Exogenous Substance P Regulates Bone Marrow Stromal Cell Migration During Mandibular Distraction Osteogenesis To Enhance Bone Formation In Rats

Small diameter sensory nerves is abundantly innervated in bone. Skeletal sensory neurons produce peripherally released a variety of neurotransmitters including substance P(SP) and calcitonin gene-related peptide(CGRP) to regulate bone formation in addition to conducting pain and sensory. We designed the following experiments in order to examing the effect of SP on DO and research its mechanism.Part 1: local injection of substance P enhanced bone formation during mandible distraction osteogenesis in rats.Objective: Sensory nerve fibers distribute substance P(SP) to innervate the medullar tissues of bone,it can accelerate for osteoblast proliferation and differentiation in vitro.However, no data showed it was used for bone formation indistraction osteogenensis. Methods: In this study, we injected 10-7M SP to distraction gap,then observed the osteogenic effects. Twenty Sprague-Dawley(SD) rats were received hemi-mandibular osteotomy and distractor implantion,after 5-day delay period then randomly assigned to 2 groups for treatment(n=10 each): SP local injected, or saline alone. The distraction period is ten days,and mandible was distracted 0.2 mm per 12 h, while 0.2ml 10-7M SP was injected to the distraction gap once a day. Micro-computed tomography(micro-CT) and histological analysis accessed bone regeneration at day15, 29. Results: There was more new bone formation in SP group at day 29 compare to control. The micro-CT images and quantitation showed more callus and more mature cortical bones formed with SP than Control group during distraction osteogenesis,bone trabecular can be observed in SP group at the final time point(day 29),while littery calcific nodules observed in saline treated group by HE staining. Conclusion: Local injected 10-7M SP to distraction gap could remarkably improved regeneration of good-quality bones and accelerated bone union during mandibular distraction in rats.Part 2: local injected substance P increased mobilization of mesenchymal stem cells during mandible distraction osteogenesis in rats.Objective: Substance P could enhance bone formation in distraction osteogenesis as the above experiment observed,However, the reason still to be unclear. This study was planned to observe mesenchymal stem cells mobilization after SP treated. Methods: Twenty Sprague-Dawley(SD) rats were received were received hemi-mandibular osteotomy and distractor implantion,after 5-day delay period then randomly assigned to 2 groups for treatment(n=10 each): SP local injected, or saline alone. The distraction period is ten days,and mandible was distracted 0.2 mm per 12 h, while 0.2ml 10-7M SP was injected to the distraction gap once a day. The Local MSCs mobilization was assessed at day 15, 29 by immunohistochemistry of Nestin, and the periphery MSCs mobilization was used flowcytometry to access the numbers of CD29 positive cells in peripheral blood 5、6、11、15、22、29 day post-operation collected through tail vein. Results: The Nestin positive cells distributed in the distraction gap in SP groups rather than located perivascularly in control group. There were more CD 29+ cells in the peripheral blood at 11、15、22day postoperation in SP groups rather than 29 day in saline groups. Conclusion: Local injected SP may lead to enhanced bone formation during mandibular distraction osteogenesis in rats by stimulate the MSCs mobilization.Part 3: SDF-1 expression in mandibular distraction osteogenesis after SP treated in ratsObjective: The concentration of SDF-1is a crucial factor for cell migration during the process of bone regeneration,so we observed the express of SDF-1 in distraction gap and peripheral blood. Methods: 30 SD rats aged 12weeks(250g ± 20g) of SP grade male rats were were received hemi-mandibular osteotomy and distractor implantion,after 5-day delay period then randomly assigned to 2 groups for treatment(n=10 each): SP local injected, or saline alone. The distraction period is ten days,and mandible was distracted 0.2 mm per 12 h, while 0.2ml 10-7M SP was injected to the distraction gap once a day. Immunohistochemistry staining was used to assesse the local SDF-1 expression at day-15 and day 6、15、29 by quantitative real time PCR, the periphery SDF-1 concentration was used ELISA kit to access in peripheral blood plasma 6、11、15、29day post-operation collected through tail vein. Results:Immunohistochemistry staining demonstrated the expression of SDF-1 was higher in SP group, quantitative real time PCR and ELISA showed that SDF-1 expression peaked at day 15, and significant difference between SP treatment and saline analysed by SPSS 14.0 software(p<0.05). Conclusion: Exogenous 10-7M SP could enhance SDF-1 expression in distraction gap and peripheral blood.Part 4: The migration of bone marrow mesenchymal stem cells enhanced by exogenous 10-7M SP in vivo.Objective: MSCs mobilization increased by Substance P to enhance bone formation during distraction osteogenesis as above experiments described but the reason of BMSCs migrated to distraction gap still to be unclear. This study wanted to reveal the effect of SP for mobilization of mesenchymal stem cells is an important process of BMSCs participate bone formation. Methods: Ten(80±20g) of SP grade male rats aged 4 weeks were obtained. BMSCs were isolated from the mandible by collagenase digestion after cut into pieces, then were identified by CD44、CD90、CD 29、CD 34、CD 45 and CXCR-4 used flowcytometry. In 1、3、5、7 day postseeding harvested culture medium and cells for ELISA and q RT-PCR to access SDF-1 and its m RNA expression and Transwell was used to access the migration. Results:Flowcytometry demonstrated that CD44、CD90、CD29 positive and CD34 CD45 negative in the surface of cultured cells, there were more CXCR4 expressd on the cell membrane for 10-7M SP interfered 7 days than control(p<0.05). SDF-1 secretion examed by ELISA and q RT-PCR showed the SDF-1 concentration was higher than control by SP treatment. There were more cells transited the membrane in SP groups in the transwell and the migrate index higher(p<0.05). Conclusion: Exogenous 10-7M SP could enhance migration of BMSCs probably due to the express of CXCR4 in cell membrane and secreted more SDF-1.Part 5: The effect of 10-7M SP on bone marrow stromal cell proliferation osteogenic activity、osteoblast differentiation in vivo.Objective: BMSCs start to proliferation and differentiation when transited to the bone defect gap, so we examed the BMSCs proliferation and differentiation irritated by 10-7M SP. Methods: The P3 stem cells were used in this study, the Brd U immunohistochemistry and cell colony formation unit(CFU)were used to test the proliferation, cell osteogenic activity and osteoblast differentiation was analysed by Alizarin red staining、alkaline phosphatase(ALP)activity assay、 q RT-PCR for ALP and Runx2 in vivo. Results: Brd U+ cell and colony units were more than control, the ALP activity and minerilization 、m RNA express of ALP、Runx2 have no statistical difference between SP groups and control. Conclusion: 10-7M SP could stimulate proliferation but have little Influence on osteogenic activity and osteoblast differentiation.