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Bacteroides gingivalis is associated with various forms of periodontal disease. To assess the role of the immune response in modulating B. gingivalis-associated periodontal disease, the effect of immunization of B. gingivalis-induced periodontal bone loss was evaluated in gnotobiotic rats. Male Sprague-Dawley rats immunized with various doses of whole cells or sham-immunized with incomplete Freund's adjuvant were monoinfected with B. gingivalis in carboxymethylcellulose by gavage. Two additional groups served as either sham-immunized or untreated germ-free controls. Forty-two days after infection, all rats were killed, periodontal bone level was assessed morphometrically and radiographically, and gingival proteinase (mammalian collagenase and acid cathepsin) activity was assessed biochemically. B. gingivalis was present in oral samples from all monoinfected rats, and no contaminating bacteria were detected in any oral or fecal sample. Animals immunized with B. gingivalis cells had elevated serum and saliva antibodies to whole cells and partially purified fimbriae from B. gingivalis. Infected sham-immunized rats had significantly more periodontal bone loss than noninfected controls, whereas the periodontal bone level in infected rats immunized with 10(10) B. gingivalis cells was similar to that of the noninfected controls. The activities of gingival collagenase and cathepsin B and L were high in sham-immunized infected rats and low in all other animal groups. In conclusion, it is possible to reduce B. gingivalis-induced periodontal tissue loss in gnotobiotic rats by immunization.

Tetracyclines have long been considered useful adjuncts in peridontal therapy based on their antimicrobial efficacy against putative periodontopathogens. However, recently these drugs were found to inhibit mammalian collagenases and several other matrix metalloproteinases (MMPs) by a mechanism independent of their antimicrobial activity. Evidence is presented that this property may be therapeutically useful in retarding pathologic connective tissue breakdown, including bone resorption. The experiments leading to this discovery are described and possible mechanisms are addressed, including the specificity of tetracyclines' anti-collagenase activity, the role of the drugs' metal ion (Zn2+, Ca2+)-binding capacity, and the site on the tetracycline molecule responsible for this nonantimicrobial property. Of extreme interest, the tetracycline molecule has been chemically modified in multiple ways, generating a new family of compounds called CMTs (chemically modified tetracyclines) that lack antimicrobial but still retain anti-collagenase activity. The first of these CMTs, 4-de-di-methylaminotetracycline, was found not to produce a major side-effect of antimicrobial tetracycline therapy--its administration to experimental animals did not result in the emergence of tetracycline-resistant microorganisms in the oral flora and gut. Numerous examples of the clinical potential of this non-antimicrobial property of tetracyclines in the treatment of periodontal and several medical diseases (e.g., sterile corneal ulcers, rheumatoid arthritis, skin bullous lesions, tumor-induced angiogenesis and metastasis) are discussed.

Previous studies demonstrated that tetracyclines (TCs) inhibited Type I (interstitial) and Type IV collagenases from different mammalian sources, but there are no studies of TCs effect on osteoblast collagenase (C'ase). The present study assessed the effect of TCs on C'ase activity from osteosarcoma cells. Semiconfluent UMR 106-01 cells were treated with minocycline or chemically modified tetracycline (CMT) at 10 micrograms/ml in the presence or absence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7)M for 24, 48, 72 and 96 hours. Media were collected at each time point and assayed following concentration, destruction of TIMP by reduction/alkylation, activation with p-aminophenylmercuric acetate (APMA), and incubation with 3H-methylated collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 hours. Collagenase activity from media was also analyzed by SDS-PAGE and fluorography. b-PTH appeared to stimulate C'ase 60-fold compared to controls; minocycline and CMT reduced PTH stimulation approximately 65% and 90%, respectively. Moreover, TCs incubated with partially purified osteoblastic collagenase directly, inhibited its activity in vitro as indicated by a lack of degradation to collagen alpha A chains. Therefore, TCs ability to inhibit bone resorption in organ culture, reported previously, may be due, in part, to reduced osteoblast collagenase activity.

Insulin-deficient, adult, diabetic rats were administrated a tetracycline (either minocycline or a chemically-modified non-antimicrobial tetracycline: CMT) by oral gavage over a 3-week period. Untreated diabetic and non-diabetic rats served as controls. On day 21, all rats received an intravenous injection of 3H-proline, as a radioprecursor of procollagen in bone, dentine and periodontal ligament (PDL) or of amelogenin in enamel; perfusion fixation with an aldehyde mixture was carried out at 20 minutes and 4 hours after isotope injection. The parietal bones (calvaria), mandibules including molars, and lower incisors of these rats were dissected and processed for light microscopic autoradiography to study 3H-proline utilization by osteoblasts, PDL fibroblasts, odontoblasts and ameloblasts. In the control rats, at 20 minutes after 3H-proline injection, silver grains of labeled precursor were detected in the osteoblasts of the periosteal surfaces of the parietal bones. At the 4 hour time period, although some radioprecursor was still present in the osteoblasts, most had progressed to the osteoid matrix. In contrast, the flattened bone-lining cells in the untreated diabetics showed minimal uptake and secretion of labeled proline at both time periods. In both minocycline- and CMT-treated diabetic rats, the labeled proline was localized in the osteoblasts and the osteoid in a pattern reminiscent of that seen in the control rats at both time periods. Of interest, CMT administration appeared to increase the labeling of the osteoid matrix more than minocycline treatment. In non-diabetic control rats, the PDL fibroblasts exhibited a polarized elongated profile and incorporated and secreted radioprecursor similar to that described for the osteoblasts in these animals. The PDL fibroblasts in the untreated diabetics lost their regular arrangement and incorporated little if any 3H-proline; once again, tetracycline administration appeared to normalize, at least in part, the structure and 3H-proline incorporation by these connective tissue cells. In contrast, diabetes and tetracycline administration did not affect the incorporation and secretion of radioprecursor by odontoblasts and secretory ameloblasts during tooth development.

Diabetes induces osteopenia, which is characterized by a deficiency of osteoid and decreased activity of osteoblasts. We recently found that tetracyclines prevent the loss of osteoid and bone matrix and the degeneration of osteoblasts in diabetic rats by a mechanism independent of their antimicrobial efficacy. However, bone remodeling requires the activity of osteoclasts as well as osteoblasts. To determine the in vivo effects of tetracycline on osteoclasts in long bones, either a tetracycline (minocycline, TC) or its chemically modified non-antibiotic analogue (CMT), 4-de-dimethylaminotetracycline, was administrated daily to streptozotocin-induced diabetic rats by oral intubation. After 21 days, the rats were perfusion-fixed with a mixture of formaldehyde and glutaraldehyde, and the humeri were dissected and processed for ultracytochemical demonstration of acid trimetaphosphatase (ACPase) activity. In untreated non-diabetic (control) rats, the osteoclasts at the zone of provisional ossification exhibited abundant mitochondria and cisterns of rough endoplasmic reticulum (RER) throughout the cytoplasm, prominent stacks of Golgi membranes, and lysosomes in the perinuclear cytoplasm, and numerous various pale vacuoles in the cytoplasmic area adjacent to well-developed ruffled border. Intense ACPase activity was observed in the Golgi saccules, lysosomes, pale vacuoles, and the extracellular canals of ruffled border. The reaction products were also noted along the resorbing bone surfaces associated with the osteoclast ruffled border. The osteoclasts in the untreated diabetic rats showed a cytoplasmic organization similar to that of the non-diabetic control rats, but showed little or no ruffled border which was replaced by a broad clear zone in some of these cells. However, most of the osteoclasts on bone matrix in the diabetics were devoid of both a ruffled border and a clear zone. ACPase activity was detected in the osteoclast cytoplasm of diabetic rat, as in the controls, but to a much lesser extent along the broad clear zone facing the resorbing bone surfaces. The osteoclasts in TC-treated diabetic rats possessed both a clear zone and a small ruffled border. However, in some cases, they lacked both structures reminiscent of the untreated diabetic cells. The osteoclasts of CMT-treated diabetic rats exhibited structural and enzymatic features essentially identical to those of the non-diabetic control rats. These results suggest that the diabetes-induced osteopenia results, at least in part, from degeneration of osteoclasts (as well as atrophic osteoblasts) and that tetracyclines may be effective in preventing these abnormalities by a mechanism not dependent on the drugs' antimicrobial properties.

A tetracycline (doxycycline) was administered daily by oral intubation to adult diabetic rats to assess the effect on skeletal tissue; the rationale--osteopenia is a complication of diabetes and tetracyclines (TCs) were recently found to inhibit pathologic collagen loss (e.g. in diabetic rat skin) by a mechanism independent of their antimicrobial efficacy. Osteopenia was induced by streptozotocin-diabetes as indicated by physical (e.g. specific gravity), chemical (e.g. calcium (Ca) content) and ultrastructural characteristics of the femurs, and TC administration (including treatment with a chemically-modified non-antimicrobial analog) appeared to prevent the development of the bone deficiency disease without affecting the severity of hyperglycemia.

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase.

Defects in neutrophil or polymorphonuclear leukocyte (PMNL) chemotaxis have been observed in a number of clinical conditions, including Down's syndrome and insulin-dependent diabetes mellitus (IDDM), which tend to be associated with severe forms of periodontal disease. In addition, impaired PMNL chemotaxis is frequently detected in individuals with localized juvenile periodontitis (LJP). The ability to monitor PMNL function in vivo at the gingival sulcus should therefore be useful as a diagnostic test. In this regard, we developed a technique which measures the response of PMNLs to a chemotactic agent, e.g., casein and N-formylmethionylleucylphenylalanine (N-FMLP) placed directly into gingival crevices. The development of the technique and its relationship to in vitro assays of chemotaxis are discussed, and data obtained from tests of the assay on control and streptozotocin-induced diabetic rats and human subjects with various periodontal diseases and IDDM are presented. As compared with healthy subjects and control animals, atypical (double peak) and reduced crevicular PMNL response patterns were observed during oral and systemic diseases. This suggests that the in vivo assay with appropriate modifications can be used diagnostically to assess PMNL migratory dysfunction and to identify individuals who may be susceptible to severe forms of periodontal disease.

An in vivo assay was recently developed to monitor the crevicular leucocyte response to chemotactic agents, e.g., casein and N-formyl peptides. This method was used to monitor humans with little or no gingival disease (C group), gingivitis (G group), chronic periodontitis (CP group) and localized juvenile periodontitis (LJP group). Casein (0.2 microliters, 2 mg/ml) was placed into an isolated gingival crevice of each subject with a calibrated wire loop and the time recorded (t = 0). Leucocytes were counted in crevicular washes (10 microliters) 15 minutes later and every 5 minutes thereafter up to t = 50 minutes. This protocol was repeated for the crevice of an adjacent tooth except that the crevicular fluid flow response to the chemotactic challenge was monitored. The C, G and CP subjects showed a similar pattern of response to the chemoattractant with a single "peak" of leucocytes at approximately t = 25 minutes. However, the peak cell count was much greater in the G and CP groups than in the C group. LJPs showed an abnormal pattern with two leucocyte peaks, one at approximately 25 minutes and the other at 45 minutes. Both peaks tended to be higher than the single peak seen in Cs but were significantly lower than that in Gs or CPs, even at similar levels of inflammation. In addition, the peak leucocyte response (to casein) in LJPs did not increase with increasing leucocyte counts in the unchallenged (resting) crevice, whereas a positive relationship was seen in the other groups of subjects. These data suggest that this new assay may provide important diagnostic information on in vivo neutrophil migration in the gingival crevice and on susceptibility to periodontal disease.

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.

The microflora of the rat gingival crevice were examined at various time intervals after inducing experimental diabetes. A variety of Gram-positive and Gram-negative cocci and short rods were isolated from the normal rat crevice. Within a week after alloxan administration, Leptotrichia buccalis was detected for the first time. Other features in the diabetic animals included an increased frequency of isolation of Proteus spp., Lactobacillus spp. and decrease of Escherichia coli. Occasionally, a decrease was also observed in Bacteroides spp. and Streptococcus spp. No difference was observed between diabetic and non-diabetic animals in the isolation of Selonomonas, Campylobacter, Bifidobacterium and Actinomyces. Plaque accumulation was markedly increased in the diabetic rats. It was concluded that the change in gingival microflora resulted from diabetes-induced alterations in the sulcular environment (such as increased substrate levels, e.g. glucose and urea, and decreased oxygen) and that the microbiological changes preceded the depending of the periodontal pocket.

Interactions between connective tissue substrates and proteinases localized to the surface of cancer cells are implicated in cancer invasion. In this report we have compared the enrichment of collagen and gelatin degrading activities and cysteine proteinase(s) in well-characterized (enzyme markers and electron microscopy) subcellular membrane fractions isolated from human small cell lung cancer lines (NCI-H69 and NCI-H82) and the RWP-1 pancreatic cancer line. With each cell line collagenolytic, gelatinolytic, and cysteine proteinase activities were enriched 5- to 128-fold in the plasma membrane fractions with differences noted between microvilli versus smooth membrane profiles. Incubation of tumor plasma membranes with methyl-3H-labeled collagen resulted in extensive degradation of the gamma, beta, alpha 1, and alpha 2 chains, suggesting the combined action of metalloproteinases. Treatment of tumor plasma membranes with the chaotropic agent, 2 M KCl, did not diminish membrane collagen- or gelatin-degrading activity, but extensively leached out the cysteine proteinase, suggesting that the latter enzyme is not an integral membrane protein. Enzyme inhibitors specific for metalloproteinases and cysteine proteinase were used to corroborate enzymatic classification. In conclusion, we have demonstrated variations in the localization of proteinases in the plasma membrane domains of different human cancer cells.

Fluids from normal and inflamed gingival flap tissues, overlying partially erupted lower third molars, were incubated on reconstituted collagen gels and examined for collagenase activity. Collagenase activity was observed only in fluids originating from tissues with pericoronitis. However, when fluids from clinically normal tissues were treated with NaSCN, some also showed collagenase activity. Only the fluids from normal-looking tissues with a history of pericoronitis showed collagenase activity after NSCN treatment. No enzyme activity was observed in treated or untreated fluids from normal tissue from patients with no history of pericoronitis. It was concluded that the fluid collagenase activity originated from the adjacent gingival tissue because (1) the activity of the untreated fluid observed in this study seemed to parallel the acitivity of the adjacent gingival tissue, based on our observations in a previous study; (2) NaSCN affected the fluid collagenase in the same way that others found it to affect gingival tissue collagenase; and (3) the disc gel electrophoretic pattern of the breakdown products of collagen produced by the GCF enzyme was similar to that produced by gingival tissue collagenase and differed from that produced by a bacterial collagenase reported to exist in human gingival fluid.

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.

In a series of experiments, Golub et al. demonstrated that tetracyclines, but not other antibiotics, can inhibit mammalian collagenases and proposed that this property could be useful in treating diseases, such as periodontal disease (but also included certain medical conditions, e.g., corneal ulcers) characterized by excessive collagen degradation (J Periodont Res 1983, 1984 and 1985; Experientia 1984; Cornea 1984). One effect was the dramatic reduction of tissue collagenase activity within the gingival crevicular fluid (GCF) of periodontal pockets after administering a standard regimen of a tetracycline (e.g., 200 mg minocycline or 1000 mg tetracycline/day). The preliminary studies described below determined the effect of (1) low-dose (LD; 40-80 mg/day) orally administered minocycline on GCF collagenase activity and on the subgingival microflora (Exp. I), and (2) tetracycline-loaded monolithic fibers (TF) on collagenase activity in vitro (Exp. II). In Exp. I, GCF collagenase activity was reduced by 45 to 80% 2 weeks after initiating LD minocycline therapy, an effect that lasted for at least several weeks after stopping drug treatment. No consistent change in the relative proportions of G(+), G(-) and motile subgingival microorganisms was detected as a result of LD treatment suggesting that the reduction in GCF collagenase activity was a direct inhibition of the enzyme by the drug. In Exp. II, 3- and 6-mm lengths of TF in vitro established tetracycline concentrations in 250 microliters of 132 micrograms/ml, from 3-mm lengths, and 265 micrograms/ml, from 6-mm lengths, after an 18-hour incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Drugs in the tetracycline family can inhibit mammalian tissue collagenase both in vitro and in vivo by a mechanism that is independent of antibiotic action. The epiphyseal cartilages of rachitic rats contain extremely high levels of collagenase (CGase), and we have used this model to study further the phenomenon of tetracycline inhibition of tissue CGase. Rickets was induced in rats by phosphate/vitamin D deficiency and parameters of gross bone morphology, bone chemistry, and serum chemistry were evaluated in both rachitic and nonrachitic animals with and without treatment with oral tetracyclines (TETs). Minocycline (or doxycycline) partially suppressed the appearance of many of the expected changes in the rachitic animals, including gross bone hardness, growth plate widening, long bone length, suppression of weight gain, and decreased bone ash content. The effects were dose dependent and were associated with marked suppression of the enhanced CGase activity. Examination of collagen breakdown products by SDS-PAGE documented that the rachitic enzyme behaved like other mammalian collagenases including in vitro inhibition with minocycline 10-20 micrograms/ml and with a nonantibiotic tetracycline. No evidence of TET osseous toxicity was noted, and, in fact, administration of TET to nonrachitic animals had a mildly favorable effect on growth and development. TET suppression of CGase can be demonstrated in a well defined model system and this form of pharmacologic enzyme inhibition can be a useful probe for delineating the role of the enzyme in connective tissue pathology.

Defects in neutrophil or polymorphonuclear leukocyte (PMNL) Chemotaxis have been observed in a number of clinical conditions, including Down's syndrome and insulindependent diabetes mellitus (IDDM), which tend to be associated with severe forms of periodontal disease. In addition, impaired PMNL Chemotaxis is frequently detected in individuals with localized juvenile periodontitis (LJP). The ability to monitor PMNL function in vivo at the gingival sulcus should therefore be useful as a diagnostic test. In this regard, we developed a technique which measures the response of PMNLs to a chemotactic agent, e.g., casein and N-formylmethionylleucylphenylalanine (N-FMLP) placed directly into gingival crevices. The development of the technique and its relationship to in vitro assays of Chemotaxis are discussed, and data obtained from tests of the assay on control and streptozotocin-induced diabetic rats and human subjects with various periodontal diseases and IDDM are presented. As compared with healthy subjects and control animals, atypical (double peak) and reduced crevicular PMNL response patterns were observed during oral and systemic diseases. This suggests that the in vivo assay with appropriate modifications can be used diagnostically to assess PMNL migratory dysfunction and to identify individuals who may be susceptible to severe forms of periodontal disease., (© 1985 American Academy of Periodontology.)

Several tetracyclines (minocycline, doxycycline, tetracycline), in levels approximating physiologic concentrations, were found to inhibit parathyroid hormone-induced bone resorption in organ culture; the specificity of this effect was demonstrated by comparison with other (non-tetracycline) types of antibiotics. The ability of tetracyclines to inhibit bone resorption is consistent with the recent proposal by Golub et al. that these antibiotics can inhibit mammalian collagenolytic enzymes by a mechanism unrelated to the drug's antibacterial efficacy, a property which could be therapeutically useful in diseases characterized by excessive collagen breakdown.

In a series of experiments, Golub et al. demonstrated that tetracyclines, but not other antibiotics, can inhibit mammalian collagenases and proposed that this property could be useful in treating diseases, such as periodontal disease (but also included certain medical conditions, e.g., corneal ulcers) characterized by excessive collagen degradation {JPeriodont Res 1983, 1984 and 1985; Experientia 1984; Cornea 1984). One effect was the dramatic reduction of tissue collagenase activity within the gingival crevicular fluid (GCF) of periodontal pockets after administering a standard regimen of a tetracycline (e.g., 200 mg minocycline or 1000 mg tetracycline/day). The preliminary studies described below determined the effect of (1) low-dose (LD; 40-80 mg/day) orally administered minocycline on GCF collagenase activity and on the subgingival microflora (Exp. I), and (2) tetracyclineloaded monolithic fibers (TF) on collagenase activity in vitro (Exp. II). In Exp. I, GCF collagenase activity was reduced by 45 to 80% 2 weeks after initiating LD minocycline therapy, an effect that lasted for at least several weeks after stopping drug treatment. No consistent change in the relative proportions of G(+), G(-) and motile subgingival microorganisms was detected as a result of LD treatment suggesting that the reduction in GCF collagenase activity was a direct inhibition of the enzyme by the drug. In Exp. II, 3- and 6mm lengths of TF in vitro established tetracycline concentrations in 250 μl of 132 μg/ml, from 3-mm lengths, and 265 μg/ml, from 6-mm lengths, after an 18-hour incubation. Although these concentrations were slightly lower than those established in the GCF of individual TF-treated periodontal pockets in vivo (Goodson et al. 1983, J Periodontol), the released tetracycline from 3- and 6-mm TFs inhibited rat polymorphonuclear leukocyte collagenase activity in vitro by 21 % and 45%, respectively (bacterial collagenase activity was also inhibited in vitro). These results indicate that TF placed into individual periodontal pockets could maintain a level of tetracycline in GCF that would effectively inhibit tissue (and perhaps also bacterial) collagenases at the lesion site., (© 1985 American Academy of Periodontology.)

The fluid in and gingival tissue lining periodontal pockets were collected from male patients undergoing periodontal therapy. The collagenolytic activity of the crevicular fluid, and the ability of the gingiva in culture to degrade (a) an exogenous collagen substrate, and (b) endogenous collagen newly synthesized and labeled with H3-hydroxyproline, were related to the severity of gingival inflammation. Although inflammation appeared to have only a slight effect on gingival collagenolytic activity and on the turnover of collagen newly synthesized in culture, a marked effect was observed on the collagenase activity in the crevicular fluid. This study suggests that the collagen destructive activity of the periodontal lesion can be assessed by monitoring crevicular fluid collagenolytic activity.

The influence of diabetes on gingival inflammation was studied through its effect on prolyl and lysyl-hydroxylase activities and on tissue-collagen content. Inflammation induced for 7 days with either endotoxin or antigen-antibody complexes reduced the activity of both enzymes by about 45 per cent, and decreased the concentration of soluble and insoluble collagens. Diabetes alone decreased the enzyme activity by more than 50 per cent but prevented the loss of the soluble and insoluble collagens which occurs during inflammation. The complex interaction of diabetes and inflammation on collagen metabolism in gingival tissue may be explained in part by abnormalities of collagen synthesis; alterations in collagen maturation and degradation, and in leukocyte function, may also occur.

To identify the mechanisms responsible for the paucity of recently synthesized collagen in connective tissues during diabetes, in vitro procollagen metabolism was studied in non-diabetic (control) and diabetic rats. Achilles tendons from the two groups were incubated for 1-8 h (35 degrees C) in medium containing [14C]proline and the radiolabeled collagen in the tissue, and that released into the media, were examined by SDS-polyacrylamide gel electrophoresis and fluorography. The bulk of the radiolabeled collagen in tendon from the diabetics was recovered as degradation products; these, but also procollagen and collagen components, were prominent in the control tissues. Moreover, the collagenous components synthesized by the diabetic rat tendons were more readily digested in vitro by trypsin than those produced by control tissues. We conclude that diabetes reduces collagen accretion in connective tissues in part due to increased intracellular degradation of procollagen.

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.

The degradation of recently synthesized collagen (probably procollagen) in rat incisor gingiva was three times greater than that in skin. Concomitantly, the formation of undegraded (intact) collagen molecules in gingiva was slower than that in skin. This high basal rate of degradation in gingiva was just slightly increased in streptozotocin-induced diabetic rats, whereas the low basal rate in skin was dramatically increased by the diabetic state. The degradation of recently synthesized collagen was measured by the relative amounts(%) of [3H]hydroxyproline-containing material in the TCA-soluble fraction of a tissue, compared with the total amount (TCA-soluble + TCA-insoluble) of [3H]hydroxyproline-containing material. Separation of the TCA fractions allowed the formation of collagen degradation products (TCA-soluble) to be viewed separately from the formation of undegraded collagen molecules (TCA-insoluble). The [3H]hydroxyproline-containing material in the TCA-soluble fraction was greatest in amount and in specific activity, 30 min after [3H] proline injection, supporting the origin of this material as being procollagen. At this time period, the relative amounts of TCA-soluble [3H]hydroxyproline-containing material were 40.3% (gingiva) and 12.7% (skin). For diabetic rats, the values were 55% and 48.8%, respectively. For the [3H]hydroxyproline-containing material in the TCA-insoluble fraction, at 30 min, the specific activity of [3H]hydroxyproline was 4.3 for gingiva and 7.4 for skin. At all other time periods, the values were also greater for skin than for gingiva, making it unlikely that the formation of intact collagen molecules occurred faster in gingiva than in skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

Gingiva from alloxan and streptozotocin-diabetic rats exhibited markedly enhanced collagenolytic activity in tissue culture. This effect was eliminated by puromycin or by repeated freeze-thawing of the tissue prior to incubation. Soluble extracts of the diabetic gingiva in situ were found to contain breakdown products of collagen similar in size to the reaction products generated by tissue collagenase. These fragments were not detected in the control tissue. This study indicates that experimental diabetes stimulates the synthesis of gingival collagenase in culture and that a similar effect occurs in vivo.

To determine if tetracyclines can inhibit human synovial collagenase from rheumatoid tissue, paired synovial tissue (or synovial fluid) was collected from 7 patients before and after oral administration of minocycline (100 mg BID) for 10 days. With each patient serving as his own control, the postminocycline collagenase activities fell an average of 67% from pretreatment values. Qualitative SDS-PAGE revealed decreased loss of alpha collagen components and reduced formation of alpha A digestion fragments. Addition of minocycline or a chemically modified tetracycline to synovial culture media in vitro profoundly inhibited collagenase activity. Further study of this action of tetracyclines could serve as a probe of the role of collagenase in rheumatoid arthritis and lead to development of agents capable of modifying the tissue destructive actions of collagenase.

Diabetes in rats inhibits the migration of neutrophils into the healing gingival crevice, an effect associated with impaired in vitro neutrophil chemotactic activity. We recently described the in vivo response of human and rat crevicular neutrophils to a chemotactic challenge and used this assay in the present study on streptozotocin-induced diabetic rats. Optimal concentrations of two chemotactic agents, casein (0.2 mul, 2 mg/ml) or N-formylmethionylleucylphenylalanine (0.2 mul, 10(-4) M), were placed into the gingival crevices of control and diabetic rats (time zero) after the resting neutrophil count was measured. After a 15-min delay, the neutrophil counts and gingival crevicular fluid flow were assessed every 5 min for another 0.5 h. The control rats (n = 14) showed an increase in neutrophil counts which reached maximum levels 30 min after the N-formylmethionylleucylphenylalanine challenge ("peak" neutrophil response) and decreased dramatically 5 min later. Diabetes of 4 days (n = 4), 14 days (n = 8), and 20 days (n = 5) duration reduced the peak neutrophil response 45, 66, and 71%, respectively. Casein produced the same response as N-formylmethionylleucylphenylalanine in control rats. Uncontrolled diabetes of 20 days duration reduced the peak neutrophil response to casein by 83%; diabetics administered insulin on a daily basis showed a reduction of only 34%. The pattern of change in gingival crevicular fluid flow in response to chemoattractants paralleled the neutrophil response. The chemotactic activity of peritoneal neutrophils was assessed in vitro with the agarose gel technique and was found to be correlated (r = 0.84; P < 0.01) with the in vivo chemotactic response in the same rats. If the same in vivo defect is observed in humans with diabetes (or with other systemic diseases associated with leukocyte dysfunction), this test could be useful diagnostically to rapidly assess neutrophil chemotaxis in lieu of in vitro assays and to identify patients who are unusually susceptible to aggressive periodontal disease.

Collagenase activity in human gingival fluid was measured using a radioactive collagen fibril assay. The activity was positively correlated with the severity of gingival disease. The fluid collagenase seemed to be controlled by alpha 2-macroglobulin, based on its activation by NaSCN, and to be present solely in the extracellular fraction. Examination of the collagen breakdown products by acrylamide gel electrophoresis indicated that the fluid collagenase was of tissue rather than bacterial origin.

Tetracyclines (including the semi-synthetic analogues, minocycline and doxycycline) are considered useful adjuncts in periodontal therapy because they suppress Gram-negative periodontopathogens. Recently, these antibiotics were found to inhibit mammalian collagenase activity, a property which may also be of therapeutic value. It has been suggested that the anti-collagenase properties of the tetracyclines are independent of their antibiotic efficacy. To advance this hypothesis further, we chemically converted tetracycline hydrochloride to its non-antimicrobial analogue, de-dimethylaminotetracycline. This chemically-modified tetracycline (CMT), although no longer an effective antibiotic, was found to inhibit the in vitro activity of collagenase from partially purified extracts of human rheumatoid synovial tissue and rachitic rat epiphysis. In a preliminary in vivo study, pathologically-excessive collagenase in skin and gingiva was induced by rendering adult male rats diabetic, and the oral administration of CMT to these rats significantly reduced the excessive collagenase activity in both tissues. Moreover, CMT administration did not affect the severe hyperglycemia in these rats but did prevent, at least in part, the diabetes-induced loss of body weight, skin weight, and skin collagen mass; these effects suggest a lack of toxicity in this animal model. A proposed clinical advantage of CMT over conventional tetracyclines, in the treatment of diseases characterized by excessive collagenolytic activity, is the lack of development of antibiotic-resistant micro-organisms during prolonged use. However, the consideration of clinical trials to support this hypothesis must await further laboratory and extensive toxicity tests.