Alteration of tooth movement by reveromycin A in osteoprotegerin-deficient mice.

INTRODUCTION: Osteoprotegerin-deficient mice develop severe high-turnover osteoporosis with porous low-density trabecular bone from an age-related increase in osteoclast activity and are useful alveolar bone models of osteoporosis or frail periodontal tissue. Bisphosphonate (BP), a first-line drug for osteoporosis, is bone-avid, causing side effects such as brittle and fragile bones and jaw osteonecrosis after tooth extraction. In orthodontics, active movement is precisely controlled by temporarily suppressing and resuming movement. BP impedes such control because of its long half-life of several years in bone. Therefore, we investigated the novel osteoclast-specific inhibitor reveromycin A (RMA), which has a short half-life in bone. We hypothesized that tooth movement could be precisely controlled through temporary discontinuation and re-administration of RMA. METHODS: Osteoprotegerin-deficient mice and wild-type mice were developed as tooth movement models under constant orthodontic force. A constant orthodontic force of 10 g was induced using a nickel-titanium closed coil spring to move the maxillary first molar for 14 days. We administered BP (1.25 mg/kg) or RMA (1.0 mg/kg) continuously and then discontinued it to reveal how the subsequent movement of teeth and surrounding alveolar bone was affected. RESULTS: Continuous BP or RMA administration suppressed osteoclast activity and preserved alveolar bone around the roots, apparently normalizing bone metabolism. Tooth movement remained suppressed after BP discontinuation but resumed at a higher rate after discontinuation of RMA. CONCLUSIONS: RMA appears useful for controlling orthodontic tooth movement because it can be suppressed and resumed through administration and discontinuation, respectively.

Novel Osteogenic Behaviors around Hydrophilic and Radical-Free 4-META/MMA-TBB: Implications of an Osseointegrating Bone Cement.

Poly(methyl methacrylate) (PMMA)-based bone cement, which is widely used to affix orthopedic metallic implants, is considered bio-tolerant but lacks osteoconductivity and is cytotoxic. Implant loosening and toxic complications are significant and recognized problems. Here we devised two strategies to improve PMMA-based bone cement: (1) adding 4-methacryloyloxylethyl trimellitate anhydride (4-META) to MMA monomer to render it hydrophilic; and (2) using tri-n-butyl borane (TBB) as a polymerization initiator instead of benzoyl peroxide (BPO) to reduce free radical production. Rat bone marrow-derived osteoblasts were cultured on PMMA-BPO, common bone cement ingredients, and 4-META/MMA-TBB, newly formulated ingredients. After 24 h of incubation, more cells survived on 4-META/MMA-TBB than on PMMA-BPO. The mineralized area was 20-times greater on 4-META/MMA-TBB than PMMA-BPO at the later culture stage and was accompanied by upregulated osteogenic gene expression. The strength of bone-to-cement integration in rat femurs was 4- and 7-times greater for 4-META/MMA-TBB than PMMA-BPO during early- and late-stage healing, respectively. MicroCT and histomorphometric analyses revealed contact osteogenesis exclusively around 4-META/MMA-TBB, with minimal soft tissue interposition. Hydrophilicity of 4-META/MMA-TBB was sustained for 24 h, particularly under wet conditions, whereas PMMA-BPO was hydrophobic immediately after mixing and was unaffected by time or condition. Electron spin resonance (ESR) spectroscopy revealed that the free radical production for 4-META/MMA-TBB was 1/10 to 1/20 that of PMMA-BPO within 24 h, and the substantial difference persisted for at least 10 days. The compromised ability of PMMA-BPO in recruiting cells was substantially alleviated by adding free radical-scavenging amino-acid N-acetyl cysteine (NAC) into the material, whereas adding NAC did not affect the ability of 4-META/MMA-TBB. These results suggest that 4-META/MMA-TBB shows significantly reduced cytotoxicity compared to PMMA-BPO and induces osteoconductivity due to uniquely created hydrophilic and radical-free interface. Further pre-clinical and clinical validations are warranted.

Biological and Physicochemical Characteristics of 2 Different Hydrophilic Surfaces Created by Saline-Storage and Ultraviolet Treatment.

OBJECTIVES: Hydrophilicity/hydrophobicity of titanium surfaces may affect osseointegration. Ordinary titanium surfaces are hydrophobic. Recently, 2 different methods of storing titanium in saline solution or treating it with ultraviolet (UV) light were introduced to generate surface hydrophilicity. This study compared biological and physicochemical properties of 2 different hydrophilic titanium surfaces created by these methods. MATERIALS: Acid-etched control, saline-stored, and UV-treated titanium surfaces were assessed by scanning electron microscopy, energy dispersive spectroscopy, and x-ray photoelectron spectroscopy. The attachment, spreading behaviors, mineralization, and gene expression of osteoblasts were examined. RESULTS: Similar microroughness was found on control and UV-treated surfaces, whereas foreign deposits were observed on saline-stored surfaces. Control and UV-treated surfaces consisted of Ti, O, and C, whereas saline-stored surfaces showed Na and Cl in addition to these 3 elements. Atomic percentage of surface carbon was higher in order of control, saline-stored, and UV-treated surfaces. Osteoblasts cultured on saline-stored surfaces showed higher levels of calcium deposition and collagen I expression than control. Osteoblasts on UV-treated surfaces showed significantly increased levels for all parameters related to cell attachment, cell spreading, the expression of adhesion and cytoskeletal proteins, mineralization, and gene expression compared with control, outperforming saline-stored surfaces for most parameters. CONCLUSION: Despite similar hydrophilicity, saline-stored and UV light-treated surfaces showed substantially different biological effects on osseointegration, associated with different surface chemistry and morphology.

Reveromycin A Administration Prevents Alveolar Bone Loss in Osteoprotegerin Knockout Mice with Periodontal Disease.

Chronic periodontal disease is characterized by alveolar bone loss and inflammatory changes. Reveromycin A (RMA) was recently developed and is a unique agent for inhibiting osteoclast activity. This study analysed the effects of RMA in an experimental mouse model of periodontitis involving osteoprotegerin (OPG)-knockout mice, specifically, whether it could control osteoclasts and reduce inflammation in periodontal tissue. We examined wild-type (WT) and OPG knockout mice (OPG KO) ligated with wire around contact points on the left first and second molars. RMA was administered twice a day to half of the mice. Using micro-computed tomography, we measured the volume of alveolar bone loss between the first and second molars, and also performed histological analysis. The OPG KO RMA+ group had significantly decreased osteoclast counts, alveolar bone loss, attachment loss, and inflammatory cytokine expression 8 weeks after ligation. Thus, RMA may reduce alveolar bone loss and inflamed periodontal tissues in patients with periodontitis.

Effect of UV-photofunctionalization on oral bacterial attachment and biofilm formation to titanium implant material.

Bacterial biofilm infections remain prevalent reasons for implant failure. Dental implant placement occurs in the oral environment, which harbors a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part of the implant structure is exposed to oral cavity and there is no effective measure to prevent bacterial attachment to implant materials. Here, we demonstrated that UV treatment of titanium immediately prior to use (photofunctionalization) affects the ability of human polymicrobial oral biofilm communities to colonize in the presence of salivary and blood components. UV-treatment of machined titanium transformed the surface from hydrophobic to superhydrophilic. UV-treated surfaces exhibited a significant reduction in bacterial attachment as well as subsequent biofilm formation compared to untreated ones, even though overall bacterial viability was not affected. The function of reducing bacterial colonization was maintained on UV-treated titanium that had been stored in a liquid environment before use. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed. Our findings confirm that UV-photofunctionalization of titanium has a strong potential to improve outcome of implant placement by creating and maintaining antimicrobial surfaces.

Lipid and fatty acid composition of mesocarp and seed of avocado fruits harvested at northern range in Japan.

The lipid and fatty acid composition of the mesocarp and seed of avocado fruit grown and harvested in Japan, which is located at the northern range of the avacado, was investigated and compared to an imported avocado purchased commercially. The potential of the avocado mesocarp as an agricultural product in Japan was also explored. Total lipids (TL) accounted for approximately 20% of the mesocarp. Further analysis showed that the neutral lipid (NL) fraction accounted for at least 95% of the TL, and almost 90% of NL was triacylglycerol. Monoenoic acids accounted for at least 65% of the total fatty acids, and oleic acid, which is regarded as an especially important functional component of avacado accounted for approximately 50% of the monounsaturated fatty acids. A comparison of the Japanese avocado cultivars and an imported avocado cultivar in the present study revealed no significant differences in the lipid and fatty acid compositions. Therefore, production of avocado fruit, which is rich in various nutritional components, is expected to be increased on a larger number of farms in Japan in the future. It is believed to be necessary to carry out further verification, such as the establishment of a cultivation technique adoptable to Japan, examination of optimal soil and land features, and cultivar selection.

Isolation of GnafC, a polysaccharide constituent of Gnaphalium affine, and synergistic effects of GnafC and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells.

After screening extensively factors in plant extracts that increase alkaline phosphatase activity, an osteoblastic differentiation marker protein in mouse calvarial osteoblast MC3T3-E1 cells, GnafC derived from Gnaphalium affine, was found to significantly enhance the alkaline phosphatase (ALPase) activity in a synergistic manner with ascorbate. GnafC was a polysaccharaide with an approximate molecular mass of 10,000 and comprised mannose, xylose, arabinose, galactose and glucose in a molar ratio of 1:2:4.3:2.5:2.7. Expression of the osteoblastic differentiation marker genes was examined by semiquantitative RT-PCR with RNAs prepared from cells at different developmental stages. With ascorbate in the culture, GnafC enhanced the expression of the ALPase and MMP13 genes from the early stage of differentiation, leading to maturation of the collagenous extracellular matrix (ECM), a prerequisite for mineralization.

Sphingosine 1-phosphate induces the production of glial cell line-derived neurotrophic factor and cellular proliferation in astrocytes.

Sphingosine 1-phosphate (S1P) is a platelet-derived bioactive sphingolipid that evokes a variety of biological responses. To understand the role of S1P in the central nervous system, we have examined the effect of S1P on the production of glial cell line-derived neurotrophic factor (GDNF) and growth regulation of cortical astrocytes from rat embryo. Moreover, we examined the possibility that the expression of GDNF is regulated differently in cultured astrocytes from the stroke-prone spontaneously hypertensive rat (SHRSP) than in those from Wistar kyoto rats (WKY). The mRNA expression was quantitated by RT-PCR based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme-linked immunosorbent assay. S1P induced the expression of GDNF mRNA and the production of GDNF protein in a dose-dependent manner in WKY astrocytes. Moreover, S1P increased cell numbers and induced the proliferation of astrocytes. In addition, the level of mRNA expression and protein production of GDNF was significantly lower in SHRSP than WKY astrocytes following exposure to S1P. These findings revealed that S1P augments GDNF protein production and cellular growth in astrocytes. Also, our results indicate that production in SHRSP astrocytes was attenuated in response to S1P compared with that observed in WKY. We conclude that S1P specifically triggers a cascade of events that regulate the production of GDNF and cell growth in astrocytes. Our results also suggest that the reduced expression of GDNF caused by S1P is a factor in the stroke proneness of SHRSP.

Black tea polyphenols, theaflavins, prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.

Tea polyphenols have been demonstrated as chemopreventive agents in a number of experimental models. However, less is known about the mechanism of chemoprevention by black tea compared with that of green tea. Some beneficial properties of theaflavins, the black tea polyphenols, were investigated in the present study. Theaflavins showed inhibitory effects on H(2)O(2)- and tert-butyl hydroperoxide (tBuOOH)-induced cytotoxicity (evaluated by tetrazolium bromide reduction), cellular oxidative stress (detected by oxidation of 2', 7'-dichlorofluorescin), and DNA damage (measured by amount of 8-OHdG and comet assay) in rat normal liver epithelium cell RL-34 cell lines. In addition, theaflavins also exhibited suppression of cytochrome P450 1A1 induced by omeprazole in the human hepatoma HepG2 cell line. Furthermore, when HepG2 cells were pretreated with omeprazole to induce CYP1A1, then exposed to benzo[a]pyrene (B[a]P), DNA damage was observed using the comet assay. However, theaflavins could inhibit this DNA damage. These results indicated that theaflavins could prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.