| Distraction osteogenesis(DO) has been widely used in the treatment of oral and maxillofacial deformities and defects. However, the exact molecular mechanism regulating osteogenesis in DO is educidated. Studies have shown that bone marrow mesenchymal stem cells(BMMSCs) play a key role in bone regeneration. As pericytes of a perivascular niche, BMMSCs must escape the stem cell niche and migrate to the bone forming areas for bone regeneration and repair triggered by bone distraction. This study aimed to explore signaling pathways regulating the migration ability of BMMSCs in DO, and investigate the effect of targeting related signaling pathways on DO. Our study provides new insights into mechanotransduction governing BMMSC migration ability, and therefore leading to novel strategies to improve therapeutic effects of DO. This study consists of three parts as follows:Part 1 Changes in expression of stress-related signaling molecules in BMMSCs under mechanical strainObjective: This part of the study was to explore the significantly changed signaling pathways in BMMSCs under mechanical strain.Method: 1.1 BMMSCs were subjected to mechanical strain using Flexcell 4000 T system, and underwent mechanical strain for 4h at magnitudes of 0.5%, 6% and 10% respectively. BMMSCs in the control group were applied with no strain. Real-time quantitative PCR analysis was performed to evaluate the gene expression. 1.2 12 healthy adult male SD rats were randomly divided into two groups. All the rats were subjected to distraction device implantation on the right mandible. After 5 days of latency, rats in the experimental group were subjected to mandibular distraction at a rate of 0.2mm / d for 10 days, while the control group underwent no distraction. All rats were sacrificed on day 15 after surgery. The bone callus specimens of the right mandible from the osteotomy zone were harvested and fixed with 4% paraformaldehyde at 4°C for 48 h. The specimens were decalcified for 4 weeks, embedded in paraffin, and then sliced longitudinally into 5 μm sections for immunohistochemistry and immunofluorescence analysis.Result: PCR results showed that genes of p38, m TOR, Raptor, p70S6 K and MMPs(MMP2, MMP9 and MMP13) were upregulated in BMMSCs subjected to mechanical strain(P<0.05). However, no significant changes were found in expression of ERK1 or ERK2. The gene expression is higher in BMMSCs subjected to 6% mechanical strain than other groups. Besides, p38 is higher in gene expression than that of m TOR. Immnofluorescent analysis showed that Nestin+/p-p38+ cell numbers were significantly higher in bone calluses of the distraction group compared with the control group(P<0.05).Conclusion:Both p38 and m TOR signaling pathways were stimulated in BMMSCs under mechanical strain. P38 signaling pathway may play a relatively more important role mediating mechanotransduction in BMMSCs in DO.Part 2 A study on the molecular pathway governing the migration ability of BMMSCsObjective: This part was to explore the impact of in vivo and in vitro mechanical strain on the migration ability of BMMSCs and the related molecular mechanism.Method: 2.1 BMMSCs were subjected to a static strain(6%, 4h) using Flexcell 4000 T system. Scratch assays and Transwell assays were performed to evaluate the impact of mechanical strain on the migration ability of BMMSCs. 2.2 P3 BMMSCs were seeded on the elastic plates, and were subjected to a static strain(6%, 4h) with or without the pretreatment of SB203580(20μM), the inbitor of p38 signaling. BMMSCs in the control group underwent no strain or pretreatment of SB203580. Transwell assays were used to evaluate the migration ability of BMMSCs in each group. 2.3 P3 BMMSCs were used. The grouping and treatment were descrbed in 2.2. After the end of the cell stretch, cellular proteins were extracted and Western Blot analysis was used to evaluate the p-p38 and MMP2 expression in BMMSCs. 2.4 24 SD rats were randomly divided into three groups. All the rats were subjected to the implantation of distraction device on the right mandibular. After 5 days of latency, the rats were subjected to continuous mandibular distraction at a rate of 0.2mm/12 h for 10 days.(A) Rats in this group were received with Anisomycin, the activator of p38 signaling, at a dose of 2.5mg / kg in 200μL DMSO per day at the distraction area for 10 days.(B) Rats in this group were received with SB203580, the inhibitor of p38 signaling, at a dose of 2.5mg / kg in 200μL DMSO at the distraction area for 10 days.(C) Rats in this groupwere received with 200μL DMSO DMSO for 10 days. All the rats were sacrificed on day 15 post surgery, the bone callus specimens of the right mandible were harvested from the osteotomy zone and fixed with 4% paraformaldehyde at 4°C for 48 h. The specimens were decalcified for 4 weeks, embedded in paraffin, and then sliced longitudinally into 5 μm sections, and were subjected to Nestin immunohistochemistry analysis.Result: Scratch assay results showed that BMMSCs in the stretch loading group moved farther than the control group(P<0.05). Transwell analysis showed that the migrated cells in the experimental group was significantly higher than that of the control(P<0.05). Western Blot analysis results showed that mechanical strain promoted p-p38 expression in BMMSCs(P<0.05). SB203580 pretreatment significantly blocked strain induced migration and MMP-2 expression of BMMSCs. Nestin immunostaining results showed that Nestin+ cells of the Anisomycin application group were much higher than the other groups(P<0.05), whereas Nestin+ cells in the SB203580 application group was significantly less than the control group(P<0.05).Conclusion: Mechanical stretch promoted BMMSCs migration ability through activating p38 singnaling pathway. This effect may be resulted from p38 mediated MMP2 expression in BMMSCs.Part 3 A study on the effect of targeting p38 signaling on DOObjective: This part of the study was designed to explore the effect of targeting p38 signaling on the osteogenesis in DO.Method: Animal grouping was described in 2.4. All the animals were sacrificed after 2 weeks of consolidation, and the mandible specimens were subjected to Micro CT and histological analysis.Result: Histological and Micro CT results showed that bone mineral density(BMD), bonevolumn/total volumn(BV/TV), and trabecular bone volumn/total bone volumn(TBV/TV) in the anisomycin group were significantly higer than those of the other groups(P<0.05), while the BMD, BV/TV and TBV/TV in the SB203580 application group were lower compared with the control group(P<0.05).Conclusion: Local injections of anisomycin may promote bone formation, possibly through mobilization of BMMSCs via activation of p38 signaling. |
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