Salivary pellicle modulates biofilm formation on titanium surfaces

Clinical Oral Investigations https://doi.org/10.1007/s00784-023-05230-9 RESEARCH Salivary pellicle modulates biofilm formation on titanium surfaces Miryam Martínez‑Hernández1 · Juan Pablo Reyes‑Grajeda2 · Matthias Hannig3 · Argelia Almaguer‑Flores1 Received: 14 May 2023 / Accepted: 18 August 2023 © The Author(s) 2023 Abstract Objectives The present study aimed to evaluate the potential of the salivary pellicle (SP) formed on titanium (Ti) surfaces to modulate the formation of a biofilm composed of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. Materials and methods Ti substrates were incubated for 2 h with a pool of saliva samples obtained from 10 systemically and periodontally healthy subjects. Enamel substrates were included as a biological reference. Scanning electron microscopy (SEM) and Raman spectroscopy analysis were used to analyze the formation of the salivary pellicle. After the SP formation, the surfaces were incubated for 12 h with a mix of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. The number of bacterial cells attached to each surface was determined by the XTT assay while bacterial viability was analyzed by fluorescence microscopy using the LIVE/DEAD® BacLightTM kit. Results The SEM and Raman spectroscopy analysis confirmed the presence of a salivary pellicle formed on the tested surfaces. Regarding the biofilm formation, the presence of the SP decreases the number of the bacterial cells detected in the test surfaces, compared with the uncover substrates. Even more, the SP-covered substrates showed similar bacterial counts in both Ti and enamel surfaces, meaning that the physicochemical differences of the substrates were less determinant than the presence of the SP. While on the SP-uncover substrates, differences in the bacterial adhesion patterns were directly related to the physicochemical nature of the substrates. Conclusions The salivary pellicle was the main modulator in the development of the biofilm consisting of representative oral bacteria on the Ti substrates. Clinical relevance The results of this study provide valuable information on the modulatory effect of the salivary pellicle on biofilm formation; such information allows us to understand better the events involved in the formation of oral biofilms on Ti dental implants. Keywords Salivary pellicle · Titanium surfaces · Dental implants · Dental enamel · Oral bacteria · Biofilm formation Introduction * Miryam Martínez‑Hernández 1 Laboratorio de Biointerfases, División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, 04510 Mexico City, Mexico 2 Laboratorio de Estructura de Proteínas, Instituto Nacional de Medicina Genómica (INMEGEN), 14610 Mexico City, Mexico 3 Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, University Hospital, Saarland University, Building 73, 66421 Homburg, Saarland, Germany Within the oral cavity, where a variety of natural and artificial surfaces are colonized with complex biofilms, saliva is essential for maintaining homeostasis [1]. Besides its function ranging from digestion to lubrication, remineralization, and beyond [2], saliva is implicated in the formation of a protein layer termed salivary pellicle (SP), formed by the adsorption of salivary components onto surfaces such as natural dentition and dental biomaterials [3]. SP contributes to basic outcomes in dentistry, including the protection of oral surfaces against wear [4], modulation of periodontal wound healing [5], and bacterial adhesion on oral surfaces [6]. Currently, dental materials have been less researched than natural teeth in terms of SP formation [7], and it is known that 13 Vol.:(0123456789) Clinical Oral Investigations the physicochemical characteristics of these biomaterials influence the composition and structure of the SP [8]. Different types of biomaterials can be placed in the oral cavity, including titanium dental implants. Dental implant therapy is a predictable therapeutic option for rehabilitating partially or fully edentulous patients, providing long-term function (10 years plus) and esthetics [9]. However, due to the abundance of microorganisms in the oral cavity, dental implants are susceptible to contamination [10], which can result in biological complications, including implant failure [11]. Once a clean implant abutment surface is exposed to the oral cavity, it is immediately covered by a salivary pellicle [12], which in turn significantly influences microbial adhesion to the Ti substratum [13, 14]. It has been reported that bacteria colonize Ti surfaces as quickly as within 30 min after dental implant placement [15] and can form a complex biofilm within a couple of weeks [16]. In this regard, there is extensive evidence that biofilm accumulation at the implant-abutment interface can result in inflammatory cell infiltration, ultimately leading to bone loss [17]. In light of the fundamental role that SP plays in periimplant health, our group previously reported that the presence of saliva inhibited the adhesion of representative oral bacteria to titanium films [18]. To deepen the understanding of the effect of salivary conditioning of titanium surfaces on the adhesion of oral bacteria, in another study by our group, different microstructured titanium substrates were incubated intraorally on periodontally healthy and periodontitis subjects, finding that the periodontal status, and therefore the salivary composition and microbiota of the volunteers who participated in this study, was more determinant in modulating the identity and proportions of oral species within biofilms formed on these substrates than the surface characteristics of Ti surfaces itself [19]. Therefore, while the formation of the oral biofilm on the microstructured Ti surfaces did not depend directly on the characteristics of the substrate as shown from the previous study [19], the amount and identity of the proteins forming part of the salivary film did depend on the physicochemical properties (e.g., roughness and wettability) of the substrates [20]. In combination, these observations support the assertion that saliva/salivary pellicle–titanium surface interactions assist in the development of oral biofilms on titanium dental implants. Studying the acquired salivary film process is important because it connects artificial and tooth surfaces with the oral environment, as well as the fact it contributes significantly to the development of severe oral diseases, including erosion, dental caries, periodontal disease, and peri-implant infection [21]. With the growing use of dental implant restorations, studies exploring SP formation on titanium substrates have become increasingly important [22, 23]. Therefore, detailed knowledge of the biological interactions occurri (...truncated)