Safety and Clinical Evaluation of a Sonic Tongue Brush.

BACKGROUND: Tongue coating consists of oral bacteria, desquamated epithelium, blood cells, and food residues and is involved in periodontal disease, halitosis, and aspiration pneumonia. Recently, a tongue brush with sonic vibration was developed to clean the tongue. This comparative study examined the extent of tongue coating, its effects on the tongue, bacterial count particularly on the posterior dorsum of the tongue, and the degree of pain using a manual tongue brush and the newly developed sonic tongue brush. MATERIALS AND METHODS: Patients' extent of tongue coating and the quantity of bacteria were analysed before and after brushing with a sonic or manual nylon tongue brush. Moreover, the impressions of the dorsum linguae were obtained before and after brushing to establish models that were observed under a stereo microscope to evaluate tongue trauma. Pain caused during the use of these brushes was evaluated based on the numerical rating scale (NRS). RESULTS: The extent of tongue coating and number of bacteria decreased in both the sonic and manual nylon brush groups after tongue cleaning; however, no significant differences were noted. Tongue trauma evaluation revealed that the tongue surface was significantly scratched in the manual brush group compared with the sonic brush group. NRS-based pain evaluation revealed no significant differences. CONCLUSIONS: The sonic brush was equally effective in removing tongue coating and bacteria compared with the manual brush. As the sonic brush does not cause tongue trauma, it may be considered a safe and effective cleaning tool of the tongue.

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.

Synergistic effect of FGF-2 and TGF-ß1 on the mineralization of human umbilical cord perivascular cells.

OBJECTIVE: Human umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) on HUCPVC mineralization. DESIGN: We prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-ß1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. RESULTS: FGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. CONCLUSION: We found that FGF-2 synergizes with TGF-ß1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

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.

Effects of One-Stage Full-Mouth Scaling and Root Planing with Azithromycin on Diabetes and Periodontal Disease: A Randomized Controlled Trial.

Recent reports show that hemoglobin A1c (HbA1c) can be lowered by improving chronic inflammation in periodontal patients with diabetes mellitus and that full-mouth scaling and root planing (FM-SRP), in combination with azithromycin (AZM) treatment, can reduce early periodontal inflammation. However, the association of FM-SRP and AZM with periodontitis and HbA1c in patients with diabetes is largely unknown. This study investigated periodontitis and HbA1c in patients with diabetes after receiving FM-SRP and AZM to evaluate which clinical parameters most reflect the diabetic condition. Fifty-one periodontal patients with diabetes mellitus were included in this study. In total, 25 patients were assigned to the FM-SRP group in which patients were treated with FM-SRP in combination with AZM, and 26 patients were assigned to the control group in which only supragingival calculus removal was performed along with the provision of oral hygiene instructions. We evaluated periodontal parameters (probing pocket depth, periodontal inflamed surface area (PISA), bleeding on probing), and periodontal bacteria and biochemical parameters (HbA1c, high-sensitive C-reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1)) at baseline (BL) and 1, 3, 6, and 9 months after treatment. Compared with BL values, the FM-SRP group showed improved clinical parameters, reduced periodontal pathogens, and significantly lower HbA1c. Inflammatory cytokines (hs-CRP, TNF-α, IL-6) were significantly reduced one month after treatment and remained low thereafter. MCP-1 did not change significantly during the experimental period. PISA showed a strong correlation with HbA1c, hs-CRP, and TNF-α. FM-SRP, in combination with AZM, produced clinical, microbiological, and HbA1c improvements in periodontal patients with previously diagnosed diabetes mellitus. Additionally, PISA was shown to be a useful index for assessing the diabetic status of patients with periodontal 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.

Effect of Locally Delivered Minocycline on the Profile of Subgingival Bacterial Genera in Patients with Periodontitis: A Prospective Pilot Study.

This prospective pilot study aimed to evaluate the effect of minocycline-HCl ointment (MO), locally delivered as an adjunct to scaling and root planing (SRP), on subgingival microflora. A total of 59 periodontitis patients received SRP as an initial periodontal therapy. In the selected periodontal pockets with probing depths (PD) of 6−9 mm, the sites that exhibited a positive reaction following a bacterial test using an immunochromatographic device were subsequently treated with MO (SRP + MO group, n = 25). No additional treatment was performed at sites showing a negative reaction (SRP group, n = 34). In addition to subgingival plaque sampling, measurement of clinical parameters including PD, clinical attachment level (CAL), bleeding on probing (BOP), plaque index and gingival index (GI) were performed at baseline and 4 weeks after the initial periodontal therapy. The subgingival microflora were assessed by terminal restriction fragment-length polymorphism analysis. Relative to baseline values, the mean scores for PD-, CAL-, BOP-, and GI-sampled sites were significantly decreased post treatment in both groups (p < 0.01). The intra-comparisons showed a significant decrease in the counts of the genera Eubacterium, Parvimonas, Filifactor, Veillonella, Fusobacterium, Porphyromonas, Prevotella, and unknown species in the SRP + MO group (p < 0.05). Inter-comparisons indicated a significant decrease in the genera Veillonella in the SRP + MO group (p = 0.01). Combination therapy of SRP and local MO induced a change in the subgingival microbial community: particularly, the number of Veillonella spp. was markedly reduced.

Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells.

Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to mesenchymal stromal cells. Here, we present the results for the characterization of the properties alkaline phosphatase-positive HUCPVCs (ALP(+)-HUCPVCs). These ALP(+)-HUCPVCs were created from HUCPVCs in this study by culturing in the presence of activated vitamin D3, an inhibitor of bone morphogenetic protein signaling and transforming growth factor-beta1 (TGF-ß1). The morphological characteristics, cell proliferation, gene expression, and mineralization-inducing ability of ALP(+)-HUCPVCs were investigated at the morphological, biological, and genetic levels. ALP(+)-HUCPVCs possess high ALP gene expression and activity in cells and a slow rate of cell growth. The morphology of ALP(+)-HUCPVCs is fibroblast-like, with an increase in actin filaments containing alpha-smooth muscle actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-ß1 stimulation possess myofibroblast-like properties that have little mineralization-inducing ability.

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.

Effect of Nd: YAG laser irradiation to the temporomandibular joint on taste threshold.

BACKGROUND/PURPOSE: The tissue absorption laser has been clinically applied to alleviate pain in various areas. It is used for pain relief from temporomandibular disease (TMD) in dentistry. Although the facial and trigeminal nerves are distributed around the temporomandibular joint, the effects of laser irradiation and absorption on the neural functions have not been directly studied. In this study, the Nd:YAG laser was applied to an area where the facial nerve passes with photonic radiation for the treatment of TMD. MATERIALS AND METHODS: Ten volunteers including seven males and three females were selected as subjects. Nd:YAG laser was irradiated area included several internal and external standard and associated acupuncture points. The chorda tympani nerve, a branch of facial nerve is distributed to the front two thirds of the tongue and is associated with the sense of taste. We evaluated the effect of laser irradiation and absorption on the taste function by means of an electric taste meter. RESULTS: No significant difference was identified in the values between before and after laser irradiation (Wilcoxon signed-rank test). CONCLUSION: It was confirmed that there was no effect on taste function while applying Nd:YAG laser irradiation around the TMD joint.

Effects of Antimicrobial Photodynamic Therapy on Organic Solution and Root Surface In Vitro.

Antimicrobial photodynamic therapy (a-PDT) is attracting attention as a new form of dental treatment. While it is primarily applied to produce an antibacterial effect, it decreases lipopolysaccharide (LPS) and protease activity. Here, we evaluated differences in the antibacterial activity of a-PDT on three types of bacteria and the effects on the organic substances (i.e., albumin and LPS). Furthermore, we investigated the effects of a-PDT on root surfaces. A FotoSan630® and toluidine blue were used to perform a-PDT in this study. We measured its antimicrobial activity against Porphyromonas gingivalis, Streptococcus mutans, and Enterococcus faecalis. Antimicrobial testing revealed strong antimicrobial action and P. gingivalis, E. faecalis, and S. mutans were almost undetectable after 50, 120, and 100 s, respectively. In organic resolution tests, albumin was significantly decreased from 1 min after a-PDT application onward, while LPS significantly decreased at 5 min after the application. The root surfaces after a-PDT were confirmed to be cleaner than the controls without suffering any damage. Depending on the bacterial species, a-PDT exhibited antimicrobial activity against various types of bacteria and sensitivity differed. Moreover, we reported that a-PDT resolves protein and LPS, enabling the formation of a healthy root surface without any damage.

Effects of Conditioned Medium from Bone Marrow Cells on Human Umbilical Cord Perivascular Cells.

Mesenchymal cells derived from human umbilical cord tissue are attracting increasing attention as a source for cell therapy. However, for applying the same in tissue engineering, it has been shown that the differentiation capacity of mesenchymal stromal cells (MSCs) is influenced by the tissue from which the cells are harvested. Thus, to explore the possibility of increasing the osteogenic capacity of MSCs derived from the perivascular tissue of the human umbilical cord (human umbilical cord perivascular cells, HUCPVCs), we cultured these cells using conditioned medium (CM) derived from cultures of human bone marrow-derived mesenchymal stromal cells (hBMMSCs). However, hBM-CM contains a wide variety of growth factors, the amounts and ratios of which are considered to vary with the cell culture stage. Thus, we aimed to evaluate the effects of hBM-CM derived from different stages of hBMMSC culture on the osteogenic capacity of HUCPVCs. The stages of hBMMSC culture were defined as follows: Stage 1 (mitogenic stage) represented the period from the start of hBMMSC culture to 70% cell confluence; Stage 2 (confluent stage) represented the period from 70% confluence to the initiation of calcified nodule formation; and Stage 3 (calcification stage) represented the period following the initiation of calcified nodule formation. An analysis of growth factors contained in the CM obtained at each stage by enzyme-linked immunosorbent assay showed that insulin-like growth factor 1 (IGF-1) was significantly elevated at Stage 2, whereas vascular endothelial growth factor (VEGF) was significantly elevated at Stage 3. HUCPVCs were cultured using the CM from each of the stages for 1, 2, or 3 weeks. RUNX2 expression was the most upregulated at week 1 and then downregulated in all the groups. The expression of collagen 1 was significantly elevated in Stage 2 HUCs at week 3. Alkaline phosphatase (ALP) activity, ALP, and alizarin staining were higher in Stage 2 HUCs and Stage 3 HUCs. The calcium content was the highest in Stage 2 HUCs. The calcium content of HUCPVC obtained by the method used in this study was six times higher than that reported in the previous study. Collectively, our results show that the CM obtained at Stage 2 was most effective in driving the osteogenic differentiation of HUCPVCs. Impact Statement Mesenchymal stromal cells (MSCs) derived from the perivascular tissue of umbilical cords are promising candidates for regenerative medicine. Because these are able to be differentiated into bone cells, cartilage cells, and adipocytes. The number of MSCs in perivascular tissue (HUCPVCs) is ∼1/300 but the number of HUCPVCs that differentiates into osteogenic cells is quite low. In order to promote osteogenic differentiation of HUCPVCs, we cultured HUCPVCs using conditioned medium collected from human bone marrow-derived mesenchymal stromal cells. Our study suggests that the use of conditioned medium can be effective on inducing osteogenic differentiation of HUCPVCs.

Effect of Azithromycin on Proinflammatory Cytokine Production in Gingival Fibroblasts and the Remodeling of Periodontal Tissue.

Previous reports have shown that azithromycin (AZM), a macrolide antibiotic, affects collagen synthesis and cytokine production in human gingival fibroblasts (hGFs). However, there are few reports on the effect of AZM on human periodontal ligament fibroblasts (hPLFs). In the present study, we comparatively examined the effects of AZM on hGFs and hPLFs. We monitored the reaction of AZM under lipopolysaccharide (LPS) stimulation or no stimulation in hGFs and hPLFs. Gene expression analyses of interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), and Type 1 collagen were performed using reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, we performed Western blotting for the analysis of the intracellular signal transduction pathway. In response to LPS stimulation, the gene expression levels of IL-6 and IL-8 in hGFs increased due to AZM in a concentration-dependent manner, and phosphorylation of nuclear factor kappa B (NF-κB) was also promoted. Additionally, AZM caused an increase in MMP-1 expression in hGFs, whereas it did not affect the expression of any of the analyzed genes in hPLFs. Our findings indicate that AZM does not affect hPLFs and acts specifically on hGFs. Thus, AZM may increase the expression of IL-6 and IL-8 under LPS stimulation to modify the inflammatory response and increase the expression of MMP-1 to promote connective tissue remodeling.

Organic resolution function and effects of platinum nanoparticles on bacteria and organic matter.

Rapid progress has been made in terms of metal nanoparticles studied in numerous fields. Metal nanoparticles have also been used in medical research, and antibacterial properties and anticancer effects have been reported. However, the underlying mechanism responsible for these effects has not been fully elucidated. Therefore, the present study focused on platinum nanoparticles (PtNPs) and examined their antibacterial properties and functional potential for decomposing organic matter, considering potential applications in the dental field. PtNPs were allowed to react with dental-related bacteria (Streptococcus mutans; Enterococcus faecalis, caries; Porphyromonas gingivalis, and endodontic and periodontal lesions). Antibacterial properties were evaluated by measuring colony formation. In addition, PtNPs were allowed to react with albumin and lipopolysaccharides (LPSs), and the functional potential to decompose organic matter was evaluated. All evaluations were performed in vitro. Colony formation in all bacterial species was completely suppressed by PtNPs at concentrations of >5 ppm. The addition of PtNPs at concentrations of >10 ppm significantly increased fragmentation and decomposition. The addition of PtNPs at concentrations of >125 pico/mL to 1 EU/mL LPS resulted in significant amounts of decomposition and elimination. The results revealed that PtNPs had antibacterial effects against dental-related bacteria and proteolytic potential to decompose proteins and LPS, an inflammatory factor associated with periodontal disease. Therefore, the use and application of PtNPs in periodontal and endodontic treatment is considered promising.

Biological responses following one-stage full-mouth scaling and root planing with and without azithromycin: Multicenter randomized trial.

BACKGROUND AND OBJECTIVE: Full-mouth scaling and root planing (FM-SRP) increases the systemic levels of inflammatory mediators via early inflammation but may be inhibited using an antimicrobial agent. This prospective intervention study evaluates the biological response and clinical effects of FM-SRP with and without systemically administered azithromycin (AZM). MATERIALS AND METHODS: A multicenter parallel randomized controlled and open-label trial. A central randomization center used computer-generated tables to allocate treatments. Sixty-three patients with moderate to severe generalized periodontitis (New American Academy of Periodontology Classification: Stage3 or 4, Grade B) were randomly assigned to receive FM-SRP with AZM (test group, n = 32) or FM-SRP without AZM (control group, n = 31). Clinical parameters and body temperature were measured, and subgingival plaque, peripheral blood, and gingival crevicular fluid were collected before and after treatment. Periodontopathic bacteria and IgG titers were measured by gingival crevicular fluid and peripheral blood. High-sensitivity assays were used to analyze systemic and local inflammatory markers, such as endotoxin, high-sensitive CRP (hs-CRP), and six inflammatory cytokines. Follow-up 6 weeks. RESULTS: The total number of bacteria and the number of Porphyromonas gingivalis and Prevotella intermedia were significantly lower in the test group after FM-SRP. IgG titers for P gingivalis significantly decreased after FM-SRP with AZM, and the body temperature increased significantly after FM-SRP without AZM. In the control group, serum hs-CRP, IFN-γ, IL-12p70, and IL-6 were significantly increased one day after treatment, but subsequently decreased below the original numerical value. In the test group, only hs-CRP showed a significant increase. CONCLUSIONS: FM-SRP resulted in similar improvements in clinical parameters with and without the use of AZM. Inflammatory mediators showed no difference between the two groups after FM-SRP treatment. The use of AZM was effective in preventing the elevation of body temperature after FM-SRP.

Potential function of TGF-ß isoforms in maturation-stage ameloblasts.

OBJECTIVES: To investigate potential functions of transforming growth factor-beta (TGF-ß) isoforms in maturation-stage ameloblasts during amelogenesis. METHODS: In vivo activation of TGF-ß was characterized by using matrix metalloproteinase 20 null (Mmp20-/-) and wild-type (Mmp20+/+) mice. Using mHAT9d cells cultured in the presence of each TGF-ß isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope. RESULTS: TGF-ß1 and TGF-ß3 were present in the enamel matrix of developing teeth which were activated by MMP20 in vivo. A genetic study revealed that the three TGF-ß isoforms upregulate kallikrein 4 (KLK4) mRNA levels but downregulate carbonic anhydrase II. Moreover, TGF-ß1 and TGF-ß2 significantly upregulated the mRNA level of amelotin, whereas TGF-ß3 dramatically downregulated the mRNA levels of odontogenic ameloblast-associated protein (ODAM), family with sequence similarity 83 member H (FAM83H), and alkaline phosphatase (ALP). Immunostaining analysis showed that the apoptosis of mHAT9d cells is induced by three TGF-ß isoforms, with TGF-ß3 being most effective. Both TGF-ß1 and TGF-ß3 induced endocytosis of amelogenin. CONCLUSIONS: We propose that TGF-ß is regulated in an isoform-specific manner to perform multiple biological functions such as gene expression related to the structure of basal lamina/ameloblasts, mineral ion transport, apoptosis, and endocytosis in maturation-stage ameloblasts.

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.

The dynamics of TGF-ß in dental pulp, odontoblasts and dentin.

Transforming growth factor-beta (TGF-ß) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-ß in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-ß1 and TGF-ß3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-ß2 is high in dental pulp. TGF-ß1 is a major isoform of TGF-ß, and latent TGF-ß1, synthesized in dental pulp, is primarily activated by matrix metalloproteinase 11 (MMP11). Activated TGF-ß1 enhances the mRNA expression levels of MMP20 and full-length dentin sialophosphoprotein (DSPP) in dental pulp cells, coinciding with the induction of odontoblast differentiation. Latent TGF-ß1 synthesized in odontoblasts is primarily activated by MMP2 and MMP20 in both odontoblasts and dentin. The activity level of TGF-ß1 was reduced in the dentin of MMP20 null mice, although the amount of latent TGF-ß1 expression did not change between wild-type and MMP20 null mice. TGF-ß1 activity was reduced with the degradation of DSPP-derived proteins that occurs with ageing. We propose that to exert its multiple biological functions, TGF-ß1 is involved in a complicated dynamic interaction with matrix metalloproteinases (MMPs) and/or DSPP-derived proteins present in dental pulp, odontoblasts and dentin.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Interleukin-4 released from human gingival fibroblasts reduces osteoclastogenesis.

OBJECTIVE: Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DESIGN: Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. RESULTS: The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. CONCLUSION: We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.