| During the process of bone formation, osteoblasts secrete a lot of growth factors suchas fibroblast growth factor-2, insulin-like growth factor-1and transforming growth factorβto regulate osteoblasts proliferation and differentiation by anticrine and paracrine ways.Among them, fibroblast growth factor-2(FGF2) plays an important role in bone and bonecollar formation. Vitro studies have shown that, the effect of FGF2on differentiation andmineralization of osteoblasts are stage-distinct and continuous treatment of FGF2couldsignificantly inhibit differentiation and mineralization of osteoblasts. FGF2inhibitsosteoblasts mineralization by altering the PPi/Pi regulatory protein expressions, however,the mechanism by which FGF2inhibits osteoblasts mineralization is still not clear.Matrix Vesicles (MVs) are membrane-invested vesicles with a diameter ranging from30to400nm and secreted into extracellular matrix by mineralization-competent cells suchas osteoblasts, chondrocytes and so on. MVs absorb Ca and Pi in the extracellular matrix toform hydroxyapatite (HA) by the functions of proteins rich in them. HA continues to growin MVs until it punctures MVs’ membrane and then localises along collagen to startmineralization. In addition to the important roles in physiological mineralization, MVs alsoplay important roles in the occurrence and development of pathologically mineralizeddiseases, for example, enhancement of MVs’ function is one of the important pathologicalmechanisms of atherosclerosis. Although MVs play important roles in mineralization, theregulatory mechanism of MVs’ function remains poorly understood.Objective：We aim to examine the effects of fibroblast growth factor-2on osteoblastsmineralization and research its extracelluar mechanism.Methods：(1) Using mineralization inducing medium containing Ascorbic acid and β -glycerophosphate to promote Saos-2cell osteo-differentiation and mineralization andestablish the cell model of mineralization.(2) Saos-2cells were divided into three groups:normal control group, osteo-induced group and FGF2group. The normal control groupwere cultured in basis medium, osteo-induced group were cultured in mineralizationinducing medium (basis medium containing50ug/ml Ascorbic acid and7.5mM β-glycerophosphate) and FGF2group were cultured in mineralization inducing group with50ng/ml FGF2. When Saos-2cells were cultured for3,6and9days, the detection of theexpression of mineralization marker genes: alkaline phosphatase (ALP), runt-relatedtranscription factor-2(Runx2) and collagenIa1(ColIa1) and qualitative, quantitativeanalysis of mineralized nodules were performed in all groups respectively.(3) MVs wereisolated from Saos-2cells of normal control group, osteo-induced group and FGF2groupwith ExoQuickTM reagents. Then an electric microscope was used to detect itsmorphology and the expression of its surface biomarkers CD63and CD9was detected byWestern Blot.(4) ALP activity of MVs was performed using p-NPP. Incubatiing inmineralizing buffer for12h, MVs could continue to uptake Ca ion and form HA mineral.After incubation, Ca2＋concentration was detected using calcium assay kit to examine vitrobiomineralization capacity of MVs.Results:(1) Quantitative analysis of mineralized nodules showed that mineralizednodules were five times more than that of control groups.(2) The expression ofmineralization marker genes: ALP, Runx2and ColIa1of Saos-2cells of FGF2group wasobviously lower than that of osteo-induced group and higher than that of control group at3,6and9days. Quantitative analysis of mineralized nodules showed that compared withosteo-induced group, mineralized nodules formation in FGF2group weresignificantlydecreased at3,6and9days respectively.(3) As seen by electric microscope,the MVs isolated from all the groups were recognized as membrane-invested vesicles withdiameters ranging from30to400nm. CD63and CD9, which are biomarkers of MVs, werepresented in MVs isolated from all the groups.(4) ALP activity analysis showed that ALPactivity of MVs of FGF2group was lower than that of osteo-induced group. Afterincubation, Calcium concentration of MVs of FGF2group was alsolower than that ofosteo-induced group. Conclusions: Long-term treatment of fibroblast growth factor-2could significantlyinhibit Saos-2cells mineralization; FGF2exert its inhibitory role by inhibiting thefuncionging of matrix vesicles. |
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