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Enamel matrix derivative (EMD) has demonstrated the potential to stimulate periodontal regeneration with mineralized tissue formation. Molecular regulators of bone metabolism include osteoprotegrin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), cyclooxygenase 2 (COX2), and core binding factor alpha 1 (Cbfa1). The role of these regulatory molecules within the context of EMD stimulation of mineralized tissue formation is unknown. Therefore, the purpose of this investigation was to explore the effects of EMD on these bone-related molecules in human periodontal ligament (PDL) cells.Human PDL-cell cultures were treated with EMD (5 to 100 microg/ml) for 24 hours. Total RNA was isolated using phenolchloroform, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers specific for OPG, RANKL, COX2, Cbfa1, and aldolase, with amplification in the exponential range for each molecule studied.The results of this study show that there is a significant (P0.05) increase in COX2 mRNA levels with EMD treatment, and no effects were noted on mRNA levels for Cbfa1. RANKL mRNA levels were significantly decreased (P0.01) up to 50% with EMD treatmentor =25 microg/ml. OPG levels showed minimal effects with EMD treatment. However, the RANKL/OPG ratio showed a 40% to 55% reduction with EMDor =25 microg/ml.This study supports a role for EMD stimulation of mineralized tissue formation consistent with periodontal regeneration by modulating regulatory molecules critical to bone metabolism at the RNA level.

The host response is a critical component in the pathogenesis of periodontitis. In fact, the clinical benefits associated with regulating the host response have been demonstrated in studies using several different classes of drugs. Biophosphates are one host-modulating class of drugs that has demonstrated this ability. These drugs are clinically effective at reducing bone resorption and have shown the ability to inhibit host degradative enzymes, specifically the matrix metalloproteinases (MMPs). Therefore, the purpose of this study was to investigate the regulatory effects of a bisphosphonate, tiludronate, on MMP levels and activity in human periodontal cells.MMP-1 and MMP-3 were assessed in cultured human periodontal ligament cells treated with a bisphosphonate, tiludronate. Reverse transcription-polymerase chain reaction was used to identify mRNA levels for both enzymes, and also for tissue inhibitors (TIMP-1). Enzyme immunoassay (EIA) and immunocytochemistry were used to assess MMP proteins in these cell cultures. Enzyme activity was assessed using FITC-conjugated substrates and quantitated using spectrophotofluorometry.Tiludronate significantly inhibited both MMP-1 and MMP-3 activity in a concentration-dependent manner. A maximal reduction in activity of 35% was achieved for each of the enzymes at a 10(-4) M concentration. Tiludronate did not have a significant effect on the mRNA levels for MMP-1, MMP-3, or TIMP-1. Similarly, there were no effects noted for either MMP-1 or MMP-3 on the protein level.This study demonstrates an inhibitory effect of tiludronate on the activity of both MMP-1 and MMP-3. These effects appear to occur without altering either mRNA or protein levels for these enzymes, supporting a possible mechanism of action that involves the ability of bisphosphonates to chelate cations from the MMPs. Furthermore, these results support the continued investigation of these drugs as potential therapeutic agents in periodontal disease.

A recent development in guided tissue regeneration procedures is the use of resorbable membranes, which eliminate the need for subsequent surgical removal. In this study we performed flap surgery in rats with (experimental) or without (control) implantation of one of the newer materials, atelocollagen. We observed the gingival epithelial cell kinetics using 3H-thymidine and examined the extent of gingival epithelium migration. Histological observations at day 1 on the experimental side demonstrated regenerated epithelium apposed to the collagen membrane with an intervening layer of necrotic tissues and/or fibrinous exudate. There was no observable proliferation of regenerated epithelium toward the root apex. On day 14, the regenerated epithelium migrated apically along the treated root surface in the control group. By contrast, on day 14 in the experimental group, the regenerated epithelium contacted the root surface at the cemento-enamel junction (CEJ). Apical to the CEJ, there was new cementum formation with inserting connective tissue fibers. Autoradiographs from day 1 experimental sides demonstrated labeled cells in the basal cell layers from oral, crevicular, and junctional epithelium. From day 1 to day 5, labeling indices of oral epithelium and regenerating crevicular epithelium on experimental sides were lower than on control sides. These histological and autoradiographic findings suggest that atelocollagen membrane inhibits apical migration of regenerating epithelium and accelerates connective tissue reattachment in part by inhibiting the mitotic function of basal epithelial cells in early stages of wound healing.

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.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 P0.05.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) (P0.001). 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 P0.001). 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 (P0.001), in contrast to the carriers who did not show any changes in the salivary levels of the enzymes after scaling.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.

Background: Transforming growth factor (TGF)-β1 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-β1 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-β1 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-β1 on proliferation and DNA synthesis in human gingival fibroblasts. We also examined effects of the chimeric ribozyme to TGF-β1 on expression of TGF-β1, type IV collagens, and fibronectin mRNAs and expression of TGF-β1 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-β1 significantly inhibited expression of TGF-β1, type IV collagen, and fibronectin mRNAs and TGF-β1 protein in human gingival fibroblasts. Mismatch ribozyme had no effect on expression of these molecules. Chimeric ribozyme to TGF-β1 also significantly inhibited proliferation and DNA synthesis in gingival fibroblasts. Conclusion: Chimeric DNA-RNA ribozyme targeting TGF-β1 may be a useful gene therapy agent for treatment of gingival hyperplasia. J Periodontol 2005;76:1265-1274.

MATRIX METALLOPROTEINASE-3 (MMP-3), or stromelysin-1, is an enzyme responsible for the degradation of a wide range of extracellular matrix proteins. Increases in MMP-3 activity have been found in several chronic inflammatory diseases, and this increased activity is thought to be mediated by interleukin-1 beta (IL-1 beta). Because IL-1 beta has been strongly associated with inflammatory periodontal disease, the purpose of this in vitro study was to investigate the role of IL-1 beta on the regulation of MMP-3 levels in cells derived from the human periodontal ligament (PDL). Human PDL cell cultures were treated with IL-1 beta at varying concentrations (0.01-1.0 ng/ml) for 24 hour prior to analysis at either transcript or protein levels. Following the isolation of total RNA, the relative levels of MMP-3 mRNA were determined using reverse transcription-polymerase chain reaction (RT-PCR) with 32P-end-labeled primers. Immunocytochemical detection of MMP-3 protein was performed using polyclonal antibodies to human MMP-3. The results of RT-PCR analysis demonstrated a concentration-dependent increase in MMP-3 mRNA expression, with IL-1 beta treatments of 0.1 and 1.0 ng/ml significantly (P0.01) increased over those cells not treated with IL-1 beta. This increase in mRNA expression was paralleled by significant (P0.001) changes at the protein level, with an average of 27.6% of the cells stained positive for MMP-3 following IL-1 beta treatment (1.0 ng/ml), compared with control cells showing no positive staining for MMP-3. In conclusion, the results of this study demonstrate that IL-1 beta upregulates MMP-3 in human PDL cells on both an mRNA and a protein level. These findings suggest possibly important roles for IL-1 beta and MMP-3 in both normal turnover and maintenance of the PDL and in the connective tissue degradation associated with periodontal disease.