The Different Effect Of BFGF And BMP-2 Pretreatment On BMMSCs’ Stemness And Osteogenic Differentiation Capability

Backgroud and ObjectivePeriodontal regeneration has been an advanced research hotspot in the field of periodontology. Bone morrow mesenchymal stem cells (BMMSCs) are pluripotent stem cells sourced from bone morrow, which are widely applied in tissue engineering. The appliance of basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) in the area of periodontal bone regeneration has been widely investigated. However, the stemness of BMMSCs will decrease during the process of proliferation and passage. Therefore, it is a research hotspot in the area of periodontal tissue engineering to gain stem cells with well proferative activity and osteogenic differentiation. This essay will investigate the different effects of bFGF and BMP-2 pretreatment on BMMSCs’ stemness and osteogenic differentiation capability and discuss the mechanism.Methods1、BMMSCs of Wistar rats were isolated and cultured. The expressions of CD34, CD44, CD45, CD90 were tested by Flow cytometry (FCM). BMMSCs were cultured till the 3rd generation and then divided into three groups and cultured for 3 days:the control group (cultured in maintenance medium); bFGF-pretreated group (cultured in maintenance medium with 20ng/ml bFGF); BMP-2-pretreated group (cultured in maintenance medium with 100ng/ml BMP-2). After the pretreatment phase, each group of cells was further cultured in maintenance medium without BMP-2 and bFGF.2、Cell proliferation of BMMSCs in three groups was determined by CCK-8 method after 1,2,3,5 days’ cultivation. FCM was used to determine the cell cycles of BMMSCs in three groups.3、The ALP activity of BMMSCs in three groups was examined by ALP kit after 3,7,14 days’ osteogenic induction cultivation. Mineralization nodules of three groups were stained by alizarin red S and quantitatively determined after 28 days’ osteogenic induction cultivation.4、The expressions of CD90 in pretreated BMMSCs in three groups were characterized by FCM.Results1、The result of FCM showed that:BMMSC lacked expression of CD34, CD45, but expressed CD44, CD90.2、The result of CCK-8 cell proliferation examination showed that:bFGF and BMP-2 pretreatment significantly increased cell proliferative activity in contrast of that of control (p<0.05), The proliferative activity of bFGF pretreated group is significantly higher than that of BMP-2 pretreated group (p<0.05). The result of cell cycle test showed that:Cell percentages in proliferative phase (S phase) increased obviously and that in resting phase(G0/G1) decreased significantly after pretreated by bFGF (p<0.05).No significant difference was found in the percentages of G0/G1,S,G2/M phase cells between BMP-2 pretreated and control groups.3、The result of ALP activity examination showed that:BMP-2 pretreatment enhanced the ALP activity at the beginning stage (3rd day) in contrast of that of control and bFGF pretreated groups (p<0.05). However, at day 7, the ALP activity of all three groups reached the top and the amount in bFGF and BMP-2 pretreated groups were quite similar (p>0.05), but higher than that in control group (p<0.05). In the later stage (14th day), bFGF pretreated group showed dramatically increased ALP activity in contrast of that of control and BMP-2 pretreated groups (p<0.05).Mineralized nodule formation was determined at Day 28 of culture. bFGF and BMP-2 pretreated groups showed similar alizarin red S staining and OD value with control group (p>0.05).4、CD90 positive cells markedly increased in bFGF pretreated group as compared to control group(p<0.05), while BMP-2 pretreatment led to slightly descending of CD90 expression.5、ConclusionsbFGF and BMP-2 pretreatment can enhance the proliferative activity of BMMSCs. The enhancing effect of bFGF may be performed by regulating cell cycle. By contrast of BMP-2, bFGF pretreatment has an advantage of maintaining the sternness of stem cells, as well as maintaining the capability of osteogenic differentiation.