Role Of TGF-β1in Strontium Ranelate-induced Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells

ObjectiveStrontium ranelate is a new anti-osteoporosis drugs, Many studies have confirmed that it can promote osteogenic differentiation of rBMSCs, In addition,It also can improve bone strength, promote blood vessel formation,and a series of other important functions. Studies have shown that the transcriptional activity of bone cell-specific transcription factor Runx2can be regulated through the extracellular signal-regulated kinase of ERK1/2and P38mitogen-activated protein kinase signaling pathway by Sr, then Runx2regulates the expression of downstream of osteogenic genes.such as,osteocalcin,osteopontin, type2collagen,then promoting the osteogenic differentiation of BMSCs.TGF-β1belongs to one of the superfamily members of transforming growth factor beta (TGF-β),the extremely wide range of biological activity,Not only can regulate the growth and differentiation of rBMSCs, But also plays an important role in osteoblast differentiation of rBMSCs,growth and proliferation of osteoblasts, including bone matrix synthesis and bone remodeling,TGF-β1is one of the preferred growth factor which regulates growth and osteoblast differentiation of rBMSCs; in addition, a variety functions of cell growth and biological. The relevant data indicate that TGF-β1may play an important role in the osteoblast differentiation process on Sr promoting rBMSCs.However,It has not been reported that the role of TGF-β1in Sr which promotes rBMSCs differentiate into osteoblast so far.This article aims to focus on:①The effects of TGF-β1expression in rBMSCs by Sr.②Whether TGF-β1play a important role in the differentiation of osteoblasts by Sr.In order to provide novel mechanism of action and experimental data on osteogenesis for Sr.Methods1.Establish isolated.cultured system on rBMSCs in vitro.Take four weeks age, male or female SD rats, removing the femurs and tibia, rBMSCs was isolated from rat bone marrow by the whole bone marrow adherent, rBMSCs was isolated from rat bone marrow by10ml syringe containing culture medium,then washed out from the bone marrow until the marrow cavity white,The cell suspension was placed in25cm2flasks,and rBMSCs was cultured at37℃,5%CO2.humidified incubator, passaged and cryopreserved After cells covered the bottom about80%.the3-5th generation cells used in the experiment. Inverted microscope to observe cell growth state,identify cultured cells that isolated from bone marrow.2.rBMSCs was induced to differentiate into osteoblasts directional.Take the third-generation rBMSCs that grew well, adjust the concentration of cells about105/ml, take4ml of the cell suspension,It was cultured by osteogenic induction medium.the experimental will start when the cells reached60%-70%confluence.3、experimental groups(1)the experimental groups of indicators of alkaline phosphatase (ALP) determination.①divided into six groups,control, Sr=0.1, Sr=1, Sr=3, Sr=5, Sr=7mM,each set five wells,rBMSCs was cultrued under the conditions of osteogenic induction medium for7days.when Sr was3mM,the ALP activity reached the maximum.②Divided into five groups, the control:the osteogenic induction medium culture;Sr group:3mM Sr+osteogenic induction medium; Sr+TGF-β1inhibitor (SB431542)+osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours,then add3mM Sr;SB431542group:SB431542+the osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours; DMSO+osteogenic induction medium:10μmol/L DMSO culture;rBMSC was cultured for7days.ALP activity was detected when rBMSCs was cultured for7days.(2)The experiments group of Alizarin red calcium nodules staining①divided into two groups, control group:osteogenic induction medium culture;Sr group:3mM Sr+osteogenic induction medium, cultured for21days.②Divided into five groups, the control:the osteogenic induction medium culture;Sr group:3mM Sr+osteogenic induction medium;Sr+SB431542+osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours,then add3mM Sr;SB431542group:SB431542+osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours; DMSO+osteogenic induction medium:10μmol/L DMSO culture; Alizarin red calcium nodules staining was detected when rBMSCs was cultured for21days.3mM Sr was used.(3) The experimental groups of TGF-β1protein expression.①divided into six groups,control, rBMSCs was cultured by different concentration.Control,Sr=0.1, Sr=1, Sr=3, Sr=5, Sr=7mM, rBMSCs was cultured under the conditions of osteogenic induction medium for7days.The expression of TGF-31protein reached the maxmun when Sr was1mM.②divided into six groups, rBMSCs was cultured by different time.control, t=1, t=3, t=5t=7t=10days group. when it was7days,The Sr concentration used in each group was1mM.The expression of TGF-β1protein reached the maxmum.③Divided into five groups, the control:the osteogenic induction medium culture;Sr group:1mM Sr+osteogenic induction medium;Sr+SB431542+osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours,then add1mM Sr;SB431542group:SB431542+the osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours; DMSO+osteogenic induction medium:10μmol/L DMSO culture;rBMSCs was cultured for7days. The expression of TGF-β1was detected by western-blot.(4) the experimental group of Runx2mRNA expression.①divided into six groups, rBMSCs was cultured by different concentration.control, Sr=1, Sr=2, Sr=5, Sr=7, Sr=10mM, rBMSCs was cultured under the conditions of osteogenic induction medium for7days.The expression of Runx2mRNA reached the maxmum when Sr=1mM.②divided into eight groups, rBMSCs was cultured by different time.control, t=0.5h, t=1h, t=2h,t=4h,t=3d,t=7d,t=14d group.The Sr concentration used in each group was1mM.The expression of Runx2mRNA reached a maxmum when it was on the7th day.③Divided into five groups,Control:osteogenic induction medium culture;Sr group:1mM Sr+osteogenic induction medium;Sr+SB431542+osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours,then add1mM Sr;SB431542group:SB431542+the osteogenic induction medium:pretreatment of rBMSCs with10μmol/L SB431542for2hours; DMSO+osteogenic induction medium:10μmol/L DMSO culture; rBMSCs was cultured for7days. The expression of Runx2mRNA was detected by western-blot.Results1.rBMSCs morphology observation. inverted phase contrast microscope observation showed that:vaccination early we can see much of round suspended cells,With prolonged incubation time,Cells were spindle-shaped or polygonal,the majority adhered within24hours, These were primary cells, when they were cultured for a week or so, the adherent cells were up to70%-80%; With prolonged incubation time and the medium was changed for much times, the cell morphology tends to single,was fibroblast-like spindle cells, and was swirling, arranged rules radially. After the integration to about80%, passaged by the ratio of1:2, re-integration to70%-80%after cells was cultured for5-7days.2. Sr increased Activity of ALP in rBMSCsrBMSCs were dealed with0、0.1、1、3、5、7mM Sr respectively for7days,The activity of ALP was detected.One-way ANOVA showed that overall were statistically differences (F=44.092, P<0.001). Each group compared with the control group could increase the ALP activity of rBMSCs (P<0.05).When Sr was up to3mM, ALP activity reached the mixmum,It has a statistically differences compared with the others(P<0.05); pretreatment of rBMSCs with10μmol/L SB431542for2hours before adding3mM Sr,rBMSC was cultured for7days.then OD of ALP was detected.One-way ANOVA showed that overall were statistically differences (F=5.053, P<0.001).ALP activity was increased when rBMSCs was cultured with3mM Sr for7days.It has a statistically differences compared with the others(P<0.01),pretreatment of rBMSCs with10μmol/L SB431542for2hours before adding3mM Sr, and this way can significantly inhibit Sr’s role in promoting the ALP activity, It has a statistically differences compared with Sr group(P<0.05), There were no statistically differences among the control, Sr+SB451542group and DMSO group(P>0.05).3. Sr increased mineralization in rBMSCsrBMSCs was dealed with0and3mM Sr for21d,Independent-Samples T Test suggest that the number of calcium nodules were significantly increased when rBMSCs was cultrued by3mM Sr for21days,compared with control (t=-9.865, P<0.001); pretreatment of rBMSCs with10μmol/L SB431542for2hours before adding3mM Sr, rBMSCs was cultured for21days.One-way ANOVA showed that overall were statistically differences (F=19.161, P<0.001).The number of calcium nodules was significantly increased,It has statistically differences compared with others(P<0.05), pretreatment of rBMSCs with10μmol/L SB431542for2hours before adding3mM Sr, and this way can significantly inhibit Sr’s role in promoting the number of calcium nodules It has a statistically differences compared with Sr group(P<0.05), There were no statistically differences among the control, Sr+SB451542group and DMSO group(P>0.05).4. Effects of Sr on expression of TGF-β1in rBMSCs.rBMSCs was dealed with0、0.1、1、3、5、7mM Sr repectively for7days,One-way ANOVA showed that overall were statistically differences (F=583.858, P<0.001).The expression of TGF-β1was significantly increased with0.1-5mM Sr,It has statistically differences compared with control (P<0.05). when Sr was up to1mM, the expression of TGF-β1reached the mixmum,It has a statistically significant compared with the others(P<0.05),when Sr was up to7mM,There was no significantly effect on TGF-β1expression compared with the control group(P>0.05);rBMSC was cultured with1mM Sr from1to10days. Welch showed that overall were statistically differences(P<0.001),Sr time-dependently increased the expression of TGF-(31, the expression of TGF-β1reached the maximum on the7th day,It has a statistically significant compared with others(P<0.01), There was no significantly effect on TGF-(31expression compared with the control group on the10th day (P>0.05); pretreatment of rBMSCs with10μmol/L SB431542for2hours before adding1mM Sr,rBMSC was cultured for7days.One-way ANOVA showed that overall were statistically differences (F=314.375, P<0.001),the expression of TGF-β1was significantly increased when Sr was up to1mM,It has statistically differences compared with others(P<0.01), pretreatment of10μmol/L SB431542for2hours before adding lmM Sr, and this way can significantly inhibit Sr’s role in promoting the the expression of TGF-β1.It has a statistically differences compared with Sr group(P<0.01), There were no statistically differences among the control, Sr+SB451542group and DMSO group(P>0.05).5. Effects of Sr on expression of Runx2mRNA in rBMSCs.rBMSCs were dealed with0、1、2、5、7、10mM Sr respectively for7days. One-way ANOVA showed that overall were statistically differences (F=985.949, P<0.001). Runx2mRNA expression were significantly increased when1-5mM Sr were used.when Sr was up to1mM, the expression of Runx2reached the mixmum,It has a statistically differences compared with control(P<0.01), the expression of Runx2significantly decreased when Sr was7mM and10mM, It has a statistically differences compared with control(P<0.01); rBMSCs was cultured with1mM Sr at range from0.5hour to14days. One-way ANOVA showed that overall were statistically differences (F=353.551, P<0.001),Sr time-dependently increased the expression of Runx2mRNA, the expression of Runx2reached the maximum On the7th day,There was significant statistically differences compared with others(P<0.01);pretreatment of rBMSC with10μmol/L SB431542for2hours before adding1mM Sr,rBMSC was cultured for7days.One-way ANOVA showed that overall were statistically differences(F=5510.983, P<0.001), the expression of Runx2was significantly increased when Sr was up tol mM,It has statistically differences compared with control(P<0.01), pretreatment of10μmol/L SB431542for2hours before adding1mM Sr, and this way can significantly inhibit Sr’s role in promoting the the expression of Runx2.It has a statistically differences compared with Sr group(P<0.01), There were no statistically differences among the control, Sr+SB451542group and DMSO group(P>0.05).Conclusion1.rBMSCs can be successfully separated,purified and cultrued by the method of bone marrow adherent culture.It is more secure and convenient.Under the conditions of osteogenic Medium culture, rBMSCs have the potential of differentiating into osteoblasts.2.Sr can promote rBMSCs differentiating into osteoblasts,this effect may be related to upregulate the expression of TGF-β1-Runx2.