Mitsuhiro Ohta

Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts

AbstractMany studies have attempted to elucidate the mechanism of the biostimulatory effects of low-level laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5–2-fold at 2–4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.

Effects of Caffeine on the Bones of Aged, Ovariectomized Rats

Caffeine is a substance which many people consume in their daily life. Caffeine’s effects on bone are still controversial. Using ovariectomized rats, the present study was conducted to determine to what extent caffeine intake affects the mechanical properties, bone minerals and histology. Aged rats were divided into 2 groups after ovariectomy. Group 1 was fed a 20% protein diet as a control, and group 2 was fed a 20% protein diet supplemented with caffeine (2 mg/100 g body weight). The respective diets were fed to the rats of each group for 90 days. Rats were then killed by heart puncture, blood was collected, and femurs were removed. In 1 group of femurs paraffin cross-sections were made at the midshaft of each bone. Total width, cortical width, total cross-sectional bone area of the midshaft, and the number of osteocytes in randomly selected areas were measured. Another group of bones was subjected to three-point bending testing until failure. Bones were then pulverized and Ca, P, Mg, Zn, Sr, Si, hydroxyproline and hexosamine contents and crystallite size were measured. Various mechanical properties, except modulus of elasticity, in the caffeine group were consistently 7–23% lower than the noncaffeine controls. Yield strain in the caffeine group was significantly less than in the noncaffeine controls. Zinc, Sr, and crystallite size of bone showed a significant decrease in the caffeine group, whereas Si contents significantly increased. Our current results indicate that routine intake of caffeine in the elderly should be regarded with some caution.

A Caffeine Diet Can Alter the Mechanical Properties of the Bones of Young Ovariectomized Rats

The general public widely consumes caffeine which is contained in various foods, beverages, and over-the-counter medications. The relationships between caffeine intake and bone fractures is controversial. Therefore, the aim of the current study was to determine what effects, if any, caffeine intake in early life exerts on mechanical properties and mineral contents of bone in growing ovariectomized rats. A total of 8 dams with pups were divided into two groups. Group 1 was fed a 20% protein diet. Group 2 was fed a 20% protein diet supplemented with caffeine (4 mg/100 g). The respective diets were fed to the dams during lactation and to the pups continuously after weaning on day 22 until the end of the experimental period. On day 32, offspring from both groups were ovariectomized. On day 52, the rats were sacrificed and the femora removed. The biomechanical properties of the femora were determined by three-point bend testing to failure at a rate of 2 mm/min, with continuous data sampling at 10 samples/s. The properties determined included the modulus of elasticity, yield load, yield stress, ultimate load, ultimate stress, and the second moment of area. The caffeine group exhibited a decrease in the various mechanical properties (ranging from approximately 7 to 20%), except for yield strain and moment of inertia. The decreases in maximum stress and elastic modulus values were significant. Calcium, magnesium, and phosphorous values for the caffeine group were significantly decreased. These results suggest that the bone in the caffeine group is weaker and less stiff, with greater deformation under applied loading. It could be concluded that caffeine intake during the early growing period affects the mechanical properties of bone.

Pemphigus Vulgaris Confined to the Gingiva: A Case Report

Pemphigus Vulgaris (PV) is an autoimmune intraepithelial blistering disease involving the skin and mucous membranes. Oral mucosa is frequently affected in patients with PV, and oral lesions may be the first sign of the disease in majority of patients. In some patients, oral lesions may also be followed by skin involvement. Therefore, timely recognition and therapy of oral lesions is critical as it may prevent skin involvement. Early oral lesions of PV are, however, often regarded as difficult to diagnose, since the initial oral lesions may be relatively nonspecific, manifesting as superficial erosions or ulcerations, and rarely presenting with the formation of intact bullae. Lesions may occur anywhere on the oral mucosa including gingiva; however; desquamtive gingivitis is less common with PV than other mucocutaneous conditions such as pemphigoid or lichen planus. This paper describes the case of a patient presenting with a one-year history of painful gingival, who is finally diagnosed as having PV.

Anti-inflammatory activities of light emitting diode irradiation on collagen-induced arthritis in mice (a secondary publication).

BACKGROUND AND AIMS Rheumatoid arthritis (RA) is an auto-immune disease afflicting multiple joints of the body, where as a result of the increase in inflammatory cytokines and tissue destructive factors such as matrix metalloproteinase (MMP)-3, deterioration of the bones and cartilages of the joints occurs. The present investigation was carried out to study the anti-inflammatory activities of light emitting diode (LED) irradiation on hind paw inflammation in collagen-induced arthritis (CIA) mice models. MATERIALS AND METHOD RA in the CIA mouse model was induced by immunization of DBA/1J mice with intradermal injections of an emulsion of bovine type II collagen and complete Freunds adjuvant. A total of 20 CIA mice were subdivided into the following groups: control group, CIA group and 2 groups of LED irradiated CIA mice (LED groups) (n=5 per group). The mouse knee joint area in the LED groups (the 570 nm and 940 nm groups) was irradiated with LED energy, three times a week for 500 s per session over 8 weeks at a dose of 5 J/cm(2). The hind paw swelling was assessed by the increase in hind paw thickness. The serum levels of the inflammatory cytokines and arthritic factor MMP-3 were determined with an enzyme-linked immunosorbent assay (ELISA). RESULTS In the LED-570 and LED-940 groups at 4 weeks after arthritis induction, the swelling inhibition index was 18.1±4.9 and 29.3±4.0 respectively. Interleukin (IL)-1β, IL-6 and MMP-3 serum levels were significantly lower in the LED-940 group. CONCLUSIONS LED irradiation, particularly in the near-infrared was effective for inhibition of the inflammatory reactions caused by RA.

Genome science-based gene expression monitoring in osteoblasts altered by low-level laser irradiation

Abstract Although the biostimulatory effects of low-level laser irradiation (LLLI) have been reported, the molecular basis of the mechanisms are not fully elucidated. To elucidate the mechanism of bone regeneration by low-level laser irradiation (LLLI), gene expressions altered in osteoblast by LLLI were identified using the subtractive gene cloning and cDNA microarray technologies. Many gene expressions in osteoblast were altered by LLLI. Many genes whose expression levels were altered by LLLI were shown, including ATP synthesis, DNA replication, signal transduction, enzymes, structural proteins, ESTs and other unknown function genes. These findings suggest that LLLI may demonstrate biostimulatory effects through the expression of many genes, and genome science-based gene expression monitoring will provide unprecedented access to elucidate the mechanism of bone regeneration stimulated by LLLI.

FENS Program for Nutrition Education in Medical Schools

Kurt Widhalma, Austria Beatriz Miranda-da-Cruza, Austria Jan Pokorny, Czech Republic Inge Tetens, Denmark Suvi M. Virtanen, Finland Daniel Lemonnier, France Helmut Oberritter, Germany Antonia Trichopoulou, Greece Brian McKenna, Ireland Nino Battistini, Italy Sigrid Bergea, Norway A. Gronowska-Senger, Poland M.D. Vaz de Almeida, Portugal Flora Correa, Portugal Bergona Olmedilla, Spain Lars H. Ellegard, Sweden U. Keller, Switzerland P.J.F. Vries, The Netherlands Christine A. Edwards, United Kingdom

Characterization of Rat Monoclonal Antibodies against Human β-Defensin-2

Defensins are a family of cationic antimicrobial peptides that participate in host defense. Human β-defensin (hBD)-2 has a potent bactericidal activity against a wide spectrum of microorganisms. Because human gingival epithelium is constantly exposed to a variety of microbial challenges, it is considered that hBD-2 has an important role in the protective mechanisms against oral bacterial infection. However, little is known about the production of hBD-2 in tissues of the oral cavity. Six rat monoclonal antibodies (MAbs) raised against chemically synthesized hBD-2 have been characterized. Rat MAbs were specific for the conformational epitopes on hBD-2, but not to hBD-1. To identify the epitope on hBD-2, a series of six overlapping peptides covering the hBD-2 whole sequence were synthesized and the immunoreactivities of six MAbs were examined. The FCPRRYK domain in hBD-2 was recognized by all six MAbs and suggested to be an epitope region. By immunocytochemistry, hBD-2 was localized focally in the epidermis ...

Isolation and Identification Methods for Slackia Exigua and Investigation of the Relationship between this Organism and Peri-Implantitis

In the genus Slackia, Slackia exigua (formerly Eubacterium exiguum) is isolated from the human oral cavity. A relationship was recently reported between S. exigua and periodontal disease, including chronic periodontitis and peri-implantitis. A suitable selective medium for the isolation of S. exigua is needed in order to assess the veritable prevalence of this organism in various lesions of the oral cavity. The purpose of the present study was to develop selective media for the isolation of S. exigua and investigate whether the monitoring of S. exigua levels is useful as a clinical indicator for the diagnosis of peri-implantitis. In order to examine the bacterium population in the oral cavity, a novel selective medium (SEXSM) was herein developed for the isolation of S. exigua. SEXSM consists of tryptic soy agar, yeast extract, hemin, Vitamin K1, L-cysteine, sheep blood, 2-phenylethanol, trimethoprim, nalidixic acid, and polymyxin B. The average growth recovery of S. exigua on SEXSM was 98.3% that on CDC blood agar. The growth of other representative oral bacteria, i.e., the genera Streptococcus, Actinomyces, Neisseria, Corynebacterium, Veillonella, Fusobacterium, and Rothia, was strongly inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of S. exigua. These primers reacted with S. exigua, but not with other representative oral bacteria. These results indicate that these primers are useful for identifying S. exigua. The proportion of S. exigua in Gingival Crevicular Fluids (GCF) collected from periodontally Healthy with Implants (HI) and Peri-Implantitis (PI) groups was examined. Colonies on SEXSM were subcultured for confirmation by a PCR analysis using the primers designed in the present study. The growth recoveries of S. exigua strains on SEXSM were very satisfactory. S. exigua in GCFs of the HI and PI groups were detected at 0.002%, and 1.7% to the total bacteria number, respectively. The selective medium, designated SEXSM, and a PCR method using the primers designed in the present study were useful for the isolation and identification of S. exigua. Moreover, the monitoring of S. exigua levels was useful as a clinical indicator for the diagnosis of periimplantitis.

Primer Design for the Identification of Ten Oral Actinomyces Species Using Multiplex PCR

Background: Actinomyces is one of the predominant genera in the oral cavity. Oral Actinomyces species play a central role in the initial stages of biofilm formation on teeth; however, limited information is currently available on the distribution of individual species in different sites or clinical conditions. Moreover, a suitable method has yet to be developed to identify oral Actinomyces species because of the phenotypic and genetic similarities between these microorganisms. Objective: Actinomyces naeslundii, A. odontolyticus, A. oris, A. georgiae, A. gerencseriae, A. graevenitzii, A. dentalis, A. johnsonii, A. israelii, and A. meyeri among the genus Actinomyces are regarded as normal human oral Actinomyces species. The purpose of the present study was to design primers to identify oral Actinomyces species using multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of the representative oral Actinomyces species. The 16S rDNA genes of the representative oral Actinomyces species were obtained from the DNA Data Bank of Japan, and a multiple sequence alignment analysis was performed with the CLUSTAL W program. Homology among the primers selected for oral Actinomyces species and their respective 16S rRNA sequences was confirmed by a BLAST search. Results: These primers were able to distinguish each oral Actinomyces species and did not display cross-reactivity with representative oral bacteria or other Actinomyces species. Moreover, we developed a multiplex PCR method with the ability to identify and differentiate oral Actinomyces species (i.e., A. naeslundii, A. johnsonii, A. oris, A. odontolyticus, A. israelii, A. georgiae, A. dentalis, A. graevenitzii, A. gerencseriae, and A. meyeri) using only two PCR tubes per sample. Conclusion: The present results indicate that our multiplex PCR method with these primers is useful for identifying the representative oral Actinomyces species. This method is easy because the use of Mighty Amp DNA Polymerase Ver.2 (Takara) means that DNA extraction may be avoided, and species identification using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of oral Actinomyces species and their involvement in oral infections to be fully clarified in future studies.