Melatonin-mediated malic enzyme 2 orchestrates mitochondrial fusion and respiratory functions to promote odontoblastic differentiation during tooth development.

Tooth development is a complex process that is tightly controlled by circadian rhythm. Melatonin (MT) is a major hormonal regulator of the circadian rhythm, and influences dentin formation and odontoblastic differentiation during tooth development; however, the underlying mechanism remains elusive. This study investigated how MT regulates odontoblastic differentiation, with a special focus on its regulation of mitochondrial dynamics. In rat dental papilla cells (DPCs), we found that MT promotes odontoblastic differentiation concurrently with enhanced mitochondrial fusion, while disruption of mitochondrial fusion by depleting optic atrophy 1 (OPA1) impairs MT-mediated differentiation and mitochondrial respiratory functions. Through RNA sequencing, we discovered that MT significantly upregulated malic enzyme 2 (ME2), a mitochondrial NAD(P)+ -dependent enzyme, and identified ME2 as a critical MT downstream effector that orchestrates odontoblastic differentiation, mitochondrial fusion, and respiration functions. By detecting the spatiotemporal expression of ME2 in developing tooth germs, and using tooth germ reconstituted organoids, we also provided in vivo and ex vivo evidence that ME2 promotes dentin formation, indicating a possible involvement of ME2 in MT-modulated tooth development. Collectively, our findings offer novel understandings regarding the molecular mechanism by which MT affects cell differentiation and organogenesis, meanwhile, the critical role of ME2 in MT-regulated mitochondrial functions is also highlighted.

Comparative study of surface electromyography of masticatory muscles in patients with different types of bruxism.

BACKGROUND: Bruxism is a rhythmic masticatory muscle activity that occurs involuntarily in a non-physiologically functional state. There is a lack of research classifying the functional status of masticatory muscles in patients with different mandibular movement types (centric clenching or eccentric grinding) of bruxism. AIM: To assess the differences of the masticatory muscle activity in patients with different types of bruxism. METHODS: A total of 21 subjects with centric bruxism (CB) and 21 subjects with eccentric bruxism (ECB) were screened from college students according to a questionnaire and their tooth wear features. Sixteen subjects with no bruxism were also recruited. The surface electromyography (EMG) signals of the temporalis anterior (TA) and superficial masseter muscle (MM) were measured in different mandibular positions and during the chewing task. The EMG amplitude and chewing cycle duration parameters were then analyzed. RESULTS: The CB group showed fewer muscle maximal motor units, with the MM being more pronounced, a higher proportion of muscle contractions to be recruited for the same load of chewing activity, and a longer chewing cycle. The ECB group showed more TA maximal motor units and higher MM activity on the non-working side in unilateral chewing. CONCLUSION: CB mainly affects the MM, and patients with CB show reduced masticatory muscle contraction efficiency and chewing cycle efficiency. ECB mainly affects the TA, and patients with ECB show enhanced contraction of non-functional lateral muscle bundles.

Treatment of condylar osteophyte in temporomandibular joint osteoarthritis with muscle balance occlusal splint and long-term follow-up: A case report.

BACKGROUND: Condylar osteophytes, a remodeling form of temporomandibular joint osteoarthritis (TMJ OA), mainly manifest as marginal angular outgrowths of the condyle. Previous researchers have advocated surgical removal of condylar osteophytes. Reports on the effect of occlusal splint on TMJ OA patients' joints have mostly focused on treatment with this splint, which can reduce the absorption of the affected condyle and promote repair and regeneration. However, the effect of the splint on the dissolution of condylar osteophytes has not yet been reported. CASE SUMMARY: A 68-year-old female patient suffered from occlusal discomfort with left facial pain for 2 years. Cone beam computed tomography showed a rare osteophyte on top of her left condyle. She was finally diagnosed with TMJ OA. The patient refused surgical treatment and received conservative treatment with a muscle balance occlusal splint. The pain experienced by the patient on the left side of her face was relieved, and her chewing ability recovered after treatment. The osteophyte dissolved, and the condylar cortex remained stable during long-term follow-up observations. CONCLUSION: The muscle balance occlusal splint could be a noninvasive means of treating condylar osteophytes in TMJ OA patients.

RORα Regulates Odontoblastic Differentiation and Mediates the Pro-Odontogenic Effect of Melatonin on Dental Papilla Cells.

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.

Suppression of osteogenic differentiation and mitochondrial function change in human periodontal ligament stem cells by melatonin at physiological levels.

N-Acetyl-5-methoxytryptamine (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM-10 nM) remains unclear. We explored the effects of 1 pM-1 µM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytometry and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis-related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and NAD+/NADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OPN) and osteocalcin (OCN) expression levels, which were restored or even up-regulated by 1 µM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and NAD+/NADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.

Changes of mitochondrial respiratory function during odontogenic differentiation of rat dental papilla cells.

Dental papilla cells (DPCs) belong to precursor cells differentiating to odontoblasts and play an important role in dentin formation and reproduction. This study aimed to explore the changes and and involvement of mitochondrial respiratory function during odontogenic differentiation. Primary DPCs were obtained from first molar dental papilla of neonatal rats and cultured in odontogenic medium for 7, 14, 21 days. DPCs, which expressed mesenchymal surface markers CD29, CD44 and CD90, had the capacity for self-renewal and multipotent differentiation. Odontoblastic induction increased mineralized matrix formation in a time-dependent manner, which was accompanied by elevated alkaline phosphatase (ALP), dentin sialophosphoprotein and dentin matrix protein 1 expression at mRNA and protein levels. Notably, odontogenic medium led to an increase in adenosine-5'-triphosphate content and mitochondrial membrane potential, whereas a decrease in intercellular reactive oxygen species production and NAD+/NADH ratio. Furthermore, odontogenic differentiation was significantly suppressed by treatment with rotenone, an inhibitor of mitochondrial respiratory chain. These results demonstrate that enhanced mitochondrial function is crucial for odontogenic differentiation of DPCs.