Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma

doi:10.1016/j.archoralbio.2014.04.001

Archives of Oral Biology

Volume 59, Issue 7, July 2014, Pages 695-703

https://doi.org/10.1016/j.archoralbio.2014.04.001 Get rights and content

Abstract

Objective

The aim of this study was to evaluate the composition of the salivary pellicle (SP) and multispecies biofilm developed on titanium nitrided by cold plasma.

Methods

Titanium discs were allocated into a control group (Ti) and an experimental group (TiN – titanium-nitrided by cold plasma). The disc surface topography was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemical composition of the disc surface was determined by X-ray photoelectron spectroscopy (XPS). Stimulated, clarified, and filtered saliva was used to form pellicles on the discs. Proteome analysis of the adsorbed SP proteins was performed by liquid chromatography–mass spectrometry (LC–MS). The surface free energy (SFE) was evaluated before and after SP formation. A multispecies biofilm composed of Actinomyces naeslundii, Streptococcus oralis, Streptococcus mutans, Fusobacterium nucleatum, Veillonella dispar, and Candida albicans was developed on the SP-coated discs. Viable microorganism counts were determined. The biomass and average thickness of biofilms were analyzed by confocal laser-scanning microscopy (CLSM) with COMSTAT software. The biofilm organization was visualized by SEM.

Results

The surface topography was similar in both groups. The SFE of the TiN group did not differ from that of the Ti group (p > 0.05), although the adsorption of pellicle proteins increased the SFE in both pellicle-coated groups (p < 0.001). Different proteins were identified on the Ti and TiN surfaces. The amount of biofilm was similar for both groups (p = 0.416), but the counts of F. nucleatum and S. oralis were higher in the TiN group (p < 0.001). Similar biofilms were characterized by the COMSTAT data, CLSM images, and SEM images.

Conclusion

The titanium nitrided by cold plasma exhibited differences in SP composition and multispecies microbial biofilm population compared to the control titanium surface.

Introduction

Since the advent of osseointegrated implants, titanium has been the preferred material for screws and prosthetic components in oral rehabilitation, due to its biocompatibility with oral tissues and surface properties (e.g., roughness, chemical composition, and topography).1, 2 These properties can be modified by treating the titanium surface to promote protein adsorption, cell signalling, and new bone formation. Treatments include turning/machine-etching, oxidization, and adding/implanting ions.³ An example of such a treatment is to nitride the titanium surface by cold plasma.4, 5

Upon exposure to the oral environment, all surfaces are immediately coated by saliva, forming a salivary pellicle (SP). Pellicle formation involves non-covalent interactions, such as hydrogen bonding, electrostatic, and Van der Waals forces, between the surface and salivary proteins. These interactions result in the selective adsorption of the salivary proteins.6, 7 The surface roughness (Ra) and surface free energy (SFE) are two of the most important properties that influence SP formation.⁸ Protein adsorption is facilitated when the surface area is increased (e.g., by roughening the surface).9, 10, 11, 12 The SFE enhances the interaction between titanium and the biologic fluids.¹¹ In situations of poor tissue recovery and bone destruction, saliva-coated surfaces present sites for microorganisms attachment.¹³

As a result of surface treatments that facilitate protein adsorption, the surface may attract microorganisms that can colonize the surface and form biofilms.¹³ Oral biofilms are structured communities of bacteria and fungi that develop on different substrata.¹⁴ Biofilm formation on titanium is similar to biofilm formation on enamel.¹⁵ It is generally initiated by species of bacteria, known as first colonizers, which are in direct contact with the coated surfaces. An example of a first colonizer is Streptococcus oralis. First colonizers interact by coaggregating with microorganisms known as second colonizers. For example, coaggregation of Fusobacterium nucleatum and Candida albicans forms a mature multispecies biofilm¹⁵ that can lead to peri-implantitis.16, 17

Previous studies have shown that biofilm formation is increased on surfaces that have been turned, machine or acid-etched,¹⁹ with the largest increase occurring when two methods of surface treatment are combined.18, 19 However, studies regarding biofilm formation on nitrided surfaces are still scarce. Thus, the aim of this study was to evaluate the composition of the SP and multispecies biofilm developed on titanium nitrided by cold plasma.

Section snippets

Experimental design

This in vitro study had a randomized and blinded design. Titanium discs (Ti – control) and titanium discs nitrided by cold plasma (TiN – experimental group) were used as substrata for SP and multispecies biofilm analyses. The surface topography was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface chemistry was analyzed by X-ray photoelectron spectroscopy (XPS).

SPs were formed on discs by immersion in stimulated and centrifuged-filtered saliva

Results

As shown in Fig. 1, the surface topographies of the Ti and TiN discs were similar. The three-dimensional (3D) surface reconstructions presented similar variations of tops, valleys, and flanks, expressed numerically by the roughness parameters in Table 1. The chemical composition analysis of the surface revealed a large peak, corresponding to nitrogen, in the TiN group. The most abundant elements detected on the surfaces of both groups were oxygen (O1s), titanium (Ti2p, Ti3s, and Ti3p), and

Discussion

The present study investigated the SP and multispecies biofilm that developed on titanium nitrided by cold plasma. Although previous studies have evaluated the effect of this surface treatment on osseointegration,4, 5 this study is the first to evaluate the SP composition and multispecies biofilm that formed on titanium nitrided surface.

The surface topographies of the Ti and TiN discs, as observed by SEM and AFM, were similar before and after surface treatment. The roughness parameters (Sa, Sdr

Conclusion

Our results show that titanium nitrided by cold plasma alters the composition of the SP and the microbial population in the multispecies biofilm that grow on this surface.

Funding

Sources of funding: FAPESP (Grants 2011/03242-0 and 2010/07894-9).

Competing interests

None declared.

Ethical approval

The study was approved by the Ethics Committee of Piracicaba Dental School (075/2011), and all participants signed an informed consent form.

Acknowledgments

The authors thank Carlos Lambert and Richard Landers for support regarding the surface treatment and its analysis. They also thank FAPESP for the scholarship of the first author (2011/03242-0) and for the financial support provided for the research (2010/07894-9).

References (32)

K. Nakanishi et al.
On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon
J Biosci Bioeng
(2001)
G.P. Rockwell et al.
Fibrinogen and albumin adsorption on titanium nanoroughness gradients
Colloids Surf B Biointerfaces
(2012)
C. Galli et al.
Creation of nanostructures to study the topographical dependency of protein adsorption
Colloids Surf B Biointerfaces
(2002)
E.C. Combe et al.
A protocol for determining the surface free energy of dental materials
Dent Mater
(2004)
P.N. Gomes et al.
Bioactivity and cellular structure of Candida albicans and Candida glabrata biofilms grown in the presence of fluconazole
Arch Oral Biol
(2011)
A.F. Paes Leme et al.
High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry
J Proteomics
(2011)
M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal Biochem
(1976)
S.J. Compton et al.
Mechanism of dye response and interference in the Bradford protein assay
Anal Biochem
(1985)
T. Thurnheer et al.
Multiplex FISH analysis of a six-species bacterial biofilm
J Microbiol Methods
(2004)
S.D. Knorr et al.
The surface free energy of dental gold-based materials
Dent Mater
(2005)

Y.J. Jang et al.

Autoinducer of Fusobacterium nucleatum as a target molecule to inhibit biofilm formation of periodontopathogens

Arch Oral Biol

(2013)

A. Wennerberg et al.

On implant surfaces: a review of current knowledge and opinions

Int J Oral Maxillofac Implants

(2010)

G. Juodzbalys et al.

Titanium dental implant surface micromorphology optimization

J Oral Implantol

(2007)

A. Wennerberg et al.

Effects of titanium implant surface topography on bone integration: a systematic review

Clin Oral Implants Res

(2009)

L. Meirelles et al.

A novel technique for tailored surface modification of dental implants – a step wise approach based on plasma immersion ion implantation

Clin Oral Implants Res

(2013)

J.S. da Silva et al.

Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding

Int J Oral Maxillofac Implants

(2011)

Cited by (27)

Proteomic profile of in situ acquired pellicle on tooth and restorative material surfaces
2023, Journal of Dentistry
Citation Excerpt :
Over the past two decades, with developments of mass spectrometry (MS)-based proteomics, high-throughput protein identification and/or quantification for the acquired pellicle have become possible [20]. In contrast to pellicle on tooth surfaces [21], less attention was paid to the pellicle on dental materials, and the majority of studies on this topic have focused on titanium surfaces [22–27]. The proteomic profile of the acquired pellicle on other dental materials, especially tooth-coloured restorative materials, under in vivo/in situ conditions remains to be elucidated.
To evaluate and compare the proteomic profile of acquired pellicle on smooth bovine tooth and tooth-coloured restorative materials, including resin composite (RC), glass ionomer cement (GIC), and casein phosphopeptide-amorphous calcium phosphate modified GIC (CPP-ACP GIC).
Two-hour in situ pellicles on tooth/materials specimens mounted in oral appliances worn by ten healthy adults were investigated. Pellicle proteins and corresponding unstimulated whole saliva were quantitatively analysed through liquid chromatography-tandem mass spectrometry.
Significantly higher amounts of protein were adsorbed onto tooth surface than restorative materials tested (4.11 ± 0.69 vs. 2.54 ± 0.38/2.98 ± 0.80/3.01 ± 0.37 µg, RC/GIC/CPP-ACP GIC). From the ten participants, 1,444 (487–1,086/person), 1,454 (645–1,051/person), 1,731 (454–1,475/person), or 1,597 (423–1,261/person) pellicle proteins were detected at least once on bovine tooth, RC, GIC, or CPP-ACP GIC, respectively, and with 1,072 (304–793/person) salivary proteins identified. Comparative quantification revealed minor differences between tooth and restorative materials pellicle profiles. High inter-individual variations in pellicle protein composition were demonstrated. Compared to the salivary protein profile, 214/57 proteins showed significantly increased/decreased abundance in pellicle formed on at least one substrate (fold change > 3.325/fold change < 0.301). Gene Ontology enrichment analysis showed some pellicle proteins detected with increased affinity to tooth/material surface were identified as being related to “calcium-dependent protein binding” or “cell-cell adhesion mediator activity”.
Similar protein quantity and composition was observed in 2 h in situ pellicles formed on different smooth restorative material surfaces. The proteomic profile of pellicles appeared distinct from that of the corresponding unstimulated whole saliva.
Host backgrounds appeared more influential on the proteomic profile of the in situ acquired pellicle than the underlying substrate characteristics among systemically and orally healthy adults. Pellicle proteins preferentially adsorbed on tooth/materials were putatively associated with calcium ion homeostasis or host-microbiota interaction.
Cross-kingdom microbial interactions in dental implant-related infections: is Candida albicans a new villain?
2022, iScience
Candida albicans, an oral fungal opportunistic pathogen, has shown the ability to colonize implant surfaces and has been frequently isolated from biofilms associated with dental implant-related infections, possibly due to its synergistic interactions with certain oral bacteria. Moreover, evidence suggests that this cross-kingdom interaction on implant can encourage bacterial growth, leading to increased fungal virulence and mucosal damage. However, the role of Candida in implant-related infections has been overlooked and not widely explored or even considered by most microbiological analyses and therapeutic approaches. Thus, we summarized the scientific evidence regarding the ability of C. albicans to colonize implant surfaces, interact in implant-related polymicrobial biofilms, and its possible role in peri-implant infections as far as biologic plausibility. Next, a systematic review of preclinical and clinical studies was conducted to identify the relevance and the gap in the existing literature regarding the role of C. albicans in the pathogenesis of peri-implant infections.
UV-photofunctionalization of a biomimetic coating for dental implants application
2020, Materials Science and Engineering C
Citation Excerpt :
The early failures due to bacterial infections are influenced by the surface properties of the biomaterials, such as the chemical composition, surface free energy and surface roughness, which are important determinants of biofilm formation on dental implants [7]. Moreover, the surface properties influence the acquired pellicle formation, which acts as a receptor for bacterial adhesion [8–10]. Streptococcus spp. are initial colonizers that are capable of providing additional sites for secondary colonizer adhesion, favoring biofilm development [11,12].
Photofunctionalization mediated by ultraviolet (UV) rays changes the physico-chemical characteristics of titanium (Ti) and improves the biological activity of dental implants. However, the role of UV-mediated photofunctionalization of biofunctional Ti surfaces on the antimicrobial and photocatalytic activity remains unknown and was investigated in this study. Commercially pure titanium (cpTi) discs were divided into four groups: (1) machined samples without UV light application [cpTi UV−]; (2) plasma electrolytic oxidation (PEO) treated samples without UV light application [PEO UV−]; (3) machined samples with UV light application [cpTi UV+]; and (4) PEO-treated samples with UV light application [PEO UV+]. The surfaces were characterized according to their morphology, roughness, crystalline phase, chemical composition and wettability. The photocatalytic activity and proteins adsorption were measured. For the microbiological assay, Streptococcus sanguinis was grown on the disc surfaces for 1 h and 6 h, and the colony forming units and bacterial organization were evaluated. In addition, to confirm the non-cytotoxic effect of PEO UV +, human gingival fibroblast (HGF) cells were cultured in a monolayer onto each material surface and the cells viability and proliferation evaluated by a fluorescent cell staining method. PEO treatment increased the Ti surface roughness and wettability (p < 0.05). Photofunctionalization reduced the hydrocarbon concentration and enhanced human blood plasma proteins and albumin adsorption mainly for the PEO-treated surface (p < 0.05). PEO UV+ also maintained higher wettability values for a longer period and provided microbial reduction at 1 h of bacterial adhesion (p = 0.012 vs. PEO UV-). Photofunctionalization did not increase the photocatalytic activity of Ti (p > 0.05). Confocal microscopy analyses demonstrated that PEO UV+ had no cell damage effect on HGF cells growth even after 24 h of incubation. The photofunctionalization of a biofunctional PEO coating seems to be a promising alternative for dental implants as it increases blood plasma proteins adsorption, reduces initial bacterial adhesion and presents no cytotoxicity effect.
Comprehensive measurements of salivary pellicle thickness formed at different intraoral sites on Si wafers and bovine enamel
2019, Colloids and Surfaces B: Biointerfaces
The salivary pellicle is a thin acellular film formed on orally exposed surfaces by adsorption of macromolecules from the oral fluids and serves as a protective layer in the maintenance of oral health. Pellicle thickness measurements are a central tool helping to understand how exogenous manipulations may influence pellicle formation. This is of particular importance for the investigation of new preventive and therapeutic approaches. In the present study we determined the kinetics of the in situ pellicle thickness formation at different intraoral sites and investigated how pellicle formation occurs in different individuals. To address the kinetic aspect, the thickness of the in situ pellicle was determined after formation periods of 3 min, 30 min and 120 min. The thickness of the pellicle was either measured on silicon wafers by ellipsometry or on bovine enamel by transmission electron microscopy. We found a physiologically important rapid pellicle formation phase within the first minutes and a slow pellicle formation phase between 30 min and 120 min. Furthermore, our results identify significant inter-individual differences both for the pellicle thickness and for the formation kinetics, indicating the consideration of individual-specific differences of the pellicle layer as an important aspect for future studies.
The impact of photofunctionalized gold nanoparticles on osseointegration
2018, Heliyon
The aims of this study were to create a new surface topography using simulated body fluids (SBF) and Gold Nanoparticles (GNPs) and then to assess the influence of UV Photofunctionalization (PhF) on the osteogenic capacity of these surfaces.
Titanium plates were divided into six groups All were acid etched with 67% Sulfuric acid, 4 were immersed in SBF and 2 of these were treated with 10 nm GNPs. Half of the TiO₂ plates were photofunctionalized to be compared with the non-PhF ones. Rat's bone marrow stem cells were seeded into the plates and then CCK8 assay, cell viability assay, immunofluorescence, and Scanning electron microscopy (SEM) were done after 24 hours. Gene expression analysis was done using real time quantitative PCR (qPCR) one week later to check for the mRNA expression of Collagen-1, Osteopontin and Osteocalcin. Alkaline phosphatase (ALP) activity was assessed after 2 weeks of cell seeding.
Our new topography has shown remarkable osteogenic potential. The new surface was the most biocompatible, and the 10 nm GNPs did not show any cytotoxicity. There was a significant increase in bioactivity, enhanced gene expressions and ALP activity.
GNPs enhances osteogenic differentiation of stem cells and Photofunctionalizing GNPs highly increases this. We have further created a novel highly efficient topography which highly enhances the speed and extent of osseointegration. This may have great potential for improving treatment outcomes for implant, maxillofacial as well as orthopedic patients.
Development of binary and ternary titanium alloys for dental implants
2017, Dental Materials
Citation Excerpt :
The chemical composition of the cpTi and Ti alloys (on the order of 1 μm3) was checked by energy-dispersive spectroscopy (EDS). X-ray photoelectron spectroscopy (XPS) analysis was used to verify the chemical composition and state of the outermost oxide layer by means of a spectrometer (Vacuum Scientific Workshop, VSW HA100) [34]. Atomic force microscopy (AFM) was used to observe the surface topography of the samples.
The aim of this study was to develop binary and ternary titanium (Ti) alloys containing zirconium (Zr) and niobium (Nb) and to characterize them in terms of microstructural, mechanical, chemical, electrochemical, and biological properties.
The experimental alloys — (in wt%) Ti–5Zr, Ti–10Zr, Ti–35Nb–5Zr, and Ti–35Nb–10Zr — were fabricated from pure metals. Commercially pure titanium (cpTi) and Ti–6Al–4V were used as controls. Microstructural analysis was performed by means of X-ray diffraction and scanning electron microscopy. Vickers microhardness, elastic modulus, dispersive energy spectroscopy, X-ray excited photoelectron spectroscopy, atomic force microscopy, surface roughness, and surface free energy were evaluated. The electrochemical behavior analysis was conducted in a body fluid solution (pH 7.4). The albumin adsorption was measured by the bicinchoninic acid method. Data were evaluated through one-way ANOVA and the Tukey test (α = 0.05).
The alloying elements proved to modify the alloy microstructure and to enhance the mechanical properties, improving the hardness and decreasing the elastic modulus of the binary and ternary alloys, respectively. Ti–Zr alloys displayed greater electrochemical stability relative to that of controls, presenting higher polarization resistance and lower capacitance. The experimental alloys were not detrimental to albumin adsorption.
The experimental alloys are suitable options for dental implant manufacturing, particularly the binary system, which showed a better combination of mechanical and electrochemical properties without the presence of toxic elements.

View all citing articles on Scopus

View full text

Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Experimental design

Results

Discussion

Conclusion

Funding

Competing interests

Ethical approval

Acknowledgments

J Biosci Bioeng

Colloids Surf B Biointerfaces

Colloids Surf B Biointerfaces

Dent Mater

Arch Oral Biol

J Proteomics

Anal Biochem

Anal Biochem

J Microbiol Methods

Dent Mater

Arch Oral Biol

On implant surfaces: a review of current knowledge and opinions

Int J Oral Maxillofac Implants

Titanium dental implant surface micromorphology optimization

J Oral Implantol

Effects of titanium implant surface topography on bone integration: a systematic review

Clin Oral Implants Res

A novel technique for tailored surface modification of dental implants – a step wise approach based on plasma immersion ion implantation

Clin Oral Implants Res

Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding

Int J Oral Maxillofac Implants