Anti-Inflammatory Effects of ß-Cryptoxanthin on 5-Fluorouracil-Induced Cytokine Expression in Human Oral Mucosal Keratinocytes.

Oral mucositis is a typical adverse effect of chemotherapy, causing oral pain that significantly reduces the patient's quality of life. ß-cryptoxanthin (ß-cry) is a carotenoid abundant in citrus fruits with antioxidant and anti-inflammatory effects. However, the ß-cry effect on oral mucositis remains unclear. We investigated the effects of 5-fluorouracil (5-FU) and ß-cry on human normal oral mucosal keratinocytes (hOMK). hOMK was seeded on a culture plate and cultured with 5-FU and ß-cry. The cell number, mRNA expression of inflammatory cytokines and matrix metalloproteinases (MMPs), and production of inflammatory cytokines in hOMK were evaluated. Additionally, the cell count and inflammatory cytokine production were analyzed when hOMK was co-stimulated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in addition to 5-FU. The numbers of hOMK significantly reduced with 5-FU stimulation, whereas it increased with ß-cry treatment. mRNA expression of interleukin (IL)-6, IL-8, metalloproteinase (MMP)-2, and MMP-9 and protein production of IL-6 and IL-8 in hOMK were augmented on 5-FU stimulation. Simultaneously, ß-cry treatment significantly suppressed IL-8 and MMP-9 mRNA expression, and IL-8 production was induced on 5-FU stimulation. Co-stimulation with P. gingivalis LPS and 5-FU enhanced IL-6 and IL-8 production in hOMK. ß-cry could enhance cell proliferation and suppress 5-FU-induced expression of inflammatory cytokines and MMP in hOMK. Thus, ß-cry can alleviate the symptoms of chemotherapy-induced oral mucositis, and its combination with oral care is effective in managing oral mucositis.

Effects of mechanical stress on human oral mucosa-derived cells.

OBJECTIVES: Placement of a denture results in the application of mechanical stress (MS), such as occlusal force, onto the oral mucosa beneath the denture. To better understand the molecular mechanism underlying MS-induced inflammation in the oral mucosa, we examined the impact of MS on human oral epithelial cells (HO-1-N-1) and human fibroblasts (HGFs) in this study. MATERIALS AND METHODS: MS was applied on HO-1-N-1 and HGFs using a hydrostatic pressure apparatus. The expression and production of inflammatory cytokines and growth factors were examined by real-time RT-PCR and ELISA. MS-induced intracellular signal transduction via MAP kinase (MAPK) was also examined. RESULTS: 1 MPa MS resulted in a significant increase in inflammatory cytokines, and 3 MPa MS resulted in a significant increase in FGF-2. MS also increased p-38 phosphorylation and the addition of a p-38 inhibitor significantly suppressed the production of inflammatory cytokines. DISCUSSION: Our study suggested that MS applied through a denture increases the production of inflammatory cytokines from oral mucosal epithelial cells and fibroblasts via the p38 MAPK cascade. These responses to MS likely lead to inflammation of the mucosal tissue beneath dentures. On other hand, up-regulation of growth factors is likely a manifestation of the biological defense mechanism against excessive MS.

Association between Helicobacter pylori infection and dental pulp reservoirs in Japanese adults.

BACKGROUND: Helicobacter pylori (H. pylori) colonize the stomach and are considered an etiological agent of gastric cancer. The oral cavity is a transmission route to the stomach, but the exact site of colonization has not yet been explicated. Our study investigated the association between H. pylori infection and presence in oral samples. METHODS: Dental pulp, supragingival plaque, and saliva from 192 patients visiting the Dentistry's outpatient clinic were collected for testing. The H. pylori ureA gene was identified via Nested PCR. Urine anti-H. pylori antibody test was utilized to detect infection. RESULTS: Twenty-five subjects were found to be antibody-positive. PCR analysis of dental pulp revealed that 23 subjects possessed the ureA gene. Twenty-one subjects were positive for both antibodies and genes in dental pulp. PCR testing revealed that 2 subjects were positive in dental plaque but negative for saliva. The subjects positive for H. pylori in dental pulp expressed clinical signs of severe dental caries. CONCLUSIONS: H. pylori infected subjects expressed H. pylori in samples from the oral cavity. The main reservoir for infection within the oral cavity was determined to be dental pulp. Moreover, H. pylori are likely transmitted from dental caries to the root canal.

Transplantation of dental pulp-derived cell sheets cultured on human amniotic membrane induced to differentiate into bone.

OBJECTIVE: The usefulness of the amniotic membrane as a cell culture substrate has led to its use in the development of dental pulp-derived cell sheets. We induced osteoblastic differentiation of dental pulp-derived cell sheets and conducted histological and immunological examinations in addition to imaging assessments for regeneration of bone defects. METHODS: Dental pulp cells were obtained by primary culture of the dental pulp tissue harvested from extracted wisdom teeth. These cells were maintained for three to four passages. Subsequently, the dental pulp cells were seeded onto an amniotic membrane to produce dental pulp-derived cell sheets. Following the induction of osteoblastic differentiation, the sheets were grafted into the subcutaneous tissue of the lower back and maxillary bone defect of a nude mouse. Histological and immunological examinations of both grafts were performed. RESULTS: Dental pulp-derived cell sheets cultured on an osteoblast differentiation-inducing medium demonstrated resemblance to dental pulp tissue and produced calcified tissue. Mineralization was maintained following grafting of the sheets. Regeneration of the maxillary bone defect was observed. CONCLUSION: Induction of osteoblastic differentiation of the dental pulp-derived cell sheets may be indicated for the regeneration of periodontal tissue.

Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration.

Transplantation of engineered three-dimensional (3D) bone tissue may provide therapeutic benefits to patients with various bone diseases. To achieve this goal, appropriate 3D scaffolds and cells are required. In the present study, we devised a novel nanogel tectonic material for artificial 3D scaffold, namely the nanogel-cross-linked porous (NanoCliP)-freeze-dried (FD) gel, and estimated its potential as a 3D scaffold for bone tissue engineering. As the osteoblasts, directly converted osteoblasts (dOBs) were used, because a large number of highly functional osteoblasts could be induced from fibroblasts that can be collected from patients with a minimally invasive procedure. The NanoCliP-FD gel was highly porous, and fibronectin coating of the gel allowed efficient adhesion of the dOBs, so that the cells occupied the almost entire surface of the walls of the pores after culturing for 7 days. The dOBs massively produced calcified bone matrix, and the culture could be continued for at least 28 days. The NanoCliP-FD gel with dOBs remarkably promoted bone regeneration in vivo after having been grafted to bone defect lesions that were artificially created in mice. The present findings suggest that the combination of the NanoCliP-FD gel and dOBs may provide a feasible therapeutic modality for bone diseases.

Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein.

Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc.

Biomechanical force induces the growth factor production in human periodontal ligament-derived cells.

Although many reports have been published on the functional roles of periodontal ligament (PDL) cells, the mechanisms involved in the maintenance and homeostasis of PDL have not been determined. We investigated the effects of biomechanical force on growth factor production, phosphorylation of MAPKs, and intracellular transduction pathways for growth factor production in human periodontal ligament (hPDL) cells using MAPK inhibitors. hPDL cells were exposed to mechanical force (6 MPa) using a hydrostatic pressure apparatus. The levels of growth factor mRNA and protein were examined by real-time RT-PCR and ELISA. The phosphorylation of MAPKs was measured using BD™ CBA Flex Set. In addition, MAPKs inhibitors were used to identify specific signal transduction pathways. Application of biomechanical force (equivalent to occlusal force) increased the synthesis of VEGF-A, FGF-2, and NGF. The application of biomechanical force increased the expression levels of phosphorylated ERK and p38, but not of JNK. Furthermore, the levels of VEGF-A and NGF expression were suppressed by ERK or p38 inhibitor. The growth factors induced by biomechanical force may play a role in the mechanisms of homeostasis of PDL.

Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways.

Effect of glycemic control on periodontitis in type 2 diabetic patients with periodontal disease.

AIMS/INTRODUCTION: Diabetes mellitus and periodontitis are closely related. A huge number of reports has addressed the effect of periodontal intervention therapy on glycemic control, but no reports have addressed the effect of glycemic intervention therapy on periodontal disease in type 2 diabetic patients. The aim of this study was to examine the effect of improved glycemic control by glycemic intervention therapy on periodontitis in type 2 diabetic patients. MATERIALS AND METHODS: A total of 35 patients underwent intervention therapy to improve glycemic control without periodontal treatment. Glycohemoglobin (HbA1c), high-sensitivity C-reactive protein (hs-CRP), bleeding on probing (BOP), probing pocket depth (PPD) and intraoral community periodontal index (CPI) codes of the World health Organization (WHO) were examined at baseline, and 2 and 6 months after the intervention therapy to improve glycemic control. RESULTS: After the improvement of glycemic control, BOP lesions improved, but deep PPD lesions and WHO CPI codes did not improve. Subanalyses showed that effective glycemic control (average HbA1c reduction 1.8%) improved BOP lesions, but did not affect deep PPD lesions and WHO CPI codes. In addition, high BOP lesions at baseline responded more effectively to glycemic intervention. Further analysis of CPI codes in all individual periodontal sites independent of WHO CPI codes in 35 patients showed that only gingival inflammation without a deep periodontal pocket improved after glycemic intervention. CONCLUSIONS: Effective glycemic control improves BOP lesions in type 2 diabetic patients with periodontitis through ameliorating inflammation at the gingival sites of periodontal tissue. This trial was registered with the University Hospital Medical Information Network (no. UMIN000007670).

ß-cryptoxanthin regulates bone resorption related-cytokine production in human periodontal ligament cells.

OBJECTIVE: ß-cryptoxanthin (ß-cry) is a type of carotenoid found in certain fruits and vegetables. Although it has been shown that ß-cry inhibits alveolar bone resorption, the molecular mechanisms for this have not yet been clarified. In the present study, we investigated the effects of ß-cry on bone resorption related-cytokine production in human periodontal ligament (hPDL) cells. DESIGN: hPDL cells were stimulated with ß-cry (1×10(-7)mol/l), mechanical stress (1 or 6MPa), and P. gingivalis. The production of interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by RT-PCR and ELISA. RESULTS: The production of IL-1ß, IL-6, IL-8, and TNF-α was not induced in hPDL cells after stimulation with ß-cry, although these cytokines were produced after stimulation with P. gingivalis. On the other hand, IL-6 and IL-8 were produced after exposure to 6MPa of mechanical stress. The production of IL-6 and IL-8 was significantly decreased by the addition of ß-cry. Furthermore, ß-cry up-regulated the production of OPG, but not RANKL. CONCLUSION: ß-cry inhibited the production of IL-6 and IL-8 induced by mechanical stress and periodontopathogenic bacteria in hPDL cells. Moreover, ß-cry up-regulated OPG production. These results suggest that ß-cry may prevent bone resorption in periodontitis.

Porphyromonas gingivalis induces myocarditis and/or myocardial infarction in mice and IL-17A is involved in pathogenesis of these diseases.

OBJECTIVES: Although an association between periodontitis and cardiovascular diseases has been suggested, the role of Porphyromonas gingivalis in cardiovascular diseases is not clear. In this study, we examined whether experimental bacteremia of P. gingivalis causes cardiovascular diseases and investigated the mechanism of pathogenesis of cardiovascular diseases induced by P. gingivalis. DESIGN: C57BL/6 mice were intravenously inoculated with 2.0 × 10(8)CFU of P. gingivalis A7436 strain. Mice were sacrificed at specified days and their hearts were collected. The collected organs were divided into two halves and used for histological evaluation and cytokine analysis. IL-17A(-/-), IFN-γ(-/-) and TNF-α(-/-) mice were also intravenously inoculated and the histological changes of hearts in mice were examined. RESULTS: Myocarditis and/or myocardial infarction were observed in mice injected with P. gingivalis. The levels of IL1-ß, IL-6, IL-17A, IL-18, TNF-α and IFN-γ mRNA increased significantly after P. gingivalis injection. In particular, high levels of IL-17A and IFN-γ mRNA expression were observed in hearts of mice after P. gingivalis injection in comparison with these levels before injection. Furthermore, the production of IL-17A was detected in hearts of wild-type mice after P. gingivalis injection. In wild-type, TNF-α(-/-) and IFN-γ(-/-) mice, moderate infiltration of neutrophils and monocytes was observed in hearts at 5 days after injection. In contrast, no inflammatory findings were observed in hearts of IL-17A(-/-) mice. CONCLUSION: We have demonstrated that an experimental bacteremia of P. gingivalis could induce myocarditis and/or myocardial infarction in mice, and IL-17A plays an important role in the pathogenesis of these diseases.

Mechanical stress enhances production of cytokines in human periodontal ligament cells induced by Porphyromonas gingivalis.

OBJECTIVE: We have previously reported that human periodontal ligament (hPDL) cells produced many kinds of cytokines as a result of bacterial stimulation, including stimulation with Porphyromonas gingivalis (P. gingivalis). However, the effects of mechanical stress on cytokine production in hPDL cells stimulated by periodontopathogenic bacteria are not clearly understood. In this study, we investigated the effects of mechanical stress on the production of inflammatory cytokines in hPDL cells induced by stimulation with P. gingivalis. METHODS: The hPDL cells were exposed to various levels of mechanical stress (1, 6, 10 and 50MPa) and costimulated with mechanical stress and P. gingivalis for 24h. Cytokine mRNA expressions were determined by RT-PCR. Cytokines in the culture supernatant were assessed by ELISA, and morphologic changes in hPDL cells were observed. RESULTS: The expressions of interleukin (IL)-6, IL-8 and tumor necrosis factor-α mRNA were observed in hPDL cells after exposure to mechanical stress. Moreover, the production of IL-6 and IL-8 increased significantly after exposure to mechanical stress ranging from 1 to 10MPa. The amount of IL-8 in the culture supernatants of hPDL cells costimulated with P. gingivalis and mechanical stress was significantly higher than the expected additive amount. The morphology of hPDL cells did not change after exposure to 6MPa, but these cells were partly detached from the Petri dish after exposure to 50MPa. CONCLUSIONS: These results suggest that local inflammation of the periodontal ligament may be induced mainly by periodontal bacteria, and mechanical stress may promote local inflammation.

Reduced masticatory function in non-elderly obese Japanese adults.

OBJECTIVES: Abnormal eating behaviors such as compulsive overeating, eating fast, chewing less, palatable soft food preferences and avoiding hard food are often observed in obese individuals, and these behaviors may affect their masticatory function, but little information of masticatory function in obese subjects are available at present. The present study investigated masticatory function in non-elderly obese Japanese adults and explored the relationships between obesity and masticatory function. METHODS: Seventy-five obese subjects (BMI ≥ 25; male: 34, female: 41) and 98 subjects with normal weight (BMI 18.5-25; male: 63, female: 35) aged 25-40 years old were enrolled in the present study. The status of masticatory function was determined using a chewing gum mixing method, a direct method of examining masticatory function, and the numbers of present teeth, untreated decayed teeth, missing teeth, and filled teeth were also examined. RESULTS: Masticatory function was significantly lower in the obese subjects both in male and female, whereas the numbers of present teeth, decayed teeth, missing teeth and filled teeth did not differ significantly between the obese subjects and the controls both in male and female. Multiple regression analysis revealed a significant correlation between obesity and reduced masticatory function after adjustment for gender, age, and numbers of decayed teeth, missing teeth, and filled teeth. CONCLUSIONS: Significantly reduced masticatory function was found in male and female non-elderly obese adults based on direct measurement of masticatory function. Multiple regression analysis suggested that obesity might induce reduced masticatory function.

IL-17 is involved in bone resorption in mouse periapical lesions.

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-gamma(-/-), TNF-alpha(-/-), and IL-17A(-/-) mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by muCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-gamma(-/-), and TNF-alpha(-/-) mice after infection with P. intermedia and P. gingivalis. However, periapical lesions were not observed in IL-17A(-/-) mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-gamma or TNF-alpha, plays an important role in the formation of periapical lesions.