A Comparative Analysis of Treatment-Related Changes in the Diagnostic Biomarker Active Metalloproteinase-8 Levels in Patients with Periodontitis

Background: Previous studies have revealed the potential diagnostic utility of aMMP-8, an active form of MMP-8, in periodontal and peri-implant diseases. While non-invasive point-of-care (PoC) chairside aMMP-8 tests have shown promise in this regard, there is a dearth of literature on the evaluation of treatment response using these tests. The present study aimed to investigate treatment-related changes in aMMP-8 levels in individuals with Stage III/IV—Grade C periodontitis compared to a healthy control group, using a quantitative chairside PoC aMMP-8 test, and to determine its correlation with clinical parameters. Methods: The study included 27 adult patients (13 smoker, 14 non-smoker) with stage III/IV-grade C periodontitis and 25 healthy adult subjects. Clinical periodontal measurements, real-time PoC aMMP-8, IFMA aMMP-8, and Western immunoblot analyses were performed before and 1 month after anti-infective scaling and root planing periodontal treatment. Time 0 measurements were taken from the healthy control group to test the consistency of the diagnostic test. Results: Both PoC aMMP-8 and IFMA aMMP-8 tests showed a statistically significant decrease in aMMP-8 levels and improvement in periodontal clinical parameters following treatment (p < 0.05). The PoC aMMP-8 test had high diagnostic sensitivity (85.2%) and specificity (100.0%) for periodontitis and was not affected by smoking (p > 0.05). Treatment also reduced MMP-8 immunoreactivity and activation as demonstrated by Western immunoblot analysis. Conclusion: The PoC aMMP-8 test shows promise as a useful tool for the real-time diagnosis and monitoring of periodontal therapy.


Introduction
Periodontitis is a chronic inflammatory disease that affects the tissues that support the teeth and is extremely prevalent in the community [1,2]. The pathogenic evolution of the dysbiotic microbial structure in the dental biofilm is among the most crucial factors in the onset and progression of periodontal disease. This process then leads to the continuation of tissue destruction as a result of the host response's non-physiologic overreaction [2]. Periodontitis, one of the most common causes of tooth loss, is not only limited to local tissues but has been linked to a variety of systemic diseases, including diabetes, cardiovascular disease, cancer, and Alzheimer's disease [3][4][5][6][7][8]. As a result, early diagnosis of the inflammatory periodontal disease process is critical in preventing tissue destruction [9,10]. and molecular forms were also assessed by IFMA and Western immunoblotting analysis, respectively.

Study Population and Design
The study design is presented in Figure 1. A total of 27 patients visiting a private clinic "Özel Fulya Agız ve Diş Saglıgı Klinigi" in Tekirdag, Turkey for their periodontal problems were recruited in the present study. The study was approved by the Biruni University Ethics Committee (2015-KAEK-71-22-06) and was carried out according to the principles of the Declaration of Helsinki. Oral and written consent was obtained from all recruited subjects. The inclusion criteria for the study were: interdental clinical attachment loss: ≥5 mm (at the site of greatest loss), detection of radiographic bone loss extending beyond 33% of the root, tooth loss due to periodontitis: ≤4 teeth (Stage III Periodontitis), ≥5 teeth (Stage IV Periodontitis). Patients with Acquired Immune Deficiency Syndrome (AIDS), uncontrolled diabetes (HbA1c > 7), and other immune-system-related chronic diseases (Crohn's disease, etc.) were excluded from the study. Pregnant or lactating females and individuals who had received periodontal treatment within the last year were also excluded. A total of 25 systemically and periodontally healthy dental students from the University of Helsinki, Finland served as healthy controls.
The present study aimed to investigate treatment-related changes in aMMP-8 levels in individuals with periodontitis using quantitative a chairside PoC aMMP-8 test and its on-line and real-time quantitative correlation with the studied clinical periodontal parameters. Consistency characteristics of the diagnostic tests were evaluated. The aMMP-8 levels and molecular forms were also assessed by IFMA and Western immunoblotting analysis, respectively.

Study Population and Design
The study design is presented in Figure 1. A total of 27 patients visiting a private clinic "Özel Fulya Ağız ve Diş Sağlığı Kliniği" in Tekirdağ, Turkey for their periodontal problems were recruited in the present study. The study was approved by the Biruni University Ethics Committee (2015-KAEK-71-22-06) and was carried out according to the principles of the Declaration of Helsinki. Oral and written consent was obtained from all recruited subjects. The inclusion criteria for the study were: interdental clinical attachment loss: ≥5 mm (at the site of greatest loss), detection of radiographic bone loss extending beyond 33% of the root, tooth loss due to periodontitis: ≤4 teeth (Stage III Periodontitis), ≥5 teeth (Stage IV Periodontitis). Patients with Acquired Immune Deficiency Syndrome (AIDS), uncontrolled diabetes (HbA1c > 7), and other immune-system-related chronic diseases (Crohn's disease, etc.) were excluded from the study. Pregnant or lactating females and individuals who had received periodontal treatment within the last year were also excluded. A total of 25 systemically and periodontally healthy dental students from the University of Helsinki, Finland served as healthy controls.

Periodontal Examination Procedure
Comprehensive periodontal examination was performed at baseline and 1 month following periodontal treatment by a single periodontist (M.K.). Probing depths (PD) were measured at six sites of each tooth with a Williams color-coded Michigan probe. Plaque index was recorded by assigning a score of 0-3 to each surface, and average oral plaque score was calculated for each patient [39]. The percentage of bleeding on probing (BoP) was determined after probing depth measurements. Gingival margin levels (GML) were determined by taking the enamel-cement junction (ECJ) into account during probing depth measurements. The areas where the free gingival margin ended at the apical of the EJC were recorded as positive values, and the areas where the free gingival margin terminated at the coronal point were recorded as negative values. Clinical attachment levels for each site were determined as the sum of GML and PD.

Periodontal Examination Procedure
Comprehensive periodontal examination was performed at baseline and 1 month following periodontal treatment by a single periodontist (M.K.). Probing depths (PD) were measured at six sites of each tooth with a Williams color-coded Michigan probe. Plaque index was recorded by assigning a score of 0-3 to each surface, and average oral plaque score was calculated for each patient [39]. The percentage of bleeding on probing (BoP) was determined after probing depth measurements. Gingival margin levels (GML) were determined by taking the enamel-cement junction (ECJ) into account during probing depth measurements. The areas where the free gingival margin ended at the apical of the EJC were recorded as positive values, and the areas where the free gingival margin terminated at the coronal point were recorded as negative values. Clinical attachment levels for each site were determined as the sum of GML and PD.

Periodontal Treatment Procedure
Periodontal treatment was carried out by a specialist periodontist (M.K.). Initially, cause-related therapy, including full-mouth scaling and root planing procedures, were performed along with oral hygiene instructions. At 2 weeks following the non-surgical phase of the periodontal therapy, periodontal sites associated with irregular bony contours, angular defects, or pockets in which a complete access with non-surgical periodontal therapy was not possible, such as grade II-III furcation defects, were treated with open flap debridement. Patients who underwent the surgical phase of treatment were prescribed amoxicillin plus clavulanic acid (1gr/day) and chlorhexidine mouth rinse (0.12%) twice a day for 7 days and recalled thereafter for suture removal. All patients were re-evaluated clinically 1 month following treatment.

Quantitative Chairside PoC aMMP-8 Analyses
Levels of aMMP-8 were measured quantitatively using rapid PoC chairside aMMP-8 kits (Periosafe ® , Dentognostics GmbH, Solingen, Germany) and a quantitative spectrometer analyzer (Oralyzer ® , Dentognostics GmbH, Solingen, Germany) on mouth rinse samples collected before treatment and 1 month following periodontal treatment. To perform a comparative analysis with the periodontitis patient group, analysis of aMMP-8 was also conducted on the healthy control group at T0 (baseline). PoC chairside aMMP-8 analyses were performed prior to clinical measurements, and manufacturer's instructions were followed. It was recommended that patients and controls not eat for 1 h before analyses. First, the patients and controls were instructed to rinse their mouths with clean water (drinking or distilled water) for 30 s and spit it out. After a waiting period of 1 min, they were told to rinse their mouths for 30 s with 5 mL of distilled water in the aMMP-8 kit (Periosafe ® ) and spit it back into the container. Then, 3-4 drops were taken from the container with a sterile syringe and poured into the well on the test cassette provided in the aMMP-8 kit. Immediately after that, the cassette was transferred to the digital spectrometer device (Oralyzer ® ) and quantitative results were obtained after 5 min. The remaining liquid in the container was transferred to Eppendorf tubes and stored at −70 • C for further laboratory analysis.

Measurement of the aMMP-8 Levels by Immunofluorometric Assay (IFMA)
The aMMP-8 level from mouth rinse samples was determined by a time-resolved immunofluorescence assay (IFMA) as described by Öztürk et al. [40]. Briefly, aMMP-8specific monoclonal antibodies 8708 and 8706 (Actim Oy, Espoo, Finland) were used in the analysis as a catching antibody and a tracer antibody, respectively. In this protocol, the diluted samples were allowed to incubate for 1 h with the Europium labelled tracer antibody. The fluorescence was measured using an EnVision 2015 multimode reader (PerkinElmer, Turku, Finland).

Western-Immunoblotting Testing Procedure
The molecular forms of MMP-8 were detected from mouth rinse samples by a modified enhanced chemiluminescence (ECL) Western blotting kit according to protocols recommended by the manufacturer (GE Healthcare, Amersham, UK) as described earlier by Rautava et al. [41]. Briefly, the proteins of mouth rinse samples were first separated by electrophoresis and then electro-transferred onto nitrocellulose membranes Protran (Whatman GmbH, Dassel, Germany). The membranes were incubated overnight with monoclonal primary antibodies anti-MMP-8 [42] and then with horseradish peroxidase-linked secondary antibody (GE Healthcare, Buckinghamshire, UK) for 1 h. The membranes were washed 4 times in TBST between each step for 15 min. The proteins were visualized using the ECL system according to protocol. The recombinant human MMP-8 (100 ng, Calbiochem, Darnstadt, Germany) was used as a positive control.

Statistical Analysis
All periodontal parameters, including probing depth, bleeding on probing, plaque index, and clinical attachment level were examined before periodontal treatment and 1 month following anti-infective periodontal treatment. Normality tests were performed to test the normality of the data before calculating paired samples t-tests. Paired-samples t-test was used to analyze the statistically significant differences between these two phases. The effect of smoking on aMMP-8 levels was tested with repeated measures ANOVA. A p < 0.05 was accepted as statistically significant value.
Receiver operating characteristic (ROC) analysis and the area under the ROC curve (AUC) were used to examine the diagnostic accuracy of aMMP-8 to classify periodontitis and periodontally healthy subjects. In order to identify optimal cut-offs from the ROC curves, the Youden Index was used for calculating diagnostic sensitivity and specificity (Se and Sp).

Study Population
A total of 27 periodontitis patients (4 = Stage III, 23 = Stage IV, 27 = Grade C) and 25 healthy control subjects were enrolled in the study. Ages of periodontitis patients ranged between 30 and 70 years. All healthy subjects were younger (age range 23 to 25 years) than the study group (p < 0.01). Demographic characteristics of periodontitis patients and healthy control subjects are shown in Table 1.

Clinical Periodontal Parameters
All periodontitis patients were subjected to non-surgical periodontal therapy followed by open flap debridement in seven of them. Statistically significant improvements following anti-infective treatment were observed for all periodontal parameters (p < 0.001) ( Table 2). Scatter plot diagrams of the relationship between probing depths, bleeding on probing, clinical attachment level, and plaque indices before periodontal treatment and after antiinfective periodontal treatment are presented in Figure 2. The clinical parameters as well Diagnostics 2023, 13, 903 6 of 20 as aMMP-8 levels of the periodontitis patients reduced to levels close to that of healthy subjects following periodontal therapy (Table 2).

Clinical Periodontal Parameters
All periodontitis patients were subjected to non-surgical periodontal therapy followed by open flap debridement in seven of them. Statistically significant improvements following anti-infective treatment were observed for all periodontal parameters (p < 0.001) ( Table 2). Scatter plot diagrams of the relationship between probing depths, bleeding on probing, clinical attachment level, and plaque indices before periodontal treatment and after anti-infective periodontal treatment are presented in Figure 2. The clinical parameters as well as aMMP-8 levels of the periodontitis patients reduced to levels close to that of healthy subjects following periodontal therapy (Table 2).  Both non-smoker and smoker subjects showed statistically significant dec terms of inflammatory clinical parameters (p < 0.001). A similar clinical healing was observed in both groups ( Figure 3).

aMMP-8 Results
A statistically significant decrease in oral rinse aMMP-8 levels following an tive periodontal treatment was observed regarding both Oralyzer ® and IFMA res in correlation with bleeding on probing (p < 0.05) (Table 2 and Figure 4). Both O and IFMA results indicated a similar pattern of decrease in terms of oral rinse levels, and it was also observed that smoking did not have a significant effect on 8 PoC testing (Figures 4 and 5) (p > 0.05).

Western Immunoblotting Analysis Results
Representative Western immunoblot analysis and aMMP-8 POC-test outcomes of MMP-8 in the studied mouth rinse samples from orally and systemically healthy and diseased study subjects are shown in Figure 7. MMP-8 was in latent form in the healthy sample ( Figure 7A, Lane 2), and in the diseased samples it was converted to active and fragmented forms (Lane 3) as analyzed by monoclonal anti-MMP-8 antibody ( Figure 7A). Negative (−, <20 ng/mL) and positive (+, ≥20 ng/mL) aMMP-8 POC-test outcomes are shown in Figure 7B. Negative (−, <20 ng/mL) and positive (+, ≥20 ng/mL) aMMP-8 POC-test outcomes are shown in Figure 7B.

Discussion
Periodontal diseases are chronic inflammatory conditions affecting the supporting tissues of the teeth [1]. One of the key enzymes involved in the breakdown of these tissues is matrix metalloproteinase-8 (MMP-8). While MMP-8 is important for normal tissue remodeling and repair, excessive or uncontrolled production of this enzyme can lead to tissue destruction and the progression of periodontal diseases. Recent studies have focused on the use of aMMP-8 as a biomarker for periodontal diseases; aMMP-8 refers to the active form of MMP-8, which is produced by neutrophils and other inflammatory cells in response to bacterial infection. Elevated levels of aMMP-8 have been linked to increased tissue destruction and disease progression in periodontal diseases, making it a valuable diagnostic and prognostic tool for these conditions [12]. Furthermore, aMMP-8 has been shown to be a more specific marker for active periodontal disease than total MMP-8, which can be found in both active and inactive forms [12,21].
The present study aimed to evaluate treatment-related changes of mouth rinse aMMP-8 levels by using PoC aMMP-8 kits and Oralyser-reader, which is a non-invasive method that rapidly and quantitatively produces chairside on-line and real-time results. Both chairside PoC aMMP-8 tests and IFMA aMMP-8 laboratory analysis confirmed that pre-treatment mouth rinse aMMP-8 levels were clearly higher than mouth rinse levels of patients after 1 month following periodontal treatment.
Our study provides valuable insights into the potential use of PoC chairside aMMP-8 tests and IFMA aMMP-8 laboratory analysis in the diagnosis and post-treatment followup of periodontal diseases. However, there are several limitations that should be considered when interpreting the results. Firstly, the small sample size could limit the generalizability of our findings. Secondly, the short follow-up period of only 1 month limits the assessment of the effectiveness of these techniques over time. On the other hand, the

Discussion
Periodontal diseases are chronic inflammatory conditions affecting the supporting tissues of the teeth [1]. One of the key enzymes involved in the breakdown of these tissues is matrix metalloproteinase-8 (MMP-8). While MMP-8 is important for normal tissue remodeling and repair, excessive or uncontrolled production of this enzyme can lead to tissue destruction and the progression of periodontal diseases. Recent studies have focused on the use of aMMP-8 as a biomarker for periodontal diseases; aMMP-8 refers to the active form of MMP-8, which is produced by neutrophils and other inflammatory cells in response to bacterial infection. Elevated levels of aMMP-8 have been linked to increased tissue destruction and disease progression in periodontal diseases, making it a valuable diagnostic and prognostic tool for these conditions [12]. Furthermore, aMMP-8 has been shown to be a more specific marker for active periodontal disease than total MMP-8, which can be found in both active and inactive forms [12,21].
The present study aimed to evaluate treatment-related changes of mouth rinse aMMP-8 levels by using PoC aMMP-8 kits and Oralyser-reader, which is a non-invasive method that rapidly and quantitatively produces chairside on-line and real-time results. Both chairside PoC aMMP-8 tests and IFMA aMMP-8 laboratory analysis confirmed that pre-treatment mouth rinse aMMP-8 levels were clearly higher than mouth rinse levels of patients after 1 month following periodontal treatment.
Our study provides valuable insights into the potential use of PoC chairside aMMP-8 tests and IFMA aMMP-8 laboratory analysis in the diagnosis and post-treatment follow-up of periodontal diseases. However, there are several limitations that should be considered when interpreting the results. Firstly, the small sample size could limit the generalizability of our findings. Secondly, the short follow-up period of only 1 month limits the assessment of the effectiveness of these techniques over time. On the other hand, the absence of periodontally healthy smokers in our study groups can be considered a limitation in comparative evaluations. Despite these limitations, our study provides important insights into the potential use of PoC chairside aMMP-8 tests and IFMA aMMP-8 laboratory analysis in the diagnosis and post-treatment follow-up of periodontal diseases.
This study utilized both the aMMP-8 PoC chairside aMMP-8 test and the aMMP-8 IFMA measurements that utilize the same monoclonal antibodies (Sorsa T et al., US patent no: US10488415B2). These techniques utilize two monoclonal, i.e., primary or catching antibody and secondary or detection [9,17,23,43,44]. Despite that, they correlate with each other; the techniques produced different values evidencing that both techniques can independently diagnose and differentiate periodontal health and disease. Both techniques can also be applied to monitor the treatment of the disease [12,24,35]. This study thus confirms and further extends the results of several previous studies demonstrating the potential benefits of POC chairside aMMP-8 and IFMA aMMP-8 laboratory analysis in terms of diagnostic distinction between periodontal health and disease [34,36,37,40,[45][46][47][48]. Furthermore, our present findings are in accordance with numerous studies linking elevated oral aMMP-8, but not total MMP-8, to active and progressive stages of periodontal and peri-implant diseases [20,23,43,[49][50][51][52][53][54][55].
It was previously shown that smokers had significantly higher levels of aMMP-8 in their saliva compared to ex-smokers or non-smokers [17,54]. When the pre-periodontal treatment results were evaluated from a diagnostic point of view, smoking was not found to significantly affect the aMMP-8 PoC testing being in agreement with previous studies on aMMP-8 in oral fluids . The sensitivity of the test was found to be 85.2% when the cut-off value was determined to be 20 ng/mL. According to a recently published study of Öztürk VÖ et al. [40], in which they included Stage III and IV periodontitis patients, diagnostic sensitivity of PoC aMMP-8 was observed as 83.9% [40]. In other studies in which periodontitis and peri-implantitis patients were included and the cut-off value was determined to be 20 ng/mL, it was observed that the aMMP-8 PoC test's sensitivity ranged between 76-90% [21].
Clinical periodontal parameters of pre-treatment and 1 month following periodontal treatment revealed statistically significant improvement as predicted and consistent with the literature. [56,57]. The quantitative chairside PoC aMMP-8 and IFMA aMMP-8 laboratory results both demonstrated a statistically significant decrease, correlating with and reflecting well with the clinical findings. There are many studies in the literature reporting a decrease in aMMP-8 levels following periodontal treatment [10,12,24,25,48,49,58]. While MMP-8 in its latent form was detected more frequently in the healthy state [53], the release of degranulated aMMP-8, its activated form, increases with periodontal and peri-implant inflammation and disease severity [12,23,54,55]. The statistical decrease in aMMP-8 levels post-periodontal treatment suggests that active tissue destruction, along with clinical disease activity, is reduced, confirming the role of MMP-8 in periodontitis pathogenesis [10,12,59].
When analyzing the clinical results, it becomes clear that factors, such as deep periodontal pockets, bleeding on probing (BOP), and oral hygiene, are strongly linked. However, despite treatment, not all patients were able to achieve complete oral health status as these parameters did not return to normal levels in all cases. Furthermore, it was observed that the post-treatment mouth rinse aMMP-8 levels (in both IFMA and PoC chairside aMMP-8 Tests) were higher than health-associated levels. In the study of Umeizudike et al., it was found that in the sixth month post-periodontal treatment, the aMMP-8 levels did not reach close to that of the healthy control group [48]. Literature data further suggests that individuals with gingivitis may have elevated aMMP-8 IFMA levels and aMMP-8 release may persist in the periodontal sites that respond poorly to treatment [25,35,36]. Periodontally and systemically healthy dental students without any periodontal disease experience and activity all had negative ([-], <20 ng/mL) aMMP-8 levels. This finding was also compatible with the literature which further affirms that Periosafe PoC aMMP-8 test negativity can be regarded as a biomarker of periodontal and peri-implant health [40]. Since this study includes a 1-month follow-up, the clinical and biochemical findings might not have reached the level of complete health due to persistent gingival inflammation and residual periodontal pockets. There are studies in the literature that suggest that the post-treatment re-evaluation period ranges from 2 weeks to 6 months (59, 60). Morrison et al. state that the severity of periodontitis can be significantly reduced during the 1-month post-periodontal treatment follow-up process. However, the oral hygiene process must be fully ensured to determine the ongoing treatment need [60].
When comparing aMMP-8 cut-off 20 ng/mL vs. 10 ng/mL [34,36], we found that especially after treatment, the periodontal health targeting was reduced with a 10 ng/mL cut-off. Deng et al. [36] used 10 ng/mL as the diagnostic cut-off value, but it should be remembered that it is not recommended by the manufacturers [21,35]. Our present results provide further support for the use of 20 ng/mL as the diagnostic cut-off value for aMMP-8 PoC tests [23,24,34,35].
Laboratory analysis of immunological inflammatory factors is considered to be the gold standard [61][62][63]. The results of PoC chairside aMMP-8 tests were consistent with those of IFMA aMMP-8 analyses, indicating that non-invasive PoC aMMP-8 analysis [12,21,34,45,64] can make a potential contribution regarding the diagnosis and periodontal therapy follow-up. However, there is a need for more longitudinal studies on the functionality of PoC chairside aMMP-8 analyses in periodontal treatment and its follow-up.

Conclusions
Observation of alarmingly high mouth rinse aMMP-8 levels in individuals with periodontitis through both point-of-care aMMP-8 and IFMA aMMP-8 analyses, and their significant decrease after anti-infective periodontal treatment, highlights the practical utility of the point-of-care aMMP-8 test for real-time diagnosis and monitoring of periodontal treatment progress.

MMP Matrix metalloproteinase PoC
Point of care IFMA Immunofluorometric assay