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Notes: Tetracyclines are now recognized to have non-antimicrobial properties with therapeutic potential - for example, these agents can inhibit pathologic collagenolysis by blocking mammalian collagenases and other matrix-degrading metal-loproteinases. In the current study, adult human subjects with moderate chronic periodontitis were administered specially formulated capsules of doxycycline, containing lower-than-usual amounts of this semisynthetic tetracycline, on a daily basis for 2 weeks prior to a full-thickness flap procedure; control subjects were administered placebo capsules. The gingiva excised during this surgical procedure were extracted, the extracts partially purified and analyzed for collagenase activity using [3H-methyl] collagen as substrate and the techniques of SDS-PAGE/fluorography or liquid scintillation spectrometry. In the absence of any drug pre-treatment, or after a 2-wk regimen of placebo capsules, the gingival extracts exhibited pathologically-excessive mammalian collagenase activity. The 2-wk regimen of low-dose doxycycline capsules reduced this activity by approximately 60–80% (p 〈 0.05 and 〈 0.01, respectively); in vitro exposure of the gingival extract to doxycycline also inhibited its collagenase activity. Collagenase activity in the crevicular fluid of periodontal pockets of an additional group of subjects was also significantly reduced, as was the severity of inflammation at the same gingival sites. The results suggest that a regimen of low-dose doxycycline capsules may provide a safe (other studies indicate that this regimen may not induce tetracycline resistance in the subgingival plaque) and effective adjunct to instrumentation therapy in the management of pathologic collagenolysis in the periodontal patient. However, further studies are necessary to confirm this hypothesis.

Notes: Recently, a new mechanism was proposed to explain the therapeutic efficacy of tetracyclines in periodontal disease— that specifically these antibiotics directly inhibit the activity of collagenase (and possibly other collagenolytic enzymes) produced by the host tissues resulting in a reduced rate of collagen breakdown (Golub et al. 1983). In the current studies, the effect of tetracyclines (minocycline, doxycycline, tetracycline) on collagenolytic enzyme activity was examined in human periodontal pockets and in several animal systems. In the clinical studies, gingival crevicular fluid (GCF) was collected on filter paper strips from individual periodontal pockets, the volume measured with the Periotron 6000, and the Gingival Index and pocket depth scored immediately thereafter at the same sites. The strips were incubated with [3H-methyi] collagen maintained in solution (or with 14C-glycine labeled collagen Fibrils), and the radiolabeled collagen degradation products measured in a liquid scintillation spectrometer, Using the techniques of SDS-PAGE and fluorography, the GCF was observed to generate collagen degradation products characteristic of those produced by mammalian collagenase, a finding consistent with previous studies. Both minocycline and tetracycline therapy reduced GCF collagenolytic activity about 70% during the initial weeks of treatment; the clinical parameters also showed improvements at the same time periods. This reduction in collagenolytic activity had largely regressed by 5 weeks after terminating tetracycline therapy and by 19 weeks after minocycline therapy, in the absence of other forms of periodontal treatment. In a preliminary study, 50 days of doxycycline treatment also reduced GCF collagenase activity, an effect which persisted for at least 2 months (but less than 1 year) after terminating its administration. In several studies involving experimental animals, the tetracyclines (1) in vivo, were found to reduce the pathologically excessive collagenase activity in gingiva and skin of diabetic rats; and (2) in vitro, directly inhibited the activity of collagenases from rat PMNL's and rabbit chondrocytes. The data in the current and our previous studies (Golub et al. 1983, Gomes, Golub & Ramamurthy 1984) continue to support the hypothesis that tetracyclines, by directly inhibiting collagenolytic enzyme(s) activity, may be therapeutically useful in treating diseases, such as periodontitis, characterized by excessive collagen breakdown.

Notes: Experimental diabetes in the rat rapidly produces a shift in the gingival crevicular microflora which is followed, within weeks, by increased production of collagenase by the gingival tissues. To assess the contribution of endogenous (hormonal or metabolic) and exogenous (altered crevicular microflora) factors on this diabetes-induced abnormality in collagenase production, the following series of experiments was undertaken: In each experiment, the germfree rats were housed in two isolators. Half of the animals in both isolators were rendered diabetic (D) with streptozotocin while the remainder were left untreated (non-diabetic (ND) controls). All of the germfree ND and D rats in one isolator of the pair were then infected with either a Gram-positive (S. mitis or A. odontolyticus) or a Gram-negative (Capnocytophaga, A. aclmomycetemcomitans or B. gingivalis) organism. In the other isolator, the ND and D rats were maintained in their germfree state. After 3 wk, the germfree and monoinfected rats were killed and the gingiva (and in one experiment, skin) were dissected. Collagenase activity produced by gingiva in culture (or extracted from gingiva or from skin) was measured using 14C-collagen fibrils (or [3Hmethyl] collagen molecules) as the substrate. Diabetes increased the collagenolytic activity in the gingiva and skin of the germfree rats. Infecting the germfree rats with a Gram-positive organism had no effect on collagenase activity generated by the gingival explants in the tissue culture system. In contrast, infection with any of the Gram-negative organisms dramatically increased the collagenase activity in the gingival tissues of the ND rats in culture, an effect not seen in the D group of rats using this system. However, monoinfection with B. gingivalis did increase collagenase activity in the extracts of gingiva (but not in skin) from both ND and D rats, with the latter group showing the highest level of enzyme activity. We suggest (i) that experimental diabetes stimulates collagenase production in gingiva by both endogenous (hormonal) and exogenous (bacterial) mechanisms, and (ii) that crevicular Gram-negative organisms stimulate collagenolysis in the adjacent gingival tissues by releasing bacterial products (e.g. endotoxins) which penetrate into the connective tissue and enhance host cell collagenase production.

Notes: Tetracyclines have been widely used as adjuncts in periodontal therapy due to the antimicrobial efficacy of these drugs. Recently, their ability to inhibit host-derived matrix metalloproteinases (collagenase and gelatinase) and bone resorption in organ culture has also been invoked as a therapeutic rationale. The current study was undertaken to determine whether tetracyclines can inhibit alveolar bone loss in vivo due to a non-antimicrobial action of these drugs. Experimental periodontitis was induced by inoculating adult, male Sprague-Dawley rats with P. gingivalis (strain 381) following kanamycin/ampicillin pretreatment. Doxycycline, non-antimicrobial chemically-modified tetracycline (CMT-1) and vehicle alone were administered daily to 3 infected groups of rats (n=6 rats per group; each group housed in a sterilized inflatable isolator) beginning 10 days after P. gingivalis inoculation. The control group (n = 6; non-infected rats) received only vehicle. After 5 weeks of daily drug administration by gastric intubation, the experiment was terminated and blood samples were taken from each animal to determine antibody levels against P. gingivalis. Plaque samples were collected from each group of animals before and after P. gingivalis inoculation and at the end of the experiment for microbiological examination. The jaws were removed from each rat, defleshed and then analyzed morphometrically and radiographically to assess bone loss. Serum antibody levels against P. gingivalis were significantly elevated in the 3 infected groups compared to the non-infected controls. This, together with the microbiologic findings, indicated that these groups of rats were infected with P. gingivalis. Significantly greater bone loss was observed in the untreated P. gingivalis-infected group compared to the controls based on both morphometric and radiographic measurements. Oral administration of doxycycline or CMT-1 to the P. gingivalis-infected rats completely inhibited this bone loss. As expected, CMT-1 did not exhibit an antimicrobial effect against P. gingivalis. Therefore, this study indicates that tetracyclines can inhibit alveolar bone loss in vivo by a mechanism which is independent of the antimicrobial efficacy of these drugs.

Notes: Diabetes increases gingival collagenase activity, an effect that may be mediated by endogenous tissue changes and exacerbated by an overgrowth of Gram-negative organisms in the gingival crevice (see Ramamurthy & Golub 1983, McNamara et al. 1982). In an attempt to reverse this collagenolytic abnormality, we administered an appropriate antibiotic, minocycline (a semisynthetic tetracycline), to diabetic rats and humans. Adult male conventional or germfree rats were made diabetic with streptozotocin, and half of these animals were administered minocycline (20 mg per day) by tube feeding for 3–4 weeks prior to sacrifice. The buccal gingiva, entire skins, and mandibles were dissected and tested for collagenolytic enzyme activity, collagen content, and alveolar bone loss, espectively. In a preliminary study, minocycline (200 mg per day) was administered for 7 days to an insulin-dependent diabetic adolescent human and an adult non-diabetic human; the twin brother of the diabetic was treated with penicillin. Gingival fluid collagenase activity was measured (using [3H-methyl] collagen as substrate in a new microassay) in 8 periodontal pockets in each subject before and after antibiotic therapy. Examination of collagenase digestion products by SDS-polyacrylamide gel electrophoresis and fluorography was also carried out. In rats, minocycline treatment: (1) suppressed the abnormally elevated collagenolytic enzyme activity in gingiva of diabetic rats, even under germfree conditions; (2) inhibited PMN leukocyte collagenase activity in vitro, an effect that was reversed by the addition of calcium ions (penicillin-streptomycin had no effect on the activity of this enzyme); and (3) retarded the abnormal loss of skin collagen and alveolar bone in diabetic rats. In a preliminary study on humans, minocycline therapy reduced the collagenase activity of gingival crevicular fluid, an effect not produced by penicillin.Our data suggests that (1) tetracycline therapy inhibits tissue collagenolytic enzyme activity by a mechanism al least in part unrelated to its antibacterial efficacy, and (2) this mechanism may provide a new therapeutic approach for suppressing excessive collagen resorption which occurs during periodontal disease and which can occur during other pathologic conditions.