Effects Of Ginkgo Bifoba Extract On The Proliferation And Osteogenic Differentiation Of BMMSCs

Background:Osteoporosis is a common bone disease worldwide and associated withbone loss and bone microstructure failure, which may lead to bonefragility and an increase in bone fracture. More than200million peopleare suffering from osteoporosis all over the world, and there are morethan88million people in our country, most of them are elderly，especiallypostmenopausal women. It has become the seventh common andfrequently occurring disease in the world. There is evidence thatosteoporosis affects the craniofacial and oral structures, so as part ofskeletal system, the jaw’s relations with systemic osteoporosis wasbecoming more and more attention. Bone density decreasing andbiomechanical nature reducing accelerated the rapid resorption of residualridge, which decreased the height and width of the alveolar ridge boneand it makes a big difficulty for restoration.So how to take effective measures to slow down the absorption ofalveolar ridge and save the tooth site are the key point to ensure thedenture restoration smoothly and aesthetically. The treatment ofosteoporosis has a long history，Scholars from different viewpoints studythe efficacy of some medications for the treatment of osteoporosis, but the side effects during treatment limited its clinical application，such asbisphosphonates，which can easily lead to osteonecrosis of the jaw (ONJ),and hormone replacement therapy (HRT),which may easily lead to theincreased risk of thrombus, the incidence of endometrial hyperplasia,breast cancer and other diseases.Therefore, traditional Chinese medicine treatment for osteoporosishas become the research focus. The occurrence of osteoporosis has closerelations with the differentiation of mesenchymal stem cells. Sinceosteoblasts and adipocytes are derived from a common multipotentialMSC progenitor, it has been proposed that the balance between osteogenesis and adipogenesis differentiation of mesenchymal stem cells(MSCs) is disrupted in osteoporosis. In recent years, stem cell therapyalso attract people’s attention. BMMSCs can differentiate into osteoblast，adipocyte，chondrocyte and so on. BMMSCs are an ideal seed cell withgood biological characteristics, they may differentiate into osteoblastsunder some specific conditions and promote the bone detect repair.Ginkgo biloba is a kind of phytoestrogen and has the ability ofdual-directional regulations in vivo，that means when the level of estrogenin the body is low, it act as a quasi estrogen; and when the levels ofestrogen in the body is higher, it has the anti-estrogen activity. So it canbe used as selective estrogen receptor modulators (SERMs), that means itcan selectively combine with the estrogen receptor on different parts of the body and organization, then show different physiological effects.When combined with the estrogen receptors on bone, osteoblast andosteoclasts，it shows the estrogen action of inhibiting osteoclast mediatedbone resorption; And when combined with estrogen receptors in breastand endometrial part, it shows anti-estrogen activities of inhibitingproliferation of breast cells and endometrial epithelial cells. However，there is few reports on how the phytoestrogen in GBE works inosteoporosis. In our experiment, different concentrations of GBE wasadded into BMMSCs to investigate whether GBE can regulate theosteogenic differentiation of BMMSCs in vitro and find out the mostappropriate concentration.Objective:In order to provide a new method for the prevention and treatmentfor osteoporosis, the objective of this study is to study the effect of GBEon proliferation and osteogenic differentiation of BMMSCs by applyingdifferent concentration GBE on the third generation of BMMSCs.Methods:1. The separation and culture of BMMSCsChoosing the third generation BMMSCs of Wistar rat, we culturedthe BMMSCs by whole marrow method and sub-cultured to pure the cell.Through the identification test to make sure the cell we separated is themultiple differentiation potential BMMSCs 2.The effect of different concentration of GBE on the proliferation ofBMMSCsApplying different concentrations of GBE on the third-generation ofBMMSCs，and evaluating its influence on the cell proliferation activitythrough CCK-8assay on the3rd，5th，7th day. The flow-cytometric analysis was also used to test the cell cycle at24h.3.The effect of GBE on the osteogenic differentiation of BMMSCsApplying the different concentrations of GBE on the third-generation ofBMMSCs, and estimate the effects of osteogenic ability of GBE byAlkalinet phosphatase method at day7and day14, Alizarin Red S at2week. Real-Time PCR was taken to test the expression of theosteogenesis related gene (BMP-2, Runx2, Col1a1) mRNAat1week.Results：1.There was no statistically significant difference between differentconcentrations of ginkgo biloba extract in the proliferation on bonemarrow mesenchymal stem cells. However, GBE could make the cells inS phase of the cell cycles.2.Alizarin red staining showed significant calcium nodules when the cellswere cultured for14days except for the control group and when GBE at150μg/mL，the calcium nodule is the most. GBE could significantlypromote the expression of alkaline phosphatase, especially when theconcentration was150μg/mL. 3.The osteogenic related genes（BMP-2，Runx2、Col1a1）mRNAexpression were also relatively higher than that of the control group andhas significant difference.Conclusion:Ginkgo biloba extract has no significant effect on the proliferation ofBMMSCs. However, it can promote the osteogenic differentiation of theBMMSCs.