Tehran University of Medical Sciences International Journal of Hematology-Oncology and Stem Cell Research 2008-2207 11 2 2017 04 03 121 132 EN Faten A.M. Abo-Aziza Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Center, Giza, Egypt Zaki A.A Department of Physiology, College of Veterinary Medicine, Cairo University, Giza, Egypt 2016 04 09 2016 05 15 Background: In the field of cellular therapy, the impact of confluence degree on harvesting 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. https://ijhoscr.tums.ac.ir/index.php/ijhoscr/article/view/592 https://ijhoscr.tums.ac.ir/index.php/ijhoscr/article/download/592/549