Calcification plays a key role in biological processes, and breakdown of the regulatory mechanism results in a pathological state such as ectopic calcification. We hypothesized that ENPP1, the enzyme that produces the calcification inhibitor pyrophosphate, is transcriptionally regulated by Nrf2, and that Nrf2 activation augments ENPP1 expression to inhibit ectopic calcification. Cell culture experiments were performed using mouse osteoblastic cell line MC3T3-E1. Nrf2 was activated by 5-aminolevulinic acid and sodium ferrous citrate. Nrf2 overexpression was induced by the transient transfection of an Nrf2 expression plasmid. ENPP1 expression was monitored by real-time RT-PCR. Because the promoter region of ENPP1 contains several Nrf2-binding sites, chromatin immunoprecipitation using an anti-Nrf2 antibody followed by real-time PCR (ChIP-qPCR) was performed. The relationship between Nrf2 activation and osteoblastic differentiation was examined by alkaline phosphatase (ALP) and Alizarin red staining. We used mice with a hypomorphic mutation in ENPP1 (ttw mice) to analyze whether Nrf2 activation inhibits ectopic calcification. Nrf2 and Nrf2 overexpression augmented ENPP1 expression and inhibited osteoblastic differentiation, as indicated by ALP expression and calcium deposits. ChIP-qPCR showed that some putative Nrf2-binding sites in the ENPP1 promoter region were bound by Nrf2. Nrf2 activation inhibited ectopic calcification in mice. ENPP1 gene expression was transcriptionally regulated by Nrf2, and Nrf2 activation augmented ENPP1 expression, leading to the attenuation of osteoblastic differentiation and ectopic calcification in vitro and in vivo. Nrf2 activation has a therapeutic potential for preventing ectopic calcification.

Background: Facial asymmetry in patients with dentofacial deformity is often associated with occlusal asymmetry, functional differences in mastication, and temporomandibular joint (TMJ) conditions. However, side-to-side differences in the stability of masticatory movement and their relationship with oral function have not been fully clarified. Therefore, this study aimed to investigate differences in masticatory movement, oral function, and the prevalence of MRI-based structural features of the temporomandibular joint between the deviated and non-deviated sides in patients with facial asymmetry. Methods: Twenty-one patients with dentofacial deformity and facial asymmetry were included in this study. Oral function was evaluated by measuring occlusal contact area, occlusal force, and masticatory performance. Masticatory movement was recorded using a mandibular movement recording system, and parameters related to the masticatory path and chewing speed were calculated. The stability of masticatory movement was evaluated using the variance of these parameters across chewing cycles. Temporomandibular joint structural features were assessed using CT and MRI. Comparisons between the deviated and non-deviated sides were performed using paired statistical tests. Results: Occlusal contact area and occlusal force were significantly greater on the deviated side than on the non-deviated side (p = 0.001 and p = 0.006, respectively), whereas no significant difference was observed in masticatory performance (p = 0.211). The deviated side showed a smaller closing angle (p = 0.005) and maximum lateral amplitude (p = 0.019), indicating a more vertical chewing pattern. The stability of masticatory movement, evaluated using the variance of masticatory path and velocity parameters, was significantly greater on the deviated side (e.g., variance of cycle axis angle, p = 0.002; variance of maximum closing velocity, p = 0.006). In addition, the prevalence of imaging-based structural features of the temporomandibular joint was significantly higher on the deviated side (p = 0.016). Conclusions: Patients with dentofacial deformity and facial asymmetry were associated with functional asymmetry between the deviated and non-deviated sides. The deviated side showed greater occlusal contact area, occlusal force, and stability of masticatory movement. These findings suggest that the deviated side may be functionally favorable for mastication and may be related to a tendency toward preferential chewing behavior. However, because the habitual chewing side was not directly evaluated in the present study, this interpretation should be considered cautiously and viewed as hypothesis-generating.

Mandibular retrognathism occurs by insufficient mandibular growth and causes several issues, such as respiratory difficulty and diminished masticatory function. At present, functional orthodontic appliances are used for stimulating mandibular growth in pediatric cases. However, the effectiveness of functional appliances is not always stable in daily practices. A more effective, reliable, and safer therapeutic method for mandibular growth promotion would be helpful for growing mandibular retrognathism patients. As we previously discovered that nutritional supplementation of myo-inositol in growing mice specifically increases mandibular endochondral growth, we performed preclinical animal experiments in rabbits in this study. Briefly, six-week-old male Japanese white rabbits were fed with or without myo-inositol supplementation in laboratory chow until 25 weeks old, and 3D image analysis using micro CT data and histological examinations was done. Myo-inositol had no systemic effect, such as femur length, though myo-inositol specifically augmented the mandibular growth. Myo-inositol increased the thickness of mandibular condylar cartilage. We discovered that the nutritional supplementation of myo-inositol during the growth period specifically augmented mandibular growth without any systemic influence, even in rabbits. Our results suggest the possibility of clinical use of myo-inositol for augmentation of the mandibular growth in growing mandibular retrognathism patients in the future.

Mandibular condylar cartilage (MCC) exhibits greater susceptibility to mechanical stress-induced degeneration than tibial articular cartilage (TAC). This study investigated whether differential epigenetic regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of antioxidant responses, is associated with distinct antioxidant capacities between these cartilage types. Cartilage tissues from 5-week-old male ICR mice (n = 16 for gene analyses, n = 8 for protein analyses) were obtained using laser microdissection. Gene and protein expression was analyzed by microarray, real-time RT-PCR, and immunohistochemistry. DNA methylation of the Nrf2 promoter was evaluated using pyrosequencing and high-resolution melting analysis. Nrf2 expression in MCC was approximately 1/10 that in TAC at mRNA level and only 5% at protein level. Downstream antioxidant enzymes (NQO1, G6PD, HO-1) showed significantly reduced expression in MCC. Oxidative DNA damage marker 8-OHdG was significantly elevated in MCC compared to TAC (20.0% vs. 10.7%, p < 0.05). The Nrf2 promoter region showed higher DNA methylation levels in MCC, confirmed by high-resolution melting analysis. Higher Nrf2 promoter methylation in MCC is associated with reduced antioxidant capacity and elevated oxidative damage. This epigenetic–antioxidant relationship may contribute to MCC’s vulnerability to mechanical stress-induced degeneration and represents a potential therapeutic target for temporomandibular joint disorders.