Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique. We therefore examined the levels of MMP-1, -3, -8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1), in GCF and saliva of patients with adult periodontitis (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF. Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1, endogenous MMP-inhibitor, are present in AP GCF, which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs, especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supraand subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were PrevotelUa intermedia (ATCC 25261), PrevoteUla buccae (ES 57), PrevoteUa oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nukeatum (ATCC 10953), Mitsuokela dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub-and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supraand subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.

In search of direct in vivo evidence of matrix metalloproteinases (MMPs) in periodontal tissue destruction, we studied the presence and localization of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL) in adult periodontitis (AP) and localized juvenile periodontitis (LJP) gingival tissue specimens by immunohistochemistry, and the activities of gelatinases by Western blot, enzymography, and activity measurements, using radioactive gelatin as substrate in gingival crevicular fluid (GCF) and saliva. In gingival tissue obtained from AP and LJP patients, polymorphonuclear leukocyte (PMN) 92-kDa MMP-9 and NGAL were seen in the connective tissue, but both the sulcular and the oral epithelia were consistently negative. Whereas PMNs located in the gingival blood vessels showed strictly cytoplasmic MMP-9 and NGAL immunoreactivities, in the case of PMN extravasation the staining reactions extended extracellularly. Gelatinase activities consisting mainly of 92-kDa gelatinase were increased in AP GCF relative to LJP GCF and periodontally healthy control GCF. Western blot with specific anti-NGAL antibodies revealed the presence of 25-kDa NGAL and its high-molecular-weight forms in AP and LJP GCF and saliva and in culture medium of oral keratinocytes, but not in gingival fibroblast culture medium. We conclude that extravasated degranulating PMNs are the major source of MMP-9 and NGAL in periodontitis gingiva, GCF, and saliva.

The aim of the present study was to characterize the eventual presence and molecular forms of gelatinase/type IV collagenase activities in gingival crevicular fluid (GCF) and saliva in different forms of periodontitis; patients with clinically healthy periodontium served as controls. Enzyme activities were monitored electrophoretically by zymography using gelatin and type IV collagen as substrates and analyzed visually and/or densitometrically. Both saliva and GCF collected from adult periodontitis, localized juvenile periodontitis and type II diabetes mellitus periodontitis patients contained species moving identically with gelatinase isolated from human neutrophils or MMP-9 (mean 98 kD), and species with mobility similar to gelatinase in fibroblast cell culture supernatants or MMP-2 (mean 76 kD). Hitherto, undescribed high molecular weight forms (mean 128 kD), were found, possibly representing polymerized or complexed enzyme active/activated in situ in the gel matrix. Small molecular forms of gelatinases (mean 51 kD and 46 kD), unable to cleave type IV collagen, were also found, most likely representing in vivo proteolytically activated, truncated enzymes. Although multiple forms of gelatinases/type IV collagenases in saliva and GCF may take part in the tissue destruction in periodontitis, their profile judged according to molecular weights does not differentiate between different forms of periodontitis.

The exact molecular mechanisms of the loosening of a dental implant are not well-known. The characteristics of implant sulci are similar to those of periodontal sulci regarding gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF). Proteolytic enzymes, matrix metalloproteinases (MMPs), participate in peri-implant tissue remodeling. Clodronate is a well-tolerated bisphosphonate-group drug currently used in bone-resorption-related diseases in humans. The mechanisms of bisphosphonate action are not clarified. Collagenase activity in diseased PISF was significantly higher than in the clinically healthy group. Immunoblotting disclosed that diseased PISF contained increased immunoreactives MMP-8 compared with the healthy PISF. The residual latent collagenase activity in the diseased PISF was activated by gold thioglucose and inhibited completely by 100 microM of doxycycline closely resembling pure neutrophil collagenase (MMP-8). The presence of MMP-8 in diseased but not in clinically healthy PISF may prove to be a useful biochemical indicator to monitor peri-implant health and disease. Pure human neutrophil collagenase (MMP-8) and the MMP-8 present in PISF and in the GCF of both loosening implants and periodontitis-affected teeth were efficiently inhibited in vitro by clodronate (50% inhibition [IC50] was achieved by 150 microM of clodronate), an osteoactive, antiresorptive bisphosphonate. Furthermore, the new finding suggests an extended and hitherto-undescribed potential for clodronate in preventing the loosening of both implants and teeth, based on a dual beneficial effect: prevention of both bone resorption/osteolysis and of soft tissue/dental ligament destruction. Potential new therapeutic indications based on the collagenase-inhibiting effect of clodronate provide potential new therapeutic indications for a variety of diseased involving connective tissue breakdown, such as periodontal disease, arthritides, and tumor invasion.

Tetracyclines have recently been shown to inhibit the activity of some but not all mammalian matrix metalloproteinases believed to mediate periodontal destruction. However, the specificity of this effect, which could have significant therapeutic implications for different periodontal diseases, has not been examined in detail. Doxycycline and 4de‐dimethylaminotetracycline (CMT‐1) have been tested in vitro for their ability to inhibit human neutrophil and fibroblast interstitial collagenases and collagenase in human gingival crevicular fluid (GCF). The GCF samples were obtained from systemically healthy and insulin‐dependent diabetic adult Periodontitis patients and from localized juvenile Periodontitis (UP) patients. The concentrations of these 2 tetracyclines required to inhibit 50% of the collagenase activity (IC50) were found to be 15 to 30 μM for human neutrophil collagenase and for collagenase in GCF of systemically healthy and diabetic adult Periodontitis patients. These concentrations approximate the tetracycline levels observed in vivo during treatment with these drugs. In contrast, human fibroblast collagenase and GCF collagenase from UP patients were both relatively resistant to tetracycline inhibition; the IC50 for doxycycline and CMT‐1 for these 2 sources of collagenase were 280 and 500 μM, respectively. Based on these and other findings, we propose the following: 1) that systemic levels of tetracycline may inhibit connective tissue breakdown by inhibiting neutrophil collagenase; 2) that tetracyclines do not inhibit fibroblast‐type collagenase, which may help explain their lack of effect on normal connective tissue remodeling; 3) that tetracycline inhibition of collagenases may serve to identify the cellular origin of the enzyme; and 4) that tetracyclines can also prevent the oxidative activation of latent human procollagenases. With regards to therapy, the anti‐collagenase property of tetracyclines may be an effective adjunct in targeting tissue breakdown in systemically healthy and diabetic adult Periodontitis patients. However, in juvenile Periodontitis the anticollagenase property of tetracyclines may be less important than the antimicrobial activity of the drug because of the relative resistance of fibroblast‐type collagenase to tetracycline inhibition. J Periodontol 1993; 64:82–88.

The aim of this study was to develop and evaluate a digital process for manufacturing of occlusal splints. An alginate impression was taken from the upper and lower jaws of a patient with temporomandibular disorder owing to cross bite and wear of the teeth, and then digitized using a table laser scanner. The scanned model was repaired using the 3DATA EXPERT software, and a splint was designed with the VISCAM RP software. A splint was manufactured from a biocompatible liquid photopolymer by stereolithography. The system employed in the process was SLA 350. The splint was worn nightly for six months. The patient adapted to the splint well and found it comfortable to use. The splint relieved tension in the patient's bite muscles. No sign of tooth wear or significant splint wear was detected after six months of testing. Modern digital technology enables us to manufacture clinically functional occlusal splints, which might reduce costs, dental technician working time and chair-side time. Maximum-dimensional errors of approximately 1 mm were found at thin walls and sharp corners of the splint when compared with the digital model.

Dental plaque is the major aetiological factor in periodontal diseases and contains several proteolytic enzymes. The origin of these proteinases is, however, poorly studied. This study was undertaken to characterize collagenase present in dental plaque of adult periodontitis patients. Vertebrate-type rather than bacterial-derived collagenase activity was detected in extracts of both supra- and subgingival dental plaque extracts of adult periodontitis patients. Dental plaque collagenase was found to exist predominantly in autoactive form. Dental plaque collagenase from periodontally healthy individuals existed in latent from. Latent dental plaque collagenase from periodontitis lesions could be activated by a 95 kD chymotrypsin-like proteinase from Treponema denticola and human leukocyte cathepsin G but not by human plasmin. Incubation of purified latent leukocyte collagenase with whole cells of Fusobacterium nucleatum, Eubacterium saburreum, Prevotella buccae and Porphyromonas gingivalis, however, did not result to the activation of the enzyme. Doxycycline in vitro inhibited dental plaque collagenase with an IC50-value of 20 microM. Dental plaque collagenase degraded more efficiently type I and II collagens than type III collagen. Western-blot analysis with specific anti-human neutrophil collagenase-antibody revealed that both in supra- and subgingival dental plaque extracts dental plaque collagenase had undergone proteolytic conversion from an 80 kD proform to a 58 kD active form which is associated with catalytic autoactivity as measured by functional collagenase assay. This reflects proteolytic activation of leukocyte collagenase in dental plaque probably by other proteases derived from potent periodontopathogenic bacteria such as T. denticola or other PMN proteases such as cathepsin G.(ABSTRACT TRUNCATED AT 250 WORDS)