The Impact of Confluence on Bone Marrow Mesenchymal Stem (BMMSC) Proliferation and Osteogenic Differentiation

Background: In the field of cellular therapy, the impact of confluence degreeonharvesting or differentiation of BMMSCs and the effect of cell-to-cell contact remain controversial. Therefore, the effect of confluence on properties of BMMSCs was studied and efficiency of confluence-associated osteogenic differentiation was identified. Materials and Methods: The impact of 20, 50, 70, 80 and 100% confluences on proliferation properties of BMMSCs, expression of ERK and p-ERK proteins and glucose consumption rate was studied. Efficiency of confluence-associated osteogenic differentiation was identified by determining calcium deposition, Alizarin Red staining, ALP activity and expression of osteopontin and osteocalcin genes. Results: There was a correlation between confluence % and BMMSCs density. Viability was declined at the lower and higher confluences. The highest CFU-F, Brd-U uptake and population doubling were obtained at 80% confluence. ERK band intensity in 100% confluent BMMSCs was lower compared to other confluences. Bands of p-ERK were highly detectable in 70% and 80% confluences. Glucose consumption rate of 70% and 80% confluences in the last days were higher than 20% and 100% confluences. Although higher osteogenic differentiation was estimated at 80% confluence using calcium deposition, Alizarin Red staining and ALP activity, it was also extended at 100% confluence Osteopontin gene was expressed among all confluences including 100% confluence, while osteocalcin gene was expressed highly in 70% confluent cells. Conclusion: We concluded that the optimum seeding density for maximal expansion and harvesting purposes is 80% confluence and for osteogenic differentiation up to 100% confluence is also acceptable.