨髓间充质干细胞作为种子细胞成为将来组织工程领域发展的趋势。目的:分析同种异体骨髓间充质干细胞在比格犬体内异位构建组织工程骨的能力,并观察骨髓间充质干细胞在体内成骨过程中的转归。方法:应用氯甲基苯甲酰氨荧光染料(CM-Dil)标记比格犬骨髓间充质干细胞,MTT法检测标记细胞的体外增殖能力。将自体或异体骨髓间充质干细胞分别接种珊瑚、β磷酸三钙支架体外成骨诱导7d后,植入比格犬背部皮下,空白支架作为阴性对照。术后3 d及1,2,4,8,12周取材,苏木精-伊红染色观察成骨情况,应用ipp软件统计分析成骨面积。PT-PCR和ELISA法检测组织工程骨成骨过程中相关基因的表达。CM-Dil组冰冻切片荧光显微镜下示踪骨髓间充质干细胞在体内的转归。结果与结论:(1)4-8周时异体组织工程骨的成骨面积小于自体组织工程骨,但到12周后2组无明显差异;(2)4周自体组织工程骨表达骨γ羧基谷氨酸蛋白高于异体组织工程骨,提示后者先于前者进入骨矿化期;(3)ELISA检测发现自体组织工程骨的碱性磷酸酶和骨钙素表达量在4周左右高于异体组织工程骨,到12周后趋于一致;(4)通过CM-Dil标记骨髓间充质干细胞发现同种异体骨髓间充质干细胞的数量相比自体骨髓间充质干细胞有明显的减少;(5)结果说明,同种异体组织工程骨在大动物体内能够异位成骨(12周),但早期(8周前)成骨效率低于自体组织工程骨,这种差别可能与骨髓间充质干细胞同种异体移植后发生免疫反应导致细胞数量减少有关。

BACKGROUND： Allogenic bone marrow mesenchymal stem cells as seed cells for tissue engineering have become the future trend of development. OBJECTIVE： To investigate the osteogenic effects of allogenic bone marrow mesenchymal stem cells and the outcome in vivo. METHODS： Bone marrow mesenchymal stem cells from beagle dogs were marked with chloromethylbenzoyl ammonia fluorescent dye（CM-Dil）, and the proliferation of labeled cells was measured using MTT assay in vitro. Autologous or allogenic bone marrow mesenchymal stem cells were inoculated into coral and β-tricalcium phosphate scaffolds for 7 days osteogenic induction and then subcutaneously implanted into the back of beagle dogs. Dogs undergoing blank scaffold implantation served as negative controls. Hematoxylin-eosin staining was used to observe new bone formation at 3 days, 1, 2, 4, 8, 12 weeks after surgery. Bone formation area was statistically analyzed using ipp software. In the CM-Dil group, frozen sections were made to trace the in vivo outcome of bone marrow mesenchymal stem cells under a fluorescence microscope. RESULTS AND CONCLUSION： The osteogenesis speed in the allogenic bone tissue engineering group was faster than that in the autologous bone tissue engineering group at 4-8 weeks after implantation, but no significant difference between the two groups was found beginning at the 12 th week. At 4 weeks after implantation, the expression of γ-carboxy glutamic acid protein in the autologous bone tissue engineering group was higher than that in the allogenic bone tissue engineering group, prompting the bone mineralization appeared earlier in the latter group than the former one. ELISA results showed that the expression of alkaline phosphatase and osteocalcin in the autologous bone tissue engineering group was higher than that in the allogenic bone tissue engineering group at 4 weeks after implantation, and then the expression showed no difference at 12 weeks. CM-Dil labeling results showed that the number of allogenic bone marrow mesenchymal stem cells was reduced significantly compared with that of autologous bone marrow mesenchymal stem cells. All these findings indicate that the ectopic osteogenesis of the allogenic tissue-engineered bone in large animals is found within 12 weeks after implantation, but the osteogenesis efficiency at early stage（within 8 weeks） is lower compared with the autologous tissue-engineered bone. This difference may be related to the post-implantation immunoreactions that lead to the reduction in cell number.