Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis.

UNLABELLED: The receptor activator for NF-kappaB ligand (RANKL) plays an important role in osteoclast formation. A recent study with animal models suggests the involvement of RANKL in the pathogenesis of this periodontal disease. However, no one has examined the level of RANKL in the body fluid of human subjects. This communication reports on the in vivo concentrations of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) of periodontal subjects with severe, moderate, and mild forms of the disease. An increased concentration of RANKL and a decreased concentration of OPG were detected in GCF from patients with periodontitis (*p < 0.05 vs. control subjects). The ratio of the concentration of RANKL to that of OPG in the GCF was significantly higher for periodontal disease patients than for healthy subjects (*p < 0.01). Taken together, these data suggest that RANKL and OPG contribute to osteoclastic bone destruction in periodontal disease. ABBREVIATIONS: GCF, gingival crevicular fluid; IL, interleukin; OPG, osteoprotegerin; RANKL, receptor activator for NF-kappaB ligand.

Cloning and functional expression of rat kidney dipeptidyl peptidase II.

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

Interleukin 1 beta, interleukin 6, beta 2-microglobulin, and transforming growth factor-alpha in gingival crevicular fluid from human periodontal disease.

Inflammatory mediators are central to the pathogenesis of periodontal diseases and may be used as markers in diagnosis. The aim of this study was to identify and quantify the various growth factors, apoptosis-related modifiers [soluble form of Fas (sFas) and bcl-2] and cytokines in the gingival crevicular fluid (GCF) of patients with different severities of periodontitis as compared with those of controls. GCF samples were taken from patients with periodontal disease and from controls. The concentrations of epidermal growth factor, transforming growth factor (TGF)-alpha, interleukin (IL)-1 beta, IL-6, interferon-gamma, beta 2-microglobulin (beta 2-MG), and apoptosis-related modifiers sFas and bcl-2 in the samples were determined by enzyme-linked immunosorbent assay. TGF-alpha was significantly lower in patients with periodontal disease than in the controls. In contrast, the concentrations of IL-1 beta, IL-6; and beta 2-MG were significantly higher in the group with severe periodontal disease than in the controls. The amount of total protein in the GCF was considerably higher in the disease group than the controls (p < 0.05). TGF-alpha, IL-1 beta, and beta 2-MG concentrations were associated (Spearman rank correlation, r < 0.05 for all) with clinical measures of disease severity (pocket depth) and inflammation (bleeding when probed). Apoptosis-related modifiers (sFas and bcl-2) could not be detected in any samples. These results suggest that the growth factor TGF-alpha and certain cytokines are associated with the presence of periodontal disease.

Subgingival plaque formation on single and polycrystal aluminum ceramics.

A study was conducted to investigate subgingival plaque formation on two types of dental implant material in the human oral cavity. Fifteen teeth affected by severe periodontitis and scheduled for extraction in five patients were selected. After thorough root planing, inlay cavities were prepared from the gingival margin to 5 mm subgingivally. Then, rectangular test specimens (polycrystal aluminum ceramic (P), single-crystal aluminum ceramic (S)) or a control dentin specimen (D) were installed in gold inlays, which were placed in the inlay cavities and left for 3, 7 or 21 d. As a consequence, 4 mm of the surface of each specimen was exposed to the environment of the periodontal pocket. After each test period, the specimens were removed from the inlays and examined by scanning electron microscopy (SEM). The results suggested that S is a more suitable dental implant material than P from the viewpoint of clinical application because of its lower degree of plaque accumulation.

Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity.

The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography also indicated a similar molecular size for the protease. The enzyme was purified to near homogeneity following anion-exchange and gel-filtration chromatography. The purified enzyme also exhibited a single protein species with a size of approximately 53 kDa. Enzyme activity was strongly dependent upon the presence of reducing agents (dithiothreitol, cysteine, and 2-mercaptoethanol) and was also stimulated in the presence of calcium ions. A comparison of the properties of the prtT gene product with comparable parameters of proteases previously purified from different strains of P. gingivalis suggested that the cloned protease represents a previously uncharacterized enzyme.

[Scaler sharpening. Effect of the cutting edge of scaler and the epoxy resin surface with several types of sharpening stones and strokes].

The purposes of this study were to determine the characteristics of a sharpened scaler and planed epoxy resin surface under different sharpening conditions. Each scaler was sharpened with the following kinds of sharpening stones and sharpening strokes by using an originally designed sharpening machine: Stones: 1) Fine India stone (ISF) 2) Arkansas stone (AS) 3) Carborundum stone (CS) 4) ISF followed by AS (ISF + AS) 5) CS followed by AS (CS + AS). Strokes: 1) up strokes (PUSH) 2) down strokes (PULL) 3) up and down strokes (RE) Epoxy resin surfaces were planed (10, 20 and 40 strokes) by sharpened scalers. Before and after these sharpening procedures for each scaler, the cutting edge of each scaler was photographed with a scanning electron microscope (SEM) to determine its roughness and the number of wire edges. The epoxy resin surface was examined with the SEM and the surface roughness was analyzed with a profilometer. The following results were obtained: 1) ISF with three types of sharpening strokes produced the roughest cutting edge. 2) ISF and CS with three types of sharpening strokes produced the largest number of wire edges. 3) PUSH with the five types of sharpening stones showed the roughest cutting edge and the largest number of wire edges. 4) ISF and CS produced more surface roughness, and AS made a smoother surface. Epoxy resin surfaces were planed effectively after 10 strokes. However, no significant differences were found in the surface roughness after 10 to 40 strokes.

[In vivo study of bacterial invasion in root planed and citric acid treated radicular surfaces of periodontally involved human teeth].

The purpose of this study was to investigate whether plaque bacteria invade the exposed radicular dentin after root planing or chemical root treatment in vivo. Eighteen caries-free human periodontally involved teeth with hopeless prognoses were studied. Fourteen teeth were scaled and root planed with hand curette type scalers. The proximal surface of each treated tooth was designated as the RP surface. The remaining half of the proximal surface was treated with citric acid (pH 1.0) for 3 minutes and was designated as the CA surface. Four untreated teeth served as controls. After 4 weeks, the teeth were extracted, and were processed for light microscopy and for scanning electron microscopy concerning bacterial invasion into the supragingival radicular dentin. The following results were obtained. 1. Radicular cementum was present on most untreated tooth surfaces. However, bacteria were never seen in the dentinal tubules. 2. Bacterial invasion into the dentinal tubules was observed in five of the 10 proximal surfaces (50% of the RP surfaces) and in nine of the 10 proximal surfaces (90% of the CA surfaces). 3. The depth (9.5 +/- 24.1 microns vs 84.6 +/- 136.3 microns) and percentage (0.8 +/- 2.1% vs 20.3 +/- 17.3%) of bacterial invasion in the dentinal tubules of the RP surfaces was lower than that of the CA surfaces. 4. Cocci and short rods were present in the supragingival dentinal tubules. 5. Since CA surfaces may accelerate bacterial invasion the citric acid treatment might be harmful in patients with inadequate plaque control.

[In vitro study of bacterial invasion in radicular dentin].

The purpose of this study was to investigate whether plaque bacteria invade exposed radicular dentin after root planing or chemical root treatment in vitro. Pieces of dentin were cut out from impacted third molars. The surface of all dentin pieces was treated with sandpaper (#240) so as to make the surface roughness of dentin pieces equal to that of the root surface after root planing (RP surface). Half of the dentin pieces were treated with citric acid (pH 1.0) for 3 minutes (CA surface). After sterilization, each dentin piece was incubated at 37 degrees C in a culture medium inoculated with either S. mutans or S. sanguis. After 1, 3, 7 and 28 days of incubation, the invasion of microorganisms into the dentinal tubules was histologically examined using a light microscope. The following results were obtained. 1. The invasion of S. mutans and S. sanguis into the dentinal tubules was observed at 1, 3, 7 and 28 days. The depth and number of bacterial invasion into the dentinal tubules were positively correlated with incubation time on CA surfaces but not with RP surfaces. 2. The depth and the number of bacterial invasion into the dentinal tubules were higher on the CA surfaces than the RP surfaces. 3. Since the citric acid treatment of scaled and root planed root surfaces may accelerate bacterial invasion from treated root surfaces, the use of citric acid might be harmful in patients with inadequate plaque control.