Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Compressive force-induced LincRNA-p21 inhibits mineralization of cementoblasts by impeding autophagy.

The mineralization capability of cementoblasts is the foundation for repairing orthodontic treatment-induced root resorption. It is essential to investigate the regulatory mechanism of mineralization in cementoblasts under mechanical compression to improve orthodontic therapy. Autophagy has a protective role in maintaining cell homeostasis under environmental stress and was reported to be involved in the mineralization process. Long noncoding RNAs are important regulators of biological processes, but their functions in compressed cementoblasts during orthodontic tooth movement remain unclear. In this study, we showed that compressive force downregulated the expression of mineralization-related markers. LincRNA-p21 was strongly enhanced by compressive force. Overexpression of lincRNA-p21 downregulated the expression of mineralization-related markers, while knockdown of lincRNA-p21 reversed the compressive force-induced decrease in mineralization. Furthermore, we found that autophagy was impeded in compressed cementoblasts. Then, overexpression of lincRNA-p21 decreased autophagic activity, while knockdown of lincRNA-p21 reversed the autophagic process decreased by mechanical compression. However, the autophagy inhibitor 3-methyladenine abolished the lincRNA-p21 knockdown-promoted mineralization, and the autophagy activator rapamycin rescued the mineralization inhibited by lincRNA-p21 overexpression. Mechanistically, the direct binding between lincRNA-p21 and FoxO3 blocked the expression of autophagy-related genes. In a mouse orthodontic tooth movement model, knockdown of lincRNA-p21 rescued the impeded autophagic process in cementoblasts, enhanced cementogenesis, and alleviated orthodontic force-induced root resorption. Overall, compressive force-induced lincRNA-p21 inhibits the mineralization capability of cementoblasts by impeding the autophagic process.

The periodontal ligament, temperature-sensitive ion channels TRPV1-4, and the mechanosensitive ion channels Piezo1 and 2: A Nobel connection.

Teeth are subject to a variety of mechanical forces and vectors. The periodontal ligament (PDL), fibrous tissue that connects the cementum of the tooth to the bony socket, plays a decisive role in transmitting force to alveolar bone via Sharpey fibers, transforming and converting these forces into biological signals. This interaction effects significant osteoblastic and osteoclastic responses via autocrine proliferative and paracrine responses. Recent discoveries of receptors for temperature and touch by the Nobel laureates David Julius and Ardem Patapoutian, respectively have a profound impact on orthodontics. Transient receptor vanilloid channel 1 (TRPV1), initially described as a receptor for temperature, has been proposed to participate in the sensing of force. TRPV4, another ion channel receptor, perceives tensile forces as well as thermal and chemical stimuli. Piezo1 and 2, the classic receptors for touch, in addition to the aforementioned receptors, have similarly been described on PDL-derived cells. In this text, we review the role of the temperature-sensitive ion channels and mechanosensitive ion channels on their biological function and influence in orthodontic treatment.

REV-ERBs negatively regulate mineralization of the cementoblasts.

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Stem cell contributions to cementoblast differentiation in healthy periodontal ligament and periodontitis.

Loss of tissue attachment as a consequence of bacterial infection and inflammation represents the main therapeutic target for the treatment of periodontitis. Cementoblasts, the cells that produce the mineralized tissue, cementum, that is responsible for connecting the soft periodontal tissue to the tooth, are a key cell type for maintaining/restoring tissue attachment following disease. Here, we identify two distinct stem cell populations that contribute to cementoblast differentiation at different times. During postnatal development, cementoblasts are formed from perivascular-derived cells expressing CD90 and perivascular-associated cells that express Axin2. During adult homeostasis, only Wnt-responsive Axin2+ cells form cementoblasts but following experimental induction of periodontal disease, CD90+ cells become the main source of cementoblasts. We thus show that different populations of resident stem cells are mobilized at different times and during disease to generate precursors for cementoblast differentiation and thus provide an insight into the targeting cells resident cells for novel therapeutic approaches. The differentiation of these stem cells into cementoblasts is however inhibited by bacterial products such as lipopolysaccharides, emphasizing that regeneration of periodontal ligament soft tissue and restoration of attachment will require a multipronged approach.

Long noncoding RNA expression profiles in intermittent parathyroid hormone induced cementogenesis.

The aim of this study was to explore the involvement of long noncoding RNAs (lncRNAs) during intermittent parathyroid hormone (PTH) induced cementogenesis. Expression profiles of lncRNAs and mRNAs were obtained using high-throughput microarray. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and coding-noncoding gene coexpression networks construction were performed. We identified 190 lncRNAs and 135 mRNAs that were differentially expressed during intermittent PTH-induced cementogenesis. In this process, the Wnt signaling pathway was negatively regulated, and eight lncRNAs were identified as possible core regulators of Wnt signaling. Based on the results of microarrray analysis, we further verified the repressed expression of Wnt signaling crucial components ß-catenin, APC and Axin2. Above all, we speculated that lncRNAs may play important roles in PTH-induced cementogenesis via the negative regulation of Wnt pathway.

Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling.

Ciliary neurotrophic factor (CNTF) was identified as a survival factor in various types of peripheral and central neurons, glia and non-neural cells. At present, there is no available data on the expression and localization of CNTF-receptors in cementoblasts as well as on the role of exogenous CNTF on this cell line. The purpose of this study was to determine if cementoblasts express CNTF-receptors and analyze the mechanism of its apoptotic regulation effects on cementoblasts. OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals). Quantified transcriptional (RT-qPCR) and translational (WB) products of CNTFRα, IL-6Rα (CD126), LIFR, p-GP130, GP130, p-ERK1/2, ERK1/2, Caspase-8, -9, -3 and cleaved-caspase-3 were evaluated. Immunofluorescence (IF) staining was applied to visualize the localization of the CNTF-receptors within cells. The apoptosis ratio was measured with an Annexin-V FITC/PI kit. The ERK1/2 antagonist (FR180204, Calbiochem) was added for further investigation by flow cytometry analysis. The CNTF-receptor complex (CNTFRα, LIFR, GP130) was functionally up-regulated in cementoblasts while cultivated with exogenous CNTF. CNTF significantly attenuated cell viability and proliferation for long-term stimulation. Flow cytometry analysis shows that CNTF enhanced the apoptosis after prolonged duration. However, after only a short-term period, CNTF halts the apoptosis of cementoblasts. Further studies revealed that CNTF activated phosphorylated GP130 and the anti-apoptotic molecule ERK1/2 signaling to participate in the regulation of the apoptosis ratio of cementoblasts. In conclusion, CNTF elicited the cellular functions through a notable induction of its receptor complex in cementoblasts. CNTF has an inhibitory effect on the cementoblast homeostasis. These data also elucidate a cellular mechanism for an exogenous CNTF-triggered apoptosis regulation in a mechanism of ERK1/2 and caspase signaling and provides insight into the complex cellular responses induced by CNTF in cementoblasts.

Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

In animal models, the administration of ciliary neurotrophic factor (CNTF) was demonstrated to reduce bone mass and to participate in bone remodeling. Cementoblasts, a cell type embedded in the cementum, are the main cells to produce and mineralize the extracellular matrix. The effect of CNTF on cementoblasts has not yet been addressed. Thus, the goal of this in vitro study was to investigate possible influences of exogenous CNTF on cementogenesis, as well as autophagy regulation and subsequent mechanisms in cementoblasts. Cementoblasts (OCCM-30) were stimulated with exogenous CNTF. Alizarin Red staining was performed to analyze the functional differentiation (mineralization) of OCCM-30 cells. The release of OPG was quantified by ELISA. The expression of cementogenesis markers (RUNX-2, OCN, BMP-7, BSP, and SPON-2) was evaluated by RT-qPCR. Western blotting (WB) was performed for the protein expression of STAT3, COX-2, SHP-2, cPLAα, cPLAß; ERK1/2, P38, and JNK. The autophagic flux was assessed using WB and RT-qPCR analysis of LC3A/B, Beclin-1, and Atg-5, and the autophagosome was investigated by immunofluorescence staining (IF). The ERK1/2 (FR180204) or STAT3 (sc-202818) antagonist was added, and the cellular response was analyzed using flow cytometry. Exogenous CNTF significantly attenuated mineralized nodule formation, impaired OPG release, and downregulated the mRNA levels of RUNX-2, OCN, BMP-7, and BSP. Moreover, CNTF induced the phosphorylation of STAT3 and activated a transient activation of SHP-2, cPLAß, ERK1/2, P38, and JNK protein. CNTF also induced autophagosome formation and promoted autophagy-associated gene and protein expressions. Additionally, the inhibition of ERK1/2 or STAT3 reversed a CNTF-induced mineralization impairment and had regulatory effects on CNTF-induced autophagosome formation. Our data revealed that CNTF acts as a potent inhibitor of cementogenesis, and it can trigger autophagy, in part by ERK1/2 and STAT3 commitment in the cementoblasts. Thus, it may play an important role in inducing or facilitating inflammatory root resorption during orthodontic tooth movement.

Hypoxia-inducible factor 1-alpha acts as a bridge factor for crosstalk between ERK1/2 and caspases in hypoxia-induced apoptosis of cementoblasts.

Hypoxia-induced apoptosis of cementoblasts (OCCM-30) may be harmful to orthodontic treatment. Hypoxia-inducible factor 1-alpha (HIF-1α) mediates the biological effects during hypoxia. Little is known about the survival mechanism capable to counteract cementoblast apoptosis. We aimed to investigate the potential roles of HIF-1α, as well as the protein-protein interactions with ERK1/2, using an in-vitro model of chemical-mimicked hypoxia and adipokines. Here, OCCM-30 were co-stimulated with resistin, visfatin or ghrelin under CoCl2 -mimicked hypoxia. In-vitro investigations revealed that CoCl2 -induced hypoxia triggered activation of caspases, resulting in apoptosis dysfunction in cementoblasts. Resistin, visfatin and ghrelin promoted the phosphorylated ERK1/2 expression in OCCM-30 cells. Furthermore, these adipokines inhibited hypoxia-induced apoptosis at different degrees. These effects were reversed by pre-treatment with ERK inhibitor (FR180204). In cells treated with FR180204, HIF-1α expression was inhibited despite the presence of three adipokines. Using dominant-negative mutants of HIF-1α, we found that siHIF-1α negatively regulated the caspase-8, caspase-9 and caspase-3 gene expression. We concluded that HIF-1α acts as a bridge factor in lengthy hypoxia-induced apoptosis in an ERK1/2-dependent pathway. Gene expressions of the caspases-3, caspase-8 and caspase-9 were shown to be differentially regulated by adipokines (resistin, visfatin and ghrelin). Our study, therefore, provides evidence for the role of ERK1/2 and HIF-1α in the apoptotic response of OCCM-30 cells exposed to CoCl2 -mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment.

Intermittent parathyroid hormone promotes cementogenesis via ephrinB2-EPHB4 forward signaling.

Intermittent parathyroid hormone (PTH) promotes periodontal repair, but the underlying mechanisms remained unclear. Recent studies found that ephrinB2-EPHB4 forward signaling mediated the anabolic effect of PTH in bone homeostasis. Considering the similarities between cementum and bone, we aimed to examine the therapeutic effect of PTH on resorbed roots and explore the role of forward signaling in this process. In vivo experiments showed that intermittent PTH significantly accelerated the regeneration of root resorption and promoted expression of EPHB4 and ephrinB2. When the signaling was blocked, the resorption repair was also delayed. In vitro studies showed that intermittent PTH promoted the expression of EPHB4 and ephrinB2 in OCCM-30 cells. The effects of PTH on the mineralization capacity of OCCM-30 cells was mediated through the ephrinB2-EPHB4 forward signaling. These results support the premise that the anabolic effects of intermittent PTH on the regeneration of root resorption is via the ephrinB2-EPHB4 forward signaling pathway.

Porphyromonas gingivalis-Lipopolysaccharide induces cytokines and enzymes of the mouse cementoblasts.

Lipopolysaccharide is a potent virulence factor of Porphyromonas gingivalis and has been implicated predominant pathogen in the development and progression of periodontal diseases. The aim of this study was to determine the effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on cementoblasts. Cementoblast (OCCM-30) were evaluated proliferation using real-time cell analyzer. In addition, total RNA was isolated at 8, 16, 24 and 72 h from 1000 ng/mL Pg-LPS treated OCCM-30 cells and mRNA expressions of pro/anti-inflammatory cytokine mediators, extracellular matrix enzymes and their tissue inhibitors and of oxidative stress enzymes were studied by real-time polymerase chain reaction. Proliferation analysis indicated that Pg-LPS slightly decreased proliferation of OCCM-30. Pg-LPS had a time-dependent impact on the expression of cytokines and enzymes. There was statistically significant up-regulation of IL-1ß and IL-10 in response to Pg-LPS at 8, 16, 24, 72 h but IL-6 expression was reduced compared to control at 8 h. While IL-8 and IL-17 expressions were determined higher than control group at 16 and 24 h, their expressions were decreased compared to control groups at 72 h (p < 0.01). While MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2 expressions increased, MMP-9 expression reduced at time-points. Also, a time-dependent up-regulation in mRNA levels for oxidative stress enzymes was detected. These results indicated that up-regulation in the transcripts of inflammation-associated cytokines and degradation enzymes were noted in the cementoblasts exposed to Pg-LPS. Cementoblasts infected with the virulence factors of periodontopathogens might also involve to the induction of inflammation and degradation of the periodontal tissues.

Periodontal ligament cells regulate osteogenesis via miR-299-5p in mesenchymal stem cells.

BACKGROUND: The periodontal ligament contains periodontal ligament cells, which is a heterogeneous cell population, and includes progenitor cells that can differentiate into osteoblasts/cementoblasts. Mesenchymal stem cells (MSCs) can differentiate into various cells and can be used for periodontal regenerative therapy. Therefore, transplanted MSCs can be affected by humoral factors from periodontal ligament cells via the transcription factors or microRNAs (miRNAs) of MSCs. In addition, periostin (POSTN) is secreted from HPL cells and can regulate periodontal regeneration and homeostasis. To clarify the regulatory mechanism of humoral factors from periodontal ligament cells, we attempted to identify key genes, specifically microRNAs, involved in this process. METHODS: Human MSCs (hMSCs) were indirectly co-cultured with human periodontal ligament cells (HPL cells) and then evaluated for osteogenesis, undifferentiated MSCs markers, and miRNA profiles. Furthermore, hMSCs were indirectly co-cultured with HPL cells in the presence of anti-POSTN monoclonal antibody (anti-POSTN Ab) to block the effect of POSTN from HPL cells, and then evaluated for osteogenesis or undifferentiated MSC markers. Moreover, hMSCs showed alterations in miRNA expression or cultured with HPL were challenged with POSTN during osteogenesis, and cells were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: hMSCs co-cultured with HPL cells showed suppressed osteogenesis and characteristic expression of SOX11, an undifferentiated MSC marker, as well as miR-299-5p. Overexpression of miR-299-5p regulated osteogenesis and SOX11 expression as observed with indirect co-culture with HPL cells. Furthermore, MSCs co-cultured with HPL cells were recovered from the suppression of osteogenesis and SOX11 mRNA expression by anti-POSTN Ab. However, POSTN induced miR-299-5p and SOX11 expression, and enhanced osteogenesis. CONCLUSION: Humoral factors from HPL cells suppressed osteogenesis in hMSCs. The suppressive effect was mediated by miR-299-5p and SOX11 in hMSCs.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

IL1ß inhibits differentiation of cementoblasts via microRNA-325-3p.

Cementum regeneration is considered the gold standard for the treatment of periodontitis. As one of the most important primary proinflammatory cytokines, interleukin 1ß (IL1ß) plays an essential role during the early stage of periodontitis and its amounts simultaneously increase dramatically during this stage. Though promising, the differentiation of cementoblasts upon IL1ß-induced inflammation of the microenvironment and the relative interaction mechanism are still unknown. Here, we found that IL1ß inhibited cementoblast differentiation and microRNA-325-3p (miR-325-3p) was increased during IL1ß-stimulated cementoblasts. Bioinformatics analysis and luciferase reporter assay demonstrated miR-325-3p targeted runt-related transcription factor 2 directly. Transfection of miR-325-3p suppressed cementoblast differentiation in vitro and the formation of cementum-like tissues in vivo. The inhibitor of miR-325-3p could mitigate the above effects induced by IL1ß. Accordingly, our finding suggests a critical role of miR-325-3p in linking inflammation to impaired cementum regeneration and provides a potential possibility for applying miR-325-3p inhibitors in the treatment of periodontitis-related bone loss.

The role of sphingosine-1-phosphate signaling pathway in cementocyte mechanotransduction.

Iatrogenic external root resorption can become a serious pathological condition with clinical tooth movement. Little is known regarding how cementum responds to mechanical loading in contrast to bone, especially under compressive stress. In the field of bone biology, several studies have established the contribution of sphingosine-1-phosphate (S1P) signaling in bone remodeling, mechanical transduction and homeostasis. As osteocytes and cementocytes share similar morphological and functional characteristics, this study aimed to investigate the mechanotransduction ability of cementocytes and to explore the contribution of S1P signaling under compressive stress induced mechanotransduction. We found that compressive stress inhibited major S1P signaling and promoted the expression of anabolic factors in IDG-CM6 cells, a novel immortalized murine cementocyte cell line. By inhibiting S1P signaling, we verified that S1P signaling played a vital role in regulating the expression of the mechanotransduction factors prostaglandin E2 (PGE2) and ß-catenin, as well as factors responsible for cementogenesis and cementoclastogenesis in IDG-CM6 cells. These results support the hypothesis that cementocytes act as key mechanically responsive cells in cementum, responding to compressive stress and directing local cementum metabolism.

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.

Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 µm in diameter, lacunae around 10-20 µm as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.

Transcription factor 7-like 2-associated signaling mechanism in regulating cementum generation by the NF-κB pathway.

Cementum regeneration is an important and challenging stage in periodontal tissue engineering and regeneration. Pathosis of the periodontium, including cementum, is important in precision diagnosis and obstinate treatment of systemic diseases, such as diabetes, leukemia, and Acquired Immune Deficiency Syndrome. Here, we found that during periodontium development, transcription factor 7-like 2 (Tcf7l2) was widely expressed in the periodontium and dental sac. In mouse cementoblast cell line (OCCM-30), the activation of NF-κB and cementoblast mineralization was significantly reduced when Tcf7l2 gene was silenced. Moreover, Tcf7l2 has a positive effect on NF-κB and cementoblast mineralization. Therefore, Tcf7l2 promotes cementum formation through the NF-κB pathway. In addition, we found a decreased expression of phosphorylated p65 and a thin layer of cementum in Tcf7l2fl/fl mice. These results suggest that Tcf7l2, which accelerates cementum formation by activating NF-κB, has great potential in the treatment of periodontitis and provide guidance for periodontal tissue regeneration.

DDIT3 regulates cementoblast mineralization by isocitrate dehydrogenase 1 through nuclear factor-κB pathway.

DDIT3 is of great importance in endoplasmic reticulum stress and is involved in many inflammatory diseases and mineralization processes. The cementum protects teeth from periodontitis and provides attachment for Sharpey's fibers of the periodontal ligament. However, the effect of DDIT3 on cementoblast differentiation remains largely unknown. In this study, we found that DDIT3 was suppressed during cementoblast differentiation. Knockdown of DDIT3 increased the messenger RNA (mRNA) and protein levels of several key osteogenic markers in vitro, including alkaline phosphatase, runt-related transcription factor 2, and osteocalcin (OCN). In addition, isocitrate dehydrogenase 1 (IDH1) was increased during cementoblast differentiation, and knockdown of DDIT3 increased the protein and mRNA levels of IDH1. Furthermore, inhibition of IDH1 could partially reduce the effect of DDIT3 on cementoblast differentiation. The DDIT3 knockdown activated nuclear factor-κB (NF-κB) transcriptional activity and upregulated the expression of p-p65 and p-IκBα. The increased osteogenic differentiation ability and IDH1 expression, as induced by the DDIT3 knockdown, could be partially turned over by the addition of NF-κB inhibitor BAY 11-7082. Overall, our data clarified that DDIT3 suppresses cementoblast differentiation through IDH1, via the NF-κB pathway.