J Orofac Sci, 2(1)2010

Journal of
OROFACIAL SCIENCES

Review
Matrix Metalloproteinases
Sahitya.Sa*, Babitha Nugalaa, Santosh Kumar B.Ba
a
Department of Periodontics, SIBAR Institute of Dental Sciences, Guntur, India.

ARTICLE INFO ABSTRACT

Article History : Matrix metalloproteinases [MMPs] are a group of enzymes that are responsible
Received : 25 March 2010 for degradation of most of the extracellular matrix proteins during organogenesis,
Accepted : 07 April 2010 growth and normal tissue turnover. The expression and activity of MMPs in
adult tissue normally is quite low, but increases significantly in various
pathological conditions that may lead to unwanted tissue destruction, such as
Key Words : inflammatory diseases, tumour growth and metastasis. They also have a marked
Matrix metalloproteinases role in tissue destructive oral diseases. Among MMPs, MMP-8 plays a major
TIMPs role in Periodontitis and is the best-known example of the unwanted tissue
Tetracyclines destruction related to increased presence and activity of MMPs at the site of
Sub Antimicrobial dose doxycycline. disease. This review focuses on the role of MMPs in periodontal disease.
©2010 SIDS.All Rights Reserved

INTRODUCTION :
The matrix metalloproteinases (MMPs) are a family matrix plate. Therefore, the enzyme was named as
of zinc and calcium dependent endopeptidases secreted interstitial collagenase (MMP-1).
by a variety of infiltrating cells i.e., neutrophils and STRUCTURE OF MMPs :
macrophages and resident cells like fibroblast, epithelia,
The MMPs share a common domain structure. The
osteoblast and osteoclast found in the periodontium.
three common domains are the pro-peptide, the catalytic
MMPs function at a neutral PH to degrade constituents
of extracellular matrix. A number of physiologic events
like embryonic development and tissue remodeling and
pathologic conditions like periodontitis, arthritis and
cancer are characterized by MMP activity1.
HISTORY :
Initially, MMPs were described by Jerome Gross and
Charles Lapiere 2(1962) who observed enzymatic activity
(collagen triple helix degradation) during tadpole tail
metamorphosis by placing a tadpole tail in a collagen
* Corresponding author :
Dr. Sahitya .S, Senior lecturer,
SIBAR Institute of Dental Sciences,
Guntur, Andhra Pradesh, India.
e- mail : sahityaveera@yahoo.com
Schematic representation of MMPs
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 J Orofac Sci, 2(1)2010
domain and the haemopexin-like C-terminal domain
which is linked to the catalytic domain by a flexible hinge
region.
CLASSIFICATION :
MMPs are divided into six subgroups based on their
substrate specificity
1. Collagenases - MMP – 1, MMP- 8, MMP - 13
2. Gelatinases – MMP -2, MMP -9
3. Stromelysins – MMP -3, MMP-10, MMP - 11
4. Membrane-type MMPs (MT-MMPs) – MMP- 14,
MMP-15, MMP- 16, MMP-17, MMP-24, MMP-25
5. Matrilysins - MMP- 7, MMP -26
6. Other MMPs- MMP -12, MMP- 18, MMP-19,
MMP-21, MMP-23A, MMP-23B, MMP-27,
MMP-28.
COLLAGENASES :
Collagenase family of MMPS currently contain 3
members,
1. Interstitial Collagenase à MMP-1
2. Neutrophil Collagenase à MMP-8
3. Collagenase 3 à MMP-13
The Collagenases play an important role in the
initiation of remodeling of an existing collagen network
as they are the only MMP’s capable of degrading the triple
helical fibrillar collagens (Type I, II, III). All three
Collagenases catalyse the cleavage of these collagens
exclusively at a single specific locus producing a
characteristic three quarters/ one quarter digestion
products that are soluble and susceptible to further
degradation by gelatinases and stromelysins3 ( Brikedal –
Hansen etal1993)
MMP-1 : preferably degrades collagen. Human
MMP-1 was first cloned and sequenced from skin
fibroblasts. Since then it has been found to be produced
by keratinocytes, endothelial cells, monocytes and
macrophages, chondrocytes, osteoblasts and various
tumour cells.
MMP-8 degrades type I collagen and is synthesized and
stored in PMN cells during their maturation in bone
marrow and released upon PMN degranulation induced
by various extracellular stimuli, like cytokines or bacterial
contact. The main substrates for MMP-8 are fibrillar
collagens. MMP-8 is catalytically more effective in
cleaving substrates (except collagen III) than MMP-1.
Increased MMP-8 levels are detected in many
inflammatory diseases like rheumatoid arthritis, chronic
lung inflammations and periodontitis.
MMP-13 degrades type II collagen and has the widest
substrate selection and is able to cleave various basement
membran components. MMP-13 cleaves type II collagen
more efficiently than type I and III and it is most effective
in cleaving gelatin.
STROMELYSINS :
The stromelysin MMP sub-family has four membranes
1. Stromelysin-1 (MMP-3)
2. Stromelysin-2 (MMP-10)
3. Stromelysin-3 (MMP-11)
4. Truncated matrilysin (MMP-7)
Stromelysin-1 was initially identified in rabbit synovial
fibroblasts as a 51 kilodalton polypeptide which, in its
purified active form showed proteolytic activity against
L-chain casein, fibronectin, gelatin, laminin, elastin,
cartilage proteoglycans, type IV collagen (Chin etal) but
no activity against type V collagen was detectable4.
Stromelysin-2 enzyme has been associated with
malignant tumors and is capable of activating
procollagenase (MMP-I) and degrading types III, IV, V
collagens, fibronectin and gelatin.
Stromelysin-3 has been associated with malignant
tumors.
Matrilysin in its active form can degrade casein, gelatins
and fibronectin but not native collagens (I, II, IV, V).
GELATINASES :
The gelatinases family contains two members-
1. 72 KD type IV collagenase & gelatinase-A - (MMP-2)
2. 95 KD gelatinase-B - (MMP-9)
These enzymes are thought to act co-operatively with
the collagenases to effect the complete degradation of
interstitial collagens, elastin, fibronectin, type IV,V,VII
collagens and gelatins but show no activity against laminin
or interstitial collagens
Difference between the two gelatinases are
demonstrated by the selective association with Tissue
inhibitors of matrix metalloproteinasesTIMP’s where in
the gelatinase A preferentiality binds to TIMP-2 where
as gelatinase-B binds to TIMP-15 (Kolkendbrock
etal 1995)
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Membrane type Metalloproteinases (MT-MMPs) :
The membrane type MMPs are the most recently
characterized MMP subfamily of four members
MT1-MMP-(MMP-14)
(Degrade collagen type I, II, III & plays an important
role in angiogenesis).
MT2-MMP - (MMP-15)
MT3-MMP - (MMP-16)
MT4-MMP - (MMP-17)
In addition to pro-gelatnase-A activation, the
MT-MMP’s are also active at degrading gelatins & type
I, II, III collagens into ¾ and ¼ products.
OTHER METALLOPROTEINASES :
In recent years number of MMP’s have been identified
and has continued to grow, although all newly
characterized MMP’s cannot be easily grouped into sub-
families described above. Recently MMP-18 has been
identified6 and its expression has been localized to placenta,
lung, pancreas, ovary, small intestine, spleen, thymus and
prostrate.
More recently pendas etal 7 isolated MMP-19
complimentary DNA (c –DNA) from a human liver
which exhibited many of the domain structural
characteristics of MMP’s
Functions of MMPs :
1. Physiological functions of MMPs :
a. Remodeling :
In the dynamic matrix, homeostasis is maintained by
a constant degradation and resynthesis of matrix
components. The activity of the MMPs is central to both
the general maintenance of the matrix and affecting the
matrix remodeling during such physiological process.
b. Wound repair :
Wound healing requires a sequential matrix
degradation, cell migration, synthesis of a provisional
matrix consisting of fibronectin, fibrin and high amounts
of collagen type II and matrix remodelling to return the
tissue to normality. MMP-9 expression has been linked
with the human mucosa, possibly participation in the
detachment of keratinocytes from the basement
membrane, migration into the wound and remodeling
of the granulation tissue matrix. Increased levels of
interstitial collagenase (MMP-1,MMP-9,MMP-3,
MMP-10) have been identified in healing burn wounds,
ulcers and cultures of chemically injured epithelium.
c. Angiogenesis :
Angiogenesis plays an important role in physiologic
morphogenesis and pathological process. The matrix
metalloproteinases play a major role in the dissolution
of the basement membrane and remodeling of the matrix
composition necessary for endothelial invasion during
angiogenesis.
2. Pathologic role of MMPs :
Various components of the extracellular matrix and
basement membrane exert a tight negative regulation on
the activities of matrix resident cells, particularly the
cellular migration and proliferation. Basement membrane
act as macro molecular filters and represent physical cell
migration. Its components such as type IV collagen and
heparin sulfate, proteoglycans help to maintain cells in a
rarely dividing quiescent state. These negative effects after
balance the stimulatory and chemotactic effects of locally
produced growth factors and cytokines and so help
maintain tissue homeostasis.
Inappropriate matrix remodeling that occurs during
the development and progression of various pathological
disorders can often swing the balance in favour of cellular
proliferation, migration and tissue destruction. The
MMPS have been implicated in mediating such
inappropriate matrix remodeling.
Role of MMPs in the pathogenesis of periodontal disease :
Significant evidence exists to show that collagenases,
along with other MMPs, play an important role in
periodontal destruction. MMPs, produced by both
infiltrating and resident cells of the periodontium, play a
role in physiological [such as tooth eruption] and
pathological [such as periodontitis] events.
Specifically, these enzymes are responsible for the
degradation of the collagen fibers attached to the root
surface, allowing for the apical migration and lateral
extension of the pocket epithelium. The clinical sequelae
of the pathological increase in collagen destruction are
loss of attachment and the formation of periodontal
pockets.
With the initiation of disease by bacteria, the associated
inflammatory response overrides its protective role and
permits destruction to occur. The earliest stages of matrix
destruction occur with the emigration of
polymorphonuclear leucocytes to make way for further
cell migration and population.
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With ongoing disease the gingival connective tissue is
flooded with lymphocytes, macrophages, and a plethora
of chemokines, lymphokines and other inflammatory
mediators. The mediators as well as the MMPs are present
in increased concentrations in the gingival crevicular fluid.
Excessive quantities of MMPs are released in inflamed
tissues, resulting in breakdown of connective tissue matrix.
The predominant MMPs in Periodontitis, particularly
MMP- 8 and MMP-9 derived from PMNs are extremely
effective in degrading type I collagen. Levels of PMN-
type MMPs have been shown to increase with severity of
periodontal disease. The release of large quantities of
MMPs in the periodontium leads to significant anatomic
disruption and breakdown of connective tissues,
contributing to the clinical signs of Periodontitis.
Bone resorption:
MMPs also play a role in bone remodeling. Along
with cathepsin k, osteoclasts express several MMPs which
together with perioblastic cell and osteoblast derived
MMPs contribute to bone resorption. MMPs are critical
for osteoclast access to the resorption site, since MMP
inhibition prevents cell migration and MMP-9 and
MMP-14 seem to be the key proteinases in this respect.
MMP-14 is located in the ruffled border of osteoclast,
possibly contributing to the osteoclast matrix interaction
that controls osteoclast attachment and detachment to
bone. MMP-13 is present in resorption lacunae and it is
essential for removal of collagen remanents left over by
osteoclast. MMP-2, 9, 13, 14 are considered important
in bone development and formation MMP-14
participates in normal bone haemostasis .
MMP activation :
MMPs are mostly produced in latent, non active form
and activation through a so called cysteine switch is
required for the enzyme function . In most cases ,
activation involves removal of the prodomain, resulting
into lower molecular weight active forms8.
Activation of MMPs is by 2 methods.
1. Non- proteolyic activation
2. Proteolytic activation.
Non-proteolytic activation of MMP proforms can
be accomplished invitro, e.g. by SH- reactive agents, such
as mercurial compounds, detergents or oxidation.
Proteolytic activation can be attained by several
proteolytic enzymes, including serine proteinases together
with other MMP. Most likely MMP activation in vivo
involves tissue and plasma proteinases and bacterial
proteinases together with oxidation stress9. Secreted
MMPs are usually activated extracellularly or at the cell
surface, the best-known example of cell surface activation
being the activation of MMP-2 in a MMP-2/TIMP-2/
MT1-MMP complex.
MMP Inhibtion :
The role of inhibitors is particularly important because
its an imbalance between the activated MMPs and their
inhibitors that leads to pathological breakdown of the
extracellular matrix to disease such as periodontitis and
arthritis10. Compensating for the deficit in the naturally
accruing inhibitors or TIMPs to block or retard the
proteolytic destruction of connective tissue is of
therapeutic significance. This can be accomplished with
the use of drugs that can
1. Inhibit the synthesis and or release of these enzymes
2. Block the activation of precursor (latent) form of their
MMPs.
3. Inhibit the activity of mature MMPs.
4. Stimulate the synthesis endogenous TIMPs or
5. Protect the hosts endogenous inhibitors from
proteolytic inactivation
The tetracycline, which may modulate many of these
matrix protective mechanisms, have been found to be
effective of MMPs mediated connective tissue
destruction in variety of pathological processes.
MMP Inhibitors
A. Endogenous inhibitors : are natural inhibitors such
as TIMPs and µ2- macroglobulins bind in a non-
covalent fashion to member of MMP family.
TIMPs probably control MMP activities
pericellularly, secreted by various cells found in serum
and human saliva, present in high concentration in
healthy sites. µ2- macroglobulin functions as a
regulation of MMPs in body fluids. During
inflammation, the high molecular wt protein may
escape the vasculature and also function in the
extracellular matrix.
TIMPs :
Form high affinity, essentially irreversible non-covalent
complex with the active form of MMPs.
1. TIMP-1 : 30 KDa glycoprotein, synthesized and
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secreted by most connective tissue and macrophages.
It binds to pro-gelatinase-B.
2. TIMP–2 : 21 KDa unglycolated proteins have 40%
sequence identity with human TIMP-1. It binds to
the proform of gelatinase A and involved in activation.
3. TIMP -3 : Isolated from chicken cells and cloned
from human and mouse sources. It almost
exclusively bonds to extracellular matrix.
4. TIMP - 4 : Recently isolated.
B. Exogenous (Synthetic) Inhibitors :
1. Zn+ and Ca2+ chelating agents :
E.g. EDTA
2. Phosphorus Containing Peptides : Produced by the
substitution of tetrahedral phosphorus atom from the
carbonyl carbon atom in a peptide substrate.
Ex : Phosphonamidate
3. Sulfur Based Inhibitors : Most potent inhibitors of
collagenases, gelatinases and stromelysins.
Ex : Mercapton derivatives.
4. Peptidyl Hydroxamic Acid Derivatives : They
Inhibits MMP – 1,2,3,7,8 and 9 in- vitro, shows
50% decline in collagenase activity
Eg : Galardin – for topical use in non-healing corneal ulcer.
Batimastat – for treatment of breast cancer, parentally
administered
Marimastat – orally administrated for treatment of
cancers including ovaries prostatic, pancreatic and GI
malignancies.
MMPs in host modulation therapy :
Recognition of the need to restore the natural balance
between tissue destruction enzymes and their inhibitors
led to the development of a disease management strategy
that focused on the modulation of MMPs involved in
the terminal catabolic component of the host response.
It is evident that TIMPs are not sufficient to down regulate
the pathologically elevated MMPs11. Therefore, the
possibility of selective MMP inhibition by synthetic
inhibitors as a method to avoid or limit the periodontal
tissue destruction was advanced. In a series of studies
carried out by Golub12, he demonstrated that most of
the MMPs are sensitive to tetracyclines and sub anti
microbial dose doxycycline (SDD).
TETRACYCLINES :
The tetracyclines are primarily bacteriostatic, inhibit
protein synthesis by binding to 30s ribosomes in
susceptible organism. Tetracyclines have been widely used
in the treatment of periodontal disease. They have the
ability to concentrate in the periodontal tissues and inhibit
the growth of microorganisms. In addition, tetracyclines
exert an anticollagenase effect that can inhibit tissue
destruction and may aid in bone regeneration.
Tetracyclines are effective in treating periodontal
diseases in part because their concentration in the gingival
crevice in 2 to 10 times that in serum. This allows a high
drug concentration to be delivered into periodontal
pockets. Tetracyclines have been found to inhibit host
derived collagenases and some other MMPs by a
mechanism independent of the well known antimicrobial
activity of these drugs. The first discovery that tetracyclines
can inhibit host derived MMPs by a mechanism
independent to their antimicrobial properties was made
in germ free rats with experimentally induced diabetes, a
model of excess collagenase activity13.
Available antimicrobial tetracyclines with these properties
are minocycline, doxycycline and tetracycline itself.
The mechanisms by which the tetracyclines inhibit
MMPs are not completely understood, but they are
thought to act via both direct and indirect mechanisms.
Tetracyclines bind to Zn or Ca associated with the MMP,
blocking the active site or inducing conformational
changes that render the proenzyme susceptible to
fragmentation during activation.
Tetracyclines inhibit connective tissue breakdown :
A. Mediated by extracellular mechanisms
1. Direct inhibition of active matrix metalloproteinases
– dependent on Ca and Zn¹¹ binding properties of
tetracyclines.
2. Inhibition of oxidative activation of pro-matrix
metalloproteinases – independent of cation binding
properties of tetracyclines.
3. They disrupt activation by promoting excessive
proteolysis of pro-matrix metalloproteinases into
enzymatically-inactive fragments – dependent on
cation binding of tetracyclines.
4. Inhibition of matrix metalloproteinases protects µ1-
proteinase inhibitor, thus indirectly decrease serine
proteinase (such as polymorphonuclear leukocytes
elastase) activity.
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B. Mediated by cellular regulation:
Tetracyclines decrease cytokines, inducible nitric
oxide synthase, phospholipase A2, prostaglandin
synthase
C. Mediated by pro-anabolic effects
1. Increase collagen production
2. Increase osteoblast activity and bone
Chemically Modified Tetracyclines(CMTs) :
The first chemically modified tetracycline was
described in 1987, soon after it was discovered that
tetracyclines could inhibit mammal-derived matrix
metalloproteinases (MMPs) by mechanisms which were
independent of the antibacterial efficacy of these drugs.
Since then, more than 30 different CMTs, in which the
4-dimethylamino group has been deleted, have been
developed14. The most prominent characteristic of these
CMTs is their loss of antibacterial activity, accompanied
by retention (or even enhancement) of their efficacy as
inhibitors of MMPs. The CMTs do not appear to result
in the side effect of antibacterial tetracyclines, namely,
the emergence of antibiotic-resistant bacteria. They have
been tested for their efficacy as inhibitors of connective
tissue breakdown, including the preservation of bone and
cartilage, in a variety of animal models of diseases,
including (but not limited to) arthritis, osteoporosis,
aortic aneurysms, periodontitis, and cancer.
There are different CMTs from CMT -1 to
CMT -9.
Modification by replacement of the carbon 11
carbonyl oxygen and the carbon 12 hydroxyl groups with
nitrogen, abolishes the anti-MMP activity, giving rise to
CMT-5, which is neither antibiotic nor anti-MMP.
Mechanism of action :
The mechanism proposed to explain these
anticollagenase properties concerned the Zn2+ and Ca2+
binding properties of the tetracyclines molecule and
explained the ability of tetracyclines to inhibit already
active collagenase or gelatinase in the extracellular matrix.
It is supported by several pieces of recent evidence. Firstly,
adding excess Zn2+ or Ca2+ ions eliminates this property.
Secondly, collagenase now appears to contain secondary
Zn2+ and Ca2+ ions outside the catalytic domain of the
enzyme, in addition to those within this domain. These
secondary ions help to maintain the conformity and
catalytic activity of the enzyme. The proposed mechanism
suggested that tetracyclines interacted with these secondary
metal ions in particular Zn2+. Thirdly, the mechanism of
its action is not associated with fragmentation of the
MMP molecule and the results of in vitro experiments
suggest a noncompetitive action of these drugs. Thus,
these findings suggest that tetracyclines other than
CMT-5 may bind secondary Zn2+ and to a lesser extent
Ca2+ in collagenase, thus altering the conformity of the
enzyme molecule and blocking its catalytic activity.
The CMT that lost its anticollagenase activity was
CMT-5 and this was the pyrazole analogue, in which the
carbon-11 carbonyl oxygen and carbon-12 hydroxy
groups were replaced by nitrogen atoms. This eliminated
the important Zn2+ and Ca2+ binding site in the tetracycline
molecule, which is active at physiological pH.
Tetracyclines and chemically modified tetracyclines
have been found to inhibit bone resorption at
concentrations within the normal therapeutic dose range
of these drugs. These appear to reduce the degradation of
osteoid by inhibition of osteoblast collagenase.
They also appear to increase bone formation by
increasing alkaline phosphatase and collagen synthesis by
these cells, they appear to decrease bone resorption by
elevation osteoclast ruffled border, decreasing osteoclast
acid production, decreasing osteoclast secretion of cysteine
proteinases and inhibiting osteoclast MMPs.
Sub-Antimicrobial Dose Doxycycline (SDD) :
Sub antimicrobial dose doxycycline [SDD] is a 20mg
dose of doxycycline [Periostat] that is approved and
indicated as an adjunct to SRP in the treatment of chronic
periodontitis. It is taken twice daily for 3 months up to
a maximum of 9 months of continuous dosing. The
20mg dose exerts its therapeutic effect by enzyme,
cytokine and osteoclast inhibition rather than by any
antibiotic effect.
Research studies have found no detectable
antimicrobial effect on the oral flora or the bacterial flora
in other regions of the body and have identified clinical
benefit when used as and adjunct to SRP.
At present, SDD is the only host modulatory therapy
specifically indicated for the treatment of chronic
periodontitis that is approved by the US Food and Drug
Administration [FDA] and accepted by American Dental
Association [ADA].Doxycycline has led to improvement
in both the periodontal health of compromised diabetic
patients and long term markers of glycemic control15 [e.g.
glycated hemoglobin].
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In addition to its antibiotic properties, the rationale
for using SDD as an HMT in the treatment of
periodontitis is that doxycycline down regulates the
activity of MMP’s by a variety of synergistic mechanisms
including – reduction in cytokine levels, stimulates
osteoblastic activity and new bone formation by up
regulating collagen production.
MMP inhibition is a promising approach in
periodontal disease treatment. Further work on other
approaches need to be evaluated.
Diagnostic kit for MMPs :
Monoclonal antibodies have been developed to detect
MMP -8 chair side. These antibodies are utilized in a
chair side dip stick test for MMP -8 that allowed the
development of a novel sensitive, specific, rapid and
practical immunological chair side dip stick test for
MMP-8 in GCF. The test bearing resemblance to
pregnancy home test kits can be performed by a dentist
without specific equipment and measures the
GCF MMP - 8 in 5 minutes. It differentiates healthy
and gingivitis sites from Periodontitis sites and reduction
of GCF MMP-8 levels can be observed after successful
periodontal treatment16.
CONCLUSION :
MMPs seem to be related to tissue destruction in
Periodontitis. MMP inhibition is a promising approach
in periodontal disease treatment. Further development
of diagnostic technology may allow the use of one or
more MMPs, TIMPs and tissue degradation product in
a combination chair side tests for Periodontitis and can
also be adapted for monitoring of other diseases.
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