Background
At present, diagnosis of periodontal diseases and evaluation of their severity are based on conventional clinical parameters and radiographic findings. However, these methods cannot rapidly detect early periodontal tissue losses and are, therefore, insufficient to determine the degrees of disease severity. Therefore, different types of adjunctive tools are introduced into clinical practice and provide more validity for correct diagnosis and proper treatment planning [
1]. Up to the present, several biomarkers have been used for diagnosis and assessment of periodontal disease status as well as prognostic prediction of periodontal disease progression because of their sensitivity and specificity. A number of previous studies have recommended gingival crevicular fluid (GCF), a serum exudate, as a source of biomolecules sampling in order to evaluate periodontal disease status [
2‐
4]. GCF constituents are composed of more than 65 components that have been reported as possible biomarkers for periodontal disease progression [
5]. These include inflammatory mediators and host-response modifiers [
6‐
8], host-derived enzymes and their inhibitors [
9‐
11] and tissue breakdown products [
12‐
14].
Among host-derived enzymes, positive association between alkaline phosphatase (ALP) and periodontal disease activity has previously been reported [
15,
16]. This membrane-bound glycoprotein, which functions as a phospho-hydrolytic enzyme, is a calcium- and phosphate-binding protein [
17]. ALP is released from neutrophils and, thus, detected in GCF collected from inflamed periodontium as well as from osteoblasts during bone formation [
18,
19]. With respect to tissue-breakdown products, several studies have investigated the levels of proteoglycans and their constituent glycosaminoglycans (GAGs) in periodontal tissues and GCF from patients with periodontal disease in comparison to those from healthy controls [
20,
21]. Major GAGs present in periodontal tissues are dermatan sulfate, hyaluronan, chondroitin sulfate (CS) and keratan sulfate. A main source of GAGs in GCF is derived from extracellular matrix degradation of periodontal tissues during the progression of periodontal disease. Consequently, raised levels of GAGs detectable in GCF directly reflect, and are associated with the destruction of periodontal tissues, especially alveolar bone, at the diseased site [
22‐
25]. Furthermore, elevated levels of CS in GCF are apparently associated with four clinical parameters of periodontal status, which serve as gold standards for periodontal inflammation and destruction [
14]. In addition to periodontal disease, CS levels can be precisely detected in physiologic tooth movement [
26,
27] and in patients with pathologic inflammatory disorders, such as degenerative joint diseases [
28,
29].
Although a number of studies have examined the relationship of each individual biochemical marker to different types and severity of periodontal diseases, there is still no study comparing the efficiency of different markers in evaluating disease severity. The aims of this study were, therefore, to investigate the levels of CS, as determined by our patented monoclonal antibody, raised against the WF6 catabolic epitope of CS [
30], and of ALP in GCF obtained from patients with various disease statuses in comparison to healthy controls, and to compare the efficiency of these two biochemical markers in assessing the severity of periodontal diseases.
Discussion
In this study, it was demonstrated that both CS and ALP levels in GCF collected from patients with different periodontal disease statuses were raised in accordance with the severity of periodontal destruction. Low but detectable CS and ALP levels were observed in the H, G, PG and PSL groups, whereas these levels were significantly elevated in the PM and PSE groups when compared with the non-destructive to slightly destructive periodontitis groups. Interestingly, a significant difference in terms of CS levels between moderate and severe chronic periodontitis sites was demonstrated, whereas no significant difference in ALP levels was found. Furthermore, the levels of both biomolecules were significantly correlated with all of four periodontal parameters, including the degrees of periodontal destruction (PD and CAL) and of inflammation (GI and PI), but stronger correlations between all parameters and CS levels than between those and ALP levels were evident. The reason we chose to study the GCF levels of CS and ALP was because it was previously demonstrated that elevated levels of these two biomolecules were closely associated with alveolar bone destruction in chronic periodontitis [
14,
35], whereas raised levels of other biomolecules, such as host-derived pro-inflammatory mediators and proteolytic enzymes, can reflect enhanced inflammation and destruction of both soft and hard periodontal tissues. Therefore, we believe that among a number of biomolecules found within GCF, CS and ALP are good candidates for this comparative study to assess the different severities of alveolar bone destruction in chronic periodontitis.
As anticipated, the average age of patients with chronic periodontitis was more than those with gingivitis and healthy controls due to the chronic nature of periodontitis, which is caused by dental plaque accumulation and persistent inflammation of nearby periodontal tissues. Nevertheless, the CS and ALP levels from the gingivitis sites of patients with chronic periodontitis (PG) were not significantly different from those of both patients with gingivitis (G) and of healthy participants (H), although the ages of patients and healthy volunteers in this study were not matched. In contrast to the age difference between the chronic periodontitis and the remaining groups, the gender distribution was not different among groups to avoid bias in the study design. Furthermore, no significant differences in clinical parameters between the H, G and PG groups were found, whereas such parameters in PSL, PM and PSE groups were enhanced according to the severity of periodontitis.
In other previous reports, the studied cohorts were mostly defined as healthy, gingivitis and chronic periodontitis [
13,
35]. However, in our study, the chronic periodontitis group was further divided into subgroups, including PG, PSL, PM, and PSE, according to the disease severity. We believe that detailed classification of chronic periodontitis according to the severity of alveolar bone destruction will better reflect the potential of biochemical markers to distinguish different disease statuses that can provide useful information and help clinicians in proper treatment planning and periodontal maintenance, while the conventional clinical parameters cannot [
36,
37].
In some previous studies, the association between ALP and periodontal disease was reported, especially in active diseased sites [
16,
35]. Significantly higher concentrations of ALP were observed in periodontitis than in healthy and gingivitis sites, and positive correlations of ALP levels with clinical parameters, including PD and GI, were reported [
16,
38]. These findings were similar to ours, and weak correlations were also demonstrated both in those studies and ours. With respect to CS levels, significantly higher levels were shown in destructive sites than in non-destructive or in slightly destructive sites, consistent with the findings from our previous study [
14]. Interestingly, even though the ALP levels were higher in the destructive sites, no significant difference in ALP levels was found between moderate and severe destruction. On the other hand, a significant difference in CS levels between moderate and severe destruction was observed, suggesting that the CS levels can be better used than the ALP levels, to differentiate the clinical severity of periodontitis, especially between moderate and severe periodontal destruction, although the levels of both biomolecules were elevated in the GCF of patients with chronic periodontitis.
It was demonstrated in this study that either CS or ALP levels were positively correlated with all four clinical parameters, which represent periodontal destruction and inflammation. The positive correlations of elevated CS and ALP levels with increased clinical severity are in line with the findings from previous studies [
14,
17,
35]. However, in this study, all four correlations between the clinical parameters and CS levels were stronger than those between clinical parameters and ALP levels, corresponding with the ability of CS levels, but not ALP levels, to differentiate between moderate and severe periodontal destruction as mentioned above. This may be because CS is derived only from destruction of host extracellular matrix [
39], whereas ALP can be derived from both bacterial cells [
40] and host cells [
41‐
43]. Moreover, it is recognized that raised CS levels are principally due to alveolar bone resorption, whereas elevated ALP levels are found to be implicated in the process of bone formation [
19] in addition to bone resorption in destructive sites [
35,
44]. Lastly, CS is a repeating disaccharide unit of GAGs and should not be cleaved by GCF proteinases, derived from both periodontal pathogens and host cells [
45], whereas ALP enzyme can be degraded by these proteinases during GCF storage. There is still a limitation of this study due to its cross-sectional design; thus, a further longitudinal study is required to monitor any alterations in the GCF levels of these two biomolecules during periodontal disease progression in each individual tooth.
Acknowledgements
We thank the Intramural Endowment Fund, Faculty of Dentistry, Chiang Mai University; the Discovery Based Development Grant (P-10-11290), National Science and Technology Development Agency, Ministry of Science and Technology; and the Thailand Research Fund to S.K. (BRG5680001) for financially supporting this study. We also thank Dr. M. Kevin O Carroll, Professor Emeritus of the University of Mississippi School of Dentistry, USA, and Faculty Consultant at Chiang Mai University Faculty of Dentistry, Thailand, for his critical reading of this manuscript.
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Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
SK: Recruitment of patients and volunteers; site selection; periodontal examination; GCF sample collections. PK: Measurement of chondroitin sulfate levels. SO: Measurement of chondroitin sulfate levels. PP: Measurement of alkaline phosphatase levels. TS: Statistical analyses. DJ: Manuscript preparation. SK: Manuscript preparation and corresponding author. We would like to declare that all authors read and approved the final version of this manuscript.