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Cementum is the least studied of all mineralized tissues and little is known about mechanisms regulating its formation. Therefore, the goal of this study was to provide new insights into the transcriptional regulation of cementum formation by determining the consequences of the deficiency of the Trps1 transcription factor in cementoblasts. We used Trps1 Col1a1 cKO (2.3Co1a1-Cre ERT2 ;Trps1 fl/fl ) mice, in which Trps1 is deleted in cementoblasts. Micro-computed tomography analyses of molars of 4-week-old males and females demonstrated significantly shorter roots with thinner mineralized tissues (root dentin and cementum) in Trps1 Col1a1 cKO compared to WT mice. Semi-quantitative histological analyses revealed a significantly reduced area of cellular cementum and localized deficiencies of acellular cementum in Trps1 Col1a1 cKO mice. Immunohistochemical analyses revealed clustering of cementoblasts at the apex of roots, and intermittent absence of cementoblasts on Trps1 Col1a1 cKO cementum surfaces. Fewer Osterix-positive cells adjacent to cellular cementum were also detected in Trps1 Col1a1 cKO compared to WT mice. Decreased levels of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme required for proper cementogenesis, were apparent in cementum, periodontal ligament, and alveolar bone of Trps1 Col1a1 cKO. There were no apparent differences in levels of bone sialoprotein (Bsp) in cementum. Quantitative analyses of picrosirius red-stained periodontal ligament revealed shorter and disorganized collagen fibers in Trps1 Col1a1 cKO mice demonstrating impaired periodontal structure. In conclusion, this study has identified Trps1 transcription factor as one of the important regulators of cellular and acellular cementum formation. Furthermore, this study suggests that Trps1 supports the function of cementoblasts by upregulating expression of the major proteins required for cementogenesis, such as Osterix and TNAP., (© 2024. The Author(s).)

Cementum is a vital component of periodontium, yet its regeneration remains a challenge. Pentraxin 3 (PTX3) is a multifunctional glycoprotein involved in extracellular matrix remodeling and bone metabolism regulation. However, the role of PTX3 in cementum formation and cementoblast differentiation has not been elucidated. In this study, we initially observed an increase in PTX3 expression during cementum formation and cementoblast differentiation. Then, overexpression of PTX3 significantly enhanced the differentiation ability of cementoblasts. While conversely, PTX3 knockdown exerted an inhibitory effect. Moreover, in Ptx3-deficient mice, we found that cementum formation was hampered. Furthermore, we confirmed the presence of PTX3 within the hyaluronan (HA) matrix, thereby activating the ITGB1/FAK/YAP1 signaling pathway. Notably, inhibiting any component of this signaling pathway partially reduced the ability of PTX3 to promote cementoblast differentiation. In conclusion, our study indicated that PTX3 promotes cementum formation and cementoblast differentiation, which is partially dependent on the HA/ITGB1/FAK/YAP1 signaling pathway. This research will contribute to our understanding of cementum regeneration after destruction., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Treatment strategies for hard tissue defects aim to establish a mineralized microenvironment that facilitates tissue remodeling. As a mineralized tissue, cementum shares a similar structure with bone and exhibits an excellent capacity to resist resorption under compression. Macrophages are crucial for mineralized remodeling; however, their functional alterations in the microenvironment of cementum remain poorly understood. Therefore, this study explores the mechanisms by which cementum resists resorption under compression and the regulatory roles of cementoblasts in macrophage functions. As a result, extracellular vesicles from compression-loaded cementoblasts (Comp-EVs) promote macrophage M2 polarization and enhance the clearance of apoptotic cells (efferocytosis) by 2- to 3-fold. Local injection of Comp-EVs relieves cementum destruction in mouse root resorption model by activating the tissue repair function of macrophages. Moreover, Comp-EV-loaded hydrogels achieve significant bone healing in calvarial bone defect. Unexpectedly, under compression, EV secretion in cementoblasts is reduced by half. RNA-Seq analysis and verification reveal that Rab35 expression decreases by 60% under compression, thereby hampering the release of EVs. Rab35 overexpression is proposed as a modification of cementoblasts to boost the yield of Comp-EVs. Collectively, Comp-EVs activate the repair function of macrophages, which will be a potential therapeutic strategy for hard tissue repair and regeneration., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Background: Intact cementum is vital for tooth stability and health. Cementoblasts, which line the root surface, are responsible for cementum formation. Recent evidence suggests that circular RNAs (circRNAs) are involved in various cellular functions and may have clinical applications. Although circHIPK3 has been shown to participate in osteogenesis, its role in cementoblast differentiation and mineralization is not well understood., Methods: The ring structure of circHIPK3 was confirmed using Sanger sequencing and an actinomycin D assay. Subcellular localization of circHIPK3 was assessed using a nucleus-cytoplasm separation assay. RT-qPCR was employed to analyze circHIPK3 expression during cementoblast differentiation and following TNF-α treatment. In vivo, periapical lesions were induced in mouse mandibular first molars to mimic an inflammatory environment, and circHIPK3 expression was evaluated. The interaction of the circHIPK3/miR-10b-5p/DOHH axis was explored through RNA pull-down assays, bioinformatics analysis, and dual-luciferase reporter assays. The effects on cementoblast differentiation and mineralization were assessed by measuring osteogenic markers, alkaline phosphatase (ALP) activity, ALP staining, and alizarin red S staining., Results: CircHIPK3 was predominantly located in the cytoplasm of cementoblasts, and its expression was significantly upregulated during cementoblast differentiation. Knockdown of circHIPK3 inhibited cementoblast differentiation and mineralization, whereas its overexpression promoted these processes. Mechanistically, circHIPK3 upregulated DOHH expression by sponging miR-10b-5p, thereby enhancing cementoblast differentiation and mineralization. The NF-κB pathway was found to act downstream of the circHIPK3/miR-10b-5p/DOHH axis in these processes. Additionally, circHIPK3 expression was significantly downregulated in inflammatory environments both in vitro and in vivo. Forced overexpression of circHIPK3 mitigated the inhibitory effects of TNF-α on cementoblast differentiation and mineralization., Conclusion: CircHIPK3 acts as a positive regulator of cementoblast differentiation and mineralization through the miR-10b-5p/DOHH/NF-κB axis, playing a crucial role in both normal and pathological cementogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: Periodontal regeneration poses challenges due to the periodontium's complexity, relying on mesenchymal cells from the periodontal ligament (hPDLSCs) to regenerate hard tissues like bone and cementum. While some hPDLSCs have high regeneration potential (HOP-hPDLSCs), most are low potential (LOP-hPDLSCs). This study analyzed hPDLSCs from a single donor to minimize inter-individual variability and focus on key differences in differentiation potentials., Design: This study used RNA-seq, genomic databases, and bioinformatics tools to explore signaling pathways (SPs), biological processes (BPs), and molecular functions (MFs) guiding HOP cells to mineralized matrix production. It also investigated limitations of LOP cells and strategies for enhancing their osteo/cementogenesis., Results: In basal conditions, HOP exhibited a multifunctional gene network with higher expression of genes related to osteo/cementogenesis, cell differentiation, immune modulation, stress response, and hormonal regulation. In contrast, LOP focused on steroid hormone biosynthesis and nucleic acid maintenance. During osteo/cementogenic induction, HOP showed strong modulation of genes related to angiogenesis, cell division, mesenchymal differentiation, and extracellular matrix production. LOP demonstrated neural synaptic-related processes and preserved cellular cytoskeleton integrity. CCKR map signaling and G-protein receptor bindings gained significance during osteo/cementogenesis in HOP-hPDLSCs. Both HOP and LOP shared common BPs related to gastrointestinal and reproductive system development., Conclusion: The osteo/cementogenic differentiation of HOP cells may be regulated by CCKR signaling, G-protein bindings, and specific hormonal regulation. LOP cells seem committed to neural mechanisms. This study sheds light on hPDLSCs' complex characteristics, offering a deeper understanding of their differentiation potential for future periodontal regeneration research and therapies., Competing Interests: Declaration of Competing Interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: Exosomes derived from stem cells are a potential cell-free tool for tissue regeneration with therapeutic potential. However, its application in cementum repair is unclear. This study aimed to investigate the effect of human periodontal ligament stem cell-derived exosomes on the biological activity of cementoblasts, the main effector cells in cementum synthesis., Materials and Methods: OCCM-30 cementoblasts were cultured with various human periodontal ligament stem cell-derived exosome concentrations. OCCM-30 cells proliferation, migration, and cementogenic mineralization were examined, along with the gene and protein expression of factors associated with cementoblastic mineralization., Results: Exosomal promoted the migration, proliferation, and mineralization of OCCM-30 cells. The exosome-treated group significantly increased the expression of cementogenic-related genes and proteins. Furthermore, the expression of p-PI3K and p-AKT was enhanced by exosome administration. Treatment with a PI3K/AKT inhibitor markedly attenuated the gene and protein expression of cementoblastic factors, and this effect was partially reversed by exosome administration., Conclusions: Human periodontal ligament stem cell-derived exosomes can promote the activity of cementoblasts via the PI3K/AKT signaling pathway, providing a scientific basis for promoting the repair process in orthodontically induced inflammatory root resorption., (© 2023 Wiley Periodicals LLC.)

Bone sialoprotein (BSP) is a multifunctional extracellular matrix (ECM) protein present in bone and cementum. Global in vivo ablation of BSP leads to bone mineralization defects, lack of acellular cementum, and periodontal breakdown. BSP harbors three main functional domains: N-terminal collagen-binding domain, hydroxyapatite-nucleating domain, and C-terminal RGD integrin-binding signaling domain. How each of these domains contributes to BSP function(s) is not understood. We hypothesized that collagen-binding and RGD domains play distinct roles in cementoblast functions. Three CRISPR/Cas9 gene-edited cell lines were derived from control wild-type (WT) OCCM.30 murine immortalized cementoblasts: 1) deletion of the N-terminus of BSP after signal peptide, including entire collagen binding domain (Ibsp ∆N-Term ); 2) deletion of exon 4 (majority of collagen-binding domain; Ibsp ∆Ex4 ); and 3) deletion of C-terminus of BSP including the integrin binding RGD domain (Ibsp ∆C-Term ). Compared to WT, Ibsp ∆Ex4 and Ibsp ∆C-Term cell lines showed reduced BSP secretion, in vitro. Abnormal cell morphology was observed in all mutant cell lines, with Ibsp ∆C-Term showing highly disorganized cytoskeleton. All mutant cell lines showed significantly lower cell proliferation compared to WT at all timepoints. Ibsp ∆N-Term cells showed reduced cell migration by 24 h. All mutants exhibited over 50 % significant reduced mineralization at days 6 and 10. While WT cells were largely unaffected by seeding density, mutant cells failed to mineralize at lower cell density. Mutant cell lines diverged from WT and from each other by dysregulated expression in 23 genes involved in mineralization, ECM, and cell signaling. In summary, disabling BSP functional domains led to profound and distinct changes in cementoblast cell functions, especially dysregulated gene expression and reduced mineralization, in a way did not align with a straightforward narrative where each functional domain caused specific, expected differences. Instead, the study uncovered a significant level of complexity in how different mutant forms of BSP affected cell functions, in vitro., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Orthodontically induced inflammatory root resorption (OIIRR) is the major iatrogenic complication of orthodontic treatment, seriously endangering tooth longevity and impairing masticatory function. Osteoclasts are thought to be the primary effector cells that initiate the pathological process of OIIRR; however, the cellular and molecular mechanisms responsible for OIIRR remain unclear. Our previous studies revealed that cementocytes, the major mechanically responsive cells in cementum, respond to compressive stress to activate and influence osteoclasts locally. For this study, we hypothesized that the sphingosine-1-phosphate (S1P) signaling pathway, a key mechanotransduction pathway in cementocytes, may regulate osteoclasts under the different magnitudes of either physiologic compressive stress that causes tooth movement or pathologic stress that causes OIIRR. Here, we show a biphasic effect of higher compression force stimulating the synthesis and secretion of S1P, whereas lower compression force reduced signaling in IDG-CM6 cementocytes. Using conditioned media from force-loaded cementocytes, we verified the cell-to-cell communication between cementocytes and osteoclasts and show that selective knockdown of S1PR1 and Rac1 plays a role in cementocyte-driven osteoclastogenesis via the S1P/S1PR1/Rac1 axis. Most importantly, the use of inhibitors of this axis reduced or prevented the pathological process of OIIRR. The intercellular communication mechanisms between cementocytes and osteoclasts may serve as a promising therapeutic target for OIIRR., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone., Methods: Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000 ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1β, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR)., Results: Both RvD1 and RvE1 (10-100 ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p < 0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2)., Conclusions: RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration., (© 2023 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

One of the major components in cementum extracellular matrix is bone sialoprotein (BSP). BSP knockout (Ibsp) mice were reported to have a nonfunctional hypo-mineralized cementum, as well as detachment and disorganization of the periodontal ligament tissue. However, studies investigating the influence of Ibsp in cementoblasts are missing yet. This study investigates the influences of Bsp in three cementoblasts cell lines (OCCM.30-WT,Ibsp ΔNterm , and Ibsp KAE ). The mRNA expression of cementoblast and osteoclast markers (Col1a1, Alpl, Ocn, Runx2, Ctsk, Rankl and Opg) and the cell morphology were compared. Additionally, a functional monocyte adhesion assay was performed. To understand the influence of external stimuli, the effect of Ibsp was investigated under static compressive force, mimicking the compression side of orthodontic tooth movement. Cementoblasts with genotype Ibsp ΔNterm and Ibsp KAE showed slight differences in cell morphology compared to OCCM.30-WT, as well as different gene expression. Under compressive force, the Ibsp cell lines presented expression pattern markers similar to the OCCM.30-WT cell line. However, Cathepsin K was strongly upregulated in Ibsp ΔNterm cementoblasts under compressive force. This study provides insight into the role of BSP in cementoblasts and explores the influence of BSP on periodontal ligament tissues. BSP markers in cementoblasts seem to be involved in the regulation of cementum organization as an important factor for a functional periodontium. In summary, our findings provide a basis for investigations regarding molecular biology interactions of BSP in cementoblasts, and a supporting input for understanding the periodontal and cellular cementum remodeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Porphyromonas gingivalis is involved in the pathogenesis of multiple polymicrobial biofilm-induced inflammatory diseases, including apical periodontitis, and it triggers pyroptosis accompanied by robust inflammatory responses. Tet methylcytosine dioxygenase 1 (TET1), an epigenetic modifier enzyme, has been is correlated with inflammation, though an association of TET1 and P. gingivalis-related pyroptosis in cementoblasts and the molecular mechanisms has not been shown. Our study here demonstrated that P. gingivalis downregulated Tet1 expression and elicited CASP11- and GSDMD-dependent pyroptosis. Additionally, Tet1 mRNA silencing in cementoblasts appeared to result in a more severe pyroptotic phenotype, where levels of CASP11 and GSDMD cleavage, lactate dehydrogenase release, and IL-1β and IL-18 production were significantly increased. Moreover, Tet1 overexpression resulted in blockade of pyroptosis activation accompanied by inflammation moderation. Further analyses revealed that TET1 modulated glycolysis, confirmed by the application of the specific inhibitor 2-deoxy-d-glucose (2-DG). The pyroptosis phenotype enhanced by Tet1 silencing was moderated by 2-DG upon P. gingivalis invasion. Taken together, these data show the effects and underlying mechanisms of TET1 on pyroptosis and inflammatory phenotype induced by P. gingivalis in cementoblasts, and provides insight into the involvement of P. gingivalis in apical periodontitis and, possibly, other inflammatory diseases., (© 2023 New York Academy of Sciences.)

Background: Cementum regeneration was regarded as the critical goal for periodontal regeneration, and M2 macrophage-based therapy was expected to be a promising strategy. However, little is known about the effects of M2 macrophages on cementoblast mineralization and tropism, especially under inflammation. Here we investigated for the first time the crosstalk between M2 macrophages and Porphyromonas gingivalis (Pg)-stimulated cementoblasts., Methods: M2 macrophages were induced with interleukin (IL)-4, and identified. CC-chemokine ligand 2 (CCL2) expression and secretion of inflammatory cementoblasts were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), immunohistochemistry for apical periodontitis (AP) mice, and by enzyme-linked immunosorbent assay. Crystal violet staining was used to observe macrophage migration. Conditional medium (CM) and transwell coculture methods were applied to evaluate the effects of M2 macrophages on cementum mineralization with or without Pg, and to explore the mechanism. Mineralization-related markers and pathway-related proteins were measured by RT-qPCR and WB., Results: M2 macrophages were identified successfully. We found an increase of CCL2 in cementoblasts and their supernatant. Also, higher CCL2 in cementoblasts was observed in the AP model. Superior recruitment of M2 macrophages to supernatant from Pg-stimulated cementoblasts or CCL2-containing medium was verified. Moreover, CM2 and Trans-M2 showed better mineralization-accelerating and rescuing effects when compared to their controls, and application of p38 inhibitor partially blocked the promotion., Conclusions: Our study demonstrated the inflammation-targeting and mineralization-promoting effects of M2 macrophages on cementoblasts, which may provide evidence for M2 macrophage-based cementum regeneration., (© 2022 American Academy of Periodontology.)

Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout ( Ibsp -/- ) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatal Ibsp -/- and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro-computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp -/- mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp -/- versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

Porphyromonas gingivalis lipopolysaccharide (PG-LPS) is an important virulence factor potentially contributing to periodontal tissue destruction. Toll-like receptor 4 (Tlr4) is a key mediator of NF-kB activation during pathogen recognition. Previous work using Tlr4-specific antibodies demonstrated a partial neutralization of PG-LPS effects on murine cementoblasts, which can affect cell function and regulate gene expression of osteoclastic markers. PG-LPS also potentially influence the inflammation process and the resorption of mineralized tissues. Yet, such inflammatory responses and cell signaling events remain to be characterized at the protein level. We thus investigated the effect of 1 and 10 µg/ml of PG-LPS, respectively, on cell morphology, cell viability, and selected key downstream molecules of the Tlr4 signaling cascade in cementoblasts. High concentrations of PG-LPS (10 µg/ml) significantly reduced cell viability after 48 h. Upon PG-LPS-stimulation, Tlr4 was significantly downregulated. Equally, IκBα, a downstream molecule, was downregulated in terms of phosphorylation and protein production. Furthermore, downstream signaling kinases, like serine/threonine kinase phospho-AKT and the mitogen-activated protein kinase (MAPK)-family, specifically phospho-ERK1/2, were significantly upregulated under high PG-LPS-concentrations. We provide new insights into PG-LPS-triggered intracellular signaling pathways in cementoblasts and thus deliver a basis for further research in PG-mediated periodontal inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

N 6 -methyladenosine (m 6 A) is the most prevalent mRNA modification which plays crucial roles in various biological processes, but its role in cementogenesis remains largely unknown. Here, using time-series transcriptomic analysis, we reveal that mRNA m 6 A demethylase Fat mass and obesity-associated protein (FTO) is involved in cementogenesis. Knocking down FTO decreases cementoblast differentiation and mineralization in both OCCM-30 cellular model and murine ectopic bone formation model. Mechanistically, we find that FTO directly binds Runt-related transcription factor 2 (Runx2) mRNA, an important cementogenesis factor, thus protecting it from YTH domain-containing family protein 2 (YTHDF2) mediated degradation, when cementoblasts are differentiating. Knocking down YTHDF2 restores the expression of Runx2 in FTO-knockdown cells. Moreover, under inflammatory conditions, TNF-α inhibits cementoblast differentiation and mineralization partly through FTO/RUNX2 axis. Collectively, our study reveals an important regulatory role of FTO/RUNX2 axis in normal and pathological cementogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Background: L-arginine (L-arg) can reduce apoptosis in a variety of cells. Cementoblast apoptosis is related to root resorption during orthodontic treatment. In the present study, we aimed to study the regulatory effect and potential mechanism of L-arg on cementoblast apoptosis and root resorption., Methods: The apoptosis-related mRNA and protein expression of murine cementoblast (OCCM-30) was assessed after L-arg treatment. To investigate the role of Sirtuin 1 (Sirt1) and autophagy in L-arg resistance to cementoblast apoptosis and root absorption, resveratrol, and EX527 were used to activate or inhibit Sirt1, and chloroquine (CQ) was used to inhibit autophagy., Results: In vitro, L-arg inhibited hypoxia-induced apoptosis in OCCM-30. Further, L-arg increased Sirt1 expression whereas Sirt1 suppression by EX527 reversed the inhibitory effect of L-arg on cell apoptosis. Sirt1 activator resveratrol increased the ratio of microtubule-associated protein light chain 3 (LC3) II/I and decreased the expression of SQSTM1/p62 (p62), suggesting autophagy activation. Autophagy enhancement could reduce apoptosis. Caspase-3 and Bax expression was decreased, and Bcl-2 expression was increased. When autophagy was inhibited by CQ, the positive effects of Sirt1 were attenuated. In vivo, L-arg application reduced root resorption in rats, as demonstrated by decreased root absorption volume. Similarly, L-arg upregulated Sirt1, which activated autophagy in the root resorption model, and less root resorption was observed in the Sirt1 activation group., Conclusion: L-arg reduced cementoblast apoptosis in hypoxia and reduced root resorption induced by loading force in rats, which may be partly mediated by Sirt1-enhanced autophagy., (© 2021 American Academy of Periodontology.)

Hypoxia often occurs in inflammatory tissues, such as tissues affected by periodontitis and apical periodontitis lesions. Mitochondrial biogenesis can be disrupted in hypoxia. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a core factor required for mitochondrial biogenesis. Cementoblasts are root surface lining cells that play an integral role in cementum formation. There is a dearth of research on the effect of hypoxia on cementoblasts and underlying mechanisms, particularly in relation to mitochondrial biogenesis during the hypoxic process. In this study, we found that the expression of hypoxia inducible factor-1α was elevated in apical periodontitis tissues in vivo. In contrast, periapical lesions exhibited a reduction of PGC-1α expression. For in vitro experiments, cobalt chloride (CoCl 2 ) was used to induce hypoxia. We observed that CoCl 2 -induced hypoxia suppressed the mineralization ability and mitochondrial biogenesis of cementoblasts, accompanied by abnormal mitochondria morphology. Furthermore, we found that CoCl 2 blocked the p38 pathway, while it activated the Erk1/2 pathway, with the former upregulating the expression of PGC-1α, while the latter reversed the effects. Overall, our findings demonstrate that mitochondrial biogenesis, especially via PGC-1α, is impaired during cementogenesis in the context of CoCl 2 -induced hypoxia, dependent on the mitogen-activated protein kinase signaling pathway., (© 2022 New York Academy of Sciences.)

Bone sialoprotein (gene: Ibsp ; protein: BSP) is a multifunctional extracellular matrix protein present in bone, cementum, and dentin. Accumulating evidence supports BSP as a key regulator of mineralized tissue formation via evolutionarily conserved functional domains, including a C-terminal integrin-binding Arg-Gly-Asp (RGD) domain implicated in extracellular matrix-cell signaling. Ablation of Ibsp in mice ( Ibsp -/- ) results in impaired bone growth and mineralization and defective osteoclastogenesis, with effects in the craniofacial region including reduced acellular cementum formation, detachment of the periodontal ligament (PDL), alveolar bone hypomineralization, and severe periodontal breakdown. We hypothesized that BSP-RGD plays an important role in cementum and alveolar bone formation and mineralization, as well as periodontal function. This hypothesis was tested by replacing the RGD motif with a nonfunctional Lys-Ala-Glu (KAE) sequence in ( Ibsp KAE/KAE ) mice and OCCM.30 murine ( Ibsp KAE ) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in Ibsp KAE/KAE mice. However, PDL volume and thickness were increased, and significantly more tartrate-resistant acid phosphatase-positive osteoclasts were found on alveolar bone surfaces of Ibsp KAE/KAE mice versus wild type mice. PDL organization was disrupted as indicated by picrosirius red stain, second harmonic generation imaging, dynamic mechanical analysis, and decreased asporin proteoglycan localization. In vitro studies implicated RGD functions in cell migration, adhesion, and mineralization, and this was confirmed by an ossicle implant model where cells lacking BSP-RGD showed substantial defects as compared with controls. In total, the BSP-RGD domain is implicated in periodontal development, though the scale and scope of changes indicated by in vitro studies indicate that other factors may partially compensate for and reduce the phenotypic severity of mice lacking BSP-RGD in vivo.

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, cPLAβ; 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, cPLAβ, 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.

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.

Remodeling of the cementum plays a crucial role in periodontal regenerative therapy, while the precise mechanism of cementogenesis has yet been adequately understood. Recent studies have indicated the connection between osteogenic differentiation and Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (Bmal1). Besides, Wnt/β-catenin signaling is proven to be an essential regulator in cementogenesis. In this study, we found a robust expression of Bmal1 in cementoblasts in the mandibular first molar of mice by immunohistochemical staining. To further explore the role of Bmal1 in cementogenesis, we examined the expression pattern of Bmal1 in OCCM-30, an immortalized murine cementoblast cell line by qRT-PCR and western blot. Our data demonstrated the upregulation of Bmal1 at both mRNA and protein levels during differentiation. Additionally, stable knockdown of Bmal1 in OCCM-30 cells resulted in downregulation of osteogenic markers such as alkaline phosphatase (Alp), osteopontin (Opn), and osteocalcin (Ocn), and reduced formation of mineralized nodules. Moreover, qRT-PCR and western blot results exhibited that the expression of β-catenin was attenuated by Bmal1 deficiency. We also found that the mRNA levels of Tcf1 and Lef1, the target transcription factors of β-catenin, were reduced by Bmal1 deficiency. In conclusion, this study preliminarily confirms that Bmal1 promotes cementoblast differentiation and cementum mineralization via Wnt/β-catenin signaling, which contributes to a potential strategy in periodontal regenerative therapy., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 Federation of American Societies for Experimental Biology.)

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 CoCl 2 -mimicked hypoxia. In-vitro investigations revealed that CoCl 2 -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 CoCl 2 -mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Cementum, a specialized bony layer covering an entire molar root surface, anchors teeth into alveolar bone. Gli1, a key transcriptional activator in Hedgehog signaling, has been identified as a mesenchymal progenitor cell marker in various tissues, including the periodontal ligament (PDL). To address the mechanisms by which Gli1 + progenitor cells contribute to cementogenesis, we used the Gli1 lacZ/+ knock-in line to mark Gli1 + progenitors and the Gli1 CreERT2/+ ; R26R tdTomato/+ line (named Gli1 Lin ) to trace Gli1 progeny cells during cementogenesis. Our data unexpectedly displayed a biphasic feature of Gli1 + PDL progenitor cells and cementum growth: a negative relationship between Gli1 + progenitor cell number and cementogenesis but a positive correlation between Gli1-derived acellular and cellular cementoblast cell number and cementum growth. DTA-ablation of Gli1 Lin cells led to a cementum hypoplasia, including a significant reduction of both acellular and cellular cementoblast cells. Gain-of-function studies (by constitutive stabilization of β-catenin in Gli1 Lin cells) revealed a cementum hyperplasia. A loss of function (by conditional deletion of β-catenin in Gli1 + cells) resulted in a reduction of postnatal cementum growth. Together, our studies support a vital role of Gli1 + progenitor cells in contribution to both types of cementum, in which canonical Wnt/β-catenin signaling positively regulates the differentiation of Gli1 + progenitors to cementoblasts during cementogenesis.

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.

The keystone pathogen Porphyromonas gingivalis (P. gingivalis) elicits inflammation and autophagy in periodontal tissues. Transcription factor CXXC-type zinc finger protein 5 (CXXC5) and various signals are sensitive to P. gingivalis invasion. Herein, we investigated the P. gingivalis-elicited autophagy activity, the contribution of CXXC5, and the involvement of signals in cementoblasts, tooth root surface cells crucial in periodontal and periapical regions. After coculture with P. gingivalis, cementoblasts exhibited inflammatory cytokine increase, light chain 3(LC3)-I/II conversion, autophagosome activation, and CXXC5 reduction. Cementoblasts with loss and gain of CXXC5 were developed. CXXC5 silencing suppressed autophagy and inflammation, thereby partially compensating for the effects of P. gingivalis, and vice versa. We then screened potential signals and verified the positive participation of Stat3/Akt/Erk networks through specific inhibitor employment. P. gingivalis and CXXC5 induced autophagy through Beclin1 and Atg5 activation. Intriguingly, Annexin V/PI assay and EdU detection revealed that P. gingivalis promoted apoptosis and repressed cell proliferation. In sum, coculture with P. gingivalis enhanced autophagy activity in cementoblasts, which was partially suppressed by CXXC5 downregulation and mediated by Jak/Stat3, PI3K-Akt, and Erk1/2 signaling. This process probably influenced cell apoptosis and proliferation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2020 Wiley Periodicals LLC.)

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-1β 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Periodontal tissues consist of cementum, periodontal ligaments, and alveolar bone, which provide indispensable support for physiological activities involving mastication, swallowing, and pronunciation. The formation of periodontal tissues requires a complex process, during which a close relationship with biomineralization is noticeable. Alveolar bone and cementum are physically hard, both of which are generated from biomineralization and possess the exact mechanical properties resembling other hard tissues. However, when periodontitis, congenital abnormalities, periapical diseases, and other pathological conditions affect the organism, the most common symptom, alveolar bone defect, is always unavoidable, which results in difficulties for current clinical treatment. Thus, exploring effective therapies to improve the prognosis is important. Matrix vesicles (MVs), a special subtype of extracellular vesicles related to histogenesis, are widely produced by the stem cells of developing hard tissues. With the assistance of the enzymes and transporters contained within them, MVs can construct the extracellular matrix and an adequate microenvironment, thus promoting biomineralization and periodontal development. Presently, MVs can be effectively extracted and delivered by scaffolds and generate hard tissues in vitro and in vivo, which are expected to be translated into therapies for alveolar bone defects. In this review, we generalize recent research progress on MV morphology, molecular composition, biological mechanism, and, in particular, the biological functions in periodontal development. In addition to the above unique roles of MVs, we further describe the available MV-related biotechnologies and achievements that make them promising for coping with existing problems and improving the treatment of alveolar bone defects.

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., (© 2020 The Authors. STEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

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 β-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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Different structures and cell types of the periodontium respond to orthodontic tooth movement (OTM) individually. Cementoblasts (OC/CM) located in the immediate vicinity of the fibroblasts on the cement have found way to the centre of actual research. Here, we identify and validate possible reference genes for OC/CM cells by RT-qPCR with and without static compressive loading. We investigated the suitability of 3 reference genes in an in vitro model of cementoblast cells using four different algorithms (Normfinder, geNorm, comparative delta-C t method and BestKeeper) under different confluences and time. Comparable to our previous publications about reference genes in OTM in rats and human periodontal ligament fibroblasts (hPDLF), Rpl22 in murine OC/CM cells appears as the least regulated gene so that it represents the most appropriate reference gene. Furthermore, unlike to the expression of our recommended reference genes, the expression of additionally investigated target genes changes with confluence and under loading compression. Based on our findings for future RT-qPCR analyses in OC/CM cells, Rpl22 or the combination Rpl22/Tbp should be favored as reference gene. According to our results, although many publications propose the use of Gapdh, it does not seem to be the most suitable approach.

Background: Previous studies reported that nicotine, which is the prominent constituent of tobacco, has negative effects on periodontium cells. However, the precise role of nicotine in cementoblast functions remains unclear. In the present study, we investigated the effects of nicotine on the functions of cementoblasts (OCCM-30) in terms of proliferation, migration, and mineralized tissue-associated gene expression., Methods: Immortalized murine cementoblasts were exposed to various concentrations (0, 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 2.5, 5, and 10 mM) of nicotine, and cementoblast proliferation was then evaluated using a real-time cell analyzer for 142 hours. Using an in vitro wound healing assay, cell migration was evaluated 2, 4, 6, and 24 hours after exposure to different concentrations of nicotine (1, 2.5, 5, and 10 mM). The mRNA expressions of bone sialoprotein (BSP), collagen type I (COL-I), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and alkaline phosphatase (ALP) were assessed in the nicotine-treated (0, 10 -3 , 10 -2 , 10 -1 , 1, 2.5, 5, and 10 mM) OCCM-30 cells by reverse transcription quantitative polymerase chain reaction at 8 and 24 hours exposure., Results: At concentrations of 1 to 10 mM, nicotine significantly reduced cementoblast proliferation (P <0.01). Exposure to nicotine at other concentrations (1, 2.5, and 5 mM) significantly reduced wound healing rates, whereas nicotine at a concentration of 10 mM immediately decreased the viability of OCCM-30 cells. Similar results were observed in inverted microscopy images at the highest nicotine concentrations. All concentrations of nicotine decreased the transcripts of BSP and COL-I in a dose- and time-dependent manner (P <0.001). Nicotine concentrations higher than 1 mM reduced the expression of OCN, RunX2, and ALP in a time-dependent manner (P <0.001)., Conclusions: This study indicated that nicotine inhibited the proliferation, migration, and mineralized tissue-associated gene expression of OCCM-30 cells. These findings suggest that nicotine negatively affects cementoblast function and the formation of new cementum, which is critical for new attachment., (© 2019 American Academy of Periodontology.)

Background: Orthodontically induced root resorption (OIRR) is considered as an undesirable and unpredictable sequel of orthodontic treatment. Recent reports demonstrated that interleukin (IL)-17/IL-34, and T cells secrete inflammatory/osteoclastogenic cytokines, which might stimulate osteoclastogenesis/bone resorption. However, little is known about the role played by IL-17/IL-34 in OIRR. The present study was aimed at investigating the odontoclastic expression pattern of IL-17 and IL-34 in resorbed cementum during different experimental tooth movements in vivo., Methods: Twenty-four 8-week-old male Wistar rats were divided into four groups: control group, optimal force group (10 g), heavy force group (50 g), and jiggling force group (compression and tension, repetition; 10 g). After 7, 14, and 21 days, the expression levels of IL-17 and IL-34 protein in the resorbed cementum were analyzed using immunohistochemical methods., Results: On day 21, the immunoreactivity for IL-17 and IL-34 in resorbed roots in the jiggling force group was stronger than that in the heavy force and optimal force groups. Moreover, the number of IL-17-positive and IL-34-positive odontoclasts was significantly increased in the jiggling force group compared with those in the other groups on day 21., Conclusions: These results suggest that jiggling forces might exacerbate OIRR compared with heavy forces, as evidenced by the increased expression of IL-17 and IL-34 in odontoclasts obtained from resorbed roots., (Copyright © 2020 World Federation of Orthodontists. Published by Elsevier Inc. All rights reserved.)

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., (© 2019 Wiley Periodicals, Inc.)

Hedgehog (Hh) signaling plays a broad role in the development of many organs including bone and teeth. It is noted that sustained Hh activity in osteoblasts negatively regulates postnatal development in mice. However, it remains unknown whether Hh signaling contributes to cementum formation. In this study, to define the roles of Hh signaling in cementum formation, we analyzed two kinds of transgenic mouse models for Hh signaling activation designed by the inactivation of Suppressor of Fused (Sufu), a negative regulator of Hh signaling, (Sufu OC ) and a forced endogenous activation of Smo (SmoM2 OC ) under the control of osteocalcin (OC) promoter-driven Cre recombinase. Interestingly, cellular cementum apposition was remarkably reduced in both mutants. Consistently, matrix formation and mineralization ability were down-regulated in OCCM-30, a cementoblast cell line, following treatment with a pharmaceutical Smo agonist. In addition, reductions in Osx expression and β-catenin activity, which are critical for cellular cementum formation, were also detected in vitro. Furthermore, the compound mutant mice designed for the stabilization of β-catenin with both Hh-Smo signaling activation in cementoblasts revealed a complete restoration of defective cellular cementum. In addition, Wnt antagonists such as Sostdc1 and Dkk1 were also induced by Smo activation and played a role in the reduction of Osx expression and β-catenin activity. Collectively, our data demonstrated that Hh signaling negatively regulates cementum apposition in a Wnt/β-catenin/Osx-dependent manner.

Objective: This study aims to uncover the role of interleukin-33 on cementoblast-mediated cementum repair., Methods: 6-8-week-old C57BL/6 mice were used to establish the model of orthodontic tooth movement. Interleukin-33 and suppression of tumorigenicity2 (ST2) expressions were immunohistochemically detected in the periodontal tissue. In vitro, cementoblast-like (OCCM-30) cells were cultured in the presence of recombinant mouse interleukin-33 protein (rmIL-33) at a 1-14 d time frame. ST2 expressions were immunofluorescently labeled and quantitatively examined. The effects of interleukin-33 on cementoblast differentiation, mineralization and proliferation were examined by alkaline phosphatase, alizarin red staining and cell counting kit-8, respectively. To further clarify the effect of interleukin-33 on cementogenesis-related protein expressions, runt-related transcription factor 2 (RUNX2), osterix, osteopontin, bone sialoprotein(BSP), osteocalcin, osteoprotegerin (OPG) and receptor activator of NF-КB ligand (RANKL) expressions were examined by western blot., Results: Orthodontic load of high magnitude induces external apical root resorption, and increases interleukin-33 expression in the periodontal tissue of mice. Cells in the cementum express ST2. Interleukin-33 initially down-regulates but later recovers ST2 mRNA and protein levels in OCCM-30 cells. Interleukin-33 abates cementoblast differentiation and mineralization, and suppresses RUNX2, osterix, BSP and osteopontin expressions in OCCM-30 cells at the later stage of the culture period. Interleukin-33 enhances RANKL expression, and reduces the ratio of OPG/RANKL in OCCM-30 cells., Conclusion: Orthodontic load of high magnitude induces interleukin-33 expression in the periodontal tissue. Interleukin-33 has a negative effect on cementogenesis via suppressing cementoblast differentiation, mineralization and cementogenesis-related protein expressions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The life history pattern of recent humans is uniquely derived in many of its aspects including an extended post-reproductive lifespan combined with short interbirth intervals. A number of theories have been proposed to explain the evolution of this unusual pattern. However most have been difficult to test due to the fragmentary nature of the hominin fossil record and the lack of methods capable of inferring such later life history events. In search of a method we tested the hypothesis that the physiologically impactful events of parturition and menopause are recorded in dental cementum microstructure. We performed histomorphological analyses of 47 teeth from 15 individuals with known life history events and were able to detect reproductive events and menopause in all females. Furthermore, we found that other stressful events such as systemic illnesses and incarceration are also detectable. Finally, through the development of a novel analytical method we were able to time all such events with high accuracy (R-squared = 0.92).

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 (PGE 2 ) and β-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., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Cementum regeneration is considered the gold standard for the treatment of periodontitis. As one of the most important primary proinflammatory cytokines, interleukin 1β (IL1β) 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 IL1β-induced inflammation of the microenvironment and the relative interaction mechanism are still unknown. Here, we found that IL1β inhibited cementoblast differentiation and microRNA-325-3p (miR-325-3p) was increased during IL1β-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 IL1β. 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., (© 2019 Wiley Periodicals, Inc.)

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/ β -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/ β -catenin pathway. CEMP1-p4 stimulation upregulated the expression of β -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 β -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 β -catenin signaling cascade.

Tooth cementum is a bone-like mineralized tissue and serves as a microbial barrier against invasion and destruction. Cementum is also responsible for tooth stability and defending pulp from outside stimuli, which is formed by cementoblasts. Although it is crucial for periodontal and periapical diseases, the mechanisms underlying the pathophysiological changes of cementoblasts and their inflammatory responses remain unclear. MiR-181b is found to modulate vascular inflammation and endotoxin tolerance. In this study, miR-181b-5p was downregulated in tumor necrosis factor-α (TNF-α)-stimulated cementoblasts, whereas proinflammatory molecules increased. The mouse periapical lesions have similar results, which imitate an inflammatory environment for cementoblasts in vivo. The bioinformatics analysis and dual luciferase reporter assay suggested that miR-181b-5p targeted interleukin-6 (IL-6). Overexpressing miR-181b-5p negatively regulated IL-6 and proinflammatory chemokine. Western blot analysis and luciferase activity reporter assay verified that miR-181b-5p weakened the NF-κB activity. Hence, miR-181b-5p moderated proinflammatory chemokine production by targeting IL-6 in cementoblasts and NF-κB signaling pathway was involved. Furthermore, miR-181b-5p promoted cementoblast apoptosis, which may enhance the resolution of inflammation. Overall, our data revealed that miR-181b-5p was a negative regulator of TNF-α-induced inflammatory responses in cementoblasts., (© 2019 Wiley Periodicals, Inc.)

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 Tcf7l2 fl/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., (© 2019 Wiley Periodicals, Inc.)

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

During cementum formation, the key roles of osterix (Osx) and inorganic pyrophosphate (PPi), mainly controlled by nucleotide pyrophosphatase 1 (Npp1; encoded by the Enpp1 gene) and progressive ankylosis protein (Ank), have been demonstrated by animal models displaying altered cementum formation. In this study, we analyzed the relationship of Osx and local PPi during cementum formation using compound mutant mice with their wildtype and corresponding single gene mutants. Importantly, functional defects in PPi regulation led to the induction of Osx expression at the cervical cementum as demonstrated by Enpp1 mutant mice and cementoblasts with the retroviral transduction of small hairpin RNA for Enpp1 or Ank. Conversely, cementoblasts exposed to inorganic PPi or with the enforced expression of Enpp1 or Ank reduced Osx expression in a concentration-dependent manner. Furthermore, the loss of Osx induced the higher expression of Npp1 and Ank at the apical region of the developing tooth root as observed in Osx-deficient mice. The activity of PPi-generating ectoenzymes (nucleoside triphosphate pyrophosphohydrolase, NTPPPHase) and the level of extracellular PPi were significantly increased in Osx-knockdown cementoblasts. However, the formation of ectopic cervical cementum was not completely diminished by inactivation of Osx in Enpp1 mutant mice. In addition, fibroblast growth factor (FGF) receptor 1 (Fgfr1) was strongly localized in cementoblasts lining the acellular cementum and involved in the inhibitory regulation of matrix accumulation and further mineralization by supporting PPi production. Taken together, these results suggest that local PPi suppresses matrix accumulation and further mineralization through an antagonistic interaction with Osx under the synergistic influence of FGF signaling during cementum formation., (Copyright © 2019 Elsevier Inc. All rights reserved.)