ATR-FTIR and Raman Spectroscopy of Peri-Implant Crevicular Fluid in Peri-Implantitis

This is an observational, analytical, cross-sectional clinical study with a within-participant split-mouth design. The study aims to characterize peri-implant crevicular fluid (PICF) obtained from peri-implant healthy and peri-implantitis sites using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and Raman spectroscopy.

Adults aged 18 years or older who have at least two dental implants, including at least one implant meeting the criteria for peri-implant health and at least one implant diagnosed with peri-implantitis, will be eligible for participation. Dental implants must have been in function for at least one year. Peri-implantitis will be diagnosed according to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

For each participant, one peri-implant healthy implant and one peri-implantitis implant will be selected for evaluation. If more than one peri-implantitis implant is eligible, the implant with the greatest probing depth and radiographic bone loss will be selected. If clinical findings are similar, random selection will be used. When more than one healthy implant is available, preference will be given to an implant located in the same jaw as the peri-implantitis implant, with random selection used when necessary.

Clinical peri-implant parameters will include plaque index, bleeding on probing, probing depth, suppuration, keratinized mucosal width, and radiographic bone loss. Implant-related characteristics, including time in function and prosthetic retention type, will also be recorded. Participant-level information including age, sex, systemic disease history, smoking status, diabetes status, and body mass index will be collected.

PICF samples will be obtained after isolation of the sampling site and removal of supragingival plaque. Sterile PerioPaper strips will be inserted into the peri-implant sulcus for 30 seconds. Samples contaminated with blood or saliva will be excluded. Collected samples will be stored at -80°C until spectroscopic analysis.

ATR-FTIR spectroscopy will be used to evaluate molecular spectral features associated with proteins, lipids, carbohydrates, nucleic acids, and other biochemical components. Raman spectroscopy will provide complementary molecular information based on light-scattering characteristics. Technical replicate measurements will be performed to improve the reliability and representativeness of the spectral data.

Spectral data will undergo exploratory and classification analyses. Principal component analysis (PCA) will be used for exploratory evaluation, and classification approaches may include partial least squares discriminant analysis (PLS-DA), support vector machine (SVM), and Random Forest. Classification performance will be evaluated using receiver operating characteristic area under the curve (ROC-AUC), sensitivity, specificity, accuracy, and confusion matrices. Validation procedures will be performed at the participant level to reduce the risk of data leakage.

The primary objective is to determine the ability of ATR-FTIR and Raman spectral profiles of PICF to distinguish peri-implant health from peri-implantitis within the same participant. Secondary analyses will evaluate associations between spectral findings and clinical or systemic parameters, compare the diagnostic performance of ATR-FTIR and Raman spectroscopy, and assess the performance of machine-learning-based classification models. Smoking and diabetes status will also be evaluated as exploratory secondary variables when the available sample size permits.