Masayuki Morishita

Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions

OBJECTIVES The purpose of this study was to evaluate the antibacterial effect of silver-zeolite (SZ) against oral bacteria under anaerobic conditions. METHODS The antibacterial activity of SZ was evaluated by determining the minimum inhibitory concentrations (MICs) using two-fold serial dilutions of SZ in Brain Heart Infusion broth. Release of Ag+ into the broth was measured by an atomic absorption technique. RESULTS SZ inhibited the growth of the bacteria tested under anaerobic conditions. The MIC of SZ ranged between 256 and 2048 micrograms/ml, which corresponded to a range of 4.8-38.4 micrograms/ml of Ag+. All strains grew in broth containing 16,384 micrograms/ml of type-A zeolite. SIGNIFICANCE These results suggested that SZ may be a useful vehicle to provide antibacterial activity to dental materials used even under anaerobic conditions such as deep in the periodontal pocket.

Correlation Between the Protease Activities and the Number of Epithelial Cells in Human Saliva

The positive significant correlation (r = 0.788, n = 37) was shown between the protease activity and the number of epithelial cells (not of leukocytes) in saliva. There was little protease activity observed in glandular saliva. It is possible to conclude from the results that a main source of salivary protease is the epithelial cells in saliva. It was also found that the oral prophylaxis reduced the numbers of epithelial cells and leukocytes and the protease activity.

The effects of oestrogen on osteocalcin production by human periodontal ligament cells

The purpose was to investigate the effects of oestradiol on the function of periodontal ligament (PDL) cells by measuring the production of osteocalcin in vitro. Cells were obtained from the healthy periodontal ligament of teeth extracted from two males and two females for orthodontic reasons. Serum-free medium was used when testing the effects of oestradiol on PDL cells. The amount of osteocalcin in the culture medium was analysed by two-step sandwich enzyme immunoassay in the presence or absence of oestradiol. It was shown that oestradiol enhanced the production of osteocalcin by PDL cells in a time- and dose-dependent manner. PDL cells obtained from both male and female donors were affected by oestradiol. It thus appears that oestradiol is one of the factors important for PDL cells to express their function.

Expression of Syndecan-2, -4, and Fibroblast Growth Factor Receptor Type 1 in Human Periodontal Ligament Fibroblasts and Down-regulation of These Membrane Proteins during Maturation in Culture

Syndecans are transmembrane heparan sulfate proteoglycans. They are known to interact with basic fibroblast growth factor (bFGF), and it has been suggested that they play important roles in the growth, morphology, and migration of a variety of cell types. We examined the expression of syndecans and fibroblast growth factor receptor type 1 (FGFR1) in periodontal ligament (PDL) cells, because these membrane proteins may play roles in the control of growth and differentiation during regeneration of PDL. Reverse-transcription/polymerase chain-reaction (RT-PCR) showed that PDL cells expressed syndecan-2 and -4 mRNAs. This was confirmed by sequence analysis of the PCR products. When PDL cells were maintained for 25 days, alkaline phosphatase (ALPase) activity gradually increased and reached a maximal level on day 20. Northern blotting analysis showed that PDL cells expressed 2.3-kb syndecan-2, 2.6-kb syndecan-4, and 2.8-kb FGFR1 mRNAs throughout the entire culture period, whereas no syndecan-1 mRNA was detectable by this method. Maximal levels of syndecan-2, -4, and FGFR1 mRNAs were observed on day 5. However, their levels were markedly decreased on days 20 and 25. Accordingly, the inhibitory effect of bFGF on ALPase activity was less on day 20 than on day 5. When PDL cells were pre-treated with heparitinase, a mitogenic response of PDL cells to bFGF was decreased. These observations indicate that PDL cells express syndecan-2, -4, and FGFR1 mRNAs, and that those levels are changed with the increase in ALPase activity in culture. The reductions in syndecan-2, -4, and FGFR1 levels may be involved in the control of growth and differentiation of PDL cells during development and regeneration.

ANALYSIS OF OESTROGEN RECEPTOR MRNA BY REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN HUMAN PERIODONTAL LIGAMENT CELLS

Periodontal ligament (PDL) cells have osteoblast-like features and are capable of differentiating into osteogenic cells. As human osteoblasts express oestrogen receptor mRNA, it is possible that PDL cells do so also, but findings have been conflicting. To determine whether they do express oestrogen receptor mRNA, the reverse transcriptase-polymerase chain reaction was performed with two different primers. Cells were obtained from a healthy periodontal ligament of premolar extracted for orthodontic reasons. The human breast adenocarcinoma cell-line MCF7 was used as a positive control. Expression of oestrogen receptor mRNA was detected in PDL cells with one of the primers but with less intensity than in MCF7 cells. Southern hybridization confirmed these results. These findings suggest that PDL cells express oestrogen receptor mRNA at low levels.

Correlations Between Salivary Protease and Supragingival or Subgingival Calculus Index

Significant positive correlations between protease activities in saliva and supragingival/subgingival calculus indices, periodontal pocket depth, or P-M-A index were confirmed. When the partial correlation was calculated, the subgingival calculus index correlated well with the sediment protease at pH 8.5, and the supragingival calculus index showed a significant correlation with the supernate protease at pH 4.5.

Effects of fluoride intake on the mineral content, acid solubility and resorption caused by experimental periodontitis of rat alveolar bone

Adult rats were given either distilled water or drinking water containing 100 parts/10(6) of fluoride. The alveolar bone of rats given fluoride for 90 days showed an increased mineral content and decreased acid solubility compared to the bone of rats given distilled water. Experimental periodontitis was initiated in both groups after 110 days of treatment to cause alveolar bone resorption. Fourteen days later, the rats were killed and it was found that the alveolar bone resorption caused by experimental periodontitis was significantly smaller in the rats given fluoride in their drinking water than in those given distilled water. The findings suggest that fluoride intake might have a protective effect on rapidly progressing alveolar bone resorption.

The concentration of salivary steroid hormones and the prevalence of gingivitis at puberty.

Gingival conditions of 1323 junior high schoolchildren aged 12-15 were examined, and 132 children who had either healthy gingivae or severe gingivitis were called to the clinic. More precise examination of gingivitis was performed by assessment of Jacksons gingivitis index (G.I.), probing depth (P.D.), and bleeding on probing. Whole saliva was collected, and the salivary concentrations of estradiol, progesterone, and testosterone were determined by radioimmunoassay. Subgingival bacterial plaque was sampled from 36 children, and total bacterial counts and morphological differentiation were performed under a phase-contrast microscope. For statistical analysis, both males and females were divided into two groups according to the concentration of each sex hormone and subgrouped by the results of clinical examinations. Chi-square analysis using 2-by-2 tables was performed to determine the relation between salivary steroid hormone levels and gingival inflammation. The results suggest that unbalanced secretion of certain hormones might be one of the factors promoting gingivitis at puberty.

Effect of a protease derived from the oral bacterium Bacteroides loescheii on the inhibition of calcium-phosphate precipitation by human parotid saliva

A protease from the culture fluid of this microorganism was purified and characterized using DEAE-Sephadex and chromatofocusing and classified as a thiol protease with a mol. wt estimated as 27,500. It abolished the inhibitory activity of parotid saliva on the precipitation of calcium phosphate from supersaturated solutions. Such proteases may contribute to dental calculus formation.

The Relationship Between Calculus Index and Protease Activity in Saliva

Protease activity is present in saliva, plaque (Soder, J Dent Res 51:389, 1972), and oral bacteria (Cowman, Perrella, and Fitzgerald, J Dent Res 55:391, 1976, and Murphy, J Dent Res 53:832, 1974). It has been suggested that the proteases might play an important role in periodontal disease. In the present study, we show that protease activity in saliva is positively correlated with age and calculus index. Whole saliva from 77 adults (40 patients with periodontal disease and 37 students in the School of Dentistry, aged 19 to 56) was collected on ice, after recording Decayed-Missing-Filled Teeth (DMFT index), oral hygiene index, debris index, and calculus index (Greene and Vermillion, J A mer Dent Assoc 61:172, 1960), and P-M-A index (Massler, Schour, and Chopra, J Periodont 21: 146, 1950). Each of the saliva samples was homogenized immediately at 400 rpm for one min with a teflon pestle homogenizer and kept at 40C. The protein concentration was determined by the method of Hartree (Anal Biochem 48:422, 1972). The protease activity was measured by a method that was essentially that of Anson (J Gen Physiol 22:79, 1938). The reaction mixture was composed of 0.25 ml of 5% hemoglobin, 0.25 ml of saliva, and 0.5 ml of 0.2 M phosphate-0.1 M citrate buffer, pH 5.5 or 0.2 M barbital buffer, pH 8.5.