Association between biological ageing and periodontitis: Evidence from a cross-sectional survey and multi-omics Mendelian randomization analysis.

AIM: To investigate the relationship and potential causality between biological ageing and periodontitis. MATERIALS AND METHODS: We obtained the National Health and Nutrition Examination Survey (NHANES) and genome-wide association study (GWAS) summary statistics as well as single-cell sequencing data. Multivariate regression analysis based on cross-sectional data, Mendelian randomization (MR) and multi-omics integration analysis were employed to explore the causal association and potential molecular mechanisms between biological ageing and periodontitis. Additionally, two-step MR mediation analysis explored the risk factors in biological ageing-mediated periodontitis. RESULTS: We analysed data from 3189 participants in the NHANES data and found that higher biological age was associated with increased risk of periodontitis. MR analyses revealed causal associations between biological age measures and periodontitis risk. Frailty (odds ratio [OR] = 2.08, 95% confidence interval [CI]: 1.04-4.18, p = .039) and GrimAge acceleration (OR = 1.16, 95% CI: 1.01-1.32, p = .033) were causally associated with periodontitis risk, and these results were validated in a large-scale meta-periodontitis GWAS dataset. Additionally, the risk effects of body mass index, waist circumference and lifetime smoking on periodontitis were partially mediated by frailty and GrimAge acceleration. CONCLUSIONS: Evidence from cross-sectional survey and MR analysis suggests that biological ageing increases the risk of periodontitis. Additionally, improving the associated risk factors can help prevent both ageing and periodontitis.

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.

DNA methylation and its potential roles in common oral diseases.

Oral diseases are among the most common diseases worldwide and are associated with systemic illnesses, and the rising occurrence of oral diseases significantly impacts the quality of life for many individuals. It is crucial to detect and treat these conditions early to prevent them from advancing. DNA methylation is a fundamental epigenetic process that contributes to a variety of diseases including various oral diseases. Taking advantage of its reversibility, DNA methylation becomes a viable therapeutic target by regulating various cellular processes. Understanding the potential role of this DNA alteration in oral diseases can provide significant advances and more opportunities for diagnosis and therapy. This article will review the biology of DNA methylation, and then mainly discuss the key findings on DNA methylation in oral cancer, periodontitis, endodontic disease, oral mucosal disease, and clefts of the lip and/or palate in the background of studies on global DNA methylation and gene-specific DNA methylation.

The value of urinary exosomal microRNA-21 in the early diagnosis and prognosis of bladder cancer.

Bladder cancer (BC) poses high morbidity and mortality, with urinary exosomal microRNA (miR)-21 showing potential value in its diagnosis and prognosis, and we probed its specific role. We prospectively selected 116 BC patients and 116 healthy volunteers as the BC and control groups, respectively. BC urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 relative expression levels were assessed. The correlations between clinical indexes and urinary exosomal miR-21, prognostic value of miR-21, and diagnostic value of the five candidate miRNAs, urine cytology, and miRNA joint diagnostic panel for BC and urinary exosomal miR-21, miR-4454, and urine cytology for Ta-T1 and T2-T4 stage BC were analyzed. Urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 were highly expressed in BC patients. miR-146a-5p, miR-93-5p, miR-663b, miR-21, miR-4454, miRNA combined diagnostic panel, and urine cytology had certain diagnostic value for BC, with miR-21, miR-4454, and miRNA co-diagnostic panel showing the highest diagnostic value. Collectively, urinary exosomal miR-21 was closely related to Tumor-Node-Metastasis staging and grading in BC patients. Urinary exosomal miR-21 had high diagnostic value for BC and Ta-T1 and T2-T4 stage BC, and had high predictive value for BC poor prognosis, providing an effective indicator for the occurrence, development, and prognostic assessment of BC.

Fibroblast growth factor pathway promotes glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in prostate cancer.

BACKGROUND: The initiation of fibroblast growth factor 1 (FGF1) expression coincident with the decrease of FGF2 expression is a well-documented event in prostate cancer (PCa) progression. Lactate dehydrogenase A (LDHA) and LDHB are essential metabolic products that promote tumor growth. However, the relationship between FGF1/FGF2 and LDHA/B-mediated glycolysis in PCa progression is not reported. Thus, we aimed to explore whether FGF1/2 could regulate LDHA and LDHB to promote glycolysis and explored the involved signaling pathway in PCa progression. METHODS: In vitro studies used RT‒qPCR, Western blot, CCK-8 assays, and flow cytometry to analyze gene and protein expression, cell viability, apoptosis, and cell cycle in PCa cell lines. Glycolysis was assessed by measuring glucose consumption, lactate production, and extracellular acidification rate (ECAR). For in vivo studies, a xenograft mouse model of PCa was established and treated with an FGF pathway inhibitor, and tumor growth was monitored. RESULTS: FGF1, FGF2, and LDHA were expressed at high levels in PCa cells, while LDHB expression was low. FGF1/2 positively modulated LDHA and negatively modulated LDHB in PCa cells. The depletion of FGF1, FGF2, or LDHA reduced cell proliferation, induced cell cycle arrest, and inhibited glycolysis. LDHB overexpression showed similar inhibitory effect on PCa cells. Mechanistically, we found that FGF1/2 positively regulated STAT1 and STAT1 transcriptionally activated LDHA expression while suppressed LDHB expression. Furthermore, the treatment of an FGF pathway inhibitor suppressed PCa tumor growth in mice. CONCLUSION: The FGF pathway facilitates glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in PCa.

Minimally Invasive Aesthetic Suturing Technique for Anterior Aesthetic Crown Lengthening Surgery: A Case Series with 24- month Follow-Up.

BACKGROUND: A minimally invasive aesthetic suturing technique was employed in aesthetic crown lengthening surgery (ACLS). The objective of this report was to evaluate the clinical and patient- reported outcomes of this technique for ACLS. METHODS: Fifteen patients who underwent ACLS were treated utilizing the described suturing technique. Clinical parameters, including plaque index (PI), gingival index (GI), bleeding index (BI), papilla index score (PIS), early wound healing index (EHI), visual analogue scale (VAS), pink esthetic score and white esthetic score (PES/WES), were recorded at baseline, immediately post-surgery and during follow-up visits spanning 5 days to 24 months. The two-sample t-test was performed to evaluate statistical significance (α = 0.05). RESULT: 100% of the patients reported a high level of satisfaction, with a stable high postoperative VAS scores. From baseline to 5-day postoperation, there was no statistically significant increase in PI, although there was a slight deterioration observed in GI (0.13Å}0.23, P＜0.05) and BI (0.49Å}0.55, P＜ 0.05). Early wound healing (EHI 1) was achieved by all patients at 5 days post-surgery. Additionally, 3 patients exhibited changes in PIS within the initial 3 months following surgery, after which, all patients attained an optimal degree of papilla filling (degree III). CONCLUSION: The application of the minimally invasive aesthetic suturing technique in ACLS demonstrates favorable outcomes in terms of patient satisfaction and long-term stability. However, the assertion of its superiority over conventional suturing methods for ACLS necessitates substantiation through rigorous investigation via well-designed randomized controlled clinical trials.

Periodontopathogen-Related Cell Autophagy-A Double-Edged Sword.

The periodontium is a highly organized ecosystem, and the imbalance between oral microorganisms and host defense leads to periodontal diseases. The periodontal pathogens, mainly Gram-negative anaerobic bacteria, colonize the periodontal niches or enter the blood circulation, resulting in periodontal tissue destruction and distal organ damage. This phenomenon links periodontitis with various systemic conditions, including cardiovascular diseases, malignant tumors, steatohepatitis, and Alzheimer's disease. Autophagy is an evolutionarily conserved cellular self-degradation process essential for eliminating internalized pathogens. Nowadays, increasing studies have been carried out in cells derived from periodontal tissues, immune system, and distant organs to investigate the relationship between periodontal pathogen infection and autophagy-related activities. On one hand, as a vital part of innate and adaptive immunity, autophagy actively participates in host resistance to periodontal bacterial infection. On the other, certain periodontal pathogens exploit autophagic vesicles or pathways to evade immune surveillance, therefore achieving survival within host cells. This review provides an overview of the autophagy process and focuses on periodontopathogen-related autophagy and their involvements in cells of different tissue origins, so as to comprehensively understand the role of autophagy in the occurrence and development of periodontal diseases and various periodontitis-associated systemic illnesses.

Macrophage-derived exosomes rescue the TNF-ɑ-suppressed osteo-/cementogenic differentiation of hPDLCs.

OBJECTIVE: Inflammatory stimuli compromise the differentiation potency of human periodontal ligament cells (hPDLCs). Macrophage-derived exosomes (M-Exo) play a role in several aspects of cellular activity. This study investigated how M-Exo contributes to the osteo-/cementogenic differentiation of hPDLCs under inflammation and the mechanism involved. METHODS: M-Exo was identified by transmission electron microscopy, western blotting (WB), and dynamic light scattering. The internalization of M-Exo by hPDLCs was observed. After M-Exo treatment, the osteo-/cementogenic markers were detected by RT-qPCR and WB, and alkaline phosphatase (ALP) activity by ALP staining. Tumor necrosis factor alpha (TNF-ɑ) was applied to simulate inflammation. The rescue effect of M-Exo on TNF-ɑ-suppressed differentiation was validated. The p38 MAPK pathway activity was tested and a specific inhibitor was applied to explore the mechanism. RESULTS: M-Exo was successfully isolated, identified and internalized by hPDLCs. M-Exo enhanced the osteo-/cementogenic differentiation of hPDLCs, as indicated by upregulated osteo-/cementogenic markers and elevated ALP activity. Moreover, TNF-ɑ inhibited the differentiation capabilities of hPDLCs, on which M-Exo showed a rescue effect. M-Exo activated the p38 MAPK pathway and SB203580 attenuated its promotion effect. CONCLUSION: This study showed that M-Exo ameliorated the TNF-ɑ-suppressed osteo-/cementogenic differentiation of hPDLCs partly through the p38 MAPK pathway.

Cryo-EM Structure of Porphyromonas gingivalis RNA Polymerase.

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in ß and ß' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development.

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.

Mitochondrial Dysfunction in Periodontitis and Associated Systemic Diseases: Implications for Pathomechanisms and Therapeutic Strategies.

Periodontitis is a chronic infectious disorder damaging periodontal tissues, including the gingiva, periodontal ligament, cementum, and alveolar bone. It arises from the complex interplay between pathogenic oral bacteria and host immune response. Contrary to the previous view of "energy factories", mitochondria have recently been recognized as semi-autonomous organelles that fine-tune cell survival, death, metabolism, and other functions. Under physiological conditions, periodontal tissue cells participate in dynamic processes, including differentiation, mineralization, and regeneration. These fundamental activities depend on properly functioning mitochondria, which play a crucial role through bioenergetics, dynamics, mitophagy, and quality control. However, during the initiation and progression of periodontitis, mitochondrial quality control is compromised due to a range of challenges, such as bacterial-host interactions, inflammation, and oxidative stress. Currently, mounting evidence suggests that mitochondria dysfunction serves as a common pathological mechanism linking periodontitis with systemic conditions like type II diabetes, obesity, and cardiovascular diseases. Therefore, targeting mitochondria to intervene in periodontitis and multiple associated systemic diseases holds great therapeutic potential. This review provides advanced insights into the interplay between mitochondria, periodontitis, and associated systemic diseases. Moreover, we emphasize the significance of diverse therapeutic modulators and signaling pathways that regulate mitochondrial function in periodontal and systemic cells.

Glycometabolic reprogramming in cementoblasts: A vital target for enhancing cell mineralization.

Cementum, a constituent part of periodontal tissues, has important adaptive and reparative functions. It serves to attach the tooth to alveolar bone and acts as a barrier delimit epithelial growth and bacteria evasion. A dynamic and highly responsive cementum is essential for maintaining occlusal relationships and the integrity of the root surface. It is a thin layer of mineralized tissue mainly produced by cementoblasts. Cementoblasts are osteoblast-like cells essential for the restoration of periodontal tissues. In recent years, glucose metabolism has been found to be critical in bone remodeling and osteoblast differentiation. However, the glucose metabolism of cementoblasts remains incompletely understood. First, immunohistochemistry staining and in vivo tracing with 18 F-fluorodeoxyglucose (18 F-FDG) revealed significantly higher glucose metabolism in cementum formation. To test the bioenergetic pathways of cementoblast differentiation, we compared the bioenergetic profiles of mineralized and unmineralized cementoblasts. As a result, we observed a significant increase in the consumption of glucose and production of lactate, coupled with the higher expression of glycolysis-related genes. However, the expression of oxidative phosphorylation-related genes was downregulated. The verified results were consistent with the RNA sequencing results. Likewise, targeted energy metabolomics shows that the levels of glycolytic metabolites were significantly higher in the mineralized cementoblasts. Seahorse assays identified an increase in glycolytic flux and reduced oxygen consumption during cementoblast mineralization. Apart from that, we also found that lactate dehydrogenase A (LDHA), a key glycolysis enzyme, positively regulates the mineralization of cementoblasts. In summary, cementoblasts mainly utilized glycolysis rather than oxidative phosphorylation during the mineralization process.

CKIP-1 mediates P. gingivalis-suppressed osteogenic/cementogenic differentiation of periodontal ligament cells partially via p38 signaling pathway.

Objectives: Casein kinase 2 interacting protein-1 (CKIP-1) is a versatile player involved in various biological processes. However, whether CKIP-1 mediates the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) under Porphyromonas gingivalis (Pg) stimulation remains unknown. Material and Methods: The effect of Pg on PDLC differentiation was first verified. CKIP-1 expression in Pg-infected PDLCs or in PDL of apical periodontitis (AP) mice was detected. The changes of CKIP-1 during PDLC differentiation was also determined. PDLC differentiation capacity in CKIP-1 knockout (KO) mice and CKIP-1-silenced PDLCs with or without Pg stimulation were further studied. Inhibitor was finally applied to verify the involvement of p38 signaling pathway in PDLC differentiation. Results: The suppression effect of Pg on PDLC differentiation was demonstrated. CKIP-1 increased in the PDL of AP mice and Pg-induced PDLCs, and decreased gradually during PDLC differentiation. Increased OSX and RUNX2 expression in PDL were observed in CKIP-1 KO mice. Also, CKIP-1 silencing facilitated and rescued Pg-inhibited PDLC differentiation. Inhibitor for p38 signaling pathway blocked CKIP-1 silencing-facilitated PDLC differentiation. Conclusions: CKIP-1 mediated the osteogenic/cementogenic differentiation of PDLCs partially through p38 signaling pathway, which may provide evidence for the regeneration of periodontal hard tissues damaged by Pg.

CKIP-1 Promotes P. gingivalis-Induced Inflammation of Periodontal Soft Tissues by Inhibiting Autophagy.

As a chronic inflammatory disease, periodontitis involves many biological processes including autophagy. At the same time, casein kinase 2 interacting protein-1 (CKIP-1) was reported to play a role in regulation of inflammation. But whether CKIP-1 and autophagy interact in periodontitis remains unclear. In this paper, our research team verified the levels of CKIP-1 expression and autophagy increase in the periodontal tissues of a ligature-induced periodontitis mouse model. And this result was also confirmed in Porphyromonas gingivalis (Pg)-induced human gingival fibroblasts (HGF) and human periodontal ligament cells (PDLC). We also showed the autophagy level in periodontal tissues is higher in Ckip-1 knockout (KO) mice than wild type (WT). At the same time, CKIP-1 knockdown lentivirus was used in PDLC and HGF, and it was found that silencing CKIP-1 significantly activated autophagy. Unfortunately, the regulatory role of autophagy in periodontitis is still unclear. Then, the autophagy agonist Rapamycin and inhibitor 3-MA were used in a periodontitis mouse model to investigate periodontal tissue destruction. We found the inflammation in periodontal tissue was reduced when autophagy activated. All these conclusions have been verified both in vivo and in vitro experiments. Finally, our research proved that silencing CKIP-1 reduces the expression of inflammatory cytokines in Pg-induced PDLC and HGF by regulating autophagy. Overall, a new role for CKIP-1 in regulating periodontal tissue inflammation was demonstrated in our study, and it is possible to treat periodontitis by targeting the CKIP-1 gene.

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 Applications and Potentials of Extracellular Vesicles from Different Cell Sources in Periodontal Regeneration.

Periodontitis is a chronic infectious disease worldwide that can cause damage to periodontal supporting tissues including gingiva, bone, cementum and periodontal ligament (PDL). The principle for the treatment of periodontitis is to control the inflammatory process. Achieving structural and functional regeneration of periodontal tissues is also essential and remains a major challenge. Though many technologies, products, and ingredients were applied in periodontal regeneration, most of the strategies have limited outcomes. Extracellular vesicles (EVs) are membranous particles with a lipid structure secreted by cells, containing a large number of biomolecules for the communication between cells. Numerous studies have demonstrated the beneficial effects of stem cell-derived EVs (SCEVs) and immune cell-derived EVs (ICEVs) on periodontal regeneration, which may be an alternative strategy for cell-based periodontal regeneration. The production of EVs is highly conserved among humans, bacteria and plants. In addition to eukaryocyte-derived EVs (CEVs), a growing body of literature suggests that bacterial/plant-derived EVs (BEVs/PEVs) also play an important role in periodontal homeostasis and regeneration. The purpose of this review is to introduce and summarize the potential therapeutic values of BEVs, CEVs and PEVs in periodontal regeneration, and discuss the current challenges and prospects for EV-based periodontal regeneration.

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.

Chitosan/silk fibroin composite bilayer PCL nanofibrous mats for bone regeneration with enhanced antibacterial properties and improved osteogenic potential.

In regenerative medicine and bone tissue engineering, various composite materials are enormously popular, but the final tissue restoration outcome is not always satisfactory. In this study, bilayer-deposited multifunctional nanofiber mats were successfully fabricated with an osteogenic side of silk fibroin/poly (ε-caprolactone) (referred to as SF/PCL) and an antibacterial side of poly (ε-caprolactone)/chitosan (referred to as PCL/CS). The PCL/CS-SF/PCL (referred to as PCSP) mats exhibited biocompatible properties, sufficient hydrophilicity and mechanical properties, as well as a higher breaking strength (3.6 MPa) than the monolayer of SF/PCL mats (1.5 MPa). The antibacterial side of PCSP mats (A-layer) demonstrated ideal antibacterial potency because the survival rate of Escherichia coli (E. coli) (approximately 25 %) and Staphylococcus aureus (S. aureus) (approximately 15 %) were both significantly lower. Subsequently, the plasmid encoding runt related transcription factor 2 (Runx2) was complexed with the osteogenic side of PCSP mats (O-layer) through polyethyleneimine (PEI), thereby enhancing both osteogenesis-related gene expression and the formation of mineralized nodules. Similarly, the implantation of PCSP+Runx2 mats effectively promoted bone tissue generation in vivo. These results indicated the excellent prospects of applying PCSP mats to bone regeneration with gene delivery.

m6 A demethylase Fto regulates the TNF-α-induced inflammatory response in cementoblasts.

OBJECTIVE: Apical periodontitis is the most frequently occurring pathological lesion. Fat mass and obesity-associated protein (Fto) is the first identified RNA N6-methyladenosine demethylase. However, whether Fto regulates apical periodontitis remains unclear. This study aimed to explore the mechanisms of Fto in the tumor necrosis factor-α (TNF-α)-induced inflammatory response. MATERIALS AND METHODS: We established an apical periodontitis model. An immortalized cementoblast cell line (OCCM-30) cells were exposed to TNF-α. Fto, Il6, Mcp1, and Mmp9 expressions were assessed by qRT-PCR. We knocked down Fto using lentiviruses and detected TNF-α-induced inflammation-related gene expressions and mRNA stability. RESULTS: Mice with apical periodontitis showed downregulation of Fto expression. OCCM-30 cells exposed to TNF-α showed an upregulation of inflammation-related genes with a decrease in Fto. Furthermore, knockdown of Fto promoted the expressions of Il6, Mcp1, and Mmp9 in TNF-α-treated OCCM-30 cells as compared with negative control cells, whereas it did not affect the mRNA stability. Interestingly, Fto knockdown activated the p65, p38, and ERK1/2 pathways, and it slightly activated the JNK signaling pathway after TNF-α administration in OCCM-30 cells. CONCLUSION: A TNF-α-induced decrease in the expression of Fto might play a critical role in the inflammatory response in cementoblasts, and knockdown of Fto might upregulate the inflammatory response.

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.