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

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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.