Effect of low-intensity pulsed ultrasound on the mineralization of force-treated cementoblasts and orthodontically induced inflammatory root resorption via the Lamin A/C-Yes associated protein axis.

AIMS: Orthodontic treatment commonly results in orthodontically induced inflammatory root resorption (OIIRR). This condition arises from excessive orthodontic force, which triggerslocal inflammatory responses and impedes cementoblasts' mineralization capacity. Low-intensity pulsed ultrasound (LIPUS) shows potential in reducing OIIRR. However, the precise mechanisms through which LIPUS reduces OIIRR remain unclear. This study aimed to explore the effects and mechanisms of LIPUS on the mineralization of force-treated cementoblasts and its impact on OIIRR. METHODS: We established a rat OIIRR model and locally administered LIPUS stimulation for 7 and 14 days. We analyzed root resorption volume, osteoclast differentiation, and the expression of osteocalcin and yes-associated protein 1 (YAP1) using micro-computed tomography (micro-CT), hematoxylin and eosin, tartrate-resistant acid phosphatase, immunofluorescence and immunohistochemistry staining. In vitro, we applied compressive force and LIPUS to the immortalized mouse cementoblasts (OCCM30). We assessed mineralization using alkaline phosphatase (ALP) staining, alizarin red staining, real-time quantitative polymerase chain reaction, Western blotting and immunofluorescence staining. RESULTS: In rats, LIPUS reduced OIIRR, as evidenced by micro-CT analysis and histological staining. In vitro, LIPUS enhanced mineralization of force-treated OCCM30 cells, as indicated by ALP and alizarin red staining, upregulated mRNA expression of mineralization-related genes, and increased protein expression of mineralization markers. Mechanistically, LIPUS activated YAP1 signaling via the cytoskeleton-Lamin A/C pathway, supported by immunofluorescence and Western blot analysis. CONCLUSION: This study demonstrates that LIPUS promotes mineralization in force-treated cementoblasts and reduces OIIRR by activating YAP1 through the cytoskeletal-Lamin A/C signaling pathway. These findings provide fresh insights into how LIPUS benefits orthodontic treatment and suggest potential strategies for preventing and treating OIIRR.

Birc5 and Nudc are screened as candidate reference genes for RT-qPCR studies in mouse cementoblast mineralization using time-series RNA-seq data.

BACKGROUND: The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh. OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization. MATERIALS AND METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking. RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes. LIMITATIONS: RefFinder's stability ranking does not consider the influence of primer efficiency. CONCLUSIONS AND IMPLICATIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.

Mitochondrial ROS participates in Porphyromonas gingivalis-induced pyroptosis in cementoblasts.

This study aimed to investigate correlation between mitochondrial reactive oxygen species and Porphyromonas gingivalis in the process of cementoblast pyroptosis. Lactate dehydrogenase activity assay, enzyme-linked immunosorbent assay, western blotting and flow cytometry analysis were utilized to explore whether Porphyromonas gingivalis triggered pyroptosis in cementoblasts. Reactive oxygen species and mitochondrial reactive oxygen species were detected using flow cytometry and fluorescence staining. The effect of mitochondrial reactive oxygen species on the Porphyromonas gingivalis-induced pyroptosis of cementoblasts was assessed by Mito-Tempo, mitochondrion-targeted superoxide dismutase mimetic. Phosphorylation levels of p65 were measured by western blotting. SC75741, a nuclear factor-kappa B inhibitor, was added to block the nuclear factor-kappa B in the Porphyromonas gingivalis-infected cementoblasts. Porphyromonas gingivalis triggered pyroptosis of cementoblasts, and an elevation in reactive oxygen species generation in the mitochondria was observed. Inhibition of mitochondrial reactive oxygen species reduced pyroptosis and nuclear factor-kappa B signaling pathway mediated the pyroptotic cell death in Porphyromonas gingivalis-infected cementoblasts. Together, our findings demonstrate that mitochondrial reactive oxygen species increased by Porphyromonas gingivalis participated in the pyroptosis of cementoblasts. Targeting mitochondrial reactive oxygen species may offer therapeutic strategies for root surface remodeling or periodontal regeneration.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

OBJECTIVE: We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. BACKGROUND: PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. METHODS: The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. RESULTS: Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. CONCLUSION: We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

Programmed cell death of periodontal ligament cells.

The periodontal ligament is a crucial tissue that provides support to the periodontium. Situated between the alveolar bone and the tooth root, it consists primarily of fibroblasts, cementoblasts, osteoblasts, osteoclasts, periodontal ligament stem cells (PDLSCs), and epithelial cell rests of Malassez. Fibroblasts, cementoblasts, osteoblasts, and osteoclasts are functionally differentiated cells, whereas PDLSCs are undifferentiated mesenchymal stem cells. The dynamic development of these cells is intricately linked to periodontal changes and homeostasis. Notably, the regulation of programmed cell death facilitates the clearance of necrotic tissue and plays a pivotal role in immune response. However, it also potentially contributes to the loss of periodontal supporting tissues and root resorption. These findings have significant implications for understanding the occurrence and progression of periodontitis, as well as the mechanisms underlying orthodontic root resorption. Further, the regulation of periodontal ligament cell (PDLC) death is influenced by both systemic and local factors. This comprehensive review focuses on recent studies reporting the mechanisms of PDLC death and related factors.

Effect of recombinant human fibroblast growth factor 21 on the mineralization of cementoblasts and its related mechanism.

OBJECTIVES: To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism. METHODS: Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor ß (TGFß)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis. RESULTS: FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells. CONCLUSIONS: rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFß/BMP signaling pathway.

Isolation and characterization of apical papilla cells from root end of human third molar and their differentiation into cementoblast cells: an in vitro study.

BACKGROUND: Periodontal regeneration, treatment of periodontal-related diseases and improving the function of implants are global therapeutic challenges. The differentiation of human stem cells from apical papilla into cementoblasts may provide a strategy for periodontitis treatment. This study aimed to evaluate the differentiation of primary human stem cells apical papilla (hSCAPs) to cementoblast cells. MATERIAL AND METHODS: SCAPs cells were isolated from human third molar and then incubated for 21 days in a differentiation microenvironment. Alkaline phosphatase (ALP) and Alizarin red S staining assays were performed to evaluate the calcium deposition and formation of hydroxyapatite in the cultured hSCAPs microenvironment. Real-time polymerase chain reaction (RT-PCR) assay was performed for cementum protein 1 (CEMP1), collagen type I (COL1), F-Spondin (SPON1), osteocalcin (OCN), and osteopontin (OPN) as specific markers of cementoblasts and their progenitors. RESULTS: ALP phosphatase activity in day 21 of treatment demonstrated a significant increase in ALP compared to the control. Alizarin red S staining assay showed that the differentiated hSCAPs offered a great amount of calcium deposition nodules compared to the control. The increased expression level of CEMP1, OCN, OPN, COL1 and Spon1 was observed in days 7, 14 and 21 compared to the control, while greatest expression level was observed in day 21. CONCLUSION: In conclusion, the differentiation microenviroment is convenient and useful for promoting the differentiation of hSCAPs into cementoblast.

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.

PD-L1, a Potential Immunomodulator Linking Immunology and Orthodontically Induced Inflammatory Root Resorption (OIIRR): Friend or Foe?

Orthodontically induced inflammatory root resorption (OIIRR) is considered an undesired and inevitable complication induced by orthodontic forces. This inflammatory mechanism is regulated by immune cells that precede orthodontic tooth movement (OTM) and can influence the severity of OIIRR. The process of OIIRR is based on an immune response. On some occasions, the immune system attacks the dentition by inflammatory processes during orthodontic treatment. Studies on the involvement of the PD-1/PD-L1 immune checkpoint have demonstrated its role in evading immune responses, aiming to identify possible novel therapeutic approaches for periodontitis. In the field of orthodontics, the important question arises of whether PD-L1 has a role in the development of OIIRR to amplify the amount of resorption. We hypothesize that blocking of the PD-L1 immune checkpoint could be a suitable procedure to reduce the process of OIIRR during orthodontic tooth movement. This review attempts to shed light on the regulation of immune mechanisms and inflammatory responses that could influence the pathogenesis of OIIRR and to acquire knowledge about the role of PD-L1 in the immunomodulation involved in OIIRR. Possible clinical outcomes will be discussed in relation to PD-L1 expression and immunologic changes throughout the resorption process.

Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia.

Recent studies have revealed that hypoxia alters the PD-L1 expression in periodontal cells. HIF-1α is a key regulator for PD-L1. As hypoxia presents a hallmark of an orthodontically induced microenvironment, hypoxic stimulation of PD-L1 expression may play vital roles in immunorthodontics and orthodontically induced inflammatory root resorption (OIIRR). This study aims to investigate the hypoxic regulation of PD-L1 in cementoblasts, and its interaction with hypoxia-induced HIF-1α expression. The cementoblast (OCCM-30) cells (M. Somerman, NIH, NIDCR, Bethesda, Maryland) were cultured in the presence and absence of cobalt (II) chloride (CoCl2). Protein expression of PD-L1 and HIF-1α as well as their gene expression were evaluated by Western blotting and RT-qPCR. Immunofluorescence was applied to visualize the localization of the proteins within cells. The HIF-1α inhibitor (HY-111387, MedChemExpress) was added, and CRISPR/Cas9 plasmid targeting HIF-1α was transferred for further investigation by flow cytometry analysis. Under hypoxic conditions, cementoblasts undergo an up-regulation of PD-L1 expression at protein and mRNA levels. Silencing of HIF-1α using CRISPR/Cas9 indicated a major positive correlation with HIF-1α in regulating PD-L1 expression. Taken together, these findings show the influence of hypoxia on PD-L1 expression is modulated in a HIF-1α dependent manner. The HIF-1α/PD-L1 pathway may play a role in the immune response of cementoblasts. Thus, combined HIF-1α/PD-L1 inhibition could be of possible therapeutic relevance for OIIRR prevention.

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.

Inhibition of osteoclastogenesis by interleukin-33 administration in the periodontal ligament under mechanical loading.

BACKGROUND AND OBJECTIVES: The molecular mechanisms mediating external root resorption are poorly understood. Interleukin-33 (IL-33) expression increased remarkably in the periodontal ligament (PDL) under orthodontic loading. The IL-33-driven responses are delicately cell type- and tissue context-dependent. It is unknown how IL-33 act on osteoclastogenesis in the context of root surface. This study aimed to investigate the effect of IL-33 on osteoclastogenesis in the PDL under mechanical loading. MATERIALS AND METHODS: C57BL/6J mice were treated with injections of phosphate buffer saline (PBS) or recombinant mouse IL-33 (rmIL-33, 6 µl, 30 µg/ml), and subjected to models of orthodontic tooth movement. Tartrated resistant acid phosphates (TRAP)-positive cells and IL-33 expressions were examined in the PDL. IL-33 release from human PDL cells (hPDLCs) was detected by ELISA. Cementoblast-like (OCCM-30) cells were cultured in the presence of rmIL-33 to examine the release of osteoclast-regulatory proteins. The effects of rmIL-33 on osteoclastogenesis were examined in vitro in cultures of bone marrow macrophages (BMMs) and in BMMs-OCCM-30 cocultures. Expressions of osteoclast-specific or -related genes and proteins were investigated in BMMs-OCCM-30 cocultures treated with or without rmIL-33, in the presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing antibody. RESULTS: Interleukin-33 expressions were upregulated in the PDL under orthodontic loading. Static compressive force enhanced expression and release of IL-33 from hPDLCs. Administration of rmIL-33 resulted in reduced number of TRAP-positive cells in the PDL, and inhibited osteoclast differentiation from BMMs in vitro. OCCM-30 cells had varied osteoprotegerin (OPG) / receptor activator for nuclear factor-κB ligand (RANKL) secretion and increased release of GM-CSF under rmIL-33 stimulation. Treatment with rmIL-33 in BMMs-OCCM-30 cocultures resulted in inhibited differentiation and decreased activity of osteoclasts, and these effects were partially reversed by GM-CSF neutralizing antibody. CONCLUSIONS: Interleukin-33 inhibits osteoclastogenesis in the PDL under orthodontic loading. The anti-osteoclastogenic effects were mediated partly by directly affecting osteoclast precursors and partly by cementoblast-mediated release of GM-CSF.

Immunorthodontics: Role of HIF-1α in the Regulation of (Peptidoglycan-Induced) PD-L1 Expression in Cementoblasts under Compressive Force.

Patients with periodontitis undergoing orthodontic therapy may suffer from undesired dental root resorption. The purpose of this in vitro study was to investigate the molecular mechanisms resulting in PD-L1 expression of cementoblasts in response to infection with Porphyromonas gingivalis (P. gingivalis) peptidoglycan (PGN) and compressive force (CF), and its interaction with hypoxia-inducible factor (HIF)-1α molecule: The cementoblast (OCCM-30) cells were kinetically infected with various concentrations of P. gingivalis PGN in the presence and absence of CF. Western blotting and RT-qPCR were performed to examine the protein expression of PD-L1 and HIF-1α as well as their gene expression. Immunofluorescence was applied to visualize the localization of these proteins within cells. An HIF-1α inhibitor was added for further investigation of necroptosis by flow cytometry analysis. Releases of soluble GAS-6 were measured by ELISA. P. gingivalis PGN dose dependently stimulated PD-L1 upregulation in cementoblasts at protein and mRNA levels. CF combined with P. gingivalis PGN had synergistic effects on the induction of PD-L1. Blockade of HIF-1α inhibited the P. gingivalis PGN-inducible PD-L1 protein expression under compression, indicating an HIF-1α dependent regulation of PD-L1 induction. Concomitantly, an HIF-1α inhibitor decreased the GAS-6 release in the presence of CF and P. gingivalis PGN co-stimulation. The data suggest that PGN of P. gingivalis participates in PD-L1 up-regulation in cementoblasts. Additionally, the influence of compressive force on P. gingivalis PGN-induced PD-L1 expression occurs in HIF-1α dependently. In this regard, HIF-1α may play roles in the immune response of cementoblasts via immune-inhibitory PD-L1. Our results underline the importance of molecular mechanisms involved in bacteria-induced periodontics and root resorption.

Transcriptome Profile of Membrane and Extracellular Matrix Components in Ligament-Fibroblastic Progenitors and Cementoblasts Differentiated from Human Periodontal Ligament Cells.

Ligament-fibroblastic cells and cementoblasts, two types of progenitor cells that differentiate from periodontal ligament stem cells (hPDLSCs), are responsible for the formation of the adhesive tissues in the tooth root. Since one of the factors that determines the fate of stem cell differentiation is the change in the microenvironment of the stem/progenitor cells, this study attempted to compare and analyze the molecular differences in the membrane and ECM of the two progenitor cells. Single cells derived from hPDLSCs were treated with TGF-ß1 and BMP7 to obtain ligament-fibroblastic and cementoblastic cells, respectively. The transcriptome profiles of three independent replicates of each progenitor were evaluated using next-generation sequencing. The representative differentially expressed genes (DEGs) were verified by qRT-PCR, Western blot analysis, and immunohistochemistry. Among a total of 2245 DEGs identified, 142 and 114 DEGs related to ECM and cell membrane molecules were upregulated in ligament-fibroblastic and cementoblast-like cells, respectively. The major types of integrin and cadherin were found to be different between the two progenitor cells. In addition, the representative core proteins for each glycosaminoglycan-specific proteoglycan class were different between the two progenitors. This study provides a detailed understanding of cell-cell and cell-ECM interactions through the specific components of the membrane and ECM for ligament-fibroblastic and cementoblastic differentiation of hPDLSCs.

Extracellular Vesicles Derived From Murine Cementoblasts Possess the Potential to Increase Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclastogenesis.

Cementum resorption, unlike bone resorption, is clinically known to occur only with limited pathological stimuli, such as trauma, orthodontic forces, and large apical periodontitis; however, the molecular mechanisms that control osteoclast formation on the cementum surface remain unclear. In this study, we focused on extracellular vesicles (EVs) secreted by cementoblasts and analyzed their effects on osteoclast differentiation. EVs were extracted from the conditioned medium (CM) of the mouse cementoblast cell line OCCM-30. Transmission electron microscopy (TEM) analysis confirmed the presence of EVs with a diameter of approximately 50-200 nm. The effect of the EVs on osteoclast differentiation was examined using the mouse osteoclast progenitor cell line RAW 264.7 with recombinant receptor activator of nuclear factor (NF)-κB ligand (rRANKL) stimulation. EVs enhanced the formation of tartrate-resistant acid phosphatase (TRAP) activity-positive cells upon rRANKL stimulation. EVs also enhanced the induction of osteoclast-associated gene and protein expression in this condition, as determined by real-time PCR and Western blotting, respectively. On the other hand, no enhancing effect of EVs was observed without rRANKL stimulation. A Western blot analysis revealed no expression of receptor activator of NF-κB ligand (RANKL) in EVs themselves. The effect on rRANKL-induced osteoclast differentiation was examined using the CM of cementoblasts in terms of TRAP activity-positive cell formation and osteoclast-associated gene expression. The conditioned medium partly inhibited rRANKL-induced osteoclast differentiation and almost completely suppressed its enhancing effect by EVs. These results indicate that cementoblasts secreted EVs, which enhanced RANKL-induced osteoclast differentiation, and simultaneously produced soluble factors that neutralized this enhancing effect of EVs, implicating this balance in the regulation of cementum absorption. A more detailed understanding of this crosstalk between cementoblasts and osteoclasts will contribute to the development of new therapies for pathological root resorption.

Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells.

OBJECTIVE: RT-qPCR is a reliable method for gene expression analysis, but the accuracy of the quantitative data depends on the appropriate selection of reference genes. A Co-culture system consisting of periodontal ligament cells (SV-PDL) and cementoblasts (OCCM-30) to investigate the crosstalk between these two cell lines under orthodontic condition is essential for experimental orthodontic setups in-vitro. Therefore, we aimed to identify a set of reliable reference genes suitable for RT-qPCR studies for prospective co-culture systems of OCCM-30 and SV-PDL cells. RESULTS: The results demonstrated that PPIB, GUSB and RPLP0 turned out to be the three most stable reference genes for OCCM-30 in the co-culture system, while PPIB, POLR2A and RPLP0 have the three highest rankings for SV-PDL cells in the co-culture system. The most stable gene combination were PPIB and POLR2A in the co-culture system. In conclusion, PPIB is overall the most stably expressed reference gene for OCCM-30 or SV-PDL cell line in the system. The combination of PPIB and POLR2A as reference genes are indicated to be the potential and mandatory to obtain accurate quantification results for normalizing RT-qPCR data in genes of interest expression in these two cell lines co-culture systems.

Effect of Low-Level Er: YAG (2940 nm) laser irradiation on the photobiomodulation of mitogen-activated protein kinase cellular signaling pathway of rodent cementoblasts.

BACKGROUNDS: Dental avulsion due to trauma, especially in young patients, is a worldwide problem, requiring tooth replacement. Delayed replantation could cause tooth loss when the cementum is severely damaged. A small number of studies has reported that photobiomodulation (PBM) therapy using Er: YAG laser irradiation activates cellular signaling responses in different cell types, resulting in a variety of favorable biological effects. The aim of this in vitro study was to evaluate the potential biostimulatory effect of low-level Er: YAG laser irradiation on the biological responses of cultured mouse cementoblasts (OCCM-30), including the mitogen-activated protein kinases (MAPKs). METHODS: OCCM-30 cells were exposed to 2940 nm Er: YAG laser irradiation for 15 s at 0.34 W (pulse duration of 100 or 1000 µs, 17 mJ/pulse) at energy densities of 1 or 2 J/cm2. Irradiated and non-irradiated OCCM-30 cells were tested for migration (Scratch assay), proliferation (MTS assay) and functional differentiation (Alizarin Red S assay). Lumican (Lum) and Fibromodulin (Fmod) gene expression, and activation of MAPKs, were assessed by RT-PCR and Western blotting, respectively. RESULTS: Low-level Er: YAG laser irradiation at 2 J/cm2 and pulse duration of 1000 µs resulted in the highest migration rate and proliferation. Moreover, the pulse duration irradiation of 100 µs increased Lum expression. Fmod expression was increased after 1000 µs pulse duration laser stimulation. Low-level Er: YAG laser irradiation increased the mineralization of OCCM-30 cells after 7 days and activated ERK1/2, P38 and JNK signaling. CONCLUSIONS: Low-level Er: YAG laser irradiation induces OCCM-30 cell migration, proliferation and differentiation, and activates the MAPK signaling pathway.

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.

Isolation of Single Cells from Mouse Periodontal Ligament.

The periodontal ligament (PDL) is an essential tissue connecting teeth and bone. It is a complex tissue specifically designed to absorb the forces of mastication; analysis of its multiple cell populations is important to understand its function and the cell changes associated with periodontal disease. Cells in the periodontal ligament are not fully understood due to their physical location and small tissue size. It is challenging to isolate thin layers of cells compared with many other more substantial tissues. Here, we provide a straightforward protocol for the isolation of periodontal ligament cells from mice.