*2.7. Collection of PICF*

PICF collection was carried out as described elsewhere [31]. The mechanical cleaning of the supragingival plaque present on the implant-supported crown was carried out. After the careful isolation of the peri-implant sites using sterile cotton, an air syringe was used for drying purposes. By introducing 1–2 mm of paper points (Periopaper, Pro Flow, Amityville, NY, USA) into the peri-implant sulcus for 30 s, the collection of PICF samples was performed. After discarding the samples contaminated with bacteria, blood, or saliva, the collection of the new specimens was carried out from the same site. An electronic calibrated gingival fluid device (Periotron 6000, Amityville, NY, USA) was utilized to measure the fluid sample volume. The elution of the collected PICF was carried out with 1 mL of phosphate-buffered saline (PBS) prior to freezing at −80 ◦C.

## *2.8. Evaluation of PICF Levels of TNF-α, MMP-1, and IL-8*

Centrifugation of PICF samples was carried out at 15,000× *g* for 15 min. Quantification of the levels of biomarkers was performed using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's guidelines (Quantikine, R&D Systems, Minneapolis, MN, USA) and calculated as pg/mL. A trained investigator (P.A.) carried out all laboratory-based analysis, (i.e., *kappa* = 0.78). In brief, the addition of 100 mL samples and standards were incorporated into the corresponding wells. Then, their incubation was performed overnight at 4 ◦C after covering them properly. Later, to discard the solution, a multi-channel pipette was utilized and cleaned four times using a 0.3 mL 1× wash solution. Post final wash, the removal of the residual liquid was carried out via aspiration. Then, the inversion of the plate was performed and blotted with sterile paper towels. Inclusion of 0.1 mL of 1× Biotinylated anti-Human CRP Detector Antibody to individual wells was carried out. Later, this was incubated at room temperature, (i.e., 25 ◦C) for 60 min. The rewashing of the solution was carried out after disposing it off. Before incubating it at 25 ◦C for 45 min, 0.1 mL of 1× HRP-Streptavidin solution was incorporated into each individual well. The solution was re-discarded and washed again. To each well, 0.1 mL of TMB One-Step Substrate Reagent was incorporated and incubation was carried out at 25 ◦C for half an hour in a dark room. The addition of 50 μL of stop solution to each well was performed before instantly taking the reading at 450 nm. Prior to each individual assay, standard curves were produced and employed for plotting outcomes. For the levels of PICF of TNF-α, MMP-1, and IL-8, the ELISA's sensitivity was 96%, 97.2%, and 96.8%, respectively.
