Matrix Metalloproteinase-8 as an Inflammatory and Prevention Biomarker in Periodontal and Peri-Implant Diseases

Levels of and especially the degree of activation of matrix metalloproteinase (MMP-8) in oral fluids (i.e., saliva, mouth rinse, gingival crevicular fluid (GCF) and peri-implantitis sulcular fluid (PISF)) increase to pathologically elevated levels in the periodontal and peri-implant diseases. This study aimed at collecting and collating data from previously published studies and determining whether active MMP-8 (aMMP-8) could serve as a biomarker for the diagnosis and prevention of periodontal and peri-implant diseases. The literature search identified a total of 284 articles. Out of 284 articles, 61 articles were found to be relevant. Data obtained from the selected studies were combined, and it indicated that aMMP-8 in oral fluids exerts the strong potential to serve as a useful adjunctive diagnostic and preventive biotechnological tool in periodontal and peri-implant diseases. aMMP-8 can be used alone or in combination with other proinflammatory and/or microbiological biomarkers.


Introduction
Periodontitis and peri-implantitis, globally common infectioninduced oral inflammatory disorders of teeth and dental implants supporting soft and hard tissue, i.e., periodontium and peri-implatium, involve destruction of both soft and hard tissues, as active periodontal and peri-implant degradation (APD). Periodontal/peri-implant tissues are mainly made up of type I collagen.
e proteolytic enzyme mainly responsible for the active periodontal/peri-implant soft and hard tissue degeneration (APD) is matrix metalloproteinase , also known as collagenase-2 or neutrophil collagenase. MMP-8 is a member of the MMP family. Structurally related but genetically distinct MMPs are Ca 2+ -and Zn 2+ -dependent endopeptidases capable of degradation of almost all extracellular matrix and basement membrane protein components both in physiologic repair and pathologic destruction of tissues, such as a breakdown of extracellular matrix in embryonic development, wound healing, and tissue remodeling [1]. e MMP family is divided into six protease groups: collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10, and MMP-11), matrilysins (MMP-7 and MMP-26), member-type MMPs (MMP-14, MMP-15, MMP-16, MMP-17, and MMP- 12), and other nonclassified MMPs, given their auxiliary contrasts [2]. Among all of these groups, the collagenase group is of particular relevance in periodontal disease as it can efficiently cleave native collagen fibers I, II, and III. MMP-8 has been categorized under the interstitial collagenase subgroup of the MMP family. Activities of MMPs are inhibited and regulated by the endogenous or natural tissue inhibitors of tissue inhibitors of MMP (TIMPs) and α2-macroglobulin [3]. e imbalance between MMPs and TIMPs often results in irreversible periodontal and peri-implant destructive pathology involving irreversible APD [3][4][5].
A number of studies that have been performed utilize point-of-care (PoC)/chair-side analysis of elevated aMMP-8 in saliva/oral fluids [15][16][17]. A study comparing a PoC immunoflow tool with the standard gold laboratory-based one concluded that concentration of aMMP-8 in oral fluids is useful in distinguishing periodontal diseases from healthy subjects [15]. Lateral flow immunoassay of aMMP-8 has been shown to have high sensitivity for at least two sites with BOP and two sites with deepened periodontal pockets [18]. Sorsa et al. demonstrated that immunofluorometric assay (IFMA) and DentoAnalyzer-PoC-test could detect aMMP-8 from GCF samples, and these methods are comparable with the chair-side/PoC aMMP-8 dip-stick test [6]. e Amersham enzyme-linked immunosorbent assay (ELISA) for total MMP-8 immunoactivities was not in line with the PoC/chair-side immune tests, specific for aMMP-8 [6]. Few studies demonstrated the associations of various periodontal pathogens in oral fluids with the levels of aMMP-8 and suggested to use in combination with aMMP-8 with other proinflammatory and microbiological biomarkers that may potentially improve the diagnostic accuracy [6,7]. e present review aimed at collecting and collating the data from published literature regarding the potential of aMMP-8 in saliva/oral fluids to be used as a biomarker and predictor for periodontal and peri-implant diseases [6,7,19,20].

Study Identification.
A literature search was performed in two electronic databases PubMed and Cochrane to identify related studies of the past 15 years. In addition to this, other relevant studies were identified by manual searching. Keyword used for study identification in all databases were "MMP-8 and periodontal inflammation," "MMP-8 and peri-implantitis," and "MMP-8 and low-dose doxycycline." e synonyms such as MMP-8, collagenase-2, and neutrophil collagenase were also searched in combination with periodontitis. e electronic search was done from November 11, 2016, to July 30, 2018.

Study Selection.
All identified studies were screened, and the selection process was done on the basis of inclusion and exclusion criteria. (1) Written in language other than English (2) Case reports (3) esis (4) Animals studies (5) Diagnosis of periodontal disease was not written (6) Experimental gingivitis

Study Selection and Data
Abstraction. e literature search identified a total of 284 articles. Out of 284 articles, data of 61 articles were selected. Data obtained from selected studies were combined and summarized in the present study (Table 1).

Sources of MMP-8 in the Oral Cavity.
A major source of MMP-8 (neutrophil-type MMP-8) in humans are degranulating triggered neutrophils, but MMP-8 (mesenchymal cell-type MMP-8) is also de novo expressed and secreted in small amounts by non-PMN-lineage cells such as epithelial cells, smooth muscle cells, fibroblasts, macrophages, and endothelial cells [74][75][76][77]. Neutrophil collagenase/ polymorphonuclear leukocyte-(PMN-) derived collagenase-2/MMP-8 differs from interstitial collagenases secreted by other cells in that it is synthesized only during the myelocyte stage of development of neutrophils in the bone marrow and stored as a latent enzyme, i.e., latent pro-MMP-8 (Mr 85 kDa) within the specific granules of PMN. Pro-MMP-8 is rapidly released from activated PMN undergoing selective subcellular granule degranulation and is then activated through the cysteine switch mechanism often, but not always, associated with selective N-terminal proteolysis to yield the active form of the enzyme (Mr 65 kDa) and activation fragments [3,[74][75][76][77].
e main source of oral salivary collagenase is PMNs that enter the oral cavity through gingival sulcus [11,75]. It is evident from the fact that collagenase was only detected in Table  1: Summary of studies related to periodontitis, peri-implantitis, and L/SDD and level of MMP-8 in oral fluids. Study     International Journal of Dentistry 9      Peri-implantitis Elevated MMP-8 could be detected in periimplantitis, oral fluid vs. healthy oral fluid International Journal of Dentistry whole oral saliva of subjects and not in secretions of major oral salivary glands. Furthermore, whole oral saliva collected from edentulous subjects did not show a significant amount of collagenase [75]. Oral fluid (GCF, PISF, mouth rinse, and saliva) collagenases exert similarity with PMN-or neutrophil-type collagenase-2 (MMP-8). It degrades type I and II collagens significantly faster than the type III collagen. Its molecular weight is 65-70 kDa, same as collagenase of the PMNs/neutrophils/MMP-8 and gingival sulcus [3,7]. It is activated by gold thioglucose, which only activates PMN/neutrophil collagenase [3,7,75,77].

Active and Latent Forms.
Most of the oral salivary collagenase found in a healthy mouth is in the latent form, whereas in case of periodontal or/and peri-implant disease patient(s), it is in active or activated (aMMP-8) form together with activation fragments [3,43,75,77]. Studies done by Gangbar et al. and Lee et al. [11,76] demonstrated that oral fluid active collagenase, but not latent, is related and reflects to progressive clinical periodontal disease activity, i.e., loss attachment or APD. aMMP-8 in oral fluids precedes, predicts, is associated with, and reflects on on-going or future/developing progressive, often hidden and subclinical, periodontal and peri-implant disease activity, i.e., CAL, APD, and active peri-implant degeneration [3,6,7,17,76,77]. Significant correlations have been found between aMMP-8 and progressing severity of periodontal and peri-implant diseases [3,4,6,7,26]. Successful periodontal and periimplant treatment significantly reduces aMMP-8 levels in oral fluids [3,6,7,17,78,79].

MMP-8 and Correlation with Periodontal Diseases.
It has been documented in several studies that salivary and oral fluids at aMMP-8 levels are higher in subjects with localized and generalized periodontitis than in healthy controls but the levels reduced after nonsurgical periodontal therapy, i.e., scaling and root planning (SRP) [12,39,41,43,53]. Furthermore, aMMP-8, but not latent/total MMP-8, levels could differentiate between periodontitis and gingivitis as well [59]. A slight increase in MMP-8 levels could be observed in case of gingivitis, which shows a decrease after dental prophylaxis or secondary preventive interventions [57].
Nwhator et al. demonstrated that aMMP-8, measured by lateral flow chair-side/PoC immunoassay (PerioSafe ® ), is directly proportional to the oral hygiene status [18]. It shows a positive correlation with chronic periodontitis and BOP but only in the presence of two or more sites having the deepened PPD of not less than 5 mm; these aMMP-8 PoC findings indicate that such deepened sites are APD affected. e sensitivity of immunoassay for a single site affected by chronic periodontitis was found to be less [18]. Levels of aMMP-8 in oral fluids have been demonstrated to correlate with clinical periodontal parameters in particularly PPD, and it also reflects the effect of treatment [12,18,44,47,54,62]. Levels of aMMP-8 are not only associated with clinical periodontal parameters status but also showed significant association with radiological parameters. aMMP-8 levels have been shown to differentiate subjects with a severe bone loss with those with a slight bone loss [53,58]. Izadi Boroujeni et al. demonstrated a sensitivity of 87% and specificity of 60% of aMMP-8 in a PoC detection of generalized chronic periodontitis [62].
In children, sites with aggressive periodontitis show higher levels of MMP than adults with chronic periodontitis [33]. Baeza et al. reported in their study that aMMP-8 levels in chronic periodontitis were elevated [69]. When aMMP-8 levels were measured by ELISA, the cutoff point was identified as 13 ng/ml chronic periodontitis case [69].
Leppilahti et al. established cutoff levels for smoking and nonsmoking periodontal patients to predict site-specific levels of treatment outcomes [56,57,59,63]. e most optimal cutoff value among smokers was 0.045, whereas for nonsmokers, the calculated value was 0.085. ese values can be helpful in longitudinal monitoring of the disease status during the maintenance period [56,57,59,63].

Study Specimens.
Oral fluids, such as mouth rinse, GCF, PISF, and saliva, have been used as specimens [3,6,7]. Mouth-rinse samples can be collected quickly, noninvasively, and the collection process is less time-consuming as compared to a collection of GCF and PISF. Mouth-rinse assay is useful for screening purposes mainly, but it does not provide exact information or identification/localization about the sites of clinically active disease. Whole saliva, variation in the salivary flow rate, use of antimicrobial medication, and smoking habits may have an impact on the results. GCF and PISF provide site-specific information, therefore useful in the personalized treatment plan of an individual [53]. Johnson et al. reported that when measured with lateral flow immunoassay, saliva showed 4.1 times higher concentration of MMP-8 in periodontal patients than periodontal healthy controls [15].
Correlation between aMMP-8 levels in serum and oral fluids have been tested in few studies [34,41,56,71]. Noack et al. reported a significant correlation between aMMP-8 concentration in the serum and severity of periodontal disease. In addition, serum MMP-8 concentration was also found to show a positive correlation with a subgingival bacterial load [71]. Differing from findings of Noack et al. [71], others on serum concentration of MMP-8 failed to find any correlation with periodontal disease [34,41]. ese varying associations can also be affected by differences in the use of clinical indices utilized to assess periodontal health and disease as well as systemic assessments of patients and healthy controls. Additionally, various mediations may affect systemic and serum aMMP-8. [34,41,56,71] Only one study reported that fibroblasts were used as a study specimen to evaluate its role in the pathophysiology of peri-implantitis. [34] When proinflammatory and matrix degrading responses of gingival and granulation tissue fibroblasts to an in vitro challenge to Porphyromonas gingivalis (P. gingivalis) were compared between subjects with healthy periodontium and patients with periodontitis and peri-implantitis lesion, MMP-8 expression was found higher in nonchallenged periimplantitis fibroblasts than in fibroblasts from healthy periodontium. is indicates that the inflammatory response was more pronounced in fibroblasts from periodontitis and peri-implantitis than in fibroblasts from periodontally healthy individuals.
ese findings suggest that the exposure of prolonged inflammation, i.e., periodontal/peri-implant disease experience and burden, can affect and promote cells' ability to express MMP-8 [34].
Passoja et al. did not find any correlation between periodontal disease and serum MMP-8 levels [34]. A study performed byÖzçaka et al. showed that the levels of MMP-8 in the serum of patients with chronic periodontitis did not significantly differ from periodontal healthy subjects [41]. Kinney et al. showed that serum levels of biomarkers did not play any significant role in the diagnosis of periodontitis [56].

aMMP-8 Level in Oral Fluids of Smokers.
According to Mäntylä et al., the mean aMMP-8 levels in smokers were found to be lower compared to non-smokers, but sites with the progressive disease show similar or higher levels of aMMP-8 in both smokers and nonsmokers [28]. Heikkinen et al. found similar results when comparing levels of aMMP-8 levels between smokers and nonsmokers, but the difference found was not statistically significant. Levels of aMMP-8 reduced after SRP but sites with exceptionally elevated aMMP-8 concentrations clustered in smokers did not show a significant decrease in aMMP-8 after SRP. ese sites with a poor response may indicate sites at elevated risk and were easily identified by the chair-side/PoC aMMP-8 test. [28,38] Baseline GCF aMMP-8 levels have been shown to predict aMMP-8 levels during maintenance of periodontitis. Particularly in smokers, high levels of aMMP-8 at the baseline indicated a poor response to periodontal treatment [60].
According to Heikkinen et al., smoking affects the biomarker values in a dose-dependent manner. Former smokers were found to have a similar level of aMMP-8 as compared to nonsmokers. Furthermore, obesity was found to be a confounder. Values of aMMP-8 among nonsmokers did not remain statistically significant when body mass index values were taken into account during analysis. However, the values were not affected in case of male smokers [38].
In contrast to these studies, Passoja et al. and Miller et al. did not find any significant correlation of smoking with an elevated aMMP-8 level in their independent studies done on   (1) and (3) before and (2) and (4) after nonsurgical periodontal treatment, scaling, and root planning (SRP). e appearance of two lines (>20 ng/ml) pointed by arrows in the figure is a considered positive test which indicates elevated risk for periodontitis. e appearance of only one line indicates successful test performance and no risk for periodontitis (aMMP-8 < 20 ng/ml) after SRP treatment. Periimplantitis (b) results based on ImplantSafe ® -PISF-strip-test; two peri-implantitis patients (1) and (3) before and (2) and (4) after peri-implantitis treatment (plastic scaling, oral hygiene instructions, and use of chlorhexidine). Two lines in the result window indicate elevated aMMP-8 in PISF and increased risk for peri-implantitis. e appearance of a single line indicates successful test performance, low aMMP-8 in PISF, and no risk for periimplantitis after treatment [78,84]. saliva and GCF, respectively [29,34]. Results of a study by Gursoy et al. showed that aMMP-8 was higher in nonsmoking periodontitis patients than controls, and in smokers', only statistically significant parameter was TIMP-1 level that could differentiate between periodontitis patients and control. e ratio of aMMP-8, measured by the IFMA method, and TIMP-1 could successfully differentiate between periodontitis and healthy smoking subjects as well. A possible explanation for this finding, according to the authors, is that MMP-8 is less effective in mediating tissue degradation in the smoker subjects. It also indicates that smoking eventually can affect the detection of the potential biomarkers of periodontal disease [37].

MMP-8 Levels before and after Nonsurgical erapy.
Gonçalves et al. demonstrated that SRP and use of systemic antibiotics effectively reduced local levels of specific MMPs in case of localized aggressive periodontitis. [54] Leppilahti et al. showed in their study that in patients who underwent azithromycin antibiotic treatment, the MMP-8 levels in GCF specifically are more stable and remain lower than a predefined cutoff level [63].
A study done by Konopka et al. showed that SRP improves all examined clinical periodontal parameters, apart from CAL. However, the GCF levels of MMP-8 after therapy in the periodontitis patient was still found to be higher than a control group [47]. In contrast to this finding, Gonçalves et al., found that level of MMP-8 in GCF was comparable to healthy sites. Most marked reduction in MMP-8 levels was noticed in a short period, i.e., 3-6 months after receiving treatment [54].
Nonsurgical therapy with and without antibiotics can reduce the level of active and total collagenase/MMP-8 [11] At the beginning of the treatment, the total collagenase activity was found similar to that of active collagenase demonstrating that most of the collagenase present at this stage was in an active form [11]. However, Konopka et al. could not find any correlation between clinical parameters and amount of humoral factors after the therapy, while they showed a correlation at the baseline with PPD and a proximal plaque index (PI) [47]. Baseline GCF MMP-8 levels strongly predict the change in level during a maintenance period [59,63]. Elevated baseline levels of GCF MMP-8 in smokers indicate a weak response to therapy [59,60,63].

Host Response Modulation.
is term is recently introduced in dentistry and means modifying destructive aspects of inflammatory host response that develops in periodontal and peri-implant tissues as a result of inflammatory outcome to chronic subgingival bacterial plaque. e purpose of this therapy was to restore a balance between proinflammatory mediators and anti-inflammatory mediators. Host modulation by low-dose-doxycycline/sub-antimicrobial-dose-doxycycline (L/SDD) medication also efficiently inhibits and reduces gingival tissue and oral fluid aMMP-8 and at the same time ceases the progression of periodontal/peri-implant tissue destruction (APD) [3,7,66]. Only L/SDD has been licensed and accepted by FDA as a host response modulator and MMP-inhibitory drug in humans for the treatment of periodontal disease until now [66]. In L/SDD, doxycycline 20 mg is given orally twice a day or 40 mg once a day to produce serum levels of doxycycline, which is too low to produce any antimicrobial effects but enough effective to inhibit/downregulate aMMP-8 [7]. In contrast to traditional dose (100 mg, once, or twice daily), L/SDD does not cause any bacterial resistance to doxycycline and does not alter normal flora, a composition of bacterial biofilm and their susceptibility to doxycycline and other antibiotics, even after long-term (up to 24 months) daily administration [66]. Furthermore, L/SDD causes a significant reduction in the levels of inflammatory mediators, mediators of collagenolysis (� aMMP-8), collagen degradation products, proinflammatory cytokines, and periodontal connective tissue destruction. [32] It has been shown to inhibit alveolar bone loss during periodontitis due to its ability to reduce gingival oxidative stress and aMMP-8 [32].
Evidence suggests that L/SDD has a strong potential for modulation of host response in beneficially aiding disease management when used as an adjunct medication to conventional mechanical therapy, SRP [27]. L/SDD reduces postsurgical BOP, PPD, and periodontal bone resorption [66]. L/SDD has been shown to support periodontal treatment like SRP as well as reduce the related systemic lowgrade inflammation [31].
Emingil et al. concluded in their study that use of L/SDD together with SRP in the chronic periodontitis patient showed better clinical results/treatment outcomes as compared to SRP alone. A significant decrease in gingival inflammation scores at 3 months, and PPD reduction at 9 months was observed in the L/SDD group compared to a placebo group and was maintained until the end of 12 months [25]. In a study, L/SDD caused 36% reduction of bone height loss, when added to periodontal maintenance [36]. Sorsa et al. concluded that L/SDD, when coupled with SRP, could inhibit the activity or decrease expression of host MMPs, especially aMMP-8, by a mechanism that is unrelated to its antimicrobial property [3,6,7].

Effect of Metal
Restorations. According to a study done by Khuslinski et al. on practically healthy subjects with intact periodontium and patients with chronic generalized periodontitis with various structural materials of dental restorations [46], the level of MMP-8 surpassed the normal only in oral fluids of patients with chronic generalized periodontitis with metal restorations. In patients with chronic generalized periodontitis with or without metal dental restorations, obtained correlation coefficients indicate triggered biochemical cascade accompanied by the activation of cytokine production in response to etiological factors. e group of patients with periodontitis and metal restorations demonstrated a reaction that is more marked.

Association of Periodontal Microorganism with MMP-8.
e presence of subgingival microorganisms, mainly Treponema denticola (T. denticola), seemed to increase the levels of salivary albumin, the total protein contain in saliva, and levels of MMP-8 in GCF. ere is a possibility that both T. denticola and Tannerella forsythia (T. forsythia) have induced a cascade-type host response with increased release and activation of MMP-8 in GCF [35,50,86]. T. denticola and P. gingivalis-derived proteases (dentosilin and gingipain, respectively) can proteolytically and efficiently activate and convert latent pro-MMP-8 to aMMP-8 [45,86,88].

MMP-8 and Genetic
Background. According to Heikkinen et al., genetic polymorphism of MMP-3 and vitamin D receptor found to be linked to initial periodontitis in Finnish adolescents, and aMMP-8 PoC/chair-side immunoassay PerioSafe ® mouth-rinse test can be used for on-line PoC detection of initial periodontitis or preperiodontitis in adolescent patients with such type of genetic predisposition.
is indicates the preventive potential of the PerioSafe ® ORALyzer ® -aMMP-8 chair-side/PoC test [70]. us, aMMP-8 mouth-rinse chair-side/PoC test positivity and 3 or more >4 mm pockets associated with the vitamin-D receptor and MMP-3 single-nucleotide polymorphisms. No association was found between single nucleotide polymorphism studied with the positivity of aMMP-8 [70].

MMP-8 in Dental
Peri-implantitis. An inflammatory reaction associated with loss of supporting bone beyond initial biological bone remodeling around a dental implant, called peri-implantitis, is commonly reported as one of the significant contributors to dental implant failure [88][89][90][91]. e etiopathogenesis in case of peri-implantitis shows considerable similarity to periodontitis and shows comparable bacterial colonization and exudate of immune cells [88]. Similar to the periodontitis, aMMP-8 levels were repeatedly found to be pathologically elevated in diseased PISF as well [21-23, 30, 35, 45, 68, 82]. Both PMN-and non-PMN-type MMP-8 isoforms particularly in active forms have been observed in PISF of peri-implantitis patients [21-23, 30, 82]. However, Wang et al. reported that MMP-8 alone was not able to differentiate peri-implantitis patients from healthy patients [67]. T. denticola and Prevotella intermedia were reported to show diagnostic ability in case of peri-implantitis [67,72,73]. Gingival inflammation showed correlation to aMMP-8 levels in PISF [22,23] [73,78,79]. Similar clinical parameters and MMP-8 levels were obtained with both zirconium and titanium abutments at the end of 1 year. However, initially, titanium abutment was reported to show higher PISF MMP-8 levels and probing depth [72].

Combining Other Biomarkers to Increase Diagnostic
Accuracy. Simultaneous measurement of more than one oral fluid marker may allow more accurate prediction of periodontal inflammatory burden [53]. Combinations of the MMP-8 biomarker and pathogens that correspond with it (such as T. denticola) may give a more accurate prediction of periodontitis as compared to a single biomarker alone [35].
Gursoy et al. concluded in their study that proportional or combined use of oral salivary biomarkers increases diagnostic accuracy, particularly in smoker subjects [37]. It was found that the MMP-8/TIMP molar ratio and the combination of two biomarkers, MMP-8 and pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), were significantly higher in detecting periodontitis compared to MMP-8 test alone [37]. A study testing accuracy of the cumulative risk score calculated (CRS) from three salivary biomarkers (i.e., P. gingivalis, IL-1β, and MMP-8) was more accurate in the diagnosis of advanced periodontitis than any of the markers alone [40]. Leppilahti et al. suggested measurement of MMP-8 and TIMP-1 to obtain higher diagnostic accuracy [42]. A study performed by Rathnayake et al. proposed use of MMP-8/TIMP-1 molar ratio as markers of periodontal disease in a larger patient population [52]. Salminen et al. proposed combination of three biomarkers, i.e., MMP-8, IL-1β, and P. gingivalis (CRS) for diagnosis of periodontitis [54] e median concentration of these three was significantly higher in the moderate to severe periodontitis group as compared to controls. In addition, Ebersole et al. reported also that salivary levels of IL-1β, IL-6, and MMP-8 provide high diagnostic accuracy for periodontitis with high sensitivity and specificity [55]. Furthermore, MMP-8 levels were higher in patients diagnosed with chronic periodontitis and diabetic, but P. gingivalis did not affect much. Unlike MMP-8, P. gingivalis values remain unaffected in edentulous subjects. P. gingivalis successfully differentiated current smokers from former smokers and however, did not show correlation with BOP [54]. erefore, using biomarkers and various pathogens in combination may improve accuracy in diagnosis; however, the complexity and costs to perform such tests routinely will increase considerably. erefore, simpler, inexpensive, and readily available tests that have been shown to be sufficient alone to detect and quantify aMMP-8, such as PerioSafe ® and ImplantSafe ® /ORALyzer ® , might be more desirable ( Figure 1) [78].

PoC Tests.
Chair-side and point-of-care (PoC) lateral flow immunotests for the detection of aMMP-8 in oral fluids are commercially available (i.e., PerioSafe ® and ImplantSafe ® ) with the detection limit of 20 ng/ml (Figure 1). e tests resemble the pregnancy home test (Figure 1). e quantitative reader-equipped ORALyzer ® PoC test of oral fluids that measure aMMP-8 is found useful in differentiating active and inactive periodontal and peri-implant sites and patients, predicting disease progression in future, and monitoring the responses to therapy during the maintenance phase [17,78]. e benefits of using these aMMP-8 tests are that these can be used in clinical settings, are easy to use, are inexpensive, and give prompt quick results with high sensitivity and specificity (i.e., the sensitivity of 90% and specificity of 70-85%) [6,7,16,17,24,28,42,44,51,62,64,70,78].
Alassiri et al. demonstrated that quantitative, PerioSafe ® and ImplantSafe ® ORALyzer ® , PoC/chair-side assays could conveniently diagnose and follow the treatment of periodontitis and peri-implantitis [17,78]. us, these tests can detect subclinical, developing periodontitis and periimplantitis and related collage degradation even before the appearance of clinical and radiographical signs [14,16,17,73,82]. ese test alarm preperiodontitis and pre-peri-implantitis and identity preventively future periodontal and peri-implantitis breakdown. ey thus make invisible destruction or onset of periodontal/peri-implant collagenolysis to be visible and detectable in an enough early and predictive manner allowing the identification and timing of the preventive interactions/treatment such as secondary prevention and/or supportive periodontal/periimplant treatment [17,61,65,73,84,85].
PerioSafe ® and ImplantSafe ® with digital readers are modern in vitro fast immunological diagnostic and prevention professional technologies/tests for examination of the oral/periodontal/peri-implant status of teeth and dental implants at different time intervals (at least once annually) to detect risk of silent or hidden periodontal, peri-implant tissue degeneration and alveolar bone loss even before they can be detected clinically or radiographically [16,17].
e PoC tests also help in time preventive treatment which is necessary for long-term success of implants, periodontal tissues, and patients. Another aspect of it is that this is also healthy and economical for the patients and society.
Regarding the chair-side/PoC-aMMP-8 lateral flow immune tests (Figure 1), the appearance of only one line indicates the negative result that reveals normal condition/a healthy status (<20 ng aMMP-8 per ml), and appearance of two lines indicates increased risk (>20 ng aMMP-8 per ml) (Figure 1) for periodontitis and/or peri-implantitis, either already existing or developing periodontitis and periimplantitis, identified by PerioSafe and ImplantSafe, respectively ( Figure 1). ese PoC tests can be used with the reader for quantitative analysis [17,78].
For quantitative analysis, dip-stick tests should be placed in the corresponding compartment of the reader. en, flap is closed, the compartment is pushed into ORALyzer ® , and check mark is pressed. e ORALyzer ® is designed in such a way that it automatically starts and measures the aMMP-8 levels after 5 min.

Summary
e current review analyzed the potential of aMMP-8 as a potential diagnostic, predictive, and preventive adjunctive biomarker/biotechnological tool for periodontal and periimplant diseases. e available evidence suggests that especially aMMP-8 in oral fluids reflect, associate, and predict well with the clinical periodontal parameters and outcomes as well as clinical disease activity of periodontitis and peri-implantitis together with evaluation of treatment outcomes [18,44,47,54]. Only few studies failed to find the correlation between clinical CAL [47], and few others reported that MMP-8 levels are not correlated with BOP [62]. In addition, aMMP-8 levels were reported to be associated with radiological parameters too [53]. Importantly when evaluating these studies, it should be kept in mind that active/activated MMP-8, not MMP-8 or total latent MMP-8, is a biomarker of active and progressive periodontal and peri-implant disease [3, 4, 6-9, 11, 17, 76, 77, 80, 81].
us, pathologically and repeatedly elevated of aMMP-8 levels in saliva show the highest sensitivity and in GCF/PISF, the highest specificity [56]. aMMP-8 levels of mouth rinse and oral saliva can be useful for screening, whereas GCF/PISF levels could predict at site-specific level treatment outcomes and may be a useful adjunct in an individual/personalized treatment and monitoring plans. us, the aMMP-8 tests represent tools for personalized medicine [56]. aMMP-8 levels reduce after nonsurgical therapy, such as SRP. Most of the studies confirmed the effect of smoking on MMP-8 level, except few [29,34]. Combination of other biomarkers (TIMP-1, IL-6, and IL-1β) and periodontal pathogens (such as T. denticola and P. gingivalis) with aMMP-8 in the detection of periodontal inflammation may increase accuracy, but aMMP-8 alone functions quantitatively very well [6,7,16,17,78]. Both IFMA and DentoELISA were found to be able to differentiate periodontitis from healthy subjects, but in general, IFMA was more accurate [6,7,16,17]. Results obtained from Amersham ELISA were not in line with IFMA and Den-toELISA. Lateral flow chair-side/PoC aMMP-8 immunoassay correlated well with clinical parameters of periodontitis but with at least two sites and extended better accuracy than BOP [18,78]. Notably, invasive aMMP-8 PoC-tests cause always bacteremia, but noninvasive aMMP-8 PoC-tests never [78].
Despite their high sensitivity and specificity, aMMP-8 PoC-assays should be mainly used as adjunct tools to the clinical examination; mouth-rinse/salivary assays are useful for screening and dip-stick for site-specific personlized medical approaches. High levels of oral fluid MMP-8 in subjects with clinically "appearing" healthy periodontium/ peri-implantium indicate silent "hidden," developing future preperiodontitis and pre-peri-implantitis indicating early preventive supportive periodontal and peri-implant treatment [16]. In the case, oral fluid aMMP-8 is not treated to be <20 ng/ml, and these preperiodontitis and pre-periimplantitis phases will often develop to be periodontitis and peri-implantitis with on-going collagenolytic APD. 22 International Journal of Dentistry Elevated oral fluid aMMP-8 thus predicts, reflects, and precedes future APD, i.e., CAL of the teeth and dental implants [3,7,17,76]. us, aMMP-8 PoC/chair-side tests make invisible hidden inflammation visible [17,78]. Our literature review results are in line with previous studies. A review done by Sorsa et al. concluded that MMP-8 is a promising candidate for diagnosis and determination of progressive periodontitis and peri-implantitis and monitoring response to therapy and further extend them also to periimplantitis and provides diagnostic tests to monitor follow treatment and adjunctive medication such as L/SDD [7].
A systematic review and a recent study were done by de Morais et al. and Alassiri et al. and they concluded the same and recommended use of MMP-8 as a quantitative biomarker of periodontal and peri-implant diseases adjunctive to clinical examination [17,78,92].

Clinical Implications.
Repeatedly pathologically elevated levels of aMMP-8, but not total/latent MMP-8, in oral fluids (mouth rinse, saliva, GCF, and PISF) show the positive correlation with the clinical and radiological parameters of periodontitis and peri-implantitis. Oral fluid aMMP-8 levels reduce after periodontal therapy, i.e., SRP combined with host modulation and/or antimicrobial medication [7,93]. Continuously and sustainably pathologically elevated oral fluid MMP-8 levels indicate and predict sites and patients with compromised disease outcomes regarding course, treatment, and maintenance. Importantly, the oral fluid PoC/chair-side tests can be utilized to predict time preventive interventions before the development of irreversible tissue destruction (APD) in periodontium and periimplantium by indentifying and alarming the preperiodontitis and pre-peri-implantitis [17,70].

Limitations of the Study
(1) Only the articles written in English language were selected. (2) A literature search was performed in two databases, and additional articles have been chosen by manual searching, but it is possible that some relevant data are left behind.

Recommendations for Future
Research. Further studies, especially longitudinal and perspective ones, should be conducted to explore the relationship between aMMP-8 with other biomarkers. Some factors such as obesity and gender reported as confounding factors should also be addressed more in detail. e relationship between serum concentration of aMMP-8 and periodontitis and periimplantitis is still not clear and needs further investigations.

Disclosure
Periodontal diagnoses and classification of periodontal disease have been performed during the international workshop for classification of periodontal diseases and conditions from October 30 to November 2, 1999 (Periodontol 1999) (type I: gingivitis, type II: chronic periodontitis, and type III: aggressive periodontitis) [85].

Conflicts of Interest
Prof. Dr. Timo Sorsa is an inventor of US-Patents 5652227, 5866432, and 6143476 on diagnostic use and method of analysis of MMP-8 and its inhibitors in oral fluids. e authors declare that there are no conflicts of interest.