Evaluation of salivary Ki-67 in health and periodontitis
Periodontitis is a multi-factorial inflammatory disease caused due to the accumulation of dysbiotic dental biofilm and featuring destruction of the teeth-supporting tissues including the loss of periodontal ligament and alveolar bone, demonstrated by the presence of gingival bleeding, periodontal pockets, clinical attachment loss (CAL) and radiographically evidenced loss of alveolar bone . The destruction of periodontal tissues mainly happens due to the host’s inflammatory and dysregulated immune response . The chronic inflammatory response in periodontal disease results from periodontal pathogens (mainly the red complex gram-negative bacteria found in subgingival dental biofilm in the susceptible host), that stimulate inflammatory cells, to produce pro-inflammatory mediators such as prostaglandin E2 (PGE2), interleukin (IL)-1β and tumor necrosis factor (TNF)-α. These pro-inflammatory mediators and virulence factors lead to the production of proteinases that destroy collagen fibers in the periodontal ligament and alveolar bone destruction by activation of osteoclasts . The sustained physiological responses initiated by periodontitis can influence a systemic inflammatory response; different biomarkers have been well-studied in the literature and have been linked to systemic inflammation . In this background, an inflammatory biomarker of interest is Ki-67. It is a nuclear protein that is found in the active phases of the cell cycle and has increased expression in different conditions such as diabetes mellitus, rheumatoid arthritis, pancreatitis, oral dysplastic conditions such as leukoplakia, erythroplakia, leukoerythroplakia, and squamous cell carcinoma . Ki-67 has a short half-life (about sixty to ninety minutes) that indicates its balanced regulation during the cell cycle . Ki-67 is a cell proliferation-specific protein first identified in the 1980s and is now considered a reliable predictive and prognostic indicator for the assessment of biopsies from cancer patients . Investigations about the role of Ki-67 as an inflammatory biomarker in periodontitis are few (Ki-67 was evaluated as a tissue proliferative marker in drug induced gingival overgrowth, in the formerly known “aggressive periodontitis” “chronic periodontitis”, “early onset periodontitis”, “adult periodontitis”, smokers with periodontitis, and periodontitis patients with type 2 diabetes mellitus, respectively), and no studies have reported its assessment in the saliva of patients having periodontitis. Saliva collection has the advantage of being non-invasive, and relatively simple and it is a multipurpose biological fluid whose analysis can reflect both the local oral, and systemic environment related to health and disease offering the foundation for a convenient patient-specific biomarker to evaluate periodontitis and other systemic diseases . Therefore, the objective of this study was to evaluate salivary Ki-67 to find out if saliva is a source of this protein and whether there is a variation in its levels in health and periodontitis. 

This cross-sectional case-control study was conducted at the College of Dental Medicine at the University of Sharjah, UAE from October 2021 to October 2022. The study protocol was approved by the Research Ethics Committee at the University of Sharjah (Reference No.: REC-21-09-28-02-S). All the clinical procedures were performed in full accordance with the revised Declaration of Helsinki and the Good Clinical Practice Guidelines. The study aim was described to all the subjects and written informed consent was obtained; they were also provided a participation information sheet before enrolment. Sample size calculation An a priori analysis was done to calculate the sample size using two-tailed t-tests with an effect size of 0.8 and α error probability of 0.05, power (1-β error probability) of 0.8, allocation ratio N2/N1 equaling 1 gave a non-centrality parameter δ = 2.884 and critical t = 2.008 with a differential of 50. This required a sample size of 26 per group, i.e., a total of 52 with an actual power = 0.807. Therefore, 52 study volunteers were equally divided into two groups: group 1 (Health)-systemically and periodontally healthy ( n = 26) and group 2 (Periodontitis)-periodontitis patients who were systemically healthy ( n = 26). Study design Individuals visiting the postgraduate clinic of Periodontology, the College of Dental Medicine at the University of Sharjah and the Clinics of the University Dental Hospital Sharjah were included in the study. An initial exam, including medical, and dental history, and clinical and radiographic examination was done to evaluate participant eligibility for recruitment in the study. Inclusion criteria were volunteers in the age group of 20 to 55 years who were systemically healthy. Health group: participants with absence or minimal bleeding on probing (BoP) < 10%, no CAL, no redness, no probing depth of more than 3 mm and no clinical swelling/edema or pus, and no bone loss evidenced in radiographs. Periodontitis group: Participants having generalized periodontitis (based on the 2017 classification) , stages II, III, IV, grade B, probing pocket depth (PPD) > 4 mm, CAL ≥ 3 mm, with BoP, and bone loss evidence on the radiographs. Exclusion criteria were individuals who were immunocompromised or with systemic diseases, requiring prophylactic antibiotics before dental treatments, tobacco smokers/smoke-less tobacco users, pregnant/breastfeeding/menopausal females, patients who underwent previous periodontal therapy and/or administered antibiotics/ anti-inflammatory agents in the last 3 months. All the clinical parameters were recorded by one periodontist after intra-examiner calibration was found to be acceptable (kappa statistic value of 0.89, 95% agreement). The following parameters were recorded on the whole dentition. Full mouth plaque (oral biofilm) score (Pl)was assessed wherein six sites per tooth were evaluated for the presence or absence of supragingival plaque using Plaqsearch ( Oraldent Ltd., Huntingdon, UK) two-tone disclosing solution. The presence  / absence (−) of plaque was recorded and  was calculated as a percentage . A visual assessment of gingival changes was done to measure the severity of inflammation using the modified gingival index (MGI) . Using a scale of 0–4, the color, texture, edema, and spontaneous bleeding of marginal and papillary gingiva were scored. Then the average score was taken. PPD, CAL measurements (recorded at six aspects per tooth: mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual) were rounded off to the nearest mm using a periodontal probe (UNC 15, Hu-Friedy ® Manufacturing Inc., Chicago, IL, USA). BoP was calculated as a percentage of sites examined. Saliva collection The saliva collection procedure followed recommendations in the literature . Before the collection of saliva, the participants in the study were told to refrain from foods that may be highly acidic or sugary, avoid caffeine, not use any medications/mouthwash, nor consume alcohol (for a minimum of twelve hours before the collection of saliva), no major meal (before one hour of saliva collection), no toothbrushing (for at least forty-five minutes prior to collection of saliva), no dental procedure done on them in the past twenty-four hours, as this may affect the assay. The saliva samples were collected at a pre-defined time of the day (8:00 a.m. to 10:00 a.m.) from every participant after rinsing with saline for thirty seconds. Thereafter they were instructed to expectorate in a basin to eliminate any food/debris and wait for ten minutes after rinsing, before saliva collection to prevent sample dilution. After this, the study volunteers were instructed to gently spit unstimulated saliva (approximately 10 to 15 ml) into sterile and plain collection tubes. The samples were then centrifuged for 20 min at 1000×g at 2–8℃. The supernatant was collected and stored at -80℃ to be used for further analysis by the assay. Analysis of Ki-67 by ELISA Quantitative determination of Human Ki-67 concentrations from saliva was done using a commercially available ELISA kit (Elabscience Biotechnology Inc., Houston, TX, USA; catalog number: E-EL-H5432) per the manufacturer’s instructions. The detection range per the manufacturer is 0.3 ~ 20ng/ml with a sensitivity of 0.19 ng/ml. The concentrations of human Ki-67 in the samples were measured in pg/ml by comparing the optical densities of the samples to the standard curve. Statistical analysis Categorical data (i.e., sex) were presented as frequencies and percentages and were analyzed using the chi-square test. Numerical data (i.e., marker and periodontal parameters) were presented as mean and standard deviation values. They were explored for normality by viewing the distribution and using Shapiro-Wilk’s test. Data were non-parametric and were analyzed using the Mann-Whitney U test. Correlations were analyzed using Spearman’s rank-order correlation coefficient with exact p-values reported for significance testing. Diagnostic accuracy in calculating the salivary Ki-67 concentrations for the predictive value was determined using receiver operating characteristic (ROC) analysis, and a z-test was used to assess the statistical significance of the AUC. The significance level was set at p < 0.05 for all tests. 

An a priori analysis was done to calculate the sample size using two-tailed t-tests with an effect size of 0.8 and α error probability of 0.05, power (1-β error probability) of 0.8, allocation ratio N2/N1 equaling 1 gave a non-centrality parameter δ = 2.884 and critical t = 2.008 with a differential of 50. This required a sample size of 26 per group, i.e., a total of 52 with an actual power = 0.807. Therefore, 52 study volunteers were equally divided into two groups: group 1 (Health)-systemically and periodontally healthy ( n = 26) and group 2 (Periodontitis)-periodontitis patients who were systemically healthy ( n = 26). 

Individuals visiting the postgraduate clinic of Periodontology, the College of Dental Medicine at the University of Sharjah and the Clinics of the University Dental Hospital Sharjah were included in the study. An initial exam, including medical, and dental history, and clinical and radiographic examination was done to evaluate participant eligibility for recruitment in the study. Inclusion criteria were volunteers in the age group of 20 to 55 years who were systemically healthy. Health group: participants with absence or minimal bleeding on probing (BoP) < 10%, no CAL, no redness, no probing depth of more than 3 mm and no clinical swelling/edema or pus, and no bone loss evidenced in radiographs. Periodontitis group: Participants having generalized periodontitis (based on the 2017 classification) , stages II, III, IV, grade B, probing pocket depth (PPD) > 4 mm, CAL ≥ 3 mm, with BoP, and bone loss evidence on the radiographs. Exclusion criteria were individuals who were immunocompromised or with systemic diseases, requiring prophylactic antibiotics before dental treatments, tobacco smokers/smoke-less tobacco users, pregnant/breastfeeding/menopausal females, patients who underwent previous periodontal therapy and/or administered antibiotics/ anti-inflammatory agents in the last 3 months. All the clinical parameters were recorded by one periodontist after intra-examiner calibration was found to be acceptable (kappa statistic value of 0.89, 95% agreement). The following parameters were recorded on the whole dentition. Full mouth plaque (oral biofilm) score (Pl)was assessed wherein six sites per tooth were evaluated for the presence or absence of supragingival plaque using Plaqsearch ( Oraldent Ltd., Huntingdon, UK) two-tone disclosing solution. The presence  / absence (−) of plaque was recorded and  was calculated as a percentage . A visual assessment of gingival changes was done to measure the severity of inflammation using the modified gingival index (MGI) . Using a scale of 0–4, the color, texture, edema, and spontaneous bleeding of marginal and papillary gingiva were scored. Then the average score was taken. PPD, CAL measurements (recorded at six aspects per tooth: mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual) were rounded off to the nearest mm using a periodontal probe (UNC 15, Hu-Friedy ® Manufacturing Inc., Chicago, IL, USA). BoP was calculated as a percentage of sites examined. 

The saliva collection procedure followed recommendations in the literature . Before the collection of saliva, the participants in the study were told to refrain from foods that may be highly acidic or sugary, avoid caffeine, not use any medications/mouthwash, nor consume alcohol (for a minimum of twelve hours before the collection of saliva), no major meal (before one hour of saliva collection), no toothbrushing (for at least forty-five minutes prior to collection of saliva), no dental procedure done on them in the past twenty-four hours, as this may affect the assay. The saliva samples were collected at a pre-defined time of the day (8:00 a.m. to 10:00 a.m.) from every participant after rinsing with saline for thirty seconds. Thereafter they were instructed to expectorate in a basin to eliminate any food/debris and wait for ten minutes after rinsing, before saliva collection to prevent sample dilution. After this, the study volunteers were instructed to gently spit unstimulated saliva (approximately 10 to 15 ml) into sterile and plain collection tubes. The samples were then centrifuged for 20 min at 1000×g at 2–8℃. The supernatant was collected and stored at -80℃ to be used for further analysis by the assay. 

Quantitative determination of Human Ki-67 concentrations from saliva was done using a commercially available ELISA kit (Elabscience Biotechnology Inc., Houston, TX, USA; catalog number: E-EL-H5432) per the manufacturer’s instructions. The detection range per the manufacturer is 0.3 ~ 20ng/ml with a sensitivity of 0.19 ng/ml. The concentrations of human Ki-67 in the samples were measured in pg/ml by comparing the optical densities of the samples to the standard curve. 

Categorical data (i.e., sex) were presented as frequencies and percentages and were analyzed using the chi-square test. Numerical data (i.e., marker and periodontal parameters) were presented as mean and standard deviation values. They were explored for normality by viewing the distribution and using Shapiro-Wilk’s test. Data were non-parametric and were analyzed using the Mann-Whitney U test. Correlations were analyzed using Spearman’s rank-order correlation coefficient with exact p-values reported for significance testing. Diagnostic accuracy in calculating the salivary Ki-67 concentrations for the predictive value was determined using receiver operating characteristic (ROC) analysis, and a z-test was used to assess the statistical significance of the AUC. The significance level was set at p < 0.05 for all tests. 

The demographic data/characteristics of parameters are in Table . The study was performed on 52 participants (i.e., 26 in each group). Eighteen (69.2%) males and 8(30.8%) females were in the periodontitis group, and in the health group, there were 11(42.3%) males and 15(57.7%) females. The difference between both groups was not statistically significant regarding sex ( p = 0.094) and age ( p = 0.728). Based on the effect size calculated from the comparisons of marker levels between both groups (d = 1.12) (i.e., large effect size), the achieved power was (0.977), and p -value = 0.001, indicating that the current sample size was adequate. The periodontitis group had significantly higher values of periodontal parameters and salivary Ki-67. Intergroup comparison of Ki-67 (pg/ml) levels is in Table ; Fig. . Subgroup analysis of Ki-67 levels in periodontitis are shown in Table . Correlations between different periodontal parameters and Ki-67 levels are presented in Table ; Figs. , and . For the periodontitis and healthy groups, all correlations were not statistically significant. Overall, there were significant positive weak correlations between Ki-67 and MGI, BoP, PPD, and significant positive strong correlation with CAL. Results of the ROC analysis of the diagnostic accuracy of Ki-67 in periodontitis prediction showed that the area under the curve (AUC) was (0.763 [95%CI (0.624:0.870]) which was statistically significant (z = 3.90, p < 0.001), sensitivity was 61.54, specificity was 88.46 and the cutoff value based on Youden was > 787.67. The ROC curve is presented in Fig. . 

Ki-67 has made its presence as a tumor marker in the past few decades. Ki-67 was discovered in the nuclei of cells in Hodgkin lymphoma. The “Ki” stands for Kiel, Germany, where the identification was done, and “67” for the number of the original clone  Ki-67 has been detected in the nucleus of proliferating cells in active phases of mitosis, however, it is not present in non-proliferating cells and is associated with apoptosis . Ki-67 has been established as an important marker of tumor proliferation and as a prognostic marker . It is a 319–358 kDa protein that is consistently detected based on the number of proliferating tumor cells that help predict the behavior of the tumor . Because Ki-67 is detected and specific to proliferative cells, various carcinomas have been investigated using this marker . About the oral environment, a systematic review and meta-analysis concluded that Ki-67 was higher in odontogenic keratocyst and malignant odontogenic tumors . Ki-67 was significantly observed in the invasive front of oral squamous cell carcinoma . Inflammation is considered a risk factor for various cancers . Also, inflammation induces the proliferation of cells , which may be relatable to a local inflammatory condition like periodontitis that is initiated by a polymicrobial etiology. Inflammation is regarded as a common platform in the association between periodontitis and cancers due to cell proliferation . It has been reported that Ki-67 is expressed lower in health as compared with inflammation and cancers . Ki‑67 has a role in inflammation, wherein its specific antibody has been employed in analyses to identify cells undergoing division and proliferation which is a typical feature of the inflammatory process . Hence, the rationale for evaluating Ki-67 in health and periodontitis is based on the premise that it is a cell proliferation marker and may be higher in inflammatory conditions, although it has been specifically and extensively studied in cancer research. From a few studies available, the literature primarily shows the evaluation of Ki-67 from periodontal tissue specimens/biopsies which are invasive procedures. Immunohistochemistry is widely accepted as the gold standard method to evaluate Ki-67 , but in this research project, saliva was chosen as the source due to the advantage of having a relatively simple, non-invasive collection procedure. One research group concluded that saliva may be more useful than serum for diagnostic and therapeutic reasons . In the current study, a significant difference was seen between the healthy and periodontitis groups for the periodontal parameters assessed, i.e., PI, MGI, BOP, PPD, CAL, and salivary Ki-67 levels. It is challenging to compare our findings as there seems to be no data about salivary Ki-67 in the literature to the best of our knowledge. The literature provides reports concerning the presence of Ki-67 in periodontal tissues. Buduneli et al., , investigated cyclosporine induced-, phenytoin induced-, and nifedipine induced- gingival overgrowth tissue specimens and compared them with oral biofilm (plaque) induced- gingivitis and systemically, periodontally healthy specimens. They noted Ki-67 was expressed significantly higher in phenytoin induced-, nifedipine induced-, followed by cyclosporine induced-gingival overgrowth, gingivitis, and healthy specimen. They found no differences in the proliferating cells and apoptosis between the gingivitis and healthy specimen. According to them, cell proliferation and apoptosis may exhibit an imbalance in drug-induced gingival overgrowth. A direct/indirect effect of the drug cyclosporin on the nuclear factor-kB (NF-kB) has been proposed. They concluded that the effect of the drug causing gingival overgrowth and a concomitant high expression of Ki-67 makes it a reliable marker of tissue growth in non-tumor lesions of the periodontal structures. In another study, Artese et al.,  examined gingival biopsies of the formerly known “aggressive periodontitis”, “chronic periodontitis”, and healthy tissue for vascular endothelial growth factor (VEGF), micro-vessel density (MVD), nitric oxide synthetase (NOS)-1 and, − 3, and Ki-67. The results were a significantly elevated expression of all the aforementioned markers, being highest in “aggressive periodontitis”, higher in “chronic periodontitis” and lowest in the healthy samples. The specific reason for including Ki-67 amongst the selected markers by these investigators was to evaluate the proliferation of endothelial cells and they observed maximum endothelial cell proliferation in the sulcular epithelium of “aggressive periodontitis” based on Ki-67 values. Also, Koulouri et al.,  reported higher numbers of cells (epithelial and fibroblast-like) with Ki-67 expression in granulation tissue of “early onset periodontitis” and “adult periodontitis” when compared with gingival tissue of “adult periodontitis”. Nagarkanti et al.,  detected higher Ki-67 expression in epithelial cells of gingival biopsies of periodontitis patients as compared with healthy specimens. Fanali et al.,  assessed Ki-67, in addition to VEGF, MVD, NOS-1, -3, CD3, CD 20, and CD68 in gingival biopsies immediately below implants welded with titanium bars and compared them with controls and observed higher expression of Ki-67 in tissues of the test group indicating higher proliferative tissue in association with the inflammatory infiltrate. An immunolocalization study by Preethi et al., , noted positive Ki-67 epithelial cells to be highest in a group of smokers with periodontitis, followed by periodontitis, smokers without periodontitis, and, lowest in health. These authors stated that the elevated expression of Ki-67 in smokers is due to the effect of tobacco products on cell proliferation and also, regenerative compensation for loss of cells and tissue because of tobacco products. Kranti et al., , reported higher Ki-67 expression in groups of patients with periodontitis and, periodontitis with controlled type 2 diabetes mellitus as compared with health. The above-mentioned studies indicate a role for Ki-67 in periodontal diseases. It also seems that Ki-67 expression may not necessarily be restricted to conditions such as cancer which has extremely high proliferative cells, but also in drug-influenced gingival overgrowth that has increased rates of proliferative cells. Tissue expression of Ki-67 in different forms of periodontitis, including periodontitis with type 2 diabetes mellitus, implies a proliferative status of the inflamed periodontal tissues. Concerning our investigation, saliva has proven to be a reliable source of Ki-67 where it was significantly increased in periodontal disease samples as compared to health. The general objective of evaluating and comparing salivary Ki-67 levels in periodontitis and health was achieved. With regard to the specific objectives of the levels of salivary Ki-67 and severity of periodontitis, an overall significant strong positive correlation was noted with CAL. No significant correlations were arrived at for the periodontitis group per se. The diagnostic accuracy of the Ki-67 concentrations based on ROC analysis stands at a cut-off value of > 787.67 pg/ml. The discussion now leads to the reasons why Ki-67 is elevated in salivary samples of periodontitis patients. It may be explained based on the factors/mediators that may upregulate Ki-67 in individuals with periodontitis. As the current investigation did not evaluate any other potential upregulators of Ki-67, some reports to validate the possibility are cited herewith. Platelet-derived growth factor (PDGF) is associated with the elevation of Ki-67 , and Pinheiro et al., , have observed high concentrations of PDGF in inflamed gingival tissues as compared with healthy controls. Nitric oxide is another mediator that upregulates Ki-67 , and it was reported earlier that elevated nitric oxide is evident in gingivitis and periodontitis . Gurkan et al.,  have reported elevated transforming growth factor (TGF)-β in the gingival crevicular fluid (GCF) and gingival tissues of periodontitis patients. TGF-β in turn is associated with upregulating Ki-67 . Hepatocyte growth factor (HGF) is also involved in the upregulation of Ki-67 and is elevated in the GCF of periodontitis cases . The aforementioned literature provides insights regarding the mediators that may be responsible for the upregulation of Ki-67. It is to be noted that these mediators have been studied in periodontal tissue and GCF samples. From the perspective of our study that examined Ki-67 in saliva, the reasons for its increased levels in periodontitis may also be due to higher salivary concentrations of those mediators that are closely related to the upregulation of Ki-67. Some of the studies regarding such mediators were detected in the saliva of periodontitis. A systematic review and meta-analysis by Chen et al.,  concluded that nitric oxide in the saliva is elevated in periodontitis patients. Similarly, PDGF is elevated in the saliva of periodontitis patients as per Wu et al., . Another interesting result put forth by Khalaf et al., , shows elevated levels of TGF-β in not only the saliva of periodontitis patients, but also in serum. HGF has also been shown to be higher in the saliva of periodontitis patients . Also, Kim et al., , have shown increased concentrations of salivary IL-1β and TNF-α (amongst others). It is known that these cytokines activate NF-kB that mediates damage to periodontal tissue . NF-κB (p50/p65) and IκB in gingival tissues , and receptor activator of NF-kB (RANK)/RANK ligand (RANKL) in saliva have been reported to be elevated in periodontitis . Elevated NF-kB may upregulate the Ki-67 levels and speed up cell proliferation. As there is an interaction between Ki-67 and NF-kB , it may be hypothesized that the higher salivary Ki-67 levels in periodontitis may also be mediated by NF-kB. Although our study did not evaluate these mediators, it is clear from the literature that potential Ki-67 upregulators are increased in the saliva of individuals who have periodontitis. Therefore, as per our results that show an elevated salivary concentration of Ki-67 in periodontitis, it is implied that it is because of the association of Ki-67 with these elevated mediators. This study had the primary objective of detecting and comparing Ki-67 in saliva of health and periodontitis which was achieved. However, the limitations are lack of a larger sample size, and that it was not longitudinal. Also, it did not include evaluation of markers related to Ki-67 that could have clarified the mechanism of action of Ki-67 vis-à-vis RANK/RANKL pathway. The inclusion of comorbidities in this study may have broadened the scope of understanding the influence of such conditions on salivary Ki-67 levels. Hence, this study has been able to provide cross-sectional information regarding salivary Ki-67 restricted to clinical inflammatory periodontal factors. Investigations evaluating inflammatory biomarkers from saliva and putative periodontal pathogens from the oral biofilm have reported noteworthy effectiveness in the diagnosis of periodontitis . A recent systematic review  has concluded that dual biomarker combinations show excellent diagnostic ability in diagnosing periodontitis. Our study may have provided better evidence by including related inflammatory mediators and microbial samples. Generally, our investigation shows that salivary Ki-67 is elevated in periodontitis compared to health. Confirmation of this observation needs future research involving assessment of salivary Ki-67 before and after periodontal therapy with a larger sample size. Furthermore, concomitant evaluation of markers closely associated with Ki-67 may provide clarity about its behavior. 

Salivary Ki-67 is a good indicator of periodontitis and may provide a pathogenic perspective and serve as a useful biomarker. This study also suggests the use of saliva as a reliable source of Ki-67 in future investigations. 

