| Background and Objection:Odontoblasts are lost when deep caries develop. However, progenitor cells present in fully developed tooth pulp are able to form functional odontoblasts, which can generate dentine/pulp-like complexes in response to carious stimuli. These undifferentiated mesenchymal cells were named dental pulp stem cells (DPSCs), and they have been cultured and extensively studied. It is reported that DPSCs can differentiate into odontoblasts, osteoblasts, adipocytes, chondrocytes and active neurons.The characteristics of dental pulp change in response to injuries or inflammation. Stromal cell-derived factor1, a member of the CXC chemokine subfamily, and its receptor CXC chemokine receptor4are distributed in inflammatory cells and microvascular endothelial cells, but not in normal pulp. The expression of IL-1, IL-8and parathyroid hormone-related protein also changes in inflamed dental pulp. Progenitor/stem cells were shown to migrate to the site of pulpal injury from their tissue of origin in extracted human molars, simulating the situation in vivo. In the case of mild caries, with moderate attrition and erosion, odontoblasts can usually survive and produce the dentin necessary to protect the underlying dental pulp tissues. However, when the initial stimuli are not treated and/or progress rapidly, primary odontoblasts are destroyed and lack the ability to produce new dentin. In these cases, undifferentiated mesenchymal cells within pulp tissues can migrate and differentiate into odontoblasts to secrete regenerative dentin.Previous studies have demonstrated the dentinogenic ability of DPSCs. However, there are limited data regarding the migration of DPSCs in deep caries, the differences between normal DPSCs and carious DPSCs (CDPSCs), or whether CDPSCs retain tissue regeneration capacity. To know the biological mechanisms of the differences between DPSCs and CDPSCs, we analysed the different proteins by2-DIGE. Mass spectroscopy (MS) based proteomics is becoming an efficient method for the detection of cellular processes at theprotein level, allowing rapid identification of proteins during differentiation.The formation of pulp-dentin complex of dental pulp stem cells is an important foundation for tooth regeneration. Stathmin is a mineralization-related protein which was blotted from normal and cariou dental pulp stem cells by proteomics. Human and rat osteoblast-like express stathmin and that this protein could play a role in regulation of the growth of these cells in response to various hormonal stimuli. There was report suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. However, there was no report about the relationship between Stathmin and the mineralization of dental pulp stem cells.This study was designed to examine the changes in characteristics, proliferation and osteogenic differentiation of CDPSCs. The aim of this study was to isolate and culture the dental pulp stem cells from carious and normal teeth and to evaluate stem cell parameters. To know the biological mechanisms of the differences between DPSCs and CDPSCs, we analysed the different proteins by2-DIGE and the function of the protein.The paper includes the following three parts:Part1:Cultued the human caries dental pulp stem cells with improved combination of explants method and enzymatic separation methodTo compare the successfulness and the growth of human caries dental pulp stem cells (hCDPSCs) cultured with three different methods. Collect24normal and caries human third molars, the dental pulp tissues were cultured by the tissue explants method, tissue-explants collagenase digestion method and the combination of explants method and enzymatic separation method, respectively. Observe the adherence of the explants, the morphology and quantity of cells under phase-contract microscopy, record the culture duration. The clones were identified by expression of Stro-1、CD90、CD105and CD29and draw the growth curve of normal DPSCs and CDPSCs. Our study indicated that human normal and caries dental pulp stem cells could be cultured by the three methods. A large number of human normal and caries dental pulp stem cells cultured by improved combination of explants method and enzymatic separation method in a shorter time, and these cells exhibited more vitality and more different morphologies. The growth rate of caries pulp stem cells is higher than that of normal pulp stem cells. The improved combination of explants method and enzymatic separation method is an ideal new method for the primary culture of human caries dental pulp stem cells in vitro, it may provide a methodological foundation for study the mechanism of the formation of the tertiary dentine when the tooth were damaged.Part2:Changes in Proliferation and Osteogenic Differentiation of Stem Cells from Deep Caries In VitroIt is suggested that dental pulp stem cells are involved in tooth regeneration and play an important role in maintaining pulp homeostasis. Previously, normal dental pulps were more widely utilized for experimental models than carious dental pulps. The aim of this study was to isolate and culture the dental pulp stem cells from carious and normal teeth and to evaluate stem cell parameters. Pulp tissues were obtained and dissociated from normal and carious teeth. Single-cell suspensions were seeded into6-well plates and purified by collecting multiple colonies. Normal dental pulp stem cells (DPSCs) and carious dental pulp stem cells (CDPSCs) were compared for morphologic appearance and for their capacity to differentiate into three lineages. Colony-forming and MTT assays, cell cycle analysis, gene expression, and ALP activity were also evaluated. Stem cells were cultured successfully from normal and carious dental pulps. CDPSCs displayed increased proliferation ability compared with DPSCs. CDPSCs also showed enhanced ALP activity, mineralization ability, and expression of osteogenesis/dentinogenesis-related genes. All cultures differentiated into three cell types. Our data suggest that caries as a local microenvironment should be taken into account when DPSCs are intended to be used for investigations and application.Part3:The proteomic analysis of CDPSCsTo better understand the mechanism of dental pulp stem cells, explore the marker of dental pulp stem cells, we analysis the DPSCs and CDPSCs by2D-DIGE (2-D Fluorescence Difference Gel Electrophoresis), By image analysis, the expression levels of20protein spots were found to be idtificantly changed between DPSCs and CDPSCs (p<0.05). Among them, the expression levels of10protein spots were increased in CDPSCs and the expression levels of10protein spots were decreased in CDPSCs. When compared with DPSCs and CDPSCs,2-DE followed by matrix-assisted laser desorption/time of flight mass spectrometry (MALDI-TOF MS) was utilized to identify20different protein, and verify partly different protein by western blot.Part4:The preliminary functional studies of Stathmin in mineralization of normal dental pulp stem cellsStathmin is a mineralization-related protein which was blotted from normal and cariou dental pulp stem cells by proteomics, there was no report about the relationship between Stathmin and the mineralization of dental pulp stem cells. To investigate the effect of Stathmin on the biological characteristics, we constructed vector of over-expressed and silienced stathmin and tranfected into DPSCs. Our results indicated that the expression of Stathmin in normal dental pulp stem cells is higher than that in carious dental pulp stem cells, and the ability of mineralization and expression of ostoegenesis-related gene of dental pulp stem cells down-regulated after silencing the expression of Stathmin. And the the ability of mineralization and expression of ostoegenesis-related gene of dental pulp stem cells increased after over expressed of Stathmin.Materials and MethodsSubjectsAll patient-related procedures (18-22years of age) utilized in this study were according to approval of the Medical Ethics Committee of Nanfang Hospital and the informed consent of the patients and it was written. Normal pulp tissues were obtained from complete wisdom teeth without caries or pulp disease (n=24). Carious pulp tissues were collected from third molars diagnosed with deep caries (n=24). The diagnosis of deep caries was determined by endodontic specialists based on clinical assessment, including the caries progressing to dental deep layer and the amount of remaining dentin thickness is less than2mm, with no history of spontaneous pain and intense. Teeth with symptom of pulpitis were excluded. Cell culture and phenotype analysis The normal pulps were separated from the crown and root, while carious pulp tissues were collected from pulp chambers. Single-cell suspensions were obtained by passing the digested tissues through a70μn cell strainer. Single-cell suspensions were seeded into6-well plates with Dulbecco modified Eagle medium containing15%fetal bovine serum,100units/ml penicillin,100mg/ml streptomycin, and50mg/ml ascorbic acid, and then incubated at37℃in5%CO2. Stem cells from normal pulp tissues were named DPSCs and those from carious pulp tissues were named CDPSCs. DPCSs and CDPSCs were enriched by collecting multiple colonies. Cell proliferation assessment Colony-forming assayTo evaluate colony-forming efficiency, single-cell suspensions of DPCSs and CDPSCs were seeded into75cm2flasks at1×105cells/ml. After7days of culture, the cells were fixed with4%Polyoxymethylene and then stained with0.1%toluidine blue. Aggregates of more than50cells were scored as colonies. The assay was repeated three times. Flow cytometry analysisCells were collected and mixed with2ml cold dehydrated alcohol at4℃for24-48h to fix cells. The cells were then washed twice and stained with propidium iodide at4℃for30minutes, and subjected to cell cycle analysis using an Elite ESP flow cytometry. The fraction of cells in the G1, S, and G2M phases of cell cycle was analyzed. Three separate experiments were performed. Growth rate MTT assayCells were seeded into96-well plates at a density of5×103cells/well and evaluated the cell proliferation rate with MTT according to the manufacturer’s instructions. MTT reduction/attenuation values for each well were measured spectrophotometrically at490nm. The MTT assay was carried out every day for7 days, and the assay was repeated in triplicate. In vitro multi-potentiality assayTo evaluate their ability to differentiate into other cell types, cells were seeded into24-well plates at a density of1×104cells/well. When they reached70%confluence, the cells were cultured in different media. For the mineralization assay, the medium was supplemented with10%fetal bovine serum,10%β-glycerophosphate10mmol/L, ascorbic acid2-phosphate5mg/mL, and0.1%dexamethasone10-5mol/L, for3weeks induction. For adipogenic differentiation, the medium was supplied with0.5mM methylisobutylxantine,0.5mM hydrocortisone, and60mM indomethacin. For chondrogenic induction, the cells were treated with chondrogenic differentiation media supplemented with10ng/mL TGF-β3for21days. After the period of differentiation, cultures were fixed in4%paraformaldehyde for20minutes. Cells that underwent osteogenic, adipogenic and chondrogenic differentiation were stained with alizarin red, oil red, and alcian blue, respectively. Every assay was repeated three times.Cell osteogenic differentiation assessmentAnalysis of alkaline phosphatase activityAt defined time points (days2,4,6,8,10, and12), the ALP activity of cells was measured using an ALP assay kit according to the manufacturer’s instructions. The amount of p-nitrophenol released by the enzymatic reaction was detected spectrophotometrically at520nm using a microplate reader. ALP activity was always expressed relative to the amount of DNA, determined with a PicoGreen dsDNA quantitation kit, and the assay was repeated3times. qRT-PCRThe total cellular RNA of cells was extracted using the Trizol Reagent, and first-stand cDNA synthesis was performed according to the manufacture’s protocol. Quantitect SYBR Green kits were utilized for detecting real-time polymerase chain reaction products using a Light Cycler Instrument. Primer sequences were designed by Invitrogen. Amplification conditions were denaturing at95℃for10minutes, followed by40cycles at95℃for10seconds, and at60℃for30seconds. Triplicate reactions and3separate experiments were performed. Western blotTotal protein was isolated and quantitated using BCA assay. The protein lysates were separated by10%SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected by chemiluminescence. The intensity of protein fragments was quantified with the Quantity One software.2-DIGEIsoelectic focusing (IEF) was performed as described by Shevchenko et al. IPG strips were used according to the manufacturer’s instructions (Amersham Pharmacia Biotech). Samples containing appropriate amounts of protein were diluted to350mL with rehydration solution9M urea,4%CHAPS,100mM DTT and0.5%(v/v) IPG buffer, with a trace of bromop henol blue, applied to strips (pH range of4-7) by overnight rehydration in a rehydration tray, and2DE was performed using9-16%second-dimension gels in an Iso-DALT apparatus until the tracking dye reached the anode end of the gels.Cell transfectionLentivirus infection:The packaged lentivirus were used to infect DPSCs in5MOI=100(multiplicity of infection). At48h transfection, the cells were bserved using a fluorescence microscope to detect green fluorescent protein (GFP). Following fluorescence-activated cell sorting, the GFP-positive cells were isolated, and the cell lines were established, which could stably overexpress Stathmin, overexpress Stathmin and silence Stathmin respectively. Statistical analysisSPSS13.0software (SPSS, San Rafael, CA) was used for statistical analysis. Data were presented as mean±SEM of at least3independent experiments. The results of MTT assay and ALP assay were analyzed by Repeated measures ANOVA. Two-tailed Student’s t test was used for comparisons of2independent groups. Statistical significance was set at P<0.05.Results:1. Cariou dental pulp stem cells were cultered by the tissue explants method, tissue-explants collagenase digestion method and the combination of explants method and enzymatic separation method. The expression of markers of mesenchymical stem cells in CDPSCs was higher than that in DPSCs (Stro-1:t=3.325, P=0.029; CD90:t=4.446, P=0.011; CD105:t=3.411, P=0.027). MTT exhibited that CDPSCs showed higher OD value than DPSCs from3day (F=7.350,P=0.000), cell cycle analysis showed that the percentage of CDPSCs in S+G2/M phase (29.94%) was higher than that in DPSCs(22.21%). Both groups of cells formed adherent clonogenic cell clusters, however, CDPSCs had higher CFU counts than DPSCs (t=9.798, P=0.001).DPSCs and CDPSCs both showed calcium deposits when incubated in mineralized culture for21days. CDPSCs produced more calcium deposits than DPSCs, as visualized by Alizarin red staining. Our data showed higher alkaline phosphatase (ALP) activity in CDPSCs than in DPSCs (t=191.741, P=0.000). The expression of osteogenesis/dentinogenesis-related genes OCN (t=3.98, P=0.003)、RUNX2(t=3.588, P=0.005)、ALP (t=6.044, P=0.000)、Osteonetin (t=2.270, P=0.047)、DSPP (t=-3.094, P=0.011)、BSP (t=5.847,P=0.000) was higher in CDPSCs than that in DPSCs. 2. The expression levels of20protein spots were found to be ignificantlychanged between DPSCs and CDPSCs (p<0.05). Among them, the expression levels of10protein spots were increased in DPSCs and the expression levels of10protein spots were decreased in DPSCs.3. The expression of Stathmin in normal dental pulp stem cells is higher than that in carious dental pulp stem cells, and the ability of mineralization and expression of ostoegenesis-related gene BSP (t=7.736,P=0.002)、OCN (t=3.670, P=0.021)、 RUNX2(t=3.309, P=0.030) of dental pulp stem cells down-regulated after silencing the expression of Stathmin. And the the ability of mineralization and expression of ostoegenesis-related gene BSP (t=-3.179,P=0.034)、OCN (t=-4.104, P=0.015)、 RUNX2(t=-3.565, P=0.023) of dental pulp stem cells increased after over expressed of Stathmin.Conclusion1. CDPSCs showed increased proliferation and mineralization ability than DPSCS.2.20protein spots were found to be idtificantly changed between DPSCs and CDPSCs.3. Stathmin regulates the mineralization of DPSCs, and expressed positive correlation. |
PDF Full Text Request