| BACKGROUNDAs a pathological phenomenon, osteoporosis is a degenerative disease. With the advent of human life extension and the coming of aging society, osteoporosis has become the serious problem which affected the quality of human life and the public health. According to a survey in2006, there were about69.44million people with osteoporosis among the population over the age of50, and about210million people who suffering lower bone mass. The severe consequence of osteoporosis is fracture. The treatment and nursing of fracture cost large amount of manpower, material and financial resources, which consequentially bring a heavy economic and psychological burden to family and society. Nevertheless, osteoporosis can be prevented and treated. The main objective of the medicine prevention and treatment is:relief body pain, improve bone mass and bone quality, and reduce the incidence of fractures. Most of the current treatment of osteoporosis medicine were base on the mechanism of inhibiting bone absorption or promoting bone formation, reducing excessive bone metabolism, maintaining normal bone mass and bone quality. Moreover, with the extensive researches of osteoporosis aetiology and molecular biology studies on mechanisms of bone metabolism, the studies about drug discovery for osteoporosis prevention and treatment have made successful progress. Strontium is one of the essential trace elements, which participates in many human physiological functions and biochemical effects. Strontium ranelate (Sr) is a new osteoporosis medicine, made by the French company Servier. It was first put on market at Ireland in November2004. In2009, it came into China market. Meanwhile, it was approved by the State Food and Drug Administration (SFDA) which can be used in treatment and prevention to osteoporosis in the group of postmenopausal women, to reduce the risk of centrum and hip fractures. It is a new generation of osteoporosis drug, representing the new develop direction of the treatment in osteoporosis. It is also the first dual role drug, which can promote bone formation and restrain bone absorption. Bone mesenchymal stem cells (BMSCs) can keep their stem cell phenotype causing multiplex differentiation. Induction of BMSCs to osteogenic differentiation can provide theoretical basis for bone tissue engineering research, fracture repair and osteoporosis problems,etc. Along with the further researches of the relationship between strontium and bone metabolism, it has been reported that Sr can promote mice BMSCs and human BMSCs differentiate to osteoblasts, as well as increasing cell alkaline phosphatase (ALP) activity and the expression of some markers of osteoblasts, such as Collagen Type I, COL-1, osteocalcin, etc. In the mean time, Sr can promote the cells’calcification. However, the specific mechanisms are still not entirely clear excepting some report on the molecular mechanism of mitogen activated protein kinase (MAPK) and calcium sensing receptor (CaSR), etc.Bone morphogenetic proteins(BMPs) is a group of glycoproteins with highly conserved structure, which have a variety of biological function. Unlike BMP-1belongs to the astacin family of metallo endopeptidases, all the rest of BMPs belong to transforming growth factor-β superfamily(TGF-β). To date, during the process of human embryonic occurrence, bone formation, haematopoiesis generation and neural development, more than20types of BMP family have been identified with all kinds of different effects. Bone morphogenetic protein-2(BMP-2) is the most extensively studied member in BMP family. The main function of BMP-2was revealled through the model analysis of the fracture healing, bone defect and spinal fusion. In the vitro model of human and animal, BMPs have been confirmed that they can stimulate stem cells differentiation to osteoblasts, and BMP-2was the most popular target. However, it has not been reported whether BMP-2was involved in the osteoblast differentiation from BMSCs when Sr was a stimulator. Therefore, this study investigated the promoting effect of osteoblast differentiation from BMSCs with the stimulation of Sr, also the most optimal concentration and time on the relevant osteogenesis markers were examined,too. Furthermore, during the osteoblast differentiation from rat BMSCs with Sr stimulation, the change of BMP-2expression and the results brought by the change were also investigated, which could provide new insight for exploring osteogenesis mechanisms of Sr.METHODS1. Acquisition of the rat BMSCsTake a4-week-old male SD rat, first killed it by cervical dislocation, then soaed it in75%alcohol for5minutes. Separated the bilateral femur and tibia under the sterile conditions, removed the surface of the muscle,fascia and other organizations,cut the proximal femur and the distal tibia,exposing the bone marrow cavity. Next the marrow cavity was flushed with DMEM sugar complete medium containing with10%fetal bovine serum,1OOU/ml penicillin and100μg/ml streptomycin. Then collected the cells, centrifuged cells at1000rpm for5minutes,discarded the supernatant, added2ml of the DMEM medium to resuspend cells and seeded cells into flasks uniformly, marked as Po,cultured in the incubator at37℃,5%CO2and80%relative humidity. After24hours,refreshed the medium to remove non-aherent cells and the impurities,5ml of fresh medium was added. Since then the medium was changed every2-3days. Morphological observation of the cells was conducted by inverted phase contrast microscope. Cells were passaged when they were closed to80%-90%fusion,since then passaged once every3-4day.2. Osteogenic differentiation of BMSCsThe third to fifth generation BMSCs were selected as the object of study. Cells were seeded in a petri dish or culture plate according to different experimental needs,and were cultured with osteogenic solution(DMEM sugar medium with10%fetal bovine serum,100U/ml penicillin,100p,g/ml streptomycin,10-8mol/L dexamethasone,10mmol/L β-glycerophosphate and50μg/ml ascorbic acid).3. Detection of alkaline phosphatase(ALP) activityThe third generation cells were selected. Cells were inoculated in24-well plates with the density of approximately1×105/ml,each group was given different treatment factors, and the ALP activity was detected by enzyme linked immunosorbent assay on the7th day. Cells were cracked fully with0.2%Triton X-100and freezing and thawing method, then were washed with PBS two times after cracked, centrifuged at12000rpm for10minutes, the supernatant was taken for detection according to the kit instructions, the results were measured with micreplate at520nm wavelength.4. Alizarin red staining for calcium nodulesThe third generation cells were selected and seeded in24mm×24mm coverslip prepositioned in six-well plates,each group was given different treatment factors, and the samples were fixed, stained, and clarified after21days according to the alizarin red staining kit instructions, calcium nodules were observed under inverted microscope,five samples were taken from each group,each sample was randomly taken from a field of vision (×100), then to compare the differences between the groups.5. Real-time PCR method for the determination of Runx2expression Select the third generation cells, seeded in25mm Petri dishes with the density of1×105/ml approximately,each dish inoculated with cell suspension of1mL,each experimental group was given different treatment,respectively.Total RNA was extracted at different time points for the gene expression determination.6. Western blotting for the determination of BMP-2expressionSelect the third generation cells, seeded in60mm Petri dishes with the density of1×105/ml approximately, each experimental group was given different treatment,respectively.Protein was extracted at different time points,quantitatived by BCA Protein Assay Kit.After separated by SDS-PAGE,total protein was transferred to PVDF membrane.Closed1.5h with5%nonfat dry milk, followed by BMP-2antibody (1:1000) overnight at4℃, washed with TBST three times, each time for10min,then secondary antibody (1:2500) were incubated1.5h, and then TBST washing three times, each time for10min.Membrane was colored with the luminescence reagent ECL,exposured in the darkroom, then use the gel imaging system to scan and analyze results.7. Statistical AnalysesAll measurement data were expressed as mean±SD.Statistical analyses were conducted with SSPSS13.0for Windows,comparing continuous variables of groups used One-way ANOVA. LSD method was used for multiple comparison for homoscedasticity,and Dunnett’s T3method was used for multiple comparisions for heterogeneity of variance. Significance was defined as P<0.05.RESULTS1. Morphological observation of BMSCsWhen the primary MSCs were inoculated, they looked round, strong refraction, mixed with a large number of blood cells surrounding and couldn’t be identified clearly. Changed the primary culture medium to remove a part of suspended blood cells after24hours, it was see that MSCs were adherent growth, some looked round, some extended to the surrounding and were triangular, polygonal and short spindle. No adherent cells were basically cleaned after several exchanges, so the adherent cells proliferated rapidly, were long spindle-shaped or fibroblast-like. These cells arranged in a certain direction, formed some significant colony. When about7-10days later, the cells colony increased, expanded, basically covered the bottom of the bottle, reaching more than80%of fusion, it was time for cells passaging. After passage, cell morphology was more single. Most of the cells showed relatively uniform spindle, evenly distributed, and were swirling, cobblestone arrangement, growing more rapidly than the primary cells. They could basically cover the bottom of the bottle about3-4days.2. Sr induced activity of ALP in BMSCsIn the differentiation process of BMSCs to osteoblasts,cells were treated with0mmol/L,0.1mmol/L,lmmol/L,3mmol/L,5mmol/L and7mmol/L Sr for7days, ALP activity difference between groups was statistically significant(F=232.089,P=0.000). ALP activity difference was statistically significant between each experimental group and control group(P<0.05); When Sr concentration was increased to3mmol/L, ALP activity difference was statistically significant compared with other groups (P=0.000), and the mean was maximum, this can be considered as that ALP activity increased to maximum.3. Sr induced mineralization in BMSCsBMSCs were treated with3mmol/L Sr for21days,the generated calcium nodules increased compared to the control group, there was statistically significance (t=-4.395,P=0.002).4. Sr induced expression of Runx2in BMSCsBMSCs were treated with Ommol/L,1mmol/L,3mmol/L,5mmol/L,7mmol/L, 10mmol/L Sr seperately for7days,the Runx2expression between each group was statistically significance(F=1120.869,P=0.000). The difference between each experimental group and control group was statistically significant(P<0.05). When Sr concentration was lmmol/L, Runx2expression difference was statistically significant compared with other groups (P<0.05), and the mean was maximum, this can be considered as that Runx2expression was the highest. BMSCs were treated with1mmol/L Sr seperately for0h,0.5h,lh,2h,4h,3d,7d and14days, the difference of Runx2expression between each group was statistically significance(F=438.015,P=0.002). The difference between each experimental group and control group was statistically significant(P<0.01). While processed cells for7days,the difference compared with all the other group was statistically significance(P<0.01),and it had the maximum mean, the Runx2expression level climbs to the highest.5. Sr induced expression of BMP-2in BMSCsBMSCs were treated with Ommol/L,0.1mmol,1mmol/L,3mmol/L,5mmol/L and7mmol/L Sr seperately for7days, BMP-2expression between each group had statistical significance(F=59.117,P=0.000).The difference between each experimental group and control group has statistical significance(P<0.01). When processed cells with1mmol/L Sr, BMP-2expression mean was the maximum, the difference compared with all the other group was statistically significance(P<0.05), this can be considered as that the BMP-2expression was the highest. BMSCs were treated with lmmol/L Sr seperately for0d,ld,3d,5d,7d and10days, the difference of BMP-2expression between each group has statistical significance(F=75.937,P=0.013), BMP-2expression of experimental groups were more than control groups, and the difference was statistically significance(P<0.05). When process cells for7days,the difference compared with all the other group has statistical significance(P<0.05),and it has the maximum mean, BMP-2expression level can be considered as the highest.6. Noggin preconditioning inhibited Sr-induced expression of BMP-2in BMSCsBMP-2expression difference was statistically significance between all the groups after different treatment(F=35.384,P=0.000). BMSCs were exposured to1mmol/L Sr for7days, BMP-2expression was more than control group, there was statistically significance(.P=0.000). Preconditioning with100ng/ml noggin for2hours, BMP-2expression decreased compared with Sr group, there was statistically significance(P=0.000), the difference between noggin group, PBS group and control group had no statistical significance (P=0.962,0.965).7. Noggin preconditioning inhibited Sr-induced activity of ALP in BMSCsALP activity difference was statistically significance between all the groups after different treatment(F=906.400,P=0.000). BMSCs were exposured to3mmol/L Sr for7days, ALP activity increased compared with control group, it had statistical significance(P=0.000). Before processing cells with Sr,preconditioning with100ng/ml noggin for2hours, ALP activity decreased, it had statistical significance(P=0.000), the difference between noggin group, PBS group and control group had no statistical significance (P=0.060,0.527).8. Noggin preconditioning inhibited Sr-induced mineralization in BMSCsMineralization difference was statistically significance between all the groups after different treatment(F=6.49,P=0.002). Processed BMSCs with3mmol/L Sr for21days, the calcium nodules number increased, this had statistical significance(P=0.002). Before processing cells with Sr, preconditioning with100ng/ml noggin for2hours, the calcium nodules number decreased, this had statistical significance(P=0.000), the difference between noggin group,PBS group and control group had no statistical significance (P=0.875,0.754).9. Noggin preconditioning inhibited Sr-induced expression of Runx2in BMSCs Runx2expression difference was statistically significance between all the groups after different treatment(F=95.196,P=0.000). Processed BMSCs with1mmol/L Sr for7days, Runx2expression increased compared with control groups, there was statistically significance(P=0.022). Before processing cells with Sr, preconditioning with100ng/ml noggin for2h, Runx2expression decreased, it had statistical significance(P=0.003), the difference between noggin group,PBS group and control group had no statistical significance (P=0.738,1.000).CONCLUSIONS1. Sr can promote BMSCs differentiat into osteoblasts, which expressed as the increase of ALP activity, mineralized nodules and the upregulation of Runx2expression during the differentiation process.2. During the process of osteoblasts differentiation from BMSCs, Sr up regulated the expression of BMP-2. Moreover, the inhibitor of BMP-2, noggin, not only inhibited Sr-induced over-expression of BMP-2, but also inhibited Sr-induced activity of ALP, mineralization and expression of Runx2. These above evidences indicated that by upregulating the expression of BMP-2may be one of the mechanisms by which Sr promotes differentiation to osteoblasts in rat BMSCs. |
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