Biomarker for Asymptomatic Apical Periodontitis in Gingival Crevicular Fluid: aMMP-8

Abstract

Objectives Asymptomatic apical periodontitis (AAP) is one of the most widespread chronic inflammatory diseases in the field of dental medicine. Active matrix metalloproteinase (aMMP-8) previously demonstrated diagnostic potential as a biomarker for AAP in gingival crevicular fluid (GCF). The aim of this study was to determine the levels and diagnostic accuracy of aMMP-8 in GCF from teeth with AAP.

Materials and Methods In this cross-sectional study, GCF samples were obtained from teeth with AAP (sample group, n = 31) and their contralateral teeth (control group, n = 31). Clinical and cone beam computed tomography (CBCT) parameters were used for the diagnosis and assessment of AAP and the determination of clinically healthy marginal periodontal tissues. One pool GCF sample per tooth was obtained from the whole crevice’s perimeter. aMMP-8 levels were determined by enzyme-linked immunosorbent assay (ELISA).

Statistical Analysis Wilcoxon signed ranks test and Spearman rank correlation coefficient (rs) were used as statistical tools. The significance level was set at p < .05.

Results The two groups demonstrated biomarker levels corresponding to a healthy marginal periodontal tissue. aMMP-8 levels were statistically and significantly higher in the samples collected from teeth with AAP. Lesions with greater volume showed correspondingly larger diameters. No statistically significant correlation between aMMP-8 levels and lesions’ volume or diameter was discovered.

Conclusion GCF composition is modified by AAP only to a minimal extent. Further research is needed to substantiate the utilization of aMMP-8 as a potential biomarker for the diagnosis of the disease as well as to explore its relationship with other biomarkers.

Keywords

asymptomatic apical periodontitis - gingival crevicular fluid - aMMP-8 - enzyme linked immunosorbent assay

Introduction

Asymptomatic apical periodontitis (AAP) is one of the most widespread chronic inflammatory diseases in the field of dental medicine.[1] AAP is an inflammatory condition of endodontic origin that affects the apical periodontium and the adjacent alveolar bone and cement. It results from the complex interplay between the endodontic biofilm and host immune-inflammatory response.[2]

The egress of bacterial toxins (lipopolysaccharide, lipoteichoic acid, enzymes, and noxious metabolic byproducts) into the periapical tissues activate the innate and adaptive immune system; thus, inducing a periapical inflammatory reaction.[3] The immunologic response is mediated by numerous cell types that produce a milieu of proinflammatory cytokines, chemokines, and neuropeptides. They induce an alteration in the physiology and metabolism of the periapical tissues. The upregulated cytokine expression results in a shift in the periodontal turnover, ultimately leading to the degradation of extracellular matrix (ECM) components, osteoclastogenesis, and osteoclast activation.[4] [5] ECM degradation of periodontal tissues is achieved by intracellular and extracellular pathways. The extracellular pathway entails remodeling by secreted proteases such as matrix metalloproteinases (MMPs).[6]

Members of the MMP family are proteolytic enzymes known to catalyze the hydrolysis of a great variety of biological macromolecules.[7] In inflammatory periodontal diseases, pathologically excessive activity on their part entails periodontal tissue destruction, which can be monitored by assessing their levels in different oral fluids.[8] [9] [10] On the basis of these biochemical or immunological findings, oral fluids have become a target for extensive research with regard to their diagnostic utilization.[11] MMPs and their regulators are potential candidates for chair-side or point-of-care (PoC) tests markers, aimed at monitoring periodontal and peri-implant diseases.[8] [12] [13] [14] [15] [16] [17] Recent studies demonstrated the diagnostic potential of MMPs found in gingival crevicular fluid (GCF) collected from teeth with AAP.[18] [19] Neutrophil collagenase, also known as matrix metalloproteinase 8 (MMP-8) and it’s active form aMMP-8, have been identified as significant collagenolytic enzymes that cause periodontal tissue destruction in periodontitis and their GCF levels are modified by AAP.[18] [20] [21]

In routine clinical practice, the traditional diagnostic methods are based on clinical–radiographic examination and are designed to evaluate the need for endodontic treatment. These verdict-oriented methods fail to fully delineate the changes observed on a cellular level. The introduction of specific and auspicious biomarkers reflecting AAP does not just provide a new, objective way of diagnosis, but can improve our knowledge of the individual disease dynamics. Therefore, the aim of this cross-sectional study was to establish whether GCF composition can be modified by AAP and assess aMMP-8 as a potential biomarker for the diagnosis of the disease.

Materials and Methods

Patient Selection

The study was approved by the Institutional Review Board of the Medical University of Plovdiv, in accordance with the ethical standards of the Declaration of Helsinki (No. Р-3326/20.12.2017). Patients referred to the Department of Operative Dentistry and Endodontics at the university for endodontic treatment with a recommendation for a preoperative cone beam computed tomography (CBCT) were screened for enrollment in the study. A total of 31 patients with a single asymptomatic periapical lesion, intact dentition, clinically healthy marginal periodontium, or gingivitis with no evidence of bone destruction were enrolled in the study. Preoperative CBCT of the teeth with AAP was indicated in all cases due to complex root canal morphology, assessment of nonhealing lesions, or endodontic treatment complications. Limited field of view (FOV) CBCT was taken in accordance with the as low as reasonably achievable (ALARA) principles and the American Association of Endodontists (AAE) and American Academy of Oral and Maxillofacial Radiology (AAOMR) joint position statement on the use of CBCT in endodontics.[22] [23] Exclusion criteria for all subjects included the presence of marginal bone loss, systemic diseases and disorders, smoking, and intake of medication that can influence periodontal tissue turnover in the last 3 months prior to the beginning of the study. Body mass index (BMI) was also taken into consideration as it may affect the GCF levels of MMP-8.[24] Informed consent was obtained from all the participants.

Radiographic Assessment of the Lesion

The sample group comprised 31 teeth diagnosed with AAP. The initial assessment of the lesions’ characteristics was performed with limited-volume FOV CBCT using dedicated software (Planmeca Romexis Dental Imaging Software; Planmeca OY, Finland). Lesion size was determined with the help of its diameter and volume. The diameter was deemed to be the most substantial cross-section perpendicular to the root surface in the apical third of the root. The volumetric measurements were calculated utilizing two different techniques–manual outlining of the lesion borders with a consequent automatic reconstruction of the area and automatic threshold volume generation. Each periapical lesion was located and assessed in the three planes (axial plane, sagittal plane, and coronal plane). The measure ellipsoid tool was used to select the area of the lesion. Automatic volume threshold generation at 300 to 350 Hounsfield units was performed ([Fig. 1]). The threshold value was the most optimal value that yielded complete outlining of the lesion. The software consequently automatically calculated the lesion volume. The second method of volume determination comprised manual outlining of the lesion in each slice. Automatic reconstruction and calculation followed ([Fig. 2]).

Fig. 1 Lesion volume determination: automatic threshold volume generation.

Fig. 2 Lesion volume determination: manual outlining of the lesion borders with a consequent automatic reconstruction.

Clinical Phase

All patients underwent initial periodontal therapy–periodontal screening and recording and calculus removal were performed, followed by oral hygiene instruction and motivation. All patient data was recorded in a medical chart that included medical and dental history, smoking habits, medication intake, and periodontal clinical parameters. Periodontal probing depth (PPD), bleeding on probing (BOP), and bleeding index (BI) were measured using a calibrated periodontal probe (UNC-15; Hu-Friedy, Chicago, IL, USA) in six sites of each tooth diagnosed with AAP (the sample group, n = 31) and its periodontally healthy contralateral tooth (the control group, n = 31). Pulp vitality testing was performed on the contralateral teeth to confirm intact pulp and periapical tissues. Patients were recalled in 2 weeks for reassessment and evaluation of the response of the initial therapy. No BOP and a probing depth of ≤ 3 mm on the sites to be sampled were a prerequisite for proceeding with the sampling procedures.

Sample Collection

Customized sample taking kits containing six GCF-stripes, one tube, and one patient form were provided by Dentognostics GmbH (Jena, Germany). One Pool GCF sample per tooth was obtained intracrevicularly from the whole crevice’s perimeter (mesiovestibular, vestibular, distovestibular, distolingual, lingual, and mesiolingual) of the teeth in the sample and control group. The teeth were isolated with cotton rolls and the sulcus was dried. Each paper strip was gently inserted in the gingival sulcus for 30 seconds ([Fig. 3]). Strips contaminated with blood or saliva were discarded and the samples were sent to the manufacturer. aMMP-8 levels were determined by laboratory-based enzyme-linked immunosorbent assay (ELISA) (DentoELISA; Dentognostics GmbH, Jena, Germany).

Fig. 3 GCF sample collection. GCF, gingival crevicular fluid.

Data was typed into a spreadsheet and SPSS software (version 18.0) (SPSS Inc.; Chicago, Illinois, United States) was used to perform the analysis. Wilcoxon signed ranks test and Spearman rank correlation coefficient (rs) were used as statistical tools. The significance level was set at p <.05.

Results

Ten female and 21 male patients were assessed in the study with a mean age of 26.4 (range 21–33). The age, gender, BMI, and the number of teeth of the study subjects are shown in [Table 1]. The clinical periodontal parameters are shown in [Table 2]. The levels of aMMP-8 were assessed in pool GCF samples collected from 31 diseased teeth and 31 control contralateral teeth. All teeth in the sample group were multirooted, 10 maxillary, and 21 mandibular. Of those, 18 were retreatment cases. Both the sample and the control group demonstrated biomarker levels corresponding to a healthy periodontal tissue ([Fig. 4]). aMMP-8 levels were not affected by age, gender, BMI, tooth location, or previous endodontic treatment. The mean levels of aMMP-8 in the sample group were 6.65 ng/mL and 3.77 ng/mL in the control group ([Table 3]). A statistically significant difference between the two groups was established as the levels of aMMP-8 were higher in the GCF, collected from teeth with AAP (Z = − 3.45, p <.001). Lesions with greater volume showed correspondingly larger diameters (r_s = 0.705, p=.000). A nonsignificant fair positive correlation was discovered between aMMP-8 levels and lesion diameter (r_s = 0.338, p=.063). No significant correlation between aMMP-8 levels and lesion volumes was established.

Table 1 Age, gender, body mass index, and number of teeth of the study subjects

Gender		Age		BMI		Number of teeth
Abbreviations: BMI, body mass index; CI, confidence interval.
Male	Female	Mean	95% CI	Mean	95% CI	Mean	95% CI
21	10	26.4	25.0,27.8	25.39	24.39,26.39	29	28, 30

Table 2 Clinical parameters of 372 tooth sites from 61 teeth

Group	Sites	PD		BI		BOP
		Mean	95% CI	Mean	95% CI	%
Abbreviations: BI, bleeding index; BOP, bleeding on probing; CI, confidence interval; PPD, periodontal probing depth.
Sample	186	2.03	1.93, 2.13	1.96	1.83, 2.09	32.3
Control	186	2.09	1.99, 2.19	1.75	1.62, 1.88	23.1
Total	372	2.06	1.99, 2.13	1.85	1.76, 1.95	27.7

Table 3 Levels of aMMP-8 in GCF pool samples from 31 patents (ng/mL)

Group	N	Mean	95% CI	Min	Max	Med
Abbreviations: aMMP, active matrix metalloproteinase; CI, confidence interval; GCF, gingival crevicular fluid.
Sample	31	6.65	5.02, 8.27	1	24	6
Control	31	3.77	2.93, 4.62	1	9	3

Fig. 4 Individual levels of aMMP-8 in control teeth and teeth with AAP. AAP, asymptomatic apical periodontitis, aMMP, active matrix metalloproteinase.

Discussion

GCF is a plasmatic extravasate collected noninvasively at the gingival margin or within the gingival crevice. It has a high-diagnostic potential as a source of biomarkers for marginal periodontal disease.[19] [20] [25] [26] [27] The biochemical analysis of GCF can be directed at components derived from the host: cytokines and other inflammatory mediators.[28] Utilizing GCF biomarkers for the assessment of marginal periodontal diseases represents a widespread concept. The biomarkers directly account for the disease’s inflammatory profile as a complement to the existing clinical–radiographic assessment.[18]

According to Baeza et al, GCF can also reflect the condition of the apical periodontium, although the extent to which AAP can modify GCF remains mostly unexplored.[18] The present study investigated aMMP-8 as a potential biomarker for the disease. Increased levels of aMMP-8 in oral fluids are associated with clinical periodontal parameters (PPD and BOP) and decrease after successful periodontal treatment.[29] [30] This presented a challenge, as any gingival inflammation would affect the detection of AAP biomarkers in GCF. We therefore suggested limiting the applicability of the method to teeth with a clinically healthy marginal periodontium in order to reduce the confounding effect of gingival inflammation. Comparing the results to a contralateral tooth with an intact apical periodontium will also provide a baseline for interpreting the results.

Previous studies validated a 20 ng/mL clinical threshold of aMMP-8 GCF levels, which indicates marginal periodontal degeneration.[31] [32] [33] [34] [35] This serves as the detection limit for the commercially available chair-side and PoC lateral flow immunotests for the detection of aMMP-8 in oral fluids (i.e., PerioSafe and ImplantSafe). Baeza et al suggests a cutoff point at 13 ng/mL in laboratory-based lateral flow immunoassay of aMMP-8 for chronic periodontitis.[18] We observed lower aMMP-8 levels in the majority of samples from the sample and control groups alike. As the sampled sites did not show any signs gingival inflammation at the time of sample collection, the results are in accordance with previously reported levels for healthy marginal periodontium. The low levels of aMMP-8 could also be attributed to the small volume of GCF produced in a clinically healthy marginal crevice. Nevertheless, aMMP-8 levels were higher in the GCF collected from teeth with AAP in comparison with the contralateral sample (p = 0.001). Therefore, the current qualitative chair-side and PoC GCF tests could not be utilized for the screening of AAP due to their high-detection threshold. Future studies should focus on laboratory-based methods as they have higher sensitivity.

The extent of bone destruction in terms of lesion diameter and volume was assessed by CBCT. In developing chronic periodontitis and peri-implantitis the collagen degradation can be detected by aMMP-8 tests prior to any clinical and radiographic sighs.[36] Given the high sensitivity of the method, we supposed larger apical lesions would be associated with higher aMMP-8 levels, even though no clinical symptoms for disease activity were present in our patients. However, only a fair positive correlation was discovered between aMMP-8 levels and lesion diameter, and the results did not reach statistical significance. One possible explanation for this finding could be the dynamics of AAP that is characterized with little lesion expansion and minimal bone resorption.[37] It is possible that GCF biomarkers will be increased in the active phase of the disease when the lesion is initially formed. Accordingly, the proposed method could be utilized in the short-term follow-up of endodontically treated teeth with no periodontal involvement to confirm the success of the intervention and monitor the development of periapical lesions.

Even though the observed impact of AAP on GCF composition seems to be minimal, our results suggest aMMP-8 in teeth with AAP reaches the gingival crevice. Beaza et al suggested two possible mechanisms of the observed phenomenon: diffusional and circulatory.[18] According to the former, the molecules might leak through the periodontal ligament. In the circulatory mechanism, the molecules enter the periapical blood vessels and reach the subepithelial plexus via the anastomoses between the vessels of the periodontal ligament and the supraperiosteal blood vessels. These findings could be significant as biomarkers found in local circulation can impact systemic circulation as well. According to recent studies, the molecules can reach the general circulation, eliciting a low-grade inflammatory response.[25] [38] [39] The results of the present study can be explained by the circulatory mechanism proposed by Baeza et al, which, in turn, can serve as an underlying mechanisms of the systemic response to periapical disease. AAP can, therefore, be a potential risk factor for inflammatory systemic diseases such as cardiovascular diseases or depression.[40] As insufficient treatment motivation is commonly observed as a result of the equivocal clinical presentation of AAP, mounting evidence of the systemic impact of the disease could improve treatment compliance.

Conclusion

The results suggested that GCF composition is modified by AAP only to a minimal extent but signifies a possible underlying mechanism of how the release of proinflammatory mediators contributes to low-grade systemic inflammation. As demonstrated in the present study, the levels of activation of MMP-8 in GCF do not increase to pathologically elevated levels in teeth with AAP. Further research is needed to substantiate the use of aMMP-8 as a potential biomarker for the diagnosis of the disease as well as explore its relationship with other biomarkers. The detection of GCF biomarkers reflecting the periapical status can aid clinical diagnosis and short-term follow-up. In line with molecular diagnostic trends, this approach can augment the current evaluation methods, promote the successful personalization of AAP treatment, and optimize the long-term prognosis of the disease.

Conflict of Interest

None declared.

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Publication History

Article published online:
12 May 2020