Background
Periodontitis is a general chronic inflammatory condition that is initiated by subgingival colonisation by periodontopathogenic bacteria, causing an inflammatory and destructive host response in susceptible patients [
1‐
3]. Diagnosis of periodontal disease is traditionally based on clinical parameters and indices that reflect a history of periodontal diseases but cannot fully reflect disease activity. Early diagnosis of ongoing tissue destruction in periodontitis is particularly important in order to prevent further attachment loss. Unlike other areas of medicine, biochemical testing is not regularly used in dentistry, despite obvious advantages [
4] for supplementing or replacing traditional diagnostic methods.
Infection of periodontal tissues by pathogenic microorganisms can trigger mechanisms of inflammatory defence that produce cytokines and enzymes prominent among which are the matrix metalloproteinases (MMPs), leading to tissue degradation and bone resorption [
3,
5]. Among the MMPs family, the active form of MMP-8 (aMMP-8) in GCF [
6] is the most prominent collagenase (collagenase 2) associated with periodontitis [
7,
8]. Measuring single-site GCF aMMP-8 levels could enable the clinician to distinguish between healthy tissues and those with gingivitis or periodontitis [
9‐
11].
Oral rinse samples consist more of GCF from periodontal pockets than saliva [
12‐
14], which contains sloughed epithelial cells from oral mucous membranes, nasopharyngeal discharge, food debris, and bacteria and their metabolic products. Thus, analysis of oral rinse may be useful in the monitoring of general periodontal disease status, and facilitate early identification of individuals at risk of periodontitis [
13].
The aim of this study is to investigate the relationship between clinical diagnostic parameters and the point-of-care testing levels of aMMP-8 in GCF and oral rinse in individuals with periodontal disease in China, and to test if the aMMP-8 levels could be effective for monitoring the status of periodontal disease.
Discussion
At present, regular measurement of attachment loss is the best choice for monitoring chronic periodontitis in the periodontal pocket [
22]. However, monitoring AL is not suitable for early detection and poor at establishing disease prognosis. Indeed, probing depth, attachment loss and X-ray analysis only reflect historical damage to the periodontal pocket, and are thus not helpful for monitoring [
23]. During the stable periods of periodontitis, the levels of aMMP-8 remained low, but the damage to the periodontal pocket and attachment loss could persist.
Similarly, the primary value of BOP and BI as a diagnostic sign is that its presence indicates that the tissues are inflamed, but it is likely that the amount of probing pressure can influence whether a site bleeds or not. Perhaps only tissue slices are able to reflect the actual pathological state, but histological diagnosis is a much difficult, expensive and time-consuming way of diagnosis.
In contrast, biomarkers serve as a convenient method for distinguishing between healthy and diseased tissues, or for learning about the response to pharmacological or other therapeutic interventions [
24]. In fact, biomarkers are potentially useful throughout the entire disease process, as they can be used at the periods of: (1) before diagnosis, for screening and risk assessment; (2) during diagnosis, for determining staging, grading and selection of appropriate therapies; (3) after diagnosis, for monitoring therapeutic outcomes and disease remission.
Our results demonstrate a clear positive correlation between aMMP-8 levels and clinical periodontal index, including PD or BI parameters, suggesting it could be used as a biomarker for diagnosis. In line with previous studies [
25], receiver operating characteristic curve analysis showed that the highest aMMP-8 threshold was 6.66, with corresponding sensitivity and specificity values of 0.8 and 0.9, respectively. These values represent a significant improvement beyond existing clinical indicators that are unable to quantify the activity of periodontitis, and facilitate early detection and diagnosis prior to the appearance of inflammatory damage to periodontal tissue. However, more thorough investigation of threshold values through longitudinal studies with larger sample size are still needed to push this in further.
Recent proteomic analyses suggested that the whole saliva contains more than 3000 different proteins belonging to various classes and that are involved in processes as diverse as cell adhesion, apoptosis, the cell cycle, enzyme regulation, responses to stimuli and immunity [
26]. Quantitative analysis of aMMP-8 in saliva and oral rinse samples has been performed previously, and higher levels were reported in people with periodontitis [
27]. It is known that MMP-8 is mainly produced by polymorphonuclear leukocytes (PMNs) [
28], but periodontal ligament fibroblasts and epithelial cells could also produce this protein. MMP-8 can promote the degradation of connective tissue extracellular matrix and accelerate the development of lesions. During inflammation, the quantity of PMNs in saliva increases, and proinflammatory cytokines in the gingival groove stimulate the further release inflammatory cytokines such as TNF-α, IL-6 via autocrine and paracrine pathways. As a result, the area of inflammation becomes enlarged, PMNs are further activated, and aMMP-8 is produced. PMNs and aMMP-8 permeating from the gingival groove eventually enter the saliva. As reported in some previous studies, tissue inhibitor of matrix metalloprotease-1 (TIMP-1) levels was screened in oral rinse samples by Amersham ELISA and a test utilizing a chromogenic substrate, and it was found that MMP-8/TIMP-1 M ratios increased together with periodontal inflammatory burden when measured by dentoELISA [
17]. Although we mainly focused on aMMP-8 in the present study, we believe that further investigations of aMMP-8 jointly with its inhibitor TIMP-1 are still needed in our future studies.
There were no significant differences in aMMP-8 levels in oral rinse samples from different groups, which was an unexpected result of the present study. The results of correlation analysis indicated that there was no significant statistical association between the two biological fluids. As we know, saliva was the main fluid in oral rinse samples, and it can also be regarded as a diagnostic fluid. But unlike GCF, it reflects the oral status more generally and more unspecificly. The source of any oral biomarker should always be taken into account when analysing its function, and MMP-8 can possibly originate from a number of different sources including GCF, and salivary glands, as well as secretion from buccal mucosa. Further exploration of the exact contribution of these potential sources to oral MMP-8 levels would be interesting but also extremely challenging.
Nevertheless, we did observe statistical significance of the MMP-8 levels in GCF samples from the healthy and diseased participants. A lack of consistency in oral rinse samples from patients with chronic periodontitis has been largely attributed to the complexity and difficulty in determining disease status using the currently available diagnostic methods [
29‐
31]. Various molecules presented in the saliva of periodontitis patients may be associated with chronic periodontitis, including enzymes such as collagenases, acute-phase proteins, and markers of bone loss [
32,
33]. Quantitative analysis of aMMP-8 in the complex saliva samples is therefore difficult and requires further investigation.
The DentoELISA aMMP-8 immune detection system can be used to measure the aMMP-8 levels in point-of-care GCF and oral rinse samples within 15 min of collection in the clinic. DentoELISA can also be used to quantify aMMP-8 levels in plasma or other clinical samples. Our results clearly demonstrate the usefulness of dentoELISA for quantitative determination of aMMP-8, and indicate that this protein may be a useful biomarker for diagnosis of periodontitis.
Some limitations need to be addressed when extrapolating our current results for further research. First, this is a cross-sectional study without longitudinal observation, hence more thorough investigation with longitudinal design are still needed to shed more light on the progression of periodontitis. Second, since the imbalance between MMP-8 and its inhibitor TIMP-1 is considered to initiate periodontal disease [
8], we think that it would be much valuable to perform further analyses on both MMP-8 and TIMP-1 in the future. Moreover, it is expected that further studies with larger sample size and more comprehensive study design will reveal more of this issue and yield better explanation of the intrinsic mechanism of the function of MMP-8 in periodontitis.