AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyz...AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P 【 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P 】 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P 【 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P 【 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P 【 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P 【 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.展开更多
基金Supported by Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre and Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)
文摘AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P 【 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P 】 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P 【 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P 【 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P 【 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P 【 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.
