The Biocompatibility of Dental Graded Nano-Glass-Zirconia Material After Aging

The Biocompatibility of Dental Graded Nano-Glass-Zirconia Material After Aging A graded nano-glass/zirconia (G/Z) system has been developed via the infiltration of nano-glass into a nano-zirconia surface, which is advantageous for robust core-veneer bonds. The aging issue is a key for yttrium-stabilized tetragonal zirconia polycrystals (Y-TZPs), and therefore, it is necessary to evaluate the influence of aging degradation on the biocompatibility of G/Z systems before their possible clinical application. Herein, such biocompatibility testing was performed with human gingival fibroblasts (HGFs) seeded onto unaged/aged G/Z and Y-TZP for 2–72 h. Assessments included an oral mucous membrane irritation test in conjunction with analyses of cell viability, cell adhesion, and oxidative stress responses. Significant metabolic decreases in aged G/Z- and Y-TZP-treated cells were observed at 72 h. G/Z did not elicit any significant differences in cell viability compared with Y-TZP over 72 h both before and after aging. The oxidative stress data for the aged G/Z- and Y-TZP-treated cells showed a significant increase at 72 h. The G/Z specimens did not elicit any significant differences in ROS production compared with Y-TZP over 72 h both before and after aging. The cell adhesion rates of both G/Z and Y-TZP increased significantly after aging. The cell adhesion rates of G/Z and Y-TZP were not significantly different before and after aging. According to the oral mucous membrane irritation test, scores for macroscopic and microscopic observations for both the aged G/Z and unaged G/Z sides were 0, demonstrating no consequent irritation. Conclusions: The excellent biocompatibility of G/Z indicates that it has potential for future clinical applications. Keywords: Dental, Zirconia, Graded, Aging, Biocompatibility Background thermal expansion coefficient and elastic modulus be- Dental zirconia-based ceramics (e.g., 3 mol% yttrium- tween the zirconia cores and veneering ceramics [4]. stabilized tetragonal zirconia polycrystals (3Y-TZPs)) ex- Consequently, in our previous study, we introduced a hibit excellent mechanical strength and superior fracture new concept for the improvement of core-veneer bond- resistance due to inherent transformation toughening ing by infiltrating a low modulus nano-sized glass with a mechanisms, and they are widely utilized for the fabrica- matching thermal expansion coefficient into the zirconia tion of prosthetic devices [1]. Zirconia core materials are surface sintered from nano-zirconia particles, thus pro- usually coated with translucent veneering porcelain to ducing elastic graded nano-glass/zirconia (G/Z) systems. cover their opaque appearance. However, layered zirco- The bond strengths of the G/Z systems to veneering nia restorations tend to fail; chipping and delamination porcelains were demonstrated to be threefold higher of the veneering ceramic have been reported as the most than those of conventional zirconia-based systems [4]. frequent reason for the failure of zirconia-based restora- The aging of Y-TZP is well acknowledged. The aging tions [2, 3]. Chipping and delamination of the veneering of Y-TZP can be induced by an oral environment, with ceramic were reported to result from mismatches of the exposure to humidity, mechanical loading, and low temperature, resulting in surface roughening, micro- * Correspondence: tlrf@jnu.edu.cn cracks, and Y-TZP particle release into the body [5, 6]. Ting Sun and Ruoyu Liu are co-first authors. In the presence of humidity and low temperature, a tet- Ting Sun and Ruoyu Liu contributed equally to this work. † ragonal to monoclinic (t-m) zirconia phase transformation Equal contributors Medical Center of Stomatology, The First Affiliated Hospital of Jinan could be triggered. The crystal volumetric expansion re- University, Guangzhou 510630, China sults in localized stress and microcracking in the material Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sun et al. Nanoscale Research Letters (2018) 13:61 Page 2 of 8 surface, allowing water to further penetrate in the interior Table 1 Chemical composition of graded nano-glass-zirconia material of the material, leading to additional phase transformation and resulting in the degradation of the mechanical proper- Material Manufacturer Main components (wt%) ties [7–9]. Additionally, it is now widely known that the Glass La O 20.0; SiO 20.0; B O 15.0; 2 3 2 2 3 composition BaO 15.0; Al O 10.0; ZrO 5.0; 2 3 2 physicochemical properties of a biomaterial such as sur- Y O 5.0; TiO 4.0; CaO 4.0 [5] 2 3 2 face roughness and chemical composition have an influ- Y-TZP powder Tosoh, Tokyo, Japan Y O 5.18, ZrO 94.82 [5] 2 3 2 ence on its biocompatibility. Thus, it is necessary to evaluate the influence of aging degradation on the bio- compatibility of G/Z. Zhang et al. [10, 11] infiltrated glass into a dense zir- presintered Y-TZP porous substrate specimens. The conia substructure and developed a graded glass- coated specimens were then infiltrated at 1350 °C for 2 h zirconia composite with superior mechanical properties. to produce a graded glass-zirconia structure. Glass infiltra- However, the biocompatibility of the graded glass- tion and densification were carried out simultaneously. zirconia composite is unknown, especially with the con- sideration of aging phenomenon. Preparation of Y-TZP Specimens Consequently, biocompatibility testing of the newly Y-TZP blanks (Weiland, Weiland Dental, Pforzheim, developed G/Z system is essential for its clinical applica- Baden-Württemberg, Germany) were designed, milled, tion due to the addition of glass materials and the conse- andsinteredtofull density using a CAD/CAMsys- quent structural changes. The induction of the G/Z tem (Zenostar, Weiland Dental, Pforzheim, Baden- system may provide a solution for failures of zirconia- Württemberg, Germany). based restorations and thus improve their success rates. Therefore, the biocompatibility testing of the G/Z sys- Cell Culture tem before and after aging will provide guidelines on Human gingival fibroblasts (HGFs) were cultured in Dul- biosafety for the clinical application of G/Z. becco’s modified Eagle’s medium (DMEM, Nutrient In the present study, the biocompatibility of the G/Z Mixture F-12) containing 10% fetal bovine serum, 1% peni- system before and after aging was evaluated. Assessments cillin/streptomycin, 1% L-glutamine, and 1% non-essential involved an oral mucous membrane irritation test in con- amino acids in a humidified atmosphere of 5% CO at junction with analyses of the cell viability, cell morph- 37 °C. The medium was changed every 3 days. Cells were ology, cell adhesion, and oxidative stress responses. removed from culture dishes by rinsing in phosphate- buffered saline (PBS) and incubated in a trypsin-EDTA so- lution. Cells were seeded on each test substrate at Methods 1×10 cells/mL in the same media for all assays. Preparation of Specimens Y-TZP is a biocompatible material already approved for Aging clinical applications, and herein, Y-TZP specimens were To stimulate the chewing conditions, mechanical aging set up as the control group. All specimens were pro- was performed in artificial saliva at 37 °C, and the load duced as uniform plates (1.5 × 1.5 × 0.2 cm). ISO 13356 was applied using a three-point flexure fixture at a 2 Hz describes the evaluation of tested specimens with a sim- frequency. The following aging profiles were used: 80 N plified geometry (bending bars) and a polished surface. load and 10 cycles for all specimens [12, 13]. Preparation of G/Z Specimens Cell Viability Glass powders were milled until nanosized particles were The viability of HGF following unaged and aged G/Z obtained with a nanometer grinding instrument (Emax, and Y-TZP exposure was determined at 2, 24, 48, and Retsch, Haan, North Rhine-Westphalia, Germany). The 72 h (exposure time) using the alamarBlue salt assay as main components and percentages (> 1 wt%) of the infil- a 10% solution in DMEM. Before the assay test, all spec- trating glass are listed in Table 1 [4]. Yttrium-stabilized imens were removed from the HGF, and then, 500 μLof zirconia powders (5.18 wt% Y O , TZ-3Y-E grade; Tosoh, alamarBlue dye was added, followed by incubation for 2 3 Tokyo, Tokyo Prefecture, Japan) were compressed under a 4 h. Aliquots (100 μL) were decanted into 96-well cell uniaxial pressure of 150 MPa for 2 min and were then culture dishes, and fluorescence intensity was deter- partially sintered at 1350 °C for 2 h in a muffle furnace. mined at excitation (530 nm) and emission (580 nm) The desired oxides were ball-milled into 200-mesh pow- wavelengths with a Synergy™ H4 Microplate Spectropho- ders. The Y-TZP substrate specimens were presintered at tometer (BioTek, Winooski, Vermont, USA). All experi- 1200 °C for 2 h, forming porous structures. The melted ments were performed in triplicate on three occasions. glass slurries were applied onto the top surface of the The cell viability was calculated as follows: viability Sun et al. Nanoscale Research Letters (2018) 13:61 Page 3 of 8 (%) = (absorbance of the treated wells) / (absorbance of Statistical Analyses the control wells). One-way analysis of variance (ANOVA) was used for the pooled (all exposure times) cell viability, oxidative stress, and cell adhesion rate data for the assessment Oxidative Stress of individual dental specimens (SPSS 22.0; SPSS Inc., Reactive oxygen species (ROS) levels of G/Z- and Y- Chicago, IL, USA). TZP-treated HGF before and after aging were identified with chemiluminescence using the Reactive Oxygen Spe- Results cies Assay Kit (Nanjing Jiancheng Bioengineering Insti- Graded Layer Structure tute, Nanjing, Jiangsu). The thickness of the graded layer was controlled to be ap- proximately 0.9–1.0 mm. The structure and SEM images Cell Adhesion of the G/Z system are shown in Fig. 1a, b.Figure 1a, b HGFs were cultured for 2 h on G/Z and Y-TZP speci- depicts a morphology consisting of traces of residual glass, men surfaces before and after aging. After fixation, cell glass-coated zirconia grains, and intergranular voids, nuclei were stained with 4′,6-diamidino-2-phenylindole which created a surface morphology ideal for increasing dihydrochloride (DAPI) (Yeasen, Shanghai, Shanghai the core-veneer bond strength. Furthermore, EDS analysis District, China). Images were obtained with an inverted of the graded layers is shown in Fig. 1c, showing with the LSM 510 fluorescence microscope (Carl Zeiss, Jena, increase in the distance from the surface, the content of Tuttlingen, Germany). The adhered cells were analyzed the Zr element increased while the contents of the Si, Al, in randomly selected areas in five sections (450 μm× and La elements decreased. Details have been described in 450 μm) at a magnification of × 200. The cell adhesion our previous study [4]. rates were determined through the number of adhered cells divided by the total number of seeded cells. Cell Viability Significant metabolic decreases in aged G/Z- and Y-TZP- Cell Morphology treated cells were observed at 72 h (P < 0.00001) (Fig. 2a). HGFs were cultured for 2 h on unaged and aged G/Z spe- No significant metabolic decrease in aged G/Z-treated cimen surfaces before and after aging. After fixation, the cells was observed at 2 h (P =0.47), 24 h (P =0.82), and cells were stained for filamentous actin (F-actin) using 48 h (P = 0.53) (Fig. 2a). No significant metabolic decrease rhodamine phalloidin (1:100 in 3% BSA in PBS). Images in aged Y-TZP-treated cells was observed at 2 h (P =0.82), were obtained with an inverted LSM 510 fluorescence 24 h (P =0.32), and 48 h (P = 0.54) (Fig. 2a). The G/Z microscope (Carl Zeiss, Jena, Tuttlingen, Germany). Sam- specimens did not elicit any significant differences in cell ples were mounted on glass coverslips using DAPI (Yeasen, viability compared with Y-TZP at 2 h (P =0.94), 24 h (P = Shanghai, Shanghai District, China) for the visualization of 0.86), 48 h (P =0.68), and 72 h (P = 0.61) of exposure be- cell nuclei. The cell morphologies on G/Z and Y-TZP sur- fore aging. The G/Z specimens did not elicit any signi- faces before and after aging were also observed via scan- ficant differences in cell viability compared with Y-TZP at ning electron microscopy (SEM) with a XL-30 ESEM 2h (P =0.98), 24 h (P =0.54), 48 h (P =0.73), and 72 h (P (Philips, Eindhoven, North Brabant, The Netherlands). = 0.50) of exposure after aging. Oral Mucous Membrane Irritation Test Oxidative Stress The oral mucous membrane irritation test was con- Oxidative stress data for the aged G/Z- and Y-TZP- ducted according to YY/T 0127.13-2009 medicine stan- treated cells showed a significant increase at 72 h (P < dards of the People’s Republic of China. Ten Wistar 0.00001, Fig. 1b). In contrast, aged G/Z-treated cells elic- male mice were selected for this test. The aged G/Z spe- ited no significant difference in ROS production at 2 h cimen was placed in one cheek pouch for each animal as (P = 0.91), 24 h (P = 0.42), and 48 h (P = 0.62). Addition- the tested material, while the unaged G/Z specimen was ally, aged Y-TZP- treated cells elicited no significant dif- placed in the contralateral side as the control. The ani- ference in ROS production at 2 h (P = 0.07), 24 h (P = mals were sacrificed after 2 weeks, and the pouches were 0.40), and 48 h (P = 0.53). The G/Z specimens did not examined macroscopically following removal of the elicit any significant difference in ROS production com- disks. Histological analyses of the buccal mucosa were pared with Y-TZP at 2 h (P = 0.16), 24 h (P = 0.79), 48 h further performed on cryosections that were stained (P = 0.14), and 72 h (P = 0.43) of exposure before aging. with hematoxylin and eosin. The average grades for all The G/Z specimens did not elicit any significant differ- macroscopic and microscopic observations were ob- ence in ROS production compared with Y-TZP at 2 h (P tained. The control group average was subtracted from = 0.27), 24 h (P = 0.17), 48 h (P = 0.07), and 72 h (P = the test group average to yield the irritation index. 0.15) of exposure after aging. Sun et al. Nanoscale Research Letters (2018) 13:61 Page 4 of 8 Fig. 1 Physical and chemical properties of G/Z. a Structural diagram. b SEM image. c EDS analysis of the functionally graded layer Cell Adhesion Cell Morphology The cell adhesion rates of both G/Z and Y-TZP in- Fluorescence images at different incubation times showed creased significantly after aging (Fig. 2c). The cell ad- that cells were attached to G/Z surfaces; however, spread- hesion ratesofunagedG/Z andY-TZP were not ing was greater on aged G/Z surfaces (Fig. 4a–c), where the significantly different (P = 0.71) (Fig. 2c). The cell ad- cells were flattened and well spread with a polygonal shape. hesion rates of aged G/Z and Y-TZP were not signifi- SEM images showed that cells cultured on aged and cantly different (P = 0.71) (Fig. 2c). The cell adhesion unaged G/Z surfaces were considerably flattened with rates of G/Z and Y-TZP showed no significant differ- extensions or elongated bodies and numerous microvilli ences after aging (P < 0.00001) (Fig. 2c). Characteristic (Fig. 5a, b). Rounded nuclei can be observed, confirming photographs of cell adhesion on Y-TZP and G/Z be- the attachment of the spread of cell cytoplasm to the fore and after aging are shown in Fig. 3a–d. specimen surface (Fig. 5a). Fig. 2 Biocompatibility of G/Z and Y-TZP before and after aging. Data represent the mean ± SD, n =5. a Cell viability of aged and unaged specimen-treated HGF. b Ros production of aged and unaged specimen-treated HGF. c Cell adhesive rates of aged and unaged specimen-treated # * HGF. Significance versus control group: P < 0.01; P < 0.05 Sun et al. Nanoscale Research Letters (2018) 13:61 Page 5 of 8 Fig. 3 Cell adhesion to G/Z and Y-TZP before and after aging. a Aged G/Z. b Unaged G/Z. c aged Y-TZP. d Unaged Y-TZP Oral Mucous Membrane Irritation Test biocompatibility of the G/Z system with the consider- Scores for the macroscopic observations for both the ation of aging was unknown. Biocompatibility tests and testing and contralateral sides were 0, demonstrating no moderate controls are essential. Consequently, a series consequent irritation. Additionally, scores from the of biocompatibility tests was conducted and compared microscopic evaluation for both sides were 0, indicating with the gold standard, Y-TZP, with the consideration of no apparent irritation reaction. Figure 6a, b demonstrates aging. Furthermore, the surface topography as well as that no histopathological changes were observed in the physical and chemical properties have been proven to be buccal mucosa treated with unaged G/Z and aged G/Z. influential for cell adhesion and viability by studies [19]. All specimens were therefore sandblasted and polished Discussion to a clinical surface roughness. Metal ceramic materials have been increasingly replaced Significant metabolic decreases in aged G/Z- and Y- by metal-free materials as the release of metal ions has TZP-treated cells were observed at 72 h (Fig. 2a), prov- been widely discussed. Various metal ions including sil- ing that aging decreases the cell proliferations for G/Z ver [14], gold [15], titanium [16], and nickel [17] of den- and Y-TZP. The influence of aging on the biocompatibil- tal prosthesis could be released into the saliva and ity of zirconia materials is controversial. A previous plasma. McGinley et al. even reported that diffused Ni study reported the decreased biocompatibility of zirconia ions from a dental Ni-Cr alloy could spread throughout after aging [20]. Meanwhile, a recent study proved the the epithelial tissue to the basal lamina and subsequently increase in the biocompatibility of aged zirconia [21]. throughout the extracellular matrix, resulting in a loss of The different influence of aging on the biocompatibility cell viability and tissue integrity [18]. Present studies might result from different aging procedures, including mostly focused on the development and improvement of the cycle, temperature, load, and frequency [22]. The in- all ceramic materials. Therefore, G/Z was introduced in fluence of aging on zirconia’s physical and chemical our previous study [4] for the improvement of the suc- property changes depends on the aggressiveness of the cess rates of zirconia-based materials. However, the aging procedure for the degradation of zirconia. To Fig. 4 Attachment, spreading, and morphology of HGF on G/Z before and after aging observed with fluorescence microscopy. a, b Aged G/Z. c Unaged G/Z. Cells were cultured for 72 h on substrates and then fixed and stained for filamentous actin (F-actin, red) and nuclei (blue) Sun et al. Nanoscale Research Letters (2018) 13:61 Page 6 of 8 Fig. 5 SEM micrographs of the HGF morphology on G/Z before and after aging 72 h post culture. a Aged G/Z. b Unaged G/Z. Original magnification: × 2000 simulate long-term intra-oral conditions, the aging pro- fluorescent staining (Fig. 4a, b) and SEM views (Fig. 5a, b) cedure used in this study was based on clinical parame- demonstrated that cells cultured on both aged and unaged ters, such as the bite load and frequency, the use of a G/Z were flattened and well spread. humid environment, and the temperature of the human Cell adhesion depends on the physicochemical proper- body [22]. ties of a biomaterial. It is well acknowledged that migra- Cell viability relies on mitochondrial activity. The de- tion and adhesion are biological parameters that are not crease of cell proliferation and the increase of ROS pro- necessarily directly linked. Cells can migrate slowly with duction might be attributed to the diffused ions very high adhesion [24, 25]. Al Qahtani et al. [26] also spreading throughout the epithelial tissue to the basal reported that the sandblasted surface of Y-TZP pre- lamina and subsequently throughout the extracellular sented higher cell adhesion but low cell proliferation matrix, resulting in a loss of cell viability and tissue in- when incubated with Saos-2 osteoblasts. The surface tegrity [6, 23]. wettability is a factor that also determines the preference The cell adhesion rates of both G/Z and Y-TZP in- of cell adhesion, through regulation of amounts of the creased after aging (Fig. 2c). Characteristic photographs protein adsorbed on the surface [27]. It was reported of cell adhesion on Y-TZP and G/Z before and after that the cells on a superhydrophilic surface even started aging are shown in Fig. 3a–d. Precise observation of cell proliferation as soon as adhesion was complete, and this attachment on G/Z was conducted. Double-labeled phenomenon was highly related to the high amounts of Fig. 6 Pathological examination of the mucosa treated with aged G/Z (a) and unaged G/Z (b) Sun et al. Nanoscale Research Letters (2018) 13:61 Page 7 of 8 the protein adsorbed on the hydrophilic surface [28]. applications. However, this study is a preliminary report, The aging abrasion of G/Z and Y-TZP provides rough and further in vivo and in vitro studies with more com- surfaces with strong wettability, allowing the strong ad- prehensive test methods are needed to confirm the hesion of cells. This type of surface will be optimal for present results. gingival adhesion around for dental abutment surfaces. Abbreviations In contrast, smooth surfaces give restricted adhesion DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride; F-actin: Filamentous properties to the materials, as appropriate for surfaces actin; FGM: HAp-Al O -ZrO ; G/Z: Graded nano-glass/zirconia; SEM: Scanning 2 3 2 electron microscopy; t-m: Tetragonal to monoclinic; Y-TZP: Yttrium-stabilized designed to prevent biofilm formation in the septic en- tetragonal zirconia polycrystal vironment of the mouth [29]. As dental prosthesis mate- rials, aging abrasion of G/Z and Y-TZP therefore Acknowledgements The authors extend their gratitude to Professor Longquan Shao for the increased the probability for biofilm formation. The cell instructive advice and suggestions on this study. adhesion rates of G/Z and Y-TZP showed no significant differences before and after aging (Fig. 2c). This finding Funding This work was supported by the National Natural Science Foundation of proved that G/Z and Y-TZP exhibit similar cell attach- China for Young Scholars (81400557); China Postdoctoral Science Foundation ment properties before and after aging, indicating the (2017M622742); Science and Technology Planning Project of Guangdong promising surface biological properties of G/Z. Province, China (2016A010103017); and scientific cultivation and innovation project of Jinan University (21617494). In vivo irritation tests are critical for the long-term ap- plication of oral medical devices. Herein, no macro- Availability of Data and Materials scopic or microscopic pathological changes were All data are fully available without restriction. observed for G/Z-treated mucosa (Fig. 6a, b). Authors’ Contributions The existence of a large amount of m-ZrO could re- TS and RYL conceived and designed the study. YLZ and XLF performed the sult in a decrease in the strength of zirconia. The reliable experiments. XNL provided the mutants. TS wrote the paper. RFL reviewed and edited the manuscript. All authors read and approved the manuscript. biocompatibility of the G/Z system might be attributed to the small phase change during the infiltration proced- Competing Interests ure, which was proven in our previous study [4]. An- The authors declare that they have no competing interests. other study proved the fair aging resistance of infiltrated Y-TZP materials. Inokoshi et al. [30] reported that Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Al O -infiltrated Y-TZP was hydrothermally stable after 2 3 published maps and institutional affiliations. aging thanks to a high amount of c-ZrO phase at the interlayer surface, although it has a higher initial mono- Author details Medical Center of Stomatology, The First Affiliated Hospital of Jinan clinic volume fraction compared to the Y-TZP. University, Guangzhou 510630, China. 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The Biocompatibility of Dental Graded Nano-Glass-Zirconia Material After Aging

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Materials Science; Nanotechnology; Nanotechnology and Microengineering; Nanoscale Science and Technology; Nanochemistry; Molecular Medicine
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10.1186/s11671-018-2479-4
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Abstract

A graded nano-glass/zirconia (G/Z) system has been developed via the infiltration of nano-glass into a nano-zirconia surface, which is advantageous for robust core-veneer bonds. The aging issue is a key for yttrium-stabilized tetragonal zirconia polycrystals (Y-TZPs), and therefore, it is necessary to evaluate the influence of aging degradation on the biocompatibility of G/Z systems before their possible clinical application. Herein, such biocompatibility testing was performed with human gingival fibroblasts (HGFs) seeded onto unaged/aged G/Z and Y-TZP for 2–72 h. Assessments included an oral mucous membrane irritation test in conjunction with analyses of cell viability, cell adhesion, and oxidative stress responses. Significant metabolic decreases in aged G/Z- and Y-TZP-treated cells were observed at 72 h. G/Z did not elicit any significant differences in cell viability compared with Y-TZP over 72 h both before and after aging. The oxidative stress data for the aged G/Z- and Y-TZP-treated cells showed a significant increase at 72 h. The G/Z specimens did not elicit any significant differences in ROS production compared with Y-TZP over 72 h both before and after aging. The cell adhesion rates of both G/Z and Y-TZP increased significantly after aging. The cell adhesion rates of G/Z and Y-TZP were not significantly different before and after aging. According to the oral mucous membrane irritation test, scores for macroscopic and microscopic observations for both the aged G/Z and unaged G/Z sides were 0, demonstrating no consequent irritation. Conclusions: The excellent biocompatibility of G/Z indicates that it has potential for future clinical applications. Keywords: Dental, Zirconia, Graded, Aging, Biocompatibility Background thermal expansion coefficient and elastic modulus be- Dental zirconia-based ceramics (e.g., 3 mol% yttrium- tween the zirconia cores and veneering ceramics [4]. stabilized tetragonal zirconia polycrystals (3Y-TZPs)) ex- Consequently, in our previous study, we introduced a hibit excellent mechanical strength and superior fracture new concept for the improvement of core-veneer bond- resistance due to inherent transformation toughening ing by infiltrating a low modulus nano-sized glass with a mechanisms, and they are widely utilized for the fabrica- matching thermal expansion coefficient into the zirconia tion of prosthetic devices [1]. Zirconia core materials are surface sintered from nano-zirconia particles, thus pro- usually coated with translucent veneering porcelain to ducing elastic graded nano-glass/zirconia (G/Z) systems. cover their opaque appearance. However, layered zirco- The bond strengths of the G/Z systems to veneering nia restorations tend to fail; chipping and delamination porcelains were demonstrated to be threefold higher of the veneering ceramic have been reported as the most than those of conventional zirconia-based systems [4]. frequent reason for the failure of zirconia-based restora- The aging of Y-TZP is well acknowledged. The aging tions [2, 3]. Chipping and delamination of the veneering of Y-TZP can be induced by an oral environment, with ceramic were reported to result from mismatches of the exposure to humidity, mechanical loading, and low temperature, resulting in surface roughening, micro- * Correspondence: tlrf@jnu.edu.cn cracks, and Y-TZP particle release into the body [5, 6]. Ting Sun and Ruoyu Liu are co-first authors. In the presence of humidity and low temperature, a tet- Ting Sun and Ruoyu Liu contributed equally to this work. † ragonal to monoclinic (t-m) zirconia phase transformation Equal contributors Medical Center of Stomatology, The First Affiliated Hospital of Jinan could be triggered. The crystal volumetric expansion re- University, Guangzhou 510630, China sults in localized stress and microcracking in the material Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sun et al. Nanoscale Research Letters (2018) 13:61 Page 2 of 8 surface, allowing water to further penetrate in the interior Table 1 Chemical composition of graded nano-glass-zirconia material of the material, leading to additional phase transformation and resulting in the degradation of the mechanical proper- Material Manufacturer Main components (wt%) ties [7–9]. Additionally, it is now widely known that the Glass La O 20.0; SiO 20.0; B O 15.0; 2 3 2 2 3 composition BaO 15.0; Al O 10.0; ZrO 5.0; 2 3 2 physicochemical properties of a biomaterial such as sur- Y O 5.0; TiO 4.0; CaO 4.0 [5] 2 3 2 face roughness and chemical composition have an influ- Y-TZP powder Tosoh, Tokyo, Japan Y O 5.18, ZrO 94.82 [5] 2 3 2 ence on its biocompatibility. Thus, it is necessary to evaluate the influence of aging degradation on the bio- compatibility of G/Z. Zhang et al. [10, 11] infiltrated glass into a dense zir- presintered Y-TZP porous substrate specimens. The conia substructure and developed a graded glass- coated specimens were then infiltrated at 1350 °C for 2 h zirconia composite with superior mechanical properties. to produce a graded glass-zirconia structure. Glass infiltra- However, the biocompatibility of the graded glass- tion and densification were carried out simultaneously. zirconia composite is unknown, especially with the con- sideration of aging phenomenon. Preparation of Y-TZP Specimens Consequently, biocompatibility testing of the newly Y-TZP blanks (Weiland, Weiland Dental, Pforzheim, developed G/Z system is essential for its clinical applica- Baden-Württemberg, Germany) were designed, milled, tion due to the addition of glass materials and the conse- andsinteredtofull density using a CAD/CAMsys- quent structural changes. The induction of the G/Z tem (Zenostar, Weiland Dental, Pforzheim, Baden- system may provide a solution for failures of zirconia- Württemberg, Germany). based restorations and thus improve their success rates. Therefore, the biocompatibility testing of the G/Z sys- Cell Culture tem before and after aging will provide guidelines on Human gingival fibroblasts (HGFs) were cultured in Dul- biosafety for the clinical application of G/Z. becco’s modified Eagle’s medium (DMEM, Nutrient In the present study, the biocompatibility of the G/Z Mixture F-12) containing 10% fetal bovine serum, 1% peni- system before and after aging was evaluated. Assessments cillin/streptomycin, 1% L-glutamine, and 1% non-essential involved an oral mucous membrane irritation test in con- amino acids in a humidified atmosphere of 5% CO at junction with analyses of the cell viability, cell morph- 37 °C. The medium was changed every 3 days. Cells were ology, cell adhesion, and oxidative stress responses. removed from culture dishes by rinsing in phosphate- buffered saline (PBS) and incubated in a trypsin-EDTA so- lution. Cells were seeded on each test substrate at Methods 1×10 cells/mL in the same media for all assays. Preparation of Specimens Y-TZP is a biocompatible material already approved for Aging clinical applications, and herein, Y-TZP specimens were To stimulate the chewing conditions, mechanical aging set up as the control group. All specimens were pro- was performed in artificial saliva at 37 °C, and the load duced as uniform plates (1.5 × 1.5 × 0.2 cm). ISO 13356 was applied using a three-point flexure fixture at a 2 Hz describes the evaluation of tested specimens with a sim- frequency. The following aging profiles were used: 80 N plified geometry (bending bars) and a polished surface. load and 10 cycles for all specimens [12, 13]. Preparation of G/Z Specimens Cell Viability Glass powders were milled until nanosized particles were The viability of HGF following unaged and aged G/Z obtained with a nanometer grinding instrument (Emax, and Y-TZP exposure was determined at 2, 24, 48, and Retsch, Haan, North Rhine-Westphalia, Germany). The 72 h (exposure time) using the alamarBlue salt assay as main components and percentages (> 1 wt%) of the infil- a 10% solution in DMEM. Before the assay test, all spec- trating glass are listed in Table 1 [4]. Yttrium-stabilized imens were removed from the HGF, and then, 500 μLof zirconia powders (5.18 wt% Y O , TZ-3Y-E grade; Tosoh, alamarBlue dye was added, followed by incubation for 2 3 Tokyo, Tokyo Prefecture, Japan) were compressed under a 4 h. Aliquots (100 μL) were decanted into 96-well cell uniaxial pressure of 150 MPa for 2 min and were then culture dishes, and fluorescence intensity was deter- partially sintered at 1350 °C for 2 h in a muffle furnace. mined at excitation (530 nm) and emission (580 nm) The desired oxides were ball-milled into 200-mesh pow- wavelengths with a Synergy™ H4 Microplate Spectropho- ders. The Y-TZP substrate specimens were presintered at tometer (BioTek, Winooski, Vermont, USA). All experi- 1200 °C for 2 h, forming porous structures. The melted ments were performed in triplicate on three occasions. glass slurries were applied onto the top surface of the The cell viability was calculated as follows: viability Sun et al. Nanoscale Research Letters (2018) 13:61 Page 3 of 8 (%) = (absorbance of the treated wells) / (absorbance of Statistical Analyses the control wells). One-way analysis of variance (ANOVA) was used for the pooled (all exposure times) cell viability, oxidative stress, and cell adhesion rate data for the assessment Oxidative Stress of individual dental specimens (SPSS 22.0; SPSS Inc., Reactive oxygen species (ROS) levels of G/Z- and Y- Chicago, IL, USA). TZP-treated HGF before and after aging were identified with chemiluminescence using the Reactive Oxygen Spe- Results cies Assay Kit (Nanjing Jiancheng Bioengineering Insti- Graded Layer Structure tute, Nanjing, Jiangsu). The thickness of the graded layer was controlled to be ap- proximately 0.9–1.0 mm. The structure and SEM images Cell Adhesion of the G/Z system are shown in Fig. 1a, b.Figure 1a, b HGFs were cultured for 2 h on G/Z and Y-TZP speci- depicts a morphology consisting of traces of residual glass, men surfaces before and after aging. After fixation, cell glass-coated zirconia grains, and intergranular voids, nuclei were stained with 4′,6-diamidino-2-phenylindole which created a surface morphology ideal for increasing dihydrochloride (DAPI) (Yeasen, Shanghai, Shanghai the core-veneer bond strength. Furthermore, EDS analysis District, China). Images were obtained with an inverted of the graded layers is shown in Fig. 1c, showing with the LSM 510 fluorescence microscope (Carl Zeiss, Jena, increase in the distance from the surface, the content of Tuttlingen, Germany). The adhered cells were analyzed the Zr element increased while the contents of the Si, Al, in randomly selected areas in five sections (450 μm× and La elements decreased. Details have been described in 450 μm) at a magnification of × 200. The cell adhesion our previous study [4]. rates were determined through the number of adhered cells divided by the total number of seeded cells. Cell Viability Significant metabolic decreases in aged G/Z- and Y-TZP- Cell Morphology treated cells were observed at 72 h (P < 0.00001) (Fig. 2a). HGFs were cultured for 2 h on unaged and aged G/Z spe- No significant metabolic decrease in aged G/Z-treated cimen surfaces before and after aging. After fixation, the cells was observed at 2 h (P =0.47), 24 h (P =0.82), and cells were stained for filamentous actin (F-actin) using 48 h (P = 0.53) (Fig. 2a). No significant metabolic decrease rhodamine phalloidin (1:100 in 3% BSA in PBS). Images in aged Y-TZP-treated cells was observed at 2 h (P =0.82), were obtained with an inverted LSM 510 fluorescence 24 h (P =0.32), and 48 h (P = 0.54) (Fig. 2a). The G/Z microscope (Carl Zeiss, Jena, Tuttlingen, Germany). Sam- specimens did not elicit any significant differences in cell ples were mounted on glass coverslips using DAPI (Yeasen, viability compared with Y-TZP at 2 h (P =0.94), 24 h (P = Shanghai, Shanghai District, China) for the visualization of 0.86), 48 h (P =0.68), and 72 h (P = 0.61) of exposure be- cell nuclei. The cell morphologies on G/Z and Y-TZP sur- fore aging. The G/Z specimens did not elicit any signi- faces before and after aging were also observed via scan- ficant differences in cell viability compared with Y-TZP at ning electron microscopy (SEM) with a XL-30 ESEM 2h (P =0.98), 24 h (P =0.54), 48 h (P =0.73), and 72 h (P (Philips, Eindhoven, North Brabant, The Netherlands). = 0.50) of exposure after aging. Oral Mucous Membrane Irritation Test Oxidative Stress The oral mucous membrane irritation test was con- Oxidative stress data for the aged G/Z- and Y-TZP- ducted according to YY/T 0127.13-2009 medicine stan- treated cells showed a significant increase at 72 h (P < dards of the People’s Republic of China. Ten Wistar 0.00001, Fig. 1b). In contrast, aged G/Z-treated cells elic- male mice were selected for this test. The aged G/Z spe- ited no significant difference in ROS production at 2 h cimen was placed in one cheek pouch for each animal as (P = 0.91), 24 h (P = 0.42), and 48 h (P = 0.62). Addition- the tested material, while the unaged G/Z specimen was ally, aged Y-TZP- treated cells elicited no significant dif- placed in the contralateral side as the control. The ani- ference in ROS production at 2 h (P = 0.07), 24 h (P = mals were sacrificed after 2 weeks, and the pouches were 0.40), and 48 h (P = 0.53). The G/Z specimens did not examined macroscopically following removal of the elicit any significant difference in ROS production com- disks. Histological analyses of the buccal mucosa were pared with Y-TZP at 2 h (P = 0.16), 24 h (P = 0.79), 48 h further performed on cryosections that were stained (P = 0.14), and 72 h (P = 0.43) of exposure before aging. with hematoxylin and eosin. The average grades for all The G/Z specimens did not elicit any significant differ- macroscopic and microscopic observations were ob- ence in ROS production compared with Y-TZP at 2 h (P tained. The control group average was subtracted from = 0.27), 24 h (P = 0.17), 48 h (P = 0.07), and 72 h (P = the test group average to yield the irritation index. 0.15) of exposure after aging. Sun et al. Nanoscale Research Letters (2018) 13:61 Page 4 of 8 Fig. 1 Physical and chemical properties of G/Z. a Structural diagram. b SEM image. c EDS analysis of the functionally graded layer Cell Adhesion Cell Morphology The cell adhesion rates of both G/Z and Y-TZP in- Fluorescence images at different incubation times showed creased significantly after aging (Fig. 2c). The cell ad- that cells were attached to G/Z surfaces; however, spread- hesion ratesofunagedG/Z andY-TZP were not ing was greater on aged G/Z surfaces (Fig. 4a–c), where the significantly different (P = 0.71) (Fig. 2c). The cell ad- cells were flattened and well spread with a polygonal shape. hesion rates of aged G/Z and Y-TZP were not signifi- SEM images showed that cells cultured on aged and cantly different (P = 0.71) (Fig. 2c). The cell adhesion unaged G/Z surfaces were considerably flattened with rates of G/Z and Y-TZP showed no significant differ- extensions or elongated bodies and numerous microvilli ences after aging (P < 0.00001) (Fig. 2c). Characteristic (Fig. 5a, b). Rounded nuclei can be observed, confirming photographs of cell adhesion on Y-TZP and G/Z be- the attachment of the spread of cell cytoplasm to the fore and after aging are shown in Fig. 3a–d. specimen surface (Fig. 5a). Fig. 2 Biocompatibility of G/Z and Y-TZP before and after aging. Data represent the mean ± SD, n =5. a Cell viability of aged and unaged specimen-treated HGF. b Ros production of aged and unaged specimen-treated HGF. c Cell adhesive rates of aged and unaged specimen-treated # * HGF. Significance versus control group: P < 0.01; P < 0.05 Sun et al. Nanoscale Research Letters (2018) 13:61 Page 5 of 8 Fig. 3 Cell adhesion to G/Z and Y-TZP before and after aging. a Aged G/Z. b Unaged G/Z. c aged Y-TZP. d Unaged Y-TZP Oral Mucous Membrane Irritation Test biocompatibility of the G/Z system with the consider- Scores for the macroscopic observations for both the ation of aging was unknown. Biocompatibility tests and testing and contralateral sides were 0, demonstrating no moderate controls are essential. Consequently, a series consequent irritation. Additionally, scores from the of biocompatibility tests was conducted and compared microscopic evaluation for both sides were 0, indicating with the gold standard, Y-TZP, with the consideration of no apparent irritation reaction. Figure 6a, b demonstrates aging. Furthermore, the surface topography as well as that no histopathological changes were observed in the physical and chemical properties have been proven to be buccal mucosa treated with unaged G/Z and aged G/Z. influential for cell adhesion and viability by studies [19]. All specimens were therefore sandblasted and polished Discussion to a clinical surface roughness. Metal ceramic materials have been increasingly replaced Significant metabolic decreases in aged G/Z- and Y- by metal-free materials as the release of metal ions has TZP-treated cells were observed at 72 h (Fig. 2a), prov- been widely discussed. Various metal ions including sil- ing that aging decreases the cell proliferations for G/Z ver [14], gold [15], titanium [16], and nickel [17] of den- and Y-TZP. The influence of aging on the biocompatibil- tal prosthesis could be released into the saliva and ity of zirconia materials is controversial. A previous plasma. McGinley et al. even reported that diffused Ni study reported the decreased biocompatibility of zirconia ions from a dental Ni-Cr alloy could spread throughout after aging [20]. Meanwhile, a recent study proved the the epithelial tissue to the basal lamina and subsequently increase in the biocompatibility of aged zirconia [21]. throughout the extracellular matrix, resulting in a loss of The different influence of aging on the biocompatibility cell viability and tissue integrity [18]. Present studies might result from different aging procedures, including mostly focused on the development and improvement of the cycle, temperature, load, and frequency [22]. The in- all ceramic materials. Therefore, G/Z was introduced in fluence of aging on zirconia’s physical and chemical our previous study [4] for the improvement of the suc- property changes depends on the aggressiveness of the cess rates of zirconia-based materials. However, the aging procedure for the degradation of zirconia. To Fig. 4 Attachment, spreading, and morphology of HGF on G/Z before and after aging observed with fluorescence microscopy. a, b Aged G/Z. c Unaged G/Z. Cells were cultured for 72 h on substrates and then fixed and stained for filamentous actin (F-actin, red) and nuclei (blue) Sun et al. Nanoscale Research Letters (2018) 13:61 Page 6 of 8 Fig. 5 SEM micrographs of the HGF morphology on G/Z before and after aging 72 h post culture. a Aged G/Z. b Unaged G/Z. Original magnification: × 2000 simulate long-term intra-oral conditions, the aging pro- fluorescent staining (Fig. 4a, b) and SEM views (Fig. 5a, b) cedure used in this study was based on clinical parame- demonstrated that cells cultured on both aged and unaged ters, such as the bite load and frequency, the use of a G/Z were flattened and well spread. humid environment, and the temperature of the human Cell adhesion depends on the physicochemical proper- body [22]. ties of a biomaterial. It is well acknowledged that migra- Cell viability relies on mitochondrial activity. The de- tion and adhesion are biological parameters that are not crease of cell proliferation and the increase of ROS pro- necessarily directly linked. Cells can migrate slowly with duction might be attributed to the diffused ions very high adhesion [24, 25]. Al Qahtani et al. [26] also spreading throughout the epithelial tissue to the basal reported that the sandblasted surface of Y-TZP pre- lamina and subsequently throughout the extracellular sented higher cell adhesion but low cell proliferation matrix, resulting in a loss of cell viability and tissue in- when incubated with Saos-2 osteoblasts. The surface tegrity [6, 23]. wettability is a factor that also determines the preference The cell adhesion rates of both G/Z and Y-TZP in- of cell adhesion, through regulation of amounts of the creased after aging (Fig. 2c). Characteristic photographs protein adsorbed on the surface [27]. It was reported of cell adhesion on Y-TZP and G/Z before and after that the cells on a superhydrophilic surface even started aging are shown in Fig. 3a–d. Precise observation of cell proliferation as soon as adhesion was complete, and this attachment on G/Z was conducted. Double-labeled phenomenon was highly related to the high amounts of Fig. 6 Pathological examination of the mucosa treated with aged G/Z (a) and unaged G/Z (b) Sun et al. Nanoscale Research Letters (2018) 13:61 Page 7 of 8 the protein adsorbed on the hydrophilic surface [28]. applications. However, this study is a preliminary report, The aging abrasion of G/Z and Y-TZP provides rough and further in vivo and in vitro studies with more com- surfaces with strong wettability, allowing the strong ad- prehensive test methods are needed to confirm the hesion of cells. This type of surface will be optimal for present results. gingival adhesion around for dental abutment surfaces. Abbreviations In contrast, smooth surfaces give restricted adhesion DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride; F-actin: Filamentous properties to the materials, as appropriate for surfaces actin; FGM: HAp-Al O -ZrO ; G/Z: Graded nano-glass/zirconia; SEM: Scanning 2 3 2 electron microscopy; t-m: Tetragonal to monoclinic; Y-TZP: Yttrium-stabilized designed to prevent biofilm formation in the septic en- tetragonal zirconia polycrystal vironment of the mouth [29]. As dental prosthesis mate- rials, aging abrasion of G/Z and Y-TZP therefore Acknowledgements The authors extend their gratitude to Professor Longquan Shao for the increased the probability for biofilm formation. The cell instructive advice and suggestions on this study. adhesion rates of G/Z and Y-TZP showed no significant differences before and after aging (Fig. 2c). This finding Funding This work was supported by the National Natural Science Foundation of proved that G/Z and Y-TZP exhibit similar cell attach- China for Young Scholars (81400557); China Postdoctoral Science Foundation ment properties before and after aging, indicating the (2017M622742); Science and Technology Planning Project of Guangdong promising surface biological properties of G/Z. Province, China (2016A010103017); and scientific cultivation and innovation project of Jinan University (21617494). In vivo irritation tests are critical for the long-term ap- plication of oral medical devices. Herein, no macro- Availability of Data and Materials scopic or microscopic pathological changes were All data are fully available without restriction. observed for G/Z-treated mucosa (Fig. 6a, b). Authors’ Contributions The existence of a large amount of m-ZrO could re- TS and RYL conceived and designed the study. YLZ and XLF performed the sult in a decrease in the strength of zirconia. The reliable experiments. XNL provided the mutants. TS wrote the paper. RFL reviewed and edited the manuscript. All authors read and approved the manuscript. biocompatibility of the G/Z system might be attributed to the small phase change during the infiltration proced- Competing Interests ure, which was proven in our previous study [4]. An- The authors declare that they have no competing interests. other study proved the fair aging resistance of infiltrated Y-TZP materials. Inokoshi et al. [30] reported that Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Al O -infiltrated Y-TZP was hydrothermally stable after 2 3 published maps and institutional affiliations. aging thanks to a high amount of c-ZrO phase at the interlayer surface, although it has a higher initial mono- Author details Medical Center of Stomatology, The First Affiliated Hospital of Jinan clinic volume fraction compared to the Y-TZP. University, Guangzhou 510630, China. Shenzhen Traditional Chinese Several studies confirmed the reliable biocompatibility 3 Medicine Hospital, Shenzhen 518033, China. Department of Stomatology, of the glass-zirconia composition. L-929 fibroblast- and Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. Saos-2 osteoblast-like cells presented good adhesion and Received: 27 November 2017 Accepted: 14 February 2018 proliferation on the surface of HAp-Al O -ZrO (FGM), 2 3 2 indicating the good biocompatibility of FGM [31]. A References glass (Na O-SiO -B O -CaO)-Hap-ZrO implant mater- 2 2 2 3 2 1. Sailer I, Gottner J, Kanel S, Hammerle CH (2009) Randomized controlled ial showed better bonding with bone than a titanium im- clinical trial of zirconia-ceramic and metal-ceramic posterior fixed dental plant material after a 3-month implantation period in prostheses: a 3-year follow-up. Int J Prosthodont 22:553–560 2. 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Nanoscale Research LettersSpringer Journals

Published: Feb 23, 2018

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