Levels of procalcitonin in saliva and peri-implant crevicular fluid in patients with peri-implant diseases and health Article in Press: Accepted Manuscript Ahmed Algohar and Ali Alqerban Archives of Oral Biology, Article 104931, Copyright © 2020 Highlights Peri-implant diseases reported higher procalcitonin in saliva and crevicular fluid. Crevicular procalcitonin was correlated with clinical parameters in peri-implantitis. Crevicular procalcitonin was correlated with bleeding in peri-implant mucositis. Abstract Objective To evaluate the levels of procalcitonin in saliva and peri-implant crevicular fluid (PICF) among healthy and peri-implant disease patients and correlate these levels with clinical and radiographic peri-implant parameters. Design Three groups of 20 participants each [Group-1: healthy, Group-2: peri-implant mucositis, and Group-3: peri-implantitis] were selected. Peri-implant plaque index, bleeding on probing, probing depth and crestal bone loss was assessed. PICF and saliva samples were evaluated for procalcitonin levels and analyzed using enzyme-linked immunosorbent assay. Kruskal-Wallis test was performed for comparisons among the study groups. Multiple comparisons were considered for Post hoc two-group comparisons using Bonferroni-corrections. The Spearman rank correlation coefficient analysis was used to analyze the correlation between procalcitonin levels of both fluids and clinical peri-implant parameters. Results Group-3 demonstrated significantly higher values for peri-implant plaque index, bleeding on probing, probing depth, and crestal bone loss as compared to Group-1 and Group-2. Participants of both Group-2 and Group-3 reported significantly increased procalcitonin levels in saliva and PICF in comparison to Group-1. Significant positive correlations were found between PICF procalcitonin levels and bleeding on probing, probing depth, and crestal bone loss in Group-3 and significant positive correlation was found between PICF and bleeding on probing in Group-2. For salivary procalcitonin levels, a significant positive correlation was observed between procalcitonin and bleeding on probing in Group-3. Conclusions The outcome of this study suggests that procalcitonin might play a role in peri-implant inflammation, and higher procalcitonin levels is suggestive of a probable surrogate biomarker for peri-implant diseases. 1 Introduction With the advancement of science and dental technology, new and improved methods have been introduced to improve modes of treatment for individuals with dental problems. Similarly, individuals who are associated with the problem of missing teeth or edentulous spaces, dental implants are considered as a safe and convenient treatment modality ( ; ). Keeping the peri-implant disease into perspective, peri-implant mucositis and peri-implantitis are the two most notable problems which have been documented due to increased frequency of dental implant placements in dental and hospital practices ( ). Peri-implant mucositis is defined as the visible inflammation of the mucosa around a functional dental implant with bleeding on gentle probing being the main clinical finding. Moreover, it is also associated with increased probing depth, erythema, and/or suppuration. Histologically, the affected site shows an inflammatory infiltrate rich in lymphocytes and plasma cells ( ). On the other hand, peri-implantitis is a plaque-associated pathological condition which is observed in the surrounding tissues of the dental implant, associated with peri-implant mucosal inflammation and subsequent loss of bone. The clinical characteristics include erythema, increased probing depths, bleeding on probing with or without suppuration, recession of the mucosal margins, and radiographic bone loss ( ). Peri-implantitis can be classified as mild, moderate, and severe by co-relating the assessed probing depths and active bone loss, as both are termed as indicators of peri-implantitis ( ; ). With respect to the histological features, the lesions of peri-implantitis show an apical extension of the soft tissue epithelium that is associated with large numbers of plasma cells, macrophages, and neutrophils. The main risk factor for peri-implant mucositis is plaque accumulation, whereas, peri-implantitis includes factors such as improper plaque control and maintenance, history of severe periodontitis smoking, and genetics ( , , ; ). It is suggested that the estimation of inflammatory biomarkers in several biological fluids is a valuable surrogate indication of the inflammatory process and it has been shown that the evaluation of gingival crevicular fluid or peri-implant crevicular fluid (PICF) from the peri-implant pocket is suitable for assessing periodontal and peri-implant diseases ( ). Saliva is a combination of complex compounds which are secreted by the mucous and salivary glands of the body ( ). It is composed mainly of proteins and other biomolecules which are responsible for maintaining the oral health of the individual. Saliva helps in various other functions including digestion, enzyme activation, lubrication, and buffering. Saliva is not only responsible for physiologic functions but is also a very good tool in assessing and diagnosing different oral and systemic diseases such as caries risk, periodontal disease, Sjogren syndrome and cancers ( ; ; , ). The severity of the disease can also be found out through salivary analysis. As it is reported in studies that salivary biomarkers such as elastase, interleukins, matrix metalloproteinases, C-reactive proteins are closely related to periodontal disease ( ; ), it can be speculated that the disease progression encountered during periodontal disease is somewhat similar to what is seen in peri-implant disease. Procalcitonin has been identified as one of the elevated proteins in response to bacterial infections and tissue injury ( ). It is a peptide precursor of hormone calcitonin protein which is involved mainly in balancing the calcium levels in the body ( ; ). Under physiologic conditions, procalcitonin is released from the C-cells of the thyroid gland and neuroendocrine cells of the lungs, in response to bacterial toxins and pro-inflammatory mediators such as interleukin-6, interleukin-1β and tumor necrosis factor-α, respectively ( ; ). Over the years, procalcitonin has been used in several clinical algorithms to reduce the excessive use of antimicrobials ( ). Regarding the detection of bacterial infections, it has emerged as a reliable biomarker ( ). The levels of procalcitonin in biological fluids may also serve as a useful indicator in diagnosing and determining the degree of several infectious cases such as sepsis, acute pancreatitis, bacteremia and bacterial meningitis ( ). Similarly, procalcitonin levels can also play an important role in the detection of inflammatory diseases in the human population ( ). After a thorough analysis of the present literature, the authors observed that no study has evaluated the salivary and PICF procalcitonin levels in peri-implant disease and health. The authors hypothesize that procalcitonin levels in the saliva and PICF may be increased in peri-implant disease as compared to healthy controls, and peri-implantitis would show higher levels in comparison to individuals with peri-implant mucositis. Therefore, the aim of our study was to investigate the procalcitonin levels in saliva and PICF of participants with peri-implant mucositis, peri-implantitis, and peri-implant health and correlate these levels with the clinical and radiographic peri-implant parameters. 2 Materials and Methods 2.1 Study Population A total of 60 non-smokers divided into three groups of 20 participants each [Group-1: healthy, Group-2: peri-implant mucositis, and Group-3: peri-implantitis] were selected. All participants were recruited from the Department of Prosthodontics, College of Dentistry, Dar Al Uloom University, Riyadh, Saudi Arabia, from December 2018 to November 2019. The present study was approved by the Medical Ethics Committee of Prince Sattam Bin Abdulaziz University, Riyadh, Saudi Arabia with protocol number: PS-000/18 and performed in accordance with the Declaration of Helsinki. Before participation, all participants were informed about the study objectives and procedures, and all participants gave written informed consent. Patients were excluded on the following criteria; any systemic diseases such as diabetes, rheumatic, thyroid disease, liver disease or immunological disease; oral or systemic bacterial sepsis; participants receiving antimicrobial therapy or any form of periodontal treatment in the previous 6 months; pregnancy; and current or former tobacco user ( ). Dental implants from each included participant were assessed clinically as well as radiographically to ascertain the suitability of the participants for the study ( ). The participants of the study had to have ≥16 teeth ( ). 2.2 Power analysis A total of 60 participants (n = 20 in each group) were selected for the present study considering a 50% difference in mean salivary procalcitonin with a total of 85% power, and alpha value set at 0.05 ( ). 2.3 Groups The selected individuals were stratified into three groups based on their peri-implant disease and conditions in accordance with the criteria selected at the 2017 World Workshop on the Classification of Periodontal and Peri‐Implant Diseases and Conditions (Bergundh et al., 2018). Group-1: (Healthy): These individuals represented with no clinical signs of inflammation, no increase in probing depth, absence of bleeding on probing and no bone loss beyond the crestal bone level after initial bone remodeling. Group-2 (peri-implant mucositis): The selected individuals showed bleeding on probing and/or suppuration on gentle probing with or without increased probing depth. Whereas no bone loss beyond the crestal bone level was observed after initial bone remodeling. Group-3 (peri-implantitis): The individuals in this group exhibited the presence of bleeding and/or suppuration on gentle probing with probing depth of ≥6 mm and radiographic level of bone ≥3 mm apical of the most coronal portion of the intraosseous part of the implant after initial bone remodeling. 2.4 Evaluation of peri-implant clinical parameters The clinical evaluation of all the study variables was done by a trained examiner (AAQ). Before conducting any clinical assessments, the examiner recorded clinical peri-implant probing depth in five patients (not included in the statistical analysis). The measurements were repeated after 1 hour of the initial assessment. Kappa scores were measured which showed excellent reliability within the examiner (к = 0.83 [83%]). Peri-implant plaque index, probing depth, and bleeding on probing were determined at four sites per implant. Using a manual periodontal probe (UNC-15 Hu-Friedy, Chicago, Illinois). Probing depth was defined as the total distance measured from the gingival crest of the margin to the base of the periodontal pocket. Whereas, the parameters plaque index and bleeding on probing reported dichotomous scoring to four sites of each implant as ‘0 – absent plaque/bleeding’ and ‘1 – present plaque/bleeding’, respectively. 2.5 Radiographic assessment of crestal bone levels Digital periapical radiographs were taken from all the participating individuals. The assessment of the radiographic films was done on a standardized computer display (Samsung SyncMaster digital TV monitor, Korea) with the help of a software (Image Tool 3.0 Program, San Antonio, Texas) ( ). The assessment of digital radiographs was carried by a calibrated examiner (AA). The crestal bone loss was defined as the linear distance measured from the implant-abutment junction to the most coronal point of the alveolar crest. 2.6 Collection and estimation of salivary procalcitonin The study volunteers were advised to avoid drinking and eating in the morning. The unstimulated saliva samples were acquired by requesting the participants to expectorate into sterile polypropylene tubes. Saliva was pooled to assess the unstimulated whole saliva flow rate in milliliters per minute (ml/min). By using the centrifugation method, the saliva samples were separated for debris. The centrifugation was done at 6000 rpm for a duration of 10 minutes at 40 °C. For the removal of debris, the clear supernatant was introduced into the Eppendorf tubes and was stored at a temperature of -80 °C until the laboratory analysis was performed. The assessment of salivary procalcitonin was performed using an enzyme-linked immunosorbent assay procalcitonin kit. 2.7 Collection and estimation of PICF procalcitonin Participants were instructed to refrain from eating, drinking, chewing gum, and brushing their teeth in the morning of the sample collection. PICF samples were obtained by inserting paper cones inside the peri-implant pocket and transferred into polypropylene tubes; clinical peri-implant measurements and any necessary interventions were then performed. Crevicular fluid was pooled to assess the PICF flow rate in microliters per minute (µl/min). Debris was separated from the supernatant by performing centrifugation at 6,000 rpm for 10 minutes at 4 °C, the clear supernatant was transferred to micro vials and stored at −80 °C until further analysis. Samples were analyzed using an enzyme-linked immunosorbent assay procalcitonin kit. 2.8 Laboratory assessment of salivary and PICF procalcitonin All laboratory assessments were performed by a trained laboratory technician (MK) who was not involved in the clinical assessments (к = 0.77 [77%]). All samples were made blinded and masked for the laboratory technician. Before using the reagents, they were equilibrated at room temperature (18-25 °C). Fifty millilitres wash buffer was prepared by combining 5 ml wash buffer PT with 45 mL of distilled water. To prepare the antibody cocktail, the capture and detector antibodies were gently mixed in Antibody Diluent. The standards were prepared according to the instruction manual provided by the manufacturer. The assay procedure was initiated by adding the samples of saliva, PICF, and 50 μL of each standard in the respective wells. After the addition of the detection antibody (50 μL) in all the wells, the specimens were incubated for 60 min. at room temperature. The wash buffer was used to rinse the plates thoroughly. 100 μL of the conjugate solution was incorporated into the wells before subjecting the specimens to incubation for 15 min.. Following the incubation period, the plates were subjected to rinsing and 100 μL of the substrate was incorporated in each well. These specimens were again sent for incubation in a dark room for 15 min. at room temperature. The termination of the reaction was done by adding 100 μL Stop Solution. The spectrophotometer was used to record the optical density at 450 nm. Furthermore, the range of detection was set at 6.25-400 pg/mL. 2.9 Statistical Analysis All statistical tests were analyzed using a specialized statistical software (SPSS for Windows v.15, IBM, Chicago, IL, USA). Data were run for normality distribution using Shapiro-Wilk test and homogeneity of variances using inferential statistic Levene’s test. For statistical analysis, Kruskal-Wallis test was performed for comparisons among the study groups. On statistically significant p-values (<0.05), post hoc two-group comparisons using Bonferroni-corrected Mann-Whitney U tests were employed. The correlations between procalcitonin levels in both saliva and PICF and peri-implant clinical parameters were analyzed using Spearman rank correlation coefficient analysis. P-values <0.05 were considered significant. 3 Results A total of 60 individuals were selected in the study, where each study group comprised of 20 participants. Both the genders participated in the study where Group-1 comprised of 7 males and 13 females, Group-2 had 9 males and 11 females, whereas, Group-3 included 12 males and 8 females, respectively. The mean age for individuals in Group-1, 2, and 3 was 34.4, 37.6, and 42.5 years, respectively. A total of 32 implants in Group-1, 27 in Group-2, and 35 in Group-3 were evaluated. The mean duration of the implants for Groups -1, -2, and -3 ranged from 38.6, 44.9, and 51.8 months, respectively. A total of 54 maxillary implants were assessed, whereas a total of 40 implants were evaluated in the mandible ( Table 1 ). Table 1 Baseline demographic, implant-related characteristics, and oral hygiene care of the study groups. Characteristics Group-1 (Healthy) Group-2 (Peri-implant mucositis) Group-3 (Peri-implantitis) Number of study participants ( n ) 20 20 20 Gender (Male/Female) 7/13 9/11 12/8 Mean age in years (±SD) 34.4 ± 4.2 37.6 ± 4.8 42.5 ± 6.4 Total number of implants included 32 27 35 Implant position (maxilla/mandible) 21/11 14/13 19/16 Duration of implants in months (mean ± SD) 38.6 ± 8.4 44.9 ± 11.5 51.8 ± 14.2 Brushing frequency (%) Once daily Twice daily 28 72 80 20 91 9 The clinical and radiographic peri-implant parameters for all the included study groups are described in Table 2 . The peri-implant plaque index and bleeding on probing scores were significantly higher in Group-2 and Group-3 compared to Group-1. A significant increase in peri-implant probing depth scores was observed in Group-3 in comparison to Group-1 (p = 0.001) and Group-2 (p = 0.01). Group-3 demonstrated significantly higher values for peri-implant crestal bone loss as compared to Groups -1 (p = 0.039) and -2 (p = 0.042). Table 2 Plaque index, bleeding on probing, probing depth and crestal bone loss among patients with peri-implant disease and health. Data are expressed in median and interquartile range. Statistical significance between groups analyzed using Kruskal-Wallis test for each independent variable and after Bonferroni adjustment. Peri-implant parameters Group-1 (Healthy) Group-2 (Peri-implant mucositis) Group-3 (Peri-implantitis) Plaque index in % 10.2 (8.3) A 28.4 (22.3) B 34.4 (18.7) B Bleeding on probing in % 9.4 (14.5) A 34.6 (21.8) B 39.2 (14.4) B Probing depth in mm 1.3 (0.6) A 1.7 (0.8) A 4.1 (0.9) B Crestal bone loss in mm 0.7 (0.5) A 1.1 (0.6) A 2.5 (0.9) B Dissimilar superscript letters indicate statistical significance at p<0.05. PICF and salivary procalcitonin values are described in Table 3 . PICF flow rate was significantly higher in Group-2 and Group-3 compared with Group-1 (P < 0.05). Patients with peri-implant disease (Group-2 and Group-3) reported significantly increased values of salivary and PICF procalcitonin levels in comparison to individuals in Group-1 (p < 0.001). However, Group-3 demonstrated significantly higher values of salivary and PICF procalcitonin in comparison to Group-1 (p = 0.0001) and Group-2 (p = 0.001). Table 3 Peri-implant crevicular fluid and salivary flow rate and levels of procalcitonin among patients with peri-implant disease and health. Data are expressed in median and interquartile range. Statistical significance between groups analyzed using Kruskal-Wallis test for each independent variable and after Bonferroni adjustment. Biomarkers Group-1 (Healthy) Group-2 (Peri-implant mucositis) Group-3 (Peri-implantitis) PICF flow rate (µl/min) 0.71 (0.41) A 0.98 (0.53) B 1.06 (0.59) B Salivary flow rate (ml/min) 0.62 (0.27) A 0.67 (0.85) A 0.65 (0.96) A PICF procalcitonin (pg/ml) 7.33 (4.18) A 42.16 (29.72) B 119.86 (74.54) C Salivary procalcitonin (pg/ml) 3.75 (2.34) A 18.08 (11.42) B 49.33 (27.91) C PICF – peri-implant crevicular fluid. Dissimilar superscript letters indicate statistical significance at p<0.05. Spearman rank correlation coefficient analysis was performed to evaluate for any correlations among levels of PICF and salivary procalcitonin, and clinical peri-implant parameters among all study groups ( Table 4 ). In Group-3, a significant positive correlation was observed between bleeding on probing ( P = 0.0128), probing depth ( P = 0.0146), and crestal bone loss ( P = 0.0013) and PICF procalcitonin levels, whereas in Group-2, the correlation was significantly positive between bleeding on probing and PICF procalcitonin levels ( P = 0.0432). In the Group-3, a significant positive correlation was observed between salivary procalcitonin and bleeding on probing ( P = 0.0011). A significant negative correlation was found between PICF procalcitonin and crestal bone loss ( P = 0.0469) in Group-3, whereas a significant negative correlation was observed between salivary procalcitonin and bleeding on probing ( P = 0.0399) in Group-2. Table 4 Spearman rank correlation analysis between peri-implant crevicular fluid and salivary procalcitonin and peri-implant parameters among patients with peri-implant disease and health. Statistical significance analyzed by Spearman rank correlation coefficient analysis. Peri-implant parameters Group-1 Group-2 Group-3 (Healthy) (Peri-implant mucositis) (Peri-implantitis) PICF Procalcitonin Plaque index Correlation coefficient 0.7894 -0.6739 -0.5745 P- value 0.3664 0.2573 0.4573 Bleeding on probing Correlation coefficient 0.8342 0.0643 * 0.1194 * P- value 1.8475 0.0432 0.0128 Probing depth Correlation coefficient 0.9643 0.7868 0.0732 * P- value 0.4387 0.5985 0.0146 Crestal bone loss Correlation coefficient -0.5734 -0.1034 * 0.3523 * P- value 0.8879 0.0469 0.0013 Salivary Procalcitonin Plaque index Correlation coefficient -0.7402 0.6749 -0.9891 P- value 0.5585 0.7435 0.6623 Bleeding on probing Correlation coefficient 0.4905 -0.1296 * 0.1812 * P- value 1.4886 0.0399 0.0011 Probing depth Correlation coefficient 0.6903 0.6853 0.8945 P- value 1.0925 0.0974 0.1127 Crestal bone loss Correlation coefficient -0.8745 -0.2344 0.6895 P- value 0.9853 0.0533 0.9231 PICF – peri-implant crevicular fluid. * Significant at p < 0.05. 4 Discussion In the present study, the authors analyzed the salivary and PICF levels of procalcitonin in patients with peri-implant diseases and health. The outcome of the current study showed elevated levels of salivary and PICF procalcitonin were observed in patients with peri-implant diseases in comparison to the healthy group, however, patients with peri-implantitis demonstrated significantly worse peri-implant parameters and a positive correlation was found between PICF procalcitonin and bleeding on probing, probing depth and crestal bone loss. C-reactive protein is a systemic biomarker that is released in response to a tissue injury during the acute phase of inflammation ( ). The active production of this biomarker takes place in the liver which is stimulated by cytokines such as tumor necrosis factor, and interleukins-1β, and interleukin-6 ( ). High levels of C-reactive protein have been associated with periodontal and peri-implant inflammation, but procalcitonin is suggested to a better indicator of disease due to its short half-life ( ). In a study done by Giannopolou et al. it was reported that procalcitonin may be suggested as more reliable in diagnosing periodontal infection rather than C-reactive protein ( ). The salivary and PICF procalcitonin both were significantly increased in the group with peri-implantitis as compared to peri-implant mucositis group and healthy group. Since we know that procalcitonin levels increase due to bacterial invasion resulting in the formation of sepsis. The results show higher values of plaque in peri-implantitis group, which supports the fact that an ample number of bacteria are present near the diseased implant site. Bacteria of gram-negative origin especially bacilli and spirochetes invade the peri-implant pocket and allow the release of endotoxins ( ). The endotoxins stimulate the respective receptors to secrete the pro-inflammatory cytokines ( ). Since, these cytokines are responsible for initiating osteoclastenogenesis and altering the expression of procalcitonin ( ), it can be assumed that the presence of increased plaque scores and abundance of cytokines in the saliva and PICF might be responsible for increased procalcitonin levels. A positive correlation between procalcitonin and clinical and radiographic peri-implant parameters (bleeding on probing, probing depth, and crestal bone loss) was observed in patients with peri-implantitis. Due to the increased concentration of plaque inside the peri-implant, the bacterial infiltrate is responsible for the release of enzymes such as matrix metalloproteinases and pro-inflammatory cytokine interleukin-1β. Studies have shown that these mediators are responsible for the periodontal breakdown which includes increase gingiva degradation, alveolar bone resorption, and periodontal attachment loss ( ). Since the procalcitonin regulation is dependent upon the secretion of these cytokines, the authors derive a hypothesis that increased value of procalcitonin might have a role in destabilizing the clinical and radiographic peri-implant parameters (bleeding on probing, probing depth and crestal bone loss). A few limitations can be listed where the number of sample size is a prominent inclusion. More research should be performed where a larger sample size should be recruited through which better results can be anticipated. The periodontal conditions of the peri-implant disease groups (data not shown) would have significantly affected the outcomes of procalcitonin. Salivary biomarkers could distinguish the “high” pro‐inflammatory responders at peri-implant mucositis sites only in subjects without inherent periodontal disease susceptibility. Pro-inflammatory cytokines should have been evaluated. The immunological analysis would have given a better picture regarding the pathological mechanism involving the peri-implant disease site. A thorough analysis of the microorganisms would have provided a clear picture to the authors regarding the involvement of bacteria at the diseased implant site. 5 Conclusion The outcome of this study suggests that procalcitonin might play a role in peri-implant inflammation, and increased procalcitonin levels is suggestive of a probable surrogate biomarker for peri-implant diseases. Ethical Approval The present study was approved by the Medical Ethics Committee of Prince Sattam Bin Abdulaziz University with reference code: PS-000/18. Funding Deanship of Scientific Research Prince Sattam Bin Abdulaziz University ( RSP#2018/44 ). Uncited references ; ; ; . Declaration of Competing Interest The authors report no declarations of interest. 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