| Objective: Distraction osteogenesis(DO)is an endogenous bone tissue engineering technique that utilizes the mechanism of bone wound healing to regenerate new bone.The osteogenic efficiency of DO is 4 to 6 times than of the adolescent growth period.In the early stage of our study,the second generation whole-genome high-throughput sequencing was performed on the canine mandibular distraction callus and the mandibular fracture tissue.The results showed that the expression of transcription factor 4(ATF4)was much higher in the MDO group than in the BF group.We hypothesized that ATF4 plays a key role in rapid osteogenesis during DO.And continuous hypoxic microenvironment in the distraction gap would act as a stress to trigger Endoplasmic Reticulum Stress(ERS)and activate PERK/eIF2α/ATF4 signaling pathways,thereby participating in the osteogenesis of bone marrow mesenchymal stem cells(BMSCs).In this study,the expression of ATF4 and HIF-1α was tested in the animal models of mandibular distraction osteogenesis and bone fracture.And BMSCs were cultured in a hypoxic microenvironment simulated in a hypoxic chamber to study the effects of appropriate hypoxic level on ERS and osteogenic differentiation of BMSCs and the role of PERK/eIF2α/ATF4 signaling pathway in osteogenic differentiation of BMSCs,so as to further clarify the molecular mechanism of rapid osteogenesis of DO and provide a more complete theoretical basis for its clinical application.Methods: 1.Establishment of animal model and hypoxia model.Exploration of the effects of hypoxic microenvironment on canine BMSCs: Firstly,the animal models of mandibular distraction osteogenesis and bone fracture were established.The expression of HIF-1α,PERK,eIF2α,ATF4,OCN,and BSP in distraction gap and fracture tissue of canine mandible was detected by qRT-PCR.Then,canine BMSCs were isolated and cultured by using density gradient centrifugation method,then the surface markers of canine BMSCs were identified by using flow cytometry.The osteogenic ability of canine BMSCs was detected by alizarin red staining assay,and the migration ability of canine BMSCs was detected by migration test.The effect of hypoxic environment on the proliferation of canine BMSCs was detected by CCK-8 assay.The effect of hypoxic environment on the osteogenic ability of canine BMSCs was detected by alizarin red test.The effect of hypoxic environment on the migration ability of BMSCs was detected by migration test.The effect of hypoxia environment on the m RNA expression of HIF-1α,PERK,eIF2α,ATF4,OCN and BSP in canine BMSCs was revealed by qRT-PCR assay.The effect of hypoxia environment on the expression of HIF-1α,t-PERK,p-PERK,t-eIF2α,p-eIF2α,ATF4,OCN and BSP in canine BMSCs was revealed by WB assay.2.Effects of ATF4 on canine BMSCs and its relationship with PERK/eIF2α/ATF4 signaling pathway: Lentivirus containing vector of small interfering-ATF4 RNA or negative control RNA was constructed and transfected to canine BMSCs.The effect of interfering ATF4 on the proliferation of canine BMSCs in hypoxic environment was detected by CCK-8.The effect of interfering ATF4 on osteogenic ability of canine BMSCs under hypoxia was detected by alizarin red assay.The effect of interfering ATF4 on the migration ability of BMSCs in hypoxia was detected by migration test.The effect of interfering ATF4 on the m RNA expression of HIF-1α,PERK,eIF2α,ATF4,OCN,and BSP in canine BMSCs in hypoxia was revealed by qRT-PCR.The effect of interfering ATF4 on the expression of HIF-1α,t-PERK,p-PERK,t-eIF2α,p-eIF2α,ATF4,OCN,and BSP in canine BMSCs under hypoxia was detected by WB assay.Results: 1.Establishment of animal model and hypoxia model.Exploration of the effects of hypoxic microenvironment on canine BMSCs: The animal models were established successfully.And the qRT-PCR data showed that the expression of HIF-1α,PERK,eIF2α,ATF4,OCN,and BSP in the distraction gap was significantly higher than in the fracture tissue(P<0.001).CCK-8 experiment showed that the proliferation of canine BMSCs was higher in the hypoxia group than in the normoxia group(P<0.01).Alizarin red assay showed that hypoxic environment could promote the osteogenic ability of BMSCs(P<0.01).Migration test showed that hypoxic environment can promote the migratory ability of canine BMSCs(P<0.001).qRT-PCR results showed that hypoxic environment promoted the gene transcription of HIF-1α,PERK,eIF2α,ATF4,OCN and BSP in canine BMSCs(P<0.001).Western Blot results showed that hypoxic environment promoted the expression of HIF-1α,t-PERK,p-PERK,t-eIF2α,p-eIF2α,ATF4,OCN and BSP in canine BMSCs(P<0.001).2.Effects of ATF4 on canine BMSCs and its relationship with PERK/eIF2α/ATF4 signaling pathway: CCK-8 assay showed that interfering ATF4 inhibited the proliferation of canine BMSCs while hypoxia rescued its ability(P<0.05).Alizarin red assay showed that interfering ATF4 inhibited the osteogenic ability of canine BMSCs while hypoxia rescued its ability(P<0.001).Migration test showed that interfering ATF4 inhibited the migratory ability of canine BMSCs while hypoxia rescued its ability(P<0.001).qRT-PCR results indicated that the expressions of HIF-1α,PERK,eIF2α,ATF4,OCN and BSP in si-NC-Hyp group were significantly higher than those in si-NC group(P<0.001).The expressions of HIF-1α,PERK and eIF2α were not significantly different between the si-ATF4 group and the si-NC group(P>0.05),while the expressions of ATF4,OCN and BSP were significantly decreased in the si-ATF4 group compared with the si-NC group(P<0.001)and rescued in the siATF4-Hyp group(P<0.05).Western Blot results showed that the expressions of HIF-1α,t-PERK,p-PERK,t-eIF2α,p-eIF2α,ATF4,OCN and BSP in si-NC-Hyp group were significantly higher than those in si-NC group(P<0.001).The expressions of HIF-1α,t-PERK,p-PERK,t-eIF2α and p-eIF2α were not significantly different between the si-ATF4 group and the si-NC group(P>0.05),while the expressions of ATF4,OCN and BSP were significantly decreased in the si-ATF4 group compared with the si-NC group(P<0.001),and rescued in the siATF4-Hyp group(P<0.001).Conclusion: 1.The distraction gap was in a hypoxic microenvironment.And the expression of ERS-related factors and osteogenic-related factors increased in the distraction gap.Hypoxic microenvironment could promote the proliferation,osteogenesis and migration of canine BMSCs.In addition,hypoxic microenvironment promoted the expression of ERS related factors and osteogenesis related factors of canine BMSCs,which triggered ERS to some extent,and further activated the PERK/eIF2α/ATF4 signaling pathway.2.Interfering ATF4 inhibited the proliferation,migration,and osteogenesis of canine BMSCs.While hypoxic microenvironment promoted the proliferation,migration,and osteogenesis of canine BMSCs,PERK/eIF2α/ATF4 signaling pathway plays an important role in it. |
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