The Role Of Enamel Matrix Derivative On The Odontoblastic Differentiation Of Human Dental Pulp Stem Cells And The Underlying Mechanisms

Objective: Dental pulp and dentin are both histologically derived from the dental papilla of tooth germs.They are biologically as a whole,namely pulpo-dentinal complex,which response connectively to the external stimulus.Odontoblasts could secrete dentin matrix and weigh heavily for the tooth development.Numerous studies have demonstrated that dental pulp stem cells(DPSCs)could proliferate and differentiate into functional odontoblasts and regenerate pulpo-dentinal complex when facing undesirable outside stimulus.Thus,the crucial step for pulp-dentin regeneration is to explore the way of inducing dental stem cells to differentiate into odontoblasts.Enamel matrix proteins(EMPs)are a complex of proteins that synthesized and secreted by inner enamel epithelial cells from enamel organ and epithelial root sheath.During the development of crown of tooth,EMPs were secreted to regulate the mineralization and maturation of enamel.When it comes to the root of tooth,EMPs were excreted to induce nearby mesenchymal cells to differentiate into odontoblasts and cementoblasts.Therefore,EMPs are one of the most considerable epithelial signals that play a crucial role throughout the tooth development.The components of EMPs are complex,including amelogenin(Am),enamelin(En),ameloblastin(Ambn),proteases,transforming growth factor ?1(TGF-?1),bone morphogenetic protein-2(BMP-2),and many other growth factors.EMPs are generally extracted from porcine tooth germs,namely enamel matrix derivative(EMD).EMD is now commercially available abroad as Emdogain and has been widely applied to clinical periodontal tissue regeneration and dental implantation.It is well documented that EMD could enhance the proliferation and differentiation of periodontal ligament stem cells(PDLSCs),osteoblasts and gingiva-derived mesenchymal stem cells(GMSCs),as well as promote periodontal hard and soft tissue regeneration.The application of EMD in periodontal tissue regeneration has gained mature experience,but its application in the field of endodontics is still in the initial and developing stage.So far,only a few studies have explored its potential for enamel remineralization and direct pulp capping in vitro.However,whether EMD could promote DPSCs odontoblastic differentiation and its underlying mechanisms remain unclear.In order to investigate the effect of EMD on odontoblast differentiation of DPSCs in vivo,an ideal cell carrier is required to create a functional and optimized microenvironment for the adhesion,proliferation and differentiation of DPSCs.Vitro Gel 3D-RGD used in our study is a ready-to-use,xeno-free tunable hydrogel designed to imitate the endogenous physiological microenvironment for in vitro and in vivo 3D culture of cells.As an injectable hydrogel,Vitro Gel 3D-RGD is suitable for in vivo study,with high porosity and good biocompatibility.Besides,favorable interplay has been found between the hydrogel and a variety of cell types,including prostate cancer cells,breast cancer cells,and bone marrow stroma cells.Mitogen-activated protein kinase(MAPK)signaling pathway is one of the vital mechanisms that take part in the regulation of cell proliferation and odonto/osteogenic differentiation.To date,numerous studies have demonstrated the regulatory role of MAPK signaling pathways in the odontoblastic differentiation of DPSCs.It has been reported that BMP-2,MTA and Biodentine can promote DPSCs odontoblastic differentiation through triggering MAPK signaling pathways.However,it is still uncertain whether the MAPK signaling pathways is involved in the EMD-induced odontoblastic differentiation of DPSCs.Since EMD is the crucial epithelial signal that activates and regulates odontoblastic differentiation during tooth development,we hypothesized that EMD might be able to reactivate the interplay between epithelium and mesenchyme,thereby promoting the odontoblastic differentiation of DPSCs and achieving the ultimate goal of pulp-dentin regeneration.In this work,we explored the influence of EMD on the odontoblastic differentiation and viability of DPSCs in vitro,and observed the potential of EMD in promoting DPSCs odontoblastic differentiation in vivo with Vitro Gel 3D-RGD served as an injectable scaffold.Then,we further explored the potential role of MAPK signaling pathways during odontoblastic differentiation of EMD-treated DPSCs to provide theoretical foundations for the application of EMD in the biological treatment of pulpal and periapical diseases.Methods:1.Isolation,characterization of human DPSCs and the effect of EMD on its cell viability1.1 DPSCs were isolated from sound dental pulp tissue and were passaged to acquire single cell cloning by serial dilution.1.2 Flow cytometric analysis was conducted to detected the expression of cell surface markers CD29,CD34,CD45,CD90,CD105,CD146,and STRO-1.And the expression of mesenchymal stem cells surface antigens STRO-1,CD105,and CD146 were further observed by immunofluorescence staining.1.3 Following odonto/osteogenic induction for 14 days,DPSCs were tested for mineralized nodules deposition by alizarin red staining.Besides,DPSCs were detected for lipid droplets by Oil red O staining after 21 days of adipogenic differentiation induction.1.4 DPSCs were treated with 25,50,and 100 ?g/mL of EMD for 1,3,5,7,and 9 days.A cell counting kit-8(CCK-8)assay was conducted to evaluated the effect of EMD on DPSCs viability.2.Effects of EMD on the odontoblastic differentiation of DPSCs in vitro and in vivo2.1 DPSCs were treated with 25,50,and 100 ?g/mL of EMD for 3,7,and 14 days.And the ALP content of DPSCs was evaluated using the Alkaline Phosphatase Assay Kit.The ALP staining assay was conducted after 7 days of incubation to evaluate the effect of EMD on the ALP activity of DPSCs.2.2 Alizarin red staining and quantification were performed to explore the effect of EMD on the calcium deposition of DPSCs at day 14 and to seek for the optimum concentration of EMD for subsequent analyses.2.3 Cells were cultured with 100?g/mL of EMD for 3,7,and 14 days.And the mRNA expression levels of odontoblastic related marker genes,including RUNX2,ALP,BSP,DMP1,DSPP,and OCN,were detected by real-time RT-PCR.2.4 The protein expression levels of RUNX2,ALP,BSP,DMP1,DSPP,and OCN,were detected by western blot analysis after 3 and 14 days of EMD treatment.2.5 DPSCs were 3D cultured in Vitro Gel 3D-RGD for 3 and 7 days.The ultrastructural feature of Vitro Gel 3D-RGD and its effect on the growth of DPSCs were observed by means of scanning electron microscopy(SEM).2.6 We conducted in vivo experiment by ectopic subcutaneous injection with Vitro Gel 3DRGD served as an injectable scaffold.After 6 weeks of operation,the specimens were collected and then sectioned for histological and immunohistochemical analyses.2.7 The effect of EMD on DPSCs odontoblastic differentiation in vivo was observed by H&E staining,Masson's trichrome staining and immunohistochemical staining of DSPP and OCN.2.8 Von Kossa staining and CD31 immunohistochemical staining were performed to detect the hard tissue constructs and neovascularization,respectively.3.Involvement the MAPK signaling pathways in the EMD-induced odontoblastic differentiation of DPSCs3.1 DPSCs were stimulated with 100 ?g/mL of EMD for 5,10,30,60,and 120 min.The expression levels of MAPK signaling pathways related proteins,including JNK,p-JNK,p38 MAPK,p-p38 MAPK,ERK1/2,and p-ERK1/2,were detected by western blot analysis.3.2 Specific pathway inhibitors SP600125,SB203580,and U0126 were applied for blocking the JNK,p38 MAPK,and ERK pathways,respectively.The effects of inhibitors alone and co-cultured with EMD on the protein levels of the respective pathway proteins were detected by western blot analysis.3.3 After blocking off the JNK,p38 MAPK,and ERK pathways with specific pathway inhibitors,the protein expression levels of RUNX2,ALP,BSP,DMP1,DSPP,and OCN,were detected by western blot analysis after 7 days of EMD treatment.3.4 After blocking off the JNK,p38 MAPK,and ERK pathways,ALP activity and ALP staining were conducted to evaluated the ALP content of DPSCs after induction with EMD for 7 days.3.5 After blocking off the JNK,p38 MAPK,and ERK pathways,alizarin red staining and quantification were performed to explore the calcium deposition of DPSCs after induction with EMD for 14 days.Results:1.Identification of human DPSCs and the effect of EMD on their cell viability.1.1 The results of flow cytometric analysis revealed that the obtained cells were negative for haemopoietic stem cells surface markers CD34 and CD45,but positively expressed mesenchymal stem cells(MSCs)surface antigens CD29,CD90,CD105,CD146,and STRO-1.Immunofluorescence staining also displayed positive expression of MSCs surface antigens STRO-1,CD105,and CD146.Besides,multilineage differentiation testing indicated that the obtained cells were successfully induced towards osteogenic and adipogenic lineages.1.2 The results of CCK-8 showed that 25,50,and 100 ?g/mL of EMD had little effect on the viability of DPSCs,and no significant differences were observed between the EMD groups and the control group among all tested time points.2.Effects of EMD on the odontoblastic differentiation of DPSCs in vitro.2.1 The ALP content of the 50 and 100 ?g/mL group were markedly higher than the OM and control group.And the ALP activity in the 100 ?g/mL group was significantly higher than that in the 50 ?g/mL group on days 3 and 14.The results of ALP staining further confirmed the same conclusion.Meanwhile,25 ?g/mL of EMD appeared to have little effect on the ALP activity of DPSCs.2.2 Similarly,50 and 100 ?g/mL of EMD also promoted the formation of mineralized nodules in DPSCs.And the 100 ?g/mL group had significantly higher calcium deposition than the 50 ?g/mL group.Among all the EMD concentrations tested,DPSCs displayed the best odontoblastic differentiation capacity with 100 ?g/mL of EMD,and therefore we chose 100 ?g/mL as the optimum concentration of EMD for subsequent analyses.2.3 The real-time RT-PCR results revealed that the mRNA expression levels of RUNX2,ALP,and BSP in the EMD group were significantly higher than the control and OM group at day 3,while the odontoblastic related genes including DSPP,DMP1,and OCN were remarkably upregulated at day 14.Furthermore,the protein levels of these genes changed in consistent with their mRNA expression levels.Compared with the other groups,the protein expression levels of RUNX2,ALP,DMP1,and OCN in the EMD group were significantly higher at both time points,while the protein levels of BSP and DSPP were significantly higher only at day 3 and 14,respectively.3.Effects of EMD on DPSCs odontoblastic differentiation and regeneration in vivo.3.1 The results of SEM imaging presented that the Vitro Gel has a high porosity and homogeneous interconnected 3D network,which allows the hydrogel system to contain large amounts of water to get reliable viscosity while maintaining its shape.Moreover,we observed that Vitro Gel 3D-RGD could well support the seeding,adhesion,and proliferation of DPSCs,unfolding that the hydrogel has favorable biocompatibility with the cells.3.2 The tissue formation following 6 weeks of subcutaneous injection supported our hypothesis that EMD might be the key epithelial signal to promote odontoblastic differentiation of DPSCs.H&E staining illustrated that more collagen matrix and mineralized tissue were formed in the EMD group compared to the control group.Similarly,Masson's trichrome staining found that the EMD group had abundant dentin matrix deposition and more newly formed blood vessels compared with the control group.Besides,the cells exposed to EMD displayed stronger positive expression of DSPP and OCN,as detected by immunohistochemical staining.3.3 Then Von Kossa staining and CD31 immunohistochemical staining were performed to detect the hard tissue constructs and neovascularization,respectively.The regenerated tissue after 6 weeks of subcutaneous injection showed remarkably higher level of mineralization and micro-vessel formation in the EMD group compared with the control group.4.EMD enhances the odontoblastic differentiation of DPSCs via activating MAPK signaling pathways.4.1 The results of western blot analysis showed that the protein expression level of p-JNK and p-ERK1/2 increased rapidly after 5 min,peaked at 10 min,and then declined gradually.In addition,the protein level of p-p38 MAPK increased immediately and reached the highest level at 5 min.The ratios of p-JNK/JNK,p-p38 MAPK/ p38 MAPK,and pERK/ERK further revealed that EMD activated the JNK,p38 MAPK,and ERK pathways in DPSCs.4.2 To further explore the involvement of MAPK signaling pathways during the EMDinduced differentiation of DPSCs,specific pathway inhibitors SP600125,SB203580,and U0126 were applied for blocking the JNK,p38 MAPK,and ERK pathways,respectively.The effectiveness of the inhibitors was verified by western blot assay.Compared with the EMD group,the protein levels of p-JNK,p-p38 MAPK,and p-ERK1/2 were significantly suppressed by their corresponding inhibitors.4.3 After blocking off the JNK,p38 MAPK,and ERK pathways with specific pathway inhibitors,the protein expression levels of RUNX2,ALP,BSP,DMP1,DSPP,and OCN,were dramatically decreased in various degrees compared with the EMD group.4.4 Specific pathway inhibitors SP600125,SB203580,and U0126 also suppressed the ALP activity and mineralized nodules deposition which were enhanced by EMD.Conclusions:1.EMD does not interfere with DPSCs cell viability but can promote odontoblastic differentiation of DPSCs in vitro.2.EMD could enhance the odontoblastic and angiogenic differentiation of DPSCs in vivo,as well as the hard tissue regeneration.3.Blocking off the JNK,ERK,and p38 pathways inhibited odontoblastic differentiation of EMD-treated DPSCs,indicating that EMD may enhance DPSCs odontoblastic differentiation via activating MAPK signaling pathways.