| Aim:Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple cell types, including osteogenic, adipogenic and chondrogenic cell lineages, and may contribute to tissue repair and regeneration. Human periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs) exhibit mesenchymal stem cells characteristics such as clonogenicity, high proliferation, when transplanted into immunocompromised rodents. PDLSCs showed the capacity to generate a cementum/PDL-like structure and contribute to periodontal tissue repair, that are capable of being used in stem-cell-mediated therapies and tissue engineering.Some studies demonstrate that a chronic inflammatory microenvironment that generates excessive cytokines may control stem cell fate and characteristics through diverse regulatory mechanisms. Periodontitis is an inflammatory disease which manifests clinically as loss of connective periodontal tissues including periodontal ligament and alveolar bone, resulting in loosening of the teeth, loss of chewing function, and ultimately tooth loss. The maintenance and regulation of normally quiescent stem cell populations is tightly controlled by the local microenvironment, according to the requirements of the host tissue. Thus, it is necessary to find out the changes in PDLSCs under inflammatory microenvironment.Inflammatory cytokines suppress tissue regeneration in bone marrow pluripotent mesenchymal stem cells.IL-1βand TNF-αare involved in the initiation and progression of chronic inflammation,have central functions in the induction of downstream responses to injury by having a chemotactic effect on other in?ammatory cells, enhancing extracellular matrix synthesis,stimulating angiogenesis and recruiting endogenous fibrogenic cells to the injury site. Moreover, IL-1βand TNF-αcan change the biological functions of stem cells. However, it has not been reported whether periodontal inflammation influences the differentiation potential of MSCs and inhibits bone regeneration.There is variety of molecular signaling pathway regulats tissue regeneration by stem cells. To achieve successful periodontal regeneration, the restoration of alveolar bone height is required. The Wnt signaling pathway plays an important role in the progression of bone regeneration. Nevertheless, the mechanism of how Wnt signaling pathway regulates osteogenic differentiation of MSCs in an inflammatory microenvironment is poorly understood. Our aim is to rescue the PDLSCs in inflammatory microenvironment through the regulation of Wnt signaling pathway.Materials and Methods:PartⅠ:Primary cultures were obtained by culturing explants of healthy periodontal tissues from 4 patients undergoing premolar and third molar extractions because of orthodontic reasons. Inflamed periodontal tissues were obtained from 7 patients diagnosed with periodontitis with alveolar bone loss (2/3) and more than 1 pocket (depth≥5 mm) extraction due to periodontitis. We isolated a mesenchymal stem cell population from the periodontal ligaments of human healthy individuals and patients with periodontitis. We tested CFU-F and the expression of mesenchymal stem-cell markers by FCM of H-PDLSCs and P-PDLSCs. We tested cell activity by MTT and used FCM to tested the proliferation index of the P-PDLSCs and H-PDLSCs. To investigate the potential of PDLSCs to undergo osteoblastic/adipogenic differentiation, P-PDLSCs and H-PDLSCs were cultured in adipogenesis-inducing or osteogenic medium for 14 days. We tested the mRNA expression ofPPARγand RUNX2 by Real time PCR.PartⅡ:To investigate whether the inflammatory microenvironment could affect the biological behavior of PDLSCs, we measured TNF-αand IL-1βexpression as both secreted cytokine oncentration in conditioned medium and mRNA expression level. H-PDLSCs were treated with IL-1β(5 ng/ mL) and TNF-α(10 ng/ mL) in basic medium for 24 h. Then, the culture medium was changed to osteogenic medium, which contained both IL-1β(5 ng/mL) and TNF-α(10 ng/ mL), and was used for H-PDLSC cultures for 7 d or 21d. The cells were then harvested and subjected to assays for in vitro osteogenic diff erentiation. Western Blot was used to detect levels of totalβ-catenin in both P-PDLSCs and H-PDLSCs .In order to begin the investigation of the mechanism of the ef fect ofβ-catenin on osteogenesis, immunofluorescence staining and real time-PCR were used for determining the protein levels(OCN,ALP,BSP) and gene expression (RUNX2,OCN COL-1)in PDLCs.PartⅢ:The expression ofβ-catenin as well as that of CaMKII and NLK, which are the key proteins of the noncanonical Wnt signaling pathway, were investigated following culture in osteogenic medium for 7 d in H-PDLSCs and P-PDLSCs. With the use of WB, we compared the levels of p38MAPK、RUNX2、COL-1BMS in H-PDLSCs and P-PDLSCs.PartⅣ:The primary group of cultures were treated with 100 ng/mL human recombinant Wnt3a, and a subset of these cultures were treated with the soluble Wnt inhibitor, DKK-1, at different concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL). Cells were seeded at a density of 5000 cells/cm2 in T25 culture flasks, maintained and expanded inα-MEM(10% FBS), and allowed to adhere overnight. The culture medium was then changed to the basal or osteogenic medium, which contained either Wnt3a or DKK-1 used for the PDLSC cultures as described above. On culture day 7, the cells were harvested and subjected to assays for in vitro osteogenic differentiation and changes of Wnt signaling pathway.Results:Part I:1. The isolated cells retained similarly typical fibroblastic spindle shape and were capable of forming CFU-F generated from single cells. The ability of P-PDLSCs to form adherent clonogenic cell clusters had no significant difference with the H-PDLSCs as shown by colony-forming efficiency analysis (P>0.05).2. FACS profiling showed that P-PDLSCs expressed stem cell markers of PDLSCs similar to those of H-PDLSC. Both P-PDLSCs and H-PDLSCs were strongly positive for CD44, CD90, and CD105 but negative for CD45. P-PDLSCs and H-PDLSCs were moderately positive for Stro-1 and CD146.3. The results showed that the growth of P-PDLSCs was better than that of H-PDLSCs from day 4 to day 8 (P < 0.05). The results of flow cytometric analysis showed that proliferation index of the P-PDLSCs cells was significantly increase relative to H-PDLSCs counterparts.4. The expression of RUNX2 and peroxisome proliferator-activated receptorγ(PPARγ) indicated significant differences between H-PDLSCs and P-PDLSCs.PartⅡ:1. The secretion of TNF-αand IL-1βwere highly increased in P-PDLSCs and H-PDLSCs after treatment with cytokines (P < 0.05,). The same trend could be seen in the gene expression levels of both TNF-αand IL-1βin PDLSCs.2. Our data revealed that levels of totalβ-catenin were increased in both P-PDLSCs and H-PDLSCs treated with cytokines.3. The inflammatory microenvironments resulted in an increased expression of cyclin D1 a target gene of theβ-catenin/LEF1 pathway.4. The results showed that the PDLSCs cultured in osteogenic medium were positive for OCN, ALP and BSP.5. RUNX2 ,OCN and COL-1were more strongly induced in H-PDLSCs cells than in P-PDLSCs cells and H-PDLSCs after treatment with cytokines relative to H-PDLSCs.PartⅢ:1. Our data revealed that level of nuclearβ-catenin was decreased in both H-PDLSCs and P-PDLSCs cultured in osteogenic medium relative to controls, indicative of decreased canonical Wnt signaling. Additionally, we found that theβ-catenin concentration in the cytoplasmic was increased in PDLSCs under osteogenic condition especially in H-PDLSCs compared with H-PDLSCs.2. When we examined the level of GSK-3βin response to osteogenic stimulation, we observed an increase in both H-PDLSCs and P-PDLSCs, while it was weakend in P-PDLSCs relative to H-PDLSCs.3. When stimulated with osteogenic medium for 7 d, CaMKII and NLK activation were observed in H-PDLSCs and P-PDLSCs, but we found that the levels of CaMKII and NLK in H-PDLSCs were higher than those in H-PDLSCs cultured in the presence or absence of osteogenic medium. 4. The basal level of p38 in H-PDLSCs was lower than that in P-PDLSCs cells cultured in the basic medium. However, the level of p-p38 was increased in H-PDLSCs than in P-PDLSCs under osteogenic condition.5. Osteogenic medium enhanced the levels of RUNX2 and COL-1of PDLSCs especially in H-PDLSCs.PartⅣ:1. H-PDLSCs and P-PDLSCs expressed theβ-catenin protein without culturing in osteogenic medium, and its expression was increased following Wnt3a treatment, particularly in the nuclear fraction. In our study, we found that treatment with Wnt3a lead to significantly increased expression of cyclin D1 and LEF1 in both H-PDLSCs and P-PDLSCs without culturing in osteogenic medium. Treatment with Wnt3a lead to significantly increased expression of cyclin D1 and LEF1 in both H-PDLSCs and P-PDLSCs. However, treatment of PDLSCs with Wnt3a under osteogenic differentiation conditions resulted in a decrease in NLK and CaMKII kinase activity. Wnt3a suppressed osteogenic differentiation in P- PDLSCs.2. PDLSCs exposed to DKK-1 for 7 d show decreased expression levels ofβ-catenin with the greatest decrease observed in the group of P-PDLSCs grown in osteogenic medium in the presence of DKK-1. Moreover, when we inhibited canonical Wnt pathway with DKK-1 under osteogenic conditions, both CaMKII and NLK activation were increased, whereas LEF1 was effectively blocked in PDLSCs. DKK-1 promotes osteogenic differentiation in P-PDLSCs.Conclusion:1. In this study we successfully isolated PDLSCs from patients with periodontitis, we found that inflammatory microenvironments induce proliferation but reduces differentiation potentials of PDLSCs.2. High levels ofβ-catenin affected the osteogenic potential of PDLSCs in inflammatory microenvironments.3. Both canonical Wnt/β-catenin and noncanonical Wnt/Ca2+ pathway could regulated the osteogenic differentiation potential of P-PDLSCs .These results confirmed the crosstalk betweenβ-catenin and the noncanonical Wnt signaling pathway in osteoblast differentiation of stem cells and the bone formation process.4.β-Catenin activated by Wnt3a suppressed osteogenic differentiation but promoted proliferation of P-PDLSCs. However,suppression ofβ-catenin by DKK-1 promotes osteogenic differentiation and suppresses proliferation of P-PDLSCs.Our results from these studies suggested the possibility that a change in theβ-catenin level could achieve a dynamic balance between the canonical and noncanonical Wnt pathway, and may lead to the recovery of the osteogenic differentiation potential of stem cells in inflammatory microenvironments.In summary, our data indicate that Wnt signaling pathway plays a important role in regulating osteogenic differentiation of MSC in inflammatory microenvironments. Given the important role of Wnt signaling for osteogenic differentiation, it was possible that agents modifying this pathway could be of value to bone regeneration in chronic inflammatory microenvironments by MSCs. |
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