Toshihide Noguchi

Gene Expression of Osteoclast Differentiation Factor Is Induced by Lipopolysaccharide in Mouse Osteoblasts Via Toll-Like Receptors

Osteoclast differentiation factor (ODF), a recently identified cytokine of the TNF family, is expressed as a membrane-associated protein in osteoblasts and stromal cells. ODF stimulates the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here we investigated the effects of LPS on the gene expression of ODF in mouse osteoblasts and an osteoblast cell line and found that LPS increased the ODF mRNA level. A specific inhibitor of extracellular signal-regulated kinase or protein kinase C inhibited this up-regulation, indicating that extracellular signal-regulated kinase and protein kinase C activation was involved. A protein synthesis inhibitor, cycloheximide, rather enhanced the LPS-mediated increase of ODF mRNA, and both a neutralizing Ab of TNF-α and a specific inhibitor of PGE synthesis failed to block the ODF mRNA increase by native LPS. Thus, LPS directly induced ODF mRNA. Mouse osteoblasts and an osteoblast cell line constitutively expressed Toll-like receptor (TLR) 2 and 4, which are known as putative LPS receptors. ODF mRNA increases in response to synthetic lipid A were defective in primary osteoblasts from C3H/HeJ mice that contain a nonfunctional mutation in the TLR4 gene, suggesting that TLR4 plays an essential role in the process. Altogether, our results indicate that ODF gene expression is directly increased in osteoblasts by LPS treatment via TLR, and this pathway may play an important role in the pathogenesis of LPS-mediated bone disorders, such as periodontitis.

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Periodontal tissue regeneration using fibroblast growth factor -2:Randomized controlled phase II clinical trial

Background The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ≥3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 µL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. Conclusions Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. Trial Registration ClinicalTrials.gov NCT00514657

MyD88 But Not TRIF Is Essential for Osteoclastogenesis Induced by Lipopolysaccharide, Diacyl Lipopeptide, and IL-1α

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Tyrosine phosphorylation is crucial for growth signaling by tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2)

[3H]Thymidine (TdR) incorporation by human osteosarcoma cell line MG‐63 was significantly stimulated at as early as 3 h after the addition of either TIMP‐1 or TIMP‐2 alone. Maximum stimulation was attained at a concentration of either 20 ng/ml (0.71 nM) TIMP‐1 or 1.0 ng/ml (46 pM) TIMP‐2. Tyrosine kinase inhibitors such as genistein, erbstatin, and herbimycin A almost completely inhibited the [3H]TdR incorporation stimulated by either of the TIMPs. However, essentially no effect was observed with H‐89, H‐7, bisindolylmaleimide and K‐252a. These inhibition studies suggest a crucial role for tyrosine kinase in the signal transduction of TIMPs. Phosphotyrosine‐containing proteins were significantly elevated by the treatment with both TIMPs. We also found that either TIMP stimulated an increase in mitogen‐activated protein (MAP) kinase activity, suggesting that MAP kinase plays a role in TIMP‐dependent growth signaling.

Low Metacarpal Bone Density, Tooth Loss, and Periodontal Disease in Japanese Women

The relationship between periodontitis and systemic bone mineral density in Japanese women is undetermined. We tested the hypothesis that periodontitis was more frequent in women with low metacarpal bone mineral density (m-BMD). Subjects were 190 Japanese women (89 premenopausal, 101 post-menopausal). Periodontal status was evaluated according to the Community Periodontal Index of Treatment Need (CPITN). M-BMD was measured by computed x-ray densitometry. The proportion of subjects with periodontitis (CPITN ≥ 3) increased as m-BMD decreased in pre-menopausal (18.2%, 36.9%, and 66.6% in the normal, borderline, and very low m-BMD groups, p < 0.02) and post-menopausal women (41.5%, 54.8%, 60%, and 68.4% in the normal, borderline, low, and very low m-BMD groups, p < 0.05). Among post-menopausal women, those with very low m-BMD had fewer teeth present than women with normal m-BMD (19.9 ± 7.2 vs. 25.1 ± 4.1, p < 0.01). These results indicate that m-BMD loss is associated with periodontitis in Japanese women, and with tooth loss after menopause.

Immortalization of Cementoblast Progenitor Cells With Bmi‐1 and TERT

A cementoblast progenitor cell line designated BCPb8 was successfully isolated from dental follicle cells immortalized with Bmi‐1 and hTERT. BCPb8 showed the potential to differentiate into cementoblasts on implantation into immunodeficient mice. BCPb8 was confirmed to be the first established cementoblast progenitor cell line and will provide a useful model for investigating cementogenesis.

Efficacy of Periodontal Disease and Tooth Loss to Screen for Low Bone Mineral Density in Japanese Women

The relationship between oral indicators and bone mineral density (BMD) has been studied by many investigators, with mixed and complex results. The purpose of the present cross-sectional study was to evaluate the associations of periodontal conditions and tooth loss with metacarpal BMD (m-BMD) in a community-based cohort and the usefulness of tooth count as a potential screening tool to detect low BMD. Subjects were 356 Japanese women (171 premenopausal, mean age 37.9 ± 8.0 years; 185 postmenopausal, mean age 63.3 ± 7.7 years). Periodontal status was evaluated by the Community Periodontal Index of Treatment Needs (CPITN). m-BMD was measured by computerized X-ray densitometry. The proportion of subjects with periodontitis (CPITN 3 or 4) increased as m-BMD decreased. The odds ratio (OR) of osteopenia or osteoporosis in relation to periodontitis was 3.2 (95% confidence interval [CI], 2.0–5.3). After adjustment for age and menopausal status, the OR was 2.0 (95% CI, 1.1–3.7). Among postmenopausal women, those having fewer than 20 teeth were 1.6 times more likely to have low m-BMD than those having more than 20 teeth (chi-square for trend in postmenopausal group, 4.27; P < 0.05). Receiver-operating curve (ROC) analysis indicated that number of teeth remaining or CPITN score had a greater than 50/50 chance to correctly identify women with osteoporosis or osteopenia, but the areas under the curve (0.72 and 0.67, respectively) are considered less than highly accurate screening tools. These results indicate that periodontitis and tooth loss after menopause may be useful indicators of m-BMD loss in Japanese women.

Lineage‐committed osteoclast precursors circulate in blood and settle down into bone

Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle–arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF‐κB (RANK)‐positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK‐positive cells expressed c‐Fms (receptors of macrophage colony‐stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK‐positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein‐2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP‐2–induced ectopic bone were bromodeoxyuridine‐negative. RANK‐positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)‐positive macrophages. When BMP disks and control disks were implanted in RANK ligand‐deficient mice, RANK‐positive cells were observed in the BMP disks but not in the controls. F4/80‐positive cells were distributed in both disks. Administration of FYT720, a sphingosine 1‐phosphate agonist, promoted the egress of RANK‐positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage‐determined QOPs circulate in the blood and settle in the bone.

Multi-center intervention study on glycohemoglobin (HbA1c) and serum, high-sensitivity CRP (hs-CRP) after local anti-infectious periodontal treatment in type 2 diabetic patients with periodontal disease

The purpose of this study was to examine whether periodontal treatment incorporating topical antibiotic therapy affects on levels of glycohemoglobin (HbA1c) and serum high-sensitivity C-reactive protein (hs-CRP) in type 2 diabetic patients with periodontal disease, and to explore the relationship between CRP and glycemic control. The whole intervention group (n=32), which underwent anti-infectious periodontal treatment, showed only transient reduction in HbA1c levels without any change in hs-CRP, while the control group (n=17) did not show any changes in HbA1c or hs-CRP. Multiple regression analysis of all subjects revealed that BMI and change in hs-CRP correlated significantly with the reduction of HbA1c at 6 months after the periodontal treatment. Based on the results of multiple regression analysis, the intervention group was subdivided into two groups: those in which hs-CRP levels decreased (CRP-D group), and those in which hs-CRP levels unchanged or increased (CRP-N group) (n=16, respectively), and re-analysis was conducted based upon these subgroups. In the CRP-D subgroup, HbA1c was significantly reduced at the end of the study, but it did not decrease in the CRP-N subgroup. The decrease of HbA1c in the CRP-D subgroup following periodontal treatment was significantly greater than that in the CRP-N subgroup. BMI of each group remained unchanged in this study at the end of the study. Thus, the results suggested that periodontal treatment with topical antibiotics improves HbA1c through reduction of CRP, which may relate to amelioration of insulin resistance, in type 2 diabetic patients with periodontal disease.