Is TIMP‐1 a biomarker for periodontal disease? A systematic review and meta‐analysis

Abstract Objective One of the most important families of proteases associated with periodontal disease is the family of the matrix metalloproteinases (MMPs). Their activity is regulated by tissue inhibitors of metalloproteinases (TIMPs), and an imbalance between MMP activity and regulation by TIMPs has been associated with the progression of periodontal disease. This strong interaction between TIMPs and MMPs might be an indication that TIMPs can be used as a biomarker to monitor periodontal disease progression in oral fluids. In particular, TIMP‐1 is a frequently studied biomarker for periodontal diseases. Therefore, the aim of this systematic review was to evaluate the scientific literature regarding TIMP‐1 concentrations in oral fluids of patients suffering from periodontitis or gingivitis in comparison to healthy individuals. Material and Methods PubMed/ MedLine and Web of Science databases were searched electronically. Studies that met the inclusion criteria were systematically evaluated and assessed for eligibility and risk of bias. Meta‐analysis was performed through the random effects model to assess the association between periodontitis/gingivitis and TIMP‐1 concentration in stimulated saliva, unstimulated saliva, and gingival crevicular fluid (GCF). Results The search strategy provided a total of 322 studies of which 10 studies met all inclusion criteria. Two studies investigated TIMP‐1 concentrations in GCF, three studies in unstimulated saliva, and five studies investigated TIMP‐1 concentrations in stimulated saliva. Three studies revealed that TIMP‐1 levels in oral fluids were significantly decreased in periodontal disease. Meta‐analysis revealed that there is no statistically significant difference between TIMP‐1 concentration in oral fluids of periodontitis/gingivitis patients in comparison to healthy individuals. Conclusions This systematic review with meta‐analysis shows that periodontal diseases are not associated with a statistically significant change in TIMP‐1 concentration in oral fluids.


| INTRODUC TI ON
Degradation of periodontal tissue is related to the activity of proteases involved in the inflammatory process. 1,2 One of the most important families of proteases associated with periodontal disease are the matrix metalloproteinases (MMPs). 3 In particular, MMP-8, MMP-9, and, to a lesser extent, MMP-14 have been studied in relation to periodontitis. 4 These MMPs are not only responsible for the degradation of the extracellular matrix during periodontitis but are also key factors in tissue remodeling processes. 5,6 The activity of MMPs is regulated by tissue inhibitors of metalloproteinases (TIMPs) which are produced and secreted by many cell types. Their production is regulated by various cytokines and growth factors. Besides MMPs, TIMPs also regulate the activity of other families such as the disintegrin metalloproteinases (ADAM and ADAMTS). 7 Therefore, TIMPs play a crucial role in important biological processes like the formation of the extracellular matrix and cell proliferation.
Upon binding to MMPs, TIMPs act like a wedge which connects to the active site of the MMP and thereby blocking the binding of substrate to MMP, resulting in reduced MMP activity. 7 An imbalance between MMP activity and regulation by TIMP has been associated with progression of periodontal disease. This imbalance results in the degradation of matrix proteins, and thereby contributes to the destruction of periodontal tissue. [8][9][10][11] The strong relation between TIMPs and MMPs suggests that TIMPs might potentially serve as a biomarker to diagnose periodontitis and monitor disease progression in oral fluids. 12,13 Of the four types of TIMPs identified in humans, TIMP-1, an inhibitor of MMP-9, has most often been associated with periodontal disease. However, so far the diagnostic value of TIMP-1 in periodontal disease has not been systematically reviewed. 7 In this context, the aim of this systematic review was to analyze the validity of TIMP-1 solely as a biomarker to diagnose periodontal disease in saliva and gingival crevicular fluid (GCF).

| MATERIAL S AND ME THODS
This systematic review was elaborated according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. 14 The PRISMA checklist is included in Table   S1. 15 The protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD42021246024. 16 F I G U R E 1 Schematic PRISMA diagram for procedural methodology 2.1 | Research strategy, selection, and inclusion and exclusion criteria An electronic database search was performed until December 31 th 2020 in the database of the National Library of Medicine (MEDLINE by PubMed) and Web of Science using a combination of medical subject headings (MeSH) terms and free text words (Appendix S1).
The resulting articles were reviewed independently by title, abstract, and full text by two reviewers (PdB and WEK). Any disagreements during the review process were resolved by discussion. Articles that met the following inclusion criteria were retrieved: studies including patients with chronic periodontitis or gingivitis diagnosed based on clinical parameters, publications in English, and studies investigating TIMP-1 concentrations in oral fluids. Publications that did not present a compatible methodology for a systematic analysis were excluded (e.g., reviews, opinions, book chapters, abstracts, and editorial letters). In vitro studies, animal studies, experiments that interfered with the expression of TIMP-1 through therapeutic methods, studies that evaluated patients with systemic diseases, studies that investigated other types of periodontitis than chronic periodontitis or gingivitis, studies investigating the systemic effect of proteases, studies that evaluated pregnant patients, and studies that evaluated children were also excluded. In addition, studies without a control group were also excluded. Where possible, sample sizes, meanvalues, and standard deviations were retrieved from the publications or calculated based on the available data. In case limited data were available, study investigators were contacted to retrieve the missing information. The whole process of literature selection was executed according to the PRISMA guidelines and is summarized in Figure 1.

| Data extraction
Information retrieved from all studies involved: authors, year of publication, number of patients diagnosed with periodontitis and number of controls, severity of the periodontal disease, criteria for diagnosis used for inclusion, TIMP-1 detection method, study results, and relevant conclusions.

| Assessment of risk of bias
The selected studies were analyzed with tools from the National Heart, Lung, and Blood Institute (NHLBI) to assess their quality. 17 First, the selected studies were classified by research design. 18 Depending on the research design, the following three risk assessment questionnaire tools were used: Controlled Intervention Studies, Observational Cohorts and Cross-Sectional Studies, and Case-Control Studies. All articles were independently assessed by two reviewers (PdB and WEK) rating each domain as 'yes', 'no', 'not applicable', or 'not reported'. The overall rating of each study could be 'good', 'fair', or 'poor'. Any disagreement on the bias risk assessment between the two reviewers was resolved by discussion.

| Statistical analysis
Statistical analyses were performed using the Cochrane Collaboration's software for preparing and maintaining Review Manager 5.4.1.. A quantitative synthesis (meta-analysis) for generating an estimate on the effect size was possible. This meta-analysis was conducted to the primary outcome: TIMP-1 concentration (ng/ mL) (mean ± SD) compared between periodontitis/ gingivitis patients and healthy individuals. In case in a study varying degrees of periodontal disease were monitored, the most severe condition was included. When in a study both chronic and acute periodontitis patients were monitored, data from the chronic patients were included in the analysis. Because of lack of identity between the included studies, the random-effects model was used to perform the metaanalysis. 19 I 2 -values higher than 50% were considered as indicative of substantial heterogeneity. P-values less than 0.05 were considered as statistically significant.

| E THIC AL RE VIE W
This study was approved by the ACTA Ethics Committee (registration number 2020113).

| Summary of the literature search and description of the included studies
The literature screening and selection process is presented in Therefore, these four articles were also excluded, based on missing data. The 10 remaining articles were included in the study and used in the meta-analyses.
The main characteristics of the included studies are described in in MMP-8/ TIMP-1 ratio in these studies was predominantly related to increased salivary MMP-8 levels in periodontitis patients and not necessarily to decreased TIMP-1 concentrations in saliva.
Only Nizam and co-workers observed a significant decrease in TIMP-1 level, whereas all three articles found a significant increase in MMP-8 concentration (Table 2).

| Quality assessment
The methodological quality of the 10 included studies was analyzed through the use of tools from the National Heart, Lung, and Blood Institute (NHLBI). Some of the items of the quality assessment tool were defined 'not reported'. Because not all these items had a relation to the focus of this study, TIMP-1 as biomarker for periodontal disease, the outcome of these items weighted less in the assessment of study quality. Among the six observational cohort studies and studies with a cross-sectional design, five were rated good and one was judged fair (Table 3). Both control intervention studies were rated fair (Table 4), due to the high number of 'not reported' items.
None of the two control intervention studies applied randomization of the study population. Of the two case control studies, one rated good whereas the other study was judged fair ( Table 5). The difference in quality is mainly due to lack of correction for potential confounders and differences in recruitment populations between the periodontal disease patients and control group participants. 12. Did the authors report that the sample size was sufficiently large to be able to detect a difference in the main outcome between groups with at least 80% power?

NR NR
13. Were outcomes reported or subgroups analyzed pre-specified (i.e., identified before analyses were conducted)?

NR YES
14. Were all randomized participants analyzed in the group to which they were originally assigned, that is, did they use an intention-to-treat analysis?

FAIR FAIR
Abbreviations: NA: Not applicable, NR: Not reported.

| Meta-analysis
For the meta-analysis, the 10 included studies were grouped on oral fluid used: stimulated saliva, unstimulated saliva, and GCF. For each oral fluid, TIMP-1 levels were compared between periodontitis/ gingivitis patients and healthy individuals. No statistically significant difference in TIMP-1 levels in stimulated saliva was observed between healthy individuals (n = 601) and participants with periodontitis (n = 412) (p = .08) (Figure 2A). Three studies showed a higher salivary level of TIMP-1 in healthy individuals, one study showed a higher level of TIMP-1 in participants with periodontal disease, and one study showed no difference in TIMP-1 levels between healthy participants and participants with periodontal disease. The heterogeneity of these studies was moderate (56%). Also, for unstimulated saliva, no statistically significant difference in salivary TIMP-1 concentration was observed between healthy individuals (n = 126) and periodontitis/ gingivitis patients (n = 128) (p = .09) ( Figure 2B). Three studies showed a higher level of TIMP-1 in healthy participants, and in one study, no difference in TIMP-1 levels between healthy participants and periodontitis/ gingivitis patients was observed. The heterogeneity between these studies was relatively high (68%). No statistically significant difference was observed between the periodontitis/ gingivitis (n = 67) and healthy (n = 35) groups (p = .35) when GCF was used as diagnostic fluid ( Figure 2C). Both studies showed a higher level of TIMP-1 in periodontitis patients, and the heterogeneity between the two studies was relatively high (62%).  (Table 2). This is reflected in the outcome of the meta-analysis which showed that the difference in TIMP-1 concentration between healthy participants and patients with gingivitis or periodontitis did not reach TA B L E 5 Quality assessment tool for Case-Control Studies statistical significance, and the heterogeneity between the studies was relatively high (Figure 2A-C). Of the 10 included studies, only two reported a significant difference between periodontitis/ gingivitis patients and healthy individuals. 5,20 These two studies used different methods for sample collection, which indicates that these changes in salivary TIMP-1 concentrations are not related to the type of collection. Additionally, we could not find significant differences concerning study population (with regard to age, gender, and inclusion of smokers) and severity or phase (acute/ chronic) of the periodontal disease between the studies that reported a significant change in TIMP-1 and the included studies who did not.

| DISCUSS ION
The high SD values indicate that there is a large variation in TIMP-1 concentration in both the periodontal disease group as in the control group (Table 2), which suggests that other confounders might be present.
A confounder known to influence TIMP-1 production is smoking; a high number of pack years and recent cessation are associated with increased salivary TIMP-1 levels. 12 The studies included Important criteria for a good biomarker are validity, reliability, and consistency. 35 Whereas TIMP-1 plays a role in a broad set of biological processes, its concentration shows a wide variation among healthy individuals which affects the consistency and reliability of F I G U R E 2 Forest plots of the meta-analysis. Comparison of TIMP-1 levels in stimulated saliva (A), unstimulated saliva (B), and GCF (C) from periodontitis/ gingivitis patients and healthy individuals using a random-effects model. Periodontal disease (PD), confidence interval (CI), and heterogeneity (I 2 ), * data provided by authors TIMP-1 as a biomarker. This is confirmed by the results presented in this systematic review with meta-analysis in which no significant changes in TIMP-1 concentrations in oral fluids were found between periodontal disease and healthy individuals. In conclusion, TIMP-1 is no reliable biomarker for screening and diagnostic purposes of periodontal disease.

ACK N OWLED G EM ENTS
No external funding, apart from the support of the authors' institution, was available for this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest related to this study.

AUTH O R CO NTR I B UTI O N S
PdB and WEK contributed to study conception and design and to the collection and data interpretation. PdB, HSB, and WEK contributed to statistical analysis and data interpretation. PdB, HSB, FB, and WEK contributed to data interpretation. All authors contributed to the manuscript draft and to critically revise the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.