| Background and purposePulpitis and periapical disease is one of the most common diseases in oral cavity.As fully mature tissue,once pulp is damaged,it will be difficult to recover.Root canal therapy can effectively alleviate the pain of patients and control the inflammation.However,teeth endodontically treated will completely lose the nutrition from the nerves and blood vessels,the teeth will change color and become brittle which are easy to postoperative fracture.Although endodontically treated teeth can prolong the retention time,it is still relatively easy to lose by comparing with healthy teeth.Regenerative endodontics is based on tissue engineering to generate new pulp tissue and replace damaged or lost ones and(or)dental pulp tissue.In a number of research methods,stem cell transplantation is an important research direction of pulp regeneration.At present,many studies have confirmed that stem cells can obtain pulp like tissue regeneration by transplanting.Although the results are very optimistic,cell transplantation has encountered a great deal of difficulties.These barriers include cell isolation;ex vivo manipulation with the potential for changing cell phenotype;and safety issues,including immunorejection,potential contamination,pathogentransmission,and potential tumorigenesis.Excessive costs associated with these issues in addition to shipping;storage;handling issues;and regulatory difficulties,including unclear pathway and the general inability to ensure batch-to-batch consistency in cell quality,cast multidimensional questions for the practicality of cell transplantation.From a dentist’s perspective,the introduction of a method of high risk and high input to the treatment of non-lethal diseases makes little sense.On the basis of summing up the latest research results at home and abroad,replacement of cells transplantation by homing of cells has become a new and hot research direction of pulp regeneration.Bone marrow stromal stem cells(BMSCs)is a kind of multipotent stem cells.It can differentiate into a variety of cells such as osteoblasts,fibroblasts,chondrocytes,adipocytes,etc.BMSCs have become a kind of pluripotent stem cells with broad application prospects.A large number of studies have indicated that bone marrow stromal stem cells can be homed to any damage parts of the body selectively and play a role in treatment,thus we assume that bone marrow stromal stem cells in the remote body stem cell niches(e.g.bone marrow)can feel stimulation,migrate with the blood flow and finally reach the tooth canal to participate in the final repair process of pulp like tissue.Stromal cell derived factor-1(SDF-1)is known to be one of main chemokines of the stem cell migration.It can bind to its receptor CXCR4,thereby affecting cell chemotaxis,cell movement,cell adhesion,secretion,angiogenesis and proliferation.SDF-1 play a key role in stem cell mobilization and homing of the peripheral circulation of hematopoietic.A series of studies have shown that,the local upregulated expression of SDF-1 induces MSCsmigrate to the injured site and repair the damage in heart,brain and liver tissue damage.In order to confirm our assumption,roots containing neutralized collagen gel with or without SDF-1 were implanted into mouse subcutaneous pockets and BMSCs labeled with GFP were transplanted into mouse via the tail vein to evaluate whether the systemic BMSCs can home and their possible role in pulp regeneration.The enhancing effect of SDF-1 on Stem cell recruitment and angiogenesis were also evaluated.Materials and methods1.The culture and transfection of bone marrow mesenchymal stem cellThe mouse mesenchymal stem cells ST2 were recovered and steadily passed.The optimal multiplicity of infection was determined after passage.The expression rate of fluorescence of BMSCs was above 90%when MOI was equal to and greater than 60,so the optimum MOI value was determined to be 60.And then the optimal screening concentration of G418 was determined.Cells in the medium containing G418 were cultured for 10 to 14 days,and the lowest concentration of G418 of cell death was 200μg/ml.The G418 maintenance concentration was determined to be 100μg/ml.BMSCs transfected by the lentiviral vector with GFP kept stable and GFP with cells expressed stably.Transfected BMSCs were purified with G418.2.The establishment of animal models and transplantation of mouse Bone marrow mesenchymal stem cell54 extracted first premolars with single canal due to orthodontic were collected and cut off the root along the crown root junction.Root canal was prepared and the apical foramen expanded to a diameter of 0.5 mm.The root was pretreated with sodium hypochlorite,EDTA and PBS.GFP + Bone marrow stromal stem cells were collected and transplanted into mouse via tail vain.Each mouse was transplanted volume 100ul,1 × 106 cells,and control mouse was injected a-MEM culture medium(serum-free)100ul.A longitudinal incision about 1cm was made on the back of the mouse,then blunt dissection was made with a hemostat,exposing the subcutaneous tissue,a subcutaneous pocket was made at last.54 male mice of 5-7 weeks were randomly divided to three groups:SDF-1 group(subcutaneous pockets containing roots with SDF-1 absorbed neutralized collagen gel and the green fluorescent protein(GFP)positive BMSCs transplantation via the tail vein),SDF-1 blank group(subcutaneous pockets containing roots with gel alone and GFP+ BMSCs transplantation)and control group(pockets containing roots with gel alone and 100ul serum-free a-MEM culture medium).The animals were sacrificed after the roots were implanted into subcutaneous pockets for 3 weeks.Root samples were embedded in paraffin and processed after external fixation and EDTA demineralization.The new pulp like tissue in the canal was observed under optical microscope by hematoxylin and eosin(HE)staining and the impact of SDF-1 on angiogenesis was evaluated through measuring the area of neovascularization by using image analysis.Sections were stained with DAPI staining solution after dewaxing hydration.The migration and differentiation of GFP+BMSCs were observed under a fluorescence microscopy directly.The impact of SDF-1 on cell homing was evaluated through measuring the number of homing GFP+ cells by using image processing software.The expression of alkaline phosphatase(ALP)and GFP in new formed tissue were detected immunohistochemically.Results1.The culture and transfection of bone marrow mesenchymal stem cellST2 cells in good condition were chosen.Green fluorescence of GFP in BMSCs was observed 48 hours after Lentiviral transfection and got the highest fluorescent light at 72-96 hours.G418(200 μg/ml)was used to purify the GFP-positive cells.The morphology of BMSCs with high level of GFP expression did not change significantly after infection,the growth state was good and the cells stably expressed GFP with passage.2.Homing of BMSCs and formation of pulp-like tissue in animal modelsHuman roots filled with scaffolds implanted into the dorsum regenerated ectopic dental pulp-like tissue in roots canal both in SDF-1 group and in SDF-1-free group.However,there was no detectable dental-pulp-like tissue regenerated in control group.H&E staining microscopically showed that root canals filled with collagen scaffold alone and without cell transplantation showed no organized cell and blood vessel.In SDF-1-free group,root canals filled with collagen scaffold alone and cell transplantation showed a small amount of cells.Residual amount of collagen scaffold exhibit grid-like morphology and some cells were arranged between the grids.A small amount of erythrocyte-filled blood vessels could be observed under high power microscope.The expression of ALP and GFP was weak in this group.Contrastingly,the root canals full of collagen gels with SDF-1 and cell transplantation yielded abundant cells and the grid-like morphology of collagen scaffold disappeared.A large number of erythrocyte-filled blood vessels could be observed under high power microscope.Total number of homing of GFP + cells and neovascularization area was significantly greater than that of SDF-1-free group.Green fluorescent protein and alkaline phosphatase was significantly stronger than that of SDF-1-free group.Conclusion1.Lentivirus carrying GFP infect BMSCs effectively.The morphology of BMSCs with high level of GFP expression did not change significantly after infection,the growth state was good and the cells stably expressed GFP with passage.2.The pulp-like tissue regeneration can be accomplished by cell homing mode.3.Systemic BMSCs could home to the root canal and participate in dental-pulp-like tissue regeneration and differentiation.4.Local application of SDF-1 can improve homing efficiency of the BMSCs and enhance vascularization. |
