Development of a hybrid bone substitute with cryopreserved auto stem cell (human bone marrow derived mesenchymal stem cell)

[实验]1.人骨髓间充质干细胞培养方法的建立和支架的制备1)人血清培养人骨髓间充质干细胞和牛血清培养人骨髓间充质干细胞的细胞化学(碱性磷酸酶活性和钙含量)比较2)支架的建立：3种不同多孔羟基磷灰石支架体内成骨能力的比较2.人骨髓间充质干细胞增殖和诱导成骨分化方法的建立1)体外：细胞化学(成骨细胞碱性磷酸酶活性和成骨细胞钙水平)2)体内：骨组织活性3)冻存增殖的hMSCs 3)杂交型骨替代物的制备冷冻的hMSCs在成骨介质(含抗坏血酸、地塞米松和甘油磷酸的α-MEM)中培养，诱导分化为成骨细胞。[结果]1)用胎牛血清培养的人骨髓间充质干细胞与人血清培养的人骨髓间充质干细胞的细胞生化活性无差异。2)3种不同支架体内的成骨能力差异无统计学意义。3)含抗坏血酸、地塞米松和甘油磷酸盐的α-MEM培养液是一种更好的成骨介质。4)冷冻的人骨髓间充质干细胞制备的杂交型骨替代物在体内观察到了活跃的成骨。[讨论]在自体血清中培养的冷冻的人骨髓间充质干细胞具有成骨潜能。如果安全性得到证实，hMSCs可能为临床应用提供可能性。

We established methods of isolating, proliferating and freezing human bone marrow-derived mesenchymal stem cells (hMSCs) and prepared hybrid-type bone substitute from the frozen hMSCs.[Experiments]1.Establishment of hMSC culture in human serum and preparation of scaffold1)Comparison of cell chemistry (alkaline phosphatase activity and Ca levels) between hMSCs cultured with human serum and those cultured with bovine serum2)Establishment of scaffold : Comparison of osteogenic potential in vivo among 3 different porous hydroxyapatite scaffolds2.Establishment of method for proliferating and inducing osteogenic differentiation from hMSCs : hMSCs were expanded in the presence of bFGF, and then cultured in the osteogenic medium(with and without glycerophosphate) to induce differentiation.1)In vitro : Cell chemistry(alkaline phosphatase activity and Ca levels in osteoblast cells)2)In vivo : Viability of bone tissues3)Freezing of proliferated hMSCs3.Preparation of hybrid-type bone substituteThe frozen hMSCs were cultured in the osteogenic medium(αMEM medium containing ascorbic acid, dexamethasone and glycerophosphate) to induce differentiation into osteogenic cell. Then, the hMCSs were seeded onto a porous hydroxyapatite to form hybrid-type bone substitute, which was implanted to nude mice to examine bone tissue viability.[Results]1)There was no difference in cellular biochemical activity between hMSCs cultured with FBS and those with human serum.2)There was difference in osteogenetic potential in vivo among the 3 different scaffolds used.3)αMEM medium containing ascorbic acid, dexamethasone and glycerophosphate was superior as osteoegenic medium.4)Active osteogenesis was observed in vivo with the hybrid-type bone substitute prepared from the frozen hMCSs.[Discussion]The frozen hMSCs cultured in autologous serum showed osteogenic potential. The hMSCs may provide possibilities for use in clinical setting if the safety is confirmed.