Functional defects in cementoblasts with disrupted bone sialoprotein functional domains, in vitro.

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.

Force-Loaded Cementocytes Regulate Osteoclastogenesis via S1P/S1PR1/Rac1 Axis.

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.

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.

Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

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}-1ß, 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.

Knockout of bone sialoprotein in cementoblasts cell lines affects specific gene expression in unstimulated and mechanically stimulated conditions.

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,IbspΔNterm, and IbspKAE). 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 IbspΔNterm and IbspKAE 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 IbspΔNterm 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.

Tet methylcytosine dioxygenase 1 modulates Porphyromonas gingivalis-triggered pyroptosis by regulating glycolysis in cementoblasts.

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-1ß 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.

High concentrations of Porphyromonas gingivalis-LPS downregulate Tlr4 and modulate phosphorylation of ERK and AKT in murine cementoblasts.

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.

Bone Sialoprotein Is Critical for Alveolar Bone Healing in Mice.

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 postnatalIbsp-/- 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.

M2 macrophages with inflammation tropism facilitate cementoblast mineralization.

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.

FTO/RUNX2 signaling axis promotes cementoblast differentiation under normal and inflammatory condition.

N6-methyladenosine (m6A) 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 m6A 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.

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.

PGC-1 alpha regulates mitochondrial biogenesis to ameliorate hypoxia-inhibited cementoblast mineralization.

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 (CoCl2 ) was used to induce hypoxia. We observed that CoCl2 -induced hypoxia suppressed the mineralization ability and mitochondrial biogenesis of cementoblasts, accompanied by abnormal mitochondria morphology. Furthermore, we found that CoCl2 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 CoCl2 -induced hypoxia, dependent on the mitogen-activated protein kinase signaling pathway.

The Bone Sialoprotein RGD Domain Modulates and Maintains Periodontal Development.

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 (IbspKAE/KAE) mice and OCCM.30 murine (IbspKAE) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in IbspKAE/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 IbspKAE/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.

Bmal1 promotes cementoblast differentiation and cementum mineralization via Wnt/ß-catenin signaling.

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/ß-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 ß-catenin was attenuated by Bmal1 deficiency. We also found that the mRNA levels of Tcf1 and Lef1, the target transcription factors of ß-catenin, were reduced by Bmal1 deficiency. In conclusion, this study preliminarily confirms that Bmal1 promotes cementoblast differentiation and cementum mineralization via Wnt/ß-catenin signaling, which contributes to a potential strategy in periodontal regenerative therapy.

L-arginine protects cementoblasts against hypoxia-induced apoptosis through Sirt1-enhanced autophagy.

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.

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.

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.

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.

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.