Lymphocyte B and Th17 chemotactic cytokine levels in peri-implant crevicular fluid of patients with healthy, peri-mucositis, and peri-implantitis implants

20 ISSN Print 0719-2460 ISSN Online 0719-2479. www.joralres.com/2020 Abstract: Peri-implantitis is one of the leading causes of implant failure and loss, and its early diagnosis is not currently feasible due to the low sensitivity of currents methods. In the current exploratory cross-sectional study, we explored the diagnostic potential of lymphocyte B and Th17chemotactic cytokine levels in peri-implant crevicular fluid (PICF) in 54 patients with healthy, peri-mucositis, or peri-implantitis implants. Periimplant crevicular fluid was collected, and the levels of the molecules under study were quantified by Luminex assay. The concentrations of CCL-20 MIP3 alpha, BAFF/BLYS, RANKL and OPG concentration in PICF were analyzed in the context of patient and clinical variables (smoking status, history of periodontitis, periodontal diagnosis, implant survival, suppuration, bleeding on probing, periodontal probing depth, clinical attachment level, mean of implant probing depth, and plaque index). Patients with peri-implantitis, appear to have an overregulation of the RANKL/BAFF-BLyS axis. This phenomenon needs to be investigated in depth in further studies with a larger sample size.


INTRODUCTION.
Peri-implantitis (PI) is one of the leading causes of implant failure and loss. It initiates with an imbalance between the bacterial biofilm and the host response at the implant surrounding mucosa, which triggers an immune-inflammatory reaction known as perimucositis and peri-implantitis. 1 The risk factors for peri-implantitis are previous history of periodontitis, smoking, diabetes, poor plaque control, and lack of regular maintenance therapy. 2 In clinics, the diagnosis of peri-implant mucositis and peri-implantitis is mostly carried out by clinical and radiographic examination.
Clinical symptoms can include bleeding on probing (BOP), increased probing depth (PD) and suppuration. Radiographic examination aids by determining bone loss. However, the sensitivity of these methods is quite low, with detection only possible once tissue damage has already occurred. Therefore, efforts are being placed on developing effective complementary diagnostic tools to aid the clinical and radiographic screening. Overall, early and precise diagnosis of periimplant diseases remains difficult and even more, the treatment protocol is not predictable, is still under revision, and not universally established.
One of the current approaches is the analysis of biomarkers in the peri-implant crevicular fluid (PICF). Biomarkers are characteristics that can be objectively measured and evaluated, which are indicators of a normal or pathological biological process, and that can be quantified with reproducibility. In recent years, several relevant biomarkers for PI have been described including interleukin-1 beta (IL-1β), plasma tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-8 (MMP-8), receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG). 3, 4 These molecules and others, such as lymphocyte B and T chemotactic cytokine (CCL-20 and BAFF/ BLYS), which are the main producers of RANKL in PI inflamed tissues, have important roles in modulating inflammatory responses, and their expression in the PICF promotes osteoclastogenesis, 3,4 which is one of the main characteristic of PI.
Based on this approach, in which we aim to develop tailored strategies for the early diagnosis and treatment of peri-implant diseases, we need to take advantage of oral fluids as a source of biomarkers to detect the risk of peri-implantitis during the early and asymptomatic stages. Furthermore, the study of biomarkers in oral fluids could represent a promising part of new predictive and diagnostic tools that combine peri-implant parameters, genetics, clinical data, and risk behaviors of the patient.
The present study aimed to explore the diagnostic potential of lymphocyte B and Th17-chemotactic cytokine levels in peri-implant crevicular fluid (PICF) of patients with healthy, peri-mucositis, and periimplantitis implants.

Study design
An exploratory cross-sectional study was conducted in the Health Care Centre of the Universidad de Los Andes and the Universidad de la Frontera, in Santiago and Temuco, respectively, Chile.
Fifty-four patients were enrolled, and their clinical, physical and periodontal data was recorded. Among the recruited patients, 17 were diagnosed as healthy implants (H), 19 patients with implants with periimplant mucositis (PM) and 18 with peri-implantitis (PI).
All subjects were systemically healthy (both sexes) and were aged between 30-78 years old. Complete full-mouth periodontal examinations were performed by a periodontist, including bleeding on probing, peri-implant probing depth, clinical attachment loss and plaque index. Exclusion parameters for the study were: chronic inflammatory diseases (diabetes, cardiovascular, chronic inflammatory, autoimmune and infectious diseases), systemic or topical antimicrobial/ anti-inflammatory therapy for the previous three months. After a periodontal and peri-implant exam, PICF samples were collected.
All clinical data for the study were recorded and after, the patients were derived to peri-implant therapy. This study was approved by the Universidad de La Frontera Scientific Ethics Committee. All patients participating in the study consented to participate by signing the appropriate informed consent form.

Diagnostic Criteria
Peri-implant health was diagnosed as absence of swelling, bleeding on probing, inflammation and suppuration; besides the absence of increased probing depth and the absence of radiographic bone loss. 5 On the other hand, peri-mucositis was described as inflammation of the peri-implant soft tissues, without bone loss, but with bleeding on probing, swelling, and suppuration in some cases. 6 Finally, peri-implantitis was characterized by the presence of inflammation, as in peri-mucositis, but with the presence of progressive bone loss. 1-7 Peri-implant crevicular fluid Collection and elution of PICF samples: Samples were collected using Periopaper TM strips (Oraflow, Smithtown, NY, USA). Briefly, the supragingival plaque was removed using curettes without contacting the marginal gingiva, and the gingival sulcus/pocket was then dried gently with an air syringe. Strips were inserted 3-5mm into the sulci/pockets for 30 seconds. Strips contaminated by saliva and/or blood were discarded.
Samples were then stored in 1.5mL tubes at -80ºC until elution. For elution of PICF, four strips per implant were placed into a 1.5ml tube containing 160 µL of phosphate buffer saline (PBS) (Corning, Mediatech Inc, NY, USA) and protease inhibitor cocktail (EDTA Complete™, mini, EDTA-free Protease Inhibitor Cocktail, Roche, USA). Tubes were vortexed and incubated on ice for 30 min, and then centrifuged at 12,000 x g for 5 min at 4°C.
The eluate was collected and placed on ice. The elution procedure was repeated and both eluates were pooled and stored at -80°C until analysis.
Luminex Assay CCL-20 MIP-3 alpha, BAFF/BLYS, RANKL and OPG concentrations in PICF samples were quantified using a custom-designed multiplex Luminex assay kit. Samples were analyzed using the multiplex assay, according to manufacturer instructions.
All samples were analyzed by duplicate. Briefly, the re-suspended microsphere cocktail (50 μl) was added to each well of a 96-well black plate. PICF eluate (50 μl) was added to each well.
The plates were carefully covered with an aluminum foil plate sealer and incubated at room temperature for 2 hr in a horizontal orbital plate-agitator at 800 ± 50 rpm.
Plates then were placed in a specially designed magnet plate holder for 1 min and the liquid was discarded. Each well was washed with 100 μl of wash buffer for 1 min in the magnet plate and then the liquid was discarded again. Biotin antibody cocktail (50 μl) was added to each well, the plate was sealed and incubated at room temperature with agitation for 1 hr.
Each well was then washed with wash buffer as previously described and diluted Streptavidin-PE (50 μl) was added. The plate was incubated for 30 min, as previously described. A new wash was performed, followed by a re-suspension of the microspheres in 100 μl of wash buffer and incubation for 2 min.
Finally, samples were detected using MAGPIX® System. The final concentration of the samples was calculated using a Milliplex Analyst (version 5.1; Merck KGaA, Darmstadt, Germany) Statistical Analysis By implant status, categorical variables were described through frequencies and percentages, and continuous variables were described through the median and interquartile range. Comparisons between biomarkers levels were explored using the Kruskal-Wallis test .
A p-value<= 0.05 was considered statically significant. The analysis was performed using STATA software (version 15.1; StataCorp, College Station, Texas, US).

RESULTS.
A total of 54 patients were recruited in the present pilot study, 17 healthy subjects, 19 patients with perimucositis, and 18 with peri-implantitis. Age, sex, smoking status, mean of periodontal probing depth, mean of clinical attachment level and suppuration did not differ significantly between healthy (H), perimucositis (PM) and peri-implantitis (PI) patients.

DISCUSSION.
A biomarker or "biological marker", refers to a broad  osteoclasts represents its secondary function. 12 In this context, the design of clusters of biomarkers could be necessary to determine the presence of the pathology, using biomarkers derived from the peri-implant biofilm, and from the destruction process of soft and hard tissues. 13 According to our results, no statistically significant differences were found in the levels of CCL20 These same authors suggest that the long use of inflammatory drug in patients with rheumatoid arthritis and osteoporosis, together with increased TNF-family cytokines might suggest that these patients are more likely to overproduce these inflammatory mediators.
However, whether this results from greater disease activity or contribute to greater disease activity remains moot. 16 Studies conducted in ligation-induced periodontitis in mice with B-cell deficiency compared to wild type controls, reported that both BAFF and APRIL support the survival of B-cells expressing RANKL, thus contributing to bone loss in the course of chronic periodontitis. 17 In this same context, in a study conducted in a cohort of patients with chronic and aggressive periodontitis, increased levels of BAFF in serum and saliva were found versus periodontally healthy patients. 18 However, results of BAFF expression in peri-implant diseases in humans have not yet been reported. The preliminary results of this study suggest that increased levels BAFF in PICF could be associated with the chronic inflammatory process of the peri-implant supporting tissues.

CONCLUSION.
Patients with peri-implantitis, apparently have an overregulation of the RANKL/BAFF-BLyS axis, which should be investigated in depth in further studies with a larger sample size.