Study Design
This observational, analytical, case-control study was carried out at School of Dental Sciences, Universiti Sains Malaysia (USM) Health Campus, Kelantan.
Ethical Consideration
The Human Research Ethics Committee of USM (JEPeM) gave the ethical permission for this study, assigning ID number USM/JEPeM/21708911.
Sample Size Estimation
A similar study was undertaken in the same population in 2017-18, where levels of MMP-9 were ascertained in human salivary samples [12]. Keeping in account that the target population and types of samples were same for this study, the same sample size was used as in the previously cited study, which was a total of 60 subjects, equally divided into control and test groups.
Data Collection
The probability, clustered sampling method was employed for this study. There were 60 patients in total, equally split between the control and test groups. Individuals with chronic periodontitis alone (and no other medical disorders) made up the control group, whereas individuals with Type-II DM and chronic periodontitis made up the test group. After evaluating their medical histories and recording their finger-prick blood glucose levels (for the control group) or assessing the HbA1c (for the test group), the participants were divided into control and test groups. Patients’ information was recorded, which included socio-demographic data, medical history and periodontal parameters.
Adults between the ages of 30-70 made up the patients who were chosen. Only individuals with a diagnosis of chronic periodontitis (defined as clinical attachment loss at two or more non-adjacent teeth) were chosen. Patients who had been clinically diagnosed with Type-II DM at least six months prior and who also met the aforementioned criteria for chronic periodontitis were chosen for the test group [13].
Women who were pregnant or nursing, those with medical conditions besides Type-II DM, and those who had undergone periodontal treatment at least three months before data collection, were excluded from the study. All the participants gave a written, signed consent.
The following order was followed with the subjects in the clinics:
- Periodontal parameters
- Saliva assemblage
- Storage of samples in -80°C freezers till laboratory analysis
The saliva samples were analyzed in the laboratory using Enzyme Linked Immunosorbent Assay (ELISA) test, using the specific ELISA kit for MMP-1 (Elabscience, Wuhan, China).
Periodontal Parameters
The periodontal parameters recorded were:
i. Plaque Score [14].
ii. Bleeding Score [15].
iii. Probing Pocket Depth (PPD) [16].
iv. Clinical Attachment Level (CAL) [17].
Inter-Examiner Reproducibility
Prior to the start of data collection, the inter-examiner reproducibility and reliability of the measurements of clinical attachment level (CAL) and probing pocket depth (PPD) were established. Two examiners had taken multiple measurements at 100 different tooth sites for various patients. The measured values were examined using the intraclass correlation coefficient (ICC) test. For PPD and CAL, the calculated correlation coefficients were 0.90 and 0.86, respectively.
As the ICC increases above 0.8, the impact of measurement error decreases until it reaches an ICC of 1.0, which indicates perfect reliability [18].
Saliva Samples Collection
Following the recording of the clinical data mentioned above, participants from both groups had saliva samples obtained to estimate MMP-1 levels. The saliva was taken by stimulation method since earlier research had shown that the flow rate of stimulated saliva was higher than the flow rate of unstimulated saliva [19]. Prior to sample collection, subjects were invited to sniff a citrus fruit (orange or lemon); it prompts the salivary glands to generate more saliva than usual. The patients were then given distilled water to drink and instructed to thoroughly clean their mouth for one minute. Then, the individuals were instructed to expectorate it. They were then instructed to spit into a 10 mL sterile tube 2 minutes after this. The patients were told to let their saliva passively run into the saliva collection tube and gather at the back of their mouth. During collection, it was advised to put the tube on ice (in an icebox). As soon as possible, the samples were moved from the icebox into the -80°C freezer until further analysis.
Preparation of Saliva Samples
A preparation of saliva samples was done before running the ELISA. To defrost all of the saliva samples without denaturing any protein indicators, they were removed from the freezers and placed on open ice for roughly 1-2 hours. The samples were centrifuged at 1,000 x g for 20 minutes at 5°C after they had completely thawed out to eliminate any debris. Supernatant was then taken out and placed in tiny 1.5mL Eppendorf tubes for storage. After conducting preliminary runs, it was discovered that the MMP-1 levels in saliva were higher than the MMP-1 ELISA Kit's detection range. To bring the MMP-1 concentration within the kit's detection range, a dilution factor of 1:100 was determined for the specimen based on the pre-run results. Each 2L part of saliva sample received 198L of Reference Standard & Sample Diluent, and 200L of diluted saliva aliquot was created for each sample. These aliquots were then kept overnight at 20°C in preparation for a lab analysis. The leftover saliva samples were properly disposed and destroyed.
Preparation of Reagents
The wash buffer solution was made first. To create 750mL of Wash Buffer, 30mL of Concentrated Wash Buffer was diluted with distilled water.
The usual working solution was then created. After centrifuging the standard at 10,000 x g for one minute, 1.0 mL of Reference Standard & Sample Diluent were added. It was left standing for ten minutes, during which time it was repeatedly turned upside down. A pipette was used to stir it well once it had completely dissolved. The working solution after reconstitution contained 2000pg/mL. This effective solution is used to create successive dilutions.
Seven 1.5mL Eppendorf tubes were used for the serial dilutions, and each one was filled with 500L of the reference standard and sample diluent. To create a 1000pg/mL working solution, 500L of the working solution was transferred to the first tube by a pipette. From this 1000pg/mL working solution, an additional 500L was pipetted and combined into the following tube. The process was repeated until there was just one tube left. The solution from the previous tube wasn't pipetted into the last tube since it is taken as a blank. The working solution concentrations obtained using this approach of serial dilutions were 2000 pg/mL, 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, and 0 pg/mL (blank).
The working solution containing the Biotinylated Detection Antibody and Concentrated HRP Conjugate was then formed. One hundred litres per well is needed for each. There should be enough for each well after diluting it from 100x to 1x workable solution. A 100-200 L additional solution was made as a safety measure. This calculation was performed using the dilution equation M1V1=M2V2.
The plate has 96 wells, and since 100 L was needed for each well, the working solution needed a total volume of 9600 L. It ended up being 9800 L after adding the extra 200 L. V1 was calculated using the dilution equation to be 98 L. Therefore, 98 L of concentrated solution and 9702 L (9800-98) of diluent were needed to create the 1 x working solution for each of the Biotinylated Detection Antibody and Concentrated HRP Conjugate. Before making the dilution, all of the tubes were centrifuged at 8,000 x g for 2 minutes.
Protocol for ELISA Assay
The first two columns were filled with standard working solution at various concentrations, 100 mL each well, two wells per concentration, added side by side. Similarly, salivary samples of 100mL each were added to the remnant wells, two samples being inserted side by side (duplication). Plate sealer was used to seal the plate, which was then incubated at a temperature of 37°C for 90 minutes.
Following the removal of the liquid from each well, 100 mL of the working solution for the Biotinylated Detection Antibody was added to each well. The plate was once more sealed, gently stirred, and incubated at 37°C for one hour.
Then, 300 L of wash buffer was added to each well after the solution from each well had been decanted. A clean piece of absorbent paper was used to pat the plate dry after the wells had been wet for 1-2 minutes. Three times in all, this washing phase was repeated. Following the cleaning, each well received 100 mL of the HRP Conjugate working solution. After applying sealer once more, the plate was incubated for 30 minutes at 37°C.
After this period of time, each well's solution was once more decanted, and five rounds of washing with wash buffer were performed. Then, each well received 90 L of the Substrate Reagent. Being light-sensitive, this step was carried out in a dark setting to shield the plate from light. Plate was wrapped in aluminium foil and sealed with a new sealant. It was then incubated once more for at a temperature of 37°C for 15 minutes. Changes in color were noted in the plate after every 5 minutes. When sufficient color change had taken place in each of the wells, 50 mL of Stop Solution were added to each well, ensuring to add it in the same order as the Substrate Reagent.
After that, the plate was put into the Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA) with the wavelength set to 450 nm, and the optical density (OD) value of each well was calculated. To get the corrected OD readings, the OD value from the blank solution well (which served as the kit's background) was removed from the OD value that was derived for each well.
Data and Statistical Analysis
SPSS version 26.0 was used to analyse all the data. Mean and standard deviation (SD) were used to summarise continuous data, and mean percentage and SD were used to summarise discrete (categorical) data. Analysis of variance (ANOVA) was used to compare continuous variables, and an independent t-test was used to determine the significance of the mean difference between the groups. Additionally, independent t-tests were used to compare categorical variables. MMP-1 levels and periodontal parameters were examined using single linear regression and multiple linear regression analysis. A P value of 0.05 was considered to be statistically significant.