The Synergistic Effects Of Stromal Cell-derived Factor-1 Combined With Exendin-4 On Periodontal Bone Tissue Regeneration

Background and Objective:Periodontitis is an oral infectious disease characterized by the destruction of the supporting tissues around teeth.The loss of periodontal ligament attachment and periodontal bone destruction caused by periodontitis has already become the main cause of tooth loss in adults.The development of periodontitis can be well controlled by existing treatment strategies.However,regeneration of the damaged periodontium,especially the periodontal bone destruction is still a challenge for clinical practice.In situ periodontal tissue engineering aims to enhance the migration,recruitment,proliferation and differentiation of functional cells around local tissues and peripheral blood circulation to regenerate periodontium and repair the defect.In situ periodontal tissue engineering might provide a new strategy for periodontal tissue regeneration.Periodontal ligament stem cells(PDLSCs)are one of adult mesenchymal stem cells(MSCs),which have the potential of self-renewal and multidirectional differentiation.PDLSCs are considered to be the most favorable source of odontogenic stem cells for periodontal regeneration.Stromal cell-derived factor-1(SDF-1)plays an important role in recruiting multiple stem cells by activating C-X-C chemokine receptor type 4(CXCR4).It provided cell sources for tissue regeneration by recruiting functional cells for migration and homing to the defect areas.However,SDF-1 itself lacks enough capacity to facilitate osteogenic differentiation of stem cells,and the promotive effect on periodontal bone tissue regeneration is unsatisfactory.As a mature hypoglycemic drug,metformin(MF)has been used in clinical for years.In addition to the capacity of blood glucose controlling,MF possesses the capacity of anti-inflammation,anti-tumor,anti-aging,and it can also promote the osteogenic differentiation of multiple stem cells.Exendin-4(EX-4),a glucagon-like peptide-1(GLP-1)analog,can upregulate bone metabolism in addition to neuroprotective,cardiac protective,anti-inflammatory and anti-oxidant effects.EX-4 plays an important role in regulating SDF-1/CXCR4 signaling pathway.EX-4 can not only enhance the recruitment of MSCs by upregulating the expression of CXCR4,but also facilitate osteogenic differentiation of MSCs.Therefore,to screen a suitable drug that could promote bone tissue regeneration,this present study first investigates the effect of MF and EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs in vitro.Moreover,the synergistic effect on CXCR4 expression was evaluated.Meanwhile,exploring the combinative effect of SDF-1 and EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs in vitro and periodontal bone regeneration of the mandibular periodontal bone defect model in vivo was explored in this study.Materials and Methods:1 Effects of MF,EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs1.1 Isolation,cultivation and identification of human PDLSCsPeriodontal ligament was stripped from healthy premolars extracted for orthodontic reasons.PDLSCs were isolated and cultured by limited dilution method,and the phenotypic characterization and expression of stem cell markers were detected by flow cytometry.1.2 Effects of MF,EX-4 on the proliferation and migration of PDLSCsCell-counting kit 8(CCK-8)and Transwell assays were performed to detect the effects of MF,EX-4 on the proliferation and migration of PDLSCs.1.3 Effects of MF,EX-4 on the osteogenic differentiation of PDLSCsThe effects of MF,EX-4 on osteogenic differentiation of PDLSCs were evaluated by alkaline phosphatase(ALP)activity,Alizarin red staining,cetylpyridinium chloride(CPC)colorimetry and quantitative real-time polymerase chain reaction(qRT-PCR).2 The synergistic effects of SDF-1 combined application with EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs2.1 Detection the CXCR4 receptor expression of PDLSCsThe expression of CXCR4 on PDLSCs were detected by immunofluorescence staining.2.2 Screening the concentration of SDF-1CCK-8 and Transwell assays were used to screen the approprite concentration of SDF-1 on PDLSCs.2.3 Screening the combined application drugThe synergistic effects SDF-1 combined with MF and EX-4 respectively on the expression of CXCR4 receptors on PDLSCs were detected by qRT-PCR,the suitable drug with stronger synergistic effects were selected for the subsequent experiments2.4 The synergistic effects of SDF-1 combined application with EX-4 on the proliferation and migration of PDLSCsCCK-8 and Transwell assays were performed to detect the synergistic effects of SDF-1 combined application with EX-4 on the proliferation and migration of PDLSCs.2.5 The synergistic effects of SDF-1 combined application with EX-4 on the osteogenic differentiation of PDLSCsThe synergistic effects of SDF-1 combined application with EX-4 on the osteogenic differentiation of PDLSCs were detected by ALP activity,Alizarin red staining,CPC and qRT-PCR analysis3 The synergistic effects of SDF-1 combined application with EX-4 on periodontal bone tissue regeneration in Wistar rat defect model3.1 Establishment of mandibular periodontal bone defect models in Wistar rats8 weeks,220-250 g weight male Wistar rats were randomly assigned into two groups:Group ?:EX-4 intraperitoneal injection group;Group?:normal saline intraperitoneal injection group.A 5×4×1 mm periodontal bone defect model was established on bilateral mandibular bones.SDF-1 loaded collagen membrane was inserted into the right defect and PBS loaded collagen membrane was inserted into the left defect.Mandibles specimens were obtained at 3 d,1 w,2 w,and 4 w after surgery for the following experiments.3.2 The synergistic effects of SDF-1 combined application with EX-4 on the mobilization of MSCs and CXCR4+cells in periodontal bone defectDouble immunofluorescence staining of CD90+/CD34-and immunofluorescence staining of CXCR4 of the paraffin section were performed to calculate the number of MSCs and CXCR4+cells in the site of periodontal bone defect to detect the synergistic effects of SDF-1 with EX-4 on the mobilization of MSCs and CXCR4+cells.3.3 The synergistic effects of SDF-1 combined application with EX-4 on the periodontal bone tissue regeneration in periodontal bone defectMicro-computed tomography(Micro-CT)were performed to detect the quantity and quality of regenerated bones by measuring bone volume/tissue volume(B V/TV),bone surface area/tissue volume(BS/TV),trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp)in the defect area.Hematoxylin&eosin(H&E)staining of the paraffin section was performed to calculate the new bone area.TRAP staining of the paraffin section was performed to calculate the number of osteoclasts in defect area among four groups.Immunohistochemical stainings of the paraffin section were performed to observe the osteogenesis-related proteins ALP,collagen type I(Col I)expression during the healing process.Results:1 Effects of MF,EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs1.1 Isolation,cultivation and identification of human PDLSCsHuman PDLSCs could be successfully cultivated by limited dilution method.The primary cells cultured in vitro showed typical spindle shape.Human PDLSCs possessed the ability of colony-forming,and were positive for MSCs-associated markers CD29,CD44 and CD73,and negative for hemopoietic stem cells(HSCs)marker CD45.1.2 Effects of MW,EX-4 on the proliferation and migration of PDLSCsResults of CCK-8 showed that 10 ?M MF had no effect on the proliferation of PDLSCs(P>0.05).50 and 100 ?M MF significantly enhanced the proliferation of PDLSCs(P<0.01).5,10,20 and 50 nM EX-4 showed no effect on the proliferation of PDLSCs(P>0.05).Results of transwell showed that compared with negative control(NC)group,all concentrations of MF and EX-4 promoted the migration of PDLSCs(P<0.01),50?M MF and 10 nM EX-4 exhibited the maximal effects(P<0.01).1.3 Effects of MF,EX-4 on the osteogenic differentiation of PDLSCsResults of ALP activity showed that compared with control group,all concentrations of MF and EX-4 significantly enhanced ALP activity of PDLSCs(P<0.05),50 ?M MF and 10 nM EX-4 exhibited the maximal effects(P<0.05).Results of Alizarin red S staining and CPC colorimetry showed that compared with control group,50?M MF and 10 nM EX-4 significantly promoted the extracellular calcium deposition of PDLSCs(P<0.001).Results of qRT-PCR showed that compared with control group,the expression of osteogenesis marker ALP,runt related transcription factor 2(Runx2)and osteocalcin(OCN)was significantly promoted by the concentration of 50 ?M MF and 10 nM EX-4(P<0.05).2 The synergistic effects of SDF-1 combined application with EX-4 on the proliferation,migration and osteogenic differentiation of PDLSCs2.1 The CXCR4 receptor expression of PDLSCsImmunofluorescence staining showed positive expression of CXCR4 on PDLSCs.2.2 Screening the concentration of SDF-1Results of CCK-8 and transwell showed that compared with other concentrations,50 ng/mL SDF-1 exhibited the maximal promoted effect on the proliferation and migration of PDLSCs(P<0.05).2.3 Screening the combined application drugqRT-PCR results showed that compared with control group,SDF-1 group and SDF-1+MF group significantly promoted the expression of CXCR4 on PDLSCs(P<0.001),there was no significant difference beteen SDF-1 group and SDF-1+MF group(P>0.05).SDF-1+EX-4 group significantly promoted CXCR4 expression compared with control,SDF-1 group and EX-4 group(P<0.01).Therefore,EX-4 was screened out for combining application with SDF-1 in subsequent experiments.2.4 The synergistic effects of SDF-1 combined application with EX-4 on the proliferation and migration of PDLSCsResults of CCK-8 showed that compared with control group,SDF-1 apparently promoted the viability of PDLSCs(P<0.001),and SDF-1 combined application with EX-4 significantly enhanced the proliferation of PDLSCs with synergistic effects(P<0.001),which was superior to single application groups(P<0.001).Results of transwell showed that compared with NC group,SDF-1,EX-4 significantly promoted the migration of PDLSCs(P<0.001),and SDF-1 combined application with EX-4 significantly enhanced the migration of PDLSCs with synergistic effects(P<0.001),which was superior to single application groups(P<0.001).2.5 The synergistic effects of SDF-1 combined application with EX-4 on the osteogenic differentiation of PDLSCsResults of ALP activity showed that compared with control group,SDF-1,EX-4 significantly promoted the ALP activity of PDLSCs(P<0.001),and SDF-1 combined application with EX-4 significantly enhanced the ALP activity of PDLSCs with synergistic effects(P<0.001),which was superior to single application groups(P<0.01)Results of Alizarin red S staining showed that compared with control group,SDF-1,EX-4 significantly enhanced the extracellular calcium deposition of PDLSCs(P<0.001),and SDF-1 combined application with EX-4 significantly enhanced the extracellular calcium deposition of PDLSCs with synergistic effects(P<0.001),which was superior to single application groups(P<0.001).Results of qRT-PCR showed that SDF-1 combined application with EX-4 significantly upregulated the expression levels of osteogenic related genes ALP,OCN,Osteopontin(OPN)of PDLSCs(P<0.05),and the synergistic effects were superior to single application groups(P<0.05)3 The synergistic effects of SDF-1 combined application with EX-4 on periodontal bone tissue regeneration in Wistar rat defect model3.1 Establishment of mandibular periodontal bone defect models in Wistar rats The experimental animals stayed a good state after awakening from surgical anesthesia without obvious inflammation and immune rejection.3.2 The synergistic effects of SDF-1 combined application with EX-4 on the mobilization of MSCs and CXCR4+cells in periodontal bone defectsDouble immunofluorescence of CD90+/CD34-results showed that the number of MSCs in SDF-1+EX-4 group were more than other groups at early stage(3 d,1 w,2 w)(P<0.05).The immunofluorescence staining of CXCR4 showed that SDF-1 combined application with EX-4 increased the number of CXCR4+cells at the early stage(3 d,1 w,2 w),and the synergistic effects were superior to single application groups(P<0.05).3.3 The synergistic effects of SDF-1 combined application with EX-4 on periodontal bone tissue regeneration in Wistar rat defect modelMicro-CT results revealed that SDF-1 combined application with EX-4 significantly promoted the quantity of regenerated bone,as well as enhanced BV/TV,BS/TV and Tb.Th at each time point(1,2,4,8 w),while the Tb.Sp dramatically declined compared with NC group and single application groups(P<0.05).H&E staining showed that the combined application group enhanced early osteogenesis during the defects healing process,and the newly formed bone volume and density were higher than NC group and single application groups(P<0.05).TRAP staining showed that the combined application of SDF-1 with EX-4 induced early bone osteoclastogenesis(3 d,1 w),the number of osteoclasts significantly increased compared with NC group and single application groups(P<0.05)Immunohistochemical staining indicated that the combined application of SDF-1 with EX-4 significantly promoted the osteogenesis-related protein of ALP(1,2 w)and Col I(2,4,8 w)expressions,and the synergistic effects were superior to single application groups(P<0.05).Conclusions:1 Human PDLSCs were successfully isolated and cultivated with limited dilution method2 MF significantly promoted the proliferation,migration and osteogenic differentiation of PDLSCs,and 50 ?M showed the maximal effect.3 EX-4 obviously promoted the migration and osteogenic differentiation of PDLSCs,and 10 nM had the optimal effect4 CXCR4 expressed positively on PDLSCs,and SDF-1 combined application with EX-4 synergistically upregulated CXCR4 expression.Combination with SDF-1 and EX-4 exhibited synergistic promotive effects on the proliferation,migration and osteogenic differentiation of PDLSCs,and the synergistic effects were significantly superior than single application.5 SDF-1 combined application with EX-4 synergistically enhanced the recruitment ability of SDF-1 to recruit more MSCs to periodontal bone defect area,provided sources of stem cells for periodontal bone regeneration.6 SDF-1 combined application with EX-4 synergistically promoted new bone formation and significantly enhanced periodontal bone regeneration in rat defects.The synergistic promotive effects were significantly superior than single application.