Lipus Promotes Periodontal Regeneratin Through SDF-1/CXCR4 Signal Pathway Mediated PDLSCs Migration And BMSCs Homing

Periodontitis is accompanied by inflammation and alveolar bone loss,and the progression of periodontitis can lead to tooth loss.To restore lost periodontal structures,a number of treatments including guided tissue regeneration,bone grafting and enamel matrix derivatives are available in animal models,as well as clinical settings.However,satisfied regeneration is hardly observed.Low-intensity pulsed ultrasound(LIPUS)is a noninvasive acoustic radiation at intensities ranging from 30 to 100 mW/cm2.LIPUS has been demonstrated to accelerate fracture healing in both experimental and clinical studies.Since LIPUS displayed beneficial effects in accelerating the fracture healing process,more and more studies have been conducted to treat periodontal disease.From a biological perspective,it has been reported that mechanical stress induces a variety of cellular events including proliferation,differentiation,and migration.LIPUS may have a similar function since it transmits mechanical energy as acoustical pressure waves into cells and tissues.Recruitment of stem cells would be a new target of periodontal treatment.Stromal cell-derived factor 1(SDF-1),also known as C-X-C motif chemokine ligand 12(CXCL12),is a type of chemokine expressed by a variety of tissues.SDF-1 can promote the recruitment and regulate the migration,proliferation and differentiation of stem/progenitor cells in tissue/organ damaging condition.The SDF-1/CXCL4 pathway plays a crucial role in injury repair.Previous study demonstrated that the SDF-1 can promote the proliferation and migration of hPDLSCs,as well as elevate the expression level of COL-I in hPDLSCs.The significant role of SDF-1 in stem cell homing and tissue regeneration has been well-demonstrated in the literature.Twist-related protein 1(TWIST1)encodes a basic helix-loop-helix transcription factor,known to contribute to epithelial-mesenchymal transition,Tumor invasion and progression,Mesenchymal cell differentiation,mesodermal and skeletal tissue development and craniosynostosis.TWIST1 may be related to the function of alveolarperiodontal ligament complex and regulates the periodontium remodeling.TWIST1 can elevate the expression level of SDF-1 in a dose-dependent manner.TWIST1 was found to directly activate SDF-1 expression,which could promote cell migration.These results suggested that TWIST1 and SDF-1/CXCR4 singnal pathway may be involved in the signal transduction of LIPUS.Therefore,in this study,we foucus on the the SDF1/CXCR4 signalling pathway,cell migration and cell homing,which might be promoted by LIPUS.In-vitro cell experiment: human periodontal ligament stem cells(hPDLSCs)were isolated from premolars.Flow cytometry and differentiation assays were used to characterize hPDLSCs.LIPUS treatment was given to hPDLSCs,and stromal cell-derived factor-1(SDF-1)expression levels were tested by real-time PCR with or without blocking the SDF-1/CXCR4 pathway by AMD3100.Enzyme-linked immunosorbent assay(ELISA)was performed to evaluate SDF-1 secretion in hPDLSCs.Wound healing and Transwell assays were conducted to assess the migrationpromoting effect of LIPUS.A potential upstream gene of SDF-1,twistrelated protein 1(TWIST1),was knocked down by siRNA transfection.The results showed that LIPUS promoted the expression of TWIST1 and SDF-1 at both the mRNA and protein levels.In addition,LIPUS enhanced the cell migration of hPDLSCs.Knockdown of TWIST1 impaired the expression of SDF-1 and cell migration of hPDLSCs.TWIST1 may be an upstream regulator of SDF-1 in hPDLSCs.Taken together,these findings indicate that the SDF1/CXCR4 signalling pathway is involved in LIPUS-promoted hPDLSCs migration,which might be one of the mechanisms for LIPUSmediated periodontal regeneration.TWIST1 might be a mechanical stress sensor during mechanotransduction.In-vivo Animal experiment: The isolated rat Bone Mesenchymal stem cells(BMSCs)were transfected by lentiviralvector-LUC-GFP.The BMSCs with stable expression of LUC and GFP were harvested and subcultured for further animal experiment.Six weeks old rats were randomly divided into blank group,periodontal defect group,periodontal defect+BMSCs group,defect+BMSCs+LIPUS group(n=5).Acute periodontal defect was established among groups.The BMSCs expressing LUC and GFP gene were injected via tail vein of rats in defect+BMSCs group and defect+BMSCs+LIPUS group.Optical in vivo Imaging was conducted on 1 day,3 days,1 week and 4 weeks after the injection to study the distribution of labeled BMSCs.The difference in periodontal healing was studied by HE and Masson staining among groups.Immunohistochemical(osteopontin OPN,Collagen Type I COLI)method was further applied to validate prior result.Optical in vivo Imaging showed no signal was detected in blank group and defect group.Fluorescence signals were detected in limbs,organs and maxillofacial region in defect+BMSCs group and defect+BMSCs+LIPUS group.Higher signal intensity were found in defect+BMSCs+LIPUS group.More new bone was found in defect+BMSCs+LIPUS group by HE and Masson staining.More positive cells were found in defect area of defect+BMSCs+LIPUS group by immunohistochemistry(OPN,COL1).The high level of OPN and COLI expression indicates the active reconstruction of new bone.Taken together,these findings indicate that LIPUS may promote BMSCs migrate to defect area,which assist healing of defect.In addition,LIPUS may promote periodontal regeneration by facilitating blood vessel and new bone formation through paracrine effect.