The Role Of Integrin-α5(ITGA5) In The Proliferation, Migration And Osteo-/odontogenic Differentiation Of Human Dental Pulp Stem Cells

As a kind of loose connective tissue, dental pulp’s blood supply is derived from the tiny blood vessels of periapical. Effective collateral blood circulation in the dental pulp is absence, so it is difficult for plup to defend the external stimulation, like inflammation. Injuries of dental pulp are usually not reversible, which could lead to necrosis and further injury of periapical tissue. To find an ideal endodontic treatment is the goal of dentists. The traditional root canal treatment (RCT) is operated to completely remove the infected pulp tissue, then cut the infected root canal wall, and fill the root canal with filling materials instead of the pulp tissue. RCT could remove most of the infection in the canal root and decrease the chance to get reinfection. However, there are some drawbacks in the traditional RCT. First, the filling material and sealing material is easy to discolor the crown of teeth, which has negative influence on the patients appearance. Second, traditional RCT decline the fracture resistance of dentine. Third, the teeth are relative to loss compared with healthy teeth even after perfect RCT.As we know, when the pulp suffered from external stimulation, undifferentiated mesenchymal cells, which exist in the pulp cavity, can differentiate into odontoblast so as to participate in the process of repairing the dental pulp tissue. To impel the recover of the damaged pulp tissue and regeneration of the necrotic pulp tissue, a better way for endodontics therapy is predicted.In2000, Gronthos first reported precursor cells with the ability of self-renewal and multi-lineage differentiation potential exist in the dental pulp of adults and named human dental pulp stem cells (hDPSCs). In general, hDPSCs are in the resting state. When external stimulation causes damage to the odontoblasts, hDPSCs can proliferate, migrate to the damaged area and differentiate into odontoblasts to form the reparative dentin, and participate in the process of repairing the injuried dental pulp tissue. The growth, proliferation, migration and differentiation to odontoblast of hDPSCs are very important for reconstructing the damaged dental pulp tissue.The regeneration of dental pulp is a kind of exploratory biology technology. That regenerates the dental pulp tissue to replace the damaged pulp tissue. The regenerated dental pulp tissue is consisted of the odontoblasts, blood vessels, nerves and connective tissue. Thus, to restore natural dental pulp tissue, odontoblasts regeneration, pulp revascularization, nerve regeneration and stent grafts are inevitable. The regeneration of odontoblasts is the core of the dental pulp tissue regeneration. As an important kind of seed cells, hDPSCs can differentiate into odontoblasts, besides, they can be induced to differentiate into dental pulp dentin complex in mice in vivo. All that further revealed its important role in the dental pulp tissue regeneration.Integrins, as a family of the glycoprotein receptors, are expressed on the surface of the membrane of all kinds of tissues in human. Integrins are involved in mediating the interaction between cells and cells, as well as cells and extracellular matrix. They also play an important role in physiological and pathological processes such as cell migration, proliferation, differentiation, signal transmission, tumor metastasis, wound healing and cellular stress to mechanical stress. As a member of integrins family, integrin alpha5(ITGA5) can combine with integrin beta1and form heterodimer to achieve physiological function. Integrin α5β1is a fibronectin (FN) receptor, signal transduction could be triggered when integrin α5β1combined with FN to activate a variety of signaling molecules and participate in physiological processes such as cell adhesion and cell skeleton reconstruction. Recent studies have confirmed that, it plays a certain role in the process of bone marrow mesenchymal cells differentiating into bone tissue.In1998, ITGA5was first reported in human dental pulp cells and proved to take part in cell adhesion process. However, little information about the function of ITGA5in hDPSCs is available. Therefore, it is necessary to get further study of the effect of ITGA5on the proliferation, migration and differentiation process of hDPSCs. It is important to investigate the role of ITGA5in hDPSCs, and clarify its function in the process of repairing and regeneration of pulp tissue.This paper is composed of three chapters:Chapter Ⅰ hDPSCs isolation,culture and identificationHealthy permanent teeth were collected from young adults, hDPSCs were isolated and cultured by combination of explants method and enzymatic separation method. The adherence of tissues were observed via a microscope, tissue blocks firmly attached to the bottom of culture flask. After5-10days’ culture, cells were observed creeping out of the pulp tissue blocks. We found that the cells of expanded culture performed fusiform and cytoplasmic translucency and could be subcultured stably. To detect the phenotype and biological characteristics of cells we cultured, cell cloning of the obtained cells were verified. Then we clarified the cells could differentiate into adipocyte, chondrocyte and osteoblast and the mesenchymal cell surface markers were positive while hematopoietic cell surface markers were negative by flow cytometry, which confirmed the obtained cells came from mesenchyme. hDPSCs were successfully isolated and extend culture, which provided a reliable source of cells for subsequent experiments.Chapter II The effect of ITGA5on proliferation and migration of hDPSCsITGA5shRNA lentivirus was constructed and was infected into human dental pulp cells. qRT-PCR and western blot were used to detect the expression of ITGA5in hDPSCs after transfection. The proliferation ability was tested by MTT assay and EdU assay and the migration ability was detected using transwell assay with ITGA5silencing. The ITGA5expression in hDPSCs could be inhibited effectively by ITGA5shRNA lentivirus.We found that the proliferation of hDPSCs was significantly reduced, and the migration was obviously declined after the inhibition of ITGA5.Chapter Ⅲ The role of ITGA5in the process of differentiation into osteo-/odontoblastThe expression of ITGA5in hDPSCs cultured with mineralization medium and with growth medium was compared by qRT-PCR. The mineralized nodules of hDPSCs with ITGA5inhibited were stained by alizarin red after21days mineralization culture. The expression of mineralization related genes, such as DSPP, DMP1, ALP, ON, OCN and BSP, were detected by qRT-PCR after knocking down ITGA5in hDPSCs by lentivirus infections. After mineralization for21days, the DSPP protein expression in hDPSCs with ITGA5silencing was tested using western blot. The results showed that the ITGA5expression in hDPSCs with mineralization medium had no significant difference with the cells cultured in the growth medium after7days. However, the ITGA5expression in mineralization cells was significantly lowered after14and21days. After21days mineralization culture, silencing ITGA5in hDPSCs could increase the number of mineralized nodules, up regulate the mineralized related genes, such as DSPP, DMP1, ALP, ON, OCN and BSP. It could also obviously enhance the expression of DSPP protein. Materials and MethodsThe isolation and culture of hDPSCsHealthy complete permanent teeth (age range from18to25) extracted due to impaction or orthodontic were collected in oral and maxillofacial surgery department of Nanfang Hospital (with patients’informed consent). After extraction, the teeth were infused in DMEM medium and sent to laboratory immediately. The teeth were splited along the groove around the cemento-enamel junction. The pulp tissue was taken out in aseptic conditions (the pulp close to the apical were removed) and placed in PBS buffer. The pulp tissue was rinsed for3times, at least. Then it was cutted into pieces about1mm×1mm×1mm, cell suspensions were obtained by passing the digested tissues, and then cultured with a-MEM,10%fetal bovine serum for medium. The cultivation condition was set at37℃and5%CO2. The cells of primary generation were seeded in96wells-plate,1～2/well.After cells colony formed, the cells were subcultured.Colony-forming assayThe cells were seeded in6wells-plate,100/well. After14days culture, the cells well fixed with paraformaldehyde and stained with crystal violet. The colonies were observed under inverted microscope (more than50cells as a clone).In vitro analysis of hDPSCs multilineage differentiation potentialhDPSCs were seeded at3”104/35mm plate and cultured to70%confluence. Then the special medium(For odontogenic differentiation, For adipogenic differentiation,For chondrogenic differentiation) were used for3weeks culture. The induced cells were fixed in70%ice-cold ethanol for20min at room temperature and then separately stained with2%Alizarin Red,Oil Red O and Alcian Blue. The results were observed under inverted microscope.Flow cytometry analysis At least1×106cells were prepared in cold PBS for each test. Cell phenotype analysis was performed by flow cytometric detection of CD29/PE, CD34/PE CD90/FITC, CD44/FITC, CD45/FITC and CD105/FITC according to the manufacturer’s instructions.qRT-PCRThe total RNA of cells was extracted using the Trizol Reagent, and first-stand cDNA synthesis was performed according to the manufacture’s protocol. SYBR Green kits were used to detect real-time polymerase chain reaction. The relative quantitative method2-ddCt was used to accurate the genes expression.Western blotThe total protein was isolated and quantitated using BCA assay. The protein lysates were separated by SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies. Immunoreactive proteins were visualized using ECL Plus.Construction of ITGA5shRNA lentivirus and cell infectionThe ITGA5-targeting sequence of oligonucleotides was designed as previously described. The negative construct consisted of a scrambled sequence with no homology to any human gene. The oligonucleotides were cloned into GV118-GFP to generate the lentiviral vectors. Recombinant lentiviral vectors and packaging vectors were then transfected into293T cells. Supernatants containing lentiviruses expressing ITGA5shRNA or scrambled shRNA were harvested72h after transfection. The lentiviruses were then purified by ultracentrifugation, and the titer of lentiviruses was determined.Cells infectionhDPSCs were transduced with the ITGA5shRNA lentivirus at multiplicity of infection (MOI) of100, and mock-infected hDPSCs cells were used as negative controls. The efficient concentration was screened.MTT assayhDPSCs were infected with ITGA5shRNA lentivirus or scrambled shRNA lentivirus. For MTT assays, the infected cells were seeded in96-well plates at2x103per well. The absorbance of each sample was analyzed at490nm using a microtiter plate reader at1d,3d,5d,7d.EdU assay5-ethynyl-2’-deoxyuridine (EdU) detection kit was used to evaluate cell proliferation.According to manufacturer’s instructions,the cells were treated with25μM EdU for6h at37℃,then fixed with dehydrated ethanol for10min.After being incubated with2mg/ml glycine for5min,the cells were treated with0.5%Triton X-100for20min and stained with1×Apollo(?) reaction cocktail for30min at RT Following wash with0.5%TritonX-100in PBS, DAPI dye was used to incubated cells at RT for10min. Images were captured under a confocal laser scanning microscope.Transwell assayThe migration activities of ITGA5shRNA group and scrambled shRNA group were assayed using transwell cell culture chambers. Following overnight serum starvation, cells were harvested and resuspended in DMEM containing0.1%FBS. Cells (2×104) were added to the upper transwell chamber. DMEM+10%FBS were added to the lower chamber. After48h the cells that migrated through the membrane were fixed in methanol and stained with0.1%crystal violet.Alizarin red stainingThe cells of the third generation were mineralization induced for21days. Then the cells were fixed with4%paraformaldehyde for20minutes, and stained for10min with alizarin red. Mineralized nodules were observed under inverted microscope. Statistical analysisThe data were analyzed and expressed as the mean±standard deviation. Statistical significance was evaluated by independent samples t-test using SPSS13.0software. Statistical significance was set at a=0.05.Results1. hDPSCs were successfully separated and culturedhDPSCs were isolated by combination of explants method and enzymatic separation method. To detect the phenotype and biological characteristics of cells we cultured, we verified that the obtained cells can form cell colony, differentiate into adipocyte, chondrocyte and osteoblast. The mesenchymal cell surface markers were positive (CD29,90.82%; CD44,99.50%; CD90,98.79%; CD105,97.92%) while hematopoietic cell surface markers were negative(CD34,0.23%; CD45,0.75%) by Flow cytometry. which confirmed the obtained cells could be considered as hDPSCs.2ITGA5was involved in proliferation and migration of hDPSCsThe ITGA5expression in hDPSCs could be inhibited effectively by ITGA5shRNA lentivirus which was tested by qRT-PCR and western blot. ITGA5shRNA group and scrambled shRNA group were set up.We found that the proliferation of ITGA5shRNA group significantly reduced by MTT assay (P<0.01) and EdU assay(P<0.01). The migration ability was obviously lowered by transwell assay after ITGA5inhibited (P<0.05).3.ITGA5controlled the process of osteo-/odontogenic differentiation in hDPSCsThe result showed that, the ITGA5expression in hDPSCs with mineralization medium had no significant difference with the cells cultured in the growth medium after7days (P>0.05). However, the ITGA5expression in mineralized cells was obviously lowered down at14and21days (P<0.05). Then two groups were set up (ITGA5shRNA group and scrambled shRNA group) to analyse the effect of ITGA5in odontoblastic differentiation. We found that after21days mineralization culture, silencing ITGA5in hDPSCs could increase the number of mineralized nodules, apparently upregulate the mineralization related genes, including DSPP、DMP1、 ALP、ON、OCN、BSP (P<0.01) and enchance DSPP protein expression obviously.Conclusion:1. hDPSCs were successfully isolated and cultured. The obtained cells had the phenotype and biological properties of mesenchymal stem cell.2. The lentivirus were successfully constructed to inhibit the ITGA5expression in hDPSCs. The proliferation and migration of hDPSCs were obviously suppressed by silencing ITGA5.3. ITGA5expression of hDPSCs induced by the mineralization medium was reduced. Knocking down ITGA5in hDPSCs distinctly promoted the osteo-/odontogenic differentiation.