The present clinical study was approved by the Ethics Committees of Tokushima University Hospital (nos. 2368 and 2719) in accordance with the Helsinki Declaration of 2013 and performed from November 2016 to August 2017. Patients who received dental implants from 3 to 9 years ago, had healthy or diseased implants with peri-implant diseases, and visited at Tokushima University Hospital for the maintenance of dental implants and treatment were recruited for the present clinical study. Thirty-five patients (10 males and 25 females; aged 68.7 ± 6.5 years) gave written informed consent after receiving an explanation of this study (Table 1). Participants with healthy and diseased dental implants did not have any systemic inflammatory diseases or a history of antibiotic therapy within 3 months. PD, BOP, and gingival index (GI) were examined as clinical indicators after the collection of PICF. GI scores were evaluated according to modifications of the standard of Löe and Silness [29]. The BL rate of alveolar bone was assessed on radiographic films according to modifications of Schei et al.’s method [30]. Diseased sites with peri-implant diseases were defined as periodontal sites with PD ≥ 3 mm, BOP negative or positive, and GI score ≥ 1. Healthy implant sites were defined as sites with PD < 3 mm, BOP negative, and GI score = 0.

PICF samples were collected from peri-implant sites using sterile paper strips according to a modified procedure of our previous method [31]. Briefly, PICF sampling sites were isolated with cotton rolls, supra-gingival plaque was removed, and sites were then very gently air-dried. Periopaper® (Oraflow Inc., NY, USA) was gently inserted into a peri-implant crevice and held for 30 s. The volume of PICF was measured using a Periotron® 8000 (Harco Electronics, Winnipeg, MB, Canada). Paper strips containing blood and pus were not used in the present study. PICF in the paper strip was extracted in 100 μl of phosphate-buffered saline (pH = 7.4) containing 0.2 μM phenylmethylsulfonyl fluoride by centrifugation and used in ELISA for calprotectin and NTx.