Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions

Background

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.

Methods

PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.

Results

PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.

Conclusions

Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.

General significance

These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.     

Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition with β-catenin signaling in inflammatory microenvironments

Inflammation can influence multipotency and self-renewal of mesenchymal stem cells (MSCs), resulting in their awakened bone-regeneration ability. Human periodontal ligament tissue-derived MSCs (PDLSCs) have been isolated, and their differentiation potential was found to be defective due to β-catenin signaling indirectly regulated by inflammatory microenvironments. Nuclear factor-κB (NF-κB) is well studied in inflammation by many different groups. The role of NF-κB needs to be studied in PDLSCs, although genetic evidences have recently shown that NF-κB inhibits osteoblastic bone formation in mice. However, the mechanism as to how inflammation leads to the modulation of β-catenin and NF-κB signaling remains unclear. In this study, we investigated β-catenin and NF-κB signaling through regulation of glycogen synthase kinase 3β activity (GSK-3β, which modulates β-catenin and NF-κB signaling) using a specific inhibitor LiCl and a phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002. We identified that NF-κB signaling might be more important for the regulation of osteogenesis in PDLSCs from periodontitis compared with β-catenin. BAY 11-7082 (an inhibitor of NF-κB) could inhibit phosphorylation of p65 and partly rescue the differentiation potential of PDLSCs in inflammation. Our data indicate that NF-κB has a central role in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. Given the molecular mechanisms of NF-κB in osteogenic differentiation governed by inflammation, it can be said that NF-κB helps in improving stem cell-mediated inflammatory bone disease therapy.     

circRNAs在成骨细胞分化中的调控作用

环状RNAs(circular RNAs,circRNAs)是一类新型内源性非编码RNAs,在调节生长发育、疾病发展等方面具有重要的生物学功能。新近研究证实,circRNAs参与调控牙周膜干细胞和骨髓干细胞等的成骨细胞分化。该文就当前circRNAs在成骨细胞分化中的最新研究进展作一综述,以帮助开发骨科疾病新疗法。     

CTHRC1 promotes osteogenic differentiation of periodontal ligament stem cells by regulating TAZ

Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.     

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.     

Double-edged-sword effect of IL-1β on the osteogenesis of periodontal ligament stem cells via crosstalk between the NF-κB,MAPK and BMP/Smad signaling pathways

Microenvironmental conditions can interfere with the functional role and differentiation of mesenchymal stem cells (MSCs). Recent studies suggest that an inflammatory microenvironment can significantly impact the osteogenic potential of periodontal ligament stem cells (PDLSCs), but the precise effects and mechanisms involved remain unclear. Here, we show for the first time that interleukin-1β (IL-1β) has dual roles in the osteogenesis of PDLSCs at concentrations ranging from physiologically healthy levels to those found in chronic periodontitis. Low doses of IL-1β activate the BMP/Smad signaling pathway to promote the osteogenesis of PDLSCs, but higher doses of IL-1β inhibit BMP/Smad signaling through the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, inhibiting osteogenesis. These results demonstrate that crosstalk between NF-κB, MAPK and BMP/Smad signaling mediates this dual effect of IL-1β on PDLSCs. We also show that the impaired osteogenesis of PDLSCs results in more inflammatory cytokines and chemokines being released, inducing the chemotaxis of macrophages, which further clarifies the role of PDLSCs in the pathogenesis of periodontitis.Approximately 90% of the population suffers from periodontitis,^{1, 2} which is characterized by chronic bacterial infections in the supporting structures of the teeth and a homeostatic imbalance between two coupled process in the periodontal system – bone resorption by osteoclasts and bone formation by osteoblasts. This disease involves interactions with bacterial products, numerous cell populations and different inflammatory mediators, and it can lead to tooth loss in adults.^{1, 2}Periodontal ligament stem cells (PDLSCs), a newly recognized sub-population of mesenchymal stem cells (MSCs), have attracted increasing attention in relation to their multipotency. As PDLSCs can easily be obtained from periodontal tissue, they are considered important for prospective cell-based therapies. Recently, PDLSCs have been shown to migrate to the site of periodontal lesions and to mediate periodontal regeneration.^{3, 4, 5} However, recent studies have found that the osteogenic capacity of stem cells is impaired in inflammatory microenvironments^6,7 and that there are complex interactions between stem cells and the microenvironment under pathological conditions. Our previous studies found that disrupted and disease-associated microenvironments could influence the characteristics and functions of MSCs.8-10 Additionally, some studies have indicated that MSCs act in an immunomodulatory manner to regulate the function and chemotaxis of immune cells and that environmental factors may determine which immunomodulatory pathways are operational in MSCs.¹¹ Thus, we assume that the mutual interactions between stem cells and inflammatory microenvironments are crucial to harnessing the regenerative potential of PDLSCs for therapeutic use.Interleukin-1 (IL-1) is a pleiotropic cytokine and a central mediator of innate immunity and inflammation.¹² In clinical studies, IL-1β has been found in increased concentrations in gingival crevicular fluid (GCF) and at sites of periodontal damage,^{13, 14} and levels of IL-1β have been reported to decrease after periodontal treatment.^{15, 16} Compared with levels at healthy sites, local IL-1β and tumor necrosis factor-α (TNF-α) levels in the microenvironments of chronic periodontitis have been found to be significantly elevated and to be associated with periodontal tissue destruction.17–19 IL-1 stimulates bone resorption by promoting osteoclast activation17^,20^,21 and mediates the osteoclastogenic effects of TNF-α by enhancing the expression of RANKL.15 In inflammatory microenvironments, IL-1 and TNF have a prominent role in the pathogenesis of periodontitis.19 Although TNF-α has activity similar to that of IL-1β, IL-1β is present at higher levels in inflamed gingival tissues, and its expression is limited to the connective tissue layer.22 Multiple studies have investigated the effect of IL-1β on osteoblast differentiation,^{23, 24} but conflicting data has been presented and the underlying mechanism of its effects remains unclear.25 A previous study has shown that the concentration of IL-1β in GCF is 145±167 pg/ml in healthy subjects and 6452±2289 pg/ml in patients with chronic periodontitis.26 In this study, we mimicked an inflammatory microenvironment using IL-1β at different concentrations that ranged from healthy physiological levels to those observed in the GCF in cases of chronic periodontitis26 and tried to establish an in vitro osteogenesis model to investigate the effects of different doses of IL-1β on PDLSCs.Previously, it has been reported that the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways have crucial roles in the regulation of inflammation and bone metabolism.²⁷²⁸ In addition, the BMP/Smad signaling pathways have important roles in the regulation of osteoblast differentiation.29 However, the roles these signaling pathways have in the osteogenesis of MSCs in inflammatory microenvironments remain unclear. In the present study, we investigated the interactions of BMP/Smad, MAPK and NF-κB signaling pathways in mediating the IL-1β-regulated osteogenic differentiation of PDLSCs. Because the resident periodontal cells can produce various inflammatory mediators that induce inflammatory cells to invade the tissue and affect bone resorption,30 we further examined the role of PDLSCs in the pathogenesis of periodontitis by determining the production of inflammatory cytokines and chemokines by PDLSCs in which osteogenesis was inhibited by IL-1β.     

Metformin facilitates the proliferation,migration, and osteogenic differentiation of periodontal ligament stem cells in vitro

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis‐related markers were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time‐dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.