Matrix Metalloproteinases (MMPs) in Periodontium: Is It a Boon or a Bane?

Abstract

Matrix metalloproteinases (MMPs) are a diverse family of endopeptidases that play a pivotal role in tissue remodeling and extracellular matrix (ECM) degradation, including in the periodontium. These enzymes are implicated in various biological processes, such as inflammation, cell proliferation, and wound healing. MMPs also contribute to remodeling the Periodontal Ligament (PDL) and alveolar bone by degrading ECMw proteins, thereby releasing growth factors beneficial for cellular repair and differentiation. Their activity is finely regulated through gene expression, proenzyme activation, and inhibition by tissue inhibitors of MMPs (TIMPs). A balanced interplay between MMPs and TIMPs is crucial for maintaining tissue homeostasis. While MMPs have beneficial roles in tissue repair and cellular functions, their dysregulation can exacerbate inflammatory responses and compromise tissue integrity. This chapter explores the dual nature of MMPs in the periodontium, examining whether they serve as a boon or a bane in periodontal health.

1. Introduction

1.1 Brief overview of matrix metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs), a group of enzymes, are members of a more prominent family of proteases requiring calcium and zinc. MMPs, called matrixins, play essential roles in reshaping tissues and breaking down extracellular matrix (ECM) proteins like collagen, laminin, elastin, proteoglycans, and fibronectin. The ECM provides structural support for the periodontium and the tissues surrounding and supporting the teeth, including the gums, connective tissue, periodontal ligaments, and the tooth socket bone [1]. By degrading ECM components, MMPs participate in tissue remodeling processes that continually rebuild the periodontium.

MMPs were first brought to scientific attention in 1962 by Woessner, who identified an enzyme in the mammalian uterus that could digest collagen. Further exploration occurred in 1966 when Jerome Gross and Charles Lapiere studied collagenolytic activity during tadpole metamorphosis. Subsequent years saw the isolation of various MMPs like MMP-2 and MMP-3 from human rheumatoid synovial fibroblasts (Nagase laboratory, Goldberg and colleagues) and rabbit synovial fibroblasts (Werb laboratory), respectively. The term “Matrix Metalloproteinases” was coined by Ed Harris, Jr., and associates in the 1980s, and each member of this family was systematically classified and assigned an enzyme number by the International Union of Biochemistry and Molecular Biology family [2].

To date, scientists have identified around 23 MMP enzymes produced in humans. These matrix metalloproteinases can be grouped into several categories depending on the extracellular matrix proteins they specifically target and break down. The major MMP classes are collagenases that degrade collagen; gelatinases that act on gelatin substrates; stromelysins with broad specificity; matrilysins that cleave matrix proteins such as fibronectin; and membrane-type MMPs that are located on cell surfaces. All MMP enzymes are first secreted as zymogens, inactive precursors of the enzymes. These precursors are later activated when the MMP catalytic function is needed [3]. Due to their complex roles, MMPs cannot be straightforwardly classified as “good” or “bad.” In periodontal health, periopathogens that invade the tissue trigger host inflammatory responses, disrupting the balance between tissue repair and destruction. Inflammatory cells like neutrophils and macrophages are elevated as part of the host’s defense mechanism and are crucial for expressing MMPs, mainly collagenases and gelatinases. Other resident cells like gingival and periodontal ligament fibroblasts also produce collagenases essential for maintaining normal tissue homeostasis. In summary, MMPs are vital enzymes that play intricate roles in both the maintenance of tissue integrity and the progression of diseases, making them critical targets for understanding and treating periodontal diseases.

In addition to their role in normal tissue homeostasis, MMPs can show destructive behavior under certain disease conditions, breaking down periodontal tissues. In chronic inflammatory diseases like periodontitis, increased expression of MMPs leads to pathological degradation of extracellular matrix components in the periodontium. This includes collagen and other proteins that anchor the junctional epithelium to the tooth surface. Excess MMP activity allows the junctional epithelium, which usually attaches the gingiva to the tooth, to detach and shift apically toward the root. MMP-mediated tissue destruction also enables lateral expansion of this epithelium. Together, these MMP-driven processes can cause connective tissue attachment loss, contributing to the progression of inflammatory periodontal diseases. Gingival inflammation or gingivitis is a reversible condition that progresses to periodontitis, which is the immune-mediated destruction of the periodontal supporting tissues, clinically manifested as increasing pocket depth, clinical attachment loss, tooth mobility, and ultimately leading to tooth loss. Irrespective of health or disease, the biological and pathological function of MMPs are controlled by various complex mechanisms through gene expression, proenzyme activation, and enzyme inhibition by endogenous inhibitors, such as tissue inhibitors of MMPs (TIMPs), which will be further discussed in this chapter. MMPs are regulated in response to inflammatory mediators, like cytokines and chemotactic molecules, exacerbating and resolving inflammatory responses by processing complement, cytokines, and other non-matrix bioactive molecules. Apart from periodontal inflammation, MMP has been associated with various chronic systemic disorders, like cardiovascular diseases, diabetes, and lung inflammation, which can exacerbate indirect ECM destruction within the periodontal tissues. Researchers have targeted to assess the potential of collagenolytic MMPs as periodontal disease biomarkers, their effect on collagen matrix degradation, and their potential as future therapeutic targets.

1.2 Importance of MMPs in tissue remodeling and cell regulation

Matrix Metalloproteinases (MMPs) were chiefly regarded as enzymes involved in breaking the extracellular matrix (ECM). Over time, it has become evident that their role extends to maintaining and regulating the structural aspects of the periodontium. These enzymes are synthesized and activated on demand by various cell types involved in tissue remodeling, including but not limited to keratinocytes, fibroblasts, endothelial cells, and a range of inflammatory cells like monocytes, lymphocytes, and macrophages. Multiple factors, such as cytokines, hormones, and cell-to-cell or cell-to-ECM interactions, can trigger MMP expression. The ECM itself is a complex structure made up of fibers (like collagen, elastin, laminin, and fibronectin), proteoglycans (such as syndecan-1 and aggrecan), glycoproteins (including tenascin, vitronectin, and entactin), and polysaccharides (e.g., hyaluronic acid). It plays a pivotal role in governing cellular activities like migration, growth, and differentiation. MMPs contribute to tissue balance by degrading these ECM components. This breakdown is intricately regulated, often in conjunction with tissue inhibitors of MMPs (TIMPs), to influence cell behavior across various conditions—developmental, physiological, and pathological, as well as during tissue repair and disease resolution. MMPs are implicated in multiple biological processes, from cell proliferation and differentiation to migration, wound healing, morphogenesis, angiogenesis, and even apoptosis. The expression of MMPs also affects the remodeling of the Periodontal Ligament (PDL) and alveolar bone, where their role extends to degrading ECM proteins. This degradation, in turn, liberates growth factors, such as insulin and fibroblast growth factors, which are bound to the ECM and beneficial for the repair, proliferation, and differentiation of native cells [4].

2. Basic biology of MMPs

2.1 General functions of MMPs

Matrix Metalloproteinases (MMPs) influence a variety of physiological and pathological undertakings, including tissue restructuring, inflammation, and wound repair [5]. Their primary role is the cleavage of matrix proteins in the extracellular environment, thereby altering cellular phenotypes and facilitating cellular migration. For instance, MMP1 or collagenase 1, produced by basal keratinocytes, has been shown to enable epithelization, highlighting its role in epithelial repair [6]. It is also expressed in endothelial cells, fibroblasts, and macrophages in response to injury, aiding in keratinocyte migration and re-epithelialization [5]. MMPs dismantle the extracellular matrix (ECM) components, such as collagen, laminin, and fibronectin, thereby enabling cell migration and proliferation. They also modulate the release and activity of growth factors and cytokines [6]. Their ability to liberate pro-apoptotic elements from the ECM is essential for physiological cell turnover.

Additionally, MMPs are pivotal in shaping tissue morphogenesis and proteolysis, influencing cell-ECM interactions that contribute to cell differentiation and tissue remodeling [7]. The basement membrane degradation by MMPs facilitates angiogenesis, which is crucial for tissue repair and wound healing. With type I collagen serving as the primary ECM component in periodontal tissues, its degradation is significant in the pathogenesis of periodontal diseases [8]. According to Sorsa et al. [9], MMP-8 plays a vital role in the breakdown and regeneration of periodontal tissues.

Equilibrium in protease activity is critical, as imbalances can lead to tissue damage, chronic inflammation, and disease progression. MMPs themselves contribute to this balance by either activating proenzymes or cleaving inhibitors like tissue inhibitors of MMPs (TIMPs) [10]. They can also act upon other proteases like plasminogen and urokinase-type plasminogen activator (uPA), thereby increasing proteolytic activity in the microenvironment.

2.2 Basic structure of MMP

MMP enzymes have several distinct functional regions in their structure: (a) an 80 amino acid propeptide segment at one end contains a variable length signaling peptide sequence. (b) The catalytic core that breaks down substrates has 170 amino acids and requires zinc. (c) An approximately 200 amino acid hemopexin-like domain binds to and aids collagen degradation. (d) Between the catalytic and hemopexin-like domains is a connecting peptide region with varying amino acid lengths that links the two domains together (Figure 1). MMP is directed to synthesize inside the cell by the N-terminal signal. MMPs are frequently produced as proenzymes, an inactive form of the enzyme, which is then cleaved to the active form by various proteinases like serine protease, plasmin, and bacterial protease. The activation process is also initiated by proteinases from the furin family and other membrane-type matrix metalloproteinases (MT-MMPs) on the surface of cells. TIMPs and specific substrates also play a significant role in the activation process. Variations do exist among the family members of MMPs. MMP-14, −15, −16, and − 24 have an additional C-terminus end. In contrast, MMP-7, −23, and − 26 lack the binding peptide and hemopexin region. Specifically, MMP-23 features an arrangement of cysteines and an immunoglobulin-like (Ig-like) domain instead of the critical region and the hemopexin domain [10].

Figure 1.

Illustrates the structure of the inactive MMP-7 zymogen (proMMP-7). In contrast to most MMPs, proMMP-7 has a simplified two-domain architecture [(A) in the diagram]. It contains a propeptide domain (pro) and a catalytic domain (cat). The propeptide region consists of three α-helical chains linked together by loop structures [(B)]. The catalytic domain houses the zinc-binding site with two zinc ions and two copper ions, as well as a globular tertiary structure formed by three α-helices, five β-pleated sheets, and several additional connecting loops (adapted from Liu et al.; 2020) [11].

2.3 Classification and types of MMPs

Table 1 demonstrates the classification of MMPs and their biological implications. MMPs are categorized into six major classes based on their structural characteristics and substrate specificity:

1. Collagenases: These break down collagen’s triple-helix structure. Members include MMP-1, MMP-8, and MMP-13.

2. Gelatinases: These primarily degrade denatured collagen or gelatin. MMP-2 and MMP-9 are the primary gelatinases.

3. Stromelysins: They have a broad substrate specificity and are known to break down proteoglycans, laminins, and other non-collagenous substrates. Members include MMP-3, MMP-10, and MMP-11.

4. Matrilysins: These also exhibit a broad range of substrate specificity but are generally smaller. The most well-known are MMP-7 and MMP-26.

5. Membrane-Type MMPs (MT-MMPs): These are anchored to the cell membrane and play a role in pericellular proteolysis. Included are MMP-14, MMP-15, MMP-16, and MMP-24.

6. Additional Unclassified MMPs: These MMPs must fit neatly into the above categories and have unique characteristics. Examples are MMP-12, MMP-19, MMP-20, and MMP-28.

Class of MMP	Common Name	MMP/Source of Secretion	Biological Implications for Periodontal Tissues	Substrate Specificity
Collagenases	Collagenase-1/interstitial collagenase/Fibroblast; collagenase	MMP-1	Degrade collagen type 1 fiber physiological remodeling in response to orthodontic forces and wound healing. Keratinocyte migration and re-epithelialization Platelet aggregation. It is upregulated in periodontal disease.	Collagen I, II, III, VII, VIII, X, XI, Gelatin, Fibronectin, Aggrecan, Entactin, Tenascin, Ovostatin, Casein
	Collagenase-2/neutrophil collagenase	MMP-8	Produced by neutrophils in response to infection and inflammation. Promotes wound healing and tissue repair. Upregulated in periodontal disease, contributing to alveolar bone resorption, and induces inflammation in response to orthodontic forces or occlusal stress.	Collagen I, II, III, Fibronectin, Aggrecan, Ovostatin
	Collagenase-3	MMP-13	ECM remodeling in response to mechanical forces (e.g., orthodontic movement) Maintains tissue integrity. Bone remodeling. It is upregulated in periodontal disease, contributing to alveolar bone resorption and inflammation. Osteoclast activation and release of bFGF.	Collagen I, II, III, IV, IX, X, XIV, Fibronectin, Laminin, Gelatin, Aggrecan, Plasminogen, Osteonectin
Gelatinases	Gelatinase A/type IV collagenase	MMP-2	Degradation and remodeling of ECM Angiogenesis tissue repair & wound healing Remodeling of Periodontal Ligament and alveolar bone Facilitate migration and activity of osteoclasts, wound healing, and tissue repair. Upregulated in inflamed periodontal tissues, temporomandibular joint disorders (TMDs), and periodontitis.	Gelatin, collagen (IV–VI, X), elastin, fibronectin
	Gelatinase B/type IV; collagenase, type V; collagenase	MMP-9	ECM remodeling in response to orthodontic movement and eruption Produced by neutrophils in response to inflammation and infection. Contributes to host defense by degrading ECM components. Facilitates Tissue repair. Upregulation in periodontal disease contributes to tissue destruction, pocket formation, and bone resorption.	Gelatin, Type V collagen, Laminin, Fibronectin
Stromelysin	Stromelysin-1/Transin,; Proteoglycanase, Collagenase activating protein (CAP)	MMP-3	ECM remodeling Macrophages and neutrophils produce it in response to inflammation and infection. Promotes tissue repair and wound healing. Alveolar bone remodeling. It is upregulated in response to orthodontic forces, occlusal stress, and periodontal disease. Contribute to tissue destruction, pocket formation, and bone resorption.	Laminin, aggrecan, gelatin, fibronectin, collagen types III, IV, IX, X, proteoglycans, and fibronectin.
	Stromelysin-2/Transin-2	MMP-10/Fibroblasts, T-lymphocytes	Involved in basal keratinocyte migration	Collagens (III–V), gelatin, casein, aggrecan, elastin, MMP-1,8
	Stromelysin-3	MMP-11/Fibroblasts	Degrade ECM components. Promote tissue repair and Wound healing angiogenesis. Alveolar bone remodeling in response to mechanical Forces. Regulate Immune Responses. Upregulation contributes to tissue destruction in periodontal diseases	collagen types I, II, and III, Fibronectin, laminin, aggrecan, gelatin,
Matrilysin	Putative Metalloprotease (PUMP-1), Martin	MMP-7/Macrophages	ECM Remodeling Tissue Repair and Regeneration Maintain epithelial barrier integrity and gingival Tissue Remodeling in response to mechanical forces and inflammation.	Collagen (IV–X), fibronectin, laminin, gelatin, aggrecan, pro-MMP-9
	Macrophage metalloelastase/Metalloelastase	MMP-12	Degrade elastin. Contribute to tissue remodeling and repair. Promotes ECM remodeling and immune cell migration during inflammation.	Elastin, Laminin, Fibronectin, Vitronectin, Type IV collagen
	Matrilysin-2	MMP-26/ B-lymphocytes	Promotes ECM Remodeling, Tissue Repair, and Regeneration. Facilitates Gingival Tissue Remodeling Contributes to ECM remodeling and immune cell migration. Dysregulation contributes to tissue destruction and pathological conditions.	Gelatin, collagen IV, pro-MMP-9
MT-MMP	MT-1 MMP−/interstitial collagenase	MMP-14/Fibroblasts, macrophages	Cell migration, tissue remodeling , tissue repair, tissue regeneration. Upregulation contributes to periodontal disease progression. Involved in the breakdown of collagen fibers within the periodontal ligament to allow tooth movement. activate proMMP-2, pro-MMP13	Type I, II, III collagens, gelatin, fibronectin, laminin, aggrecan, a2-macroglobulin, pro-TNF-a, fibrinogen, pro-MMP-2, −13, −20
	MT2-MMP/gelatinase A	MMP-15/Fibroblasts, macrophages	Degrade gelatin, collagen type IV, in the basement membrane. Contributes to tissue remodeling during tooth eruption, orthodontic tooth movement, and wound healing. Upregulated Periodontal Disease is upregulated in response to inflammatory signals contributing to periodontal tissue destruction. Regulate proMMP-9 (pro gelatinase B) and amplify the proteolytic activity in the ECM of the periodontium.	Fibronectin, laminin, aggrecan, perlecan, pro-TNF-a, pro-MMP-2
	MT3-MMP	MMP-16/Fibroblasts, macrophages, vascular smooth muscles	Cell-Matrix Interactions Influence processes like cell adhesion, migration, and signaling within the periodontal tissues. Contribute to tissue remodeling by activating proMMP-2 (gelatinase A), which contributes to the degradation of collagen and gelatin. Promote Angiogenesis. Influence immune cell migration and cytokine signaling during inflammation	Collagen III, gelatin, casein., gelatin, fibronectin, laminin, a2-macroglobulin, pro-MMP-2
	MT-4 MMP	MMP-17/Eosinophils, lymphocytes, and monocytes	ECM Remodeling. It helps in immune cell migration and promotes interactions between periodontal ligament fibroblasts and other cells within the tissues. Modulate immune cell behavior and cytokine signaling. Contribute to the progression of periodontal inflammation. Influence angiogenesis within the periodontal tissues by activating pro-angiogenic factors and their effects on vascular endothelial cells. Contributes to the regulation of other MMPs	Gelatin, fibrinogen, fibrin, pro-TNF-a
	MT5 –MMP	MMP-24/Brain-specific
	MT6-MMP	MMP-24/peripheral blood leukocytes
Other MMPs	Stromelysin-3	MMP-11		does not cleave collagen, aggrecan, fibronectin, laminin
	Metalloelastase	MMP-12/Macrophages	Macrophage migration.	Collagen: IV, elastin, fibronectin, gelatin, laminin
	RASI-1	MMP-19 (RASI)		collagen: IV, fibronectin, aggrecan, COMP, laminin, gelatin
	Enamelysin	MMP-20/Endothelial cells mature human odontoblasts		collagen: V, aggrecan, amelogenin, COMP
	—	MMP-21		a1-anti-trypsin
	CA-MMP	MMP-23/reproductive tissues		unknown
	MT6-MMP, Leukolysin	MMP-25		collagen: IV, gelatin, fibronectin, laminin, fibrin
	Epilysin	MMP-28/keratinocytes	Tissue hemostasis and wound repair	unknown

Table 1.

Classification of MMPs, its biological implication, and substrate specificity.

These classes are not mutually exclusive, and some MMPs may have overlapping functions and substrate specificity [5, 12, 13, 14].

2.4 Regulation of MMPs: activation and inhibition

MMPs are predominantly activated by several factors, including specific components of the extracellular matrix, low pH conditions, elevated temperature, the serine protease plasmin, bacterial proteases, oxidative stress conditions, and the furin family of proteases as well as other membrane-type matrix metalloproteinases (MT-MMPs). These factors result in the cleavage of the inactive zymogen form of MMPs into their active enzymatic form. Conversely, MMP activities are kept in check through a series of regulatory cascades. Regulation can occur at multiple levels, such as during gene transcription, the activation of the zymogen forms, and inhibition by tissue inhibitors of metalloproteinases (TIMPs). TIMPs are naturally occurring proteins that specifically inhibit MMP activities, thereby balancing their proteolytic action. Furthermore, MMPs can also be modulated through their interaction with cell surface components and other extracellular molecules [6]. Overall, the regulation of MMPs is a complex interplay of activation and inhibition mechanisms that control their biological functions.

Transcriptional Regulation: The regulation of MMP expression at the transcriptional level is complex and varies across different MMPs and tissues. A multitude of signaling molecules, including cytokines and growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor, tumor necrosis factor (TNF-α), keratinocyte growth factor (KGF), and transforming growth factor (TGF-β), among others like interleukins and interferons, play roles in transcriptional activation. These molecules exert their influence via several signaling pathways, including NF-κB, MAPK, and Smad-dependent pathways, as well as through integrin-mediated activation of the focal adhesion kinase (FAK) or the activation of the Wnt signaling pathway. Subsequently, activated transcription factors like AP-1 (Activator Protein-1), NF-κB (Nuclear Factor-κB), and SP-1 (Specificity Protein 1) bind to specific regions in the promoter sequences of MMP genes, thereby facilitating or inhibiting MMP gene expression. The balance between proinflammatory and anti-inflammatory signals has a decisive impact on regulating MMP gene expression, ultimately influencing the outcome of various biological processes [7].

Zymogen Activation: Activation of pro-MMPs, the zymogen or inactive form of MMPs, is a highly localized event thought to primarily occur in the immediate pericellular region. Pro-MMPs require cleavage of their inhibitory propeptide or prodomain to become active, which shields the enzyme’s active site. This activation is contingent upon a conformational change in the prodomain, which disrupts the cysteine switch–zinc interaction. Three primary mechanisms have been identified for this process: (1) Direct cleavage of the pro-domain by partially activated MMP intermediates or other active MMPs. For example, the activation of pro-MMP-2 involves a combined action of TIMP-2 and MMP-14; (2) Induction of conformational changes at allosteric sites within the prodomain; (3) Chemical modification of the free cysteine in the prodomain by reactive oxygen species or by non-physiological agents. Other factors that can lead to activation include pH variations and binding to specific activators, such as the role of MT-MMPs in activating pro-MMP-2 and pro-MMP-9 [15].

Inhibition by Tissue Inhibitors of Metalloproteinases (TIMPs): TIMPs are endogenous inhibitors that halt the process of extracellular matrix degradation by forming tight complexes with active MMPs. Four types of TIMPs have been identified, ranging from TIMP-1 to TIMP-4. These TIMPs are essential regulators of MMP activity by limiting their proteolytic function. A balanced equilibrium between MMPs and TIMPs is imperative for maintaining tissue homeostasis. By attaching to active forms of MMPs, TIMPs effectively prevent ongoing proteolytic activities, thus preserving the integrity of the extracellular matrix.

Substrate Specificity: The choice of substrates targeted by MMPs is highly influenced by the structure of their catalytic domain and other regions within the enzyme. As a result, different MMPs display unique substrate preferences. The extracellular matrix contains a diverse array of potential substrates, and the specific activity of MMPs can be determined by the tissue or disease context in which they operate. The requirement for calcium ions in the enzymatic activity of MMPs is evident from inhibiting these enzymes by metal chelators like EDTA and tetracyclines [5].

Cell Surface Localization: Some MMPs, notably the membrane-type MMPs (MT-MMPs), are anchored to cell membranes, localizing their activities to specific cellular compartments or microenvironments. This spatial regulation ensures that the proteolytic activity of MMPs is precisely controlled.

Feedback Regulation: MMP activity also operates under a feedback mechanism. The proteolytic degradation of matrix elements can release bioactive fragments that modulate further MMP expression and activity. Additionally, some MMPs can cleave and activate latent cytokines and growth factors, which then influence the behavior of adjacent cells.

Interaction with Other Molecules: The expression and activation of MMPs can be modulated through interactions with other molecules, including growth factors, cytokines, components of the extracellular matrix, and cell surface receptors. These interactions add another layer of complexity to the regulation of MMP activity.

Overall, regulating MMPs involves various factors to ensure their activity is appropriately balanced for maintaining tissue function and integrity. Dysregulation of this equilibrium can contribute to pathological conditions, such as inflammation and periodontal tissue degradation.

2.5 MMP activation cascade

Matrix metalloproteinases (MMPs) are activated in a cascade-like manner, particularly in connective tissues. Under normal physiological conditions, MMPs are expressed at low levels. The activation process is often triggered by various factors, including host inflammatory cells, lipopolysaccharides (LPS), toxins, and metabolic byproducts byproducts from gram-negative anaerobes like A. actinomycetemcomitans and P. gingivalis. These triggers elicit an inflammatory response, generally thought to be initiated by the toxins, enzymes, and metabolites released by potent periodontopathogenic bacteria in dental plaque. In response to these proinflammatory cues, various cells within the periodontal tissues—including periodontal ligament cells, gingival fibroblasts, monocytes/macrophages, gingival sulcular epithelial cells/oral keratinocytes, osteoblasts/osteoclasts, and endothelial cells—are activated. These activated cells express and secrete an array of proinflammatory molecules like interleukin-1, interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), prostaglandins (PGE2), cysteine proteinases, and MMPs. Concurrently, chemoattractant signals are activated, facilitating the transmigration of leukocytes and monocytes/macrophages to the site of inflammation, thereby amplifying tissue destruction [9]. The cascade of MMP activation involves several key steps. Membrane-bound MMP-14 activates proMMP-13, degrading type I collagen, a significant component of the cementum, periodontal ligament (PL), and alveolar bone extracellular matrix. MMP-13 further activates proMMP-9, which can, in turn, trigger proMMP-2 and proMMP-13. MMP-2 and MMP-9 are responsible for processing the gelatin that results from the activity of collagenases like MMP-13. Autoactivation of MMP-13 occurs via self-proteolysis, adding another layer of complexity to the activation cascade. Furthermore, polymorphonuclear cells (PMN) generate reactive oxygen species (ROS), including HOCl and H₂O₂, which can modify the balance of proteases and antiproteases. These ROS can activate latent MMPs while simultaneously inactivating the tissue inhibitors of MMPs (TIMP)-1. This complex cascade of events ensures that MMPs are precisely regulated and their activity is tightly controlled (Figure 2).

Figure 2.

Illustration of the activation cascades of different matrix metalloproteinases (MMPs) that degrade connective tissue in periodontitis (adapted from Franco et al.; 2017) [16]. The tooth structure and supporting tissues are shown in (A), including radicular cementum, periodontal ligament (PL), and alveolar bone. Proteolytic activation steps are depicted by arrows connecting inactive MMP zymogens (full circles) and activated MMPs (partial circles). (B) Shows MMP-14 on the cell surface activating proMMP-13, degrading the abundant type I collagen in the extracellular matrix of the periodontal ligament and alveolar bone. Active MMP-13 can also activate proMMP-9, which in turn activates proMMP-2. MMP-2 and MMP-9 process gelatin generated from collagen breakdown. In addition, MMP-13 undergoes autoactivation. Reactive oxygen species from immune cells stimulate MMP activation and tissue inhibitor of MMP (TIMP) inactivation, shifting the protease/antiprotease balance.

3. MMPs in healthy periodontium

3.1 Role in tissue remodeling

Periodontal tissues undergo continuous physiological remodeling to adapt to functional demands and maintain overall tissue integrity. The extracellular matrix (ECM) elements, such as collagen, elastin, and proteoglycans, are regularly broken down and degraded by matrix metalloproteinases (MMPs). This enzymatic activity facilitates the turnover of older or damaged matrix components, providing space for the synthesis and deposition of new matrix molecules. MMPs are vital in healthy conditions and are pivotal in remodeling the periodontal ligament (PDL) under diseased states.

MMPs are also critical players in inducing biochemical changes within the PDL during orthodontic tooth movement. This leads to the reorganization of PDL fibers and new ECM deposition. Mechanical forces trigger the expression of MMPs, particularly at sites experiencing compression and tension. Concurrently, tissue inhibitors of metalloproteinases (TIMPs) are expressed in response to mechanical forces, mainly to downregulate MMP activity, especially at tension sites. The specific role of MMPs in osteoclastic bone resorption remains to be fully elucidated. However, it is understood that demineralization of the bone inorganic matrix by acid and degradation of the organic matrix, along with cathepsin K, are involved [17]. Studies have shown elevated expression levels of MMP-1, −2, −3, −7, −8, −12, and −13 in gingival crevicular fluid (GCF) at both compression and tension zones due to the influence of orthodontic forces. Collagenase-1 (MMP-1) and collagenase-2 (MMP-8) are particularly adept at breaking down the native triple helix structure of interstitial collagen, thereby initiating cellular remodeling [18].

In the context of periodontal tissue injuries, whether due to inflammation, trauma, or infection, MMPs are pivotal in the breakdown of damaged ECM. They create an environment conducive to wound healing by attracting immune cells and growth factors to the injury site. MMPs, especially MMP-8 and MMP-13, have been highlighted as crucial elements in tissue remodeling and are considered markers of periodontal repair in diseased tissues [19].

3.2 Maintenance of periodontal tissue homeostasis

In a healthy periodontal setting, the symbiotic interactions between host cells and pathogenic bacteria trigger a protective immune response essential for maintaining periodontal equilibrium [20]. Within this framework, fibroblasts in the periodontal ligament emerge as key players, responsible for synthesizing collagen fibers and showing a remarkable ability to proliferate and migrate, vital characteristics for efficient periodontal wound healing. MMP-13, primarily expressed by fibroblasts during the healing process in human gingival tissues, plays a unique role in the extracellular matrix (ECM) turnover. Research findings suggest that MMP-13 and MMP-1 may have differentiated functions regulating ECM, particularly in gingival wound repair [21]. MMP-1, for example, influences the migration of keratinocytes and, through the specific cleavage of collagen types I and III, may facilitate fibroblast migration [21]. The quick recycling of collagenous extracellular matrix (ECM) near and within gingival granulation tissue, especially when stimulated by TGF-1, appears to be facilitated by the activity of MMP-13. This enzyme likely plays an essential role in maintaining a fragile balance between the deposition and degradation of ECM during gingival wound healing. Unveiling the cell-specific mechanisms that differentially regulate MMP-13 expression in dermal versus gingival fibroblasts could open new avenues for treating tissue fibrosis [22].

Matrix metalloproteinase (MMP) activity is rigorously controlled to avert rapid tissue degradation. The integrity and repair of periodontal tissues hinge on a well-regulated equilibrium between MMPs and their natural inhibitors. Two primary categories of proteins, tissue inhibitors of metalloproteinases (TIMPs) and α2-macroglobulin, serve as natural checks on MMP activity. Specifically, α2-macroglobulin, a protein predominantly produced in the liver and found in various tissue fluids, can irreversibly inhibit MMPs, thus contributing to tissue homeostasis [23].

The role of mechanical forces and paracrine and juxtracrine signaling with nearby bone and immune cells is well recognized in influencing osteoclast and osteoblast activities in bone remodeling. Hormonal signals in the bloodstream and immune factors further modulate these processes. In this context, MMP-2, MMP-9, MMP-13, and MMP-14 are critical players in bone formation and development [24]. Specifically, MMP-14 activation of transforming growth factor-beta (TGF-β) wards off apoptosis and enhances osteoblast survival as they trans-differentiate into osteocytes, thereby supporting healthy bone homeostasis [25]. Moreover, MMP-14’s role in maintaining osteocyte viability is integral to normal bone repair and growth [26]. Apart from MMPs, tissue inhibitors of metalloproteinases (TIMPs) also exert regulatory control over other proteinase families, such as ADAM and ADAMTS. TIMPs are thus central to various biological events, including creating the extracellular matrix and cellular proliferation. Regarding orthodontic treatments, TIMPs can have a consequential impact on osteoblast behavior and the aberrant reconstruction of periodontal vessels, potentially affecting the efficacy of the treatment. The broad spectrum of cells within the body releases TIMP-1, which has a somewhat narrower range of inhibition than other TIMPs. It selectively binds and inhibits nearly all MMPs, except for MMP-14, MMP-16, MMP-18, MMP-19, MT1-MMP, MT2-MMP, MT3-MMP, and MT5-MMP, displaying particular affinity for MMP-9 and its precursor, pro-MMP-9. Meanwhile, TIMP-2 suppresses the activity of MMP-2, MMP-9, MMP-14, and MT1-MMP; TIMP-3 inhibits MMP-2 and MMP-9; and TIMP-4 acts against MMP-2, MMP-26, and MT1-MMP.

3.3 Contribution to oral epithelial turnover

Matrix metalloproteinases (MMP-1, −2, −7, −9) are instrumental in facilitating the migration of oral epithelial cells by degrading components of the extracellular matrix (ECM). This creates conduits for cellular movement, which is pivotal for processes like wound healing, tissue repair, and average cell turnover. The cleavage of type 1 collagen by MMP-1, for instance, has been shown to drive the migration of keratinocytes during the re-epithelialization phase of wound healing [27]. MMPs also play a critical role in shaping the integrity of the ECM, which serves as a barrier. Disruption of this barrier due to dysregulated MMP activity could contribute to initiating various oral diseases. These enzymes modulate cellular functions and influence intercellular and cell-to-matrix communications through their interactions with the ECM [28].

Moreover, MMPs have been identified as regulators of apoptosis, a form of programmed cell death that primarily involves caspase protease activity [29]. In certain pathological conditions like oral lichen planus, MMPs are thought to induce factors that lead to the apoptosis of basal keratinocytes in the oral mucosa [30]. Research has shown that the absence of the underlying basement membrane, and consequently of MMPs, leads to the upregulation of apoptosis-related genes and subsequent cell death. This has been evidenced in studies where epithelial cells were separated from their basement membrane [6].

4. Pathological implications of MMPs in periodontium

4.1 MMPs in periodontal diseases

4.1.1 Role of MMPs in gingivitis and periodontitis

In healthy and inflamed periodontal tissues, the predominant cells—fibroblasts, keratinocytes, endothelial cells, and macrophages—respond to stimuli like growth factors, cytokines, and substances from microbial flora by activating the transcription of one or more MMP genes. Cytokines, such as IL-1 and TGF-alpha, are believed to play a significant role in controlling the expression of these MMP genes in periodontal tissues. Collagenases and gelatinases have received particular attention in the study of periodontal diseases, as type I collagen is the primary component of the periodontal extracellular matrix (ECM). The initiation of tissue degradation occurs when an imbalance between MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), tilts toward ECM degradation. In such cases, the fibrogenic response is insufficiently rapid to counterbalance the proteolytic activity of the ECM [6].

Conversely, tissue proliferation is observed when fibrogenesis outpaces ECM degradation [31]. In chronic periodontitis, oxidative stress and toxic bacterial byproducts lead to the overexpression of MMPs. This skews the homeostatic equilibrium between active MMPs and TIMPs, favoring increased MMP activity. A complex interplay of host-derived and microbial proteolytic enzymes contributes to the degradation of vital ECM components, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. Elevated levels of these enzymes in gingival tissues and oral biofluids, such as gingival crevicular fluid (GCF), have provided valuable insights into the mechanisms of tissue destruction within the periodontium [9].

Polymorphonuclear cells (PMNs), the first cells to be recruited to inflamed periodontal tissues, play a crucial role in phagocytosing periodontal pathogens. In addition, they release various antimicrobial substances, including neutrophil elastase and MMPs, in response to bacterial challenges. MMP-8, or neutrophil collagenase, is particularly noteworthy. This enzyme is produced during bone marrow maturation and stored within neutrophils. When released during neutrophil degranulation, in response to inflammatory stimuli such as TNF-alpha, IL-1, and lipopolysaccharides (LPS), it initiates cascades that ultimately result in the degradation of periodontal tissues [32]. Studies have shown that active MMP-8 (aMMP-8) plays a direct role in the pathological destruction of periodontal connective tissues. Besides being produced by PMNs, MMP-8 can also be expressed by monocytes, macrophages, plasma cells, endothelial cells, fibroblasts, and epithelial cells during inflammatory processes. Moreover, its activity is not limited to collagen degradation; MMP-8 can also enhance the recruitment of leukocytes to the site of inflammation. Elevated expression levels of MMP-8 have been detected in saliva and GCF, and these have been correlated with clinical parameters of periodontal destruction in patients with chronic periodontitis [33].

MMP-8 is particularly salient in cases involving type II diabetes with periodontitis as opposed to systemically healthy patients, showing an uptick in collagen degradation [34] attributed to higher levels of proinflammatory cytokines [35]. This proteinase is also crucial for alveolar bone resorption and can activate pro-MMP-9, thus contributing to further tissue destruction in chronic periodontitis patients. A correlation has been observed between severe alveolar bone loss and upregulated levels of salivary MMP-8, as opposed to cases involving mild bone loss in chronic periodontitis patients [36, 37]. Collagenases, like MMP-1, MMP-8, and MMP-13, are predominantly produced by periodontal ligament fibroblasts and cells residing in the gingiva.

Gelatinases, notably MMP-2 and MMP-9, are mainly produced by neutrophils and macrophages and play a significant role in destructive periodontal diseases. These enzymes cleave collagen IV found in the basement membrane, which destroys both gingival connective tissue and the periodontal ligament (PDL), contributing to the onset of periodontitis. Studies show that epithelial cells generate increased matrix metalloproteinases (MMP) levels. This surplus production prompts migration of the junctional epithelium toward the tooth’s apex and breaks down surrounding connective tissue. Meanwhile, other cell types like fibroblasts, endothelial cells, and osteoblasts generate MMP-2 while it remains inactive. Overexpression of MMP-9 is linked with increased tissue breakdown and is considered an indicator of the severity of periodontal disease. Matrix metalloproteinases (MMPs) are critical mediators in osteoclast activity and recruitment, facilitating the release of various cytokines and growth factors from the bone matrix. Specifically, MMP-9 is responsible for the release of TGF-b, while MMP-14 modulates the release of RANKL. These MMPs also influence messenger binding to cellular receptors, further highlighting their regulatory role. Inhibition of MMP-9 and MMP-14 obstructs osteoclast migration to resorption sites, emphasizing their critical function in bone metabolism.

The ruffled border of osteoclasts is mainly associated with MMP-14, which enhances osteoclast-matrix interactions and modulates both attachment and detachment of osteoclasts to the bone substrate. During bone remodeling, MMP-13 (collagenase-3) is expressed predominantly by osteoblasts, periosteal cells, and fibroblasts. MMP-13 is instrumental in the initial phase of complete bone resorption by targeting collagen I for proteolysis. Following this cleavage, gelatinases such as MMP-2 and MMP-9 further degrade denatured collagen fragments. In resorption lacunae, MMP-13 is pivotal in clearing the residual collagen left behind by osteoclasts [38]. This enzyme also influences a cascade of events vital for bone remodeling, including the release of pro-MMP-9 from osteoclasts, the deactivation of galectin-3, inhibition of osteoclastogenesis, and facilitation of signaling pathways involving nuclear factor B and TGF-1. Such orchestrated activities suggest that MMP-13 and other MMPs play a consequential role in the progression of periodontal disease.

4.1.2 Role of MMPs in peri-implant disease

Implant-supported oral rehabilitation has proven beneficial for enhancing patient quality of life, but it is not without its complications, including peri-implant mucositis and peri-implantitis. Peri-implant mucositis involves inflammation in the connective tissue surrounding a dental implant, gradually losing the supporting jawbone. Differences in an individual’s immune response can affect levels of matrix metalloproteinases (MMPs) like MMP-1, MMP-7, and MMP-8 detected in the fluid of an implant site showing signs of mucositis. In particular, MMP-8, also called collagenase-2, is an early biomarker indicating the start of experimentally induced mucositis lesions around dental implants when plaque accumulation triggers an inflammatory response [39]. Recent research identifies MMP-8 as the primary Collagenase in active peri-implantitis, with its concentration in peri-implant crevicular fluid reflecting the active stage of inflammatory peri-implant disease [40]. Contrasting this, a systematic review reported increased MMP-1, −8, and −13 levels in peri-implant sulcular fluid, correlating with elevated annual vertical bone loss [19]. Recent evidence has also pointed to elevated levels of MMP-8 and MMP-9 immediately post-implantation, decreasing during the healing phase. This observation suggests a direct relationship between these MMPs, the degree of osseointegration, and the overall healing process [19].

Furthermore, the presence of lesions around dental implants appears to trigger the upregulation of proinflammatory markers and metalloproteinases, which could contribute to the chemotaxis of active osteoclasts and alter the bone remodeling landscape around the implant process [19]. Pathogens like P. gingivalis exacerbate the situation by inducing MMP-1 up-regulation in fibroblasts from peri-implantitis sites, which may substantially escalate the progression of peri-implantitis [41]. The presence of P. gingivalis in tandem with Titanium (Ti) wear particles can notably accelerate the inflammatory processes in the peri-implant tissues. Notably, genetic predispositions may also influence the osseointegration process and implant longevity. The MMP-1 gene’s G-1607GG polymorphism has been correlated with early implant failure [42], while promoter polymorphisms in MMP-13 (−77 A G) and MMP-8 (C-7997) have been linked to loss of osseointegration and implant loss, respectively [43]. Table 2 illustrates various studies demonstrating the expression of MMPs and TIMPs in periodontal disease. Identifying such genetic markers could enable clinicians to predict early implant failure, offering pathways for preemptive action in susceptible patients.

MMPs &TIMPs	Sample/method of estimation	Relevant findings	Conclusion	Author & year
MMP-8, 14 TIMP-1	Saliva/IFMA and ELISA	Higher MMP-8, & TIMP-1 concentrations in periodontitis subjects	MMP-8 and TIMP-1 may be used for the diagnosis of advanced periodontitis.	Gursoy et al. [44]
MMP-8,14, TIMP-1	GCF/IFMA	High MMP-8 was found in active and inactive sites in periodontitis subjects.	MMP-8 could be helpful as a potential biomarker.	Hernandez et al. [45]
MMP-2, 3,8,9; TIMP-1, −2	Plasma/ELISA	Higher levels MMP-3, MMP-8, and MMP-9 in periodontitis patients circulating MMP-8 and MMP-9 decreased after periodontal therapy	Periodontal therapy decreases inflammation—estimation of MMP-8.-9 helps to determine the inflammatory status of periodontal tissue.	Marcaccini et al. [46]
MMP-8, 9; TIMP-1, 2	GCF/ELISA	Higher levels of MMP-8, TIMP-2, MPO, and MMP-9 in periodontitis patients. A decrease in levels was observed during periodontal therapy.	Dysregulated MMP-8, TIMP-2, MPO, and MMP-9 result in periodontal disease progression.	Marcaccini et al. [47]
MMP-8, 9	GCF/ELISA	Higher levels of MMP-8 and − 9 in periodontitis patients.	MMP-8 and − 9 serve as biomarkers of periodontal disease and aid in early diagnosis of periodontitis.	Rai et al. [48]
MMP-8, TIMP-1	Oral rinse/IFMA, ELISA	MMP-8, TIMP, and MMP-8/TIMP-1 are higher with the severity of periodontal inflammatory burden.	MMP-8, together with TIMP-1, could be a potential diagnostic marker.	Leppilahti et al. [49]
MMP-14, TIMP-2	Gingival tissue/western blot analysis.	MMP-14 and TIMP-2 increased in patients with type 2 DM with chronic periodontitis and chronic periodontitis	MMP-14 and TIMP-2 impact the progression of periodontal inflammation associated with type 2 DM.	Kim et al. [50]
MMP-8, 9, 13	Saliva/ELISA	MMP-8, MMP-9, and MMP-13 were higher in subjects with generalized periodontitis	MMP-8 is a promising biomarker candidate for identifying the loss of alveolar bone.	Gursoy et al. [36]
MMP-1, 2,3,8,12,13	GCF/ fluorometric kits	Sustained reduction in MMP-1, MMP-8, MMP-9, MMP-12, and MMP-13 levels up to 6 months after non-surgical periodontal therapy.	Treatment of LAgP with SRP and systemic antibiotics effectively reduced levels of MMPs in African-American individuals.	Goncalves et al. [51]
MMP-8	Saliva/ ELISA	MMP-8 levels were higher in untreated periodontitis, diabetes mellitus, and untreated periodontitis with diabetes mellitus	Diabetes patients had higher levels of salivary MMP-8 regardless of periodontal inflammation. This aspect needs to be considered during periodontal diagnosis since both diseases have been shown to affect protein expression in saliva.	Costa et al. [34]
MMP-8	Saliva/ELISA	MMP-8 levels were higher in subjects with T. denticola and T. forsythia	T. denticola and T. forsythia induce a cascade of host response with increased MMP-8 in GCF.	Yakob et al. [52]
MMP-8	Periodontal tissue/immunohistochemistry	Increasing MMP-8 levels were found in patients with periodontal disease alone, diabetes, and patients with both disorders.	A rising pattern in MMP-8 protein expression levels in chronic diseases.	Hardy et al. [35]
MMP-8	GCF and oral rinse samples/ELISA	Higher aMMP-8 levels in periodontitis patients.	aMMP-8 levels estimation helps in the early diagnosis of periodontal disease and inflammation.	(Yuan, Liu, and Zheng [53]
MMP-2, 8,9 Salivary MMP-8	GCF and saliva/ELISA	Higher salivary MMP-8, crevicular MMP-9, and MMP-2 were observed in periodontitis compared to gingivitis and healthy groups.	MMP-8, crevicular MMP-2- 2, and 9 are biomarkers of periodontal disease and aid in early detection of periodontitis or gingivitis.	Rai et al. [33]
MMP-9	gingival tissues/indirect immunofluorescence	Lower levels of MMP-9 staining were detected in epithelium not exposed to inflammation.	MMP-9 could contribute to gingival epithelial changes in response to periodontal infection.	Smith et al. [54]
MMP-13 TIMP-1	GCF/	It increased MMP-13 activity in periodontitis subjects.	MMP-13 could contribute to the progression of chronic periodontitis and proMMP-9 activation.	Hernandez Rios et al. [37]
MMP-2,9 TIMP-1	GCF/ELISA	Higher concentrations of MMP-9 were observed in patients with periodontitis.; MMP-2 levels were slightly diminished in periodontitis subjects. The lowest concentrations of TIMP-1 were observed in patients with periodontitis, and the concentrations increased after periodontal treatment.	The reduction in TIMP-1 concentrations in disease situations suggests a breakdown of the balance between the amount of MMPs and their inhibitor.	Maeso, Bravo and Bascones [55]
MT1-MMP, TIMP-2	Gingival tissues/ Western-blot	Increased expression of MT1-MMP and TIMP-2 in periodontitis-affected gingival tissues	MT1-MMP and TIMP-2 contribute to collagen remodeling in periodontal disease.	Oyarzun et al. [56]

Table 2.

Studies demonstrating the expression of MMPs and TIMPs in periodontal disease.

4.2 Role of microbial pathogens in activation and expression of MMP

A cascade of inflammatory events is triggered by pathogens like Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia (T. forsythia), and Treponema denticola (T. denticola), which are the critical pathogens of the oral biofilm. Treponema denticola secretes specific proteases that activate pro-MMP-2, produced by periodontal ligament cells in an inactive form. The release of active MMP-2 will result in fibronectin degradation, inducing apoptosis or inhibition of osteoblast differentiation. As part of the tissue repair process, this tissue damage can cause MMP expression to be increased [13]. Pathogens trigger the host immune system, resulting in an inflammatory reaction. As part of the tissue remodeling process, inflammation frequently results in the upregulation of many MMPs. Studies have reported that exaggerated host response by periodontal pathogens T. denticola and T. forsythia could induce increased expression of MMP-8 and MMP-9 in the GCF of periodontitis patients [52, 57].

For instance, lipopolysaccharides (LPS) from gram-negative bacteria can stimulate host cells and release inflammatory mediators like MMPs [58]. The effect of P. gingivalis on fibroblasts from periodontitis and peri-implantitis lesions exhibited a pronounced inflammatory response, which could accelerate the progression of peri-implantitis and periodontitis [59]. P. gingivalis promotes monocyte migration by stimulating MMP-9 production, resulting in tissue deterioration. Periodontitis patients have elevated MMP-9 expression, which predicts disease activity [57]. Once impaired in chronic conditions, the integrity of the host tissues could trigger the release of growth factors, cytokines, and other signaling molecules that induce MMP production. Actinomycetemcomitans LPS can activate the p38alpha MAPK and Jun N-terminus Protein-Serine Kinase (JNK) pathways, increasing the activator protein-1 (AP-1) and nuclear factor kappa-B (NFkappaB) activities, which in turn accelerates up the expression of MMP-2, MMP-3, TIMP-1, and urokinase-type plasminogen activator (uPA). This might hasten the destruction of periodontal connective tissue [60]. Pathogens can activate host proteases that activate pro-MMPs (inactive precursor forms of MMPs) into their active forms. Some pathogens may use MMPs to facilitate their migration and invasion through tissues.

4.3 MMP regulation in response to periodontal therapy

Non-surgical periodontal therapy profoundly reduces inflammation, exhibited clinically and through a reduction in tissue-destructive proteases like MMPs. Reductions in the level of MMP-8 have been reported in patients with chronic periodontitis and those with diabetes-related periodontitis. These reductions have been observed following antibiotics, scaling and root planning treatment, and MMP inhibitors as adjunct medications. Kinane et al. reported decreased levels of MMP-8 in the gingival crevicular fluid (GCF) of patients with chronic periodontitis 3 months after undergoing non-surgical periodontal therapy. Persistently elevated levels of MMP-8 in GCF samples indicate a significant risk of a poor response to periodontal treatment [52].

Furthermore, MMP-8, TIMP-2, MPO, and MMP-9 reductions were observed in the GCF of patients with chronic periodontitis 3 months after periodontal therapy [47]. Studies have also demonstrated a reduction in the levels of MMP-3 and MMP-13 in the GCF after periodontal treatment, underscoring their significant roles in periodontal destruction [19, 61]. As such, MMP-3 and MMP-13 could serve as valuable inflammatory biomarkers for diagnosing the severity of periodontal disease. Significant reductions in the levels of MMP-8, MMP-2, and MMP-9 after non-surgical periodontal therapy have been found to correlate well with the improvement of periodontal parameters, suggesting their utility as biomarkers for monitoring during periodontal recall visits [33].

4.4 MMP’s role as oral biomarkers

Conventional periodontal screening has relied on clinical and radiographic assessments. However, the role of biomarkers in biological fluids is emerging as a prospective screening technique that bridges the gap between systemic disorders and periodontal disease. Tests with specific sensitivity and specificity are essential for this approach to gain traction in routine dental practice. Matrix metalloproteinase-8 (MMP-8) has emerged as one of the most promising biomarkers. It is found in gingival crevicular fluid, peri-implant sulcular fluid, and saliva and can be used to predict, diagnose, and monitor the progression of episodic periodontitis and peri-implantitis. When used alone or in combination with interleukin-1beta (IL-1β) and Porphyromonas gingivalis, MMP-8 can serve as an alternative to traditional periodontal examination for assessing overall risk in large-scale public health surveys [62].

In patients with chronic periodontitis, elevated MMP-8, MMP-9, and MMP-13 levels in gingival crevicular fluid (GCF) have been proposed as potential biomarkers for disease progression [19]. Increased MMP expression has been linked not only to periodontal disease but also to systemic conditions. For example, MMP-8 levels were higher among periodontitis smokers than non-smokers [63]. MMPs are also implicated in gestational health. Elevated GCF levels of MMP-8 and MMP-9 in women with severe periodontitis during early pregnancy have been associated with gestational diabetes [64].

Similarly, decreased MMP-8 levels in peri-implant crevicular fluid (PICF) have been linked to periodontal health, while elevated levels are associated with a heightened risk of peri-implantitis [65]. Quantitative estimation of Matrix Metalloproteinase-8 (MMP-8) levels using chair-side devices has proven to be a pivotal tool in distinguishing between inactive and active sites in both periodontal and peri-implant diseases [66]. Concurrently, in conjunction with clinical evaluations, the assessment of salivary MMP-8 and MMP-9 levels has shown promise in monitoring the periodontal health of patients undergoing orthodontic treatment [67]. Particularly, salivary MMP-9 stands as a more reliable marker for predicting periodontal inflammation during orthodontic procedures, offering new avenues for mitigating periodontal risks during these treatments. Moreover, a strong correlation has been established between the levels of red-complex anaerobic periodontal bacteria and salivary markers, such as MMP-8, MMP-9, and osteoprotegerin, all of which can serve as effective predictors for the severity of periodontal disease [68]. Given these advancements, the future of periodontal disease identification appears increasingly promising. Employing salivary and biofilm biomarkers may facilitate the prediction of periodontal disease progression or stability in larger patient cohorts, thus enabling clinicians to deliver more targeted and timely interventions.

5. Therapeutic implications

5.1 MMP inhibitors as potential therapeutic agents in periodontal diseases

Tissue Inhibitors of Metalloproteinases (TIMPs): TIMP-1, TIMP-2, TIMP-3, and TIMP-4 are endogenous inhibitors in humans that play a crucial role in regulating MMP activity. These inhibitors restore the extracellular matrix, tissue, and cell remodeling processes, all mediated by MMPs. Various mechanisms come into play in the functioning of MMP inhibitors: substitution of zinc ions, calcium chelation at the active terminal, interaction with zymogens before their activation, or coating of the target substrate. TIMPs, due to their high selectivity, can control the enzymatic activity of MMPs without being affected by heat or proteolytic denaturation. Their affinity varies across different MMPs [69]. TIMPs are also regulated at the transcriptional level by cytokines, growth factors, and chemokines. Apart from their inhibitory function, TIMPs can interact with proforms of MMPs and have been found to bind directly to cell surface receptors, exerting functions beyond MMP inhibition.

Synthetic Inhibitors of MMPs: To enhance selectivity and stability, synthetic MMP inhibitors have been developed for medical applications. Tetracyclines, particularly sub-antimicrobial doses of 20 mg doxycycline (Periostat), have been approved by the FDA for treating chronic inflammatory periodontal diseases due to their anti-collagenase activity [70]. Clinical trials have confirmed the safety and efficacy of Periostat as an adjuvant treatment [71]. Another promising compound is 6-deoxy-6-demethyl-4-dedimethylamino tetracycline, also known as COL-3 or CMT-3. This non-antimicrobial derivative of tetracycline exhibits its action by various mechanisms, including direct inhibition of activated MMPs and modulation of cellular mechanisms [72]. Subantimicrobial-dose doxycycline has been shown to suppress inflammatory biomarkers like cytokines, chemokines, and MMPs, with clinical improvements persisting up to 3 months post-discontinuation [71, 73, 74]. Studies have also demonstrated the safety and efficacy of chemically modified curcumin in managing chronic periodontal diseases. It has been found to suppress excessive MMP-2, MMP-9, and the activated form of MMP-8 in diseased gingival tissues [75, 76]. The field of periodontal therapeutics is increasingly acknowledging the potential of both natural and synthetic MMP inhibitors. These agents inhibit the destructive actions of MMPs and have other cellular and molecular regulatory functions. Their roles in modulating the periodontal environment and mitigating disease progression make them valuable additions to the toolkit of periodontal disease management.

5.2 Challenges and adverse effects of MMP inhibition

Maintaining a balanced MMP-TIMP ratio is essential for preserving healthy periodontal structures and preventing periodontal diseases. While MMP inhibitors offer promise as potential therapeutic agents, their development and clinical implementation have faced hurdles. Numerous clinical trials involving synthetic MMP inhibitors have yet to yield expected outcomes due to the complex roles and interactions of MMPs in both health and disease. Thus, balancing the benefits of MMP inhibition against potential adverse effects remains a challenge that researchers and clinicians continue to address. Endogenous TIMPs are unsuitable for pharmacological applications due to their short half-lives, antigenicity, and non-selectivity. Their limited therapeutic utility arises from a short in vivo half-life, immunogenicity, poor stability, and low availability [72]. Tetracycline derivatives have shown promise but come with challenges such as poor aqueous solubility, low oral bioavailability, and non-selectivity, leading to a higher rate of adverse effects with inconsistent and unsatisfactory clinical outcomes. Broad-spectrum synthetic Inhibitors like Marimastat, Batimastat, and Ilomastat have been extensively investigated but have faced issues with high toxicities and limited clinical success. Adverse effects related to musculoskeletal discomfort and inflammation have been observed following both short-term (e.g., Marimastat) and long-term treatment (e.g., BMS-275291) [38].

5.3 Potential for targeted MMP therapies

A more nuanced understanding of the complexities and diversities of cellular pathways mediated by MMPs is essential for developing targeted MMP therapies. Future MMP inhibitors (MMPIs) must ideally have a single-target function, high selectivity, and specificity and preferably act topically. Traditional MMPIs were developed to target the active site zinc and were comprised of a zinc-binding group (ZBG) and a substrate-based peptide backbone to chelate the zinc ion and inactivate the enzyme. Unfortunately, the first generation of MMPIs failed in clinical trials due to limited selectivity and broad-spectrum inhibition. Protein engineering has been employed to develop MMPIs that are highly specific and selective. This innovation has helped overcome the difficulties of generating potent monoclonal antibody-based MMPIs. One groundbreaking strategy focuses on exosites, also known as “hotspots,” which are specific inhibitory sites located away from the conserved catalytic cleft. Targeting these exosites is an alternative approach to modulating MMP selectivity [77]. Researchers have generated inhibitory antibodies with TIMP-like binding mechanisms that specifically target the activated forms of gelatinases (matrix metalloproteinases 2 and 9). These antibodies were developed using a unique vaccination strategy based on principles of molecular mimicry [78]. The high affinity of a monoclonal antibody for its target, coupled with a therapeutic scaffold having excellent pharmacological properties, provides the possibility of extreme potency and selectivity. Such innovative MMPIs, developed through specialized protein engineering and directed evolution techniques, present a novel avenue for understanding the intricate mechanisms behind beneficial and detrimental tissue proteolysis [79].

6. Future directions and concluding remarks

6.1 Emerging research on MMPs and periodontal health

Personalized treatment plans could offer more precise and efficient therapies by assessing each patient,s unique MMP profile. An imbalance between MMPs and TIMPs can lead to ECM degradation, while an imbalance favoring TIMPs results in ECM deposition. New avenues for periodontal treatment are opening up with MMPs as novel targets. Research should focus on multiple therapeutic options, including small molecule inhibitors, monoclonal antibodies, and nanoparticles, to improve periodontal health. Recent advancements include selective synthetic inhibitors designed for specific MMPs, such as synthetic cyclic peptide CTT (HWGFTLC), which appears to target gelatinases selectively [13]. Numerous innovative approaches are also being explored, ranging from TIMP analogs to function-blocking antibodies.

6.2 Potential for biomarker development in periodontal disease diagnosis

Early detection of periodontal disease could be facilitated by monitoring MMP levels. Integrating active-matrix metalloproteinase-8 (aMMP-8) as a biomarker into the new classification system for periodontitis may prove particularly beneficial [80]. Further, a point-of-care (PoC) mouth rinse test for aMMP-8 has been shown to help identify ongoing periodontal breakdown and as an adjunct diagnostic tool in prediabetes/diabetes screenings [81]. Lateral-flow PoC/chair-side tests like PerioSafe and ImplantSafe have also been developed, aiding in the early detection of periodontal deterioration among patients with systemic diseases.

6.3 Conclusive thoughts on the dual nature of MMPs in periodontium

MMPs in the periodontium serve a dual role: they maintain tissue homeostasis and contribute to tissue degeneration. Monitoring MMP levels can help assess the severity of the disease, predict the success of treatment plans, and evaluate the efficacy of various interventions. While targeting MMPs presents exciting therapeutic possibilities, it is important to remember that the complex regulatory mechanisms governing MMPs still need to be fully understood. Their functions could change based on the disease stage and individual patient characteristics. More research is required to understand the specific roles of different MMPs in various aspects of periodontal health and disease. Matrix Metalloproteinases play a pivotal role in the multifaceted landscape of periodontal health and disease. Understanding the specific functions of MMPs can help develop targeted therapies to manage periodontal diseases better and promote overall oral health.