Synovial microenvironment in temporomandibular joint osteoarthritis: crosstalk with chondrocytes and potential therapeutic targets.

Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines.

The emerging role of the semaphorin family in cartilage and osteoarthritis.

In the pathogenesis of osteoarthritis, various signaling pathways may influence the bone joint through a common terminal pathway, thereby contributing to the pathological remodeling of the joint. Semaphorins (SEMAs) are cell-surface proteins actively involved in and primarily responsible for regulating chondrocyte function in the pathophysiological process of osteoarthritis (OA). The significance of the SEMA family in OA is increasingly acknowledged as pivotal. This review aims to summarize the mechanisms through which different members of the SEMA family impact various structures within joints. The findings indicate that SEMA3A and SEMA4D are particularly relevant to OA, as they participate in cartilage injury, subchondral bone remodeling, or synovitis. Additionally, other elements such as SEMA4A and SEMA5A may also contribute to the onset and progression of OA by affecting different components of the bone and joint. The mentioned mechanisms demonstrate the indispensable role of SEMA family members in OA, although the detailed mechanisms still require further exploration.

The role of YAP/TAZ on joint and arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common forms of arthritis with undefined etiology and pathogenesis. Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ), which act as sensors for cellular mechanical and inflammatory cues, have been identified as crucial players in the regulation of joint homeostasis. Current studies also reveal a significant association between YAP/TAZ and the pathogenesis of OA and RA. The objective of this review is to elucidate the impact of YAP/TAZ on different joint tissues and to provide inspiration for further studying the potential therapeutic implications of YAP/TAZ on arthritis. Databases, such as PubMed, Cochran Library, and Embase, were searched for all available studies during the past two decades, with keywords "YAP," "TAZ," "OA," and "RA."

The role of neuropilin in bone/cartilage diseases.

Bone remodeling is the balance between osteoblasts and osteoclasts. Bone diseases such as osteoporosis and osteoarthritis are associated with imbalanced bone remodeling. Skeletal injury leads to limited motor function and pain. Neurophilin was initially identified in axons, and its various ligands and roles in bone remodeling, angiogenesis, neuropathic pain and immune regulation were later discovered. Neurophilin promotes osteoblast mineralization and inhibits osteoclast differentiation and its function. Neuropolin-1 provides channels for immune cell chemotaxis and cytokine diffusion and leads to pain. Neuropolin-1 regulates the proportion of T helper type 17 (Th17) and regulatory T cells (Treg cells), and affects bone immunity. Vascular endothelial growth factors (VEGF) combine with neuropilin and promote angiogenesis. Class 3 semaphorins (Sema3a) compete with VEGF to bind neuropilin, which reduces angiogenesis and rejects sympathetic nerves. This review elaborates on the structure and general physiological functions of neuropilin and summarizes the role of neuropilin and its ligands in bone and cartilage diseases. Finally, treatment strategies and future research directions based on neuropilin are proposed.

Loss of PA28γ exacerbates imbalanced differentiation of bone marrow stromal cells during bone formation and bone healing in mice.

Proteasome activator subunit 3 (PA28γ) is a member of the proteasome activator family, which mainly regulates the degradation and stability of proteins. Studies have shown that it plays crucial roles in lipid formation, stemness maintenance, and blood vessel formation. However, few studies have clarified the association between PA28γ and bone diseases. Herein, we identified PA28γ as a previously unknown regulator of bone homeostasis that coordinates bone formation and lipid accumulation. PA28γ-knockout mice presented with the characteristics of low bone mass and accumulation of lipids. Suppressed expression of PA28γ restrained the osteogenic differentiation and enhanced the adipogenic differentiation of bone marrow stromal cells (BMSCs). Overexpression of PA28γ promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. Mechanistically, PA28γ interacted with Wnt5α, and the two interactors appeared to be positively correlated. PA28γ mainly activated the downstream Wnt/ß-catenin signaling pathway, which affects BMSCs differentiation homeostasis. Deletion of Wnt5α significantly delayed the promotion of osteogenic differentiation and partially alleviated the inhibitory effect of adipogenic differentiation of BMSCs in the PA28γ-overexpressing group. Furthermore, we demonstrated that PA28γ-knockout mice had an inhibited rate of bone healing in a drill-hole femoral bone defect model in vivo. Therefore, our results confirm the effects of PA28γ on bone formation and bone defect repair, indicating that PA28γ mainly interacts with Wnt5α to activate the Wnt/ß-catenin signaling pathway regulating BMSCs differentiation homeostasis. Our results reveal the function of PA28γ in bone diseases and provide a new theoretical basis for expanding the treatment of bone diseases.

Long non-coding RNA LncTUG1 regulates favourable compression force-induced cementocytes mineralization via PU.1/TLR4/SphK1 signalling.

Orthodontic tooth movement (OTM) is a highly coordinated biomechanical response to orthodontic forces with active remodelling of alveolar bone but minor root resorption. Such antiresorptive properties of root relate to cementocyte mineralization, the mechanisms of which remain largely unknown. This study used the microarray analysis to explore long non-coding ribonucleic acids involved in stress-induced cementocyte mineralization. Gain- and loss-of-function experiments, including Alkaline phosphatase (ALP) activity and Alizarin Red S staining, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence analyses of mineralization-associated factors, were conducted to verify long non-coding ribonucleic acids taurine-upregulated gene 1 (LncTUG1) regulation in stress-induced cementocyte mineralization, via targeting the Toll-like receptor 4 (TLR4)/SphK1 axis. The luciferase reporter assays, chromatin immunoprecipitation assays, RNA pull-down, RNA immunoprecipitation, and co-localization assays were performed to elucidate the interactions between LncTUG1, PU.1, and TLR4. Our findings indicated that LncTUG1 overexpression attenuated stress-induced cementocyte mineralization, while blocking the TLR4/SphK1 axis reversed the inhibitory effect of LncTUG1 on stress-induced cementocyte mineralization. The in vivo findings also confirmed the involvement of TLR4/SphK1 signalling in cementocyte mineralization during OTM. Mechanistically, LncTUG1 bound with PU.1 subsequently enhanced TLR4 promotor activity and thus transcriptionally elevated the expression of TLR4. In conclusion, our data revealed a critical role of LncTUG1 in regulating stress-induced cementocyte mineralization via PU.1/TLR4/SphK1 signalling, which might provide further insights for developing novel therapeutic strategies that could protect roots from resorption during OTM.

The role of WWP1 and WWP2 in bone/cartilage development and diseases.

Bone and cartilage diseases are often associated with trauma and senescence, manifested as pain and limited mobility. The repair of bone and cartilage lesion by mesenchymal stem cells is regulated by various transcription factors. WW domain-containing protein 1 (WWP1) and WW domain-containing protein 2 (WWP2) are named for WW domain which recognizes PPXY (phono Ser Pro and Pro Arg) motifs of substrate. WWP1and WWP2 are prominent components of the homologous to the E6-AP carboxyl terminus (HECT) subfamily, a group of the ubiquitin ligase. Recently, some studies have found that WWP1 and WWP2 play an important role in the pathogenesis of bone and cartilage diseases and regulate the level and the transactivation of various transcription factors through ubiquitination. Therefore, this review summarizes the distribution and effects of WWP1 and WWP2 in the development of bone and cartilage, discusses the potential mechanism and therapeutic drugs in bone and cartilage diseases such as osteoarthritis, fracture, and osteoporosis.

AFF4 regulates osteogenic potential of human periodontal ligament stem cells via mTOR-ULK1-autophagy axis.

Scaffold protein AF4/FMR2 family member 4 (AFF4) has been found to play a role in osteogenic commitment of stem cells. However, function of AFF4 in human periodontal ligament stem cells (hPDLSCs) has not been studied yet. This present study aims to investigate the biological effect of AFF4 on osteogenic differentiation of hPDLSCs and potential mechanistic pathway. First, AFF4 expression profile was evaluated in conditions of periodontitis and osteogenic differentiation of hPDLSCs by immunohistochemical staining, western blot and qRT-PCR. Next, si-RNA mediated knockdown and lentiviral transduction mediated overexpression of AFF4 were adopted to explore impact of AFF4 on osteogenic capacity of hPDLSCs. Then, possible mechanistic pathway was identified. At last, pharmacological agonist of autophagy, rapamycin, was utilized to affirm the role of autophagy in AFF4-regulated osteogenesis of hPDLSCs. First, AFF4 expressions were significantly lower in inflamed periodontal tissues and lipopolysaccharides-treated hPDLSCs than controls, and were up-regulated during osteogenic differentiation of hPDLSCs. Next, osteogenic potential of hPDLSCs was impaired by AFF4 knockdown and potentiated by AFF4 overexpression. Moreover, AFF4 was found to positively regulate autophagic activity in hPDLSCs. At last, rapamycin treatment was shown to be able to partly restore AFF4 knockdown-suppressed osteogenic differentiation. Our study demonstrates that AFF4 regulates osteogenic potential of hPDLSCs via targeting autophagic activity. The involvement of AFF4 in periodontal homeostasis was identified for the first time.

LITTIP/Lgr6/HnRNPK complex regulates cementogenesis via Wnt signaling.

Orthodontically induced tooth root resorption (OIRR) is a serious complication during orthodontic treatment. Stimulating cementum repair is the fundamental approach for the treatment of OIRR. Parathyroid hormone (PTH) might be a potential therapeutic agent for OIRR, but its effects still lack direct evidence, and the underlying mechanisms remain unclear. This study aims to explore the potential involvement of long noncoding RNAs (lncRNAs) in mediating the anabolic effects of intermittent PTH and contributing to cementum repair, as identifying lncRNA-disease associations can provide valuable insights for disease diagnosis and treatment. Here, we showed that intermittent PTH regulates cell proliferation and mineralization in immortalized murine cementoblast OCCM-30 via the regulation of the Wnt pathway. In vivo, daily administration of PTH is sufficient to accelerate root regeneration by locally inhibiting Wnt/ß-catenin signaling. Through RNA microarray analysis, lncRNA LITTIP (LGR6 intergenic transcript under intermittent PTH) is identified as a key regulator of cementogenesis under intermittent PTH. Chromatin isolation by RNA purification (ChIRP) and RNA immunoprecipitation (RIP) assays revealed that LITTIP binds to mRNA of leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and heterogeneous nuclear ribonucleoprotein K (HnRNPK) protein. Further co-transfection experiments confirmed that LITTIP plays a structural role in the formation of the LITTIP/Lgr6/HnRNPK complex. Moreover, LITTIP is able to promote the expression of LGR6 via the RNA-binding protein HnRNPK. Collectively, our results indicate that the intermittent PTH administration accelerates root regeneration via inhibiting Wnt pathway. The lncRNA LITTIP is identified to negatively regulate cementogenesis, which activates Wnt/ß-catenin signaling via high expression of LGR6 promoted by HnRNPK.

Photobiomodulation Therapy and Pulp-Regenerative Endodontics: A Narrative Review.

Regenerative endodontic procedures (REPs) were used to recover the dental pulp's vitality in order to avoid the undesirable outcomes of conventional endodontic treatment and to promote dentinal formation, especially for immature permanent teeth. Photobiomodulation therapy (PBMT) exhibits photobiological and photochemical effects for improving the root canal's environmental conditions by compensating for oxidative stress and increasing the blood supply to implanted stem cells and improving their survival. Basic research has revealed that PBMT can modulate human dental pulp stem cells' (hDPSCs) differentiation, proliferation, and activity, and subsequent tissue activation. However, many unclear points still remain regarding the mechanisms of action induced by PBMT in REPs. Therefore, in this review, we present the applications of laser and PBMT irradiation to the procedures of REPs and in endodontics. In addition, the effects of PBMT on the regenerative processes of hDPSCs are reviewed from biochemical and cytological perspectives on the basis of the available literature. Furthermore, we consider the feasibility of treatment in which PBMT irradiation is applied to stem cells, including dental pulp stem cells, and we discuss research that has reported on its effect.

A CBCT Investigation of the Sella Turcica Dimension and Sella Turcica Bridging in Different Vertical Growth Patterns.

This study aimed to compare the sella turcica dimensions and sella turcica bridging (STB) via cone-beam computed tomography in different vertical patterns and then analyze the link between the sella turcica and vertical growth patterns. The CBCT images of 120 skeletal Class I subjects (an equal proportion of females and males; mean age of 21.46 years) were divided into three vertical growth skeletal groups. Student's t tests and Mann-Whitney U tests were used to assess the possible diversity in genders. The link between sella turcica dimensions and different vertical patterns was explored by one-way analysis of variance, as well as Pearson and Spearman correlation tests. The prevalence of STB was compared using the chi-square test. Sella turcica shapes were not linked to gender, but statistical differences were observed among different vertical patterns. In the low-angle group, a larger posterior clinoid distance and smaller posterior clinoid height, tuberculum sellae height, and dorsum sellae height were determined, and the incidence of STB was higher (p < 0.01). Sella turcica shapes were linked to vertical growth patterns, mainly involving the posterior clinoid process and STB, which could be used as an index to assess vertical growth trends.

Cyclic di-adenosine monophosphate regulates the osteogenic and adipogenic differentiation of hPDLSCs via MAPK and NF-κB signaling.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that can be recognized by infected host cells and activate the immunoinflammatory response. The purpose of this study is to demonstrate the effect of c-di-AMP on the differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mechanisms. In the present study, we find that the gingival crevicular fluid (GCF) of patients with chronic periodontitis has a higher expression level of c-di-AMP than that of healthy people. In vitro, c-di-AMP influences the differentiation of hPDLSCs by upregulating Toll-like receptors (TLRs); specifically, it inhibits osteogenic differentiation by activating NF-κB and ERK/MAPK and promotes adipogenic differentiation through the NF-κB and p38/MAPK signaling pathways. Inhibitors of TLRs or activated pathways reduce the changes induced by c-di-AMP. Our results establish the potential correlation among bacterial c-di-AMP, periodontal tissue homeostasis and chronic periodontitis pathogenesis.

Cellular communication network factor 1 interlinks autophagy and ERK signaling to promote osteogenesis of periodontal ligament stem cells.

OBJECTIVES: To investigate the effects of cellular communication network factor 1 (CCN1), a critical matricellular protein, on alveolar bone regeneration, and to elucidate the underlying molecular mechanism. BACKGROUND: In the process of orthodontic tooth movement, bone deposition on the tension side of human periodontal ligament stem cells (hPDLSCs) ensured high efficiency and long-term stability of the treatment. The matricellular protein CCN1 is responsive to mechanical stimulation, exhibiting important tasks in bone homoeostasis. However, the role and mechanism of CCN1 on alveolar bone remodeling of hPDLSCs remains unclear. METHODS: The expression and distribution of CCN1 in rat periodontal ligament were detected by immunofluorescence staining and immunohistochemical staining. ELISA verified the secretion of CCN1 triggered by stretch loading. To examine the mineralization ability of hPDLSCs induced by CCN1, Western blotting, qRT-PCR, ARS, and ALP staining were performed. CCK-8 and cell migration assay were performed to detect the cell proliferation rate and the wound healing. PI3K/Akt, MAPK, and autophagy activation were examined via Western blotting and immunofluorescence. RESULTS: Mechanical stimuli induced the release of CCN1 into extracellular environment by hPDLSCs. Knockdown of CCN1 attenuated the osteogenesis of hPDLSCs while rhCCN1 enhanced the expression of Runx2, Col 1, ALPL, and promoted the mineralization nodule formation. CCN1 activated PI3K/Akt and ERK signaling, and blockage of PI3K/Akt signaling reversed the accelerated cell migration triggered by CCN1. The enhanced osteogenesis induced by CCN1 was abolished by ERK signaling inhibitor PD98059 or autophagy inhibitor 3-MA. Further investigation demonstrated PD98059 abrogated the activation of autophagy. CONCLUSION: This study demonstrated that CCN1 promotes osteogenesis in hPDLSCs via autophagy and MAPK/ERK pathway.

Progranulin is essential for bone homeostasis and immunology.

Intercellular communication or crosstalk between immune and skeletal cells is considered a crucial element in bone homeostasis modulation. Progranulin (PGRN) is an autocrine growth factor that is structured as beads-on-a-string and participates in multiple pathophysiological processes, including atherosclerosis, arthritis, neurodegenerative pathologies, cancer, and wound repair. PGRN functions as a competitor that binds to tumor necrosis factor receptor 1 (TNFR1), thereby blocking the TNF-α pathway. PGRN is regarded as an agonist of chondrogenesis and osteogenesis, delaying the progression of inflammation through the TNFR2 pathway. The exploitation of PGRN may bring benefits for inflammatory bone diseases and the stabilization of bone homeostasis. The PGRN-modified analog Atsttrin possesses three TNFR-binding fragments and thereby exerts superior therapeutic effects on multiple preclinical animal models compared to PGRN. In this review, we highlight the emerging roles of PGRN in bone formation, as well as in physiological and TNF-α-mediated inflammatory conditions revealed in recent discoveries. We address potential therapies for the treatment of inflammatory bone conditions, such as periodontitis, by the use of PGRN and its derivative Atsttrin.

Connective tissue growth factor promotes cementogenesis and cementum repair via Cx43/ß-catenin axis.

BACKGROUND: Orthodontic tooth movement inevitably induces cementum resorption, which is an urgent problem for orthodontists to confront. Human periodontal ligament stem cells (hPDLSCs) exert an important role in the orthodontic tooth movement and exhibit multidirectional differentiation ability in cementum regeneration. Connective tissue growth factor (CTGF) is an important extracellular matrix protein for bone homeostasis and cell differentiation. The purpose of our study was to explore the role of CTGF in cementum repair and cementogenesis and to elucidate its underlying mechanism. METHODS: A cementum defect model was established by tooth movement with heavy forces, and the cementum repair effect of CTGF was observed via micro-CT, HE staining and immunohistochemical staining. RT‒qPCR, western blotting (WB), alizarin red staining and ALP activity experiments verified the mineralization ability of hPDLSCs stimulated with CTGF. The expression of Cx43 in periodontal ligament cells was detected by WB and immunofluorescence (IF) experiments after CTGF stimulation in vivo and in vitro. Subsequently, the mineralization ability of hPDLSCs was observed after application of CTGF and the small interfering RNA Si-Cx43. Additionally, co-intervention via application of the small interfering RNA Si-CTGF and the Cx43 agonist ATRA in hPDLSCs was performed to deepen the mechanistic study. Next, WB, IF experiments and co-immunoprecipitation were conducted to confirm whether CTGF triggers the Cx43/ß-catenin axis to regulate cementoblast differentiation of hPDLSCs. RESULTS: Local oral administration of CTGF to the cementum defects in vivo facilitated cementum repair. CTGF facilitated the cementogenesis of hPDLSCs in a concentration-dependent manner. Cx43 acted as a downstream effector of CTGF to regulate cementoblast differentiation. Si-Cx43 reduced CTGF-induced cementoblast differentiation. The Cx43 agonist ATRA restored the low differentiation capacity induced by Si-CTGF. Further mechanistic studies showed that CTGF triggered the activation of ß-catenin in a dose-dependent manner. In addition, co-localization IF analysis and co-immunoprecipitation demonstrated that Cx43 interacted with ß-catenin at cell‒cell connections. Si-Cx43 attenuated the substantial expression of ß-catenin induced by CTGF. The Cx43 agonist reversed the inhibition of ß-catenin induced by Si-CTGF. IF demonstrated that the nuclear importation of ß-catenin was related to the immense expression of Cx43 at cell‒cell junctions. CONCLUSIONS: Taken together, these data demonstrate that CTGF promotes cementum repair and cementogenesis through activation of the Cx43/ß-catenin signalling axis.

Activation of the pattern recognition receptor NOD1 in periodontitis impairs the osteogenic capacity of human periodontal ligament stem cells via p38/MAPK signalling.

OBJECTIVES: Nucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism. METHODS: Histological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q-PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1-regulated osteogenesis. RESULTS: NOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri-DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co-incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri-DAP-induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri-DAP in hPDLSCs. CONCLUSIONS: Our study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

Canonical Wnt/ß-catenin signaling has positive effects on osteogenesis, but can have negative effects on cementogenesis.

BACKGROUND: To date, therapeutic approaches for cementum regeneration are limited and outcomes remain unpredictable. A significant barrier to improve therapies for cementum regeneration is that the cementocyte and its intracellular signal transduction mechanisms remain poorly understood. This study aims to elucidate the regulatory mechanism of Wnt pathway in cementogenesis. METHODS: The effects of canonical Wnt signaling were compared in vitro using immortalized murine cementocyte cell line IDG-CM6 and osteocyte cell line IDG-SW3 by quantitative real-time polymerase chain reaction, Western blot, confocal microscopy, alkaline phosphatase (ALP) assay, and Alizarin red S staining. In vivo, histological changes of cementum and bone formation were examined in transgenic mice in which constitutive activation of ß-catenin is driven by Dmp1 promoter. RESULTS: Expression of components of the Wnt/ß-catenin pathway were much greater in the IDG-SW3 cells compared with the IDG-CM6 cells resulting in much lower expression of Sost/sclerostin in the IDG-SW3 cells. In the IDG-CM6 cells, low dose Wnt3a (20 ng/ml) had a modest effect while high dose (200 ng/ml) inhibited runt-related transcription factor 2, osterix, ALP, and osteopontin in contrast to the IDG-SW3 cells where high dose Wnt3a dramatically increased mRNA expression of these same markers. However, high Wnt3a significantly increased mRNA for components of Wnt/ß-catenin signaling pathway in both IDG-CM6 and IDG-SW3 cells. In vivo, constitutive activation of ß-catenin in the Dmp1-lineage cells in mice leads to bone hyperplasia and cementum hypoplasia. CONCLUSION: These findings indicate that Wnt signaling has distinct and different effects on the regulation of long bone as compared with cementum.

Connective tissue growth factor promotes cell-to-cell communication in human periodontal ligament stem cells via MAPK and PI3K pathway.

BACKGROUND: Cell-cell communication is an essential process to respond to biological stimuli and sustain the micro environmental homeostasis of human periodontal ligament stem cells (hPDLSCs). Connective tissue growth factor (CTGF), a critical secreted matrix protein, exhibits significant tasks in regulating the cell-cell and cell-matrix interactions. This study aimed to explore the relationship between CTGF and cell communication and the underlying mechanism. METHODS: qRT-PCR was used to detect CCN family, connexin, and pannexin family expression in hPDLSCs. Stimulation with CTGF, cell migration assay was performed to examine the wound repair. The scrape loading/dye transfer assay was employed to access lucifer Yellow molecules transfer efficiency mediated by cell-cell communication. Connexin43 (Cx43), Pannexin1 (Panx1), MAPK, and the PI3K/Akt signaling pathway proteins were examined via Western blotting. Immunofluorescence was applied to visualize the localization of specific proteins within cells. Corresponding pathway inhibitors were applied to hPDLSCs to detect Cx43, Panx1 expression, and intercellular communication induced by CTGF. RESULTS: Our result showed that CTGF was the second most expressed CCN family member in hPDLSCs. Cx43, and Panx1 were the most widely expressed gap junction hemichannels in hPDLSCs. CTGF enhanced hPDLSCs migration in a dose-dependent manner. CTGF promoted cell-cell communication by up-regulating Cx43 and Panx1. CTGF induced Akt, JNK, and p38 phosphorylation and subcellular relocation. Inhibiting corresponding pathways reduced Cx43 expression, thereby weakening CTGF-induced cell-cell communication. However, the Panx1 expression in CTGF-treated hPDLSCs mainly depended on PI3K/Akt signaling. CONCLUSION: We provided novel evidence that CTGF promoted cell-cell communication in hPDLSCs through MAPK and PI3K pathway.

The virulence factor GroEL directs the osteogenic and adipogenic differentiation of human periodontal ligament stem cells through the involvement of JNK/MAPK and NF-κB signaling.

BACKGROUND: GroEL, a bacterial metabolite, is an important stimulator of inflammation. The aim of this study is to confirm the effect of the virulence factor GroEL on differentiation potential of periodontal ligament (PDL) stem cells (PDLSCs) and the potential mechanisms. METHODS: PDLSCs were obtained from extracted human premolars. GroEL was administered to osteogenic- and adipogenic-induced hPDLSCs. Alkaline phosphatase (ALP) staining, Alizarin Red staining and Oil Red staining were performed. Gene and protein expression were separately measured by qPCR and Western blotting. The expression and localization of activated signaling factors were confirmed by immunofluorescence staining. The inhibitors of myeloid differentiation factor 88 (MyD88, an adaptor protein of TLRs), JNK/MAPK and NF-κB signaling were used to verify their specific effects. RESULTS: First, we found that GroEL inhibited the osteogenic differentiation and enhanced the adipogenic differentiation of hPDLSCs. Next, we found that GroEL increased the expression of TLR2 and TLR4 and GroEL activated JNK/MAPK and NF-κB signaling, which can be blocked by inhibition of MyD88. Finally, we found that inhibition of MyD88 restored GroEL-induced osteogenic and adipogenic differentiation and blocking JNK/MAPK or NF-κB signaling partly restored GroEL effects. CONCLUSION: In the current study, we revealed a potential interaction between bacteria and host cells by showing that GroEL directs the osteogenic and adipogenic differentiation of hPDLSCs by the involvement of JNK/MAPK and NF-κB signaling. This study provides evidence that bacterial products can influence the differentiation of stem cells and reveals potential effect of GroEL on the context of tissue regeneration.

CTGF facilitates cell-cell communication in chondrocytes via PI3K/Akt signalling pathway.

PURPOSES: Gap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism. MATERIALS AND METHODS: qPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK-8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell-cell communication. RESULTS: The CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up-regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF. CONCLUSIONS: For the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.