Effect Of CGRP On Proliferation And Osteogenesis In Rat Bone Marrow Mesenchymal Stem Cells

Background and Purposes:In contrast to the conventional approaches of periodontal anti-inflammatory therapy, the regenerative therapeutic procedures aimed at repairing lost periodontal tissues, including alveolar bone, periodontal ligament and root cementum, which remain more challenging.At present, the clinical and scientific research methods of periodontal tissue regeneration only stay in the level of regeneration of the "form", but it can not attain "function" regeneration. In the point of view of developmental biology, the physiological growth of every tissues and organs is accompanied with the synchronous neural growth.Nerve not only can serve as a nerve conductor, but also can secrete some neurotransmitters which regulate the growth of tissues and cells.Based on this principle, adding neural factors as mediators in the process of periodontal tissue regeneration to achieve the functional regeneration of periodontal tissuewill be an important topic which is worthy of continuous exploring and researching.The major manifestation of periodontitis is progressive destruction of the alveolar bone which leads to loose and missing of teeth.Alveolar bone is the most active bone tissue. The function of neural factors in the process of alveolar bone reconstructioncannot be ignored.Preliminary experiments found that the regenerated alveolar is relatively less dense and more porous after cutting off inferior alveolar nerve.Calcitonin gene related peptide is a neuropeptide produced by nervous system. It is considered to be an important neurotransmitter in signaling pathways of osteogenesis. But the function of CGRP during stem cell osteogenetic differentiation has not been elucidated. Bone marrow mesenchymal stem cells are frequently uesed seed cells in tissue engineering, which have good proliferate capacity,multi-lineage differentiation ability,directional migration ability and are easy to obtain. So this experiment will use themas the research object.On the basis of above-mentioned research backgroud, this experiment explored the function of CGRP in Rat-BMSCs during the process of osteogenetic differentiation in order to provide theoretical support to further study about the role of neural factors in periodontal tissue regeneration.Methods:1. The effect of different concentrations of CGRP on rBMSCs proliferation.1) Bone marrow mesenchymal stem cells were cultured in vitro.Realtime-PCR and Western blot were conducted to detect the protein and mRNA expression level of Ramp1 and CRLR which are CGRP receptors.2) CCK-8 method were adopted to test the effect of different concentrations of CGRP(0,10-6mol/L,10’7mol/L,10-8mol/L,10-9mol/L) in rBMSCs proliferation and identify the concentration of 8-37 which wasthe antagonist of CGRP.2. The effect of CGRP on osteogenic differentiation of rBMSCs.1) Rat-BMSCs were mineralized induced in vitro.Cells were divided into four groups randomly, which were control group,CGRP group(10-8mol/l),8-37group (10-7mol/l), CGRP+8-37group (10-7mol/l 8-37+10-8mol/l CGRP).2) Alkaline phosphatase and Western blot were conducted to detect the protein of Runx2、BSP、ALP, and real-time PCR was applied to measure the mRNA expression level ofRunx2、BSP、ALP.3) Alizarin red staining and calciumion concentration measurement were used to test the differentiation and osteogenesis ability of rBMSCs in the later period.Results:1. The primary and passages of rBMSCs were in good conditions.2. The protein and mRNA of Ramp1 and CRLR which were the receptors of CGRP could be found in rBMSCs.3.10-9～10-6mol/l CGRP could increase the CCK-8 activity compared with control groups (P<0.05), While the function of 10-8～10-6mol/l CGRP were more obvious. So we took 10-8 mol/l as the experimental concentrations which were the minimum effective concentration.It was found that tenfold to hundredfold of receptor antagonists reversed the effects of CGRP (P< 0.05), and there was no statistical difference between tenfold to hundredfold of receptor antagonists. So we selected tenfold of receptor antagonist as proper concentration.4. Expression levels of osteoblast-related genes and protein, including Runx2,ALP, BSP, were tested at 1 and 5 day. The expression levels of genes and proteins in control group were lower than which in CGRP group,8-37 group and CGRP+8-37 group(P<0.05). Meanwhile, the expression levels of CGRP group was the highest (P<0.05).5. After 3 weeks, the mineral nodules stained with the calcium-specific Alizarin Red revealed that CGRP group exhibited largest areas covered by mineral nodules compared with others. Meanwhile, the mineral nodules in PLAP-1-transfected rBMSCs were fewest.The concentration of calcium ion in control group was the lowest (P<0.05). And the concentration of calcium ion in CGRP group was higher than others (P<0.05). No significant morphological changes were observed in 8-37 group and CGRP+8-37 group.Conclusion:1. CGRP receptors can be found in Bone marrow mesenchymal stem cells.2. Appropriate concentration of CGRP promotes the proliferation and osteogenic differentiation of BMSCs.