Periodontal disease, peri-implant disease and levels of salivary biomarkers IL-1β, IL-10, RANK, OPG, MMP-2, TGF-β and TNF-α: follow-up over 5 years

Abstract Objective The aim of this study was to evaluate the levels of salivary biomarkers IL-1β, IL-10, RANK, OPG, MMP-2, TG-β and TNF-α in individuals with diagnosis of peri-implant mucositis in the absence or presence of periodontal and peri-implant maintenance therapy (TMPP) over 5 years. Material and Methods Eighty individuals diagnosed with peri-implant mucositis were divided into two groups: one group that underwent periodontal and peri-implant regularly maintenance therapy, called GTP (n=39), and a second group that received no regular maintenance GNTP (n=41). Each participant underwent a complete periodontal and peri-implant clinical examination. Collection of saliva samples and radiographic examination to evaluate peri-implant bone levels were conducted at two times: initial examination (T1) and after 5 years (T2). The salivary samples were evaluated through ELISA for the following markers: IL-1β, IL-10, RANK, OPG, MMP-2, TGF and TNF-α. Results A higher incidence of peri-implantitis was observed in the GNTP group (43.9%) than in the GTP group (18%) (p=0.000). All individuals (n=12) who presented peri-implant mucositis and had resolution at T2 were in the GTP group. After 5 years, there was an increase in the incidence of periodontitis in the GNTP group compared to the GTP group (p=0.001). The results of the study revealed an increase in the salivary concentration of TNF-α in the GNTP group compared to the GTP group. The other salivary biomarkers that were evaluated did not show statistically significant differences between the two groups. Conclusions The salivary concentration of TNF-α was increased in individuals with worse periodontal and peri-implant clinical condition and in those with a higher incidence of peri-implantitis, especially in the GNTP group. Longitudinal studies in larger populations are needed to confirm these findings and elucidate the role of this biomarker in peri-implant disease.


Introduction
The infectious-inflammatory disease occurring around implants is known as peri-implant disease (PID) and it may manifest itself as peri-implantitis mucositis or periimplantitis. Peri-implant mucositis (MP) is characterized by inflammatory infectious disease that results in reversible inflammation of peri-implant soft tissues and peri-implantitis by the loss of soft and hard tissues around the implants. 1 Studies have attempted to clarify the role of cytokines in this immuno-inflammatory response, but the literature presents conflicting and scarce data regarding the concentration of potential markers of periodontitis (PE) and peri-implantitis 2,3 (PI) and their role in the progression of these diseases. 4,5 In addition, cytokines, chemokines, enzymes of cellular destruction and the molecules produced as a consequence of tissue destruction in PE and PID are released and can be identified in saliva. 6,7 Recent studies indicate that the use of immunological markers may aid in the diagnosis of health and PID. The advantage of using saliva instead of blood or gingival crevicular fluid analysis is that it is a non-invasive collection method, has high availability, is painless and does not require special equipment for collection. 8 It is a fact that the literature includes numerous The literature highlights that TMPP decreases biological complications and increases the success of long-term implants. [9][10][11] . In this sense, the objective of this study was to compare the salivary concentrations These individuals were divided into 2 groups related to regularity TMPP: one group that performed TMPP regularly, that is, at least one visit/year (GTP=39), and one that did not perform TMPP regularly, that is, less than one visit/year (GNTP=41). The periodontal and peri-implant clinical data of these individuals were previously reported. 13 In T1 and T2, the following parameters for the teeth and implants in periodontal/peri-implant examinations were recorded: clinical attachment level (CAL), periodontal probing depth (PS) and periimplant probing depth (PSi), bleeding on periodontal probing (BOP) and bleeding on peri-implant probing (BOPi), periodontal (PL) and peri-implant (PLi) plaque index. 14,15 In the implants, the presence of peri-implant suppuration (Si) was also evaluated, and radiographic measurements were also conducted to evaluate bone levels. The methodology for collecting these clinical data was described in detail before. 12 Salivary sample collection was performed at the time of clinical evaluations at T1 and T2 and will be described later.
The procedure and the research were explained in detail to each participant, and free and informed consent was obtained. Additionally, all 80 individuals evaluated for periodontal and peri-implant parameters were referred for free periodontal/peri-implant maintenance treatment in each scheduled visit. This study was approved by the Research Ethics Committee under protocol number 05650203000-10.

Inclusion and exclusion criteria
This study adopted the same inclusion and exclusion criteria as the original data published. 12

Results
The diagnosis of periodontal and peri-implant disease of the sample at T1 and T2 are presented in  (Table 3). In addition, all individuals (n=12) who manifested MP at T1 but were presented as healthy at T2 were in the GTP group (Table 4)   concentration of TNF-α was significantly higher in individuals in the GNTP group who developed PI after 5 years. Our findings are in agreement with those of several authors who found increased salivary cytokine levels in cases of PE 18,20,21 and PID. 22 Another aspect of PID and cytokines was the focus of one study. 23 The authors showed that after measuring the concentration of IL-1β in total saliva, saliva of the parotid gland and Notably, the results of our study showed that the individuals who had resolved MP with peri-implant health belonged to the GTP group, and the prevalence of PI in the GNTP group (43.9%) was higher than in the GTP group (18%) (p<0.001). Similar findings were found in the literature 24 , one study 25 reported that in 47 individuals with a history of PE followed for 7.9 years, the prevalence of PI was of 31.9% among the participants who underwent TMPP and 52.2% among those who did not undergo TMPP (p=0.102).
The authors noted that lack of adherence to a regular periodontal/peri-implant maintenance program is correlated with a high incidence of peri-implant bone loss and implant loss. Hence, three main factors may have greatly influenced our findings: smoking, presence of PE and the absence of TMPP. Smoking is considered a major risk factor for PID. 19 In this study, smokers were excluded from the sample in order to minimize this confounding factor in data analysis.
In the GTP group, 12 individuals with MP in T1 were diagnosed as healthy at T2. This result is in agreement with studies on the influence of preventive maintenance in the control of PE and MP. The findings of recent systematic reviews and meta-analysis 14 showed that TMPP is important in preventing and reducing the occurrence of PE and MP. Another systematic review study with meta-analysis stressed the importance of peri-implant maintenance, as it is the best way to prevent PI is MP control. 26 The literature shows that professional follow-up in maintenance consultations prevents the development of PE, which is a risk factor for PI. 27 Another study has shown that TMPP reduces the occurrence of PI in individuals with a history of PE, and the lack of TMPP is correlated with a higher incidence of peri-implant bone loss in individuals with and without a history of PE. 28 According to the literature, 29 the primary objective for avoiding the occurrence of complications with implants is based on a reinforcement of plaque control and reduction of risk factors, such as smoking and adjustment of prostheses, which hinder good local hygiene.
In addition to periodic maintenance, the concentrations of periodontal and peri-implant biomarkers have been studied in an attempt to predict their progression. In one study 20   TNF-α concentration was low at the beginning of PE and increased as it progressed. In the present study, a similarity to the previously mentioned study was observed: the cytokine in question was low in the MP group and increased in the PI implant group. Thus, the TNF-α biomarker was highlighted as a potential tool for the prognosis and progression of PE and PI.
An important aspect of our study related to saliva concentration was discussed in a previous study 30 .
The authors observed that total salivary cytokines may represent only a fraction of the total content in saliva and that cytokines can be negatively affected (diluted) by salivary components (mucin), which decreases the detection power of the ELISA assay.
Another study showed that mucin had already been found to be increased in saliva in cases of chronic PE compared to periodontally healthy cases. 31 The literature showed that, when using ELISA for salivary analysis, a correction must be made by the protein such that the test can be considered reliable. 32 This concern over the influence of salivary protein was considered in our study, since the concentrations of the cytokines were normalized by the salivary protein to attempt to decrease the salivary viscosity effect at the collection time points (T1 and T2).
In addition to the absence of significant differences in the salivary concentration of IL-1β, IL-10 TGF-β and RANK/OPG between T1 and T2, it is worth noting that the hypothesis of this study was finding increased levels of IL-1β, IL-10, TGF-β and RANK/OPG in the GNTP group compared to the GTP group, especially in individuals who developed PI. However, one study has also observed that the salivary levels of this biomarker are not able to differentiate MP from PI. 33