BMAL1-Downregulation Aggravates Porphyromonas Gingivalis-Induced Atherosclerosis by Encouraging Oxidative Stress.

RATIONALE: Atherosclerotic cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerotic cardiovascular diseases are considered as chronic inflammation processes. In addition to risk factors associated with the cardiovascular system itself, pathogenic bacteria such as the periodontitis-associated Porphyromonas gingivalis (P gingivalis) are also closely correlated with the development of atherosclerosis, but the underlying mechanisms are still elusive. OBJECTIVE: To elucidate the mechanisms of P gingivalis-accelerated atherosclerosis and explore novel therapeutic strategies of atherosclerotic cardiovascular diseases. METHODS AND RESULTS: Bmal1-/- (brain and muscle Arnt-like protein 1) mice, ApoE-/- mice, Bmal1-/-ApoE-/- mice, conditional endothelial cell Bmal1 knockout mice (Bmal1fl/fl; Tek-Cre mice), and the corresponding jet-legged mouse model were used. Pgingivalis accelerates atherosclerosis progression by triggering arterial oxidative stress and inflammatory responses in ApoE-/- mice, accompanied by the perturbed circadian clock. Circadian clock disruption boosts P gingivalis-induced atherosclerosis progression. The mechanistic dissection shows that P gingivalis infection activates the TLRs-NF-κB signaling axis, which subsequently recruits DNMT-1 to methylate the BMAL1 promoter and thus suppresses BMAL1 transcription. The downregulation of BMAL1 releases CLOCK, which phosphorylates p65 and further enhances NF-κB signaling, elevating oxidative stress and inflammatory response in human aortic endothelial cells. Besides, the mouse model exhibits that joint administration of metronidazole and melatonin serves as an effective strategy for treating atherosclerotic cardiovascular diseases. CONCLUSIONS: P gingivalis accelerates atherosclerosis via the NF-κB-BMAL1-NF-κB signaling loop. Melatonin and metronidazole are promising auxiliary medications toward atherosclerotic cardiovascular diseases.

The osteoclastic activity in apical distal region of molar mesial roots affects orthodontic tooth movement and root resorption in rats.

The utilization of optimal orthodontic force is crucial to prevent undesirable side effects and ensure efficient tooth movement during orthodontic treatment. However, the sensitivity of existing detection techniques is not sufficient, and the criteria for evaluating optimal force have not been yet established. Here, by employing 3D finite element analysis methodology, we found that the apical distal region (A-D region) of mesial roots is particularly sensitive to orthodontic force in rats. Tartrate-resistant acidic phosphatase (TRAP)-positive osteoclasts began accumulating in the A-D region under the force of 40 grams (g), leading to alveolar bone resorption and tooth movement. When the force reached 80 g, TRAP-positive osteoclasts started appearing on the root surface in the A-D region. Additionally, micro-computed tomography revealed a significant root resorption at 80 g. Notably, the A-D region was identified as a major contributor to whole root resorption. It was determined that 40 g is the minimum effective force for tooth movement with minimal side effects according to the analysis of tooth movement, inclination, and hyalinization. These findings suggest that the A-D region with its changes on the root surface is an important consideration and sensitive indicator when evaluating orthodontic forces for a rat model. Collectively, our investigations into this region would aid in offering valuable implications for preventing and minimizing root resorption during patients' orthodontic treatment.

Dental professionals' use of personal protective equipment during COVID-19: a cross-sectional study in China.

Background: Appropriate use of personal protective equipment (PPE) could significantly reduce the risk of viral transmission and infection. This study aimed to assess the use of PPE among dentists during the COVID-19 pandemic in China, explore its influencing factors, and provide some practical recommendations. Methods: An online cross-sectional survey was conducted among 384 Chinese dentists in September 2022. The questionnaire comprised a series of questions about demographic characteristics, compliance with proper PPE use, personal barriers to use, and exposure risk estimation. Results: Of the 384 respondents, 57.3% had unacceptable compliance with the proper use of PPE during COVID-19. Medical surgical mask is the most common for dental professionals to wear (93.8%), followed by goggles or face shield (63.8%), and isolation gown (53.1%). Unexpectedly, only 63.3% of respondents always change masks with guidelines. The condition for changing goggles/face shields and isolation gowns is even worse (45.6 and 37.0%, respectively). Visual barriers, physical discomfort, complex procedures, and heavy workload were the most common personal barriers to use. According to the results of Chi-square test and correlation analysis, PPE use compliance was associated with age, years of practice, medical institution type, and exposure risk estimation. Conclusion: Chinese dental professionals need to improve their compliance with the proper use of PPE, especially those in the 31-40 age group, with 11-15 experience years and working in private dental clinics. Increasing compliance with PPE may be achieved by addressing personal barriers to use, human resource shortages, and perceptions of exposure risk.

Insight into the roles of melatonin in bone tissue and bone­related diseases (Review).

Bone­related diseases comprise a large group of common diseases, including fractures, osteoporosis and osteoarthritis (OA), which affect a large number of individuals, particularly the elderly. The progressive destruction and loss of alveolar bone caused by periodontitis is a specific type of bone loss, which has a high incidence and markedly reduces the quality of life of patients. With the existing methods of prevention and treatment, the incidence and mortality of bone­related diseases are still gradually increasing, creating a significant financial burden to societies worldwide. To prevent the occurrence of bone­related diseases, delay their progression or reverse the injuries they cause, new alternative or complementary treatments need to be developed. Melatonin exerts numerous physiological effects, including inducing anti­inflammatory and antioxidative functions, resetting circadian rhythms and promoting wound healing and tissue regeneration. Melatonin also participates in the health management of bone and cartilage. In the present review, the potential roles of melatonin in the pathogenesis and progression of bone injury, osteoporosis, OA and periodontitis are summarized. Furthermore, the high efficiency and diversity of the physiological regulatory effects of melatonin are highlighted and the potential benefits of the use of melatonin for the clinical prevention and treatment of bone­related diseases are discussed.

The biological function of BMAL1 in skeleton development and disorders.

BMAL1 is a core component of the circadian clock loop, which directs the sophisticated circadian expression of clock-controlled genes. Skeletal Bone development is a complex biological process involving intramembranous ossification, endochondral ossification and bone remodeling, as well as specific cells, such as mesenchymal cells, osteoblasts, osteoclasts, chondrocytes, etc. Growing evidences suggest that BMAL1 is indispensable for hard tissue development, including bone, cartilage and teeth. Loss of BMAL1 in animals can inhibit bone and cartilage development, and result in abnormal bone mass. In mesenchymal cells, BMAL1 defect inhibits osteoblastic and chondrocytic differentiation. Inactivation of BMAL1 also can promote the differentiation and formation of osteoclasts and increase bone resorption. Specifically, preclinical data demonstrate that the abnormity of BMAL1 expression is associated with skeletal disorders such as skeletal mandibular hypoplasia, osteoarthritis, osteoporosis, etc. In this review, we systemically describe the impact of BMAL1 in skeletal development and homeostasis, and devote to searching new therapy strategies for bone disorders.

Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner.

Cardiovascular disease is still the leading cause of mortality worldwide. Vascular endothelial dysfunction is viewed as the initial step of most cardiovascular diseases. Many studies have indicated that periodontal pathogens, especially Porphyromonas gingivalis, are closely correlated with vascular endothelial homeostasis, but the function of P. gingivalis and the underlying mechanisms are still elusive. To illuminate the effects and elucidate the mechanisms of P. gingivalis on endothelial structural integrity, we developed P. gingivalis infection models in vivo and in vitro. Endothelial cell proliferation, differentiation and apoptosis were detected. Here, we showed that P. gingivalis can impair endothelial integrity by inhibiting cell proliferation and inducing endothelial mesenchymal transformation and apoptosis of endothelial cells, which reduce the cell levels and cause the endothelium to lose its ability to repair itself. A mechanistic analysis showed that TLR antagonist or NF-κB signalling inhibitor can largely rescue the damaged integrity of the endothelium caused by P. gingivalis, suggesting that TLR-NF-κB signalling plays a vital role in vascular endothelial homeostasis destroyed by P. gingivalis. These results suggest a potential intervention method for the prevention and treatment of cardiovascular disease.

Role of the oral microbiota in cancer evolution and progression.

Bacteria identified in the oral cavity are highly complicated. They include approximately 1000 species with a diverse variety of commensal microbes that play crucial roles in the health status of individuals. Epidemiological studies related to molecular pathology have revealed that there is a close relationship between oral microbiota and tumor occurrence. Oral microbiota has attracted considerable attention for its role in in-situ or distant tumor progression. Anaerobic oral bacteria with potential pathogenic abilities, especially Fusobacterium nucleatum and Porphyromonas gingivalis, are well studied and have close relationships with various types of carcinomas. Some aerobic bacteria such as Parvimonas are also linked to tumorigenesis. Moreover, human papillomavirus, oral fungi, and parasites are closely associated with oropharyngeal carcinoma. Microbial dysbiosis, colonization, and translocation of oral microbiota are necessary for implementation of carcinogenic functions. Various underlying mechanisms of oral microbiota-induced carcinogenesis have been reported including excessive inflammatory reaction, immunosuppression of host, promotion of malignant transformation, antiapoptotic activity, and secretion of carcinogens. In this review, we have systemically described the impact of oral microbial abnormalities on carcinogenesis and the future directions in this field for bringing in new ideas for effective prevention of tumors.

Circadian BMAL1 regulates mandibular condyle development by hedgehog pathway.

OBJECTIVE: Chondrogenesis and endochondral ossification in mandibular condyle play crucial roles in maxillofacial morphogenesis and function. Circadian regulator brain and muscle arnt-like 1 (BMAL1) is proven to be essential for embryonic and postnatal development. The goal of this study was to define the functions of BMAL1 in the embryonic and postnatal growth of mandibular condylar cartilages (MCC). MATERIALS AND METHODS: Micro-CT, TUNEL staining and EdU assay were performed using BMAL1-deficient mice model, and in vitro experiments were performed using rat chondrocytes isolated from MCC. RNA sequencing in mandibular condyle tissues from Bmal1-/- mice and the age-matched wild-type mice was used for transcriptional profiling at different postnatal stages. RESULTS: The expression levels of BMAL1 decrease gradually in MCC. BMAL1 is proved to regulate sequential chondrocyte differentiation, and its deficiency can result in the impairment of endochondral ossification of MCC. RNA sequencing reveals hedgehog signalling pathway is the potential target of BMAL1. BMAL1 regulates hedgehog signalling and affects its downstream cascades through directly binding to the promoters of Ptch1 and Ihh, modulating targets of hedgehog signalling which is indispensable for endochondral ossification. Importantly, the short stature phenotypes caused by BMAL1 deficiency can be rescued by hedgehog signalling activator. CONCLUSIONS: Collectively, these results indicate that BMAL1 plays critical roles on chondrogenesis and endochondral ossification of MCC, giving a new insight on potential therapeutic strategies for facial dysmorphism.

BMAL1 deficiency promotes skeletal mandibular hypoplasia via OPG downregulation.

OBJECTIVES: Skeletal mandibular hypoplasia (SMH), a common type of developmental deformities, results in impaired aesthetics of facial profile, occlusal dysfunction and poor life quality. In this study, BMAL1 deficiency leads to SMH formation, and we aim to investigate the mechanism by which BMAL1 deficiency induces SMH. MATERIALS AND METHODS: Circadian rhythm-disordered mouse models were constructed by placing animals in a jet lag schedule of 6-h light advance every 7 days for 4 or 8 weeks. The OPG expression was evaluated by histomorphometry, immunohistochemistry and western blot analysis. The mechanism by which BMAL1 affects OPG expression was investigated by chromatin immunoprecipitation and luciferase reporter assays. The phenotypes caused by BMAL1 knockout can be rescued by exogenous supplementation with OPG. RESULTS: We demonstrate that the expressions of BMAL1 and OPG decreased in SMH patients. Circadian rhythm-disordered mice and Bmal1-/- mice exhibited decreased expression of OPG, reduced bone mass and bone size of mandibles. Our results revealed that BMAL1 bound directly to the Opg promoter and upregulated its expression, thus inhibiting osteoclast differentiation. BMAL1 deficiency increased osteoclast differentiation by downregulating OPG expression. In vitro, the enhancement effect of osteoclast differentiation caused by BMAL1 knockdown was significantly reversed by exogenous supplementation with OPG. Importantly, bone loss caused by BMAL1 knockout can be partially reversed by injecting OPG Intraperitoneally. CONCLUSIONS: These results indicate that the circadian clock plays a critical role in the growth and development of mandible by regulating OPG expression, and present a potential therapeutic strategy to prevent SMH.

BMAL1 Deficiency Contributes to Mandibular Dysplasia by Upregulating MMP3.

Skeletal mandibular hypoplasia (SMH), one of the common types of craniofacial deformities, seriously affects appearance, chewing, pronunciation, and breathing. Moreover, SMH is prone to inducing obstructive sleep apnea syndrome. We found that brain and muscle ARNT-like 1 (BMAL1), the core component of the molecular circadian oscillator, was significantly decreased in mandibles of juvenile SMH patients. Accordingly, SMH was observed in circadian-rhythm-disrupted or BMAL1-deficient mice. RNA sequencing and protein chip analyses suggested that matrix metallopeptidase 3 (MMP3) is the potential target of BMAL1. Interestingly, in juvenile SMH patients, we observed that MMP3 was obviously increased. Consistently, MMP3 was upregulated during the whole growth period of 3-10 weeks in Bmal1-/- mice. Given these findings, we set out to characterize the underlying mechanism and found BMAL1 deficiency enhanced Mmp3 transcription through activating p65 phosphorylation. Together, our results provide insight into the mechanism by which BMAL1 is implicated in the pathogenesis of SMH.

Hypoxic Preconditioning Enhances Dental Pulp Stem Cell Therapy for Infection-Caused Bone Destruction.

The use of biological repair in infectious bone defects has been a major challenge for clinicians. With potential for bone regeneration, stem cell therapy could be an effective biological restoration measure for infection-caused bone destruction. In this study, we propose a new stem cell therapy strategy for infectious bone defect repair through systemic transplantation of human dental pulp stem cells (hDPSCs). Hypoxic preconditioning (HP) is thought to be able to enhance duration of survival and therapeutic potency of engrafted stem cells; therefore, we examined the role of HP on hDPSC therapeutic efficacy. Our results show that HP significantly enhanced hDPSC survival rate and osteogenic differentiation. hDPSCs were all CXCR4 positive under hypoxic pretreatment and their migration in response to SDF-1 was increased in vitro. hDPSC migration increase can be abolished after application of CXCR4 antagonist, AMD3100. In a mouse apical periodontitis bone destruction model, after transplantation of hypoxic preconditioned hDPSCs through intravenous injection, upregulated hDPSC recruitment and recovery of alveolar bone mass were observed in infected periapical tissue, and osteogenesis and bone mineralization were enhanced. Significantly, in periapical lesions, we found increased SDF-1 production and CXCR4/CFSE+ colabeled cells. Together, our results suggested that hypoxic preconditioned hDPSCs are capable of repairing infectious bone defects through the SDF-1/CXCR4 axis. Our investigation provides a novel infection-caused bone loss therapeutic strategy using hDPSC transplantation, and HP is an effective way of improving hDPSC survival rate, recruitment, and osteogenesis.

Morphological and microtensile bond strength evaluation of three adhesive systems to caries-affected human dentine with chemomechanical caries removal.

OBJECTIVES: To evaluate the influence of chemomechanical caries removal on bond strengths of three adhesive systems to caries-affected human dentine. METHODS: 45 extracted human third molars with occlusal moderate caries were used. One half of each cavity was prepared with chemomechanical method and the other half with bur as the control. Three adhesive systems, Prime&Bond NT (Dentsply, Germany) without any previous conditioning, an etch-and-rinse adhesive (One-Step, Bisco, USA) and a self-etching adhesive (Adper Prompt-L-Pop, 3M ESPE, USA), were applied. The compomer or resin crowns were, then, built up. The prepared specimens were sectioned for scanning electron microscopy (SEM) examination and microtensile bond strength (MTBS) testing. Data were analysed using two-way ANOVA with subsequent application of Student-Newman-Keuls test at p<0.05. RESULTS: Opened dentinal tubules and less smear layer were visible by SEM after chemomechanical caries removal, whilst an obvious smear layer covering the dentine surface with occluded tubules was exhibited after bur excavation. Resin tags were seen only in specimens with chemomechanical excavation when Prime&Bond NT system was applied. Statistical analysis of the values of MTBS failed to show significant difference between caries removal methods. However, the values of MTBS for One-Step system achieved with two caries removal methods were both significant higher than other adhesive systems. CONCLUSIONS: Chemomechanical caries removal did not influence the bond strengths of the adhesive systems used in this study to caries-affected human dentine. Highest bond strength was achieved with application of etch-and-rinse adhesive system.