| Maxillofacial fractures and defects are frequently-occurring diseases in oral surgery.However,the healing of maxillofacial bone defects is an extremely complex process,and bone repair activities mainly include:osteoclast-guided bone resorption,osteoclast-osteogenic coupling,and restoration of new bone by osteoblasts.How to accelerate the healing of maxillofacial defects from these three stages has always been a compelling difficult.Maxillofacial bone is a vastly vascularized tissue.There is rich blood supply in bone to maintain its nutrition.Its blood supply accounts for about 10%of cardiac output.However,due to closed fractures,inflammation,infection,tumor,many local or systemic factors can cause interruption or reduction of blood flow in bone tissue,thereby causing tissue hypoxia.After jaw trauma,the jaw tissue is in a state of hypoxia,and the hypoxia-inducible factor HIF-1αmight be crucial for bone repair.Our research group’s previous research found that HIF-1αcan accelerate bone remodeling by activating osteoclasts.Based on this,we put forward the hypothesis that the jaw defect area is in a state of hypoxia,and HIF-1αregulates the healing process of the mandible by affecting the resorption function of osteoclasts and its coupling effect on osteoblasts.Therefore,in this study,we established a mouse model of unilateral mandibular defect and constructed osteoclast-specific knockout HIF-1αmodel mice to study the phenotypic changes in the healing process of mandibular defects.RNA sequencing screened the possible signaling pathways and osteoclast-osteogenesis coupling factors,and studied the role and molecular mechanism of HIF-1αin osteoclasts in the process of jaw healing from two aspects:phenotype and mechanism.This study consists of the following five parts:Part Ⅰ Expression of HIF-1αin osteoclasts in the process of mandibular healing in miceObjective:To construct a wild type（WT）mouse model of mandibular bone defect,and to observe the expression of HIF-1αin osteoclasts in the mouse mandibular bone defect model.Methods:Male WT mice of 6-week-old C57BL/6J genetic background were used to establish a unilateral mandibular bone defect model.The mandibular samples were collected 3 days,7 days and 14 days after modeling,fixed and decalcified for paraffin sectioning.Trap and immunofluorescence staining were used to observe the distribution of osteoclasts in mouse bone defects and the expression of Ctsk and HIF-1αimmunofluorescence co-localization.PCR technology was used to detect the gene expression of tnfrsf11a,csf1r,mmp9,ctsk,HIF-1α,and the expression of HIF-1αprotein.Results:1.The osteoclast-related genes tnfrsf11a,csf1r,trap,mmp9 and ctsk were highly expressed after mandibular bone defect,and the genes and proteins of HIF-1αrelated to hypoxia were all highly expressed.2.TRAP staining showed that many multinucleated TRAP-positive osteoclasts were recruited in the bone defect area 7 days after operation for bone resorption;immunofluorescence co-localization results showed that high-expressing HIF-1αosteoclasts appeared in the bone defect.Conclusion:Seven days after the operation of the mandibular defect in mice,the HIF-1αgene and protein were highly expressed in the bone defect area,the osteoclasts were highly active at the fractured end of the bone,and a large number of HIF-1αpositive osteoclasts were recruited to the bone defect.Part Ⅱ Effects of intra-osteoclast knockout of HIF-1αon the healing process of mouse mandibleObjective:To explore the effect of HIF-1αin osteoclasts on the healing of the mandible in mice.Methods:The Ctsk-cre tool mouse with the genetic background was used to cross with HIF-1αflox/flox mouse（Ctrl）to obtain HIF-1αflox/flox;Ctsk-cre mice were targeted for osteoclasts Conditional knockout（Cko）model of HIF-1α,unilateral mandibular bone defect model was established in Ctrl group mice and Cko group mice at 6 weeks of age,and the mandibles were harvested at 1,3,and 5 weeks after surgery.The collected mouse mandible samples were fixed,decalcified,and embedded in paraffin.The histological sections were subjected to Trap staining,immunohistofluorescence staining,HE staining,ALP staining,and OCN immunohistochemical staining.Some mice were injected intraperitoneally with calcein and xylenol orange every other week after modeling,and the collected mouse mandible samples were fixed,dehydrated,embedded in resin,and stained with DAPI and von Kossa on hard tissue sections.Results:1.Compared by Ctrl group,the bone healing process of the mice in the Cko group was significantly delayed.The thickness of the trabecular bone in the Cko group was smaller,the degree of separation of the trabecular bone was larger,and the number of trabecular bones was higher.The ratio of bone volume decreased.2.Compared by Ctrl group,the Trap-positive area of the mandibular defect in the Cko group was significantly reduced,and the number of multinucleated osteoclasts in the trabecular bone was significantly reduced.Ctsk and HIF-1αco-localized yellow signals around the trabecular bone in the Ctrl group,but no co-localization signal was found in the mandibular defect of the Cko group.3.The result of HE stains shown trabecular bone area in mandibular defect mice in the Cko was less,the space between the trabecular bone was larger,the defect area was not filled with connective tissue,and a small amount of bone fragments were not absorbed.ALP shown that the mandibular defect in the Ctrl group was heavily stained with alkaline phosphatase aggregation,while the Cko group stained lighter and smaller.Immunohistochemical staining of OCN showed that OCN staining in Cko was weaker than Ctrl group.4.Fluorescence double-labeling showed that the new woven bone with red signal in the Ctrl group was reticular,and area was larger than Cko group.The new bone tissue in the Cko group was progressive in layers,and there was no obvious woven bone morphology.Von Kossa shown that defect in the Ctrl was basically filled by woven bone,while in the Cko group,the defect was less deeply stained with hard tissue,and defect filled by connective tissues,and the defect healing was significantly delayed.Conclusion:Conditional knockout of HIF-1αin osteoclasts affects bone resorption in defect area,delays healing process of mandibular defects.Hypoxia-inducible factor-1αis beneficial to the healing process of mandibular bone defects.Part Ⅲ Effects of HIF-1αon the Biological Function of OsteoclastsObjective:To investigate the effects of osteoclast biological function after knocking out HIF-1αin osteoclastsMethods:The monocytes of 4-week-old mice in the Ctrl group and the Cko group were extracted to induce osteoclast differentiation,and the expression of osteoclast-related genes and proteins ctsk and mmp9,as well as hypoxia-related genes HIF-1αwere detected by cobalt chloride,a hypoxia mimetic.The differentiation of osteoclasts was detected by Trap staining,the formation of pseudopodia of osteoclasts was detected by immunofluorescence,bone resorption of osteoclasts was detected by bone resorption experiment,and the morphological changes of osteoclasts were observed by scanning electron microscope.Results:1.The expression of ctsk,mmp9 and HIF-1αgenes in osteoclasts in the Ctrl group were increased under the stimulation of hypoxia mimics,while the hypoxia mimics had no significant effect on the Cko group.2.In the Ctrl+Co Cl2 group,the osteoclasts number was larger,the cell surface area was larger,the Trap positive area was the largest,the osteoclast differentiation was the most active,and the bone resorption area was the largest,followed by the Ctrl group without Co Cl2.In the Cko group,no matter whether the addition of Co Cl2 had no significant effect on the differentiation of osteoclasts,the osteoclasts number in the Cko group was less,the cell surface area was reduced,the number of nuclei was about three to five,the bone resorption area was small,and the osteoclasts were small.Cell differentiation is affected.3.The osteoclasts in the ko group had short and round pseudopodia,while the osteoclasts in the ctrl group extended multiple pseudopodia with irregular shape and large body surface area.The bone lacuna was scanned after the osteoclasts on the bone surface were washed away.It could be seen that the bone lacuna in the Ctrl group had a larger area and deeper absorption lacuna compared with the ko group,and a large number of pits appeared on the bone surface with rough surface.Conclusion:HIF-1αmay regulate osteoclast differentiation and modulates bone resorption,and regulates the formation of pseudopodia and cytoskeleton of osteoclasts during the differentiation process.Part Ⅳ Trasnscriptomic analysis of HIF-1αknockdown in osteoclastsObjective:To explore the related mechanism of HIF-1αon the biological function changes of osteoclastsMethods:RNA-seq sequencing technology was used to further screen target genes,R language software package was used to draw differentially expressed genes,and top GO was used for GO enrichment analysis.According to the KEGG enrichment analysis results of differentially expressed genes,cytoskeleton-related Rho,Rock1pathway analysis,Rho and Rock1 staining by immunofluorescence,PCR detection of gene expression,and WB detection of protein expression.Results:1.The mRNA expression of osteoclasts in Cko group and Ctrl group changed in a total of 1320 mRNAs,480 mRNAs were up-regulated and 840 mRNAs were down-regulated in Cko group.2.GO analysis showed significant changes in the cytoskeleton,signal connections and cell surface receptors in knockout HIF-1αosteoclasts,and KEGG pathway analysis showed significant changes in local adhesion,cytoskeleton and cell adhesion molecule pathways.3.Signal intensity of Rho and Rock1 in Ctrl group was higher and there were a lot of strong positive signals at the cell fusion and pseudopodia,and a lot of filamentous processes around the cell membrane.The expression of Rho and Rock1 in the KO group was basically around the nucleus,with few pseudopodia and almost no signal of Rho and Rock1.PCR and WB results shown expression of Rho and Rock1 in the Ctrl group was higher.Conclusion:HIF-1αregulates pseudopodia formation and cytoskeleton of osteoclasts through HIF-1α/Rho/Rock1 during differentiation.Part Ⅴ Research of HIF-1αon the regulation of osteoclast-osteogenesis coupling Objective:To explore the role of HIF-1αin osteoclasts in regulating osteoclast-osteogenic couplingMethods:The osteoclast supernatants of the Ctrl group and the Cko group were collected as conditioned medium to co-culture bone marrow stromal cells（BMSC）,and the migration and osteogenic differentiation changes of BMSC were observed.The osteoclast-osteogenesis coupling factor cardiotrophin-1（CT-1）was screened by Elisa technology and RNA-seq,and exogenous CT-1 was used to rescue the effect of knockout of HIF-1αin osteoclasts.Treatment of decreased osteogenic capacity and delayed bone healing of BMSC.Results:1.Compared with the supernatant of the Cko group,the supernatant of the Ctrl group enhanced the migration of BMSC and the expression of osteoblast differentiation-related genes（alp and runx2）.ALP and alizarin red staining showed that the supernatant of the Cko could not improve osteogenic differentiation capacity.2.The secreted factor CT-1 related to osteoclast-osteogenesis coupling in Cko group in Elisa experiment was significantly decreased.The expression changes of CT-1 gene and protein were detected by PCR and Western Blot.Ctrl group was higher.Cell immunofluorescence staining showed that CT-1 was normally expressed in osteoclasts,but after knocking out HIF-1α,the expression of CT-1 in osteoclasts was significantly reduced.It was found by tissue immunofluorescence staining that after mandibular osteotomy,expressions of CT-1 in mandibular defect area of Ctrl was higher than Cko.3.Alizarin red staining showed that CT-1 could significantly enhance the osteogenic differentiation ability of BMSC.Osteoclast supernatant in Cko group+CT-1 could rescue the decreased osteoclast coupling ability caused by knockout of HIF-1αin osteoclasts.By labeling CT-1 protein with FITC,CT-1 protein can be taken up by BMSC and promote their migration and differentiation.Conclusion:HIF-1αpromotes osteoclast secretion coupling factor CT-1,which acts on BMSCs to promote their migration and osteogenic differentiation.Exogenous CT-1 can rescue osteoclasts caused by knockout of HIF-1α.Decreased bone coupling ability. |
