Yuichi Mine

Impact of titanium ions on osteoblast-, osteoclast-and gingival epithelial-like cells

PURPOSE To investigate the effects of titanium (Ti) ions on the cell viability, the cell differentiation and the gene expressions related to bone resorption including Receptor Activator of NF-kappaB Ligand (RANKL) and Osteoprotegerin (OPG) in the tissues around dental implants, the osteoblast-, osteoclast-, and gingival epithelial-like cells were exposed to Ti ions. METHODS An MTS assay was carried out to evaluate the viabilities of osteoblast-like MC3T3-E1, osteoclast-like RAW264.7 and epithelial cell-like GE-1 cells. The gene expressions in these cells were analyzed by the use of RT-PCR and real-time quantitative RT-PCR. RESULTS Ti ions in the concentration range 1-9 ppm had little effect on the viabilities of MC3T3-E1, RAW264.7 and GE-1, whereas 20 ppm Ti ions significantly decreased the viabilities of all cells. Analyses of RT-PCR and real-time quantitative RT-PCR data revealed that Ti ions at 9 ppm remarkably inhibited the expressions of Runx2, Osterix and type I collagen in MC3T3-E1. In RAW264.7, Ti ions showed no effects on the levels of mRNAs for TRAP and cathepsin K enhanced by RANKL. Ti ions at the range of 1-9 ppm showed no effects on the levels of mRNAs for RANKL and OPG in GE-1, while Ti ions at 9 ppm enhanced the expression of these genes in MC3T3-E1. CONCLUSIONS These results, taken together, suggested that Ti ions show the biological effects, both on the viabilities of osteoblast and osteoclast and on the differentiation of either the osteoblastic or osteoclastic cells, which may influence the prognosis of dental implants.

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three‐dimensional (3D) clinostat system simulating microgravity to culture MC3T3‐E1 and RAW264.7 cells. Long‐term exposure (7 days) of MC3T3‐E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen αI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of β3‐integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long‐term 3D clinostat exposure inhibits the differentiation of MC3T3‐E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL‐dependent cellular fusion of RAW264.7 cells, suggesting that long‐term mechanical unloading suppresses bone formation and resorption.

Titanium ion induces necrosis and sensitivity to lipopolysaccharide in gingival epithelial-like cells.

Gingival epithelial-like cells (GE-1) were cultured and used to examine the cellular responses of gingival tissues to varying concentrations of titanium (Ti) ions. Titanium ions at concentrations of more than 13 ppm significantly decreased the viability of GE-1 cells and increased LDH release from the cells into the supernatant, but had no significant effect on their caspase 3 activity. These data suggest that a high concentration of Ti ions induced necrosis of the GE-1 cells. Titanium ions at a concentration of 5 ppm significantly increased the level of CCL2 mRNA expression in GE-1 cells exposed to lipopolysaccharide derived from Porphyromonas gingivalis in a synergistic manner. Moreover, the mRNA expression levels of TLR-4 and ICAM-1 in GE-1 cells loaded with Ti ions at 9 ppm were significantly enhanced as compared with those in GE-1 cells without Ti stimulation. We suggest that Ti ions are in part responsible for monocyte infiltration in the oral cavity by elevating the sensitivity of gingival epithelial cells to microorganisms. Taken together, these data indicate that Ti ions may be involved in cytotoxicity and inflammation at the interfaces of dental implants and gingival tissue.

Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell–cell fusion during RANKL-mediated osteoclastogenesis

To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and β2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.

Involvement of ERK and p38 MAPK pathways on interleukin-33-induced RANKL expression in osteoblastic cells

The receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) system is a well‐known key factor in osteoclast differentiation, and osteoblastic lineage cells are the major sources of RANKL and OPG in local bone tissue. Recently, a new molecule from the interleukin (IL)‐1 family, IL‐33, was identified. Here, we report the possible involvement of IL‐33 in RANKL and OPG expression, and the signaling pathways that are required for maximal IL‐33‐induced RANKL expression in MC3T3‐E1 osteoblastic cells. Stimulation with IL‐33 increased the mRNA expression and secretion of RANKL in MC3T3‐E1 cells. The IL‐33‐induced RANKL mRNA expression was inhibited by an anti‐IL‐33 monoclonal antibody. Furthermore, ERK and p38 MAPK inhibitors, but not a JNK inhibitor, suppressed IL‐33‐induced RANKL mRNA expression. On the other hand, IL‐33 had no effect on OPG mRNA expression and protein secretion. These results taken together suggest that IL‐33 stimulates RANKL expression through mechanisms dependent on the ERK and p38 MAPK pathways in MC3T3‐E1 cells.

Titanium Immobilized with an Antimicrobial Peptide Derived from Histatin Accelerates the Differentiation of Osteoblastic Cell Line, MC3T3-E1

The objective of this study was to evaluate the effect of titanium immobilized with a cationic antimicrobial peptide (JH8194) derived from histatin on the biofilm formation of Porphyromonas gingivalis and differentiation of osteoblastic cells (MC3T3-E1). The titanium specimens (Ti) were immobilized with JH8194, according to the method previously described. The colonization of P. gingivalis on JH8194-Ti was significantly lower than that on control- and blocking-Ti. JH8194-Ti enhanced the mRNA expressions of Runx2 and OPN, and ALPase activity in the MC3T3-E1, as compared with those of control- and blocking-Ti. These results, taken together, suggested the possibility that JH8194-Ti may be a potential aid to shorten the period of acquiring osseointegration.

Evaluation of trabecular bone formation in a canine model surrounding a dental implant fixture immobilized with an antimicrobial peptide derived from histatin.

JH8194 induces osteoblast differentiation, although it was originally designed to improve antifungal activity. This suggests that JH8194 is useful for implant treatment. Therefore, the aim of this study was to evaluate the osseointegration capacity of JH8194-modified titanium dental implant fixtures (JH8194-Fi). The implants were randomly implanted into the edentulous ridge of dog mandibles. Healing abutments were inserted immediately after implant placement. Three weeks later, peri-implant bone levels, the first bone-to-implant contact points, and trabecular bone formation surrounding the implants were assessed by histological and digital image analyses based on microcomputed tomography (microCT). The histological analysis revealed an enhancement of mature trabecular bone around the JH8194-Fi compared with untreated fixtures (control-Fi). Similarly, microCT combined with analysis by Zed View™ also showed increased trabecular bone formation surrounding the JH8194-Fi compared with the control-Fi (Student’s t-test, P < 0.05). JH8194 may offer an alternative biological modification of titanium surfaces to enhance trabecular bone formation around dental implants, which may contribute to the transient acquirement of osseointegration and the long-term success of implant therapy.

Bovine milk fermented with Lactobacillus rhamnosus L8020 decreases the oral carriage of mutans streptococci and the burden of periodontal pathogens

AIM   The aim of this study was to find the oral isolate of lactobacilli, which has the potential to inhibit either periodontal, cariogenic, or fungal pathogens in vitro, and to examine the effects of bovine milk fermented with the isolate on the oral carriage of cariogenic and periodontal pathogens. METHODS   The inhibitory effects of the supernatant of Man-Rogosa-Sharpe broth, in which each of 42 oral isolates of lactobacilli grown, was examined. One isolate, Lactobacillus rhamnosus L8020, that showed the potential to inhibit either periodontal, cariogenic, or fungal pathogens in vitro, was used to examine the effects of fermented milk on the oral carriage of cariogenic and periodontal pathogens, which was examined by a placebo-controlled and cohort trial using 50 participants. RESULTS   Edible yogurt containing Lactobacillus rhamnosus L8020 significantly reduced the oral carriage of mutans streptococci (P < 0.01) and four periodontal pathogens examined: Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Fusobacterium spp. (P < 0.01), but the phenomenon were not observed with the placebo yogurt (P > 0.05). CONCLUSION   These results suggest that yogurt with Lactobacillus rhamnosus L8020 could reduce the risk of dental caries and periodontal disease.

Inhibition of RANKL-dependent cellular fusion in pre-osteoclasts by amiloride and a NHE10-specific monoclonal antibody

The functions of Na+/H+ exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL‐stimulated control cells, amiloride decreased the number of large TRAP‐positive osteoclast cells (OCs) with ≥10 nuclei and increased the number of small TRAP‐positive OCs with ≤10 nuclei during sRANKL‐dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose‐ and time‐dependent manners. NHEs 1–9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti‐mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP‐positive OCs, but increased the number of small TRAP‐positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti‐NHE10 MAb prevented sRANKL‐promoted cellular fusion. The anti‐NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy.