Influence of osteoporosis and mechanical loading on bone around osseointegrated dental implants: A rodent study.

This study aimed to evaluate the influence of estrogen deficiency and mechanical loading on bone around osseointegrated dental implants in a rat jaw model. The maxillary right first molars of 36 rats were extracted. One week later, the rats were divided into an unloaded group and a loaded group; short head implants and long head implants were inserted respectively. Nine weeks after implantation, the rats were further subjected to ovariectomy (OVX) or sham surgery. All animals were euthanized 21 weeks after OVX. Micro-computed tomography, histological and histomorphometrical evaluation were undertaken. Systemic bone mineral density and bone volume fraction decreased in OVX groups compared with the sham controls. Histomorphometrical observation indicated that unloaded OVX group showed significantly damaged osseointegration and bone loss versus the loaded OVX group. Both the bone density (BD) inside the peri-implant grooves and the percentage of bone-to-implant contact (BIC) were lower in the OVX groups than in the sham-surgery groups, although mechanical loading increased the BIC and BD in the loaded OVX group compared with the unloaded OVX group. An increased number of positive cells for tartrate-resistant acid phosphatase was observed in the OVX groups versus the sham controls. The percentage of sclerostin-positive osteocytes was lower under loaded compared with unloaded conditions in both the OVX groups and the sham controls. In conclusion, estrogen deficiency could be a risk factor for the long-term stability of osseointegrated implants, while mechanical loading could attenuate the negative influence of estrogen deficiency on bone formation and osseointegration.

The Significance of Posterior Occlusal Support of Teeth and Removable Prostheses in Oral Functions and Standing Motion.

The purpose of this study was to evaluate the effect of posterior occlusal support of natural teeth and artificial teeth on oral functions and standing motion. Patients who had been treated with removable prostheses were enrolled as the subjects. Their systemic conditions (body mass index (BMI) and skeletal muscle mass index (SMI)) were recorded. The subjects were classified into two groups according to a modified Eichner index: B1-3 (with posterior occlusal support) and B4C (without posterior occlusal support). Maximum occlusal force (MOF), masticatory performance (MP), and standing motion (sway and strength) were evaluated for cases with and without removable prostheses. There were no significant differences in BMI and SMI between the B1-3 group and the B4C group. The subjects with removable prostheses demonstrated significantly higher values in MOF, MP, and sway and strength than the subjects without removable prostheses. The comparison of oral functions between the B1-3 group and the B4C group revealed that the positive effect of posterior occlusal support of natural teeth and removable prostheses and the significant positive effects of posterior occlusal support on standing motion were partly observed in these comparisons. Posterior occlusal support of natural teeth and even of removable prostheses may contribute to the enhancement of oral functions and standing motion.

Production of cloned transgenic silkworms by breeding non-diapausing parthenogenetic strains.

Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.

Effect of a Single Injection of Benidipine-Impregnated Biodegradable Microcarriers on Bone and Gingival Healing at the Tooth Extraction Socket.

Objective: A dihydropyridine-type calcium channel blocker, benidipine (BD), is extensively used in hypertension therapy. In vitro study reported BD promoting bone metabolism. We evaluated the effect of sustained release of BD-loaded poly(lactic-co-glycolic acid) (PLGA) microcarriers on the promotion of bone and gingival healing at an extraction socket in vivo. In addition, the effect of BD on osteoblasts, osteocytes, fibroblasts, and epithelial cells was evaluated in vitro. Approach: The maxillary first molar of rats was extracted. Next, PLGA microcarriers containing BD were directly injected into the gingivobuccal fold as a single dose. After injection, bone and soft-tissue healing was histologically evaluated. Effect of BD on proliferation, migration, and gene expression of gingival and bone cell was also examined in vitro. Results: After tooth extraction, BD significantly augmented bone volume and density, and also epithelial wound healing. During in vitro studies, BD promoted significant proliferation and migration of fibroblasts and epithelial cells. Real-time RT-PCR revealed that BD upregulated messenger RNA expression of Ahsg (alpha 2-HS glycoprotein) and Csf2 (colony-stimulating factor 2) in osteoblasts. Innovation: The prevention of bone and soft-tissue reduction associated with tooth extraction has been eagerly anticipated in the field of dentistry. This study first reported the effect of BD on extraction socket healing. Conclusion: A single dose of topically administered BD-loaded PLGA microcarriers promoted bone and soft-tissue healing at the extraction site of tooth.

Mechanical loading induced osteocyte apoptosis and connexin 43 expression in three-dimensional cell culture and dental implant model.

Osteocytes are thought to act as stress sensors, and are known to display a gap junction-mediated stress-transfer mechanism. To demonstrate the stress-related function of osteocytes, cells of an osteocyte-like cell line derived from murine long bone osteocyte Y4 (MLO-Y4) were cultivated in a three-dimensional culture and subjected to cyclic loading from a titanium plate. This application of physiological loading using a titanium plate significantly increased connexin 43 (Cx43) expression, the number of dead and apoptotic cells, and receptor activator of nuclear factor κB ligand expression. Furthermore, the conditioned medium from the loaded osteocytes induced alkaline phosphatase activity in bone marrow cell culture. In addition, we immunohistologically determined whether bone metabolism increased as a result of the occlusal force in the bone surrounding the titanium implants in a rat model. Increased Cx43 expression and apoptotic osteocytes were observed in the loading group as well as a significantly increased number of tartrate-resistant acid phosphatase-positive cells. These findings indicate that stress from the implant adversely affected the osteocytes, which may promote osteoclastic and osteoblastic cell formation around the implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 815-827, 2019.

Evaluation of pyrogallol-induced cytotoxicity in catalase-mutant escherichia coli and mutagenicity in Salmonella typhimurium.

We evaluated pyrogallol cytotoxicity using Escherichia coli strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b)) and wild-type (Cs(a)), and pyrogallol mutagenicity by Ames test. Pyrogallol was more toxic to Cs(b) rather than to Cs(a) (p < 0.05), while catalase, superoxide dismutase and ascorbic acid decrease the toxic effect. Pyrogallol also showed mutagenic effect (mutagenic index = 3.8 for 10 micromol pyrogallol/plate) while ascorbic acid (19.4% reduction, p < 0.001) and naringin (35.1% reduction, p < 0.001) played a protective role against it. Pyrogallol cytotoxicity and mutagenicity seem to be attributable, at least in part, to reactive oxygen species formation. This study also suggests that newly established catalase mutant E. coli is probably useful in hazard identification of oxidative chemicals.

Development of a new assay for evaluation of l-3,4-dihydroxyphenylalanine cytotoxicity in catalase-mutant Escherichia coli.

The present study aimed to assess whether a newly constructed, catalase-deficient Escherichia coli strain that express mammalian catalase gene could be used to identify oxidative stress-generating chemicals. We tested l-3,4-dihydroxyphenylalanine (l-DOPA), a well-known agent that induces reactive oxygen species. We found that l-DOPA exposure reduced the survival of catalase-mutant E. coli in a dose-dependent manner, especially in the strains with lower catalase activities, implying the usefulness of these strains in assessment of oxidative chemicals.

Effects of mask fitness and worker education on the prevention of occupational dust exposure.

To decrease the incidence of pneumoconiosis, we examined dust protective mask performance and its relation to pulmonary function as well as the effects of worker education on the proper wearing of masks. One hundred and seventy-eight workers from 15 factories subject to dust exposure participated in this study. All participants were interviewed to obtain relevant personal information and underwent both a mask leakage and a pulmonary function test. The mask leakage was expressed as a percentage, with under 10% leakage indicating that the dust protective mask worked efficiently. In addition, 23 workers from 2 factories were educated on how to wear masks properly. The average mask leakage was 24.3%, and 58% of workers wore ineffective masks. Though pulmonary function was almost normal, the percent vital capacity (%VC) tended to be lower depending on the mask leakage. Mask education, which was very easy and took only a short time, dramatically decreased average mask leakage from 32.1% to 10.5% (p0.001). Educating workers to wear masks properly might prevent the worsening of pulmonary function in response to dust exposure. Appropriate mask fitness by education could be useful in preventing the development of pneumoconiosis.

Cytotoxicity of lawsone and cytoprotective activity of antioxidants in catalase mutant Escherichia coli.

Lawsone is an active naphthoquinone derivative isolated from henna (Lawsonia inermis L.), a widely used hair dye. Previous study on the toxicity of lawsone remains unclear since the involvement of oxidative stress and the kind of ROS (reactive oxygen species) involved have not been fully resolved yet. This present study reports the cytotoxic effects of lawsone and henna. We carried out CAT assay (a zone of inhibition test of bacterial growth and colony-forming efficiency test of transformant Escherichia coli strains that express mammalian catalase gene derived from normal catalase mice (Cs(a)) and catalase-deficient mutant mice (Cs(b))), Ames mutagenicity assay and H(2)O(2) generation assay. Lawsone generated H(2)O(2) slightly in phosphate buffer system and was not mutagenic in Ames assay using TA 98, TA 100 and TA 102, both in the absence and presence of metabolic activation. Lawsone exposure inhibited the growth of both Cs(a) and Cs(b) strains in a dose-dependent manner. Mean zone diameter for Cs(a) was 9.75+/-0.96 mm and 12.75+/-1.5 mm for Cs(b). Natural henna leaves did not show toxic effects, whereas two out of four samples of marketed henna products were shown toxicity effects. Catalase abolished zone of inhibition (ZOI) of marketed henna products, eliminated ZOI of lawsone in a dose-dependent manner and low concentration of exogenous MnSOD and Cu/ZnSOD eliminated the toxicity. Histidine and DTPA, the metal chelator; BHA and low concentration of capsaicin, the inducer of NADH-quinone reductase, effectively protected Cs(a) and Cs(b) against lawsone in this study. We suggest that lawsone cytotoxicity is probably mediated, at least in part, by the release of O(2)(-), H(2)O(2) and OH(-).

Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori.

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.

Practicality of Veterans Specific Activity Questionnaire in evaluation of exercise capacity of community-dwelling Japanese elderly.

OBJECTIVE: The aim of this study is to determine whether a questionnaire-based method using the Veterans Specific Activity Questionnaire (VSAQ) is a practical tool for the development of a safe exercise program to prevent a reduction in physical performance. METHODS: One hundred and twenty-one senior residents of Yakage, Okayama, agreed to voluntarily participate in this study. They were asked to complete a questionnaire for information on age, sex, subjective health status, exercise habits and VSAQ. We investigated the relationship between age and exercise capacity predicted by VSAQ (predicted metabolic equivalents (METs)). In addition, for 36 out of the 121 participants, we performed a 6-min walk distance test (6MD) and investigated whether its results correlate with the predicted METs. Furthermore, we prepared a modified VSAQ and examined its practicality in the evaluation of the exercise capacity of Japanese elderly (n=50). RESULTS: We found that the predicted METs correlate well with age. Habitual exercise and subjective health status did not affect the predicted METs. A significant correlation was observed between the predicted METs and the results of 6MD (r=0.56, p<0.001). We also found that certain activities included in the original VSAQ are unfamiliar to Japanese elderly; thus, we made a few modifications to the original VSAQ in order to evaluate the physical fitness of Japanese elderly. The number of inadequate answers was reduced by employing the modified VSAQ. CONCLUSION: These findings imply that the modified VSAQ is useful in evaluating the exercise capacity of Japanese elderly adequately and is a practical scale for safe exercise.

Application of artificial insemination technique to eupyrene and/or apyrene sperm in Bombyx mori.

The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33 degrees C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm.

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm, Bombyx mori.

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.