The Effect Of SP On The Co-culture Vascular Endothelial Cells And Osteoblasts Induced From BMSCs

The repairing problems of clinical bone defects and nonunion caused by a variety ofreasons, which were the hot pints and difficulty researched in the orthopedic academia.Currently，the clinical commonly used approaches which include autogenous bone graftsand allografts, but its related technologies have some varying degrees of shortcomings.Therefore, it can not achieve the real clinical applications. With the research deepening ofbone tissue engineering, this provides new ideas and methods for clinical bone defectsrepairing. Bone tissue engineering materials composited seed cells and nerve factor whichused for the repairing of bone defects, with good plasticity and osteogenic effects, so theclinical application is highly anticipated.Fracture healing as a complex physiological process, involved the synergies of a variety ofcells and cytokines. Growth factors as an important material of the occurrence, development and maturation of human tissues and organs, provide the necessarynutritional foundation for the regeneration of tissue engineered bone, the effect ofneurotransmitters on bone metabolism is particularly prominent. Substance P (SP) is aneuropeptide which was first discovered neurotransmitter, neuropeptide SP has beenconfirmed from sensory nerve fibers, effect on osteoblasts, and was throughout the entirehealing process. However, there are differences in present experimental study, the effectsof SP on osteoblastic of bone metabolism is not clear. According to the literature, theosteoblasts and vascular endothelial cells co-culture system in vitro, can promote theproliferation of osteoblasts and the vascularization of tissue engineered bone regenerationprocess, which contributes to strengthen the repairing of bone defect of bone tissueengineering. To this end, the experiment by establishing endothelial cells (ECs) andosteoblasts (OB) co-culture system derived by bone marrow stromal cells (BMSCs) invitro, observe the optimal concentration and effects of SP on the co-culture cells system, todefinite the effect of SP function on osteoblasts and endothelial cells co-culture system, toexplore the prospects of SP in bone defect repair.AimTo study the effect of SP (substance P) on the co-culture endothelial cells (EC) andosteoblasts (OB) from rabbit BMSCs in vitro. To find the optimal concentration and theeffects of SP on the common seed cells to to provide experimental support of theapplication of SP for promoting tissue engineering to repair bone defects.MethodsFirstly, the BMSCs were obtained from new-born rabbits by using gradient centrifugemethod and cultured in vitro. After three passages on BMSCs，which were induced intovascular endothelial cells and osteoblasts and detected the cell-associated qualitativedetection for identification. After being induced for7days，the two types cellwere directlyco-culture in a ratio of1to2，the co-culture vascular endothelial cells and osteoblasts inthe second generation were used,and then SP with different concentrations1×10-12-1×10-6mol/L was added(experimental group). While the second generation of co-culture cells without SP was used as a control(control group).The function of SP was assessed byCCK-8examination assay and the growth curve was drawn at1,3,5,7days, then countedthe cells. Determine the ALP activity and BGP (osteocalcin) activity.Secondly, respectively establish ECs (Induced endothelial cells) and OB (Inducedosteoblasts) culture system, or co-culture system directly or indirectly at the ratio of1:2,the cells were simultaneously added with1×10-8mol/L SP. After cultured48h, analyze thecells cycle by flow cytometry to detect the cell proliferation and activity; and respectivelymeasure the expression of ALP (Alkaline phosphatase) through ELISA, and detect the OC(osteocalcin) gene expression of osteoblasts mRNA by real-time quantitative PCR analysis.The cell culture system without SP as the control groups, to compare the changing of cellfunction with SP.Results1. We can directly see the obvious effects of SP from CCK-8graph on the first day,the effect of the low concentration(1×10-12mol/L) was not obvious; various concentrationsof SP were promoting the co-culture cells after3days, the effect was growing with timegoing on. The cell proliferation effect was more obvious in the1×10-8mol/L concentration,there were significant difference between groups, cell hyperplasia. The results suggest thatthe expression of alkaline phosphatase activity，which indicated that the effects of SP wassignificantly increased three days later. The effect of low concentrations (1×10-12mol/L) isnot obvious, but the effect of SP on1×10-8-1×10-6mol/Lwere obvious. In osteocalcin(BGP) expression assay, compared to the control group, various concentration of SP in theexperimental group were improved after3days, the effect was more obvious in the1×10-8mol/L concentration, there were significant difference between groups.2. Compared to OB cultured alone, the G1phase of the cell cycle was significantlyreduced of indirect co-culture system，and S phase cell block (P<0.05); the ALPexpression was elevated (P<0.05) and OC mRNA was significantly improved (P<0.001).Compared to OB cultured alone, the ALP expression and OC mRNA of direct co-culturesystem were both significantly improved (P<0.001). Compared to indirect co-culture system, the ALP expression and OC mRNA of direct co-culture system was also elevated(P<0.05). After added in SP, the experimental group of OB cultured alone compared to thecontrol group, the cells in the G1phase of cell cycle was reduced and was increased in theG2/M phase (P<0.05); the ALP expression was elevated, OC mRNA was also improved(P<0.05). The experimental group of co-culture system compared to the control group, thecells in the G1phase of cell cycle was significantly reduced (P<0.001), G2/M and S phasecell block; the ALP expression was elevated, OC was also increased (P<0.05).ConclusionsIn direct co-culture system in vitro, the effect of SP-promoting on the new seed cellswas significantly. SP can improve activity of co-culture osteoblasts. There were goodcellular compatibility between the endothelial cells and osteoblasts induced from BMSCswhen the two kinds of cells were co-culture. SP can promote the activity of OB culturedalone, and can also significantly promote the activity and proliferation of co-culturesystem.