2.Definition and Ideal Criteria of Biomarker
3.Need for a Periodontal Diagnostic Indicator/ Uses
4. Host response in periodontal disease
5. Test to detect biomarker
6. Classification of biomarkers in periodontal diseases
1) GCF biomarkers of periodontal disease activity
2) Salivary Biomarkers of periodontal disease activity
3)Bone remodelling and bone resorption biomarkers
4) Proteonomic markers
7.Serum diagnostics/ Marker for periodontal disease
9. Future Directions
Periodontitis is a group of inflammatory diseases that affect the connective tissue attachment and supporting bone around the teeth. It is widely accepted that the initiation and the progression of periodontitis are dependent on the presence of virulent microorganisms capable of causing disease. Although the bacteria are initiating agents in periodontitis, the host response to the pathogenic infection is critical to disease progression1. After its initiation, the disease progresses with the loss of collagen fibers and attachment to the cemental surface, apical migration of the junctional epithelium, formation of deepened periodontal pockets, and resorption of alveolar bone. If left untreated, the disease continues with progressive bone destruction, leading to tooth mobility and subsequent tooth loss. Periodontal disease afflicts over 50% of the adult population in the United States, with approximately 10% displaying severe disease concomitant with early tooth loss2.
A goal of periodontal diagnostic procedure is to provide useful information to the clinician regarding the present periodontal disease type, location and severity. These findings serve as a basis for treatment planning and provide essential data during periodontal maintenance and disease monitoring phases of treatment. Traditional periodontal diagnostic parameters used clinically include probing depths, bleeding on probing, clinical attachment levels, plaque index, and radiographs assessing alveolar bone level. The strengths of these traditional tools are their ease of use, their cost-effectiveness, and that they are relatively noninvasive. Traditional diagnostic procedures are inherently limited, in that only disease history, not current disease status, can be assessed. Clinical attachment loss readings by the periodontal probe and radiographic evaluations of alveolar bone loss measure damage from past episodes of destruction and require a 2 to 3mm threshold change before a site can be identified as having experienced a significant anatomic event .
Advances in oral and periodontal disease diagnostic research are moving toward methods whereby periodontal risk can be identified and quantified by objective measures such as biomarkers. There are several key questions regarding current clinical decision making: How can clinicians assess risk for periodontal disease? What are the useful laboratory and clinical methods for periodontal risk assessment? And What can be achieved by controlling periodontal disease using a risk profile? Risk factors are considered modifiers of disease activity. In association with host susceptibility and a variety of local and systemic conditions, they influence the initiation and progression of periodontitis and successive changes on biomarkers3 . Biomarkers of disease in succession play an important role in life sciences and have begun to assume a greater role in diagnosis, monitoring of therapy outcomes, and drug discovery. The challenge for biomarkers is to allow earlier detection of disease evolution and more robust therapy efficacy measurements. For biomarkers to assume their rightful role in routine practice, it is essential that their relation to the mechanism of disease progression and therapeutic intervention be more fully understood .
Diagnostic tools to measure periodontal disease at the molecular, cellular, tissue, and clinical levels4
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There is a need for the development of new diagnostic tests that can detect the presence of active disease, predict future disease progression, and evaluate the response to periodontal therapy, thereby improving the clinical management of periodontal patients. The diagnosis of active phases of periodontal disease and the identification of patients at risk for active disease represent challenges for clinical investigators and practitioners .
1. A substance that is measured objectively and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention4 .
2. It is defined as a “ parameter that is objectively measured and evaluated as an indicator of normal biological or pathological processes or pharmacological response to a therapeutic intervention4 .”
Many chronic conditions are the result of complex diseases. It is typical for all complex diseases that they vary in the age of onset, are linked to multiple biological pathways, and that multiple genetic and environmental factors are involved. Examples of complex diseases are rheumatoid arthritis, Crohn’s disease, and periodontitis.
On the basis of our current understanding of the complexity of periodontitis the identification of one single diagnostic marker for all forms of periodontal disease seems illusionary. Nevertheless, researchers have been searching actively for unequivocal markers of periodontitis in gingival crevice fluid to develop a simple test, to be used chair-side, to determine whether a patient suffers from periodontitis and needs therapy, as opposed to another patient who needs no intervention even though he has gingivitis5.
Ideal criteria of biomarker:
Ideal criteria for a biomarker are5,
1.It should have great sensitivity, specificity and accuracy in reflecting diseases.
2.It must be able to clearly reflect the early stage of disease.
3.It must be easily detected without complicated medical procedures/ easy to use.
4.It must minimize false positive and negative.
5.It should have cost effective method for screening.
CHAPTER 3-NEED FOR A PERIODONTAL DIAGNOSTIC INDICATORS
A periodontal diagnostic tool provides pertinent information for differential diagnosis, localization of disease and severity of infection. These diagnostics in turn serve as a basis for planning treatment and provide a means for assessing the effectiveness of periodontal therapy. Current clinical diagnostic parameters that were introduced more than 50 years ago continue to function as the basic model for periodontal diagnosis in clinical practice today. They include probing pocket depths, bleeding on probing, clinical attachment levels, plaque index, and radiographs that quantify alveolar bone levels6. Albeit easy to use, cost-effective and relatively noninvasive, clinical attachment loss evaluation by the periodontal probe measures damage from past episodes of destruction and requires a 2- to 3-mm threshold change before a site can be deemed as having experienced significant breakdown. Recent revisions in the design of automated periodontal probes have improved the accuracy and long-term tracking of disease progression. Furthermore, the use of subtraction radiography also offers a method to detect minute changes in the height of alveolar bone. However, both of the above-mentioned techniques are most often seen in the research setting and seldom in clinical practice. In addition to these limitations, conventional disease diagnosis techniques lack the capacity to identify highly susceptible patients who are at risk for future breakdown. Researchers are confronted then with the need for an innovative diagnostic test that focuses on the early recognition of the microbial challenge to the host. Optimal innovative approaches would correctly determine the presence of current disease activity, predict sites vulnerable for future breakdown, and assess the response to periodontal interventions. A new paradigm for periodontal diagnosis would ultimately affect improved clinical management of periodontal patients.
CHAPTER 4- HOST RESPONSE IN PERIODONTAL DISEASE
The immune system protects the host against the infection. Healthy individuals defend themselves against antigenic stimuli via innate or acquired immunity. Immune responses to pathogens ideally should accomplish their objectives with minimal injury to normal host tissues. However prolonged, exuberant or inappropriate immune responses are often capable of inducing tissue injury and significant host pathology7. Following periodontal infection the initial response typically is recruitment and migration of polymorphonuclear leucocytes to the site of periodontal infection. If polymorphonuclear leucocytes successfully eliminate the pathogens and their by products via phagocytosis and intercellular killing mechanisms, the clinical result may be limited to gingivitis. However if these mechanisms are evaded and inflammation continues in host tissues then a transition from gingivitis to periodontitis is facilitated. The production of antibody by plasma cells at this stage can limit the infection, not if the action of polymorphonuclear leucocytes and antibodies are not sufficient for bacterial clearance then antigens and antigenic products can result in macrophage and T- lymphocyte activation.
Innate immunity includes cells of non-hematopoietic origin (especially epithelial derivatives), plasma factors (such as complement) and myeloid cells of hematopoietic origin (especially phagocytes). Phagocytes such as macrophages and neutrophils have surface receptors that recognize and bind certain surface molecules of bacteria, which can prime or prolong the life of the phagocytes and occasionally promote phagocytosis (engulfment of the bacterium). However, phagocytosis usually requires opsonization (coating the bacterium with some recognizable, self-derived molecule), especially, antibodyand/or complement. This will also stimulate the production of cytokines, which are chemical mediators used as signals in intercellular communication. Pathogens induce the release of cytokines by phagocytes that trigger a complex set of responses that are collectively referred to as inflammation. Within connective tissues, the initial phases of an inflammatory response is characterized by the infiltration of acute inflammatory cells, especially neutrophils, followed within 30 minutes by chronic inflammatory cells, especially macrophages and lymphocytes. This is referred to as the natural history of inflammation. In the periodontium, there are two distinct areas of leukocyte infiltration: that is, the (i) junctional epithelium/gingival crevice and (ii) the subjacent connective tissues. Because of the brevity of acute inflammatory infiltration, the subjacent connective tissues will almost always be dominated by chronic inflammatory cells. Because of the selectivity of transepithelial migration, the junctional epithelium/ gingival crevice is always dominated by neutrophils8.
Specific immunity however is induced upon exposure to foreign substance, recognizes distinct macromolecules and increases in magnitude and capability with each successive antigenic exposure. Specific immunity can be characterize as being either cell mediated (via T lymphocytes) or humoral ( mediated by antibodies). Antibodies belong in the third fastest migrating group of serum globulin the gamma globulins. The immunoglobulin (Ig) refers to the immunity conferring portion of the gamma globulin fraction9.
Cells are B lymphocytes which give rise to plasma cells. Mediators are 5 antibody isotypes (IgG, IgM, IgA, IgD, andIgE).
Cell mediated immunity:
Cells are T lymphocytes, monocytes or macrophages. Mediators are interleukin or cytokines( such as IL-1α, IL-1β, IL-8and TNF-α).
Pathways to specific immunity
Along the afferent pathway leading to specific immunity, the activation of lymphocytes depends critically on several key interactions with inflammatory cells of innate immunity. In particular, innate immune cells are required for (i) antigen processing and presentation, (ii) co-stimulation and (iii) provision of early differentiative signals (cytokines such as interleukin- 1). In many instances, the final effector mechanism is also dependent upon cells of innate immunity. Lymphocytes or their products play an important role in the self-recognitive targeting of the effector mechanisms, adding a “smart bomb’-like quality to the immune response; often enabling the effector mechanisms to overcome microbial evasive strategies. For example, many bacteria have capsules that enable them to evade successful opsonization by complement alone. Therefore, more specific recognition by components of adaptive immunity (lymphocytes) is sometimes required to assist the phagocytes recognize and eliminate such pathogens8.
Lymphocytes are unique among human cells in that they possess antigen receptors. These receptors (i) specifically recognize oligomeric structures (mainly the primary sequence) and (ii) exhibit a clonal distribution: the antigen receptor of one lymphocyte is specific for a different antigen than the antigen receptor of a second lymphocyte. Lymphocytes achieve such antigen diversity of their receptors by rearranging the genes for these receptors during their development in the bone marrow and thymus. In this manner, a few hundred genes can be rearranged to code for antigen receptors with millions of different specificities. Thus, although each lymphocyte bears receptors with only a single specificity on its surface, the pool of lymphocytes contains cells with millions of specificities8.
CHAPTER 5-TEST TO DETECT BIOMARKERS
d.DNA probe technology
f.Polymerase chain reaction
Culture methods are considered gold standard against which the other microbiological identification methods have been compared. Generally plaque samples are cultivated under anaerobic conditions and the use of selective and non-selective media with several biochemical and physical tests allows the identification of different putative pathogens. The main advantage of this method is that the clinician can obtain relative and absolute counts of the cultured species. Moreover it is the only vitro method able to assess for antibiotic susceptibility of the microbes. These are able to grow and multiply those bacteria which are suited to grow on the culture medium used with necessary growth requirements10.
Culturing has one unique advantage over the other microbiological identification methods, it permits the assessment of antibiotic sensitivity. However it also has some significant limitations including its inability to detect low levels of microorganisms, high cost,labor intensiveness, prolonged period of time before results can be obtained and inability or difficulty in growing several bacterial species11.
Dark field or phase contrast microscopy has been suggested as an alternative to culture methods on the basis of its ability to assess directly and rapidly the morphology and motility of bacteria in a plaque sample. However most of the main putative periodontal pathogens including Aa, Pg, Tf, Ekinella corrodens(Ec) and Eubacterium species are non-motile and therefore this technique is unable to identify these species. Microscopy is also unable to differentiate among the various species of Treponema. Therefore Darkfield microscopy seems as unlikely candidate as a diagnostic test of destructive periodontal diseases10.
These visual techniques can determine the relative proportions of coccal and filamentous shaped organisms12. Short rod like organisms such as P.gingivalis will usually appear coccoid in shape and motile organisms such as spirochetes, can be easily identified based on their shape and movements. Since individual species can not be identified, the main usefulness of microscopic identification comes from observing a shift in the appearance of the flora with periodontal therapy. Successful treatment will result in a change from a highly pathogenic flora, which is densely populated and dominated by motile and rod like organisms to a more healthful one, which is sparsely populated, coccal and non-motile. A significant benefit of microscopic evaluation of plaque is its ability to be performed in the office. It may also have some value in patient education. However it does not help in selection of an antimicrobial agent when desired as a part of therapy. Further more routine use of darkfield microscopy for treated periodontitis patients during periodontal maintainence has not been shown to help predict recurrence of disease.
Immunological assays employ antibodies that recognize specific bacterial antigens to detect target micro-organisms. This reaction can be revealed using a variety of procedures including direct and indirect immunofluroscent (microscopy) assays(IFAs) flow cytometry, enzyme linked immunosorbant assay, membrane assay and latex agglutination.
Direct IFA employs both monoclonal and polyclonal antibodies conjugated to a fluroscein marker that binds with the bacterial antigen to form a fluroscent immune complex detectable under a microscope.
Indirect IFA employs a secondary fluoroscein conjugated antibody that reacts with the primary antigen-antibody complex. Both Direct and Indirect IFAs are able to identify the pathogen and quantify the percentage of the pathogen directly using a plaque smear. IFA has been usede mainly to detect Aa and Pg.
Cytoflurography or flow cytometry for the rapid identification of oral bacteria involves labelling bacterial cells from a patient plaque sample with both species-specific antibody and a second fluroscein conjugated antibody. The suspension is then introduced into the flow cytometer , which separates the bacterial cells into an almost single cell suspension by means of a laminar flow through a narrow tube. The sophistication and cost involved in this procedure precludes its wide use.
Enzyme linked immunosorbent assay: It is similar in principle to other radioimmunoassay but instead of the radioisotope an enzymatically derived color reaction is substituted as a label. The intensity of the color depends on the concentration of the antigen and is usually read photometrically for optimal quantification. ELISA has been used primarily to detect serum antibodies to periodontal pathogens, although it has also been used in research studies to quantify specific pathogens in subgingival samples using specific monoclonal antibodies.The use of highly specific immunological techniques such as immunofluroscence or ELISA can detect individual bacterial species. Proved useful to detect the presence and relative proportions of selected bacterial species. Use specific antibodies which bind to the selected bacterial antigens and are then detected by direct or indirect immunofluroscence .In the ELISA assay the primary antiboby is detected through a calorimetric reaction which is catalysed through an enzyme, usually horseradish peroxidase or alkaline phosphatise linked to the antiboby. They can only detect species for which a suitable antibody is available.
Latex agglutination is a simple immunologic assay based on the binding of protein to latex. Latex beads are coated with the species specific antibody and when these beads come in contact with the microbial cell surface antigens or antigen extracts, cross linking occurs, in agglutination or clumping is then visible, usually in 2-5 minutes. Because of their simplicity and rapidity, these assays have great potential for chairside detection of periodontal pathogens10.
d.Nucleic acid probes:
A probe is a known nucleic acid molecule (DNA or RNA) from a specific micro-organism artificially synthesized and labelled for its detection when placed with a plaque sample. DNA probes used segment of a single stranded nucleic acid, labelled with an enzyme or radioisotope that is able to “hybridize” to the complementary nucleic acid sequence and thus detect the presence of target micro-organism. Hybridization refers to the pairing of complementary DNA strands to produce a double stranded nucleic acid. The nucleotide base pair relationship is so specific that strands cannot anneal unless the respective nucleotide strand sequences are complementary10. The probe sequences may be whole genomic randomly cloned sequences of nucleic acids or synthetic oligonucleotides ( also known as 16S rRNA probes). Of the three it is the oligonucleotide probes that have the greatest specificity and lowest cross-reactivity because they target genes specific to bacterial species. Whole genomic probes and random cloned probes may contain sequences common to multiple species. Nucleic acid probes used for microbiological analysis of plaque have been confirmed to have greater sensitivity than culture methods. In addition viability of microorganisms is not a requirement of nucleic acid probe analysis that may be an advantage when plaque sample analysis may be delayed because of lengthy transportation from clinic to laboratory13.
An enzymatic assay has been developed that detects bacteria that possess trypsin like enzymes such as T. forsythensis, Treponema denticola and P.gingivalis. when a plaque sample containing any combination of these three bacteria is placed on a paper strip impregnated with a colorless substrate N-benzoyl-DL-arginine-2-napthylamide(BANA), the BANA substrate breakdown and produces a blue-black color whose intensity is proportional to the total amount of the three organisms. While this chairside test is unable to distinguish between the relative proportions of the three bacteria and cannot identify the presence of other oral microorganisms, the BANA test has been shown in studies to be well correlated with pocket depth in periodontally diseased sites. Its utility as a diagnostic method is however uncertain due to its low reliability to predict clinical assessment of disease progression. It may have more value when performed in combination with other chairside microbiological tests such as microscopy14.
f.Polymerase chain reaction:
It has emerged as the most powerful tool for the amplification of genes and their RNA transcripts. This technique developed in 1985, is the single technique used almost universally to study DNA and RNA obtained from a variety of tissue sources. PCR allows large quantities of DNA to be obtained in a simplified and automated manner. PCR typically begins with the isolation of DNA from a fresh tissue specimen. By heating the complementary double strands, DNA splits into single stranded forms intended to act as the template dictating the nucleotide sequence in vitro. The amplification is followed using a DNA polymerase that requires a primer or known short oligonucleotide sequence corresponding to the border of the region that is amplified. For obtaining amplified fragments of constant length and in large quantities a second primer complementary of the opposed chain must be used to anneal the template and flank the region of interest. This amplification can be performed several times known as cycles. In each cycle the process of complementary chain denaturation, primer hybridization and primer extension by means of the polymerase take place. With each cycle there is an exponential increase in the quantity of DNA. Throughtout this process the temperature during the cycle is critical to control the double chain denaturationand the stability of the hybridization between the model fragment and the primer.
Standard PCR technology although demonstrating high sensitivity and specificity for the identification of the target periodontal pathogens is unable to quantify them accurately in critical samples and therefore its role as a routine clinical diagnostic tool is limited10.
CHAPTER 6- CLASSIFICATION OF BIOMARKERS IN PERIODONTAL DISEASE ACTIVITY
Much effort has been made in recent years to identify risk factors responsible for initiation and progression of periodontal diseases. Mounting evidences indicate that gingivitis and periodontitis are caused by various host responses which are associated with the continuous presence of microorganisms in the gingival crevice. They may cause disease either by decreasing the host defence capability or by triggering a variety of local inflammatory responses. Increasingly attention has been directed to assaying various by products of the host bacterial interactions like Aspartate aminotransferase, Lactate dehydrogenase, Arylsulfatase, cathepsins, β-glucoronidase etc.
Biomarkers can be categorized into five broad categories,
1.Gingival crevicular fluid biomarkers of periodontal tissue activity
1.Gingival crevicular fluid biomarkers of periodontal tissue activity:
Gingival crevicular fluid provides a non-invasive means of studying the host response factor by change of constituents in the fluid. The inflammatory exudates from gingival microcirculation crosses inflammaed periodontal tissue and en route collects molecules of potential interest from the local inflammatory reaction. Therefore a fluid offers a great potential source of factors like enzymes that may be associated with tissue destruction15. It contains a rich array of cellular and biochemical factors which have been shown to indicate the metabolic status of various tissue components of the periodontium. Such factors are now finding value as potential diagnostic or prognostic markers of the periodontium in health and disease16.
Table 3 Classification of GCF markers
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1.Products and mediators of inflammation:
a) Matrix Metalloproteinases(MMPs):
Matrix metalloproteinases (MMPs) form the most important family of proteinases that participate in the normal turnover of periodontal tissues as well as their degradative aspects during periodontal diseases. Most cells in normal and inflamed periodontal tissues synthesize various MMPs that together have the capacity in concert to initiate and complete the degradation of connective tissue matrices. In addition, as described below, some MMPs act as metabolic regulators by processing bioactive molecules to modulate cell responses. As MMPs can potentially destroy tissues, their activity is strictly controlled at different levels. First, specific inhibition of MMPs can be mediated by the four members of the tissue inhibitor of metalloproteinase (TIMP) family, proteins that regulate the extracellular activity of MMPs18. Altered TIMP expression is also known to occur in many disease processes and affects the processing of extracellular matrix and growth factors to modulate cell behavior. Second, MMPs are synthesized as latent zymogens. Activation of MMP zymogens is a critical step for regulating MMP activity and hence the composition, structure, and function of periodontal connective tissue matrices. Third, most MMPs are secreted from cells as a soluble proform. A distinct group of MMPs called membrane-type MMPs (MT-MMPs) however, are not released but exert their activity on the cell surface. For some soluble MMPs, activation occurs at the cell surface following proteolytic cleavage by MT-MMPs, often in a TIMP dependent pathway. For other MMPs, activation occurs in the extracellular environment in an activation cascade initiated by tissue proteinases, such as plasmin, kallikrein, and tryptase, a process that is often amplified by the activated MMPs functioning as pro-MMP activators. Therefore, an improved understanding of the structural basis of MMP function may point to new avenues for the therapeutic treatment of periodontitis. Notably, studies of substrate recognition and cleavage, MMP inhibition, and the domain–domain interactions that occur inthe activation and association of MMPs and TIMPs with the cell membrane and in the matrix are of considerable importance. With the publication of the sequence of much of the human genome it is now appears that the MMP gene family encodes a total of 24 homologous proteinases (MMPs 1–3 and 7–28) and three pseudogenes. MMPs are unified by a common global fold and mechanism of catalysis, but are distinguished within the family by selectivity for different substrates. Since many substrates are cleaved after interaction with MMP substrate-binding exosite domains lying outside the catalytic domain, it is important to understand the structural basis for MMP activity. Another significant research challenge is elucidating the in vivo substrates of MMPs and their role in vivo. Even though MMPs are known to degrade a variety of proteins and proteoglycans in vitro, this degradative action cannot be applied directly to their actual functions under the complex conditions of living tissues. New strategies are needed to search for additional MMP substrates since sequence information and peptide-bond specificity determined from screening of either synthetic or phage libraries does not necessarily identify or provide information on the in vivo substrates of MMPs. Indeed, many new functions for MMPs have been recently determined that do not include degradation of extracellular matrix molecules. It now appears that MMPs have the ability to also process with great precision bioactive molecules such as growth factors and cytokines, cell surface receptors and adhesion molecules. Hence, this positions MMPs as critical regulators of inflammation and immune cell function, as well as of extracellular matrix cell behavior. Until the actual functions of specific MMPs are known it is very difficult to interpret their measured levels in tissue fluids, such as GCF.
Structure/function of MMPs:
Like many extracellular proteins, MMPs are multidomain mosaic proteins that share similar primary, secondary, and tertiary structures. Functionally, the most important domain of MMPs is the catalytic domain. Amongst the MMPs for which structural information is available, the basic structural fold, in which only small differences are apparent, appears almost identical. Typically these lie in the ‘specificity loop’ that defines the prime side of the lower lip of the catalytic cleft, but some other differences are also apparent on the upper lip of the cleft on the non-primed side. Nonetheless, what is remarkable is how similar the catalytic domain and active sites appear amongst the different MMPs. Accordingly, subtle differences in the catalytic domain are critically important for determining fine substrate specificity preferences. However, substrate preferences are intensely modified by exosites, which introduces a new substrate-binding interaction surface close to the active site cleft19. The basic fold of the catalytic domain is a fivestranded b-sheet with three a-helices. The most important of these helices, helix B, forms the base of the active-site cleft and contains the HExxHxxGxxH Zn2π ion binding motif. Further downstream is an invariant methionine turn, which positions the hydrophobic side-chain of the methionine under the catalytic Zn2π. The catalytic domain is also stabilized by one structural Zn2π ion on the upper surface in the ‘S-loop’ and by two or three Ca2π ions, depending on the MMP. The active-site glutamate binds and polarizes a water molecule which co-ordinates with the active-site Zn2π and functions as the nucleophile for peptide-bond hydrolysis. During cleavage, a tetrahedral transition-state intermediate forms in which the negatively charged oxygen is stabilized by co-ordination with the catalytic Zn2π ion. The active-site specificity subsites differ between MMPs to accommodate different peptide backbones of substrates around the scissile bond. A crucial molecular determinant of MMP substrate specificity is the side chain of the amino acid residue (P1ƒ) immediately after the scissile bond . The S1ƒ specificity pocket of MMPs accommodates this side chain and so the size and chemical characteristics of the S1ƒ subsite are important in determining peptide bond preference for cleavage. For example, the small S1ƒ pocket of MMP-1 and MMP-7 restrains substrate preference to small hydrophobic residues at P1ƒ. In contrast, other MMPs, such as MMP-2, MMP-3 MMP-8 MMP-9 and MMP-13 have large S1ƒ pockets and can accommodate a more diverse range of amino acids at P1ƒ.