| Objective: Bone is under the control of many factors including the nervous system.Recent studies showed that Semaphorin3 A,a chemorepellent,can adjust bone mass.But the mechanism remains elusive.Schwann cell,serves as a pivotal role after nerve injury,interacts with Semaphorin3 A under many physiological and pathological circumstances.The aim of this study is to primarily investigate the role of neural cells in the promotion of osteogenesis by Semaphorin3 A,to explore the exact function nervous system has in homeostasis of bone and the factors affecting this process,therefore to provide theoretical basis in prevention and treatment of bone-related diseases.Methodology:1.Establishment of co-culture system of human osteoblastic cell MG63 and human Schwann cell.Cell proliferation was assessed on day 1,3 and 5.The ratio of MG63:Schwann cell with the best cell vitality was then selected.2.Add Semaphorin3 A to MG63,Schwann cell and 1:1 co-culture system respectively to reach final concentration of 0,25,50,and 100 ng/m L.Then observe cell viability,cell death at day 1,3 and 5,to see if Semaphorin3 A exerts any influence on proliferation and apoptosis of these three groups of cells.3.Add Semaphorin3 A to MG63,Schwann cell and 1:1 co-culture system respectively to reach final concentration of 0,25,50,and 100 ng/m L.Then observe cell viability at 6,12,and 24 hours,to see if Semaphorin3 A is cytotoxic to these three groups of cells.4.Use Real-time PCR to analyze expression of Semaphorin3 A receptors in MG63 and Schwann cell.5.Osteogenic differentiation of MG63 was tested in both MG63 single cell culture and 1:1 co-culture system under various concentration of Semaphorin3 A.Alkaline phosphatase(ALP)assay,ECM calcium nodule staining and osteogenic gene expression by q RT-PCR were conducted after culturing for 7 and 14 days.6.Results were analyzed statistically using SPSS,difference between groups were determined by one way analysis of variance(ANOVA),and statistical significance of difference was considered when p < 0.05.Results: 1.The ratio of MG63 : Schwann cell at 1:1 showed the best cell vitality at all timepoints examined,thus 1:1 of MG63 : Schwann cell was selected as the proportion used in the following test.2.Semaphorin3 A had no effect on cell proliferation of MG63 and 1:1 co-culture system at all times.For Schwann cell,no difference in proliferation was observed at day 1 and 3,however at day 5,semaphorin3 A inhibited proliferation of Schwann cell.3.Semaphorin3 A had no effect of cytotoxicity on 1:1 system,and had no effect in apoptosis on 1:1 co-culture system.4.Receptors of Semaphorin3 A expressed in MG63 were Neuropilin 1,Neuropilin 2,Plexin a1,Plexin a2 and Plexin a3.Schwann cell expressed Neuropilin 2 and Plexin a3.5.Alkaline Phosphatase assay showed that at day 7 and 14,ALP level in MG63 single cell culture was inhibited with increasing concentration of Semaphorin3 A,however,ALP level of MG63 in the 1:1 co-culture was enhanced when concentration of Semaphorin3 A increased.6.ECM calcium nodule staining suggested that at day 14,calcium deposition in MG63 single cell culture was inhibited with increasing concentration of Semaphorin3 A,however,calcium deposition of MG63 in the 1:1 co-culture was enhanced when concentration of Semaphorin3 A increased.7.Osteogenic gene expression quantified by q RT-PCR showed that at day 7 and 14,expression of osteogenic genes RUNX2/OCN in MG63 single cell culture were inhibited as concentration of semaphorin3 A increased,while expression of osteogenic genes RUNX2/OCN in MG63 of 1:1 co-culture were promoted.Conclusion: In this study,it was confirmed that under low concentration of Semaphorin3 A,osteogenic differentiation of MG63 was inhibited.However,in the presence of Schwann cell,low concentration of Semaphorin3 A reversely enhanced osteogenic differentiation of MG63.Therefore the conclusion of this study is that Semaphorin3 A can enhance osteogenesis of osteoblastic cells via interaction with neural cells in vitro. |
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