7.mmp

role of mmp in periodontics,what are mmps,classification

1. MATRIX METALLOPROTEINASES PUNIT PG STUDENT

2. CONTENTS INTRODUCTION HISTORY STRUCTURE OF MMPS CLASSIFICATION Collagenases Gelatinases Stromelysins Matrilysins Membrane type MMPs ACTIVATION OF LATENT METALLOPROTEINASES TO EFFECTUAL PROTEINASES BIOLOGICAL ACTION OF MMPS

3. REGULATION OF MMPS 1) Transcriptional regulation 2) Precursor activation 3) Substrate specificity 4) MMP inhibitors ROLE OF MMP IN PERIODONTAL DISEASE SAMPLING SOURCES FOR MMP DIAGNOSTIC TEST FOR MMPS CONCLUSIONS REFERENCES

4. INTRODUCTION MMPs also known as matrixins Matrix metalloproteinases (MMPs) are a large family of zinc and calcium-dependent endopeptidases , which are responsible for the tissue remodeling and degradation of the extracellular matrix (ECM), including collagens, elastins , gelatin, matrix glycoproteins , and proteoglycan . ( Woessner ,1991)

5. HISTORY Discovered by Jerome Gross and Charles M. Lapiere (1962) while studying the degradation of triple-helical collagen during the metamorphosis of a tadpole tail . The collagen was cleaved by an enzyme known as interstitial collagenase. This enzyme was first isolated from human skin in the inactive form, pro MMP (also called MMP zymogen ) by Eisen and Jeffrey in 1968.

6. L ater found in both invertebrates and plants. In 1990, it was discovered cysteine was responsible for regulating the enzyme in its inactive form. After the complete sequencing of the human genome, it was determined that twenty two different genes encoded a set of all human MMPs. (Lohi,2001)

7. MMP numbering is usually determined by the order of their discovery, MMP-1 being the first. MMP nomenclature often includes a characteristic name, for example gelatinase , stromelysins etc. MMP-2 was the first purified from a malignant murine sarcoma cell line and also first identified type IV collagenase. MMP-7 has a high affinity for elastin and was isolated from a mixed tumor. MMP-8 was first cloned from the peripheral leukocytes of a patient with chronic granulocytic leukaemia from which messenger RNA (mRNA) was extracted.

8. MMP-9 was cloned from the HT1080 fibrosarcoma cell line. MMP-13 was first discovered from carcinoma of human breast and later cloned from interleukin-1 (IL-1) stimulated chondrocytes . MMP- 14 was first identified from the tumor cell surface and it increases invasiveness of cultured carcinoma cells. MMP- 18 was the first identifiable amphibian collagenase. MMP- 26 was cloned from a cDNA library of human endometrial tumor. MMP-28 was first discovered from human testis and cDNA library of keratinocyte .

9. STRUCTURE OF MMPs Matrix metalloproteinases are structurally similar, but differ in substrate specificity, in that each MMP has the ability to degrade a particular subset of matrix proteins.

10. Domain 1- A propeptide region containing the conserved cysteine , which is part of the " cysteine switch" that confers latency. Domain 2-A catalytic core domain at the N-terminus, which is the minimum sequence necessary for enzymatic activity, and contains the Zn++-binding site. Domain 3-A C-terminus region that contains hemopexin -like repeats, which are involved in substrate binding.

11. Domain 4. A proline -rich hinge region. Domain 5. A gelatin binding domain in the gelatinases that is composed of fibronectin like repeats. Domain 6. Membrane Insertion Extension Most MMPs have their C terminus at the end of the hemopexin domain, the four MT-MMPs have a further extension that governs insertion of these proteases into the cell membrane

12. CLASSIFICATION Based on the substrate specificity MMPs are classified into the following types: Group MMPs Nomenclature Group substrate Collagenases MMP-1 Collagenase 1, fibroblast collagenase Types I, II, III, VII, X, XI collagens, gelatin, entactin , aggrecan , tenascin , perlecan , vitronectin , α 2Ma MMP-8 Collagenase 2, neutrophil collagenase Types I, II, III, VII, X collagen, gelatin, aggrecan , entactin , tenascin , tissue factor pathway inhibitor, α 2Ma MMP-13 Collagenase 3 Type I, II, III, IV, VI, VII, X, IX, XIV collagen, gelatin, fibronectin , entactin , aggrecan , tenascin

13. Group MMPs Nomenclature Group substrate Gelatinases MMP-2 Gelatinase A Types I, III, IV, V, VII, X, XI collagens , gelatin, elastin , fibronectin , laminin , aggrecan , vitronectin MMP-9 Gelatinase B Types I, IV, V, VII, X, XI, XIV, XVII, gelatin, elastin , fibronectin , laminin , aggrecan , vitronectin , decorin , plasminogen , proTNF - α

14. Group MMPs Nomenclature Group substrate Stromelysins MMP-3 Stromelysin 1 Types III, IV, V, IX, X, XI collagens , elastin , proteoglycans , laminin , fibronectin , gelatin, fibrin/ fibrinogen, . aggrecan , vitronectin , perlecan , decorin , proIL-1bc, plasminogen , Ecadherin , α2Ma, proTNF - α MMP-10 Stromelysin 2 Types III, IV, V, IX, X, XI, proteoglycans , laminin , fibronectin , gelatin, aggrecan , elastin , fibrin/ fibrinogen, vitronectin MMP-11 Stromelysin 3 Unknown

15. Group MMPs Nomenclature Group substrate Matrilysins MMP-7 Matrilysin 1, PUMP-1 Elastin , proteoglycans , laminin , fibronectin , gelatin, types I, III, IV, V, IX, X, XI collagens, fibrin/fibrinogen, tenascin , vitronectin , pro α - defensin , decorin , E- cadherin , plasminogen , proTNF - α . MMP-26 Matrilysin 2 Fibronectin , fibrinogen, gelatin, type IV collagen,,laminin-1.

16. Group MMPs Nomenclature Group substrate Membrane type MMPs MMP-14 MT1-MMP Types I, II, III collagens, gelatin, fibronectin , tenascin , perlecan , nidogen , vitronectin,factor XII, fibrin, proTNF α , laminin , cartilage proteoglycan core protein, α 2Ma, . . MMP-15 MT2-MMP Laminin , fibronectin , tenascin , nidogen , entactin , gelatin, aggrecan , vitronectin , proTNF-ab , transglutaminase MMP-16 MT3-MMP Gelatin, type III collagen, perlecan, fibronectin , vitronectin , aggrecan , transglutaminase . MMP-17 MT4-MMP Gelatin, fibrin/fibrinogen, α 2Ma, proTNF - α . MMP-24 MT5-MMP Proteoglycan , type I collagen, fibronectin , laminin . MMP-25 MT6-MMP Gelatin, type IV collagen, fibronectin

17. Group MMPs Nomenclature Group substrate OTHERS MMPs MMP-12 Macrophage elastase MMP-12 Types I, IV collagen, (Metallo elastase) aggrecan, decorin, gelatin, elastin , fibronectin , fibrin/fibrinogen, laminin , proteoglycan , vitronectin , plasminogen , α 2Ma, MMP-18 Type I collagen MMP-19 (No trivial name) Type I and IV collagens, fibronectin , gelatin, tenascin , casein, laminin , entactin , aggrecan . MMP-20 Enamelysin Amelogenin, casein, gelatin, lysin) ( Ename fibronectin , type IV, XVIII collagens, laminin , tenascin C, aggrecan MMP-27 Type II collagen, gelatin, fibronectin MMP-28 Epilysin Casein

18. Collagenases MMPs 1, 8 and 13 are the major secreted collagenases that initiate degradation of fibrillar collagens (type I, II, III). The fibrillar collagens are cleaved at a specific site to produce triple helical fragments, which denature spontaneously to gelatin at 37 °C.

19. Matrix metalloproteinase-1 Also known as Interstitial collagenase Matrix metalloproteinase-1 cleaves type III collagen and is expressed in various normal cells, such as keratinocytes , fibroblasts, endothelial cells, macrophages and chondrocytes . Activated in vivo by proteinases ( eg . Plasmin,trypsin )

20. Matrix metalloproteinase-8 Also known as Collagenase-2 and neutrophil collagenase. MMP-8 cleaves type I and II collagens and is synthesized by maturing leukocytes in bone marrow, stored intracellularly in granules and released in response to extracellular stimuli.

21. Matrix metalloproteinase-13 Also known as Collagenase -3 Matrix metalloproteinase-13 was originally cloned from a breast tumor and cleaves preferentially type II collagen and also gelatin more effectively than other collagenases . It is expressed during bone development and gingival wound repair as well as in pathological situations, such as in squamous cell carcinomas (SCC) of different organs, chondrosarcomas and melanoma. (Kerkela,2003)

22. Gelatinases Gelatinase A is expressed in a variety of normal cells, including fibroblasts, keratinocytes , endothelial cells and chondrocytes . Gelatinase B is produced by keratinocytes and macrophages. Gelatinases are able to degrade type IV, V, VII, X, XI, and XIV collagens, gelatin, elastin , proteoglycan and fibronectin and have a vital role in cancer invasion.

23. Matrix metalloproteinase-2 Also known as ( gelatinase A/72-kDa gelatinase /type IV collagenase) . It is synthesized as a 72 kDa proenzyme that is proteolytically processed to the 66 kDa active form.

24. Matrix metalloproteinase-9 Also known as ( gelatinase B, 92-kDa gelatinase /type IV collagenase). Exhibits a broad range of substrate specificity for native collagens including types IV, V, VII, X, as well as gelatin, elastin , proteoglycan . Secreted as 92 kDa proenzyme and can be activated by cathepsin G and MMP-3.

25. Stromelysins MMP-3 (stromelysin-1) MMP-10 (stromelysin-2) MMP-11 (stromelysin-3)

26. Matrix metalloproteinase-3 Also known as (stromelysin-1and transin ). Matrix metalloproteinase-3 is expressed by keratinocytes , fibroblasts and chondrocytes . Cleaves number of subtrates incuding cartilage proteoglycans ,collagen types II,III ,IV, V and IX, as well as fibronectin and laminin.

27. Matrix metalloproteinase-10 Also known as (stromelysin-2and transin-2) Matrix metalloproteinase-10 was initially identified in an adenocarcinoma cDNA library, but can also be detected in SCCs of the head, neck and lung

28. Matrix metalloproteinase-11 Also known as (stromelysin-3) MMP-11 is expressed in mesenchymal cells located close to epithelial cells during physiological and pathological tissue remodeling. It is expressed in most invasive human carcinomas and is associated with a poor clinical outcome in breast cancer. Does not cleave extracellular matrix proteins such as collagen and elastin .

29. Matrilysins Matrix metalloproteinase-7 Also known as matrilysin /PUMP . Matrix metalloproteinase-7 was originally identified as the small putative uterine metalloproteinase (PUMP). Unlike most other MMPs expressed only in response to injury, MMP-7 is expressed by non-injured, non-inflamed mucosal epithelium in many tissues.

30. Matrix metalloproteinase-7 is up regulated in many tumors, especially of epithelial origin, such as breast, lung and skin cancers. MMP-7 can also inhibit tumor angiogenesis by producing angiostatin .

31. Matrix metalloproteinase-26 Also known as matrilysin-2 . Matrix metalloproteinase-26 is detected in placenta and uterus and is also widely expressed in diverse tumor cell lines and in malignant tumors. ( Birkedal-Hansen,1993)

32. Membrane type MMP Four type I transmembrane proteins (MMP-14, -15, -16, and -24). Two glycosylphosphatidylinositol-anchored proteins (MMP-17 and -25).They all have a furin recognition sequence at the C-terminus of the propeptide . They are therefore activated intracellularly and active enzymes are likely to be expressed on the cell surface. All MT-MMPs, except MT4-MMP (MMP-17) can activate proMMP-2. MT1-MMP (MMP-14) has collagenolytic activity on collagens I, II, and III

33. Matrix metalloproteinase-12 Also known as macrophage metalloelastase . Main subtrates are elastin , fibronectin,gelatin , laminin . MMP-12 was initially found in alveolar macrophages of cigarette smokers and is the most effective MMP against elastin . ( Massova,1998)

34. Matrix metalloproteinase-19 is widely expressed in tissues, including placenta, lung pancreas, ovary, spleen and intestine. Matrix metalloproteinase-19 is capable of degrading many components of the ECM and basement membrane (BM), but cannot activate any pro-MMPs.

35. MMP-20 or enamelysin is exclusively be expressed in ameloblasts and odontoblasts of developing teeth and helps in degrading amelogenin . MMP-28 expressed at high levels particularly in testis and in injured epidermis. ( Kahari V, 1997).

36. ACTIVATION OF MMP IN CELL SUFACE Two pathways are now well characterized that lead to the activation of metalloproteinases at or near the cell surface. One pathway (a) involves the generation of the serine proteinase plasmin from plasminogen; this is brought about by the binding of plasminogen activator (PA) to its membrane receptor ( uPAR ). Plasmin can activate most prometalloproteinases . The second pathway (b) involves membrane-bound metalloproteinases that act on certain prometdoproteinases (in the case of gelatinase A it is a progelatinase AITIMP-2 complex that becomes bound. There is likely to be overlap and interaction between the two pathways, specific to each situation. Activation may also take place away from the cell surface if appropriate proteolytic activity is available for modifying the proenzyme forms of metalloproteinases.

37. Plasmin , a serine proteinase derived from plasminogen by the action of plasminogen activator ( uPA ), has been found to be effective in collagenase and stromelysin - 1 activation in cell model systems and is strongly implicated in many situations in vivo. This activation process takes place near or at the cell surface where plasminogen activator is bound to its receptor. There is little evidence that plasmin itself is an effective proteinase in matrix degradation. (Murphy G,1992)

38. Stromelysins can be activated by many proteinases with different substrate specificity, and in in vitro systems stromelysin-1 is required for efficient activation of collagenase, which otherwise has low activity. Stromelysin can also be an efficient activator of gelatinase -B (MMP-9). (Ogata Y,1992)

39. Proteinases such as plasmin are not, however, efficient activators of gelatinase A, and the precise activation mechanism for gelatinase A is still being explored. Activation could be achieved by incubating progelatinase A with fibroblast membranes and involves the Gterminal domain binding to the cell surface. (Ward RV,1994)

40. Sato et al. (1994) and Cao et al. (1995) provided evidence that the membrane component was a new type of membrane-bound metalloproteinase (MT1 metalloproteinase, MMP-14). Further, an interaction between a complex of progelatinase A and TIMP-2 and the membrane-bound MT1 metalloproteinase seems to be involved ( Strongin , 1995), as well as intermolecular autolytic cleavage (Atkinson SJ,1995).

41. The membrane bound metalloproteinase gives a second mechanism for activation that results in active enzyme being produced near or at the cell surface . Activation near the cell surface may result in proteolytic degradation being shielded from the effects of inhibitors. ( Basbaum , 1996) MT1 metalloproteinase can also process procollagenase-3 to active enzyme . ( Knauper , 1996)

42. Biological action of MMPs Known substrates include most of the ECM components ( fibronectin , vitronectin , laminin , entacin , tenascin , aggrecan etc.) The collagens (types I,II,III,IV,V,VI,VII,VIII,IX,X,XIV) have all been shown to be subtrates of different MMPs, with greatly different efficacies. In addition to CT and ECM components, proteinase inhibitors such as α 1-proteinase inhibitor, antithrombin –III and α 2-macroglobulin are selectively cleaved by MMPs.

43. The matrix metalloproteinase (MMP) family of extracellular proteinases regulates development and physiologic events. These enzymes are important for cell migration, invasion, proliferation, and apoptosis. They regulate many developmental processes, including branching morphogenesis, angiogenesis, wound healing, and extracellular matrix degradation. MMP activity may be required during development and normal physiology in several ways: (1) To degrade ECM molecules and allow cell migration (2) To alter the ECM micro-environment and result in alteration in cellular behavior (3) To modulate the activity of biologically active molecules by direct cleavage, release from bound stores, or the modulating of the activity of their inhibitors

44. Up regulators of MMPs production: IL-1 β TNF- α Epidermal growth factor Platelet derived growth factor (PDGF) TGF- α

45. Down regulators of MMPs expression: Interferon γ TNF- β Glucocorticoids

46. REGULATION OF MMPS The activity of MMP against extracellular matrix substrates is regulated at 4 “gates”. 1) By transcriptional regulation of MMP genes; 2) By precursor activation; 3) By differences in substrate specificity; and 4) By MMP inhibitors

47. Transcriptional regulation At the level of transcription, cytokines and growth factors like TNF- α , IL-1, FGF and PDGF can induce the production of MMPs, depending on the situation and cell type. Certain hormones ( parathormone , progesterone, glucocorticoids ), chemical agents ( phorbol esters) as well as cell–cell and cell–matrix interactions can induce or repress the expression of MMPs. All these extracellular stimuli regulate MMP activity through the activator protein-1 binding site, which is situated in the proximal promoter region in inducible MMP genes ( MMPs -1, -3, -7, -9, -10, -12, -13 ,1 4 and -19 )

48. Activation of precursors The latency of MMP precursors appears to be maintained, at least in part, by a coordinate bond which links an unpaired Cys residue in the propeptide to the active site Zn++. Disruption of the Cys -Zn++ bond is a prerequisite to activation and may be achieved in a number of different ways: 1) by interaction or modification of the Cys residue by organomercurials , metal ions, thiol reagents, and oxidants;

49. 2) by conformational change of the Polypeptide backbone induced by certain chaotropic agents (KI ; and 3) by excision of a portion of the propepeptide by proteolytic enzymes ( trypsin , plasmin , chymotrypsin , neutrophil elastate , cathepsin B, and plasma kallikrein ). The enzyme subsequently catalyzes 1 or more autolytic cleavages to generate the fully-processed form.

50. Substrate Specificity A certain level of regulation of MMP activity is encoded at the level of the substrate. Although the enzymes have somewhat overlapping substrate specificities, there are also notable differences, particularly with respect to the cleavage of collagens. Virtually all of the enzymes cleave gelatin and fibronectin at some rate, and most cleave type IV and V collagens at sufficiently high temperatures.

51. Inhibitors of MMPs MMPs can be inhibited by the ubiquitous plasma proteinase inhibitor alpha 2-macroglobulin, the major group of inhibitors of the metalloproteinases in tissues is the tissue inhibitors of metalloproteinases Four separate gene products in this family have now been identified. Different inhibitors include- Alpha 2- macroglobulins Tissue inhibitors of metalloproteinases Inhibiting antibodies Synthetic inhibitors

52. α 2-macroglobulins Active MMP are captured by alpha 2-macroglobulins by a unique venus -fly-trap mechanism activated by cleavage of a bond in the “bait region”. This cleavage leads to hydrolysis of a labile internal thiol -ester bond and covalent cross-linking of a nascent glutamyl residue to lysyl side chains exposed on the surface of the attacking proteinase .

53. Tissue inhibitors of metalloproteinases (TIMPs) The first TIMP was described in 1975 as a protein, in culture medium of human fibroblasts and in human serum, which was able to inhibit collagenase activity. Currently, four TIMPs (TIMP 1–4) are known: TIMP-1 ,TIMP-2 , TIMP-3 and TIMP-4. TIMPs appear to regulate matrix degradation both by proteinase elimination and by blockage of autolytic MMP activation. (DeClerck,1991)

54. TIMPs are widely distributed in tissues and fluids and are expressed by many cell types including fibroblasts, keratinocytes , monocytes / macrophages,and endothelial cells. TIMP-1 is a 28 .5kDa glycosylated protein . TIMP-1 forms complexes with active collagenase, but not procollagenase

55. TIMP-2, a unglycosylated protein of 21.5 kDa and is expressed by fibroblasts and endothelial cells and perhaps by other cells as well. It regulates the activation of pro-MMP-2 by binding to its c-terminal region.

56. TIMP -3 is a 27 kDa glycoprotein,expressed by a variety of cells. It has an affinity for components of the ECM. TIMP-3 Prevents the activation of pro MMP -2 by MT2-MMP.

57. Inhibitors of MMPs fall into three pharmacologic categories: Collagen peptidomimetics and nonpeptidomimetics , Peptidomimetic MMP Inhibitors Batimastat . Marimastat Nonpeptidic MMP Inhibitors BAY 12–9566. AG3340. BMS-275291 Tetracycline derivatives, Doxycycline. Col-3 ( metastat ). Bisphosphonates.

58. EXOGENOUS (SYNTHETIC) MMP INHIBITORS Agent Class MMP Inhibition Batimastat Peptido -mimetic MMP-1, 2, 3, 7, 9 Marimastat Peptido -mimetic MMP-1, 2, 3, 7, 9, 12 AG3340 ( prinomastat ) Nonpeptido -mimetic MMP-2, 3, 9, 13, 14 BAY 12-9566 Nonpeptido -mimetic MMP-2, 3, 9 MMI270 Nonpeptido-mimetic MMP-1, 2, 3, 9, 13, 14 COL-3 ( metastat ) Tetracycline derivative MMP-2, 9 BMS-275291 Nonpeptido-mimetic MMP-2, 9 CP-471,358 Nonpeptido-mimetic MMP-2, 3, 8, 9, 12, 13, 14 AE-941( neovastat ) Shark cartilage extract MMP-1, 2, 7, 9, 12, 13

59. Peptidomimetic MMP Inhibitors These compounds are pseudopeptide derivatives reversibly binds at the active site of the MMP in a stereospecific manner and chelates the zinc atom on the enzyme activation site Batimastat Batimastat (the first MMP inhibitor) is a nonorally bioavailable low-molecular weight hydroxamate . Because of its poor solubility, it is administered intraperitoneally and intrapleurally . Batimastat is relatively well tolerated, except in patients without malignant ascites, in whom intraperitoneal administration of the agent resulted in significant abdominal pain . Systemic toxicity has been mild and of no clinical consequences. Plasma levels of batimastat peaked 24–48 hours after intracavitary administration and were characterized by a prolonged elimination half-life of approximately 3–4 weeks

60. Marimastat is a synthetic low-molecular-weight MMP inhibitor that, in contrast to batimastat , is orally bioavailable, with an absolute bioavailability of 20%–50%.It contains a collagen-mimicking hydroxamate structure that chelates the zinc ion at the active site of MMPs. Like batimastat , marimastat is relatively nonspecific, inhibiting the activity of MMP-1, -2 , -3, -7, and - 9 NON PEPTIDE INHIBITORS BAY 12–9566 is an orally bioavailable biphenyl compound that is a potent inhibitor of MMP-2, -3, and -9. The drug has been very well tolerated, resulting in only mild thrombocytopenia and minor elevations in liver function tests, of no clinical consequences. AG3340 is a nonpeptidic collagen-mimicking MMP inhibitor and inhibits MMP -2, - 9, -3, and - 13.

61. BMS-275291 is an orally bioavailable MMP Inhibitor and demonstrated potent inhibitory activity against MMP-2 and MMP-9 CHEMICALLY MODIFIED TETRACYCLINES: In a series of in vitro experiments addressing mechanisms, Golub and co-workers investigated the specificity of anti- collagenolytic activity of TCs and located the site on the TC molecule responsible for the host-modulating, non-antimicrobial properties (Golub et al. 1987, 1991)

62. CMT-1 , 4-dedimethyl amino tetracycline, CMT-2 , tetracyclinenitrile ; CMT-3 , 4-dedimethylamino sancycline ; CMT-4 , 4-dedimethylaminochlorotetracycline; CMT-5 , tetracycline pyrazole ; CMT-6 , 4-dedimethylamino 4-hydroxytetracycline; CMT-7 , 12_-deoxy-4-dedimethyl amino tetracycline; CMT-8, 4-dedimethylamino doxycycline.1992a,b)

63. Regarding the site of the TC molecule responsible for its anti-MMP activity, these investigators produced the first series of chemically modified TCs (CMTs) by removing the dimethylamino group from the carbon-4 position,the side-chain, which is required for the antimicrobial activity of TCs F ew other modifications were made to the core tetracycline structures by addition or deletion of functional groups and eight different analogues were initially synthesized. Numerous preclinical studies were pursued once this new role of TCs was known, and most of these studies have demonstrated that TCs or CMTs can inhibit MMP activity and connective tissue breakdown in vivo and in vitro in various experimental models .

64. CMTs have several potential advantages over conventional TCs; notably, their long-term systemic administration does not result in gastrointestinal toxicity, and higher plasma concentrations can be reached for prolonged time periods, which allows for less frequent administration regimens. Bisphosphonates As MMP Inhibitors Bisphosphonates may directly inhibit the activity of several MMPs as tiludronate inhibit MMP-1 and MMP-3 but did not affect MMP production in periodontal ligament cells. Chlodronate was able to inhibit MMP-8 in periimplant sulcus fluid

65. Role of Matrix metalloproteinase in periodontal disease Matrix metalloproteinases play key roles in the degradation of the extracellular matrix, basement membrane as well as in the modification of cytokine action and activation of osteoclasts. The expression and activity of MMPs in non- inflamed periodontium is low but is drastically enhanced in the inflammatory conditions.

66. The evidence that matrix metalloproteinases are involved in tissue destruction in human periodontal disease has been summarized by Birkedal -Hansen (1993) and includes the following: 1) cells isolated from normal and inflamed gingiva are capable of expressing a wide variety of matrix metalloproteinase in culture 2) several matrix metalloproteinases can be detected from the gingiva in vivo; and 3) matrix metalloproteinase-8 and matrix metalloproteinase- 3 are readily detected in gingival crevicular fluid from gingivitis and periodontitis patients.

67. Both resident gingival and periodontal ligament fibroblasts produce collagenases that are thought to be involved in normal tissue turnover. Inflammatory cells such as neutrophils and macrophages produce MMPs, with neutrophils being the major source of collagenase and gelatinase in inflammatory diseases such as periodontitis.

68. Epithelial cells can also produce elevated levels of these enzymes, which may facilitate the apical migration and lateral extension of the junctional epithelium and the subsequent loss of connective tissue attachment. Inflammatory cells, particularly neutrophils , are thought to play a particularly important role in the MMP-mediated periodontal destructive lesion .

69. Matrix Metalloproteinases and Bone Resorption Osteoclast express, along with cathepsin K, several MMPs, which together with periosteoblastic cell and osteoblast -derived MMPs contribute to bone resorption. ( Parikka ,2005) Osteoblast -derived collagenase (MMP-13) seems to be main responsible for degrading the nonmineralized osteoid layer covering bone surfaces, exposing the mineralized matrix to osteoclasts .

70. MMPs are critical for osteoclast access to the resorption site, since MMP inhibition prevents cell migration ( Blavier , 1995) and MMP-9 and -14 seem to be the key proteinases in this respect. ( Engsig ,2001). MMP-14 is located in the ruffled border of osteoclasts, possibly contributing to the osteoclastmatrix interaction that controls osteoclast attachment and detachment to bone . (Sato ,1997)

71. MMPs may also contribute to osteoclastic bone resorption by regulating osteoclast recruitment and activity by, for example, releasing cytokines and growth factors (such as TGF-b by MMP-9 or RANKL by MMP-14) from bone matrix, or by regulating the messenger binding to the receptors. [ Delaisse ,2003]

72. The importance of osteoblastic MMPs on bone development and formation has only recently been realized. At least MMP-2, -9, -13, and -14 are considered important in bone development and formation . (Johansson N,1997) MMP-14 also participates in normal bone homeostasis: MMP-14-activated TGF- β inhibits apoptosis and maintains osteoblast survival during osteoblast trans-differentiation into osteocyte . (Karsdal,2002) MMP-14 is also essential in maintaining osteocyte viability ( Holmbeck K,2005 ) , thus significantly contributing to normal bone development and repair.

73. Sampling sources for matrix metalloproteinases Saliva and gingival crevicular fluid are potential sample sources for matrix metalloproteinases or their inhibitors. Both sources differ in that the saliva sample is a general oral sample, whereas the gingival crevicular fluid is a site-specific sample.

74. Matrix metalloproteinases in gingival crevicular fluid Collagenase activity increased with disease severity in gingival crevicular fluid, that is adult periodontitis and localized juvenile periodontitis levels were greater than gingivitis levels, which were greater than normal . ( Larive et al, 1986) Ingman et al. (1993) demonstrated that saliva samples did not reflect periodontal tissue destruction clearly, as they found only a low amount of total collagenase activity in saliva of untreated localized juvenile periodontitis patient, and the level was comparable to normal patients .

75. According to Haerian et al. (1995) , there are significantly higher levels of tissue inhibitor of matrix metalloproteinase in gingival crevicular fluid from healthy as compared with diseased periodontitis sites.

76. Lee et al. (1995) suggested that performing functional testing for actual collagenase activity may be the best method of detecting destructive periodontitis. This type of anlaysis is more complex than enzyme-linked immunosorbent assay detection of matrix metalloproteases and would be unlikely to be easily transferred to a chairside test kit. In terms of deciding what would be the best candidate matrix metalloproteinase to consider as a diagnostic tool for active periodontitis .

77. Romanelli et al. (1999) has highlighted the importance of matrix metalloproteinase- 8. This metalloproteinase is detected in its active form in gingival crevicular fluid associated with periodontitis, whereas it is mostly found in the latent form in gingivitis. Distinct forms of latent and active gingival crevicular fluid matrix metalloproteinase- 8 have been noted in gingival crevicular fluid of periodontitis patients, which appear to result from a unique activation mechanism that occurs in periodontitis.

78. Nomura et al. (1998) have recently considered the relative importance of multiple matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in periodontal disease. They employed a principal component analysis that revealed that 87% of periodontal disease activity could be explained by matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 and -2.

79. Diagnostic test for MMPs Mantyla et al,2003 develop monoclonal antibodies for MMP-8 which can be utilized in a chair-side test for MMP-8. This leads to the development of a chair-side dip-stick test for MMP-8 and it is useful in detecting MMP-8 levels in GCF and peri -implant sulcular fluid (PISF). This test measures the GCF MMP-8 level in 5 minutes and can be performed by a dentist rapidly and without any specific equipment. It differentiates healthy and gingivitis sites from periodontitis sites. Also reduction in MMP-8 levels can be observed after successful periodontal treatment.

80. CONCLUSION MMPs are the important family of endopeptidases capable of degrading ECM and basement membrane leading to the periodontal diseases. These MMPs are mainly responsible for the degradation of collagen fibers. High levels of activity are found mainly in gingival crevicular fluid in inflammatory conditions like periodontitis, cancer etc. Therefore detection and reduction of these levels are important for inhibiting the progressive lesions. So, the therapeutic interventions should focus on reducing the levels of activity MMPs.

81. REFERENCES Newman, klokkevold , Takei, Caranzaa – clinical periodontology – 10 th edition. New man, klokkevold , Takei, Caranzaa – clinical periodontology – 11 th edition . Role of Matrix Metalloproteinases in Human Periodontal Diseases. J Periodontol 1993; 64:474- 484. Modulation of matrix metalloproteinase activities in periodontitis as a treatment strategy. Periodontology 2000, Vol. 24, 226–238. Regulators of tissue destruction and homeostasis as diagnostic aids in periodontology . Periodontology 2000, Vol. 24, 215–225. Matrix Metalloproteinases & Implication in Periodontitis- A Short Review. Journal of Dental & Allied Sciences 2013;2(2):66-70

82. Role of matrix metalloproteinases (MMPS) in periodontitis its management. Journal of Indian Academy of Dental Specialist Researchers Vol. 1:Issue 2; Jul-Dec 2014 The role of matrix metalloproteinases in the oral environment. Acta Odontologica Scandinavica , 2007; 65: 113. Matrix metalloproteinases – an overview. Research and Reports in Biology 2010:1 1–20. Matrix metalloproteinases : the most important pathway involved with periodontal destruction. Braz J Oral Sci. October-December 2005 - Vol. 4 - Number 15

83. Thank you