Matrix metalloproteinases: Contribution to pathogenesis, diagnosis and treatment of periodontal inflammation

Abstract

Matrix metalloproteinases (MMPs) form a family of enzymes that mediate multiple functions both in the tissue destruction and immune responses related to periodontal inflammation. The expression and activity of MMPs in non‐inflamed periodontium is low but is drastically enhanced to pathologically elevated levels due to the dental plaque and infection‐induced periodontal inflammation. Soft and hard tissue destruction during periodontitis and peri‐implantitis are thought to reflect a cascade of events involving bacterial virulence factors/enzymes, pro‐inflammatory cytokines, reactive oxygen species and MMPs. However, recent studies suggest that MMPs can also exert anti‐inflammatory effects in defence of the host by processing anti‐inflammatory cytokines and chemokines, as well as by regulating apoptotic and immune responses. MMP‐inhibitor (MMPI)‐drugs, such as doxycycline, can be used as adjunctive medication to augment both the scaling and root planing‐treatment of periodontitis locally and to reduce inflammation systematically. Furthermore, MMPs present in oral fluids (gingival crevicular fluid (GCF), peri‐implant sulcular fluid (PISF), mouth‐rinses and saliva) can be utilized to develop new non‐invasive, chair/bed‐side, point‐of‐care diagnostics for periodontitis and dental peri‐implantitis.

• Chair‐side diagnostics
• doxycycline
• gingival crevicular fluid
• inflammation
• matrix metalloproteinase
• periodontal disease

Abbreviations
AP‐1	=	activating protein‐1
(CA)n	=	microsatellite containing tandem repeats of deoxy (cytidine, adenine) sequences
ECM	=	extracellular matrix
EDTA	=	ethylenediamine tetraacetic acid
FDA	=	Food and Drug Administration
IFMA	=	time‐resolved immunofluorometric assay
LPS	=	lipopolysaccharide
RUNT	=	RUNT‐domain containing transcription factor
RUNX	=	RUNX‐related protein containing transcription factor
TGF	=	transforming growth factor
TPA	=	tetradecanoylphorbol acetate

Introduction

Four decades ago, the first collagenolytic enzyme was discovered 1. This collagenase (MMP‐1) was responsible for the involution of the tadpole tail during amphibian morphogenesis 1. Today, it is recognized that the matrix metalloproteinases (MMPs) constitute a multigene family of over 25 structurally related but genetically distinct secreted or cell surface‐associated proteolytic enzymes that can process or degrade numerous extracellular, pericellular and non‐matrix substrates 2–8. In order to classify the MMP superfamily it is important to recognize their specific characteristics. Each MMP consists of a specific sequence of domains with several different motifs 2–8. This domain structure includes the signal peptide, the propeptide domain, the catalytic domain, and the C‐terminal hemopexin‐like domain, which are present in almost all MMPs 2–8. However, certain MMPs have additional domains, such as a transmembrane or a cytoplasmic domain 2–8. To date 25 members of the MMP family have been identified and characterized, including 22 found in the human genome. Structural similarities of the MMPs suggest that they have originated from a common ancestor by a series of gene duplication 2–8. MMP genes coding for MMP‐1, ‐3, ‐7, ‐8, ‐10, ‐12, ‐13, ‐20, ‐21 and ‐28 are located in the chromosome 11 at 11q21‐23, chromosome 10, and chromosome 17, respectively, and the others are located in chromosomes 1, 8, 12, 14, 16, 20 and 22 2–6. Since MMPs can collaboratively degrade almost all tissue and structural components of connective tissue matrices and basement membranes, their activities are strictly controlled at different levels 2–8. In addition, as described in detail below, certain MMPs can act as metabolic regulators by processing various non‐matrix bioactive molecules to modulate various cellular, immunologic, intracellular, apoptotic and anti‐inflammatory responses 3–5.

Key messages

• Matrix metalloproteinases (MMPs) are among the key mediators of irreversible tissue destruction in periodontitis and peri‐implantitis

• MMPs can be inhibited by doxycycline in periodontitis with beneficial clinical outcome

• MMPs can be diagnostically utilized in point‐of‐care bed/chair‐side tests

• MMP inhibition by doxycycline seemingly also exert systemically beneficial anti‐inflammatory effects

Structural and functional features of matrix metalloproteinases (MMPs)

For an enzyme to be classified as an MMP 1–8, two conserved motifs are required, namely the prodomain and the catalytic domain. The prodomain usually consists of about 80 amino acids and contains a conserved cysteine residue which can bind to the zinc atom in the catalytic domain. The disruption of this bond, usually mediated as a result of cleavage(s) of the prodomain, is required for activation of the latent proenzyme to its catalytically competent form. Serine and cysteine proteinases of mammalian and microbial origins as well as other MMPs can proteolytically process the prodomain, which results directly form this limited proteolysis, or from a subsequent autocatalytic process which results in the lower molecular weight active form(s) 2–8. The importance of calcium ions for the optimal catalytic competence of MMPs is reflected by the inhibition of the enzyme activity by metal chelators such as EDTA and the tetracyclines 2–8.

In addition to the conserved pro‐ and catalytic domains, MMPs contain various other functional domains mediating substrate specificity, recognition and interaction with other proteins and molecules 2–8. All MMPs except MMPs ‐7, ‐23 and ‐26 have a proline‐rich hinge region and a hemopexin‐like C‐terminal domain 2–8. The hemopexin domain is involved in the binding of tissue inhibitors of metalloproteinases (TIMPs) and in the cleavage of the native triple helical collagen 2–8. Both ends of the hemopexin domain contain cysteine residues, which by formation of a disulfide bridge folds the domain into a four‐bladed propeller structure 4. The two gelatinases, MMP‐2 and ‐9, contain repeats of fibronectin type II modules that are responsible for the gelatin‐binding properties 8. Membrane‐type MMPs (MT‐MMPs) and MMP‐11, ‐21, ‐23 and ‐28 contain a furin‐cleavage site between the propeptide and catalytic domain, which is proteolytic activation site for the furin‐like enzymes 8. Four of the six membrane‐type MMPs (MT1‐, MT2‐, MT3‐ and MT5‐MMP) contain a single transmembrane and a single cytosolic domain, and MT4‐ and MT6‐MMPs are connected to the cell surface with a C‐terminal hydrophobic extension which acts as a glycosyl phosphatidyl inositol (GPI)‐anchoring signal 9. These domains direct the MT‐MMPs onto a cell surface 9,10.

Collagenases

MMP‐1 (collagenase‐1) is produced as two different forms, mainly as a 52 kDa less‐glycosylated protein but also as a 57 kDa glycosylated form. After removal of the propeptide, the active 42 and 48 kDa MMP‐1 species can digest fibrillar interstitial collagens at a single specific site, Gly₇₇₅‐Ile₇₇₆ in α1(I)‐chain and Gly₇₇₅‐Leu₇₇₆ in α2(I)‐chain producing the classical αA (3/4)‐ and αB (1/4)‐cleavage products 1,2,6. Mutations in these collagenase‐cleavage sites can render the collagen fibrils resistant to MMP‐1 and other collagenases (MMP‐8, ‐13) 2,6. MMP‐1 has a wide range of other substrates, including various structural extracellular matrix (ECM) components as well as soluble non‐matrix mediators and other MMPs 2, 6.

Collagenase‐2 (MMP‐8), also called neutrophil collagenase, has previously been thought to be expressed solely by leukocytes of the polymorphonuclear (PMN) lineage cell line 2,6,11. MMP‐8 is synthesized during the maturation of PMNs in the bone marrow; it then becomes glycosylated and is prestored in the subcellular specific granules, from where it can be released by selective degranulation in response to appropriate triggering stimuli 2,6,11–14. The neutrophil (PMN)‐type MMP‐8 is more glycosylated than other MMPs including the fibroblast‐type MMP‐8 11,15; these carbohydrate moieties of PMN‐type MMP‐8 are believed to act as targeting signals directing its subcellular location into intracellular specific granules of PMNs 2,6,11,15 and may explain the sensitivity of PMN‐type MMP‐8 to activation and inactivation by reactive oxygen species 11,14,16. MMP‐8 can also be produced (de novo expressed) by articular chondrocytes 17,18, and during various inflammatory diseases such as bronchitis, asthma, periodontitis and arthritis by resident synovial and gingival fibroblasts, epithelial cells/keratinocytes, odontoblasts, oral cancer cells, monocyte/macrophages and plasma cells 15,19–27. Like MMP‐1, MMP‐8 also has wide substrate specificity; and its activities overlap with the other collagenases (MMP‐1 and ‐13) 2,6,11,28. Recently, MMP‐8 has been shown to exert unexpected anti‐inflammatory defensive and protective characteristics against the spread of experimental skin cancer and inflammatory lung diseases probably by processing anti‐inflammatory cytokines and chemokines as well as regulating inflammatory cell apoptosis and immune response 29–31.

The third member of the collagenase subfamily is collagenase‐3 (MMP‐13) which was originally cloned from a human breast cancer cDNA library 32. MMP‐13 seems to have a very restricted expression but broad substrate specificity and is a catalytically very efficient enzyme 6,33–36. MMP‐13 is expressed during bone development as well as in fetal and gingival/oral wound healing 21,27,34–36. Enhanced MMP‐13 levels have been shown to be expressed in many pathological tissue destructive conditions like arthritis, periodontal disease, chronic ulcers, atherosclerosis and many types of malignant tumours 6,21,27,32–38. MMP‐13 expression is also related to the invasive capacity of various malignant cell lines and tumours 6,32,35,38. The ability of MMP‐13 to digest type II collagen ten times more efficiently than types I or III suggests that MMP‐13 also is involved in the pathophysiology of the arthritides 33,36,37. In addition, MMP‐13 can also process chemokines and can be inhibited by tetracyclines and bisphosphonate alendronate 5,6,34–36.

Gelatinases

The two gelatinases, 72 kDa gelatinase A (MMP‐2) and 92 kDa gelatinase B (MMP‐9) 2–8, have rather similar substrate specificity 8. Both progelatinases can be isolated as complexes with TIMPs; proMMP‐2 binds to TIMP‐2 and proMMP‐9 to TIMP‐1 2–8. MMP‐2 has a characteristic activation mechanism on the cell surface involving MT‐MMPs and involving also TIMP‐2 2–10, whereas human proMMP‐9 is sensitive to activation by a serine proteinase, human trypsin‐2 and oxidants 8,11.

92 kDa MMP‐9 can be expressed by various cell lines such as keratinocytes, osteoclasts, eosinophils, neutrophils and macrophages 2–11. Increased MMP‐9 production and activation has been detected in many kinds of inflammatory and malignant diseases 8 such as periodontitis, peri‐implantitis and pericoronitis as well as various malignant tumours 8,39–43. MMP‐9 expression and activation can be induced by many inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), interferon‐γ (IFN‐ γ), IL‐2, but also decreased by IL‐10 and activation IL‐4 8,42,43. Correspondingly, its production and activation can be controlled with the ‘biological’ drugs exemplified by the etanercept‐type TNF‐blockers 42.

Stromelysins

The stromelysin subfamily consists of stromelysin‐1 (MMP‐3) and stromelysin‐2 (MMP‐10) 2–6. Their main substrates are basement membrane components such as laminins and type IV collagen but not interstitial collagens 2–6. Stromelysins can also cleave various ECM components and activate other proMMPs 2–6. MMP‐3 appears to have many roles; for example, it can induce tissue remodelling and destruction, apoptosis or promote differentiation 2–6. MMP‐3 has also been shown to be involved in the gingival tissue and crevicular fluid cooperative MMP‐activation cascades in periodontitis 28,43,44.

Membrane‐type MMPs

Membrane‐type MMPs (MT‐MMPs) differ from the other MMPs in that they are bound to the cell surface 9,10,45. Six different MT‐MMPs have been identified and characterized, MT1‐, MT2‐, MT3‐ and MT5‐MMPs have a transmembrane and a C‐terminal cytosolic domain 2–5,9,10,45,46, whereas MT4‐MMP and MT6‐MMP are bound to the cell surface with a C‐terminal hydrophopic extension that acts as a glycosyl phosphatidyl inositol (GPI) anchor 2–5,9,10.

MT‐MMPs can modify the pericellular environment by degrading various ECM components 9,10,45. MT‐MMPs can digest native type I–III collagens 45, and can activate other MMPs like proMMP‐2, proMMP‐8 and proMMP‐13 45–47. MT1‐MMP expression is regulated by various cytoskeleton‐ECM interactions (45–59). MT1‐MMP plays also an important role in the angiogenesis and in tumour growth and spread 45. MT5‐MMP has been detected in brain and MT6‐MMP in PMNs 48,49, and increased expression of the MT‐MMPs has been found in many malignant tumours, and in inflammatory conditions such as arthritis and periodontitis 49–54. Soluble or shedded form(s) of MT1‐MMP (MMP‐14) and MT6‐MMP (MMP‐25) have been detected in culture media of human renal mesangial cells, breast cancer cells, gingival fibroblasts, periodontitis‐affected human gingival crevicular fluid (GCF), inflamed bronchoalveolar lavage fluid and tear fluid 47,54–58.

Tissue inhibitors of matrix metalloproteinases (TIMPs)

TIMPs participate in the regulation of the ECM metabolism 2–5,35,59–61. The TIMP family consists of four members (TIMPs 1–4). TIMPs share some important structural features and an ability to inhibit MMPs, but exert also specific or selective distinct biochemical characteristics and expression profiles 2–5,35,59–61.

TIMPs are in general variably glycosylated proteins with molecular masses of 21–34 kDa, their nucleotide sequence homology is close to 25%. TIMPs contain 12 conserved cysteine residues capable of pairing to 6 disulphide bridges, of which 3 are located in the N‐terminal and 3 in the C‐terminal domain 2–5,35,59–61. The conserved N‐terminal domain of TIMPs is usually responsible for their biological activities and MMP binding 2–5,61. Disulfide bonds in the N‐terminal domain are required for the MMP inhibitory activity 2–5,61. The C‐terminal domains participate in the complex formation with the proMMPs 2–5,35,59–61. TIMPs 1–4 can inhibit all MMPs but TIMP‐1 is a relatively ineffective inhibitor of MMP‐19 and membrane‐type MMPs. TIMPs ‐1, ‐2 and ‐4 are secreted extracellular proteins, whereas TIMP‐3 is bound to the extracellular matrix 2. TIMPs ‐1, ‐2 and ‐3 are expressed by several cell types, and the TIMP‐4 expression has been found in various tissues including brain, heart, ovary and skeletal muscle 2–5,35,59–61. The primary function of TIMPs is regarded to be MMP inhibition, but TIMPs can exert also other functions, which may include MMP transportation and stabilization, MMP focalization to the cell surface via MT‐MMP binding, inhibition of angiogenesis, promotion of bone‐resorbing activity, growth factor‐like activity, and pro‐ and anti‐apoptotic functions 2,62,63. All these natural functions of TIMPs cannot be obtained with the synthetic MMP‐inhibitors 59–63.

Regulation of MMP activity and expression

MMP activity is regulated at multiple levels, including transcription, secretion/degranulation activation of the zymogen, inhibition of activity, extracellular inhibitors, localization either inside or outside the cell and internalization as well as by clearance 2–8,11–16,28,35,59,64.

Activity and expression of most MMPs are maintained at undetectable low or quiescent levels in intact normal tissues 2–8,35,59,64. The expression of MMP genes is regulated by transcription factors, co‐activators and co‐repressor proteins 2–8, 59. Transcriptional activation can be stimulated by a variety of pro‐inflammatory cytokines, hormones and growth factors, such as TNF‐α, IL‐1, IL‐6, epidermal growth factor, platelet‐derived growth factor and fibroblast growth factor 2–8,28,34,35,57,59,61,64–69. For example, CD40‐ligand can induce MMP‐1 expression, insulin MMP‐12 expression, hypoxia the expression of MMP‐2, MT1‐MMP and MMP‐13, and hyperoxia the expression of MMP‐8 and ‐9 2–8,34,35,59,61,64–69. Activation of latent proMMPs can occur intracellularly, at the cell surface by MT‐MMPs and in the extracellular space by other proteases 2–8,45–47. Plasmin and trypsins are regarded to be important serine proteinase activators of MMPs in vivo2–8,11–16,70–72. Cysteine proteinases can activate proMMPs released from gingival fibroblasts and proforms of serine proteinases 57. Bacterial and candidal proteinases as well as reactive oxygen species can activate proMMPs 11,14,16,59,61,73–75. Activated MMPs can further participate in the processing and activation of other MMPs in mutual activation cascades 2–8,45–47. Human trypsin‐2 is one of the potential candidates to be the initial activating serine proteinase of proMMP‐activation cascade 70–72. Furthermore, human trypsin‐2 can also directly degrade native interstitial type I and II collagens 71,72. In most cases, proMMP activation involves the direct removal of the prodomain, resulting in about 10 kDa lower molecular weight active forms 2,6,8,70–73, but recent studies have also shown that certain MMPs can be active while in full‐size or even complex forms evidently due to the interaction(s) with substrates, ligands, inhibitors and oxidants 16,39,59,76,77.

MMPs in periodontal inflammation

Bacterial dental plaque is currently considered to be the major risk factor for the development of periodontal disease 28,64,73,75,78. Periodontal destruction is due to uncontrolled inflammatory response to the bacterial insult consisting of a group of Gram‐negative anaerobic periodontopathogenic bacteria organized as a biofilm 28,64,73,75,78. The mechanisms involved in the initiation and progression of periodontal diseases are not clearly understood 28,64,75. The toxins, enzymes and metabolites of potent periodontopathogenic bacteria present in the dental plaque are considered to be the initiating factors of the host immune response which is primarily responsible for the tissue destruction. It is the host cells which produce and release proinflammatory mediators (cytokines and chemokines) as well as reactive oxygen species, prostaglandins and various proteinases 11–13,28,57,75,79. Lipopolysaccharides (LPS) derived from bacterial membrane have the capacity to activate host epithelial cells to express and release proinflammatory cytokines such as interleukin‐1, interleukin‐8, tumour necrosis factor‐α, prostaglandins and proteases 11–15,28, 57, 64, 75. LPS represents one of the many microbe‐derived ligands for the toll‐like receptors, which are involved in the microbe‐induced inflammatory responses in periodontitis and other diseases 64,75. Chemoattractant signals are initiated and transmigrating recruited leukocytes and monocyte/macrophages to amplify inflammation at the local site 28,64. The next stage of circulus vitiosus of periodontal inflammation follows; resident periodontal ligament and other cells in the periodontium, such as gingival fibroblasts, monocyte/macrophages, gingival sulcular epithelial cells/oral keratinocytes, osteoblasts/osteoclasts and endothelial cells, are activated in response to various pro‐inflammatory stimuli to express and secrete pro‐inflammatory cytokines, cysteine proteinases and MMPs 12,13,15,28,57,64,74,75,78.

The involvement of cytokines in MMP transcription

Three important regulatory mechanisms can control the mode and extent of MMP expression in the inflamed periodontium: regulation of transcription levels and secretion; activation of the proenzyme in the extracellular milieu and on the cell surfaces; and, finally, regulation/inhibition of MMP activity by their endogenous inhibitors (TIMPs) 2–8,11–13,28,35,64,80. In addition, MMPs are degraded and targets of clearance 2–8,28,59,61,64,80,81. MMP genes are expressed when required in a physiological extracellular matrix remodelling or in the pathological tissue destruction 2–8,28,59,64,80,81. Furthermore, certain inductive MMPs, such as MMP‐8 and ‐9, have recently been shown to protect against the spread of experimental lung inflammation and skin cancer 28–31,59,79. MMP‐1 and ‐2 are known to be constitutively expressed at low levels in the periodontium 2–8,28,64,65. However, the expression of inductive MMPs can be up‐ or down‐regulated by pro‐ and anti‐inflammatory cytokines, extracellular matrix proteins, bacterial virulence factors and enzymes, cell stress, cell shape changes, cellular contacts and communication, and by phorbol esters 2–8,28,35,59,64,65,80,81. Furthermore, MMP expression can be induced or repressed by MMPs' own substrate(s) either in native intact and/or cleaved/processed forms with various outcomes 4–6,59,64,80,81. For example, type I collagen can act as a ligand for the discoidin‐domain containing receptor‐like tyrosine kinases, which can up‐regulate MMP‐1 expression when activated by intact type I collagen and which become inactivated if the type I collagen ligand is degraded by collagenases 59, 80–83.

Many MMP, including MMP‐1, ‐3, ‐7, ‐9, ‐10, ‐12 and ‐13 gene promoters, comprise activating protein‐1 (AP‐1) consensus elements, such as AP‐1 binding site, polyoma virus enhancer activator or PEA1, tetradecanoylphorbol acetate (TPA)‐responsive element or TPE, one of which is located at about ‐70. The AP‐1 binding site is activated upon interaction with AP‐1 consisting of certain activated and complexed transcription factors such as the products of immediate early genes Fos and Jun. Other important MMP promoters include polyoma enhancer activator 3 site, which is not found in MMP‐2, binding Ets erythroblastosis 26, transformation‐specific protein first discovered in the E26 avian erythroblastosis virus, nuclear factor Kappa B binding sites binding NF Kappa B, and RUNX‐2 binding site, only found in MMP‐13 promoter, and binding RUNT domain factor‐2 2–8,65–69,80,81,84. These MMP promoters can be up‐regulated by various proinflammatory cytokines, growth factors, the extracellular matrix metalloproteinase inducer (EMMPRIN) and bacterial virulence factors 2–8,28,64–69,80,81,84. Resident cells, i.e. gingival fibroblasts, epithelial cells/keratinocytes, osteoblasts/osteoclasts, periodontal ligament cells, in the periodontium together with recruited inflammatory cells, i.e., neutrophils, monocytes/macrophages and plasma cells, can thus be both stimulated or down‐regulated by various cytokines 2–8,20,28,43,44,57,64–69,80,81,84. For example, transforming growth factor (TGF)‐β can suppress the MMP‐1, ‐3 and ‐8 gene transcription but can also induce the MMP‐13 gene expression 2–8,20,28,34–36,65–69,80,81,84.

IL‐1β and TNF‐ α can up‐regulate MMP‐3, ‐8 and ‐9 gene and protein expression by gingival fibroblasts, and IL‐1β and TNF‐α exert the same effect on osteoblast MMP‐13 28,43,44,57,64,65–69. On the other hand IL‐6 does not exert effects on keratinocyte MMP expression, but TGF‐1β can repress the transcription of the most MMP genes by keratinocytes but can up‐regulate MMP‐2 35,36,65,68,69. Furthermore, IL‐8 and bacterial virulence factors have been shown to be essential to recruit and trigger extravasating neutrophil influx to the sites of periodontal inflammation and can further promote neutrophil degranulation to release large amounts of oxidatively activated presynthetized and prepacked both MMP‐8 and ‐9 11–16,64,85,86; this can occur with or without de novo induction of MMP‐8 and ‐9 mRNA expression 22,23.

Recent studies have revealed exocites, i.e. MMP substrate binding, processing and cleavage sites locating outside the catalytic domain and active site of MMPs; the exocites can cleave non‐collagenous/non‐matrix MMP substrates including certain pro‐ and anti‐inflammatory cytokines and chemokines 2–8,59,64,80. Exocites thus broaden the substrate and tissue targeting profile of MMPs 2–8,59,64,80. The exocite processings and cleavages have been found to result usually in small‐size molecules or peptides 59,64,80, the effects of which may in fact be clearly more potent than the full‐size parent or native molecule 3,5,59. In this regard, IL‐8 expressed by periodontitis‐affected gingival sulcular epithelial cells can provoke the release of MMP‐8 and ‐9 by recruited neutrophils and is also a potential target of the released MMPs 59,64,80,85,86. Thus, proteolysis of various non‐matrix matrix substrates such as cytokines, chemokines, receptors, adhesion molecules, complement components and serpins is currently receiving as much attention as the traditional roles of MMP‐8 and ‐9 in the degradation of the extracellular matrix and basement membrane during pathogenesis of both inflammatory and malignant diseases 2–8,11,28–31,59,64,79,80,85,86. Therefore, the levels and involvement of MMPs can no longer be regarded as crucial solely because of tissue destructive activity, but must also be considered crucial for the protective anti‐inflammatory immune processes 2–8,28,59,64.

Matrix metalloproteinase promoter region polymorphisms associated with periodontitis and peri‐implantitis

MMP gene expression levels can be affected by the genetic variation(s), and promoter region polymorphisms can thus be involved in the development and progression of various malignant 87–91 and also inflammatory diseases such as periodontal disease, dental peri‐implantitis, osteoporosis, coronary heart disease and multiple sclerosis 92–109.

An insertion or deletion of guanine at position ‐1607 in the human MMP‐1 promoter region generates two different alleles: one with single guanine C (G) and the other with two guanines (2G). The 2G allele together with adjacent adenosine generates a core binding site, 5′‐GGA ‐3′, for Ets transcription factors located immediately adjacent to the AP‐1 site causing a 37‐fold increase in the transcriptioned activity of MMP‐1 87. It has been shown that the 2G allele significantly increases the transcription of MMP‐1 relative to the 1G allele 87. The 2G allele displays enhanced MMP‐1 transcription in both tumour cells and normal fibroblasts, and the presence of this allele has been associated with the enhanced development of ovarian cancer and metastatic melanomas 87–92. The presence of the 2G allele has recently been associated with severe chronic periodontitis and early dental implant failures (peri‐implantitis) 93–95. However, in Japanese and Czech populations no association between the corresponding MMP‐1 polymorphism and periodontitis could be found, suggesting that the genetic variation in different races may explain only part of the various outcomes 96,97.

Two functional genetic polymorphisms have been detected in the MMP‐9 promoter 98,99, namely a SNP (single nucleotide polymorphism) at position ‐1562 and a d(CA)n microsatellite repeat at position ‐90. The SNP is a C‐to‐T substitution that increases the transcriptional activity of MMP‐9 98,99. The C‐1562T polymorphism has been found to be associated with the coronary atherosclerosis 99. The (CA)n is a multi‐allelic microsatellite polymorphism, where the common form is the (CA)₁₄. The (CA)₁₄‐repeat has only 50% of the transcriptional activity of the MMP‐9 promoter when compared to the (CA)₂₁‐repeat allele 89. An association between the (CA)n repeats and abdominal aortic aneurysm and intracranial aneurysm has been found, but there have also been variant results 100–103. No associations have been found between MMP‐9 promoter polymorphisms with the susceptibility or severity of periodontal diseases or peri‐implantitis 104.

A 5A/6A polymorphism has been found in the MMP‐3 (stromelysin‐1) promoter. This SNP has been found to be associated with atherosclerosis 105,106. The frequency of the 5A allele is clearly enhanced in the affected individuals relative to the control subjects, and the risk of acute myocardial infarction in individuals carrying one or two copies of the 5A allele has been estimated to be 2.25‐fold 107,108. Itagaki et al. 96 did not find support for the hypothesis that MMP‐1 or MMP‐3 promoter polymorphisms could influence the susceptibility to or progression of periodontitis. Holla et al. 109 found that polymorphisms in the MMP‐2 promoter did not contribute significantly to the susceptibility to periodontitis, and de Souza et al. 104 found that polymorphisms in the TIMP‐2 promoter did not allow any conclusions regarding enhanced susceptibility to or severity of periodontitis.

Taken together, there are potential reasons why MMP‐1, ‐2, ‐3 and ‐9 promoter polymorphisms do not show significant influence on the susceptibility to periodontitis or dental peri‐implantitis 93–98,105,108,109. In the case the cells carrying the genetic variations of MMPs would pathologically excessively up‐regulate their MMP expressions leading to MMP over‐production, the enhanced prevalence of systemic collagenolytic tissue destructive disorders such cancers, arthritides, skin diseases, ulcers, eye and lung diseases in addition to local periodontitis could be expected 2–8,28,64,110,111. However, periodontitis patients are usually systemically rather healthy although enhanced periodontal tissue destruction involving locally up‐regulated and activated gingival tissue and GCF MMPs has been associated to certain systemic diseases such as unbalanced diabetes and cardiovascular diseases 112,113.

Recent MMP single gene knock‐out studies have evidenced that the affected mice do not show any particular diseases 114,115. Since the MMP members share the most common extracellular matrix and non‐matrix substrates, and eventually can compensate for lost functions, it can be concluded that single gene polymorphism(s) of a certain individual MMP may be not enough to exert significant effects on periodontal disease. Overall, the MMP transcription at the sites of periodontal inflammation is regulated and affected by the combined action of multiple local factors including bacteria, microbial virulence factors, enzymes and metabolites, host's adhesion and immune molecules, hormones, cytokines and growth factors etc. 11–13,28,64,65. Thus, in addition to genetic factors there are distinct and multiple local molecular factors involved in the periodontal soft and hard tissue destruction; shifting from bacterial infection into host cell behaviour, including various cytokine‐, serine proteinase‐/serpin‐, MMP‐ and TIMP‐cascades 28,33,65. Noteworthy, certain MMPs such as MMP‐8 and ‐9 have recently been found to exert, in addition to their surrogate tissue destructive properties, also unexpected protective and defensive anti‐inflammatory properties 28–31,59,64,79.

Characteristics and diagnostic utilization of MMPs present in the diseased periodontal tissues, gingival crevicular fluid (GCF), and pharmacological inhibition of MMPs in periodontitis

GCF has been found to contain large amounts of serum proteins, inflammatory mediators, host tissue and cell degradation products as well as microbial metabolites and enzymes 7,28,64. Especially proteinases such as MMPs, as well as neutrophil elastase and cathepsin G, are thought to play a central role in the regulation of periodontal tissue turnover in health and disease. These enzymes in GCF or in peri‐implant sulcular fluid (PISF) are considered to reflect periodontal and dental peri‐implant health and disease 7,28,64,116,117. The collection and analysis of GCF and PISF samples could provide a useful non‐invasive means to assess and monitor the pathophysiological status of the periodontium and dental peri‐implant tissues in a site‐specific manner 28,64,116,117. Thus, high expectations have been placed on GCF and PISF enzymes in the search for molecular indicators or markers to guide clinicians towards early chair‐side and point‐of‐care detection and monitoring of periodontal and dental peri‐implant health and disease 7,28,64,116,117.

It has been reported that the predominant MMPs in periodontitis‐affected GCF and peri‐implantitis‐affected PISF are PMN‐derived MMP‐8 and ‐9 7,116–123. However, the resident cells (sulcular epithelial cells/keratinocytes, gingival and periodontal ligament fibroblasts, endothelial cells, monocyte/macrophages, plasma cells, osteoblasts/osteoclasts etc.) in the periodontium likely also (albeit to a smaller extent) contribute MMP‐8 and ‐9 during periodontitis and peri‐implantitis 11–14,21–24,26–28,40–44, 64. Diseased GCF and PISF contain elevated levels of MMP‐3, MT1‐MMP (MMP‐14), MT6‐MMP (MMP‐25), MMP‐7, and MMP‐26 in active forms relative to periodontally healthy GCF and PISF 21,39–44,54,120–122; whereas MMP‐1 and ‐2 have been detected only in low levels in diseased GCF and PISF 7,28,64,65. The pathologically elevated and activated MMPs can form and act in co‐operative cascades in the periodontitis‐affected gingival tissue and GCF 28,42–44. Furthermore, markedly enhanced expression of MMP‐13 (collagenase‐3) has been demonstrated in the gingival tissue sections from chronic periodontitis patients suggesting MMP‐13 expression is important in the proliferation and intrusion of the activated epithelium into the periodontal connective tissues and in the progression of attachment loss and deepening of periodontal pockets 21,27,28,64.

Thus, the predominant MMPs present in the inflamed gingival tissue, gingival crevicular fluid (GCF), saliva/mouth‐rinse‐samples as well as dental peri‐implant sulcular fluid (PISF) are PMN‐derived inductive MMP‐8 and ‐9 and epithelial or bone cell‐derived MMP‐13, rather than constitutively expressed MMP‐1 and MMP‐2 7,27,28,64,65,116–123. Levels and degree of activation of PMN‐derived MMP‐8 and ‐9 and the bone‐ or epithelial cell‐derived MMP‐13 have been shown to increase with increasing periodontal disease severity and activity and to decrease following periodontal treatment (scaling and root planing) 7,28,64,116–123. Disturbances between MMP and TIMP ratios have been implicated in the ethiopathogenesis of periodontitis 7,28,44,64. Taken together, the breakdown of gingival collagen fibres in periodontal soft and hard tissues (alveolar bone resorption) by the cooperative action and cascade of both MMPs and cytokines contributes the detectable clinical signs and outcome of periodontitis and peri‐implantitis including enhanced pocket formation, attachment loss, bone resorption, gingival recessions, increased tooth/dental implant mobility and finally tooth/dental implant loss 7,28,64. In this regard a systemic pharmaceutical capable of down‐regulating the pathologically elevated levels of MMPs could be very useful as an adjunctive treatment/medication in periodontitis or dental peri‐implantitis 7,120,123,124.

A number of MMP inhibitors have been synthesized, but the major drawbacks of these molecules are the timing of the medication and adverse side effects 125. The fact that only a limited number of compounds have reached advanced clinical trials in spite of extensive efforts by almost all major pharmaceutical companies indicates that the development of MMP inhibitors is very demanding and challenging 125,126. In this regard, the ideal selectivity profile of MMP inhibitors and the target MMPs for various malignant and inflammatory disease states are unclear 28,59,64,123,125,126. Musculoskeletal syndrome has been reported among the most common side effect in clinical studies using MMP inhibitors, and events such as joint pain, stiffness, oedema, skin discoloration, and reduced skin mobility have been observed 59,125. In arthritis and periodontitis the excessive inhibition of MMPs surprisingly exacerbates rather than alleviates the disease 59,125,127. In this regard, Bjornsson et al. 127 recently described the effects of a potent and efficient broad‐spectrum hydroxamic peptide‐based MMP‐inhibitor, Batimastat, on periodontal destruction in a rat model. Surprisingly, instead of reducing the severity of experimental periodontitis, Batimastat actually enhanced the periodontal tissue destruction 127. What has not been sufficiently appreciated is that a minimal, but not pathologically excessive, level of MMPs seems to exert protective and anti‐inflammatory functions 29–31,79. Thus, we have proposed that ‘leaky’, less efficient MMP‐inhibitors, such as the tetracycline‐based MMP‐inhibitors, may be safe and effective because they only reduce the pathologically excessive MMPs but do not reduce MMP levels/activities below those required for physiological or anti‐inflammatory functions 7,123,124,128, whereas the potent and efficient synthetic MMP‐inhibitors, such as Batimastat, excessively reduce these MMPs below the physiologic or protective basal levels eventually resulting in clinically significant and harmful side effects 59,125,127. Some effective and selective in vitro MMP inhibitors, such as cyclic peptides produced in phage display libraries, are still in preclinical testing 126. Tetracyclines, especially doxycycline, which, in addition to their antimicrobial activity, can act as MMP inhibitors, an effect which is independent of their antimicrobial activity 7,120,123,124. Doxycycline inhibits MMPs at many levels: 1) inhibits the catalytic activity, 2) suppresses MMP gene expression, 3) prevents the proteolytic and oxidative activation of MMPs and 4) prevents the oxidative and MMP‐dependent inactivation of serpins such as α1‐antitrypsin 7,15,120,123,124. Presently Periostat® (doxycycline hyclate, CollaGenex Pharmaceuticals Inc.) is the only collagenase inhibitor approved by the Food and Drug Administration (FDA) and is clinically employed for the adjunctive treatment of periodontitis 123,124,129. This ‘low‐dose’ doxycycline formulation, used at 20 mg b.i.d., has been shown to have lost its antibiotic/anti‐bacterial activity 7,123,124,129. A low‐dose or sub‐antimicrobial dose of doxycycline medication (LDD or SDD) does not promote musculoskeletal syndrome side effects, is capable of inhibiting the pathologically elevated GCF and gingival tissue collagenase activity without causing their complete inhibition, and does not generate the development of microbial resistance to antibiotics 7,123,124,128,129.

Studies have demonstrated that low‐dose doxycycline (LDD) used as a systemic adjunctive medication to scaling and root planing (SRP) for periodontitis patients significantly improves the clinical outcome and attachment and reduces pocket depth, slowing the progression of periodontitis when compared with SRP alone 7,123,124,129. Furthermore, extensive literature has shown that systemic diseases such as diabetes and cardiovascular diseases share links between biomarkers of systemic inflammation (C‐reactive protein [CRP] IL‐6, TNF‐α and MMP‐9) and periodontitis 112,113,130,131. Severe periodontitis patients have significantly elevated serum CRP levels relative to patients with moderate or mild periodontitis 130,131. Moreover, Golub et al. 132 have proposed a ‘two‐hit’ model of periodontitis wherein the second ‘hit’, systemic inflammation, co‐induces periodontal tissue destruction in concert with the first ‘hit’, the microbial products such as LPS. Cardiology research groups have repeatedly in large‐scale clinical studies demonstrated that plasma CRP and other biomarkers (i.e., IL‐6, TNF‐α and MMP‐9) can be utilized as predictors of future cardiovascular diseases including heart attacks 133,134. In this regard, our group has reported that low‐dose doxycycline medication, but not placebo, can significantly (but not completely) 128 reduces the plasma levels of these systemic pro‐inflammatory biomarkers including also MMPs in patients with acute coronary syndromes (ACS) 135,136 (Figure 1). A prospective, randomized double‐blind pilot study of 6‐month of LDD—or placebo—treatment was performed to reduce systemic inflammation and prevent conditions promoting plaque rupture 135,136. LDD medication was shown to cause greater reductive effect on plasma CRP values (but no effect of placebo) of ACS patients when baseline plasma CRP values are ⩾5 µg/mL (high‐sensitivity CRP), and little or no effect of LDD medication was observed when baseline CRP values are ⩽5 µg/mL (Figure 1). Furthermore, Western immunoblotting and zymographic analysis 22,23,70,135,136 of plasma samples revealed that LDD medication decreased MMP‐8 and ‐9 levels by 30% and 50%, respectively, after 6‐month therapy, whereas placebo (6 months) had no statistically significant effect (Figure 1). In addition, the major species of MMP‐8 present in human plasma is the PMN‐type MMP‐8 isoform (15, Sorsa T, Golub LM, Brown DL, Konttinen YT, et al., unpublished data).

Figure 1. Effects of 6‐month regimen of low‐dose doxycycline (LDD; B = before, A = after) or placebo on plasma levels of pro‐inflammatory biomarkers in patients (n = 30) with acute coronary syndromes (ACS). (a): MMP‐8; (b): MMP‐9; (c): C‐reactive protein (CRP). MMP‐8 and ‐9 values represent densitometric scanning units and CRP represent µg/mL, and are expressed as means + SDs. (MMP = matrix metalloproteinases.)

A major issue, when considering the use of low‐dose doxycycline (LDD) MMP‐inhibitory medication therapy, is the duration of the therapy and detection or monitoring the link between the clinical and biological outcomes 123,124,129. The classical periodontal diagnosis determines mainly the previous periodontal tissue destruction, i.e. the disease outcome rather than disease activity 137. Evaluation of periodontal disease activity before significant destruction, and measurement of successful treatment or disease arrest, would allow the treatment to be directed to the right patient, possibly even to the site of high disease activity, at the right moment 116,117,137. There is a need for a convenient chair‐side, point‐of‐care test for diagnosis and monitoring of periodontal diseases 28,64,137. MMP‐8 in GCF is a potential candidate for such a test 27,28,44,64,116–120. We have recently developed monoclonal antibodies for MMP‐8 15 to be 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 point‐of‐care dip‐stick test for MMP‐8 in GCF and peri‐implant sulcular fluid (PISF) 15,116,117,120–123,138. This test resembles pregnancy home test kits 116,117,138, can conveniently be performed by a dentist without specific equipment, and measures the GCF MMP‐8 levels in 5 minutes 138 (Figure 2). Clinicians prefer such rapid (<10 min from start to finish) chair‐side point‐of‐care tests 28,64,116,117,137,138. The test can differentiate healthy and gingivitis sites from periodontitis sites 138. The test stick results (Figure 2) were found to be in good agreement with the quantitative MMP‐8 immunofluorometric assay (IFMA) analysis from GCF collected from periodontally healthy individuals and from gingivitis and periodontitis patients when all positive test results (+ to +++) were analysed (Figure 3). The agreement was very good when the Kappa‐statistics (κ = 0 0.81) were calculated, and the chair‐side GCF MMP‐8 test provided a sensitivity of 0.83 and specificity of 0.96 138. The elevated test‐stick (++ to +++) results and MMP‐8 IFMA analysis from GCF during the maintenance phase of periodontal treatment (SRP) preceded the clinical attachment loss (Figure 4). The dip‐stick technology (Figure 2) allows the use of one or more MMPs, TIMPs and tissue degradation products 28,64,116–123,138 in a combination test. GCF MMP‐8 level testing is a useful tool to monitor the beneficial effects of adjunctive sub‐antimicrobial doxycycline medication for periodontitis patients 139–141. This rapid point‐of‐care test developed for periodontitis is a useful tool also for monitoring of peri‐implantitis 116,117,121,122. It can be adjusted to monitor the follow‐up of orthodontic tooth movement 142,143. Evidently the principle of this chair‐side point‐of‐care MMP‐8 test could be adapted for the arthritic, eye and lung diseases 22,23,144–149. In addition to GCF 138–141, the effect of the MMP inhibitory medication can be followed by utilizing both MMP activity assays and immunoassays from salivary, oral rinse, tear fluid and serum/plasma samples 135,136,138,144–149 (Figure 1).

Figure 2. Test positive result of the MMP‐8 specific chair‐side dip‐stick is confirmed with the appearance of the blue line in the catching zone (left, indicated by arrows). (MMP = matrix metalloproteinases.)

Figure 3. Scattergram of periodontitis patients' sites (n = 133) at baseline; correlation of MMP‐8 concentration (assessed by IFMA, µg/L) and MMP‐8 specific chair‐side dipstick test results (0 = negative; 1 = +; 2 = ++; 3 = +++). (IFMA = time‐resolved immunofluorometric assay; MMP = matrix metalloproteinases.)

Figure 4. MMP‐8 concentration in GCF (µg/L, bars) collected from 5–8 distinct sites from periodontitis patients (n = 8) and assessed by IFMA and MMP‐8 specific chair‐side dipstick test (−; +; ++ and +++) precede loss of attachment (AL, mm) during maintenance phase of periodontal treatment (scaling and root planing). MMP‐8 concentrations and AL are represented as means. (GCF = gingival crevicular fluid; IFMA = time‐resolved immunofluorometric assay; MMP = matrix metalloproteinases.)

Conclusions

Overall, the excessive levels of PMN‐derived MMP‐8 and ‐9 are more associated with the progression, course and treatment of periodontitis and can contribute to the disease ethiopathogenesis in different ways, through cleavage(s) of fibrillar collagens, basement membrane and other matrix macromolecules, inactivation of proteinase inhibitors (α₁‐antitrypsin and α₂‐macroglobulin) and non‐matrix substrates including chemokines, such as CXC chemokine, a neutrophil chemoattract 3,5,28,59,64. However, lower levels of these MMPs can exert anti‐inflammatory protection by regulating the inflammatory cell and apoptotic responses 30,31,79. In this respect, recently unexpected inhibitory effects of MMP‐8 in experimental skin cancer progression have been reported 29. MMP‐8 gene deletion in mice increased tumour development after carcinogen treatment, and genetic manipulation of metastasic cells to overproduce MMP‐8 resulted in decreased metastasis dissemination 29,150. In summary, these observations suggest that MMPs associated with periodontal inflammation seem to act in cascades 28,43,44,64 and to exert multiple functions regulating both the surrogate tissue destruction and protective immune functions 28–31,64,79,151, and in oral fluids such as GCF and PISF these MMPs can be utilized as diagnostic tools 116,117,138. Synthetic MMP‐inhibitors such as doxycycline and CMT‐3 (6‐demethyl‐6‐deoxy‐4‐dedimethyl‐amino‐tetracycline—which has been chemically stripped—free of this antibiotic activity) can be used to support both the periodontal treatment (scaling and root planing) 7,120,123,124,129,139–141 and reduce systemic inflammation 135,136.

Acknowledgements

The research in our laboratories has been supported grants from the Academy of Finland and the HUCH‐EVO (TYH 5306, TYH 6104 and TI 020Y0002) Foundation.