Mandibular Endochondral Growth Is Specifically Augmented by Nutritional Supplementation with Myo-Inositol Even in Rabbits.

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.

Oral angiotensin-converting enzyme inhibitor captopril protects the heart from Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice.

Although angiotensin converting enzyme (ACE) inhibitors are considered useful for the treatment of human heart failure, some experimental failing-heart models have shown little beneficial effect of ACE inhibitors in animals with poor oral health, particularly periodontitis. In this study, we examined the effects of the ACE inhibitor captopril (Cap; 0.1 mg/mL in drinking water) on cardiac dysfunction in mice treated with Porphyromonas gingivalis lipopolysaccharide (PG-LPS) at a dose (0.8 mg/kg/day) equivalent to the circulating level in patients with periodontal disease. Mice were divided into four groups: 1) Control, 2) PG-LPS, 3) Cap, and 4) PG-LPS + Cap. After1 week, we evaluated cardiac function by echocardiography. The left ventricular ejection fraction was significantly decreased in PG-LPS-treated mice compared to the control (from 66 ± 1.8 to 59 ± 2.5%), while Cap ameliorated the dysfunction (63 ± 1.1%). The area of cardiac fibrosis was significantly increased (approximately 2.9-fold) and the number of apoptotic myocytes was significantly increased (approximately 5.6-fold) in the heart of PG-LPS-treated group versus the control, and these changes were suppressed by Cap. The impairment of cardiac function in PG-LPS-treated mice was associated with protein kinase C δ phosphorylation (Tyr-311), leading to upregulation of NADPH oxidase 4 and xanthine oxidase, and calmodulin kinase II phosphorylation (Thr-286) with increased phospholamban phosphorylation (Thr-17). These changes were also suppressed by Cap. Our results suggest that the renin-angiotensin system might play an important role in the development of cardiac diseases induced by PG-LPS.

Effects of the angiotensin-converting enzyme inhibitor captopril on occlusal-disharmony-induced cardiac dysfunction in mice.

Occlusal disharmony is known to affect not only the oral cavity environment, but also the autonomic nervous system in the heart. Since the renin-angiotensin system (RAS) inhibitor captopril (Cap) is one of the first-line drugs for preventing cardiac remodeling in patients with heart failure, we hypothesized that Cap might prevent cardiac dysfunction induced by occlusal disharmony. Here, to test this idea, we used our bite-opening (BO) mouse model, which was developed by cementing a suitable appliance onto the mandibular incisor. Mice were divided into four groups: (1) Control, (2) BO, (3) Cap, and (4) BO + Cap. After 2 weeks, we evaluated cardiac function by echocardiography and confirmed that cardiac function was significantly decreased in the BO group compared to the control, while Cap ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and oxidative stress-induced myocardial damage in the BO group were significantly increased versus the control, and these increases were suppressed by Cap. Cardiac dysfunction induced by BO was associated with dual phosphorylation on PKCδ (Tyr-311/Thr-505), leading to activation of CaMKII with increased phosphorylation of RyR2 and phospholamban. Our results suggest that the RAS might play an important role in the development of cardiac diseases induced by occlusal anomalies.

Early diagnosis of aortic calcification through dental X-ray examination for dental pulp stones.

Vascular calcification, an ectopic calcification exacerbated by aging and renal dysfunction, is closely associated with cardiovascular disease. However, early detection indicators are limited. This study focused on dental pulp stones, ectopic calcifications found in oral tissues that are easily identifiable on dental radiographs. Our investigation explored the frequency and timing of these calcifications in different locations and their relationship to aortic calcification. In cadavers, we examined the association between the frequency of dental pulp stones and aortic calcification, revealing a significant association. Notably, dental pulp stones appeared prior to aortic calcification. Using a rat model of hyperphosphatemia, we confirmed that dental pulp stones formed earlier than calcification in the aortic arch. Interestingly, there were very few instances of aortic calcification without dental pulp stones. Additionally, we conducted cell culture experiments with vascular smooth muscle cells (SMCs) and dental pulp cells (DPCs) to explore the regulatory mechanism underlying high phosphate-mediated calcification. We found that DPCs produced calcification deposits more rapidly and exhibited a stronger augmentation of osteoblast differentiation markers compared with SMCs. In conclusion, the observation of dental pulp stones through X-ray examination during dental checkups could be a valuable method for early diagnosis of aortic calcification risk.

Vidarabine, an anti-herpes agent, improves Porphyromonas gingivalis lipopolysaccharide-induced cardiac dysfunction in mice.

In this work, we examined the involvement of type 5 adenylyl cyclase (AC5) in cardiac dysfunction induced in mice given Porphyromonas gingivalis lipopolysaccharide (PG-LPS) at a dose equivalent to the circulating levels in periodontitis (PD) patients. Cardiac function was significantly decreased in mice given PG-LPS compared to the control, but treatment for 1 week with the AC5 inhibitor vidarabine ameliorated the dysfunction. Cardiac fibrosis and myocyte apoptosis were significantly increased in the PG-LPS group, but vidarabine blocked these changes. The PG-LPS-induced cardiac dysfunction was associated with activation of cyclic AMP/Ca2+-calmodulin-dependent protein kinase II signaling and increased phospholamban phosphorylation at threonine 17. These results suggest that pharmacological AC5 inhibition may be a promising approach to treat PD-associated cardiovascular disease.

Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice.

Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 ± 0.5 to 58 ± 1.2%) and remained low at 4 weeks (57 ± 1.0%). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

Vidarabine, an anti-herpes agent, prevents occlusal-disharmony-induced cardiac dysfunction in mice.

We recently reported a positive relationship between occlusal disharmony and cardiovascular disease via activation of ß-adrenergic signaling in mice. Furthermore, inhibition of type 5 adenylyl cyclase (AC5), a major cardiac subtype in adults, protects the heart against oxidative stress. Here, we examined the role of AC5 in the development of occlusal-disharmony-induced cardiovascular disease in bite-opening (BO) mice, prepared by cementing a suitable appliance onto the mandibular incisor. We first examined the effects of BO treatment on cardiac function in mice treated or not treated for 2 weeks with vidarabine, which we previously identified as an inhibitor of cardiac AC. Cardiac function was significantly decreased in the BO group compared to the control group, but vidarabine ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but vidarabine blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as increased activation of the Ca2+-calmodulin-dependent protein kinase II/receptor-interacting protein 3 signaling pathway. These data suggest that AC5 inhibition with vidarabine might be a new therapeutic approach for the treatment of cardiovascular disease associated with occlusal disharmony.

Effects of chronic Porphylomonas gingivalis lipopolysaccharide infusion on cardiac dysfunction in mice.

OBJECTIVE: Periodontitis (PD) is a chronic inflammatory disease of tooth-supportive tissue. An association between PD and cardiovascular disease (CVD) has been established. Although PD is generally accepted as a risk factor for CVD, the existence of a relationship remains debatable. Possible mechanisms include the release of inflammatory mediators such as lipopolysaccharide (LPS), which may spread systemically and promote CVD. METHODS: To compare the effects of lipopolysaccharide derived from Porphylomonas gingivalis (PG-LPS) on cardiac muscle in mice, mice were treated for 1 week with/without PG-LPS at a dose equivalent to the circulating level in PD patients (0.8 mg/kg/day). RESULTS: Cardiac function in terms of left ventricular ejection function was significantly decreased at 1 week compared to that in the control (from 66 ± 0.5% to 57 ± 1.1%). Compared to the controls, the number of apoptotic myocytes and the area of fibrosis were significantly increased by approximately 2.7-fold and 14-fold, respectively. The impairment of cardiac function appeared to involve the activation of cAMP/PKA signaling and cAMP/calmodulin kinase II signaling (CaMKII), leading to cardiac fibrosis, myocyte apoptosis and heart failure. CONCLUSIONS: Our results indicate that cAMP/PKA and cAMP/CaMKII signaling may be a new therapeutic target for the treatment of cardiovascular diseases in patients with periodontitis.

Oxidized Low-Density Lipoprotein Promotes In Vitro Calcification.

Calcification plays an important role in the human body in maintaining homeostasis. In the human body, the presence of a high amount of oxidized low-density lipoprotein (ox-LDL) is a consistent feature of the local areas that are common sites of ectopic calcification, namely dental calculus, renal calculus, and the areas affected by arteriosclerosis. Hence, ox-LDL may have some effect on calcification. Scanning electron microscopy (SEM) observation revealed a high amount of amorphous calcium phosphate (ACP) when ox-LDL was included in the solution. In the in vitro experiment, the highest amount of precipitation of calcium phosphate was observed in the solution containing ox-LDL compared to the inclusion of other biomaterials and was 4.2 times higher than that of deionized water for 4.86 mM calcium and 2.71 mM phosphate. The morphology of calcium phosphate precipitates in the solution containing ox-LDL differed from that of the precipitates in solutions containing other biomaterials, as determined by transmission electron microscopy (TEM). Through the time course observation of the sediments using TEM, it was observed that the sediments changed from spherical or oval shape to a thin film shape. These results indicate that sediments acquired a long-range order array, and the phase transitioned from non-crystalline to crystalline with an increased time and density of ACP. Thus, it is concluded that ox-LDL promoted ACP precipitation and it plays an important role in ectopic calcification.

Effects of occlusal disharmony on cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage in mice.

Occlusal disharmony leads to morphological changes in the hippocampus and osteopenia of the lumbar vertebra and long bones in mice, and causes stress. Various types of stress are associated with increased incidence of cardiovascular disease, but the relationship between occlusal disharmony and cardiovascular disease remain poorly understood. Therefore, in this work, we examined the effects of occlusal disharmony on cardiac homeostasis in bite-opening (BO) mice, in which a 0.7 mm space was introduced by cementing a suitable applicance onto the mandibular incisior. We first examined the effects of BO on the level of serum corticosterone, a key biomarker for stress, and on heart rate variability at 14 days after BO treatment, compared with baseline. BO treatment increased serum corticosterone levels by approximately 3.6-fold and the low frequency/high frequency ratio, an index of sympathetic nervous activity, was significantly increased by approximately 4-fold by the BO treatment. We then examined the effects of BO treatment on cardiac homeostasis in mice treated or not treated with the non-selective ß-blocker propranolol for 2 weeks. Cardiac function was significantly decreased in the BO group compared to the control group, but propranolol ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but propranolol blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as inhibition of Akt/mTOR signaling and autophagic flux. These data suggest that occlusal disharmony might affect cardiac homeostasis via alteration of the autonomic nervous system.

Role of ß-adrenergic signaling in masseter muscle.

In skeletal muscle, the major isoform of ß-adrenergic receptor (ß-AR) is ß2-AR and the minor isoform is ß1-AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the ß-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic ß1- or ß2-AR activation with a specific ß1-AR agonist, dobutamine (DOB), or a specific ß2-AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic ß1-AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic ß2-AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic ß1-AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic ß2-AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the ß1-AR pathway is deleterious and the ß2-AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.

Effects of chronic Porphyromonas gingivalis lipopolysaccharide infusion on skeletal muscles in mice.

Periodontitis, which is caused by various oral organisms, predominantly affects adults, and is one of the main causes of tooth loss, as well as leading to progression of numerous systemic diseases. However, its relationship to sarcopenia (aging-associated degenerative loss of skeletal muscle mass and function) remains unclear. The aim of this study was to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on skeletal muscle in mice, and to establish the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) for 4 weeks. This treatment significantly decreased the weight of fast-twitch skeletal muscles (masseter and tibialis anterior muscles), but not that of slow-twitch skeletal muscle (soleus muscle). The area of fibrosis was significantly increased in masseter muscle, but remained unchanged in the other two muscles. The number of apoptotic myocytes was significantly increased (approximately eightfold) in masseter muscle. These data suggest that persistent subclinical exposure to PG-LPS might reduce the size of fast-twitch skeletal muscle, but not slow-twitch skeletal muscle. Masseter muscle appears to be especially susceptible to the adverse effects of PG-LPS, because muscle remodeling (muscle fibrosis and myocyte apoptosis) was induced solely in masseter muscle. Thus, periodontitis might be one of the major causes of oral sarcopenia.

Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.

HSPA1A is upregulated in periodontal ligament at early stage of tooth movement in rats.

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction.