Platr3/NUDT21/NF-κB Axis Mediates P. gingivalis-Suppressed Cementoblast Mineralization.

Porphyromonas gingivalis (P. gingivalis) is one of the major pathogens causing periodontitis and apical periodontitis (AP). Long noncoding RNA (lncRNA) can regulate cellular mineralization and inflammatory diseases. The aim of this study was to investigate the role and mechanism of lncRNA in P. gingivalis-stimulated cementoblast mineralization. In vivo, C57BL/6 mice were divided into the healthy, the AP, and AP + P. gingivalis groups (n = six mice per group). Micro computed tomography, immunohistochemistry staining, and fluorescence in situ hybridization were used to observe periapical tissue. In vitro, cementoblasts were treated with osteogenic medium or P. gingivalis. Pluripotency associated transcript 3 (Platr3), interleukin 1 beta (IL1B), and osteogenic markers were analyzed by quantitative real-time polymerase chain reaction and western blot. RNA pull-down and RNA immunoprecipitation assays were used to detect proteins that bind to Platr3. RNA sequencing was performed in Platr3-silenced cementoblasts. In vivo, P. gingivalis promoted periapical tissue destruction and IL1B expression, but inhibited Platr3 expression. In vitro, P. gingivalis facilitated IL1B expression (P < 0.001), whereas suppressed the expression of Platr3 (P < 0.001) and osteogenic markers (P < 0.01 or 0.001). In contrast, Platr3 overexpression alleviated the repressive effect of P. gingivalis on cementoblast mineralization (P < 0.01 or 0.001). Furthermore, Platr3 bound to nudix hydrolase 21 (NUDT21) and regulated the nuclear factor-κB (NF-κB) signaling pathway. Knocking down NUDT21 suppressed osteogenic marker expression and activated the above signaling pathway. Collectively, the results elucidated that Platr3 mediated P. gingivalis-suppressed cementoblast mineralization through the NF-κB signaling pathway by binding to NUDT21.

Carbon Nanotubes Induce Mineralization of Human Cementoblasts.

INTRODUCTION: Carbon nanotubes (CNT) are 1 of the allotropes of carbon with unique properties. CNT shows good bone-tissue compatibility and has been reported to induce osteogenesis; therefore, it is regarded as an ideal material in a wide range of applications. However, the therapeutic effect of CNT-containing materials in the healing of apical periodontal tissue is unknown. The purpose of this study was to clarify the effect of CNT on the proliferation and mineralization of the human cementoblast cell line (HCEM). METHODS: The proliferation of HCEM cells with CNT stimulation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed from 24-72 hours. Calcium deposition levels were evaluated by alizarin red S staining on days 7 and 10, and mineralization-related gene expression was examined by quantitative real-time polymerase chain reaction on days 3, 7, and 10. Scanning electron microscopy was used to observe the culture with CNT on day 14. RESULTS: CNT showed no cytotoxicity to HCEM cell proliferation. Treatment was performed with mineralization medium, CNT-induced HCEM mineralization on day 7, and increased calcium deposition on days 7 and 14. Messenger RNA expression of alkaline phosphatase was significantly increased throughout the experimental period, and bone sialoprotein was significantly increased on day 3 by CNT, whereas no effect was found on mRNA expression of type I collagen. CNT was observed in attachment to the cell surface on day 14. CONCLUSIONS: CNT promotes the mineralization of HCEM cells, indicating the potential as a new bioactive component for apical periodontal tissue regeneration materials through the regulation of cementoblast mineralization.

CXXC5 mitigates P. gingivalis-inhibited cementogenesis by influencing mitochondrial biogenesis.

BACKGROUND: Cementoblasts on the tooth-root surface are responsible for cementum formation (cementogenesis) and sensitive to Porphyromonas gingivalis stimulation. We have previously proved transcription factor CXXC-type zinc finger protein 5 (CXXC5) participates in cementogenesis. Here, we aimed to elucidate the mechanism in which CXXC5 regulates P. gingivalis-inhibited cementogenesis from the perspective of mitochondrial biogenesis. METHODS: In vivo, periapical lesions were induced in mouse mandibular first molars by pulp exposure, and P. gingivalis was applied into the root canals. In vitro, a cementoblast cell line (OCCM-30) was induced cementogenesis and submitted for RNA sequencing. These cells were co-cultured with P. gingivalis and examined for osteogenic ability and mitochondrial biogenesis. Cells with stable CXXC5 overexpression were constructed by lentivirus transduction, and PGC-1α (central inducer of mitochondrial biogenesis) was down-regulated by siRNA transfection. RESULTS: Periapical lesions were enlarged, and PGC-1α expression was reduced by P. gingivalis treatment. Upon apical inflammation, Cxxc5 expression decreased with Il-6 upregulation. RNA sequencing showed enhanced expression of osteogenic markers, Cxxc5, and mitochondrial biogenesis markers during cementogenesis. P. gingivalis suppressed osteogenic capacities, mitochondrial biogenesis markers, mitochondrial (mt)DNA copy number, and cellular ATP content of cementoblasts, whereas CXXC5 overexpression rescued these effects. PGC-1α knockdown dramatically impaired cementoblast differentiation, confirming the role of mitochondrial biogenesis on cementogenesis. CONCLUSIONS: CXXC5 is a P. gingivalis-sensitive transcription factor that positively regulates cementogenesis by influencing PGC-1α-dependent mitochondrial biogenesis. Video Abstract.

Activation of periodontal ligament cell cytodifferentiation by juxtacrine signaling from cementoblasts.

BACKGROUND: New cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell-cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts. METHODS: To analyze the role of human cementoblast-like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein-tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co-cultures). We sorted co-cultured HPDLs and analyzed their characteristics, including the expression of cementum-related genes. In addition, we cultured HPDLs and HCEMs in a non-contact environment using a culture system composed of an upper insert and a lower well separated by a semi-permeable membrane (indirect co-cultures), and similar analysis was performed. Gene expression of integrin-binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless-type (Wnt) signaling on the differentiation of HPDLs into cementoblasts. RESULTS: Direct co-culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast-related genes in HPDLs, whereas indirect co-culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co-culture. CONCLUSION: Our results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.

Hyaluronic acid enhances cell migration, viability, and mineralized tissue-specific genes in cementoblasts.

BACKGROUND/OBJECTIVES: It has been repeatedly demonstrated that cementum formation is a crucial step in periodontal regeneration. Hyaluronic acid (HA) is an important component of the extracellular matrix which regulates cells functions and cell-cell communication. Hyaluronic acid/derivatives have been used in regenerative periodontal therapy, but the cellular effects of HA are still unknown. To investigate the effects of HA on cementoblast functions, cell viability, migration, mineralization, differentiation, and mineralized tissue-associated genes and cementoblast-specific markers of the cementoblasts were tested. MATERIALS AND METHODS: Cementoblasts (OCCM-30) were treated with various dilutions (0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) of HA and examined for cell viability, migration, mineralization, and gene expressions. The mRNA expressions of osteocalcin (OCN), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), collagen type I (COL-I), alkaline phosphatase (ALP), cementum protein-1 (CEMP-1), cementum attachment protein (CAP), and small mothers against decapentaplegic (Smad) -1, 2, 3, 6, 7, ß-catenin (Ctnnb1) were performed with real-time polymerase chain reaction (RT-PCR). Total RNA was isolated on days 3 and 8, and cell viability was determined using MTT assay on days 1 and 3. The cell mineralization was evaluated by von Kossa staining on day 8. Cell migration was assessed 2, 4, 6, and 24 hours following exposure to HA dilutions using an in vitro wound healing assay (0, 1:2, 1:4, 1:8). RESULTS: At dilution of 1:2 to 1:128, HA importantly increased cell viability (p < .01). HA at a dilution of 1/2 increased wound healing rates after 4 h compared to the other dilutions and the untreated control group. Increased numbers of mineralized nodules were determined at dilutions of 1:2, 1:4, and 1:8 compared with control group. mRNA expressions of mineralized tissue marker including COL-I, BSP, RunX2, ALP, and OCN significantly improved by HA treatments compared with control group both on 3 days and on 8 days (p < .01). Smad 2, Smad 3, Smad 7, and ß-catenin (Ctnnb1) mRNAs were up-regulated, while Smad1 and Smad 6 were not affected by HA administration. Additionally, HA at dilutions of 1:2, 1:4, and 1:8 remarkably enhanced CEMP-1 and CAP expressions in a dilution- and time-dependent manner (p < .01). CONCLUSIONS: The present results have demonstrated that HA affected the expression of both mineralized tissue markers and cementoblast-specific genes. Positive effects of HA on the cementoblast functions demonstrated that HA application may play a key role in cementum regeneration.

Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts.

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, ß-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, ß-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, ß-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L-1 of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L-1 ß-glycerolphosphate, 75 µmol L-1 ascorbic acid and 10 nmol L-1 dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and 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.

The Proteomes of Oral Cells Change during Co-Cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens.

BACKGROUND: Changes in the proteome of oral cells during periodontitis have rarely been investigated. This lack of information is partially attributed to the lack of human cell lines derived from the oral cavity for in vitro research. The objective of the present study was to create cell lines from relevant oral tissues and compare protein expression in cells cultured alone and in cells co-cultivated with periodontitis-associated bacterial strains. METHODS: We established human cell lines of gingival keratinocytes, osteoblastic lineage cells from the alveolar bone, periodontal ligament fibroblasts, and cementum cells. Using state-of-the-art label-free mass spectrometry, we investigated changes in the proteomes of these cells after co-cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens for 48 h. RESULTS: Gingival keratinocytes, representing ectodermal cells, exhibited decreased expression of specific keratins, basement membrane components, and cell-cell contact proteins after cultivation with the bacterial strains. Mesodermal lineage cells generally exhibited similar proteomes after co-cultivation with bacteria; in particular, collagens and integrins were expressed at higher levels. CONCLUSIONS: The results of the present study will help us elucidate the cellular mechanisms of periodontitis. Although co-cultivation with two periodontitis-associated bacterial strains significantly altered the proteomes of oral cells, future research is needed to examine the effects of complex biofilms mimicking in vivo conditions.

Clock genes are expressed in cementum and regulate the proliferation and mineralization of cementoblasts.

Circadian clock genes are present in the ameloblasts, odontoblasts, and dental pulp cells. The cementum plays a vital role in connecting the roots of teeth to the alveolar bone by anchoring the periodontal ligament. The present study aimed at confirming the existence of clock genes and describing the potential regulatory effects of REV-ERBα in the cementum. The tooth-periodontal ligament-alveolar bone complexes of 6-week-old mice were analyzed using immunohistochemistry. OCCM-30 cells, an immortalized cementoblast cell line, were synchronized with dexamethasone. We used RT-PCR to detect the expression of clock genes in the absence or presence of SR8278, an effective antagonist of REV-ERBα. We performed a cell counting kit-8 (CCK-8) assay to determine the effect of SR8278 on cell proliferation. RT-PCR and Western blot were used to measure the expression of mineralization-related markers in mineralization-induced OCCM-30 cells, with or without SR8278 treatment. Finally, we used Alizarin red staining, and ALP staining and activity to further verify the effect of SR8278 on mineralization of OCCM-30 cells on macro-level. In our study, clock protein expression was confirmed in the murine cementum. Clock genes were shown to oscillate continuously in OCCM-30 cells. SR8278-induced inactivation of REV-ERBα inhibited the proliferation but promoted the mineralization of OCCM-30 cells. The present study confirmed the presence of clock genes in the cementum, where they potentially participate in cell proliferation and mineralization. Our findings may inspire new research directions for periodontal regeneration via clock gene manipulation.

Effects of intermittent parathyroid hormone on cementoblast-mediated periodontal repair.

OBJECTIVES: To investigate the effects of intermittent parathyroid hormone on cementoblast-mediated periodontal repair in the context of orthodontic-induced root resorption. MATERIALS AND METHODS: The rat model of orthodontic-induced root resorption was established. Sixty rats were randomly allocated into the experiment group (n = 30) and the control group (n = 30), either receiving a daily subcutaneous injection of recombinant human PTH or placebo vehicle. Enzyme-linked immunosorbent assay, Micro-computed tomography, hematoxylin and eosin staining, and immunohistochemistry staining were performed to detect the periodontal repair. In vitro, OCCM-30 cells were exposed to intermittent PTH (incubated with PTH for the first 6 h in each 24-h cycle). After three cycles, flow cytometry assay, alkaline phosphatase staining, and Alizarin red staining were performed. Quantitative real-time polymerase chain reaction and Western blotting were employed to further determine the effects of intermittent PTH. RESULTS: Intermittent PTH-responsive repair enhancement was detected with the expression of bone sialoprotein, osteocalcin, collagen-1, and alkaline phosphatase significantly upregulated. Increased expressions of cementoblastic proteins were positively correlated to cycles of PTH administration. The proportion of cementoblasts in S and G2/M phases was increased; namely, intermittent PTH promoted cementoblast cell proliferation. CONCLUSIONS: Intermittent parathyroid hormone administration promotes cementoblast-mediated cementogenesis during periodontal repair in a time-dependent manner.

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.

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.

Expression profile analysis of long noncoding RNA and messenger RNA during mouse cementoblast mineralization.

BACKGROUND AND OBJECTIVE: Emerging evidence has uncovered that long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) exert biofunctions on cellular mineralization and bone formation. In this study, we aimed to identify lncRNA-mRNA expression profiles and expression patterns, and explore their underlying biofunctions during cementoblast mineralization. MATERIALS AND METHODS: Cementoblasts were cultured in mineralized medium for 0, 7, and 14 days. We used quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) to detect expression levels of osteocalcin (OCN), bone sialoprotein (BSP), and Osterix (Osx). Alkaline phosphatase (ALP) staining and alizarin red staining (ARS) were conducted to detect ALP activity and number of mineralized nodule. Total RNA was extracted from cells and used for high-throughput sequencing. EBSeq package was applied to analyze differentially expressed genes. Mfuzz R package was used to identify gene expression patterns. The weighted gene co-expression network analysis (WGCNA) was performed to explore co-expressed mRNAs of differentially expressed lncRNAs (DElncRNAs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were adopted by Clusterprofile R package. RESULTS: Cementoblasts were successfully induced by osteogenic medium. Compared with those on day 0, 384 DElncRNAs and 4255 differentially expressed mRNAs (DEmRNAs), respectively, were found on day 7. Meanwhile, 645 DElncRNAs and 4717 DEmRNAs were detected on day 14. Both DElncRNAs and DEmRNAs were classified into six clusters with different expression patterns. DEmRNAs and co-expressed mRNA of DElncRNAs were predominantly related to cell process, binding, phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, hypoxia-inducible factor-1 (HIF-1) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and hippo signaling pathway. CONCLUSION: The results demonstrated that both noncoding and coding RNAs were involved in the process of mineralization in cementoblasts, which may provide a new database for further study.

Polycaprolactone scaffolds as a biomaterial for cementoblast delivery: An in vitro study.

OBJECTIVE: To define the potential of polycaprolactone (PCL) scaffold for cementoblast delivery. BACKGROUND: Dental cementum is critical for tooth attachment and position, and its regenerative capabilities remain unpredictable. METHODS: PCL scaffolds were manufactured by the electrospinning technique at 10% and 20% (w/v) and seeded with cementoblasts (OCCM-30). Scaffolds were characterized for their morphology and biological performance by scanning electron microscopy (SEM), confocal and conventional histology, cytocompatibility (PrestoBlue assay), gene expression (type I collagen - Col1; bone sialoprotein - Bsp; runt-related transcription factor 2 - Runx-2; alkaline phosphatase - Alpl; osteopontin - Opn; osteocalcin - Ocn, osterix - Osx), and the potential to induce extracellular matrix deposition and mineralization in vitro. RESULTS: Overall, data analysis showed that PCL scaffolds allowed cell adhesion and proliferation, modulated the expression of key markers of cementoblasts, and led to enhanced extracellular matrix deposition and calcium deposition as compared to the control group. CONCLUSION: Altogether, our findings allow concluding that PCL scaffolds are a viable tool to culture OCCM-30 cells, leading to an increased potential to promote mineralization in vitro. Further studies should be designed in order to define the clinical relevance of cementoblast-loaded PCL scaffolds to promote new cementum formation.

PGC-1α attenuates TNF-α-induced inflammatory responses in OCCM-30 cells.

BACKGROUND AND OBJECTIVES: Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis and oxidative metabolism, has been associated with many inflammatory diseases. However, little is known about the function and mechanism of PGC-1α in cementoblasts under periodontitis. Our study aimed to investigate the effects of PGC-1α in immortalized cementoblast cell line OCCM-30 under TNF-α stimulation. MATERIALS AND METHODS: OCCM-30 cells were cultured and exposed to TNF-α, and PGC-1α expression was assessed by Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Chemical inhibitors targeting various signaling pathways including NF-κB, p38 MAPK, Akt, and p53 were used to identify the regulatory mechanism involved. ZLN005 was used to upregulate PGC-1α and the subsequent alteration of inflammatory cytokines expression under TNF-α stimulation were examined by qRT-PCR and Elisa. PGC-1α siRNA was employed to further verify the role of PGC-1α in inflammatory response. Dual-reporter gene assays were performed to examine the transcriptional activity of p65, and the phosphorylation level of p65 was evaluated by western blotting. Immunofluorescence assays and nuclear and cytoplasmic extractions were performed to check the nuclear translocation of p65. Coimmunoprecipitation studies were also performed to check whether there is direct binding between p65 and PGC-1α. RESULTS: TNF-α suppressed PGC-1α expression in OCCM-30 cells. Blocking p38 MAPK pathways restored the expression of PGC-1α. ZLN005 can upregulate PGC-1α in OCCM-30 cells. The upregulation of PGC-1α by ZLN005 inhibited TNF-α-induced proinflammatory cytokine expression, which was impaired by the transfection of PGC-1α siRNA. Knocking down PGC-1α also partially restored the ZLN005-decreased transcriptional activity of p65. However, the phosphorylation level and nuclear translocation of p65 were not significantly affected by PGC-1α. It was found that p65 was bound to PGC-1α in OCCM-30 cells stimulated by TNF-α, and the binding was increased upon ZLN005 treatment. CONCLUSIONS: PGC-1α can attenuate TNF-α-induced inflammatory responses in OCCM-30 cells.

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.

Effects of long noncoding RNA H19 on cementoblast differentiation, mineralisation, and proliferation.

OBJECTIVE: Cementum which is a layer of thin and bone-like mineralised tissue covering tooth root surface is deposited and mineralised by cementoblasts. Recent studies suggested long noncoding RNA H19 (H19) promotes osteoblast differentiation and matrix mineralisation, however, the effect of H19 on cementoblasts remains unknown. This study aimed to clarify the regulatory effects of H19 on cementoblast differentiation, mineralisation, and proliferation. MATERIAL AND METHODS: An immortalised murine cementoblast cell line OCCM-30 was used in this study. H19 expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) during OCCM-30 cell differentiation. OCCM-30 cells were transfected with lentivirus or siRNA to up-regulate or down-regulate H19, then the levels of runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), osteocalcin (Bglap) were tested by RT-qPCR or western blot. Alizarin red staining, ALP activity assay and MTS assay were performed to determine the mineralisation and proliferation ability of OCCM-30 cells. RESULTS: H19 was dramatically increased during OCCM-30 cell differentiation. Overexpression of H19 increased the levels of Runx2, Sp7, Alpl, Ibsp, and Bglap and enhanced ALP activity and the formation of mineral nodules. While down-regulation of H19 suppressed the above cementoblast differentiation genes and inhibited ALP activity and mineral nodule formation. However, the proliferation of OCCM-30 cells was not affected. CONCLUSIONS: H19 promotes the differentiation and mineralisation of cementoblasts without affecting cell proliferation.

CircRNA Lrp6 promotes cementoblast differentiation via miR-145a-5p/Zeb2 axis.

BACKGROUND AND OBJECTIVE: Cementum is a part of the periodontium and anchors periodontal ligaments to the alveolar bone. Cementoblasts are responsible for the cementum formation via matrix deposition and subsequently mineralization. Thus, exploring novel mechanisms underlying the function of cementoblast contributes to the treatment of cementum damage. Recently, circRNA Lrp6 (circLRP6) has been of interest due to its active role in cell differentiation, but its potential role in cementoblast differentiation remains unclear. Herein, we attempted to elucidate the role of circLRP6 in cementoblast differentiation and clarify any associated mechanisms. MATERIAL AND METHODS: The mRNA expressions of circLRP6, miR-145a-5p, zinc finger E-box binding homeobox 2 (Zeb2), runt-related transcription factor 2 (Runx2), osteopontin (Opn), and bone sialoprotein (Bsp) were evaluated by qRT-PCR. The protein levels of Zeb2 were measured by Western blot. Bioinformatic analysis and dual-luciferase reporter assays were used to test the potential binding targets of miR-145a-5p. The differentiation potentials of the cementoblasts were assessed by Alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin red S (ARS) staining, and quantification. RESULTS: In this study, circLRP6 was significantly upregulated in cementoblast differentiation. Furthermore, circLRP6 knockdown inhibited ALP levels, reduced calcium nodule formation and the expression of Runx2, Opn, and Bsp. Mechanically, bioinformatic analysis and dual-luciferase reporter assays confirmed miR-145a-5p was a potential binding target of circLRP6. miR-145a-5p can negatively regulate cementoblast differentiation. Subsequently, bioinformatic analysis and dual-luciferase reporter assays confirmed Zeb2 was a potential miR-145a-5p target. miR-145a-5p overexpression resulted in a downregulation of Zeb2. Furthermore, Zeb2 inhibition partially reversed the effect of circLRP6 during cementoblast differentiation. CONCLUSION: Taken together, circLRP6 appears to modulate cementoblast differentiation by antagonizing the function of miR-145a-5p, thereby increasing Zeb2. This study serves as a stepping stone for the potential development of an approach to promote cementum formation.

Physicochemical Properties, Cytocompatibility, and Biocompatibility of a Bioactive Glass Based Retrograde Filling Material.

The ideal retrograde filling material that is easy to handle, has good physicochemical properties, and is biocompatible has not yet been developed. The current study reports the development of a novel bioactive glass based powder for use as a retrograde filling material that is capable of altering the consistency and hardening rate of mixtures when mixed with existing bioactive glass based cement. Furthermore, its physicochemical properties, in vitro effects on human cementoblast-like cells, and in vivo effects on inflammatory responses were evaluated. The surface of the hardened cement showed the formation of hydroxyapatite-like precipitates and calcium and silicate ions were eluted from the cement when the pH level was stabilized at 10.5. Additionally, the cement was found to be insoluble and exhibited favorable handling properties. No adverse effects on viability, proliferation, and expression of differentiated markers were observed in the in vitro experiment, and the cement was capable of inducing calcium deposition in the cells. Moreover, the cement demonstrated a lower number of infiltrated inflammatory cells compared to the other materials used in the in vivo mouse subcutaneous implantation experiment. These findings suggest that the retrograde filling material composed of bioactive glass and the novel bioactive glass based powder exhibits favorable physicochemical properties, cytocompatibility, and biocompatibility.