Regulation Of Calcitonin Gene-related Peptide On Cross-talk Between Osteoblasts-like And Endothelial Cells

Bone repair is a complex event that involves the interaction and coordination ofvariety many cells, and regulated by biochemical and mechanical signals. Calcitoningene-related peptide (CGRP) generated by peripheral nerve, is widely distributed in thecentral and peripheral neuronal systems and exhibits numerous biological activities inmammals, especially in osteogenese and angiogenses. In vitro experiments, CGRP has beenpromoted that can rise proliferation of osteoblasts, presence CGRP receptors on osteoblastsurface, and has a large number of downstream cell signaling pathways. But in theseexperimental studies, the majority only explored CGRP on single osteoblasts or osteoclastcells to expand the biological role of cells in fracture healing, and ignored the effectsmulti-cell interaction between cells. Therefore, these studies are unable to explainedCGRP’s osteogenesis in vivo multi-cell environment correct.In vivo identification of regulators of bone remodeling is complex and nearlyimpossible because of many other factors in callus, such as cytokines, growth factors, andthe bone matrix itself. The co-culture system using OB and VECs provided a more simpleand useful method to explore these problems. Several studies have shown that the cross-talkbetween cells occurs at two levels, the first is completed direct role in cell-to-cell via themembrane protein, and the second is the indirect role through autocrine and paracrinemanner to complete the cell. CGRP positive fibers develop along the blood vessel growth inthe process of fracture healing callus, and play an important biological role in angiogenesisand the formation, which involved in the description that CGRP join in the regulation onCross-talk between the OB-VECs.Therefore, we propose that CGRP exerts a biological effect on the OB-VEC co-culturesystem, and in this study, we focus on its regulation of OB differentiation. We establishedOB-VEC co-culture system to identified osteoblast differentiation via the detection of osteogenetic production, and explore the mechanism of regulation of CGRP on thecross-talk between the OB-VECs by detection the secretion of VECs cell factor andexpression these cell factor receptor on OB. At last, this study can elaborated themechanism of neural regulation on the blood vessels and osteoblasts cross-talk.Materials and Methods1. Cell culture: MG-63and HUVECs cells (ATCC) were cultured in Dulbecco’smodified Eagle’s Medium supplemented with10%fetal calf serum. Cells were subculturedevery72h in a humidified5%CO2incubator. Cells from passages6–8were used in theexperiments.2. Establishment of MG-63-HUVECs co-culture.2.1Direct contact co-culture conditions: MG-63were mixed with HUVECs inDMEC medium (10μg/mL PBS) and plated in12multiwell dishes at1×105cells/well，co-cultured24h and observed under an inverted microscope.2.2Idirect contact co-culture conditions: A0.4μm transwell membrane insert wasused to separate HUVECs from MG-63. After MG-63cells were plated in a12multiwell at5×5×105cells/well and cultured for24h, the transwell was added, and then HUVECs wereadded to the insert chamber at5×105cells/well.3. Detection the differentiation of MG-63in indirect co-culture sondition:3.1Detection of CollagenⅠ mRNA in MG-63: Cells were indirect co-cultured for24h,48h,72and96h, mRNA samples in every group were extracted and via a Real timePCR to find the effect of co-culture on the expression of CollagenⅠmRNA in MG-63.3.2Detection of Osteocalcin production in supernatants: Cells were indirectco-cultured for24h,48h,72and96h, supernatants in every group were extracted and viaELISA to find the effect of co-culture on the expression of oc in MG-63.4. Effect of CGRP on the differentiation of MG-63in indirect co-culturesondition:4.1Detection of CollagenⅠ mRNA in MG-63: The groups of CGRP was dividedinto dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), mRNA samples in every group were extracted and via a Real time PCR to findthe effect of CGRP on the expression of CollagenⅠmRNA in MG-63. 4.2Detection of Osteocalcin production in supernatants: The groups of CGRP wasdivided into dose-effect groups (100nM，10nM，1nM和0.1nM) and time-groups (0h，24h，48h，72h和96h), supernatants in every group were extracted and via ELISA to find theeffect of CGRP on the expression of oc in MG-63.5. Regulation of CGRP on VEGF-A in MG-63-HUVECs Cross-talk:5.1Detection of VEGF-A production in supernatants:MG-63, HUVECs, andMG-63-HUVECs indirect co-culture cells were induced by CGRP for0h,12h,24h,36h and48h, mRNA samples and supernatants in every group were extracted, and via Real timePCR or ELISA to find the effect of CGRP on the expression of VEGF-A and VEGF-AmRNA in cells.5.2Expression of VEGF1/2on MG-63s: The CGRP groups (CGRP) were induced by10nM CGRP for48hours, and the control group (CON) was treated with DMEC medium.Co-culture groups (CO) were indirect co-cultured with HUVECs and without CGRP for48hours, and the experimental groups (EX) were indirect co-cultured with HUVECs and with10nM CGRP. Expression of VEGFR1/2in MG-63cells were detected byimmunofluorescence after induced for48h.Results:1. MG-63-HUVECs co-culture system.1.1Direct co-culture: MG-63and HUVECs cells were co-cultured in accordancewith1:4,1:2,1:1,2:1and4:1for24h. MG-63:HUVECs got the best growth condition as in1:1proportion, which MG-63and HUVECs cells intermingled with growth, and HUVECscell grew as nodular and MG-63cells grew around these endothelial cell nodules. AsMG-63-HUVECs co-culture system were labeled by the DiI fluorescent,24h, the HUVECsgrew as nodular, the MG-63cells grew around endothelial cell nodules;48h, cell necrosiswere found in the center of HUVECs nodules;72h, HUVECs were separated from MG-63cells layer, MG-63cells invades into endothelial nodules;96h, HUVECs formed tube-likestructures, the MG-63cells completely distributed the bottom of the bottle.1.2Indirect co-culture: MG-63cells were indirect co-cultured with HUVECs cells,its Collagen Ⅰ mRNA expression was significantly increased, expression of Collagen ⅠmRNA in co-cultured groups was1.32of the control group,(P <0.05);48h achieve maximum,1.57of the control group (P <0.01). OC production in supernatants groupsincreased with time, and the co-culture groups was higher than that in the control groups,24h, OC in co-culture groups was1.54of the control group,(P <0.01);48h,1.48of thecontrol group (P <0.01);72h,1.35of the control group (P <0.05).2. Effect of CGRP on MG-63in indirect co-culture system.Different concentrations of CGRP effect on MG-63-HUVECs co-culture for48h,Collagen Ⅰ mRNA in and OC expression in MG-63cells both increased. In10nM CGRPgroup, expression of Collagen Ⅰ mRNA and OC in MG-63cells increased the mostobvious,1.94(P <0.01) and2.02(P <0.01).MG-63-HUVECs cells were induced by10nM CGRP, Collagen Ⅰ mRNA expressionin MG-63cells presented an increasing trend, reached peak for the control group1.98at48h,(P <0.01); OC production in supernatants contented of an increasing trend since24h(P <0.01), peaked2.91for the control group at96h.3. Regulation of CGRP on the VEGF-A in MG-63-HUVECs co-culture:3.1Effect of CGRP on the production of VEGF-A:Induced by10nM CGRP, in HUVECs groups, VEGF-A production were raised, andreached peak for the control group2.31at48h, VEGF-A mRNA were raised, peaked2.71for the control group at24h; in MG-63groups, VEGF-A production were decreased from36h, and decline to he lowest level,0.66of control groups at48h; CGRP induced co-culturegroup, VEGF-A mRNA expression in HUVECs increased significantly after24h, andpeak for the2.42control group at24h.3.2Effect of CGRP on the expression of VEGFR1/2in MG-63:CGRP and co-culture environment both increased VEGFR1expression in MG-63cells.Induced by the10nM CPGR for48hours, through cross-comparison, VEGFR1expressionin CO group was higher than the CON group,0.186:0.124(p <0.01); VEGFR1expressionin EXP group was significantly higher than the CON group,0.201:0.124(p <0.01), and washigher than CGRP group,0.201:0.118(p <0.01).The CGRP and co-culture environment both increased the expression of VEGFR2inMG-63cells. Induced by the10nM CPGR for48hours, through cross-comparison,VEGFR2in CGRP and CO groups were significantly increase compared to the CON group,0.177:0.081(p <0.01) and0.154:0.081(p <0.01); VEGFR2in EXP groups noticeable higher than the CON group,0.194:0.081,(p <0.01), and significantly higher than the COgroup,0.194:0.154(p <0.05).Conclusion:1. MG-63cells and HUVECs cells can be co-cultured in high glucose DMEM mediumdirectly, and the1:1grow ratio is the dominant proportion. Vascular endothelial cells growinto cells noduls, and become tube-like structures with time. MG-63cells grow aroundthese cells noduls.2. In indirect MG-63-HUVECs co-culture environment, Collegen I mRNA expressionand secreted OC increased, and promoted the differentiation and maturation of the MG-63cells.3. In co-culture system, CGRP can increase Collagen Ⅰ mRNA and OC expression inMG-63cells, whose optimal concentration is10nM. Under induction of CGRP orco-culture with HUVECs, Collagen Ⅰ mRNA and OC expression in MG-63cells can beincreased, and promoted the differentiation and maturation of the MG-63cells.4. CGRP can promote the VEGF-A mRNA expression in HUVECs cell, and raised theVEGFR1/2in MG-63in MG-63-HUVECs co-culture system, VEGFR2more significant.But CGRP can not effect on the VEGF-A production in supernatants of MG-63-HUVECsco-culture.