|Year : 2023 | Volume
| Issue : 5 | Page : 463-468
Comparative estimation of C-terminal cross-linked telopeptide of type I collagen levels in gingival crevicular fluid in diabetic and nondiabetic patients with chronic periodontitis: A prospective clinical study
Anushree Ningombam1, Nipun Dhalla1, Pooja Palwankar1, Anjana Goyal2, Ruchi Pandey1
1 Department of Periodontology, Manav Rachna Dental College, FDS, MRIIRS, Faridabad, Haryana, India
2 Department of Biochemistry, Manav Rachna Dental College, FDS, MRIIRS, Faridabad, Haryana, India
|Date of Submission||08-Jun-2023|
|Date of Decision||28-Aug-2023|
|Date of Acceptance||01-Sep-2023|
|Date of Web Publication||30-Oct-2023|
Dr. Anushree Ningombam
Singjamei makha Khongnang Pheidekpi, Imphal, Manipur 795 008
Source of Support: None, Conflict of Interest: None
Aim: To estimate C-terminal cross-linked telopeptide (CTx) levels in GCF, using ELISA in diabetic and nondiabetic patients with chronic periodontitis. Materials and Methods: A total of 42 patients were recruited: 21 type II diabetic patients with chronic periodontitis (group A) and 21 nondiabetic patients with chronic periodontitis (group B). Scaling and root planing (SRP) were done in groups, followed by collection of GCF samples and recording of clinical variables. Measurements for clinical indicators of periodontal health were taken on 0 day (baseline), first and after third month. The Ctx level of GCF was taken at baseline and after 3 months using enzyme-linked immunosorbent assay (ELISA). GPower software (version 3.0) was used. Paired t tests and Student’s t tests as well as parametric tests were used to analyze the results. The threshold for significance was fixed at P < 0.05. Result: Following SRP, there was a significant reduction in Ctx levelsand an improvement in clinical parameters in both groups. There is a negative correlation uniting CTx quantity with an improvement in periodontal conditions in both diabetic and nondiabetic patients. Conclusion: CTx levels in both diabetic and nondiabetic candidates with chronic periodontitis infection were reduced after nonsurgical periodontal therapy.
Keywords: Chronic periodontitis, CTX, diabetes, GCF
|How to cite this article:|
Ningombam A, Dhalla N, Palwankar P, Goyal A, Pandey R. Comparative estimation of C-terminal cross-linked telopeptide of type I collagen levels in gingival crevicular fluid in diabetic and nondiabetic patients with chronic periodontitis: A prospective clinical study. J Int Oral Health 2023;15:463-8
|How to cite this URL:|
Ningombam A, Dhalla N, Palwankar P, Goyal A, Pandey R. Comparative estimation of C-terminal cross-linked telopeptide of type I collagen levels in gingival crevicular fluid in diabetic and nondiabetic patients with chronic periodontitis: A prospective clinical study. J Int Oral Health [serial online] 2023 [cited 2023 Dec 2];15:463-8. Available from: https://www.jioh.org/text.asp?2023/15/5/463/388785
| Introduction|| |
Chronic periodontitis (CP), an inflammatory disease of the teeth-supporting tissues, is instigated by specific microorganisms, resulting in a progressive degradation of periodontal tissues and various outcomes such as clinical attachment loss (CAL), pocket formation, and recession. Once the disease sets in, the collagen fibers lose their components to the cemental surface, leading to the periodontal pockets formation and the eventual breakdown of alveolar bone. Biochemical signaling facilitates inflammation, connective tissue breakdown, and alveolar bone turnover, contributing to clinical morbidity. During the bone turnover process, biomolecules found to be increased in GCF of periodontally afflicted subjects, act as indicators of disease progression. Biological markers are substances measured objectively and are known indicators of bacterial, physiological, and pharmacologic responses to curative interventions.
About 90% of the organic matrix of ossified tissues is composed of type I collagen compound, thereby playing a significant role in osseous tissue. Therefore, in many osteolytic and osseous metabolic diseases, remnants of collagen breakdown become crucial indicators of bone turnover. Postgenetic translation, collagen molecules undergo alterations, producing cross-linked telopeptides and cross-links that are believed to be especially linked to the resorption of ossified tissues. While these remnants were formerly evaluated in urine, current scientific and economic emphasis leans toward developing and evaluating methods for their measurement in serum. Several such indicators include free gamma-carboxyglutamic acid, hydroxyproline, pyridinoline, deoxypyridinoline, type I collagen remnants, and C- or N-terminals telopeptides (CTx, NTx), among others such as bone sialoprotein.
The collection of GCF has been classified into intracellular and extracellular methods, involving various techniques such as the use of micropipettes, absorbing paper, preweighed threads, and crevicular washings. Analysis of GCF helps determine active disease progression or regression. Levels of prostaglandin E2, including interleukin-1 in the crevicular fluid of gingiva, are found to be higher in type I diabetics with gingivitis or periodontitis compared to nondiabetic individuals.
During osteoclast-mediated resorption, the N-terminal along with C-terminal telopeptide remnants of type I collagen are released into the blood and removed by kidney filtration. In individuals with Crohn’s disease, high CTx levels are associated with reduced bone mass and are highly effective in identifying bone metastases. Consequently, the C-terminal cross-linked telopeptide of type I collagen (CTx) is established as a possible biomarker for the indirect assessment of bone resorption. CTx is a biochemical indicator reflecting the deterioration of the C-terminal end of type I collagen telopeptide and is released when the intact bone collagen is cleaved by cathepsin-K. These remnants of bone collagen serve as the primary indicators of bone resorption as a result of osteoclastic activity. CTx, thus, has become the preferred biomarker for assessing bone resorption.
There is growing evidence of a connection between systemic disorders such as cardiovascular disease, respiratory tract infections, diabetes, insulin resistance, gastrointestinal, Alzheimer’s disease, and periodontitis. Elevated glycated hemoglobin levels are associated with severe periodontitis. In advanced diseases, diabetes with active resorption of bone, CTx can be judged for the evaluation of severity as well as activity of the disease., Thus, we aimed to compare the CTx levels in GCF between diabetic and nondiabetic patients with CP, pre- and post-nonsurgical periodontal therapy.
| Materials and Methods|| |
Study design and groups
The study was conducted in the Department of Periodontology after the approval of institutional ethical committee to estimate CTx level in type II diabetic and nondiabetic patients with CP using the ELISA sandwich technique. A sample size of 42 subjects (21 subjects each with 84 sites in each group), aged about 35–65 years, PPD of 4–6 mm, and having CAL of 1–3 mm, was decided after statistical analysis and was recruited from the OPD upon fulfillment of the inclusion criteria.
Patients who had undergone oral prophylaxis or taken antibiotics in the past 3 months, pregnant or lactating patients, patients who consumed any kind of tobacco, and had any systemic disease other than type II diabetes were excluded. Patients received an information sheet about the study, oral explanations of its purpose in a language they could understand, and explanations of its design, potential risks, and benefits.
Those in group A included type II controlled diabetics who have CP with glycated hemoglobin (HbA1c) levels <7%. Those in group B included nondiabetics who have CP with HbA1c levels of 4%–6% (in accordance with the guidelines of the American Diabetic Association). Patients were screened for diabetes using the routine random blood sugar test conducted in the department using a glucometer. They were asked to report the HbA1c test results for participation.
A pooled sample of GCF was collected from each quadrant (total of 4 sites) with the deepest pocket. For this purpose, impressions were taken and customized occlusal stents of acrylic were made and placed on the prepared casts to minimize distortions and the clinical parameters were noted.
Measurements of modified gingival (MGI), plaque (PI) (Turesky-Gilmore-version Glickman’s of the Quigley and Hein), probing pocket depth (PPD), and CAL were taken at 0 day (baseline), first and after third months. At each recall appointment, oral maintenance instructions were reiterated, and scaling was performed again as necessary part of maintenance.
Collection of GCF and CTx estimation
As per inclusion criteria, GCF was collected from the selected sites with the help of calibrated capillary micropipettes using the intrasulcular method of collection and stored in the Eppendorf tube at –80 °C for estimation of CTx level. Each of the Eppendorf tubes was named as per the enrolment number and stored in a container containing the registration details of each patient from both groups. The biochemical analyst was blinded to the groups by coding the Eppendorf tubes before the analysis and they were decoded after. The Sandwich-ELISA principle is used. This kit includes a micro-precoated ELISA plate with a specific antibody to human CTX-I. (Source: Elabscience. User Manual for Human CTX-I ELISA Kit. Catalog No: E-EL- H0835. 1st Edition; 2022. Elabscience Biotechnology Inc.)
GPower software, Germany (version 3.0), was used. With an alpha of 0.05 and a power of 80, it was determined that a total sample size of 42 (21 in each group) was necessary. Data were first tabulated in Microsoft Excel and then a descriptive statistical analytic test was performed using an independent t test using SPSS 21.0. The analysis was done by calculating the mean value and standard deviation for the continuous variables. Chi-square test analysis was performed to compare the data between groups. The Shapiro–Wilk test was performed to check the uniform distribution of the data.
| Results|| |
On intragroup comparison, the overall mean changes in the modified gingival index from baseline to third month were 0.48 ± 0.16 and 0.58 ± 0.19 for groups A and B, respectively. The overall mean changes in the plaque index from baseline to third month were 1.27 ± 0.25 and 1.20 ± 0.24 for groups A and B, respectively. The overall mean changes in PPD from baseline to third month were 1.48 ± 0.49 and 1.55 ± 0.59 for groups A and B, respectively. The overall mean changes from baseline to third month were 1.43 ± 0.50 and 1.51 ± 0.66 for groups A and B, respectively. The statistically significant (P value < 0.05) changes were noticed [Table 1] and [Table 2].
|Table 1: Intragroup and intergroup changes in mean MGI and PI at different time intervals|
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|Table 2: Intragroup and intergroup changes in mean PPD and CAL at different time intervals|
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On intergroup comparison of the modified gingival index, the P value remained nonsignificant from baseline where the P value is 0.068 to third month where P value is 0.671. On intergroup comparison of plaque index the P value remained nonsignificant from baseline where P value is 0.764 to third month where P value is 0.105. On intergroup comparison of PPD, the P value remained nonsignificant from baseline where P value is 0.201, to third month where P value is 0.603. Similarly, on intergroup comparison of clinical attachment level, the P value remained nonsignificant from baseline where P value is 0.253 to third month where P value is 0.534 [Table 1] and [Table 2].
The mean change in CTx levels from baseline to third month was 0.145 ± 0.05 and 0.133 ± 0.06 for groups A and B, respectively, which was statistically significant but on intergroup comparison between baseline to third month, CTx levels did not show significant changes [Table 3].
|Table 3: Intragroup and intergroup changes in mean CTx at different time intervals|
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| Discussion|| |
Periodontitis is a polymicrobial disease that is inflammatory in nature and is associated with several systemic diseases, including diabetes mellitus, a metabolic condition defined and distinguished by hyperglycemia. In diabetics, there is a sustained state of hyperglycemia that causes macro- and micro-vascular problems as well as inevitable structural and functional damage to multiple principal organs. It has also been shown an impact on the periodontal tissues leading to periodontitis and has been labeled as the sixth consequence.
Hyperglycemia has been proven to affect bone turnover and osseous healing because it prevents osteoblastic cells from proliferating and hampers the rate of collagen synthesis.
Diabetes- and periodontitis-specific indicators of inflammation are a sign of the presence of the disease. The release of gingival crevicular fluid is closely related to the growth and regression of an inflammatory lesion in the periodontal tissues. By-products of tissue metabolism and mediators of local tissue degeneration are carried by the serum exudate, also known as GCF, as it enters the sulcus via the microcirculation of the tissues of the gingiva.
A key objective of periodontal diagnostics is the early detection of adverse outcomes in tissue supporting the teeth that occur during the start or progression of the disease. To aid in the early diagnosis of alveolar bone loss, type I collagen fragments have been examined in a variety of sources including saliva, mouth rinse, and crevicular fluid from patients with periodontal infection. The main bone-degrading cells are osteoclasts, and cathepsin-K is essential for their capacity to resorb collagen. This telopeptide of type I collagen with a cross-linked C-terminus (CTx) is produced as a by-product of tissue degradation by cysteine protease, which functions best in an acidic pH.
The collagen-related degradation marker CTX-I is used to estimate the resorption of bone. There are presently limited studies that demonstrate a difference between healthy controls and patients with periodontitis in terms of salivary CTX-I, with numerous studies showing that patients with CP have higher CTX-I levels.
There has been mounting evidence in the past 10 years that non-surgical periodontal therapy enhances diabetic control. The monitoring of diabetic patients and the occurrence of such problems may be improved by the long-term beneficial effects of periodontal treatment on glycemic management. The foundation for developing treatment standards will be proving that proper periodontal treatment improves the outcomes of diabetes patients.
In this investigation, we examined the changes in CTX-1 volume with type II diabetes CP as well as in subjects with CP but without diabetes, who were getting nonsurgical periodontal therapy. Forty-two patients (21 subjects in each group) from the outpatient department were enrolled, who exhibited a pocket depth of 4–6 mm and a clinical attachment level of 1–3 mm. A group included type II controlled diabetics who have CP with glycated hemoglobin A1c levels <7%. Those in group B included nondiabetics who have CP with glycemic hemoglobin A1c levels of 4%–6%.
On intergroup comparisons of plaque scores (P = 0.383), modified gingival scores (P = 0.065), PPD (P = 0.672), and clinical attachment gain (P = 0.647) showed nonsignificant changes from baseline up to third month. In contrast, on the intragroup comparison, these parameters showed significant changes from baseline to third month. The participants showed good oral hygiene maintenance and a change in overall plaque score from baseline to third month. The observed reductions in plaque scores are similar to those deduced by Bukleta et al.  demonstrating the beneficial aspect of tooth cleaning in maintaining periodontal health and metabolic control.
The outcome of our study is consistent with several examinations where reductions in gingival bleeding, or gingival indices in both diabetic as well as CP patients were reported, ,,,, which can be attributed to the reduction in pathogenic bacteria and oral biofilm responsible for inflammation. It has been suggested for diabetics that glucose levels improve as a result of gingival inflammation and bleeding being reduced confirming a favorable response of diabetic patients to NSPT.23
The reduction in PPD may be ascribed to thorough treatment provided in the form of SRP combined with the effect of oral health advice given when the study was initiated. The participants showed good oral hygiene maintenance and overall lessening in pocket depth from baseline through third month can be attributed to the reduction in pathogenic bacteria and oral biofilm responsible for pocket formation. In a similar study by Quintero et al., they discovered a substantial decline in PPD with SRP and oral hygiene instructions along with warm saline rinses, demonstrating the benefits of SRP in combination with just warm saline rinses to effectively control inflammation and improve pocket depth.
The overall reduction in CAL at different time points in the intragroup comparison can be attributed to the effective response to the treatment given and the maintenance by the participants as well as the reinforcement of oral hygiene in every visit which correlates with the reduction in inflammation, the elimination of plaque and PPD after NSPT and it coincides with the findings of Quintero et al. There was no gain of statistical magnitude in CAL in the intergroup comparison which is in accordance with the nonsignificant decrease in clinical attachment level from 0 day, 6 weeks, and 3 months in a study by Izuora KE et al., Shashikumar et al., and Ibrahim et al.,,
On the intragroup comparison, a significant difference was observed in the CTx levels. This decrease in CTx levels can be ascribed to the effectiveness of scaling and root planing rendered to both groups combined with maintenance therapy provided at subsequent visits, and the proven evidence of better glycemic control after NSPT, in various studies. It can be assumed that improved control of both type II diabetes and CP may have resulted in a reduction of alveolar bone loss and a sequential reduction of CTx levels. However, when a comparison was done between the groups, no substantial differences could be found in CTx levels from baseline to third month (P = 0.465). This lack of significance in the levels, when the intergroup comparisons are made in relation to both clinical and biochemical parameters may be in accordance with a statement provided by Lee et al., that adults without systemic complications who have well-controlled diabetes are treated similarly to patients without diabetes.
The clinical criteria are positively correlated with the CTx levels in GCF, which rise proportionately to the progression of periodontal disease. Decreased levels of CTx were seen at the end of 3 months in both groups, proving a negative correlation to improvement in periodontal conditions. The current investigation had some limitations, as it lasted 3 months, a more thorough assessment and judgment of the findings would have been possible with a longer study period. The radiographic analysis of bone loss at baseline and after 3 months was not compared. Better outcomes might have come from a larger sample size and further research should be conducted.
| Conclusion|| |
Despite the study’s limitations, it can be said that nonsurgical periodontal therapy can improve periodontal conditions and lower levels of CTx, a marker of bone resorption, in both Type II diabetics with well-controlled blood sugar levels and people with CP who are not diabetic.
We express our gratitude to Department of Periodontology, Manav Rachna Dental College, MRIIRS, Faridabad, Haryana, for their invaluable support for the research.
Financial support and sponsorship
Conflicts of interest
The authors have no conflicts of interest regarding this research.
Patient declaration of consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/ her clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Anushree Ningombam, Nipun Dhalla: Concept, Design of study, data collection, data interpretation and manuscript writing and editing.
Pooja Palwankar, Ruchi Pandey: Data collection and analysis, manuscript writing.
Anjana Goyal: Data analysis and writing.
All the authors approved the final version of the manuscript for publication. The manuscript has been read and approved by all the authors.
Ethical policy and Institutional Review board statement
Ethical approval no. MRDC/IEC/2020/13.
Data availability statement
The data set used in this study is available on request.
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[Table 1], [Table 2], [Table 3]