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

我们建立了人骨髓间充质干细胞（hMSCs）的分离、增殖和冷冻方法，并将冷冻后的hMSCs制备杂交型骨代用品。人血清hMSC培养的建立及支架的制备1)人血清培养的hMSC与牛血清培养的hMSC细胞化学（碱性磷酸酶活性和钙水平）的比较2)支架的建立：3种不同多孔羟基磷灰石支架在体内成骨潜能的比较2。hMSCs增殖诱导成骨分化方法的建立：在bFGF存在下扩增hMSCs，然后在成骨培养基（含和不含甘油磷酸盐）中培养诱导分化。1)体外：细胞化学（成骨细胞碱性磷酸酶活性和钙水平）2)体内：骨组织活力3)增殖hmscs的冷冻将冷冻后的hMSCs在含抗坏血酸、地塞米松和甘油磷酸盐的αMEM成骨培养基中培养，诱导分化为成骨细胞。然后，将hMCSs植入多孔羟基磷灰石，形成混合型骨替代物，植入裸鼠体内，检测骨组织活力。[结果]1)胎牛血清培养的hMSCs与人血清培养的hMSCs细胞生化活性无差异。2) 3种不同支架的体内成骨潜能存在差异。3)含抗坏血酸、地塞米松和甘油磷酸盐的αMEM培养基成骨效果较好。4)冷冻hmcs制备的混合型骨替代物在体内观察到活性成骨。【讨论】自体血清中冷冻培养的hMSCs具有成骨潜能。如果安全性得到证实，hMSCs可能会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.