Surface attachment is the first step in biofilm formation, and the ability of bacteria to adhere to surfaces and develop a biofilm is directly influenced by electrostatic interactions between the bacteria and the chemical composition of material surfaces. Here, we investigated the influence of physical and chemical characteristics of titanium (Ti) and zirconia (ZrO2) as implant abutment surfaces on the bacterial adhesion phase and compared the results to bovine enamel (BE) simulating a human tooth. To achieve this goal, we used 2 common pathogens of the oral cavity, Streptococcus mutans UA140 and Porphyromonas gingivalis 33277. To investigate the influence of material surfaces on bacterial adhesion, we studied the surface free energy as well as the topography by atomic force microscopy, and the chemical elements composition by scanning electron microscopy equipped with an energy dispersive X-ray spectroscope. Our results indicated a hydrophobic characteristic for all of the materials; however, the presence of polar and nonpolar components could aid in understanding why greater numbers of bacteria had adhered to BE compared to the other surfaces. Our confocal microscopy data support the proposition that electrostatic interactions, indeed, affected the initial adhesion phase. Within the limitations of a laboratory study, the results revealed bacterial adhered on BE and no bacteria could be observed by confocal images on Ti and ZrO2 implant abutment surfaces.

Introduction

The long-term success of dental implants is thought to be strongly dependent on the presence of healthy peri-implant tissue.1  Although implant failure is a multifactorial process,2  peri-implantitis has been proposed to be one of the most critical factors implicated in the loss of implants.3,4  In this context, the physical and chemical characteristics of implant abutment surfaces have been shown to directly affect bacterial adhesion.5 

To date, titanium has been considered the gold standard due to its excellent properties, providing it with a dominant position among the available abutment and implant materials.6  Esthetic features favored the introduction of ceramic implant abutment materials.7  Similarly to titanium, zirconia is also biocompatible, highly resistant to corrosion, and provides great strength and toughness.7,8  Although, the effect of implant and abutment surfaces on bacterial adhesion and biofilm formation has been reported in both in vivo and in vitro studies,911  there is no unified consensus regarding how different materials affect bacterial colonization.5,12,13  Titanium and zirconia are hydrophobic materials. Hydrophobic attractive forces and electrostatic charge interactions between surfaces and bacteria have been proposed to play a key role in biofilm formation.14,15  Most bacteria have many ionizable groups on their surfaces,1618  which confer a net negative charge, particularly during the early stationary phase of cell growth.19  However, the charge that is present on the cell surface of some types of bacteria can create a hydrophobic effect via nonpolar molecules and result in an affinity for other hydrophobic surfaces.20  This characteristic explains why some bacterial species preferentially interact with certain materials and why the findings reported in the literature are inconsistent depending on the type of bacterial species assessed. Given the complex relationship between bacteria and material surfaces and the clinical relevance of this topic, this study was conducted to investigate the influence of the physical and chemical properties of 2 different implant abutment materials, titanium and zirconia, on bacterial adhesion. In addition, bovine enamel (BE) was used as a control, simulating the human cervical tooth surface.

Materials and Methods

Disc preparation and surface analysis

Standardized disc-shaped specimens with a diameter and thickness of 8 mm and 2 mm were obtained from machined pure titanium and yttrium-stabilized zirconia (Conexão Sistemas de Próteses Ltda, São Paulo, SP, Brazil). Enamel discs of the same dimensions were prepared from bovine incisors and used as a control. The enamel was cut with a diamond drill for glass thread (Dinser Diamond Tools Ltda, São Paulo, Brazil) coupled to the drill bench vise (Schulz - FSB model 16 - chuck taper DIN 238-B18, São Paulo, SP, Brazil) and then subjected to surface polishing utilizing a procedure with waterproof paper grains of 800, 2500, and 4000 (T469-SF-Noton, Saint-Gobam Abrasives Ltd, Jundiaí, São Paulo, SP, Brazil).

The surface roughness of all of the discs was quantitatively analyzed using a portable roughness analyzer (Mitutoyosurftest SJ-401, Mitutoyo Corporation, Kawasaki, Japan). To standardize the surface roughness measurements, we developed a device with 2 parallel slots to define the central area of the discs and a circular space to stabilize the samples during the readings. For each disc, 2 measurements were performed on each side, and to verify the reliability of the results, the analyses were repeated twice. The reproducibility of the readings was assessed by calculating the intraclass correlation coefficient (ICC) with a confidence interval of 95%.

To analyze the surface topography, we used 3 samples of each material and 3 randomized sites per sample (TopScan 3D), measuring an area of 2 × 2 μm and 10 × 10 μm (Figure 1). A quantitative characterization of the nanoscale surface topography and roughness was conducted using atomic force microscopy (Agilent AFM system, Model 5500, Agilent Technologies, Chandler, Ariz). Contact-mode topographic images were obtained with an applied force constant of 42 N/m, resonance frequency of 320 kHz and silicon tips with a nominal apical radius of less than 8 nm. Next, we examined the chemical element composition of 3 discs from each material by scanning electron microscopy (FEG-MEV; JEOL 7500F model) equipped with an energy dispersive X-ray spectroscope (EDX) (JSM-5900LV, Joel Ltd, Tokyo, Japan).

Figure 1

Micrographs taken by an atomic force microscopy to describe the surfaces: (a) bovine enamel, (b) Ti, (c) ZrO2.

Figure 1

Micrographs taken by an atomic force microscopy to describe the surfaces: (a) bovine enamel, (b) Ti, (c) ZrO2.

To evaluate the wetting and surface tension, we used 5 discs of each material, and the measurement was repeated 3 times for each sample. The contact angle value determined for each wet agent was calculated using the Young–Laplace equation and then inserted into specialized drop-shape analysis software to perform the measurements (SCA-SOFTWARE / OCA-20, DataPhysics Instruments GmbH, Filderstadt, Baden-Württemberg, Germany). The SFE was obtained using the concept of polar and dispersion components according to the method described by Owen.21 

Bacterial conditions and qualitative assessment of bacterial adhesion

Porphyromonas gingivalis 33277 was cultured in blood agar supplemented with 0.1% hemin and menadione, for 48 hours at 37°C, under anaerobic condition (85% N2, 10% H2, 5% CO2). P. gingivalis colonies were transferred to a new tube containing fresh BHI broth medium supplemented by hemin and menadione and maintained for 24 hours (a period previously defined by the growth curve analysis) inside anaerobic chamber, and then the cultures were adjusted to OD600nm1.4 before the experiment. Streptococcus mutans UA140 was cultured in TH medium plates prepared by adding 1.5% (wt/vol) agar to the medium and incubated at 37°C, under anaerobic condition. One colony of S. mutans was diluted in TH broth medium and overnight cultures were diluted to an OD600nm = 0.02 in fresh TH medium containing 0.5% (wt/vol) sucrose. Eight-hundred microliters of each culture was transferred to a 24-well plate containing the discs of each material. The discs were completely immersed in the prepared bacterial suspensions and incubated at 37°C under anaerobic conditions. S. mutans were analyzed after 16 hours incubation, while the discs that were immersed in the P. gingivalis culture were analyzed after 48 hours.

In order to evaluate the density of the bacteria adhered on Ti and ZrO2 disc surfaces, after 16 and 48 hours incubation, the discs were transferred to a new 12-well plate and washed 3 times with 1 mL of sterile phosphate-buffered saline (PBS) to remove unattached bacteria. The bacteria were then labeled with 0.01 mM of Syto-9 and 0.06 mM of propidium iodide (PI), LIVE/DEAD stain BacLight Bacterial Viability Kit (Invitrogen Corporation, Grand Island, NY), for 15 minutes, according to the manufacturer's instructions. For visualization, the discs were placed on a glass cover slip, and the adhesion of the bacteria was visualized with a 40x oil-immersion (Plan NeoFluar NA 1.3 oil) objective using an LSM 510 confocal laser-scanning microscope (Version 4.2, Carl Zeiss MicroImaging Co, Ltd, Jena, Germany).

Results

Physical and chemical characteristics

The roughness average (Ra) was used as a physical parameter, and the values for the Ti and ZrO2 discs, respectively, were as follows: 0.21 μm (±0.06) and 0.22 μm (±0.03). For the BE discs, only discs with a roughness range between 0.05 and 0.1 μm were included in the study. Table 1 shows the average surface roughness (Ra) and maximum surface roughness (Rmax) as determined using AFM and an optical profilometer.

Table

Average value, surface roughness (Ra), peak to valley height (Maximum), median and average peak spacing (Rms) measured for the different surfaces: Ti, ZrO2, bovine enamel (BE) in nanometer (nm)

Average value, surface roughness (Ra), peak to valley height (Maximum), median and average peak spacing (Rms) measured for the different surfaces: Ti, ZrO2, bovine enamel (BE) in nanometer (nm)
Average value, surface roughness (Ra), peak to valley height (Maximum), median and average peak spacing (Rms) measured for the different surfaces: Ti, ZrO2, bovine enamel (BE) in nanometer (nm)

The presence of Ti, as unique element contained in Ti discs, was confirmed by elemental analysis using an energy dispersive spectroscopy. For ZrO2, we detected zirconium (Zr), oxygen (O), and a small amount of carbon (C) in its chemical composition. And, to the BE, hydroxyapatite constituents were found: calcium (Ca), phosphor (P), and O were found. To analyze the physical and chemical characteristics of Ti, ZrO2 and BE, we dropped 4 liquids of different polarities on each material and measured the contact angle between them. Among the surfaces studied, BE presented hydrophilic and lipophilic characteristics, in contrast, Ti and ZrO2 presented the highest contact angle values with water and the lowest with hydrophobic wet agents (Figure 2). The SFE was defined by the intersection between a straight line and the y-axis (Figure 3a, b, c). ZrO2 materials presented the highest SFE value, and BE showed the lowest.

Figure 2

Effect of material surface on the wettability. In each material, 4 liquids were used: water, ethylene glycol, polyethylene glycol, and diiodomethane. Red indicates Ti; blue, ZrO2; green, BE.

Figure 2

Effect of material surface on the wettability. In each material, 4 liquids were used: water, ethylene glycol, polyethylene glycol, and diiodomethane. Red indicates Ti; blue, ZrO2; green, BE.

Figure 3

Energy-free surface (mN/m) analysis in surfaces: (a) BE; (b) Ti, (c) ZrO2.

Figure 3

Energy-free surface (mN/m) analysis in surfaces: (a) BE; (b) Ti, (c) ZrO2.

Effect of surface hydrophobicity on bacterial adhesion

To identify the bacterial adhesion structure and viability of the cells that had adhered onto the different surfaces, we performed high-resolution analyses using CLSM. A wetting behavior effect of the 3 different materials on bacterial adhesion was confirmed by confocal microscopy analysis. Higher magnification confocal imaging (×40) revealed that bacterial colonization on BE was clearly characterized by larger, taller, and more widespread microcolonies in comparison to the Ti and ZrO2 surfaces for both S. mutans (Figure 4) and P. gingivalis (Figure 5). In the case of S. mutans, a small number of bacterial cells was observed on the ZrO2 discs, but no bacteria were detected on the Ti discs.

Figures 4 and 5

Figure 4. Biofilm of S. mutans UA159 were grown on different surfaces in TH medium, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The figure shows cross-section images of biofilms after 16 hours developed on bovine enamel (BE) surfaces. Dead cells were stained red, and live cells were stained green. Figure 5. Biofilms of P. gingivalis 33277 were grown on different surfaces in BHI medium, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The figure shows cross-section images of biofilms after 48 hours developed on BE surfaces. Dead cells were stained red, and live cells were stained green.

Figures 4 and 5

Figure 4. Biofilm of S. mutans UA159 were grown on different surfaces in TH medium, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The figure shows cross-section images of biofilms after 16 hours developed on bovine enamel (BE) surfaces. Dead cells were stained red, and live cells were stained green. Figure 5. Biofilms of P. gingivalis 33277 were grown on different surfaces in BHI medium, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The figure shows cross-section images of biofilms after 48 hours developed on BE surfaces. Dead cells were stained red, and live cells were stained green.

Discussion

The physical and chemical characteristics of material surfaces have been shown to have a direct impact on the adhesion phase in bacteria.22  Numerous studies have reported a relationship between the types of substrate and the bacterial species,12,13,23,24  and some of them have shown that the initial phase is highly influenced by a surface roughness above a threshold of 0.2 μm.25  Hence, to minimize the effect of roughness in our study, we only used discs with mean roughness values equal to or less than 0.22 μm. Furthermore, we introduced BE simulating the cervical of a human tooth, as a control to investigate the effect of the chemical composition on bacterial adhesion and to improve our understanding of the opposite effect on Ti and ZrO2 materials. For control group, we selected only the discs with a superficial roughness ranging between 0.05 and 0.1 um, close to a human tooth. 26  Both S. mutans and P. gingivalis27  adhered better on BE than on Ti or ZrO2 surfaces. This preference in adhesion was confirmed by confocal images and could be explained by the presence of intermolecular interactions between chemical groups on the cell walls of bacteria and polar and nonpolar regions of BE surfaces. Different intermolecular forces can dictate how bacteria interact with different surfaces.28  Depending on the characteristics of these forces, the interaction can be stronger or weaker. The cell wall of S. mutans consists of a thick layer of peptidoglycan that is covered mainly by neutral and acidic polysaccharides, several proteins and teichoic acid.29  Surface proteins on S. mutans confer the charge to these Gram-positive bacteria. The wetting behavior of BE with liquids of different polarities revealed that this type of material has polar and nonpolar characteristics, and consequently, it provides improved electrostatic interactions between the material and the bacteria.30  The hydrophobic regions of S. mutans can also interact with Ti and ZrO2; however, the forces exhibited by the nonpolar molecules are weak. This weak interaction could have allowed the easy detachment of the bacteria from the Ti and ZrO2 discs during the rinsing step.

In the case of Gram-negative bacteria, these species possess a thinner peptidoglycan layer and an outer membrane that consists of proteins, phospholipids and lipopolysaccharides (LPS).31  Although the presence of LPS confers a negative net charge, which has been found to contribute hydrophilic characteristics,32  the degree to which LPS plays a role in these hydrophilic features is largely dependent on the structural components, which vary between different species.33,34  According to our results, the high hydrophobic and lipophilic characteristics of Ti and ZrO2 surfaces, which were evaluated based on the contact angle, could have influenced the ability of P. gingivalis to adhere to these materials. However, the charges present on the BE surfaces could have enhanced the electrostatic interaction with the bacterial cells.

Interactions between bacteria and materials are also influenced by salivary proteins. Although the salivary pellicle has a definitive impact on bacterial adhesion,35,36  in the current study, a salivary pellicle was not used to more distinctively identify the effects of the chemical characteristics of the material surfaces on bacterial adhesion. The composition of the materials and their physicochemical properties may modulate initial bacterial adhesion37  and, consequently, affect the microbial quality. However, our results cannot be generalized because we examined the impact of the material surfaces using 2 different species of bacteria and must consider the various contributions affecting the final results. These contributing factors include the intermolecular interactions between different species of bacteria, the salivary pellicle and the pH of the oral cavity, which can cause imbalances in the microbial community.

We believe that the results and concepts presented here will improve understanding regarding the inherent characteristics of materials that affect bacterial adhesion and can be helpful in the design of implant abutment material surfaces to prevent plaque accumulation.

Conclusion

Within the limitations of a laboratory study, the present results suggest that bacterial adhesion on Ti was lower than that on ZrO2, independent of the bacterial species or Gram-positive or Gram-negative status, but it was consistently higher on BE than on abutment materials.

Abbreviations

    Abbreviations
     
  • BE

    bovine enamel

  •  
  • EDS

    energy-dispersive X-ray spectroscopy

  •  
  • LPS

    lipopolysaccharides

  •  
  • SFE

    surface free energy

Acknowledgments

Erica Dorigatti de Avila received funds from São Paulo Research Foundation (FAPESP), Grant number: 2011/05106-6, Coordination for the Improvement of Higher Education Personnel (CAPES) and Lemann Foundation. The authors thank Conexao Sistema de Protese for donating the pure titanium and zirconia stabilized with yttrium discs used in this study.

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