The Effects Of Pro-inflammatory Cytokines On The Proliferation And Osteogenic/dentinogenic Differentiation Of Stem Cells From Apical Papilla

Background and objectiveIn recent years, several clinical reports have shown that immature permanent teeth containing pulpitis or periapical periodontitis can undergo apexogenesis of developing roots and continuous root development. Many studies indicate that SCAP are important to the root dentinogenesis and continuous root development.In2006, stem cells from apical papilla (SCAP) are found in apical papilla of immature permanent teeth and have been isolated and characterized by Sonoyama et al, which are a population of early mesenchymal stem cells with a high degree of proliferation, self-renewal and multi-differentiation potential that can differentiate to osteogenic, adipogenic, nerve, cartilage and myogenic cells under the specific culture conditions. SCAP are isolated from the developing tissue and appear to have a greater proliferation, doubling capacity, telomerase activity, cell migration and regeneration differentiation potential than dental pulp stem cells (DPSCs). Several studies support the hypothesis that SCAP may be the source of primary odontoblasts that are responsible for the root dentinogenesis and continuous root development. The characteristics of SCAP make it not only to be a cell model of dentinogenic differentiation mechanism in root development study, but also to be the extremely outstanding source of early dentinogenic stem cells which can be used in pulp/dentin regeneration and root regeneration, and it has a widely prospect for clinic application.Many internal mechanisms and microenviromental factors can influence their proliferation and differentiation. Inflammation produces a large number of inflammatory cytokines to change the microenvironment in which influence stem cell mediated tissue regeneration and repair through the effects. The occurrence and development of pulpitis, periapical periodontitis is a complex physiological processes which composed of various cytokines. Pro-inflammatory cytokines can be rapidly produced by inflammatory cells in dental tissues when contacting with pathogens, and are able to trigger potent inflammatory responses, such as TNF-a, IL-1β are high expression and have functions in the induction of downstream responses to change the chatacteristics of stem cells. And some studies also showed that persistent chronic inflammation environmental stimulation can reduce the proliferation and migration ability of stem cells, and inhibit the regeneration potential by the change of intrinsic pathway. There is a certain correlation between lesions and inflammatory factor when the immature permanent teeth containing pulpitis or periapical periodontitis. The expression of inflammatory factors in different lesions are not the same, and may have different effects on the survival rate, regeneration differentiation ability of SCAP, then influence the level of the root dentinogenesis and continuous root development.The SCAP of immature teeth containing pulpitis and periapical periodontitis are exposed to unfavorable conditions, such as pro-inflammatory cytokines released from necrotic or inflammatory cells. Then, we hypothesized that some changes in their characteristics could happen. However, the changes involved in SCAP characteristics under pro-inflammatory cytokines stimuli are not completely understood. Therefore, in this study, we cultured and purified SCAP as cell model in vitro and selected TNF-a, IL-8, IL-1β as a model inflammatory signal simulating the inflammatory microenvironment in vivo. And the purpose of this study was to investigate the effects on the proliferation and differentiation of SCAP challenged with TNF-a, IL-8and IL-1β, so as to clear regulatory role of inflammatory microenvironment on the proliferation and differentiation of SCAP. Then it provided a theoretical basis to further clarify its function in the process of the repairing, regeneration mechanism of periapical tissues, and also provided a certain experimental basis for the further study of the biological characteristics, osteogenic/dentinogenic differentiation mechanism, pulp/dentin regeneration and root regeneration tissue engineering of SCAP.Methods1. Isolation and identification of stem cells from apical papillaWe obtained the normal human impacted third molars or orthodontic teeth with immature roots during the course of orthodontic treatment. The apical papilla was separated as a whole from the tip of the root under sterile conditions. Then the SCAP were isolated and cultured by tissue block method, and the cells were collected and prepared for limiting dilution procedures to obtain single-colony-derived strains. The morphological characteristics and growth situation of the primary cells and subculture cells of SCAP were observed under an inverted microscope. The expression of the vimentin, CD24, STRO-1and cytokeratin were detected by using immunofluorescence technique. The proliferation activity of SCAP was measured by MTT assay and then the cell growth curve was drawn. The colony forming unit-fibroblastic (CFU-F) forming efficiency of SCAP was detected by staining with crystal violet. And the cell cycle analysis was determined by using a cell cycle assay kit with the flow cytometry.The apical papilla stem cells were cultured under conditioned medium (mineralized induced liquid, adipogenic and chondrogenic induction liquid liquid) respectively.After induction for3weeks, alkaline phosphatase (ALP) activity staining, alizarin red staining, oil red-O staining and alcian blue staining were used to verify whether it possess multil-differentiation ability respectively. After mineralized induction for3weeks, the expression of osteogenic/dentinogenic related markers alkaline phosphatase, dentin matrix protein-1(DMP-1), dentin sialophosphoprotein (DSPP) expression of SCAP were detected by immunofluorescence technique.2. The effects of inflammatory cytokines on the proliferation of stem cells from apical papillaThe cells were seeded in the96-well plate with standard medium. After24hours, they were serum-deprived overnight, then replaced with fresh a-MEM medium (2%FBS) and treated with TNF-α, IL-8, IL-1β (0,1,2.5,5,10, and50ng/mL) for4days, with or without10ng/mL TNF-a,10ng/mL IL-8or5ng/mL IL-1βfor different time periods (1,3,5,7,9days), experiments were performed in triplicate and were repeated three times. Culture medium was replaced at24h interval. The cell proliferation was assessed with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer’s instructions. According to the above packet with10d incubation, the effects of colony-forming efficiency were detected by crystal violet staining. According to the above packet with2d incubation, the effects of Cell cycle analysis were determined by using a cell cycle assay kit with the flow cytometry. Experiments were performed in triplicate and were repeated three times.3. The effects of inflammatory cytokines on the osteogenic/dentinogenic differentiation of stem cells from apical papillaSCAP were plated in6-well plates with standard medium for24hours, then the medium was replaced with osteogenic/dentinogenic differentiation medium containing10%FBS,50g/mL ascorbic acid,10mM glycerophosphate and the cells were stimulated with or without10ng/mL TNF-a,10ng/mL IL-8or5ng/mL IL-1β. After induction for14days, the cultures were measured with alkaline phosphatase activity staining and alizarin red staining, then ALP staining and alizarin red staining was measured quantitatively by ALP activity assay and colorimetric assay. According to the above packet with mineralized induction for3,7,14days, then extraction of total RNA, using qRT-PCR method to detect the expression of osteogenic/dentinogenic related genes ALP, DSPP, DMP-1. Experiments were performed in triplicate and were repeated three times.Result1. Isolation and identification of stem cells from apical papillaCells started to grow from the dental papilla tissue at approximately day5. SCAP were capable of forming adherent clonogenic cell clusters, and retained fibroblastic spindle morphology. Purified SCAP were cultured and obtained by limited dilution cloning assay and further expand to culture. The passaged cells presented fibroblast like shape, greater growth ability and arranged in spiral when the cells reached90%confluence. Immunocytofluorescence staining showed that SCAP were positive for vimentin, the early mesenchymal stem cell marker STRO-1and the specific marker CD24, and negative for cytokeratin. In the MTT assay, the growth curve of SCAP was observed from days1to10and like "S" shape through incubation phase, logarithmic growth phase, plateau phase. The crystal violet staining result showed that the cells presented colony like growth under microscopic and the colony-forming efficiency was determined as18.1%-21.3%. The analysis of cell cycle showed G0/G1phase cells68.12%, G2/M5.73%, S26.16%, the majority of cells were in the quiescent stage and early stage S synthesis, the high proportion of stage S indicated strong proliferation ability. After osteogenic/dentinogenic induction for3weeks, ALP staining and alizarin red staining indicated that extensive amounts of mineralized nodules formation of SCAP were observed under inverted microscope, and immunocytofluorescence staining showed that the cells were positive for ALP, DSPP DMP-1. In addition, some SCAP developed into oil red O-positive lipid fat cells after adipogenesis induction for3weeks and also were positive for alician blue staining after chondrogenic induction for3weeks.2. The effects of inflammatory cytokines on the proliferation of stem cells from apical papillaThe dose-response studies showed that10ng/mL TNF-a,10ng/mL IL-8and5ng/mL IL-1βp had the most optimal effect on proliferation of SCAP in comparison with the control, and higher amounts did not increase proliferation further. The time-response studies showed that the proliferation was increased for cells treated with10ng/mL TNF-a,10ng/mL IL-8and5ng/mL IL-1β at day3and day5, but longer time stimulating decreased the proliferation (P<0.05).10ng/mL TNF-a,10ng/mL IL-8and5ng/mL IL-1β stimulation promoted cell cycle progression and improved the numbers of CFU-F formation of SCAP.3. The effects of inflammatory cytokines on the osteogenic/dentinogenic differentiation of stem cells from apical papillaAfter3weeks of SCAP cultured with differentiation medium containing10ng/mL TNF-a or5ng/mL IL-1β, the ALP activity and mineralized nodule formation of these groups decreased significantly in comparison with the controls, and the expression of ALP mRNA was also significantly down regulated (P<0.05). In contrast,10ng/mL IL-8showed no significant difference. At the same time, after mineralization induction, DSPP mRNA and DMP-1mRNA expression in each experimental group of SCAP were significantly increased at7days (P<0.05). The expression of DMP-1mRNA in experimental group was decreased (P<0.05), while the expression of DSPP mRNA in each group had no obvious change.ConclusionIn this study, the primary SCAP was obtained from the apical papilla by tissue block method successfully and the purified SCAP were cultured and obtained by limited dilution cloning assay. The characteristics of mesenchymal stem cells of SCAP were assessed by immunofluorescence staining. The osteogenic/dentinogenic, adipogenic and chondrogenic induction experiment indicated that SCAP are capable of multi-differentiation potential. Under inflammatory cytokines TNF-α, IL-1β, IL-8simulation, the results showed that it can promote cell proliferation and regulate osteogenic/dentinogenic differentiation of SCAP. The results of our study provided experimental basis for the further study of the osteogenic/dentinogenic differentiation of SCAP under inflammatory microenvieronment, and also provided a theoretical basis to further clarify its function in the process of the repairing, regeneration mechanism of periapical tissues.