The aim of this study was to develop in vitro biofilms on SLA titanium (Ti-SLA) and zirconium oxide (ZrO 2 ) surfaces and to evaluate the effect of antiseptic agents on the number of putative periodontal pathogenic species.
An in vitro biofilm model was developed on sterile discs of Ti-SLA and ZrO 2 . Three antiseptic agents [chlorhexidine and cetyl-pyridinium-chloride (CHX/CPC), essential oils (EEOOs) and cetyl-peridinium-chloride (CPC)] were applied to 72-h biofilms, immersing discs during 1 min in the antiseptic solution, either with or without mechanical disruption. Viable bacteria [colony forming units (CFU/mL)] were measured by quantitative polymerase chain reaction (qPCR) combined with propidium monoazide. A generalized lineal model was constructed to determine the effect of the agents on the viable bacterial counts of Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis and Fusobacterium nucleatum on each surface.
The exposure to each antiseptic solution resulted in a statistically significant reductions in the number of viable target species included in the in vitro multi-species biofilm, on both Ti-SLA and ZrO 2 (p < 0.001) which was of up to 2 orders for A. actinomycetemcomitans , for P. gingivalis 2 orders on Ti-SLA and up to 3 orders on ZrO 2, and, for F. nucleatum up to 4 orders. No significant differences were found in counts of the tested bacteria between in vitro biofilms formed on both Ti-SLA and ZrO 2 , after topically exposure to the antimicrobial agents whether the application was purely chemical or combined with mechanical disruption.
A. actinomycetemcomitans , P. gingivalis and F. nucleatum responded similarly to their exposure to antiseptics when grown in multispecies biofilms on titanium and zirconium surfaces, in spite of the described structural differences between these bacterial communities.
Biofilms are complex microbial communities developed on solid surfaces exposed to a wet environment . In the oral cavity, different biofilms may be encountered attached to different solid oral surfaces, including teeth, prosthetic devices and dental implants . The formation and maturation of biofilms on dental implant surfaces have been associated with the etiology of peri-implant mucositis and peri-implantitis, in a similar manner as the subgingival biofilm is associated with gingivitis and periodontitis .
Despite the similarities between biofilms on tooth and implant surfaces, some specific features might be attributed to the specific implant surface characteristics . Previous in vivo and in vitro investigations have reported that surface characteristics such as roughness, surface free energy, wettability and degree of sterilization may affect biofilm formation and its bacterial three-dimensional distribution, although there is still controversy on the relevance of these differences. Recent studies evaluating biofilms on abutments, with different surface composition and topography, have shown that there is a correlation between surface roughness and viable biomass within the biofilm . There is, however, controversy on which are the key factors guiding biofilm formation on implant surfaces, since in some studies using in vitro biofilm models, surface roughness seems to be the main factor , while in others, surface free energy, rather than roughness, seems to be the key factor determining initial bacterial adhesion . Similarly, a positive correlation between surface roughness and bacterial colonization has been found in some models , while in others, certain titanium topographies seemed to inhibit bacteria adhesion together with the promotion of bone tissue formation . Also, titanium purity, and not only surface topography, may influence early bacterial colonization . Our research group, using an in vitro multi-bacterial species biofilm, has reported significant differences in biofilm thickness and three-dimensional structure, when comparing titanium and zirconium surfaces, with a higher number of initial and early colonizers ( Streptococcus oralis , Actinomyces naeslundii and Veillonella parvula ) on zirconium than on titanium surfaces . These results are coincident with recent studies by de Avila et al. , reporting quantitative and qualitative differences between biofilms formed on titanium versus zirconium surfaces.
Antimicrobial agents, such as chlorhexidine (CHX), essential oils (EEOOs) or cetyl-pyridinium chloride (CPC), combined with mechanical debridement, are the gold standard therapy in the treatment of peri-implant mucositis and in the secondary prevention of peri-implantitis . However, there is controversy whether implant micro-surface topography and chemistry influence the antimicrobial effect of these antimicrobial agents. This in vitro study was, therefore, aiming to assess whether the number of specific bacterial pathogens growing on in vitro biofilms over SLA titanium and zirconium oxide surfaces, were differentially affected when exposed to different antiseptic agents (alcohol-free EEOOSs, CPC and CHX/CPC).
Material and methods
Bacterial strains and culture conditions
Standard reference strains of S. oralis CECT 907T, V. parvula NCTC 11810, A. naeslundii ATCC 19039, Fusobacterium nucleatum DMSZ 20482, Aggregatibacter actinomycetemcomitans DSMZ 8324 and Porphyromonas gingivalis ATCC 33277 were used. These bacteria were grown on blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg L −1 hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L −1 menadione (Merck, Darmstadt, Germany) in anaerobic conditions (10% H 2 , 10% CO 2 , and balance N 2 ) at 37 °C for 24–72 h.
Sterile discs of 5 mm of diameter made of two different surface materials were used: (1) titanium with a SLA grade 2 surface (Ti-SLA) (Sand-blasted, Large grit, Acid-etched; Straumann; Institut Straumann AG, Basel, Switzerland), and (2) sterile zirconium oxide (ZrO 2 ), with a rough micro surface obtained after chemical treatment with a hot solution of hydrofluoric acid, according to a proprietary process of Institut Straumann AG (Institut Straumann AG, Basel, Switzerland). The resulting rough surface topography of ZrO 2 discs has a S a value of 0.55 mm (standard deviation, SD = 0.01) with a rough surface topography similar the Ti-SLA surface implants when evaluated with scanning electron microscopy (SEM), although zirconium surfaces seemed to have a flatter profile with less porosity [ S a value of Ti-SLA surface of 1.17 mm (SD = 0.04)] .
Un-stimulated saliva was obtained from healthy volunteers in 10 mL aliquots at least 1.5 h after eating, drinking or tooth brushing. Each saliva sample was treated with 2.5 mmol L −1 DL–Dithiothreitol (Sigma) for 10 min with continuous stirring in order to reduce salivary protein aggregation. It was then centrifuged (10 min, 4 °C and 12,000 rpm) and the obtained supernatant was diluted (1:1) with phosphate buffered saline (PBS; pH = 7.4). The sample was then filtered and sterilized through a 0.22 μm pore size Millex GV low-protein-binding filter X50 (Millipore, Millipore Corporation Bedford, USA) and stored at −20 °C. The efficacy of this protocol was validated by plating processed saliva samples onto supplemented blood agar plates for 72 h at 37 °C, when confirmed by lack of any bacterial growth on either aerobically or anaerobically incubated plates.
Biofilms were generated using the method described by Sánchez et al. with slightly different bacterial concentrations when preparing the bacterial suspension. Briefly, planktonic cultures of each bacteria were grown anaerobically in a protein-rich medium containing brain–heart infusion (BHI) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 2.5 g L −1 mucin (Oxoid), 1.0 g L −1 yeast extract (Oxoid), 0.1 g L −1 cysteine (Sigma), 2.0 g L −1 sodium bicarbonate (Merck), 5.0 mg L −1 hemin (Sigma), 1.0 mg L −1 menadione (Merck) and 0.25% (v/v) glutamic acid (Sigma). Upon mid-exponential phase, the cells were mixed in modified BHI medium containing 10 3 colony forming units (CFU) mL −1 for S. oralis , 10 5 CFU mL −1 for V. parvula and A. naeslundii, and 10 8 CFU mL −1 for F. nucleatum, A. actinomycetemcomitans and P. gingivalis . Ti-SLA and ZrO 2 sterile discs were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany). Each well was inoculated with 1.5 mL mixed bacteria suspension prepared and incubated in anaerobic conditions (10% H 2 , 10% CO 2 , and balance N 2 ) at 37 °C for 72 h. Plates containing only culture medium were also incubated to check for sterility.
Before SEM analysis, three discs of each material (Ti-SLA and ZrO 2 ) covered with biofilms grown in vitro for 72 h were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde for 4 h at 4 °C. After fixation, the discs were washed twice in PBS and again twice in sterile water (immersion time per washed, 10 min) and dehydrated through a series of graded ethanol solutions (50, 60, 70, 80, 90 and 100%; immersion time per series, 10 min). Then, the samples were critical point dried, sputter-coated with gold and analyzed by electron microscopy JSM 6400 (JSM6400; JEOL, Tokyo, Japan), with back-scattered electron detector and an image resolution of 25 KV. Analyses were carried out at ICTS National Centre of Electronic Microscopy (Campus of International Excellence Moncloa, University Complutense, Madrid, Spain).
Exposure to antimicrobial compounds
The following commercially available antiseptic mouth rinse formulations were used: (1) 0.12% CHX and 0.05% CPC without alcohol (CHX/CPC) (Perio-Aid tratameiento ® ; Dentaid, Cerdanyola, Spain), (2) a combination of four EEOOs (thymol 0.06%, eucalyptol 0.09%, methyl salicylate 0.06% and menthol 0.01%) without alcohol (Listerine ® Zero; Johnson & Johnson, Madrid, Spain), an (3) 0.05% CPC without alcohol (Oral B-ProExpert, Procter & Gamble, Weybridge, Surrey, UK). PBS was used as a negative control solution.
To monitor the bactericidal action of the three tested mouth rinses on 72 h biofilms, the discs were immersed during 1 min in the antiseptic solution and in PBS as control, with and without mechanical disruption by means of agitation through vortex at room temperature, which provided constant stirring at 90 rpm. After this exposure, the discs were sequentially rinsed in 2 mL of sterile PBS (immersion time per rinse, 10 s), three times, to remove the antiseptic solutions.
In each experiment, the three antimicrobial agents and the control solutions were tested with and without mechanical disruption (agitation). These experiments were repeated three times on different days using fresh bacterial cultures with trios of biofilms for each independent outcome variables.
After the antimicrobial treatment, biofilms were disrupted by vortex for 2 min in 1 mL of PBS. To discriminate between DNA from live and dead bacteria, propidium monoazide (PMA) (Biotium Inc., Hayword, CA, USA) was used. The use of this PMA dye has shown the ability to distinguish between viable and irreversibly damaged cells and hence when combined with quantitative polymerase chain reaction (qPCR) to detect the DNA from viable bacteria . PMA was added to sample tubes containing 250 μL of disaggregated biofilm cells, at a final concentration of 100 μM. Following an incubation period of 10 min at 4 °C in the dark, the samples were subjected to light-exposure for 20 min, using a 550 W halogen light source, placed 20 cm above the samples. During this exposure, the sample tubes were laid horizontally on ice to avoid excessive heating. After PMA photo-induced DNA cross-linking, the cells were centrifuged at 12,000 rpm for 3 min prior to DNA isolation. To control for any possible influence of the experimental process on bacterial viability, the same procedure (incubation at 4 °C and exposure to light source) but without the exposure to PMA, was used as negative control.
Bacterial DNA was isolated from all biofilms using a commercial kit (MolYsis Complete5; Molzym GmbH & CoKG, Bremen, Germany), following manufacturer’s instructions (the protocol for bacterial DNA extraction was followed from step 6, avoiding preliminary steps) and the hydrolysis 5′nuclease probe assay qPCR method was used for detecting and quantifying the bacterial DNA. The qPCR amplification was performed following a protocol previously optimized by our research group, using primers and probes targeted against 16S rRNA gene [obtained through Life Technologies Invitrogen (Carlsbad, CA, USA) and Applied Biosystems (Carlsbad, CA, USA) .
Each DNA sample was analyzed in duplicate. Quantification cycle (Cq) values, previously known as cycle threshold (Ct) values, describing the PCR cycle number at which fluorescence rises above the baseline, were determined using the provided software package (LC 480 Software 1.5; Roche Diagnostic GmbH; Mannheim, Germany). Quantification of viable cells by qPCR was based on standard curves . The correlation between Cq values and CFU/mL was automatically generated through the software (LC 480 Software 1.5; Roche).
All assays were developed with a linear quantitative detection range established by the slope range of 3.3–3.6 cycles/log decade, r 2 > 0.997 and an efficiency range of 1.9–2.0.
Measures to avoid carryover DNA were established. In spite of this, when non-template control (NTC) was detectable, the limit of detection was established on the last value of the standard curve that holds five cycles of difference with NTC.
The following independent variables were considered: (1) the material surface (Ti-SLA and ZrO 2 ), (2) the mechanical disruption applied by constant agitation (with or without), (3) type of antiseptic/control (PBS, CHX/CPC, EEOOs or CPC), and (4) their interaction. The number of viable bacteria present on in vitro biofilms formed on SLA titanium and zirconium oxide surfaces and measured as viable CFU/mL of A. actinomycetemcomitans , P. gingivalis and F. nucleatum was the primary dependent outcome variable.
An experiment-level analysis was performed for each study parameter (n = 9 or 72 for qPCR results). Shapiro–Wilk goodness-of-fit tests and distribution of data were used to assess normality. Data were expressed as means and 95% confidence intervals (CI).
In order to compare the effect of material surface, type of antiseptic with or without mechanical disruption, and their interaction on the main outcome variable (CFU/ml) a general lineal model was constructed for each bacterium ( A. actinomycetemcomitans , P. gingivalis and F. nucleatum ) using the method of maximum likelihood and Bonferroni corrections for multiple comparisons.
Results were considered statistically significant at p < 0.05. A software package (IBM SPSS Statistics 21.0; IBM Corporation, Armonk, NY, USA) was used for all data analysis.