Abo-Aziza Faten A M, A A Zaki
Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Center, Giza, Egypt.
Department of Physiology, College of Veterinary Medicine, Cairo University, Giza, Egypt.
Int J Hematol Oncol Stem Cell Res. 2017 Apr 1;11(2):121-132.
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. 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. 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. 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.
在细胞治疗领域，汇合度对骨髓间充质干细胞（BMMSCs）收获或分化的影响以及细胞间接触的作用仍存在争议。因此，本研究探讨了汇合度对BMMSCs特性的影响，并确定了汇合相关成骨分化的效率。研究了20%、50%、70%、80%和100%汇合度对BMMSCs增殖特性、ERK和p-ERK蛋白表达以及葡萄糖消耗率的影响。通过测定钙沉积、茜素红染色、碱性磷酸酶（ALP）活性以及骨桥蛋白和骨钙素基因的表达来确定汇合相关成骨分化的效率。汇合百分比与BMMSCs密度之间存在相关性。在较低和较高汇合度时活力下降。在80%汇合度时获得最高的集落形成单位 - 成纤维细胞（CFU-F）、Brd-U摄取和群体倍增。与其他汇合度相比，100%汇合的BMMSCs中ERK条带强度较低。p-ERK条带在70%和80%汇合度时高度可检测。最后几天70%和80%汇合度的葡萄糖消耗率高于20%和100%汇合度。尽管使用钙沉积、茜素红染色和ALP活性评估在80%汇合度时成骨分化较高，但在100%汇合度时也有增强。骨桥蛋白基因在包括100%汇合度在内的所有汇合度中均有表达，而骨钙素基因在70%汇合的细胞中高表达。我们得出结论，为实现最大扩增和收获目的，最佳接种密度为80%汇合度，而成骨分化时高达100%汇合度也是可以接受的。
