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Biological and physicochemical implications of the aging process on titanium and zirconia implant material surfaces
RESEARCH AND EDUCATION
Biological and physicochemical implications of the aging process on titanium and zirconia implant material surfaces José Francisco S. S. da Rocha, DDS, MSc,a Erica D. de Avila, DDS, PhD,b Maria Sílvia M. Rigolin, DDS, MSc, PhD,c Paula A. Barbugli, DDS, MSc, PhD,d Danny O. M. Marin, DDS, MSc, PhD,e Francisco A. Mollo Junior, DDS, MSc, PhD,f and Janaina H. Jorge, DDS, MSc, PhDg The oral cavity represents an aggressive environment because of mechanical stimulus, the action of saliva, different temperature and pH conditions, and blood components, all of which can favor additional bacterial adhesion to the implant material and alter surface characteristics.1,2 The quality and type of timerelated material degradation and its implications for a biological system will depend on the material used and its location in the oral cavity. Among the materials examined for dental implant fabrication, titanium (Ti) is still considered the gold-standard material because of its biocompatibility and excellent corrosion resistance.2-11 However, the biological aging of Ti is associated with the natural process of degradation, which
ABSTRACT Statement of problem. Changes in physicochemical properties because of implant material aging and natural deterioration in the oral environment can facilitate microbial colonization and disturb the soft-tissue seal between the implant surfaces. Purpose. The purpose of this in vitro study was to investigate the effect of aging time on the physicochemical profile of titanium (Ti) and zirconia (ZrO2) implant materials. Further microbiology and cell analyses were used to provide insights into the physicochemical implications of biological behavior. Material and methods. Disk-shaped specimens of Ti and ZrO2 were submitted to roughness, morphology, and surface free energy (SFE) analyses before nonaging (NA) and after the aging process (A). To simulate natural aging, disks were subjected to low-temperature degradation (LTD) by using an autoclave at 134 ºC and 0.2 MPa pressure for 20 hours. The biological activities of the Ti and ZrO2 surfaces were determined by analyzing Candida albicans (C. albicans) biofilms and human gingival fibroblast (HGF) cell proliferation. For the microbiology assays, a variance analysis method (ANOVA) was used with the Tukey post hoc test. For the evaluation of cellular proliferation, the Kruskal-Wallis test followed by Dunn multiple comparisons were used. Results. Ti nonaging (TNA) and ZrO2 nonaging (ZNA) disks displayed hydrophilic and lipophilic properties, and this effect was sustained after the aging process. Low-temperature degradation resulted in a modest change in intermolecular interaction, with 1.06-fold for TA and 1.10-fold for ZA. No difference in biofilm formation was observed between NA and A disks of the same material. After 48 hours, the viability of the attached HGF cells was very similar to that in the NA and A groups, regardless of the tested material. Conclusion. The changes in the physicochemical properties of Ti and ZrO2 induced by the aging process do not interfere with C. albicans biofilm formation and HGF cell attachment, even after long-term exposure. (J Prosthet Dent 2020;-:---)
a
Masters graduate, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. Postdoctoral Research Fellow, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. c Postdoctoral Research Fellow, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. d Technical Assistant, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. e Assistant Professor, Department of Dentistry, Federal University of Santa Catarina (UFSC), Florianópolis, Brazil. f Associate Professor, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. g Associate Professor, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. b
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Changes in physicochemical properties because of implant material aging and natural deterioration in the oral environment can disturb local and systemic health. This perspective highlights the relevant effects of aging time on the composition, processing, and surface preparation of dental implant materials.
occurs as a consequence of the material’s interaction with the physiological body fluids and hydrocarbon accumulation onto the substrate.12 Although the surface of titanium is considered one of the most chemically stable, it is negatively affected by proteins upon oxidation.13,14 In addition, the amount of hydrocarbon adsorbed on the Ti surface interferes with the attachment of negatively charged blood proteins to the implant surface and reduces the degree of new bone formation surrounding the implant.15 Esthetic requirements have led to the use of zirconia as a prosthetic material.16 Nonetheless, the mechanical properties of zirconia are significantly compromised by aging kinetics.16,17 Zirconia exists in 3 crystal phases, monoclinic, tetragonal, and cubic, depending on the local conditions and can undergo transformation under the stress and humidity associated with temperature change.18-22 During the aging time, the increase in internal stresses in the presence of water initiates zirconia phase transformation from tetragonal to monoclinic. This phenomenon, known as lowtemperature degradation (LTD), originates at the surface and spreads inside the material leading to microcracks.23,24 In addition to biomechanical consequences, overall aging of material surfaces can affect their physicochemical properties and favor or hinder biofilm colonization.25-31 The roughness, wettability, and surface free energy (SFE) of implant materials affect their interactions with biological fluids and tissues.11,12,32,33 A roughness limit of approximately 0.2 mm has been defined, above which no further significant changes occur in regard to biofilm accumulation.34 As a consequence of the wetting properties, the materials will display a particular SFE. This physical phenomenon, caused by intermolecular interactions at an interface, is translated to the chemical nature of the biological organism on the substrate.35-38 Although the effect of aging on the biomechanical properties of commercial implant materials has been investigated,4-6,15-17,32 information regarding the importance of aging kinetics on a biological system is lacking. The purpose of this in vitro study was to provide insights concerning the effect of aging time on the
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physicochemical properties of the material surfaces of Ti and ZrO2 by testing the hypotheses that aging time affects the physicochemical properties of Ti and ZrO2 material surfaces, which, in turn, favors Candida albicans (C. albicans) biofilm formation, as well as the response and viability of human gingival fibroblast (HGF) cells. MATERIAL AND METHODS Disk-shaped specimens of titanium (Ti) and zirconia stabilized with yttrium (ZrO2) (Conexao Sistema de Protese Ltd) were obtained with a final size of 8 mm in diameter and 2 mm in thickness.7,8,11 The disks were then cleaned as described by de Avila et al8 before further experiments. A total of 72 Ti and 72 ZrO2 disks were allocated into 4 groups: Ti nonaging (TNA) (n=36), Ti aging (TA) (n=36), ZrO2 nonaging (ZNA) (n=36), and ZrO2 aging (ZA) (n=36). To simulate natural aging by LTD, experimental disks were submitted to a thermocycle in an autoclave (Bench-top autoclaves-25; Phoenix Luferco) at 134 C and 0.2 MPa pressure for 20 hours.39,40 Ethical approval for this study was obtained from the university (CAAE 68576317.0.0000.5416). Unstimulated whole human saliva was collected from 2 healthy adult volunteers. The saliva was pooled and clarified by centrifugation at 5000 g for 10 minutes at 4 ºC. The supernatant was passed through a 0.2-mm filter. The resulting saliva was stored at -80 ºC until use. The salivary pellicle was formed on the Ti and ZrO2 disks before and after the aging step for wetting, SFE, and biological assessment to simulate the oral environment. To achieve this, disks from each control and experimental group were placed into a 24-well plate (Thermo Fisher Scientific) submerged in 500 mL of saliva and incubated at 37 C for 60 minutes.
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Figure 2. Qualitative 2D laser scanning confocal microscopy images. A, TNA (original magnification ×10). B, TNA (original magnification ×63). C, TA (original magnification ×10). D, TA (original magnification ×63).
Profilometer and confocal laser scanning microscopy (CLSM) were used to determine the differences in the topography of the surfaces after the aging process. The contact angle and SFE were also measured to characterize the surfaces in all groups, but for these analyses, the measurements were performed by using NA and A salivacoated specimens. Before further experiments, the surface roughness of all specimens was measured by using a profilometer (Mitutoyo Surftest SJ-401; Mitutoyo Corp), according to de Avila et al.8 After the aging process, the disks were again submitted to the roughness analyses to compare both outcomes. A CLSM (Zeiss LSM 800) was used to characterize the 2-dimensional (2D) surface topography of the Ti and ZrO2 disks before and after the aging process. Images were recorded at ×10 and ×63 magnifications by using laser excitation at 640 nm. The experiment was performed in duplicate for each NA and A group. To investigate the influence of aging time on SFE behavior, 4 NA and A saliva-coated disks were selected, and deionized water and diiodomethane were used as liquids of distinct polarities. For the contact angle measurement, 2 mL of different wetting agents were dropped
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onto each surface, and the wettability of the solid surface was measured by using the sessile drop technique (raméhart Model 200; ramé-hart Instrument Co).41 The average value from each disk was calculated and entered into a specialized software program for the SFE calculation (DROPimage Advanced; ramé-hart Instrument Co) according to the equation described by Owens and Wendt42: Ysv=Ysl+Ylve2(Ysd.Yld)0.5e2(Ysp.Ylp)0.5, where Y is the interfacial surface tension; the subscripts s, l, and v denote solid, liquid, and vapor; the superscript d indicates the dispersion forces component; and the superscript p indicates the polar forces component. C. albicans ATCC 90028 was cultured as described by Pellissari et al.43 The NA and A saliva-coated Ti and ZrO2 disks were washed once with PBS and transferred to a 24-well plate (TPP tissue culture plates) containing 750 mL of C. albicans at a concentration of 1×106 cell/mL. The plate was incubated at 37 C with shaking at 75 rpm for 90 minutes. The disks were then washed twice with PBS to remove nonadherent fungal cells and transferred to a plate with Roswell Park Memorial Institute (RPMI) 1640 R6504 (Sigma-Aldrich Inc). The plate was incubated at
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Figure 2. (Continued) E, ZNA (original magnification ×10). F, ZNA (original magnification ×63). G, ZA (original magnification ×10). H, ZA (original magnification ×63). TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
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Figure 3. A, Contact angle values of distilled water (DW) and diiodomethane (D) on saliva-coated NA and A Ti and ZrO2 disks. B, Surface free energy between TNA and TA (white bar, Ti) and between ZNA and ZA (dark gray bar, ZrO2). TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
37 C on a rotatory shaker (75 rpm) for 48 hours, and the medium was refreshed after each 24 hours. The RPMI medium was then removed at the end of the incubation period, and the disks were gently placed into a 24-well plate and washed once with PBS before further
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processing. The PBS washed disks were placed into Falcon tubes containing 4.5 mL of sterile PBS. The tubes were exposed to ultrasound for 5 minutes to complete the detachment of C. albicans cells. Then, 100 mL of the fungal culture was transferred to an Eppendorf tube da Rocha et al
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1200000 1000000 800000 600000 400000 200000 0
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Groups Figure 4. Quantitative measurement of CFU/mL showing C. albicans biofilm formation on tissue culture polystyrene (white bar, C+) and saliva-coated NA and A Ti (light gray bar, Ti) and ZrO2 disks (dark gray bar, to ZrO2). Data shown as mean ±standard deviation. *P<.001. TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
containing 900 mL of fresh medium for the serial dilution procedure. Twenty-five microliters of each sampling well were dropped on fresh sabouraud dextrose agar (SDA) (Acumedia Manufacturers Inc) with chloramphenicol agar plate and incubated at 37 C for 48 hours. After the incubation period, the colony-forming units per milliliter (CFU/mL) were determined by using a digital colony counter (CP 600 Plus; Phoenix Ind Com Equipamentos Científicos Ltda). The experiment was performed in triplicate on 3 different occasions (n=9). C. albicans biofilm was also investigated by scanning electron microscopy (SEM) (JSM-6610LV; JEOL Ltda). The control and experimental PBS washed disks were fixed with 2.5% glutaraldehyde for 30 minutes at room temperature. The disks were gradually dehydrated for 5 minutes and covered with 20 nm of gold-sputtered film (Q150R; Quorum Technologies). Images were acquired at ×250 magnification from different areas of the slides by SEM. C. albicans inoculated onto the polystyrene plate served as positive controls for biofilm formation. For this analysis, SEM was performed in duplicate for each control and experimental group. The C. albicans cultures used in this study were also inoculated directly onto the polystyrene plate to serve as positive controls. Additionally, NA and A saliva-coated Ti and ZrO2 disks in addition to wells without C. albicans were incubated with sterile medium to serve as background controls. HGF cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (Lonza Walkersville, Inc) with 1.0 g/L glucose, 10% fetal bovine serum (FBS) (Gibco-BRL), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 C. The NA and A saliva-coated Ti and ZrO2 disks were washed once with PBS buffer solution and transferred to 24-well black plates (TPP tissue culture plates) containing 500 mL da Rocha et al
of DMEM. Then, 3×104 cells were plated onto each control and experimental disk, and the plate was maintained at 37 C for 24 hours under 5% CO2. In sequence, the medium was removed, and the same volume of fresh medium containing 10% alamarBlue (Invitrogen) was added to each sampling well. The fluorescence signals were measured after 48 hours by using a microplate fluorometer (Fluoroskan Ascent FL; Thermo Fisher Scientific) at excitation of 544 nm and emission wavelengths of 590 nm. The HGF cultures used in this study were also inoculated directly onto the polystyrene plate to serve as positive controls. Additionally, NA and A Ti and ZrO2 disks, as well as wells without cells, were incubated with sterile medium to serve as background controls. The experiment was performed in triplicate with 3 biological repetitions (n=9). To confirm the impact of aging time on cell behavior, HGF cells were cultured on each control (TNA and ZNA) and experimental (TA and ZA) disk, and cell morphology was analyzed by SEM after 48 hours. Fixation and specimen preparation protocols followed the same sequence described for the microbial biofilms. For the microbiology and cellular experiments, control disks were subjected to sterilization by gamma irradiation (IPEN) (Institute of Nuclear Energy Research). For the experimental groups, TA and ZA, the sterilization process was achieved by the autoclaving process itself. Based on determining that the roughness, wetting, and SFE data set had a normal distribution (D’AgostinoPearson omnibus test), statistical comparisons between NA and A groups were performed by using the 2-tailed paired t test. When the material surfaces were considered, the comparisons were completed by using the 2tailed unpaired t test. For the microbiology assays, normal distribution and homoscedasticity were confirmed, and a variance analysis method (ANOVA) was used with the Tukey post hoc test. For the evaluation of cellular proliferation, as the data were not normally distributed and as P<.05, the Kruskal-Wallis test followed by Dunn multiple comparisons was used. All statistical analyses were performed by using a statistical software program (GraphPad Prism, v5.0c; GraphPad Software) (a=.05). RESULTS Roughness analyses confirmed that only Ti and ZrO2 disks with roughness values below 0.2 mm had been selected for this study. No differences were observed before and after aging simulated by LTD (Fig. 1). The effect of the aging process on surface topography was also studied by using 2D images obtained by CLSM (Fig. 2). Qualitative information revealed that the natural grooves inherent to the Ti machined surfaces remained in the TA group. Regarding ZrO2, the images
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Figure 5. Scanning electron microscopy (SEM) images (original magnification ×250) revealing that colonization of Ti surfaces is clearly covered with C. albicans compared with sparse colonization on ZrO2 material. A, Positive control. B, Titanium nonaging (TNA). C, Zirconia nonaging (ZNA). D, Titanium aging (TA). E, Zirconia aging (ZA).
showed a domain structure in a ceramic grain pattern, which was assumed to be from sintering. The surface morphologies of NA and A specimens did not differ from each other and were in good agreement with their Ra value. A contact angle less than 90 degrees signifies that wetting of the material is favorable and that fluid will spread over a large area on the surface. Thus, it is possible to assume that saliva-coated TNA
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and ZNA disks displayed hydrophilic and lipophilic properties that were sustainable even after the aging process. However, a mild change from 39.7 C to 29.8 C was observed for ZrO2 before and after the disks had undergone LTD (Fig. 3A). Considering the wetting properties of solid surfaces, SFE testing demonstrated that the aging process resulted in a modest change in the intermolecular interactions of Ti
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Cell Proliferation (Fluorescence excitation/emission [544/590nm] )
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observed between Ti and ZrO2, regardless of the aging time. Fibroblasts were confluent on both materials; however, Ti seemed to control the position of cells in an organized pattern on the surface, with a better flat spindle-shaped cell alignment (Fig. 7).
2000 1500 1000
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C+
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Groups Figure 6. Effect of aging process on HGF cell viability after 48 hours of culture by using quantitative measurement of alamarBlue staining on tissue culture polystyrene (green bar, C+), Ti disks (orange and gray bars, Ti), and ZrO2disks (blue and yellow bars, to ZrO2). Data are shown as mean ±SD. Statistically significant differences are indicated as P<.05. TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
and ZrO2, which were 1.06-fold for TA and 1.10-fold for ZA (Fig. 3B). The surface properties of Ti and ZrO2 after the aging process induced by autoclaving were examined to determine any influence on C. albicans biofilm formation. The data demonstrated no differences in biofilm formation between saliva-coated NA and A disks in the same material group (Fig. 4). With respect to the type of material, regardless of the aging variable, CFU/ mL disclosed a statistically significant difference (P<.001) in overall biofilm with 2.8-fold fewer viable cells/mL on the ZNA surface than TNA and 4.0-fold fewer viable cells per mL on the ZA surface than TA. Higher magnification imaging by SEM revealed that the colonization of the Ti surface was covered with C. albicans embedded in extracellular material compared with the sparse colonization on the ZrO2 material. ZrO2 seemed to interfere with C. albicans attachment, revealing considerably less fungi on the disks. Consistent with the quantitative outcomes, when the aging process was considerable, the area coverage and the frequency of hyphal elements appeared to be similar between saliva-coated NA disks and A in the same material group (Fig. 5). The effect of aging time was also evaluated for mammalian cells. The proliferation assay revealed that after 48 hours of incubation, the viability of the attached HGF cells was similar between both saliva-coated NA and A groups in the same material group (Fig. 6). A detailed qualitative assessment of the attached cells with SEM confirmed no difference in HGF cell spreading and morphology between saliva-coated NA and A disks compared with their respective material counterparts. A discreet difference in overall HGF cell morphology was
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An autoclave method was used to estimate the behavior of implant materials in the oral cavity, and roughness was not affected by the aging process. However, LTD reduced the contact angle between water and the material, and the surface became more hydrophilic. Although a disruption of energy at the surface was confirmed after the disks were autoclaved, the research hypothesis that there is a relationship between overall superficial modifications and the amount of C. albicans accumulation and fibroblast proliferation was rejected. An aging method with temperature and humidity stimuli at 0.2 MPa and 134 C was used to induce monoclinic phase transformation of ZrO2, as recommended by the International Organization for Standardization (ISO Standard 13356:2008) for the use of zirconia in dental implants.39 The data revealed that aging time did not affect the roughness of either material. In the case of ZrO2, the absence of a topographic effect was not expected.5 The progress of the transformation can lead to surface degradation, and an increase in the transformation area could therefore result in material loss and increasing surface roughness.21,22 Thus, the technique to measure the peak value from both materials is a limitation of the present study. It is possible that the surface roughness machine was less accurate in calculating irregularities created after autoclave processing. However, this technique has been accepted by the scientific community. This means that even if a more accurate technique had been used, the difference itself would not have been clinically relevant. Microscopy images also revealed no difference in surface topography between NA and A saliva-coated Ti and ZrO2 disks. A significant change from hydrophilic to more hydrophilic surfaces was confirmed for Ti and ZrO2 substrates. The temperature and pressure during autoclaving can affect the oxidation states and induce the reduction of Ti in the oxide layer from Ti4+ (more stable and less polar) to the Ti3+.37,38 As previously mentioned, oral conditions associated with humidity and temperature can induce phase transformation of ZrO2.5,6,20,40 Roughness, wettability, and SFE seem to play important roles in the process of microbial adhesion on material surfaces.7,8,11,34 Nonspreading on high-energy surfaces may occur regardless of whether the SFE of such liquids is comparable with the solid chemical nature.41 As C. albicans species exhibit hydrophobicity on hyphae, which causes stronger C. albicans adhesion to THE JOURNAL OF PROSTHETIC DENTISTRY
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Figure 7. Scanning electron microscopy (SEM) images (original magnification ×250) revealing discreet difference in overall HGF cell morphology between Ti and ZrO2 regardless of aging time. HGF cells presented nonorganized pattern on ZrO2 surface, with notable alteration in cell alignment (red arrow). A, Positive control. B, Titanium nonaging (TNA). C, Zirconia non-aging (ZNA). D, Titanium aging (TA). E, Zirconia aging (ZA).
hydrophobic substrata,30,31 a significant difference was expected in overall biofilm formation on the NA surface. Surprisingly, the results revealed similarity in the biofilm formed on NA and A disks even after 48 hours of incubation. However, when both materials were compared, a notable statistical difference in overall biofilm could be observed with 2.8-fold fewer viable cells/mL on ZNA than the TNA surface and 4.0-fold fewer viable cells/mL on ZA than the TA surface. Considering that the oral
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environment contains more than 600 microbial phylotypes,1 findings using only a single pathogenic fungal species should be interpreted with caution. The results demonstrated that the changes in hydrophilicity and SFE did not disturb the proliferation and morphology of fibroblast cells. Thereby, aging time decreases the bioactivity and osteoconductivity of titanium by decreasing the amount and quality of cell attachment.12,26,38 Here, contrary to expectation, the contact
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angle between water and TA was significantly lower than that to water and TNA, whereas the fluorescence intensity, representative of cell proliferation, was similar. In the case of fibroblasts, contradictory results suggest that cellular adhesion cannot be completely explained by the surface wettability. In the cell-based experiments, a discreet difference in cell shape and orientation was observed for the ZrO2 material, regardless of the aging process. The qualitative assays revealed a more mature morphology defined by elongated and aligned cells on Ti substrates, indicating better attachment and cell surface affinity between HGF and Ti. Further investigations should provide detailed information regarding the aging effect on titanium and ZrO2 material composition and structure by using multispecies biofilm models and tissue culture to evaluate the inflammatory response. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. Changes in hydrophilicity and SFE on commercial implant abutment materials induced by the aging process do not interfere with C. albicans biofilm formation, an effect that is also maintained for mammalian cells. 2. The aging process of the Ti and ZrO2 surfaces was not toxic to HGF cells, even after long-term exposure. 3. The Ti topography pattern seemed to affect cell behavior, as a more mature morphology defined by elongated and aligned cells was recorded on the Ti substrate. REFERENCES 1. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770-83. 2. Choi SH, Jeong WS, Cha JY, Lee JH, Lee KJ, Yu HS, et al. Overcoming the biological aging of titanium using a wet storage method after ultraviolet treatment. Sci Rep 2017;7:3833. 3. Correa DRN, Kuroda PAB, Lourenco ML, Buzalaf MAR, Mendoza ME, Archanjo BS, et al. Microstructure and selected mechanical properties of aged Ti-15Zr-based alloys for biomedical applications. Mater Sci Eng C Mater Biol Appl 2018;91:762-71. 4. Papanagiotou HP, Morgano SM, Giordano RA, Pober R. In vitro evaluation of low-temperature aging effects and finishing procedures on the flexural strength and structural stability of Y-TZP dental ceramics. J Prosthet Dent 2006;96:154-64. 5. Pereira GKR, Muller C, Wandscher VF, Rippe MP, Kleverlaan CJ, Valandro LF. Comparison of different low-temperature aging protocols: its effects on the mechanical behavior of Y-TZP ceramics. J Mech Behav Biomed Mater 2016;60:324-30. 6. Pereira GKR, Silvestri T, Amaral M, Rippe MP, Kleverlaan CJ, Valandro LF. Fatigue limit of polycrystalline zirconium oxide ceramics: Effect of grinding and low-temperature aging. J Mech Behav Biomed Mater 2016;61:45-54. 7. de Avila ED, Avila-Campos MJ, Vergani CE, Spolidorio DM, Mollo Fde A Jr. Structural and quantitative analysis of a mature anaerobic biofilm on different implant abutment surfaces. J Prosthet Dent 2016;115:428-36. 8. de Avila ED, de Molon RS, Lima BP, Lux R, Shi W, Junior MJ, et al. Impact of physical chemical characteristics of abutment implant surfaces on bacteria adhesion. J Oral Implantol 2016;42:153-8. 9. de Avila ED, de Molon RS, Palomari Spolidorio DM, de Assis Mollo F Jr. Implications of surface and bulk properties of abutment implants and their degradation in the health of periodontal tissue. Materials (Basel) 2013;6:5951-66.
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Acta Biomater 2017;54:469-78. 15. Att W, Hori N, Takeuchi M, Ouyang J, Yang Y, Anpo M, et al. Timedependent degradation of titanium osteoconductivity: an implication of biological aging of implant materials. Biomaterials 2009;30:5352-63. 16. Denry I, Kelly JR. Emerging ceramic-based materials for dentistry. J Dent Res 2014;93:1235-42. 17. Chevalier J, Loh J, Gremillard L, Meille S, Adolfson E. Low-temperature degradation in zirconia with a porous surface. Acta Biomater 2011;7:2986-93. 18. Hallmann L, Ulmer P, Wille S, Kern M. Effect of differences in coefficient of thermal expansion of veneer and Y-TZP ceramics on interface phase transformation. J Prosthet Dent 2014;112:591-9. 19. Scherrer SS, Cattani-Lorente M, Vittecoq E, de Mestral F, Griggs JA, Wiskott HW. Fatigue behavior in water of Y-TZP zirconia ceramics after abrasion with 30 mum silica-coated alumina particles. Dent Mater 2011;27: 28-42. 20. Amaral M, Valandro LF, Bottino MA, Souza RO. Low-temperature degradation of a Y-TZP ceramic after surface treatments. J Biomed Mater Res B Appl Biomater 2013;101:1387-92. 21. Cattani-Lorente M, Durual S, Amez-Droz M, Wiskott HW, Scherrer SS. Hydrothermal degradation of a 3Y-TZP translucent dental ceramic: A comparison of numerical predictions with experimental data after 2 years of aging. Dent Mater 2016;32:394-402. 22. Lughi V, Sergo V. Low temperature degradation -aging- of zirconia: A critical review of the relevant aspects in dentistry. Dent Mater 2010;26:807-20. 23. Ritter JE. Predicting lifetimes of materials and material structures. Dent Mater 1995;11:142-6. 24. Zhang Y, Lawn B. Long-term strength of ceramics for biomedical applications. J Biomed Mater Res B Appl Biomater 2004;69:166-72. 25. Pereira G, Amaral M, Cesar PF, Bottino MC, Kleverlaan CJ, Valandro LF. Effect of low-temperature aging on the mechanical behavior of ground YTZP. J Mech Behav Biomed Mater 2015;45:183-92. 26. Miyauchi T, Yamada M, Yamamoto A, Iwasa F, Suzawa T, Kamijo R, et al. The enhanced characteristics of osteoblast adhesion to photofunctionalized nanoscale TiO2 layers on biomaterials surfaces. Biomaterials 2010;31:3827-39. 27. Esfahanizadeh N, Motalebi S, Daneshparvar N, Akhoundi N, Bonakdar S. Morphology, proliferation, and gene expression of gingival fibroblasts on laser-lok, titanium, and zirconia surfaces. Lasers Med Sci 2016;31:863-73. 28. Nevins M, Kim DM, Jun SH, Guze K, Schupbach P, Nevins ML. Histologic evidence of a connective tissue attachment to laser microgrooved abutments: a canine study. Int J Periodontics Restorative Dent 2010;30:245-55. 29. Rutkunas V, Bukelskiene V, Sabaliauskas V, Balciunas E, Malinauskas M, Baltriukiene D. Assessment of human gingival fibroblast interaction with dental implant abutment materials. J Mater Sci Mater Med 2015;26:169. 30. Beaussart A, Alsteens D, El-Kirat-Chatel S, Lipke PN, Kucharíková S, Van Dijck P, et al. Single-molecule imaging and functional analysis of Als adhesins and mannans during Candida albicans morphogenesis. ACS Nano 2012;6:10950-64. 31. Glee PM, Sundstrom P, Hazen KC. Expression of surface hydrophobic proteins by Candida albicans in vivo. Infect Immun 1995;63:1373-9. 32. Kamo M, Kyomoto M, Miyaji F. Time course of surface characteristics of alkali- and heat-treated titanium dental implants during vacuum storage. J Biomed Mater Res B Appl Biomater 2017;105:1453-60. 33. Spriano S, Sarath Chandra V, Cochis A, Uberti F, Rimondini L, Bertone E, et al. How do wettability, zeta potential and hydroxylation degree affect the biological response of biomaterials? Mater Sci Eng C Mater Biol Appl 2017;74:542-55. 34. Song F, Koo H, Ren D. Effects of material properties on bacterial adhesion and biofilm formation. J Dent Res 2015;94:1027-34. 35. Bollen CM, Papaioanno W, Van Eldere J, Schepers E, Quirynen M, van Steenberghe D. The influence of abutment surface roughness on plaque accumulation and peri-implant mucositis. Clin Oral Implants Res 1996;7: 201-11. 36. Rigolin MSM, de Avila ED, Basso FG, Hebling J, de SCCA, Mollo Junior FA. Effect of different implant abutment surfaces on OBA-09 epithelial cell adhesion. Microsc Res Tech 2017;80:1304-9. 37. de Avila ED, Lima BP, Sekiya T, Torii Y, Ogawa T, Shi W, et al. Effect of UVphotofunctionalization on oral bacterial attachment and biofilm formation to titanium implant material. Biomaterials 2015;67:84-92.
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38. Hori N, Iwasa F, Tsukimura N, Sugita Y, Ueno T, Kojima N, et al. Effects of UV photofunctionalization on the nanotopography enhanced initial bioactivity of titanium. Acta Biomater 2011;7:3679-91. 39. Wille S, Zumstrull P, Kaidas V, Jessen LK, Kern M. Low temperature degradation of single layers of multilayered zirconia in comparison to conventional unshaded zirconia: phase transformation and flexural strength. J Mech Behav Biomed Mater 2018;77:171-5. 40. Pereira GKR, Venturini AB, Silvestri T, Dapieve KS, Montagner AF, Soares FZM, et al. Low-temperature degradation of Y-TZP ceramics: A systematic review and meta-analysis. J Mech Behav Biomed Mater 2015;55:151-63. 41. Zisman WA. Relation to the equilibrium contact angle to liquid and solid constitution. Adv Chem 1964:1-51. 42. Owens DK, Wendt RC. Estimation of the surface free energy of polymers. J Appl Polym Sci 1969;13:1741-7. 43. Pellissari CV, Pavarina AC, Bagnato VS, Mima EG, Vergani CE, Jorge JH. Cytotoxicity of antimicrobial photodynamic inactivation on epithelial cells when co-cultured with Candida albicans. Photochem Photobiol Sci 2016;15: 682-90.
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Corresponding author: Dr Erica D. de Avila Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara São Paulo State University (UNESP) Rua Humaita, 1680, 14.801-903, Araraquara São Paulo BRAZIL Email: [email protected] Acknowledgments The authors acknowledge the Conexão Sistemas de Próteses Ltda for donating specimens, CAPES (Coordination for the Improvement of Higher Education Personnel) for the award of the scholarship, and Erica Dorigatti de Avila for her support in the preparation of the manuscript. Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.11.024
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Biological and physicochemical implications of the aging process on titanium and zirconia implant material surfaces José Francisco S. S. da Rocha, DDS, MSc,a Erica D. de Avila, DDS, PhD,b Maria Sílvia M. Rigolin, DDS, MSc, PhD,c Paula A. Barbugli, DDS, MSc, PhD,d Danny O. M. Marin, DDS, MSc, PhD,e Francisco A. Mollo Junior, DDS, MSc, PhD,f and Janaina H. Jorge, DDS, MSc, PhDg The oral cavity represents an aggressive environment because of mechanical stimulus, the action of saliva, different temperature and pH conditions, and blood components, all of which can favor additional bacterial adhesion to the implant material and alter surface characteristics.1,2 The quality and type of timerelated material degradation and its implications for a biological system will depend on the material used and its location in the oral cavity. Among the materials examined for dental implant fabrication, titanium (Ti) is still considered the gold-standard material because of its biocompatibility and excellent corrosion resistance.2-11 However, the biological aging of Ti is associated with the natural process of degradation, which
ABSTRACT Statement of problem. Changes in physicochemical properties because of implant material aging and natural deterioration in the oral environment can facilitate microbial colonization and disturb the soft-tissue seal between the implant surfaces. Purpose. The purpose of this in vitro study was to investigate the effect of aging time on the physicochemical profile of titanium (Ti) and zirconia (ZrO2) implant materials. Further microbiology and cell analyses were used to provide insights into the physicochemical implications of biological behavior. Material and methods. Disk-shaped specimens of Ti and ZrO2 were submitted to roughness, morphology, and surface free energy (SFE) analyses before nonaging (NA) and after the aging process (A). To simulate natural aging, disks were subjected to low-temperature degradation (LTD) by using an autoclave at 134 ºC and 0.2 MPa pressure for 20 hours. The biological activities of the Ti and ZrO2 surfaces were determined by analyzing Candida albicans (C. albicans) biofilms and human gingival fibroblast (HGF) cell proliferation. For the microbiology assays, a variance analysis method (ANOVA) was used with the Tukey post hoc test. For the evaluation of cellular proliferation, the Kruskal-Wallis test followed by Dunn multiple comparisons were used. Results. Ti nonaging (TNA) and ZrO2 nonaging (ZNA) disks displayed hydrophilic and lipophilic properties, and this effect was sustained after the aging process. Low-temperature degradation resulted in a modest change in intermolecular interaction, with 1.06-fold for TA and 1.10-fold for ZA. No difference in biofilm formation was observed between NA and A disks of the same material. After 48 hours, the viability of the attached HGF cells was very similar to that in the NA and A groups, regardless of the tested material. Conclusion. The changes in the physicochemical properties of Ti and ZrO2 induced by the aging process do not interfere with C. albicans biofilm formation and HGF cell attachment, even after long-term exposure. (J Prosthet Dent 2020;-:---)
a
Masters graduate, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. Postdoctoral Research Fellow, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. c Postdoctoral Research Fellow, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. d Technical Assistant, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. e Assistant Professor, Department of Dentistry, Federal University of Santa Catarina (UFSC), Florianópolis, Brazil. f Associate Professor, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. g Associate Professor, Department of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, São Paulo State University (UNESP), Araraquara, Brazil. b
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Changes in physicochemical properties because of implant material aging and natural deterioration in the oral environment can disturb local and systemic health. This perspective highlights the relevant effects of aging time on the composition, processing, and surface preparation of dental implant materials.
occurs as a consequence of the material’s interaction with the physiological body fluids and hydrocarbon accumulation onto the substrate.12 Although the surface of titanium is considered one of the most chemically stable, it is negatively affected by proteins upon oxidation.13,14 In addition, the amount of hydrocarbon adsorbed on the Ti surface interferes with the attachment of negatively charged blood proteins to the implant surface and reduces the degree of new bone formation surrounding the implant.15 Esthetic requirements have led to the use of zirconia as a prosthetic material.16 Nonetheless, the mechanical properties of zirconia are significantly compromised by aging kinetics.16,17 Zirconia exists in 3 crystal phases, monoclinic, tetragonal, and cubic, depending on the local conditions and can undergo transformation under the stress and humidity associated with temperature change.18-22 During the aging time, the increase in internal stresses in the presence of water initiates zirconia phase transformation from tetragonal to monoclinic. This phenomenon, known as lowtemperature degradation (LTD), originates at the surface and spreads inside the material leading to microcracks.23,24 In addition to biomechanical consequences, overall aging of material surfaces can affect their physicochemical properties and favor or hinder biofilm colonization.25-31 The roughness, wettability, and surface free energy (SFE) of implant materials affect their interactions with biological fluids and tissues.11,12,32,33 A roughness limit of approximately 0.2 mm has been defined, above which no further significant changes occur in regard to biofilm accumulation.34 As a consequence of the wetting properties, the materials will display a particular SFE. This physical phenomenon, caused by intermolecular interactions at an interface, is translated to the chemical nature of the biological organism on the substrate.35-38 Although the effect of aging on the biomechanical properties of commercial implant materials has been investigated,4-6,15-17,32 information regarding the importance of aging kinetics on a biological system is lacking. The purpose of this in vitro study was to provide insights concerning the effect of aging time on the
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physicochemical properties of the material surfaces of Ti and ZrO2 by testing the hypotheses that aging time affects the physicochemical properties of Ti and ZrO2 material surfaces, which, in turn, favors Candida albicans (C. albicans) biofilm formation, as well as the response and viability of human gingival fibroblast (HGF) cells. MATERIAL AND METHODS Disk-shaped specimens of titanium (Ti) and zirconia stabilized with yttrium (ZrO2) (Conexao Sistema de Protese Ltd) were obtained with a final size of 8 mm in diameter and 2 mm in thickness.7,8,11 The disks were then cleaned as described by de Avila et al8 before further experiments. A total of 72 Ti and 72 ZrO2 disks were allocated into 4 groups: Ti nonaging (TNA) (n=36), Ti aging (TA) (n=36), ZrO2 nonaging (ZNA) (n=36), and ZrO2 aging (ZA) (n=36). To simulate natural aging by LTD, experimental disks were submitted to a thermocycle in an autoclave (Bench-top autoclaves-25; Phoenix Luferco) at 134 C and 0.2 MPa pressure for 20 hours.39,40 Ethical approval for this study was obtained from the university (CAAE 68576317.0.0000.5416). Unstimulated whole human saliva was collected from 2 healthy adult volunteers. The saliva was pooled and clarified by centrifugation at 5000 g for 10 minutes at 4 ºC. The supernatant was passed through a 0.2-mm filter. The resulting saliva was stored at -80 ºC until use. The salivary pellicle was formed on the Ti and ZrO2 disks before and after the aging step for wetting, SFE, and biological assessment to simulate the oral environment. To achieve this, disks from each control and experimental group were placed into a 24-well plate (Thermo Fisher Scientific) submerged in 500 mL of saliva and incubated at 37 C for 60 minutes.
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Figure 2. Qualitative 2D laser scanning confocal microscopy images. A, TNA (original magnification ×10). B, TNA (original magnification ×63). C, TA (original magnification ×10). D, TA (original magnification ×63).
Profilometer and confocal laser scanning microscopy (CLSM) were used to determine the differences in the topography of the surfaces after the aging process. The contact angle and SFE were also measured to characterize the surfaces in all groups, but for these analyses, the measurements were performed by using NA and A salivacoated specimens. Before further experiments, the surface roughness of all specimens was measured by using a profilometer (Mitutoyo Surftest SJ-401; Mitutoyo Corp), according to de Avila et al.8 After the aging process, the disks were again submitted to the roughness analyses to compare both outcomes. A CLSM (Zeiss LSM 800) was used to characterize the 2-dimensional (2D) surface topography of the Ti and ZrO2 disks before and after the aging process. Images were recorded at ×10 and ×63 magnifications by using laser excitation at 640 nm. The experiment was performed in duplicate for each NA and A group. To investigate the influence of aging time on SFE behavior, 4 NA and A saliva-coated disks were selected, and deionized water and diiodomethane were used as liquids of distinct polarities. For the contact angle measurement, 2 mL of different wetting agents were dropped
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onto each surface, and the wettability of the solid surface was measured by using the sessile drop technique (raméhart Model 200; ramé-hart Instrument Co).41 The average value from each disk was calculated and entered into a specialized software program for the SFE calculation (DROPimage Advanced; ramé-hart Instrument Co) according to the equation described by Owens and Wendt42: Ysv=Ysl+Ylve2(Ysd.Yld)0.5e2(Ysp.Ylp)0.5, where Y is the interfacial surface tension; the subscripts s, l, and v denote solid, liquid, and vapor; the superscript d indicates the dispersion forces component; and the superscript p indicates the polar forces component. C. albicans ATCC 90028 was cultured as described by Pellissari et al.43 The NA and A saliva-coated Ti and ZrO2 disks were washed once with PBS and transferred to a 24-well plate (TPP tissue culture plates) containing 750 mL of C. albicans at a concentration of 1×106 cell/mL. The plate was incubated at 37 C with shaking at 75 rpm for 90 minutes. The disks were then washed twice with PBS to remove nonadherent fungal cells and transferred to a plate with Roswell Park Memorial Institute (RPMI) 1640 R6504 (Sigma-Aldrich Inc). The plate was incubated at
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Figure 2. (Continued) E, ZNA (original magnification ×10). F, ZNA (original magnification ×63). G, ZA (original magnification ×10). H, ZA (original magnification ×63). TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
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Figure 3. A, Contact angle values of distilled water (DW) and diiodomethane (D) on saliva-coated NA and A Ti and ZrO2 disks. B, Surface free energy between TNA and TA (white bar, Ti) and between ZNA and ZA (dark gray bar, ZrO2). TA, titanium aging; TNA, titanium nonaging; ZA, zirconia aging; ZNA, zirconia nonaging.
37 C on a rotatory shaker (75 rpm) for 48 hours, and the medium was refreshed after each 24 hours. The RPMI medium was then removed at the end of the incubation period, and the disks were gently placed into a 24-well plate and washed once with PBS before further
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processing. The PBS washed disks were placed into Falcon tubes containing 4.5 mL of sterile PBS. The tubes were exposed to ultrasound for 5 minutes to complete the detachment of C. albicans cells. Then, 100 mL of the fungal culture was transferred to an Eppendorf tube da Rocha et al
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containing 900 mL of fresh medium for the serial dilution procedure. Twenty-five microliters of each sampling well were dropped on fresh sabouraud dextrose agar (SDA) (Acumedia Manufacturers Inc) with chloramphenicol agar plate and incubated at 37 C for 48 hours. After the incubation period, the colony-forming units per milliliter (CFU/mL) were determined by using a digital colony counter (CP 600 Plus; Phoenix Ind Com Equipamentos Científicos Ltda). The experiment was performed in triplicate on 3 different occasions (n=9). C. albicans biofilm was also investigated by scanning electron microscopy (SEM) (JSM-6610LV; JEOL Ltda). The control and experimental PBS washed disks were fixed with 2.5% glutaraldehyde for 30 minutes at room temperature. The disks were gradually dehydrated for 5 minutes and covered with 20 nm of gold-sputtered film (Q150R; Quorum Technologies). Images were acquired at ×250 magnification from different areas of the slides by SEM. C. albicans inoculated onto the polystyrene plate served as positive controls for biofilm formation. For this analysis, SEM was performed in duplicate for each control and experimental group. The C. albicans cultures used in this study were also inoculated directly onto the polystyrene plate to serve as positive controls. Additionally, NA and A saliva-coated Ti and ZrO2 disks in addition to wells without C. albicans were incubated with sterile medium to serve as background controls. HGF cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (Lonza Walkersville, Inc) with 1.0 g/L glucose, 10% fetal bovine serum (FBS) (Gibco-BRL), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 C. The NA and A saliva-coated Ti and ZrO2 disks were washed once with PBS buffer solution and transferred to 24-well black plates (TPP tissue culture plates) containing 500 mL da Rocha et al
of DMEM. Then, 3×104 cells were plated onto each control and experimental disk, and the plate was maintained at 37 C for 24 hours under 5% CO2. In sequence, the medium was removed, and the same volume of fresh medium containing 10% alamarBlue (Invitrogen) was added to each sampling well. The fluorescence signals were measured after 48 hours by using a microplate fluorometer (Fluoroskan Ascent FL; Thermo Fisher Scientific) at excitation of 544 nm and emission wavelengths of 590 nm. The HGF cultures used in this study were also inoculated directly onto the polystyrene plate to serve as positive controls. Additionally, NA and A Ti and ZrO2 disks, as well as wells without cells, were incubated with sterile medium to serve as background controls. The experiment was performed in triplicate with 3 biological repetitions (n=9). To confirm the impact of aging time on cell behavior, HGF cells were cultured on each control (TNA and ZNA) and experimental (TA and ZA) disk, and cell morphology was analyzed by SEM after 48 hours. Fixation and specimen preparation protocols followed the same sequence described for the microbial biofilms. For the microbiology and cellular experiments, control disks were subjected to sterilization by gamma irradiation (IPEN) (Institute of Nuclear Energy Research). For the experimental groups, TA and ZA, the sterilization process was achieved by the autoclaving process itself. Based on determining that the roughness, wetting, and SFE data set had a normal distribution (D’AgostinoPearson omnibus test), statistical comparisons between NA and A groups were performed by using the 2-tailed paired t test. When the material surfaces were considered, the comparisons were completed by using the 2tailed unpaired t test. For the microbiology assays, normal distribution and homoscedasticity were confirmed, and a variance analysis method (ANOVA) was used with the Tukey post hoc test. For the evaluation of cellular proliferation, as the data were not normally distributed and as P<.05, the Kruskal-Wallis test followed by Dunn multiple comparisons was used. All statistical analyses were performed by using a statistical software program (GraphPad Prism, v5.0c; GraphPad Software) (a=.05). RESULTS Roughness analyses confirmed that only Ti and ZrO2 disks with roughness values below 0.2 mm had been selected for this study. No differences were observed before and after aging simulated by LTD (Fig. 1). The effect of the aging process on surface topography was also studied by using 2D images obtained by CLSM (Fig. 2). Qualitative information revealed that the natural grooves inherent to the Ti machined surfaces remained in the TA group. Regarding ZrO2, the images
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Figure 5. Scanning electron microscopy (SEM) images (original magnification ×250) revealing that colonization of Ti surfaces is clearly covered with C. albicans compared with sparse colonization on ZrO2 material. A, Positive control. B, Titanium nonaging (TNA). C, Zirconia nonaging (ZNA). D, Titanium aging (TA). E, Zirconia aging (ZA).
showed a domain structure in a ceramic grain pattern, which was assumed to be from sintering. The surface morphologies of NA and A specimens did not differ from each other and were in good agreement with their Ra value. A contact angle less than 90 degrees signifies that wetting of the material is favorable and that fluid will spread over a large area on the surface. Thus, it is possible to assume that saliva-coated TNA
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and ZNA disks displayed hydrophilic and lipophilic properties that were sustainable even after the aging process. However, a mild change from 39.7 C to 29.8 C was observed for ZrO2 before and after the disks had undergone LTD (Fig. 3A). Considering the wetting properties of solid surfaces, SFE testing demonstrated that the aging process resulted in a modest change in the intermolecular interactions of Ti
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Cell Proliferation (Fluorescence excitation/emission [544/590nm] )
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observed between Ti and ZrO2, regardless of the aging time. Fibroblasts were confluent on both materials; however, Ti seemed to control the position of cells in an organized pattern on the surface, with a better flat spindle-shaped cell alignment (Fig. 7).
2000 1500 1000
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C+
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and ZrO2, which were 1.06-fold for TA and 1.10-fold for ZA (Fig. 3B). The surface properties of Ti and ZrO2 after the aging process induced by autoclaving were examined to determine any influence on C. albicans biofilm formation. The data demonstrated no differences in biofilm formation between saliva-coated NA and A disks in the same material group (Fig. 4). With respect to the type of material, regardless of the aging variable, CFU/ mL disclosed a statistically significant difference (P<.001) in overall biofilm with 2.8-fold fewer viable cells/mL on the ZNA surface than TNA and 4.0-fold fewer viable cells per mL on the ZA surface than TA. Higher magnification imaging by SEM revealed that the colonization of the Ti surface was covered with C. albicans embedded in extracellular material compared with the sparse colonization on the ZrO2 material. ZrO2 seemed to interfere with C. albicans attachment, revealing considerably less fungi on the disks. Consistent with the quantitative outcomes, when the aging process was considerable, the area coverage and the frequency of hyphal elements appeared to be similar between saliva-coated NA disks and A in the same material group (Fig. 5). The effect of aging time was also evaluated for mammalian cells. The proliferation assay revealed that after 48 hours of incubation, the viability of the attached HGF cells was similar between both saliva-coated NA and A groups in the same material group (Fig. 6). A detailed qualitative assessment of the attached cells with SEM confirmed no difference in HGF cell spreading and morphology between saliva-coated NA and A disks compared with their respective material counterparts. A discreet difference in overall HGF cell morphology was
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An autoclave method was used to estimate the behavior of implant materials in the oral cavity, and roughness was not affected by the aging process. However, LTD reduced the contact angle between water and the material, and the surface became more hydrophilic. Although a disruption of energy at the surface was confirmed after the disks were autoclaved, the research hypothesis that there is a relationship between overall superficial modifications and the amount of C. albicans accumulation and fibroblast proliferation was rejected. An aging method with temperature and humidity stimuli at 0.2 MPa and 134 C was used to induce monoclinic phase transformation of ZrO2, as recommended by the International Organization for Standardization (ISO Standard 13356:2008) for the use of zirconia in dental implants.39 The data revealed that aging time did not affect the roughness of either material. In the case of ZrO2, the absence of a topographic effect was not expected.5 The progress of the transformation can lead to surface degradation, and an increase in the transformation area could therefore result in material loss and increasing surface roughness.21,22 Thus, the technique to measure the peak value from both materials is a limitation of the present study. It is possible that the surface roughness machine was less accurate in calculating irregularities created after autoclave processing. However, this technique has been accepted by the scientific community. This means that even if a more accurate technique had been used, the difference itself would not have been clinically relevant. Microscopy images also revealed no difference in surface topography between NA and A saliva-coated Ti and ZrO2 disks. A significant change from hydrophilic to more hydrophilic surfaces was confirmed for Ti and ZrO2 substrates. The temperature and pressure during autoclaving can affect the oxidation states and induce the reduction of Ti in the oxide layer from Ti4+ (more stable and less polar) to the Ti3+.37,38 As previously mentioned, oral conditions associated with humidity and temperature can induce phase transformation of ZrO2.5,6,20,40 Roughness, wettability, and SFE seem to play important roles in the process of microbial adhesion on material surfaces.7,8,11,34 Nonspreading on high-energy surfaces may occur regardless of whether the SFE of such liquids is comparable with the solid chemical nature.41 As C. albicans species exhibit hydrophobicity on hyphae, which causes stronger C. albicans adhesion to THE JOURNAL OF PROSTHETIC DENTISTRY
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Figure 7. Scanning electron microscopy (SEM) images (original magnification ×250) revealing discreet difference in overall HGF cell morphology between Ti and ZrO2 regardless of aging time. HGF cells presented nonorganized pattern on ZrO2 surface, with notable alteration in cell alignment (red arrow). A, Positive control. B, Titanium nonaging (TNA). C, Zirconia non-aging (ZNA). D, Titanium aging (TA). E, Zirconia aging (ZA).
hydrophobic substrata,30,31 a significant difference was expected in overall biofilm formation on the NA surface. Surprisingly, the results revealed similarity in the biofilm formed on NA and A disks even after 48 hours of incubation. However, when both materials were compared, a notable statistical difference in overall biofilm could be observed with 2.8-fold fewer viable cells/mL on ZNA than the TNA surface and 4.0-fold fewer viable cells/mL on ZA than the TA surface. Considering that the oral
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environment contains more than 600 microbial phylotypes,1 findings using only a single pathogenic fungal species should be interpreted with caution. The results demonstrated that the changes in hydrophilicity and SFE did not disturb the proliferation and morphology of fibroblast cells. Thereby, aging time decreases the bioactivity and osteoconductivity of titanium by decreasing the amount and quality of cell attachment.12,26,38 Here, contrary to expectation, the contact
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angle between water and TA was significantly lower than that to water and TNA, whereas the fluorescence intensity, representative of cell proliferation, was similar. In the case of fibroblasts, contradictory results suggest that cellular adhesion cannot be completely explained by the surface wettability. In the cell-based experiments, a discreet difference in cell shape and orientation was observed for the ZrO2 material, regardless of the aging process. The qualitative assays revealed a more mature morphology defined by elongated and aligned cells on Ti substrates, indicating better attachment and cell surface affinity between HGF and Ti. Further investigations should provide detailed information regarding the aging effect on titanium and ZrO2 material composition and structure by using multispecies biofilm models and tissue culture to evaluate the inflammatory response. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. Changes in hydrophilicity and SFE on commercial implant abutment materials induced by the aging process do not interfere with C. albicans biofilm formation, an effect that is also maintained for mammalian cells. 2. The aging process of the Ti and ZrO2 surfaces was not toxic to HGF cells, even after long-term exposure. 3. The Ti topography pattern seemed to affect cell behavior, as a more mature morphology defined by elongated and aligned cells was recorded on the Ti substrate. REFERENCES 1. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770-83. 2. Choi SH, Jeong WS, Cha JY, Lee JH, Lee KJ, Yu HS, et al. Overcoming the biological aging of titanium using a wet storage method after ultraviolet treatment. Sci Rep 2017;7:3833. 3. Correa DRN, Kuroda PAB, Lourenco ML, Buzalaf MAR, Mendoza ME, Archanjo BS, et al. Microstructure and selected mechanical properties of aged Ti-15Zr-based alloys for biomedical applications. Mater Sci Eng C Mater Biol Appl 2018;91:762-71. 4. Papanagiotou HP, Morgano SM, Giordano RA, Pober R. In vitro evaluation of low-temperature aging effects and finishing procedures on the flexural strength and structural stability of Y-TZP dental ceramics. J Prosthet Dent 2006;96:154-64. 5. Pereira GKR, Muller C, Wandscher VF, Rippe MP, Kleverlaan CJ, Valandro LF. Comparison of different low-temperature aging protocols: its effects on the mechanical behavior of Y-TZP ceramics. J Mech Behav Biomed Mater 2016;60:324-30. 6. Pereira GKR, Silvestri T, Amaral M, Rippe MP, Kleverlaan CJ, Valandro LF. Fatigue limit of polycrystalline zirconium oxide ceramics: Effect of grinding and low-temperature aging. J Mech Behav Biomed Mater 2016;61:45-54. 7. de Avila ED, Avila-Campos MJ, Vergani CE, Spolidorio DM, Mollo Fde A Jr. Structural and quantitative analysis of a mature anaerobic biofilm on different implant abutment surfaces. J Prosthet Dent 2016;115:428-36. 8. de Avila ED, de Molon RS, Lima BP, Lux R, Shi W, Junior MJ, et al. Impact of physical chemical characteristics of abutment implant surfaces on bacteria adhesion. J Oral Implantol 2016;42:153-8. 9. de Avila ED, de Molon RS, Palomari Spolidorio DM, de Assis Mollo F Jr. Implications of surface and bulk properties of abutment implants and their degradation in the health of periodontal tissue. Materials (Basel) 2013;6:5951-66.
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Corresponding author: Dr Erica D. de Avila Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara São Paulo State University (UNESP) Rua Humaita, 1680, 14.801-903, Araraquara São Paulo BRAZIL Email: [email protected] Acknowledgments The authors acknowledge the Conexão Sistemas de Próteses Ltda for donating specimens, CAPES (Coordination for the Improvement of Higher Education Personnel) for the award of the scholarship, and Erica Dorigatti de Avila for her support in the preparation of the manuscript. Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.11.024
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