Elastase as an indicator of periodontal disease progression.

THIS STUDY SOUGHT TO EVALUATE the ability of gingival crevicular fluid (GCF) elastase to predict attachment and bone loss in human periodontitis. Thirty subjects who were medically healthy and had a history of progressive periodontitis were studied with an automated probe. Five sites in each patient were monitored bi-monthly for a 6-month period for attachment loss. Subtraction radiography was utilized at the beginning and end of the study to monitor bone loss. GCF elastase was measured at 0 month and then bi-monthly by collecting GCF on paper strips impregnated with PMN leukocyte elastase substrate inserted into the gingival crevice for 15 seconds. After 8 minutes of reaction time, the strips were scored relative to fluorescent standards in an ultraviolet view box. Strips were then eluted in methanol and total elastase measured by spectrofluorometry. Total elastase was significantly higher in sites demonstrating progressive attachment loss than in inactive sites (2.81 +/- .29 versus 2.03 +/- .07, P less than 0.0005) and sites demonstrating bone loss (2.32 +/- .17 versus 2.01 +/- .08 P less than 0.05). When considering the joint presence of bone loss and attachment loss of 1.0 mm or greater in the 6-month period following a visual elastase kit score of 2 or greater, the test kit shows a sensitivity and specificity of 82% and 66%, respectively. This study demonstrated that GCF elastase levels are significantly higher in sites demonstrating progressive periodontal attachment and bone loss assessed 6 months later and may serve as a predictor of future bone and attachment loss.

The effect of enamel preparation on the tensile bond strength of orthodontic composite resin.

The bonding of orthodontic brackets to enamel surface using bis-GMA composite resin is usually accomplished by first cleaning the tooth surface then etching with phosphoric acid. This study compared the tensile bond strength of composite resin applied to a tooth surface which had been cleansed with an air-powder polisher to that of the same resin applied to a surface cleansed using a rubber cup and pumice. A wire loop apparatus was attached to bonded orthodontic brackets and pulled in tension in order to test the adherence of the bracket to the tooth. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to evaluate the tooth surface to determine whether sodium bicarbonate material remained after the cleaning operation. All data was analyzed by the one way analysis of variants, the Student-Newman-Keuls test and Duncan's multiple comparison test. No statistical differences were found between the tensile strength of the bonds on the teeth cleansed with the air-powder polisher and those cleansed with a rubber cup and pumice. However, a double exposure of the tooth to phosphoric acid may lower the tensile bond strength by a significant amount.

Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase.

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form.

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).

Retinoic acid stimulates degradation of interstitial collagen fibrils by rat mucosal keratinocytes in vitro.

A large body of evidence suggests that retinoids modulate the phenotypic expression of epithelial cells of skin and mucous membranes. The purpose of this study was to investigate the effect of retinoic acid on keratinocyte-mediated collagen breakdown. Keratinocytes derived from the ventral surfaces of the tongues of 4-6 week-old male Wistar rats were established in culture under conditions which are restrictive to growth of fibroblasts, and they were eventually cloned by limiting dilution. The cells were seeded (100,000 cells/cm2) in dishes coated with 3H-labeled, reconstituted type I collagen fibrils and incubated in serum-free medium over a 3-5 day period. Dissolution of the collagen fibrils was monitored by the release of radioactivity to the culture medium. Unstimulated cells metabolized the collagen rather slowly, but addition of retinoic acid in concentrations from 10(-6)M to 10(-8)M resulted in marked acceleration of the degradative process, with complete solubilization of the collagen fibrils in four or five days. The effect of 10(-6)M retinoic acid was of the same order of magnitude as that obtained by addition of a proteolytic activating system either in the form of plasmin or of plasminogen, which is converted to catalytic plasmin by endogenous activators. The effects of retinoic acid and plasminogen/plasmin, however, were not additive. Keratinocytes rendered vitamin A-deficient by cultivation in sera from deficient rats were clearly less effective in degrading the collagen substrate than were "sufficient" cells. Addition of retinoic acid (10(-7)M) enhanced collagen breakdown in both sets of cultures and partially restored the collagenolytic activity of the deficient cells.