| Pulpal and periapical diseases is one of the most common dental diseases that seriously harms human beings.Conventional root canal treatment can preserve the teeth,but it’s lose their resistance after the pulp is lost,the fragility increases,and the sensory function is lost,especially the undeveloped young permanent teeth.Dental pulp regeneration is the best way to obtain a biological treatment effect on a tooth.Mesenchymal stem cells(MSCs)play a very important role in the regeneration of dental pulp.BMSCs has been proved that could migrate to the damaged parts of the body,and this migration is related to various chemokines.Stromal cell derived factor-1(SDF-1)is the most important one.Due to the unique histological structure and environment of the pulp,the regeneration and restoration of the pulp tissue faces greater challenges.In order to promote the homing of endogenous stem cells to involve in pulp regeneration,the SDF-1/CXCR4 axis chemotaxis MSCs for homing and participating dental pulp regeneration.The homing of the MSCs cells and it’s biological effects was explored to provide new ideas for biotreatments of the pulpal and periapical diseases in the future.The subject is divided into the following 4 chapter: Chapter one :Chemotaxis effects of SDF-1 on Rat CXCR4-Bone Marrow Mesenchymal Stem Cells(BMSCs)-In Vitro Study.Oobjective: This study aimed to isolate,culture and identifing rat bone marrow mesenchymal stem cells,and detecting the CXCR4 expression,in order to evaluate the chemotactic effect of SDF-1 on BMSCs in vitro,and lay the foundation for further research.Methods: Through adherence method.The isolation,cultivation and purification of rat bone marrow mesenchymal stem cells was carried out through the adherence method.The cell morphology were observed,the growth curve were detected by CCK-8,and the cell surface markers were identified by flow cytometry.Respectively,osteogenic and adipogenic differentiation were Conducted.Immunohistochemistry was used to detect the expression of CXCR4 in bone marrow mesenchymal stem cells.Transwell chamber was used to evaluate the migration of BMSCs induced by SDF-1 in vitro.Results: The results showed that the cell morphology was spindle shaped and the shape was homogeneous.The cell colonies were arranged radially,and the proliferation rate was fast.The average passage time was three days,and it could be continuously and steadily passaged to the 10 generation.The growth of bone marrow mesenchymal stem cells was active.The cell surface markers of the third generation of bone marrow mesenchymal stem cells CD29,CD44 showed positive expression,while CD34,CD45 showed negative expression.The oil red O staining and alizarin red staining were positive after theconduction of adipogenic and osteogenesisa.CXCR4 was expressed in bone marrow mesenchymal stem cells.The results of the effect of SDF-1 on the migration of rat BMSCS in vitro show that,the number of BMSCs migration increased significantly after adding SDF-1,the difference was statistically significant(P<0.05),and the cell migration number was the highest at 50 ng/ml.The blocking of CXCR4 using 5μg/m L AMD3100 reduced the migrated cell number(P<0.05).Conclusion: The cultivated cells are multipotential mesenchymal stem cells and they can express CXCR4.SDF-1 significantly promoted the migration of BMSCs.The intensity of chemotaxis was related to the concentration of SDF-1.The effect was the strongest at 50 ng/ml.The blocking agent AMD3100 of CXCR4 could significantly inhibit the migration of BMSCs.However,the migration of cells cannot be completely blocked.Chapter two : Effect of periapical injury on the expression of CXCR4 in bone marrow cellsObjective: This study aimed to explore the relationship between local injury and systemic responses of stem cell mobilization,by evaluating the change of CXCR4 expression in bone marrow cells after periapical injury.Methods: Seventy-two Sprague-Dawley rats were used and randomly divided into 3 groups: control group(n=8),model group(n=8),and periapical injury group(56).The periapical injury group was further divided into 7 subgroups according to the time points of 12 h,1d,2d,4d,7d,14 d,and 21 d postinjury.After the establishment of non-vital root canal model,the experimental animals(periapical injury group)were subjected to periapical injure to induce bleeding.The specimen were harvested at different time point.The expression of CXCR4 in bone marrowwas detected by immunohistochemistry.Results: Immunohistochemistry showed that CXCR4 cells in the femur were mainly distributed in the red pulp at the proximal femur.All the groups and time points expressed CXCR4 cells in the bone marrow,which were mainly distributed in blood vessels.Compared with the blank control group,the blood vessels in the bone marrow of the model group and the experimental group had different degrees of expansion in 7 different time periods,and a large number of CXCR4 cells were accumulated in the dilated blood vessels,which were positively expressed by CXCR4.The percentage of the area occupied by the cells in the visual field was significantly increased in the experimental group at 12 h,1d,4d,7d,14 d and 21d(P<0.05).Although the expression at 2d was enhanced,there was no significant difference compared with the blank control group and the model group(P>0.05).Comparison of 7 time points in the experimental group showed that the expression of CXCR4 was significantly higher than that of 2d at 4d and 21 d.There was no significant difference in expression between the blank group and the model group.Conclusion: CXCR4 cells are mainly distributed in the red pulp of the femur and mainly located in the blood vessels.After periapical injury,CXCR4 cells accumulating increasingly in the blood vessels with vasodilatation.The results suggest that the injury of the periapical tissue can trigger a series of changes in the bone marrow,providing a basis for the storage and release of endogenous stem cells,for the involvement of tissue regeneration and repair.Chapter three: Tracing and homing of BMSCs overexpressing CXCR4 and CXCR4 genes silencingObjective: Using gene technology to intervene the expression level of CXCR4 in BMSCs and providing basic conditions for tracing BMSCs to observe the biological effects and Influencing factor of SDF-1/CXCR4 axis on the homing of peripheral blood overexpressing CXCR4 and CXCR4 genes silencing BMSCs in vivo after the perapical injury or root canal SDF-1 gel implantion.Methods: 1、The Intervention of the expression level of CXCR4 in BMSCs:The CXCR4 gene overexpressed lentiviral vector and CXCR4 gene silencing shCXCR4 lentiviral vector were labeled with GFP green fluorescence and Mcherry red fluorescence,and transfected into the third generation of male BMSCs.The fluorescence expression of the cells was observed under inverted microscope,using qPCR and Immunohistochemical to detect CXCR4 mRNA and CXCR4 protein expression levels after transfection of BMSCs2、The biological effects and Influencing factor on the homing of BMSCs :1)Same individuals rats with left and right contrapose: 4 female rats,left and right mandibular first molar were subjected to SDF-1 gel implantation,and periapical injure to induce bleeding,respectively.Rats were divided into two groups,2 in each group,CXCR4-BMSCs overexpressed group(CXCR4-BMSCs),CXCR4-BMSCs suspension was injected through the tail vein,and CXCR4 gene silencing BMSCs group(shCXCR4-BMSCs)was injected with shCXCR4-BMSCs suspension.After 24 hours,the rats were sacrificed and the mandible,heart,lung,liver,spleen and kidney were collected.After the sample was processed,the male SRY chromosome was detected by in situ hybridization,and the effects of different treatment methods on the migration of CXCR4-BMSCs and shCXCR4-BMSCs in vivo were observed.2)Different individuals rats contrapose: 10 female rats with only right mandibular first molar were included.(1).6 rats were randomly divided into 2 groups;SDF-1 group and revascularization group,3 rats each.The right mandibular first molar of each group were subjected to SDF-1 gel implantation or periapical injure to induce bleeding,then the tail vein were injected with CXCR4-BMSCs.(2).4 rats were randomly divided into 2 groups;revascularization and SDF-1 group group,3 rats each.The right mandibular first molar were manipulated,then were injected with BMSCs.After 24 h,all rats mandible were harvested.Samples were processed,the migration of male CXCR4+ cells in the tooth root canal and periapical tissue of different individual rats were observed by in situ hybridization.All rats mandibula were processed for HE staining.Results: 1、The Intervention of the expression level of CXCR4 in BMSCs:Recombinant GFP-NC,GFP-shCXCR4,Mcherry-NC and Mcherry-CXCR4 lentiviruses were transfected into BMSCs for four days,and fluorescence was observed under fluorescence microscope,lentiviral transfection with fluorescent protein(Mcherry or GFP)and CXCR4 gene lentiviral transfected cells can successfully express fluorescent protein(Mcherry or GFP),indicating that the virus is successfully transfected into BMSCs.The results of qPCR and immunohistochemistry showed that compared with the negative control and the normal group,the expression level of CXCR4-mRNA was significantly upregulated in the overe xpressed CXCR4 group(P<0.01),the CXCR4 silencing group was down-regulated(P<0.05).There was no significant change in the control groups compared with the normal group(P>0.05).Similarly,the expression level of CXCR4 protein in the overexpression group was significantly up-regulated(P ≤ 0.01),and the expression of CXCR4 in the silence group was significantly down-regulated(P < 0.01).There was no significant change between the two negative control groups and the normal group(P > 0.05).2、The biological effects and Influencing factor on the homing of BMSCs :2.1 Same individuals rats with left and right contrapose:2.1.1 Histological appearance : After 24 h of periapical injury,HE staining show there were a large number of inflammatory cell in the root canal and periapical tissue.After implantation of SDF-1 hydrogel collagen scaffold for 24 h,small number of inflammatory cells were found in the root canal,no inflammatory cells seen in apex area of the root.2.1.2 In situ hybridization detection of SRY mRNA :24 h after injection of cells,CXCR4-BMSCs carrying SRY gene was observed in both periapical lesions and intra root canal,but the number of CXCR4-BMSCs cells in periapical lesion side was higher than that in contralateral tooth(implantated with SDF-1 gel).However,in shCXCR4-BMSCs group,BMSCs carrying the SRY gene were not found in the periapical or root canal in the rats,which BMSCs were CXCR4 gene silenced.2.2 Different individuals rats contrapose:The distribution of CXCR4-BMSCs and BMSCs in the periapical and root canal was detected by in situ hybridization.In different individuals,whether it was apical injury or root canal SDF-1 gel implantation,a large number of brown coloured CXCR4-BMSCs were found in both root canal and the periapical tissue,and the cells homing in periapical tissue more than root canals.The number of BMSCs that normally expressed CXCR4 was less in the periapical and root canal than in the CXCR4-BMSCs group.3、Distribution of CXCR4-BMSCs and shCXCR4-BMSCs in whole body organsSRY chromosome detection results showed that CXCR4 gene over expression BMSCs were aggregated in lung and kidney,while only a small number were found in the heart,liver and spleen tissues.shCXCR4-BMSCs were mainly found in the spleen,followed by the kidney and lung,while shCXCR4-BMSCs were not found in the heart and liver tissues.Conclusion: 1、successfully constructed lentivirus vector-CXCR4 gene over expression and CXCR4 gene silencing of bone marrow mesenchymal stem cells,and laid the foundation to use in situ hybridization to detect male SRY for tracking the migration of BMSCs in vivo2、Whether it is apical periodontal injury or artificial implantation of SDF-1,it is can promote the homing of CXCR4 cells from peripheral blood to the root canal and periapical tissue3、In same individuals rats,if there are multiple reasons in the same body for the increase of local SDF-1 concentration in many places may cause interference with each other,and the chemotaxis of the highest concentration of SDF-1 is the strongest.4、Inflammatory response may enhance the sensitivity of CXCR4-positive cells to SDF-1 chemotaxis,and enhancing the chemotaxis effects5、chemotaxis of SDF-1 is affected by the expression level of CXCR4 in chemotactic cells.The higher the expression level,the higher the homing ability.SDF-1 does not have chemotactic homing effect on cells that do not express CXCR4.6、The distribution of CXCR4-BMSCs and shCXCR4-BMSCs in systemic organs is different.CXCR4-BMSCs are mainly distributed in lung and kidney,shCXCR4-BMSCs are mainly distributed in lung and spleen.Chapter four :Histological Evaluation of the Role of SDF-1 in Dental Pulp RegenerationObjective: To compare the histological characteristics of pulpless root canal newborn tissue induced by periapical hemorrhage and root canal implantation of SDF-1,and to explore the role and mechanism of SDF-1 in dental pulp regeneration.Methods:A total of 48,8 week old female rats(200-240g)were randomly divided into 4 groups,12 rats in each group.After the establishment of non-vital root canal model,the left mandibular first molar were subjected to SDF-1 gel implantation,the right mandibular first molar were subjected to periapical injure to induce bleeding.The rats tail vein were respectively injected with 1ml of BMSCs cell suspension(n=12),1ml of AMD3100 pretreated BMSCs cells suspension(n=12),1ml of DMEM medium(n=12),the fourth groups had no tail vein injection(n=12).Rats were killed after 15 days(n=6)and 30 days(n=6),and the mandible were harvested.Samples were processed for SRY chromosome detection by in situ hybridization.The homing and migration of male CXCR4+ cells in the root canal and periapical tissue were observed.HE staining was used to observe the change of the characteristics of the new tissues in the root canal.Results: HE staining :After 15 days of periapical hemorrhage,except for the blank control group,inflammatory cell infiltration was observed in the periapical tissues of the other three groups.BMSCs group,F12 group and blank group showed bone like cells in the root canal,blue staining matrices of dentin/bone like tissue masses were visible in the root canal of AMD3100 group.After 15 days of the implantation of SDF-1 gel in the left mandibular first molars root canals,inflammatory cell infiltration was observed in the apical segment of the root canal of BMSCs group,AMD3100 group and blank group.A new tissue containing blood vessel is seen in the root canal of BMSCs group and AMD3100 group.In blank group,vascular-like reticular structure is seen with in the new tissue in the root canal.30 days after apical hemorrhage,different degrees of inflammatory cell infiltration were observed in the right periapical tissue.The connective tissue-like structure containing blood vessels was observed in the root canal of the BMSCs group.dentin/bone tissue like structure was visible in the root canal of the AMD3100 group.Blue matrix of bone-like tissue is seen in the root canal of the F12 group.Dentin-like tissue structures were visible in the root canal of the blank group.30 days after the implantation of the SDF-1 gel,a loose connective tissue-like structure containing blood vessels-like was observed in the root canal of BMSCs group.A bone-like tissue structure can be seen in the root canal of AMD3100 group.The root canal of the F12 group is filled with a loose matrix structure.A loose matrix structure containing blood vessels-like can be seen in the root canal of the blank group.In situ hybridization results show that,15 days after BMSCs were injected into the tail vein,SRY chromosome-carrying male BMSCs migrated to the root canals and periapical tissues in both injured and SDF-1 mandibular first molars.the SRY chromosome-carrying cells were not found in the root canals or periapical tissues of other groups.30 days after tail vein injection of BMSCs,BMSCs cells carrying SRY chromosome migrated to the root canals and periapical tissues in both injured and SDF-1 mandibular first molars,a large number of BMSCs carrying male rat characteristic genes were found in periodontal tissues and root canals of the right injured tooth,while the number of migrated BMSCs in the root canals of the left tooth(implantated with SDF-1 gel)was relatively less.In other groups,SRY chromosome-carrying cells were not found in the root canals and in the root canals or periapical tissues.Conclusions:1、SDF-1/CXCR4 bioaxis can chemotaxis distant stem cells toward the root canals and participate in tissue regeneration and repair.2 、SDF-1/CXCR4 bioaxis may be more propitious to the formation of vascularized connective tissue within the root canal.Total conclusion:1.Bone marrow mesenchymal stem cells express CXCR4,and SDF-1 has a chemotactic effect on bone marrow mesenchymal stem cells.The intensity of chemotaxis is related to the concentration of SDF-1.2.Local stimulating in the periapical can dilating the blood vessels in the red pulp of the femur bone marrow,and the number of cells expressing CXCR4 in the blood vessels increase.3.Using lentiviral transfection,CXCR4 overexpression and silencing of bone marrow mesenchymal stem cells can be successfully constructed and successfully homing to the periapical tissue and root canal.4.Whether through the periapical stimulation of bleeding or root canal implantation of SDF-1,BMSCs which is expressing CXCR4 in peripheral blood can homing to the root canal for participating dental pulp regeneration,the perapical injury in the same body can be more chemotaxis for BMSCs to reache the periapical canal and root canal,but preliminary results showed that implantation of SDF-1 in the root canal was more propitious to vascularized neonatal tissue regeneration.5.The chemotaxis of SDF-1 is affected by a variety of factors:(1)cell CXCR4 expression level;(2)inflammatory response;(3)whether there are multiple lesions or inflammation.6.The distribution of CXCR4-BMSCs and shCXCR4-BMSCs in systemic organs is different.CXCR4-BMSCs are mainly distributed in lung and kidney,shCXCR4-BMSCs are mainly distributed in lung and spleen.7.The SDF-1/CXCR4 bioaxis can play a role in the homing of stem cells into the root canal during the process of pulp regeneration,but the mechanism of the proliferation and differentiation of homing stem cells needs further study... |
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