Yanli Ding

Human Neutrophil Collagenase MMP-8 in Peri-implant Sulcus Fluid and its Inhibition by Clodronate

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 pM 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 uM 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 diseases involving connective tissue breakdown, such as periodontal disease, arthritides, and tumor invasion.

Membrane Components of Treponema denticola Trigger Proteinase Release from Human Polymorphonuclear Leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis.

We studied the in vivo effect of long‐term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix melalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase‐like, cathepsin G‐like and trypsin‐like activities) of saliva (n= 10) and gelatinase, lactoferrin and TIMP‐1 of saliva (n=10) and serum (n= 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS‐PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75‐kD and active 65‐kD MMP‐8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP–1‐levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP‐8) activity during long‐term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long‐term doxycycline/tetracycline treatment.

Inhibition of Matrix Metalloproteinase-1 by Dichloromethylene Bisphosphonate (Clodronate)

Abstract. Interstitial collagenase present in human jaw cyst extract and purified human fibroblast-type collagenase (MMP-1) were both efficiently inhibited in vitro by clodronate, an osteoactive, antiresorptive bisphosphonate. The IC50 of clodronate to inhibit MMP-1 is 150 μM. These findings suggest an extended and hitherto undescribed properties for clodronate/biphosphonates in prevention and treatment of tissue degradation in both bone and soft tissue destructive diseases.