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1. ACTIONS OF MMPS

2. MATRIX METALLOPROTEINASES (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become activated in the pericellular environment by disruption of a Zn++- cysteine bond which blocks the reactivity of the active site.

3. METABOLIC DEGRADATION OF EXTRACELLULAR MATRIX • Periodontal disease is initially characterized by destruction of ECM subjacent to J.E including basement membrane followed by breakdown of deeper C.T including P.L and alveolar bone. Degradation of ECM involve 4 distinct pathways:

4. MMP precursor activation Degradation of Collagen fibrils Lysosomal cathepsins Plasminogen dependent pathway Serine protienases (pericellular) MMP pathway (pericellular) Phagocytic pathway Cathepsins (intracellular) Osteoclastic pathway Cathepsins (pericellular)

5. Breakdown of collagen occurs during inflammation, tissue breakdown, remodelling and tissue repair or wound healing. process occurs in 2 different pathways: intracellular extracellular. In non-pathological conditions phagocytosis and intracellular digestion of collagen fibrils is a process observed in gingiva and P.L. In pathological conditions such as periodontal disease balance between synthesis and degradation is disrupted. In early gingivitis many of collagen fibrils in gingiva are broken down to make space for infiltrating inflammatory cells. this process changes to firm, pink gingiva into swollen loose and reddish tissue

6. Progression into periodontitis occurs when collagen fibrils of P,L and alveolar bone broken down. • ECM not only consists of collagen fibrils but also proteoglycans and fibronectin which must be removed first in order for the collagenase to have access to collagen substrate.

8. MMP’S produced by both infiltrating and resident cells of periodontium play role in : physiological Pathological events.

9. PHYSIOLOGICAL ACTIONS • Embroyonic development. • Post-partum uterus. • Tissue remodelling. • Salivary gland morphogenesis. • Tooth eruption. • Angiogenesis-MMPS play a major role in dissolution of basement membrane and remodelling of matrix composition necessary for endothelial composition necessary for endothelial invasion during angiogenesis. • Wound repair-wound healing requires sequential matrix degradation ,cell migration ,synthesis of provisional matrix consisting of fibronectin, fibrin and high amounts of collagen type-2 and matrix remodelling. • MMP-9 expression has been linked with human mucosa, possibly participation in detachment of keratinocytes from the basement membrane, migration into wound and remodelling of granulation tissue matrix. • Increased levels of interstitial collagenases, gelatinases and stromelysins have been identified in healing burn wounds, ulcers, and cultures of chemically injured epithelium.

10. PATHOLOGICAL ACTIONS • In appropriate MMP activity constitutes part of pathogenic mechanisms associated with variety of diseases includes: • Destruction of cartilage and bone in rheumatiod and osteoarthritis. • Invasive tumour growth and angiogenesis. • Degradation of myelin-basic protein in neuro inflammatory disease. • Opening of blood brain barrier following brain injury. • Loss of aortic valve during aneurisms • Tissue destruction in gastric ulceration • Liver fibrosis. • ARDS. • break down of C.T in periodontal disease.

11. ROLE OF MMP IN HUMAN PERIODONTAL DISEASES • Loss of periodontal attachment is assosciated with extensive breakdown of collagen fibers in the periodontal tissues. • Involment of MMPS in pathological tissue destruction is well documented. • Evidence that MMP’s are involved in tissue destruction in human periodontal disease has been summarized by BIRKEDAL-HANSEN includes following: • 1)cells isolated from normal and inflammed gingiva are capable of expressing a wide variety of MMP in culture. • 2)several MMP’s can be detected from the gingiva in vivo. • 3)MMP-8 and MMP-3 are readily detected in GCF from gingivitis and periodontitis patients.

12. 4)detection of elevated levels of active rather than latent collagenase in the fluid of periodontal pocket and in extracts of adjacent inflammed gingival tissue- Golub l JDR 1987,and the presence of MMP mRNA in cells of periodontal lesion such as -periodontal ligament and gingival fibroblasts • keratinocytes. • Endothelial cells. • Osteoblasts. • And even osteoclasts.

14. 5)CELLULAR SOURCE: • in earlier studies cellular source of MMP is derived from the host and not from the periodondopathic bacteria. • Gangbar etal have established that the source of procollagenase in patients with periodontal disease and inflammation is PMN leukocytes, abundant components of acute and chronic inflammation, which agrees with the findings of sorsa etal. • Pinchback etal demonstrated using immunohistochemistry that MMP-1 and TIMP-1 in advanced destructive tissue were associated with the vasculature of c.t.

15. MMP activity and periodontal disease • In periodontal disease MMP’S play key roles in degradation of extracellular matrix, basement membrane, and protective serpins as well as in the modification of cytokine action and activation of osteoclasts. • P.gingivalis and A.A.Comitans produce bacterial collagenases, these proteinases are not believed to be major importance in periodontal collagen breakdown. • One way to differentiate between mammalian and bacterial collagenase is by their different modes of collagenolysis: • Mammalian collagenase cleaves the undenatured triple helical collagen molecule at single site resulting in ¾ and ¼ fragments. because of their reduced melting temperature ,spontaneously denature at body temperature and are cleaved by other proteolytic enzymes, particularly gelatinase.

16. Bacterial collagenase attacks collagen substrate at multiple sites resulting in more than 200 peptide fragments. • Reason: why mammalian collagenase attack one specific site ¾ of distance from amino terminal end of helical collagen molecule is : • 1)relatively low hydroxyproline content and reduced helical stability at that site. • 2)presence of collagenase susceptible gly-leu and gly-ileu peptide bonds at that site.

17. EVIDENCE OF COLLAGENOLYSIS IN CHRONIC PERIODONTITIS • Collagenases(collagenase-1,-2,-3) catalyze an initial and specific cleavage of collagen at the intrahelical 775Gly-776Ile bond ¾ and ¼ fragments. these fragments denature at body temp by undergoing helix to coil transition. • This was analyzed by using an antibody, which recognizes the collagenase produced neoepitope at ¾ carboxy terminal fragment of type 1 collagen (COL1-3/4C) SORSA.T etal jpr 2003.

18. Collagenase-1 is not found in periodontal pocket epithelium, but is present in very low concs in GCF- INGMAN T jcp 1996. • Collagenase-2 has been detected in sulcular epithelium, although it mainly derives from GCF neutrophils. its activity in GCF correlates with periodontal tissue bone loss –lee w etal jpr 1995. • Collagenase-3 is expressed in pocket epithelium is elevated in GCF and its conc diminishes after trt. Moderate COL1-3/4C was found in c.t in lamina propria close to the sulcular and junctional epithelium. this study suggests that Host cell derived collagenases are, in chronic periodontitis ,able to cleave across triple helical collagen fibrils insitu.

19. BIRKEDAL-HANSEN JP 1993 –imbalance between activated MMPS and their host derived endogenous inhibitors leads to pathological breakdown of the extracellular matrix during periodontitis and numerous other diseases. • These enzymes are responsible for degradation of collagen fibers attached to the root surface allowing for the apical migration and lateral extension of pocket epithelium. • Clinical sequale of pathological increase in collagen degradation are :loss of attachment formation of periodontal pockets

20. MMP’S IN GCF • GCF MMP-1 levels have been found to be much higher in localized juvenile periodontitis compared with adult periodontitis and healthy subjects ,while MMP-8 is the main collagenase in GCF ,saliva and inflammed gingiva in adult periodontitis- sorsa.t etal jpr 1988. • Collagenase activity was absent in GCF of normal and gingivitis sites in dogs. • But high levels were found in GCF of localized.juvenie.p compared with normal pts- ingman.t etal jcp 1996. • MMP-3 levels in G.C.F of gingivitis were significantly higher than in control sites.

21. During inflammation MMP-1 is produced and remains in gingival tissue, where as majority of MMP-8 is released into periodontal pocket. • MMP-8 is mostly derived from PMNs in which it is stored in specific granules and released when triggered by periodontopathogenic bacteria or their virulence factors (sorsa etal 1992) • PMN-derived MMPs (MMP-8,MMP-9) have been shown to be associated with active phase of periodontitis (westerlund etal 1996).

22. Recent studies indicate that MMP-8 may not originate exclusively from PMN leukocytes. • Evidence from cell culture suggests that upon exposure of fibroblasts to LPS and cytokines such as IL-1β and TNF-α,the genes for destructive MMP production are turned on. • Under these conditions fibroblasts are capable of expressing MMP-8 as well.- GOLUB L 1994. • Decario a jpr 1998 –bacterial tiol protienases derived from p.gingivalis can induce production of MMPS and a collagen degrading phenotype not only In fibroblasts but also in epithelial cells.

23. MMP-8 exists in elevated amounts in G.C.F collected from inflammed periodontal pockets, and is predominently converted to active 60kda form by plaque host and microbial derived proteases. (sorsa, mantyala etal 2003) • After periodontal trt significant reduction occurs in MMP-8 G.C.F levels, where as in G.C.F of healthy individuals MMP-8 is virtually undetectable. • In gingivitis MMP-8 is mostly in its latent inactive form.

24. MMP-2,8 &13 are expressed in gingival sulcular epithelium affected by periodontitis. • MMP-13 is also expressed in inflammed periodontal tissues by basal cells of gingival pocket epithelium .in these cells MMP synthesis can be induced de novo by pro-inflammatory cytokines and growth factors (ravanti etal 1999).

25. Haerian etal show that there are significantly higher levels of TIMP in G.C.F from healthy as compared with diseased periodontitis sites. • High MMP levels and low TIMP levels are associated with periodontitis.

26. MMP- DENTAL PLAQUE AND ORAL FLUIDS • Dental plaque contains collagenase (MMP-8) and gelatinase(MMP-9). • Host MMPS originate from whole saliva which is a mixture of fluids including saliva from major and minor salivary glands as well as G.C.F in dentate individuals. • Saliva contains MMP-8& -9, and also TIMP. • Conditions associated with increase caries incidence like sjogrens syndrome increase collagenase and MMP-9 activities are observed in whole saliva. • After radiation therapy for head and neck cancer salivary MMP-9 activity is increased together with decreased salivary pH and flow rate.

27. MMPS –GINGIVAL ENLARGEMENT • Excessive accumulation of epithelial and connective tissue elements, especially collagen, non-collagenous proteins and proteoglycans due to an metabolic fibrotic process, leads to gingival enlargement. • Collagen content increases, with the loss of type I and elevated levels of type III collagen. • Phenytoin and cyclosporin A suppress expression of MMP-1, TIMP-l, and cathepsin L, in a time dependant manner by suppression of mRNA expression encoding MMP-1, TIMP-1, cathepsin L. • Cyclosporin A inhibits the collagenase activity of fibroblasts, monocytes and release of MMP-8 by the neutrophils. Phenytoin has a stronger inhibitory effect on the expression of TIMP-1. • The decreased ability of protein degradation by lysosomal enzymes Is, atleast one of the factors in the pathogenesis of drug induced gingival enlargement.

28. ROLE OF MMP IN BONE RESORPTION • It has been suggested that osteoblasts express FIB-CL when stimulated by bone resorbing agents- otsuka etal, civikelli R etal 1989. • Osteoclastic bone resorption is initiated by an osteoblast response to resorptive signals such as PTH, which includes expression of FIB-CL (MMP-13) and other MMPS and results in dissolution of unmineralized collagenous osteoid layer .osteoblasts vacate the surface as newly recruited osteoclasts move in. • Osteoclasts do not appear to express MMP ,but utilize a distinct acidic cathepsin –dependent mechanism for dissolution of unmineralized matrices. • It is possible that expression of MMP is an early key event in bone resorption and this fact explains the reason why bone resorption is so highly sensitive to IL-1 and TNF-α.

29. Tests for diagnosis • Measurement of pyridinoline-containing fragments of type-1 collagen (such as c-terminal telopeptide region of type-1 collagen) released into G.C.F during periodontal bone resorption may prove to be better diagnostic marker of active disease than measurement of protienase activity.- glannobile etal jcp 1995. • Mantyala, sorsa.t jpr 2003- developed monoclonal antibodies for MMP-8 to be utilized in chair side dip stick test for MMP-8 in G.C.F and peri-implant sulcular fluid. • The test bearing resemblance to pregnancy home test kits can be performed by dentists with out specific equipment and measures G.C.F MMP-8 levels in 5 min. it differentiates healthy and gingivitis sites from periodontitis sites and reduction of G.C.F MMP-8 levels can be observed after successful periodontal trt.

30. MMP-1 polymorphism and chronic periodontitis: • De souza AP etal jcp 2003- 1G/2G polymorphism in promoter of MMP-1 could be risk factor for severe chronic periodontitis .this polymorphism can be used as a genetic marker for severe periodontitis.

31. MMP INHIBITORS 1)Endogenous or natural inhibitors: TIMPS α2-macroglobulin. 2) Exogenous or synthetic inhibitors: hydroxamic acid derivatives. Bisphosphanates Marimastat Batimastat Tetracyclines

32. TETRACYCLINES AND MMPS • One of the aim of periodontal therapy is use of drugs for modulating host response mechanisms in order to suppress or inhibit soft tissue destruction and alveolar bone resorption. • Tetracycline can inhibit host derived MMP by mechanism independent of antimicrobial properties of drug. • A series of 10 different chemically modified tetracyclines have been identified of which 9 are found to retain their anticollagenase but lost their antimicrobial property.CMTC-5 found to have lost its anticollagenase activity

33. Tetracyclines can inhibit C.T breakdown by various mechanisms: 1)mediated by extracellular mechanisms: a) direct inhibition of already active MMPs-dependent on ca+2 and zn+2 binding properties of tetracyclines. b) prevent oxidative conversion of pro MMPs in ECM into active MMPs by scavenging of PMN generated reactive oxygen metabolites. c) tetracyclines disrupt activation by promoting excessive proteolysis of pro MMPs into enzymatically inactive fragments. d) inhibition of MMP protects ,α1 protienase inhibitor, thus indirectly ,decreases serine protienases (such as PMN elastase activity) and protecting elastase susceptible substrates such as elastic fibres ,fibronectin, proteoglycans and TIMP.

34. 2)MEDIATED BY CELLULAR REGULATION: a) tetracyclines inhibit the activation of pro TNFα there by leading to decrease in the formation of powerful cytokines TNF β. b) Tetracyclines inhibit production of cytokines, inducible nitric oxide synthase, phospolipase A2,prostaglandin synthase. effects on protein kinase c, calmodulin. 3)MEDIATED BY PRO ANABOLIC EFFECTS: • Tetracyclines increase collagen production. • Increase osteoblastic activity and bone formation.

35. A major advantage of tetracyclines as inhibitors of periodontal breakdown is due to fact that these drugs have been used safely for several years and particularly doxycycline has been found to be higly conc in G.C.F at levels 5-10 times greater than in serum and these antibiotics show substantivity because they bind to tooth structure and are slowly released as still active agents. • Advantage of low dose doxycycline (introduced by GOLUB etal and approved by US food and drug administration for human use in 1998)and CMTC is that both regimens lack antimicrobial efficacy and hence can be given for prolonged periods with out emergence of drug resistence.

36. CONCLUSION • The role of inhibitors of matrix metalloproteinases is particularly important because it is the imbalance between the activated matrix metalloproteinases and their endogenous inhibitors that leads to the pathologic breakdown of the extracellular matrix in periodontitis • Preclinical and clinical studies have demonstrated that inhibition of the MMPS as a host modulatory approach, results in favorable changes in the biochemical markers of the disease as well as clinical markers of therapeutic efficacy. • The host modulatory agents such as MMP inhibitors as an adjunct to conventional treatment can enhance and make clinical therapeutic responses more predictable in a susceptible patient

37. THANK U