Salivary enzyme levels after scaling and interleukin-1 genotypes in Japanese patients with chronic periodontitis.

BACKGROUND: Saliva has been used as a diagnostic fluid in medicine and dentistry. It is easy to collect using non-invasive methods. The intracellular enzymes present in saliva have been studied as markers of periodontal disease. The purpose of this study was to determine the salivary enzyme levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) after scaling and to clarify the influence of interleukin (IL)-1 genotypes on these enzyme levels. METHODS: Forty-nine Japanese patients with chronic periodontitis (24 men and 25 women; mean age: 55.1 years) were enrolled in this study. Measurements of clinical parameters including probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) and collections of stimulated whole mixed saliva were performed at baseline and 4 weeks after scaling. After evaluation of salivary AST, ALT, and LDH levels, DNA was extracted from various cells in whole saliva. IL-1A+4845 G/T genotype was determined by polymerase chain reaction amplification, followed by enzyme digestion and electrophoresis. Statistical analysis was performed by the Wilcoxon signed-rank and Mann-Whitney U tests. A significant difference was set at P <0.05. RESULTS: Mean PD, CAL, and BOP values significantly decreased after scaling (mean +/- SE: 3.2 +/- 0.1 mm to 2.6 +/- 0.1 mm in PD; 3.9 +/- 0.2 mm to 3.3 +/- 0.2 mm in CAL; and 41% +/- 4% to 18% +/- 3% in BOP) (P <0.001). The values of AST, ALT, and LDH were 77.0 +/- 7.5, 43.9 +/- 5.5, and 753.4 +/- 96.5 (units per liter [U/l]) at baseline, and significantly decreased to 55.5 +/- 6.5, 30.0 +/- 5.5, and 394.7 +/- 34.0 (U/l) after scaling, respectively (P = 0.01, P = 0.006, and P <0.001). The carriage rate of the IL-1A+4845 allele 2 was 24.5%. No difference was noted in the decrease in PD, CAL, and BOP after scaling between the carriers (N = 12) and non-carriers (N = 37) of IL-1A+4845 allele 2. However, the IL-1A allele 2 non-carriers displayed a significant decrease in salivary AST and ALT levels (P <0.001), in contrast to the carriers who did not show any changes in the salivary levels of the enzymes after scaling. CONCLUSIONS: These results documented that salivary AST, ALT, and LDH levels reflect inflammation and destruction of periodontal tissue, suggesting clinically useful markers following periodontal therapy. In addition, although IL-1A+4845 alleles may not influence clinical parameters, they may influence post-scaling values of salivary AST and ALT.

Regeneration therapy for oral disease.

The aim of this paper is to provide a review of the current understanding of the mechanisms, cell and factors required for regeneration and restoration of periodontal tissue around natural teeth. Periodontal regeneration is a complex multifactorial process involving cell populations: periodontal ligament cells, bone cells, gingival fibroblasts and epithelial cells. This paper describes bone graft, guided tissue regeneration and enamel matrix derivative with the application of growth factors.

Screening of periodontitis with salivary enzyme tests.

The purpose of this study was to determine the usefulness of salivary biochemical markers for the screening of periodontal disease and examine the agreement between the results of saliva enzyme tests and those of probing depth. The present study included a total of 187 subjects who underwent annual medical check-ups at the Comprehensive Health Care Center, Honjo, Saitama Prefecture, Japan. Periodontal pocket probing was performed with a WHO probe, and various enzymes and biochemical parameters in saliva were measured. For lactate dehydrogenase (LDH), the proportions of the five isoenzymes were calculated. To decide the cut-off point for each enzymatic activity, receiver operating characteristic curves (ROC curves) were constructed and the points of minimum difference between sensitivity and specificity were decided. Among the biochemical markers tested, salivary LDH level had the highest sensitivity and specificity (sensitivity 0.66, specificity 0.67), while salivary levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN) also had sensitivity and specificity above 0.60. Among the LDH isoenzymes, LDH4 and LDH5 dominated in whole saliva samples. Salivary LDH may be a feasible and useful parameter for the screening of periodontal disease, while salivary AST and BUN also appear to be potentially useful for this purpose.

Inhibition of growth of human gingival fibroblasts by chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1.

BACKGROUND: Transforming growth factor (TGF)-beta1 is involved in the pathogenesis of both drug-induced gingival overgrowth and hereditary gingival fibromatosis. Ribozymes enzymatically cleave target mRNAs and are expected to be utilized as the basis of novel nucleic acid-based therapies. We designed a chimeric DNA-RNA ribozyme targeting TGF-beta1 mRNA and examined its effect on growth of gingival fibroblasts in culture. METHODS: Chimeric DNA-RNA hammerhead ribozyme with sequence complementary to the loop structure of human TGF-beta1 mRNA was used. We evaluated transfer of the chimeric ribozyme by hemagglutinating virus of Japan (HVJ)-envelope into cultured human gingival fibroblasts in vitro and rat gingival tissues in vivo. We then examined effects of the chimeric ribozyme to TGF-beta1 on proliferation and DNA synthesis in human gingival fibroblasts. We also examined effects of the chimeric ribozyme to TGF-beta1 on expression of TGF-beta1, type IV collagens, and fibronectin mRNAs and expression of TGF-beta1 protein in human gingival fibroblasts. RESULTS: Chimeric ribozyme was sufficiently distributed into human fibroblasts in vitro and rat gingivae in vivo. Chimeric ribozyme to TGF-beta1 significantly inhibited expression of TGF-beta1, type IV collagen, and fibronectin mRNAs and TGF-beta1 protein in human gingival fibroblasts. Mismatch ribozyme had no effect on expression of these molecules. Chimeric ribozyme to TGF-beta1 also significantly inhibited proliferation and DNA synthesis in gingival fibroblasts. CONCLUSION: Chimeric DNA-RNA ribozyme targeting TGF-beta1 may be a useful gene therapy agent for treatment of gingival hyperplasia.

Proliferation and tube formation of periodontal endothelial cells.

Angiogenesis is indispensable to guide a regeneration of good periodontal tissue in the wound healing after periodontal surgery. Hepatocyte growth factor is well known for a strong angiogenic factor and it may play important roles in the periodontal tissue during periodontal wound healing. In exploring the promotion of angiogenesis in the periodontal ligament, proliferative and tubulogenic responses of endothelial cells to hepatocyte growth factor and to soluble factors secreted by fibroblasts were investigated. Pavement-shaped cells isolated from a human periodontal ligament were identified as the endothelial cell by their granular immunoreactivity for factor VIII. The proliferation of the endothelial cells was accelerated by the addition of hepatocyte growth factor or fibroblast-conditioned medium, and far more by adding both than either. The endothelial cells seeded on the agar containing both hepatocyte growth factor and fibroblast products formed a dense network in a shorter time than on the agar containing either. The endothelial cells in the dense network took a tube-like structure with lumen and were covered with laminin. These results suggest that hepatocyte growth factor administered into the regenerating periodontal tissue may promote, synergistically with local factors produced by the activated fibroblast, the proliferation and tubulogenesis of the remaining endothelial cells.

Effects of application of platelet releasate in periodontal regeneration therapy.

The alpha granules of platelets contain various growth factors, which display in vitro and in vivo activities known to be important in wound healing. Biologically active proteins from platelets include platelet-derived growth factor, transforming growth factor-beta, and insulin-like growth factor, as well as other less well-described angiogenic and differentiated protein factors. The purpose of this study was to evaluate histologically and histometrically the new tissue formation on furcation treatment with platelet-derived factor releasate (PR) in beagle dogs. Class II furcations were created in the mandibular premolars of eight adult male beagle dogs. Periodontal regenerative treatments were then performed using collagen sponge graft material with and without the topical application of PR. PR was prepared fresh from blood drawn from dogs before treatments. A histologic evaluation of the effect on new tissue formation was then performed by comparing periodontal tissue regeneration of sites treated with and without PR. Four and 12 weeks after the flap operations, histologic sections were processed and histologically analyzed. The evaluated parameters were bone, cementum and connective tissue attachment regeneration, length of epithelium, resorption, and ankylosis. Histologically evaluated, the regeneration of new cementum was greater at the collagen sponge with PR sites compared to the control sites throughout the entire research period (P < .01). Four and 12 weeks after the flap operations, the amount of new bone in the sites treated with PR was greater than that in the control group (P < .05). These results suggest that collagen sponge graft material with PR promotes new attachment on the periodontal tissue regeneration treatments.

Salivary biomarkers for predicting the progression of chronic periodontitis.

OBJECTIVE: Predicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis. DESIGN: Eighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann-Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied. RESULTS: Counts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p<0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30). CONCLUSION: Our findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.

The efficacy of povidone-iodine products against periodontopathic bacteria.

A total of 8 strains of 6 bacterial species, Porphyromonas gingivalis ATCC33277 and TDC286, Actinobacillus actinomycetemcomitans ATCC29523 and JP2, Fusobacterium nucleatum No. 2, Tannerella forsythensis ATCC43937, Prevotella intermedia ATCC25611 and Streptococcus anginosus ATCC33397, were treated with povidone-iodine (PVP-I) gargle (PVP-I: 0.47 and 0.23% w/v) or chlorhexidine gluconate (CHG) gargle (CHG: 0.002% w/v) for 15, 30 or 60 s, after which they were inoculated into various media, cultured and counted for residual bacteria. At both concentrations, PVP-I gargle reduced the viable cell count of all 8 bacterial strains to below the measurable limit within 15 s. By contrast, there were more than 1,000 viable colonies 60 s following treatment with the CHG gargle. The results demonstrate that povidone-iodine gargle has rapid bactericidal activity against the causative bacteria of periodontal disease.

Large-scale investigation of genomic markers for severe periodontitis.

The purpose of the present study was to investigate the genomic markers for periodontitis, using large-scale single-nucleotide polymorphism (SNP) association studies comparing healthy volunteers and patients with periodontitis. Genomic DNA was obtained from 19 healthy volunteers and 22 patients with severe periodontitis, all of whom were Japanese. The subjects were genotyped at 637 SNPs in 244 genes on a large scale, using the TaqMan polymerase chain reaction (PCR) system. Statistically significant differences in allele and genotype frequencies were analyzed with Fisher's exact test. We found statistically significant differences (P < 0.01) between the healthy volunteers and patients with severe periodontitis in the following genes; gonadotropin-releasing hormone 1 (GNRH1), phosphatidylinositol 3-kinase regulatory 1 (PIK3R1), dipeptidylpeptidase 4 (DPP4), fibrinogen-like 2 (FGL2), and calcitonin receptor (CALCR). These results suggest that SNPs in the GNRH1, PIK3R1, DPP4, FGL2, and CALCR genes are genomic markers for severe periodontitis. Our findings indicate the necessity of analyzing SNPs in genes on a large scale (i.e., genome-wide approach), to identify genomic markers for periodontitis.

Single nucleotide polymorphisms associated with aggressive periodontitis and severe chronic periodontitis in Japanese.

Periodontitis is a common inflammatory disease causing destruction of periodontal tissues. It is a multifactor disease involving genetic factors and oral environmental factors. To determine genetic risk factors associated with aggressive periodontitis or severe chronic periodontitis, single nucleotide polymorphisms (SNPs) in multiple candidate genes were investigated in Japanese. We studied 134 patients with aggressive periodontitis, 117 patients with severe chronic periodontitis, and 125 healthy volunteers without periodontitis, under case-control setting, and 310 SNPs in 125 candidate genes were genotyped. Association evaluation by Fisher's exact test (p < 0.01) revealed statistically significant SNPs in multiple genes, not only in inflammatory mediators (IL6ST and PTGDS, associated with aggressive periodontitis; and CTSD, associated with severe chronic periodontitis), but also in structural factors of periodontal tissues (COL4A1, COL1A1, and KRT23, associated with aggressive periodontitis; and HSPG2, COL17A1, and EGF, associated with severe chronic periodontitis). These appear to be good candidates as genetic factors for future study.