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Enhancing the Bioactivity of Yttria-Stabilized Tetragonal Zirconia Ceramics via Grain Boundary Activation Jinhuan Ke, Jiandong Ye, and Fupo He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03405 • Publication Date (Web): 19 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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ACS Applied Materials & Interfaces

Enhancing the Bioactivity of Yttria-Stabilized Tetragonal Zirconia Ceramics via Grain Boundary Activation

Jinhuan Ke a,b, Fupo Hec, Jiandong Ye a,b,d a

﹡

School of Materials Science and Engineering, South China University of Technology,

Guangzhou 510641, China b

National Engineering Research Center for Tissue Restoration and Reconstruction,

Guangzhou 510006, China c

School of Electromechanical Engineering, Guangdong University of Technology,

Guangzhou 510006, China d

Key Laboratory of Biomedical Materials of Ministry of Education, South China

University of Technology, Guangzhou 510641, China

ABSTRACT Yttria-stabilized tetragonal zirconia (Y-TZP) has been proposed as a potential dental implant due to its good biocompatibility, excellent mechanical properties, and distinctive aesthetic effect. However, Y-TZP cannot form chemical bonds with bone tissue because of its biological inertness, which affects the reliability and long-term efficacy of Y-TZP implants. In this study, to improve the bioactivity of Y-TZP ceramics while maintaining their good mechanical performance, Y-TZP was modified by grain boundary activation via the infiltration of a bioactive glass (BG) sol into the

﹡

Corresponding author. E-mail address: [email protected] (J. Ye) 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

surface layer of Y-TZP ceramics under different negative pressures (atmospheric pressure, 0.05 and 0.1 kPa), followed by gelling and sintering. The in vitro bioactivity, mechanical properties and cell behavior of the Y-TZP with improved bioactivity were systematically investigated using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS), electron probe microanalysis (EPMA) and Raman spectroscopy. The results of the bioactivity test conducted by immersing Y-TZP in simulated body fluid (SBF) showed that a bone-like apatite layer was produced on the entire surface. The mechanical properties of the modified Y-TZP decreased as the permeation negative pressure in the BG infiltration process increased relative to those of the Y-TZP blank group. However, the samples infiltrated with the BG sol under 0.05 kPa and atmospheric pressure still retained good mechanical performance. The cell culture results revealed that the bioactive surface modification of Y-TZP could promote cell adhesion and differentiation. The present work demonstrates that the bioactivity of Y-TZP can be enhanced by grain boundary activation, and the bioactive Y-TZP is expected to be a potential candidate for use as a dental implant material.

KEYWORDS: Y-TZP; BG infiltration; grain boundary activation; bioactivity; mechanical properties; cellular response; dental implant material

1. INTRODUCTION Since dental implants were first introduced by Brånemark et al. over 50 years 2 ACS Paragon Plus Environment

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ago, they have been well accepted as a reliable treatment for missing teeth caused by periodontal disease, dental caries and trauma, etc.

1

Dental implants are produced

using metals, ceramics or polymers. Although some metals, alloys and organic polymer materials can partially satisfy the requirements for dental restorative materials, some deficiencies remain. Polymer materials have disadvantages of poor wear resistance and low mechanical strength. Conversely, metals, particularly pure titanium and its alloys, are currently the leading dental implants because of their excellent

mechanical

osseointegration

2-3

properties,

corrosion

resistance,

biocompatibility

and

. However, the increasing clinical application of metal dental

implants is accompanied by increasing problems. First, gingival recession may cause aesthetic problems due to the inherent grayish color of titanium and its alloys. In addition, toxic metal ions will slowly be released from the implants into the oral environment. These toxic metal ions, on the one hand, may induce bone resorption, causing failure of the oral implants; on the other hand, after these metal ions accumulate around soft tissue, lymph nodes or other locations of the mouth, they may become potential allergens

4-7

. Therefore, further research on non-metallic implant

materials is of great significance for developing oral implants. With the increasing aesthetic requirements for dental implants, Y-TZP has received increasingly more attention from the scientific and medical communities as a potential dental implant material. Y-TZP is characterized by good biocompatibility, excellent mechanical properties and a unique aesthetic effect

4,8-10

. Moreover, in

contrast to traditional metal implants, Y-TZP is capable of resisting corrosion and 3 ACS Paragon Plus Environment


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maintaining long-term chemical stability in the human electrolytic environment, without deteriorations in its mechanical strength. In 1988, partially stabilized zirconia was successfully used as an implant material by Akageawaet et al.

4

in an animal

experiment. The success rate of commercial zirconia dental implants (CeraRoot, Senden, and Nobel Biocare) that have been applied clinically ranges from 76.6% to 95.0% 2,11-12. Different studies have shown that zirconia could be a good replacement for titanium in dental implants. However, Y-TZP is biologically inert.

13

When Y-TZP is implanted in vivo, no

chemical bonds form between the implant and adjacent bone tissues; the implant is surrounded by fibrous tissues, which impede the osseointegration process. Osseointegration, which stimulates bone healing and new bone formation, is a critical process for the success of an implant 14. As an implant, the bioinert Y-TZP is subject to premature failure, which is caused by micro-motion and loosening. It is known that a bioactive material can firmly bond with living bone by forming an apatite layer on its surface after implantation in a bony site. Consequently, enhancing the bioactivity of Y-TZP is expected to greatly improve implant fixation through the formation of chemical bonds between bone and the implant. The bioinert material can obtain good surface bioactivity via the formation of organic functional groups or the deposition of a thin bioceramic coating via several methods 15. These methods include the micro-arc oxidation of zirconium 16, CO2-irradiation 17, chemical etching 18, ion implantation 19, surface coating

20-22

, Al2O3 cyclone sand blasting

biomimetic mineralization

25

23

, dopamine modification

24

,

, and incorporating bioactive materials such as calcium 4 ACS Paragon Plus Environment

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phosphate, bioactive glass, and wollastonite

26-28

. However, incorporating bioactive

constituents into zirconia to form composites will lead to a significant decrease in mechanical performance. Although a surface coating cannot affect mechanical performance, the adhesion strength between the coating and substrate is not satisfactory; under the complex environment in the oral cavity, the implants are prone to loosening and peeling, caused by the dissolution and desquamation of the bioactive coating due to the permeation and corrosion of saliva. In this study, to improve the bioactivity of Y-TZP ceramics while maintaining their good mechanical performance, surface bioactivity was imparted to Y-TZP by grain boundary activation via the infiltration of a BG sol into the surface layer of Y-TZP ceramics, followed by gelling and sintering processes. The phase composition, microstructure, bioactivity, mechanical properties and cell behavior of the Y-TZP with improved bioactivity were systematically investigated.

2. MATERIALS AND METHODS 2.1. Materials Preparation 2.1.1. Preparation of Bioactive Glass Sol The BG used in this study contains 58 wt% SiO2, 9 wt% P2O5, and 33 wt% CaO 29. Tetraethoxysilane (TEOS, Si(OC2H5)4), triethylphosphate (TEP, OP(OC2H5)3) and calcium nitrate (Ca(NO3)2·4H2O) were used as the SiO2, P2O5 and CaO precursors, respectively. First, TEOS was hydrolyzed with a 0.1 M HNO3 solution as a catalyst, followed by the sequential addition of TEP and calcium nitrate in 45-min 5 ACS Paragon Plus Environment


intervals. The reaction solution was maintained under constant stirring during the hydrolysis process. Subsequently, the solution was stirred for an additional 1 h to achieve a complete hydrolysis reaction and dissolution of calcium nitrate and to obtain the BG sol. 2.1.2. Preparation of Presintered Zirconia Green Compacts High-purity Y-TZP (3 mol% Y2O3) powder was commercially obtained from Jiangxi Size Material Co. Ltd. The Y-TZP powder was loaded into stainless steel molds after granulation, and uniaxially pressed under 10 MPa by a pressing machine. Y-TZP disks (10 mm  2 mm) and bars (5 mm  6 mm  60 mm) were obtained. The disks and bars were enclosed in latex sheaths and subjected to cold isostatic compaction under 100 MPa, and then they were presintered at 900C for 2 h. The presintered samples were sequentially polished using 500#, 800#, and 1200# carborundum abrasive papers. After ultrasonic cleaning in ethanol for 10 min, the samples were removed and air dried. 2.1.3. Sol Infiltration To study the influence of the sol penetration depth on the microstructure and mechanical properties of Y-TZP, the as-presintered Y-TZP samples were randomly divided into 4 groups (groups A, B, C, and D). For group A (control group), the presintered Y-TZP sample was only sintered at 1450°C without sol infiltration. For groups B, C and D, the presintered Y-TZP samples were subjected to sol infiltration for 5 min under atmospheric pressure, 0.05 kPa and 0.1 kPa, respectively, to regulate the penetration of the BG sol into the surface layer of the Y-TZP samples. 6 ACS Paragon Plus Environment

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Then, the samples of groups B, C and D were placed in a confined environment with a certain humidity (40%-60%) at room temperature for 24 h to complete the sol-gel transformation process. Finally, the samples of groups B, C and D were sintered at 1450C. Groups A, B, C and D are labeled TZP, TZP-BGap, TZP-BG0.05, and TZP-BG0.1, respectively, in this article.

2.2. Material Characterization 2.2.1. Phase Analysis and Morphology Observation X-ray diffraction (XRD, X'Pert PRO, PANalytical B.V., the Netherlands) with CuKα radiation (40 kV, 40 mA) was used to determine the phase compositions of the samples. Diffraction patterns were recorded from 10° to 90° using a scan speed of 1/min and a step size of 0.05. The polished surfaces of the samples were observed by scanning electron microscopy (SEM, Merlin, CarlZeiss AG, Germany). For the morphology observations, the polished samples were thermally etched at 1200°C for 15 min. The Ca elemental distribution in the BG permeated surface was studied by electron probe microanalysis (EPMA, EPMA-1600, Shimadzu, Japan) on the polished cross sections of the samples. 2.2.2. Density and Porosity Determination Archimedes principle was employed to measure the open porosity and density of the samples. The mass of the dry samples was recorded as W1. After soaking in deionized water under vacuum for 2 h at room temperature (20-30°C), the samples were suspended in water using a fine copper wire. The weight of the suspended 7 ACS Paragon Plus Environment


samples in water was recorded as W2. The samples were removed from the water, and their surface was wiped with a water-saturated towel. The mass of the samples filled with water was recorded as W3. Bulk density (D1), relative density (D2) and porosity (P) were calculated as follows: D1=W1·ρ/(W2-W3); D2=D1/D0; P=(W2-W1)/(W2-W3) where D0 is the theoretical density of Y-TZP (6.10 g/cm3) and ρ is the density of water (1 g/cm3). 2.2.3. Characterization of Mechanical Properties The flexural strength was measured using a universal material testing machine (Instron 5967, Instron, USA) with a three-point bending test according to ASTM C 1116-94. The specimen size was 3 mm  4 mm  36 mm. The span was 30 mm, and the rate of loading was 0.5 mm/min. The flexural strength of the samples was calculated using the following formula: σ=3PL/2bh2 where σ is the flexural strength (MPa), P is the fracturing load (N), L is the span between the fulcrum below (mm), B is the width of the sample (mm), and h denotes the height of the sample (mm). The fracture toughness was tested using the single notched beam method according to ASTM C-1421. The specimen size was 3 mm × 4 mm × 36 mm. The span was 30 mm, and the movement speed of the crosshead was 0.5 mm/min. A notch with an incision depth equal to 1.62.4 mm and incision width of 0.25 mm was made using a cutting machine. The formula for calculating the fracture toughness was as 8 ACS Paragon Plus Environment

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follows: K IC 

3PL a  Y (a ) w 2bh 2

Where KIC is the fracture toughness (MPa·m1/2), P is the maximum load when the sample broke (N), L is the span (mm), b is the width of the sample (mm), a is the notch depth (mm), w is the height of the sample (mm), and Y is the stress intensity shape factor. 2.2.4. Characterization of Bioactivity The bioactivity was evaluated by immersing the samples in the 1.5  simulated body fluid (SBF) solution, which was prepared according to the formula developed by Kokubo et al. 30. In brief, NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2 and Na2SO4 were dissolved in distilled water. The solution was buffered at pH 7.4 using tris-hydroxymethyl-aminomethane and HCl at 36.5°C. The composition of the 1.5 × SBF solution was as follows: Na+, 213.0; K+, 7.5; Mg2+, 2.25; Ca2+, 3.75; Cl−, 221.7; HCO3−, 6.3; HPO42−, 1.5; and SO42−, 0.75 mM. All samples were ultrasonically cleaned for 5 min, followed by heat sterilization at 120°C for 2 h prior to immersion in SBF. Each sample was immersed in the SBF solution under dynamic conditions at 36.5°C. The ratio of the sample surface area to the SBF volume was 0.1 cm-1. The SBF was refreshed daily such that apatite formation would not be inhibited due to the lack of ions, as suggested by Kokubo et al. 30. After soaking in SBF for a scheduled amount of time, the samples were removed from the SBF solution, carefully washed with distilled water, and air dried. An SEM equipped with EDS was used for the morphological and elemental 9 ACS Paragon Plus Environment


characterizations of the deposited coatings. Raman (LabRAM Aramis, HORIBA JobinYvon, France) and XRD were used to determine the structures and phase compositions of the coatings, respectively. 2.2. 5. In Vitro Biological Investigation Mouse bone marrow-derived mesenchymal stem cells (mBMSCs; ATCC, Cat. no. CRL-12424) at passage 7 were used to assess the cell behavior of the samples. mBMSCs were cultured in cell culture flakes in an incubator with 5% CO2 and 95% relative humidity at 37°C. The complete culture medium, which was prepared using high-glucose Dulbecco's modified Eagle's medium (H-DMEM; Gibco, no. 11965-092) with 10 vol% fetal bovine serum (FBS; HyClone, Cat. no. NWJ0473), was used for cell culture. The medium was refreshed every two days. All samples ( 8 mm  2 mm) were heat sterilized at 120°C for 2 h prior to cell seeding. The sterile samples were transferred into 48-well plates, and 500 μL of complete culture medium was added to each well to pre-wet the samples. After 2 h, the medium was wiped off, and mBMSCs in the culture medium were added to each well with a cell density of 1.5 × 104 cells per well. To evaluate the morphology of the cells, after 2 days of cell culture, the cells on the samples were immobilized using a 4 vol% glutaraldehyde solution at 4°C for 30 min at room temperature. After washing once with PBS, the cells were permeabilized for 5 min with 0.1% Triton X-100 (Sigma-Aldrich, USA). The cell-sample constructs were washed 3 times with PBS; then, the cell cytoskeletons and nuclei of mBMSCs were stained with Alexa Fluor®488-phalloidin (AAT Bioquest, USA) and 10 ACS Paragon Plus Environment

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4,6-diamidino-2-phenylindole

(DAPI,

Sigma-Aldrich,

USA),

respectively,

in

accordance with the manufacturers’ protocols. Finally, a confocal laser scanning microscope (SP5, Leica, Germany) was used to capture fluorescent images of the cytoskeletal organization and nuclei of mBMSCs. A Live/Dead kit (Biotium, USA) was used to evaluate the cell viability after being cultured for 1 and 3 d. After the samples were washed 3 times with PBS, a Calcein-AM/PI solution was added to each well, followed by incubating in a thermostatic water bath away from light at 37°C for 15 min. Then, the samples were rinsed with PBS to remove the staining solution. A fluorescence microscope (Eclipse Ti-U, Nikon, Japan) was used to observe the cell viability. The metabolic activity of cells was determined using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) assay. First, the cell-seeded samples were transferred to a new 48-well plate after incubation for 1, 3 and 7 d. Then, 250 μL of CCK-8 reagent solution was added to each well. After incubating for 1 h, 100 mL of supernatant was transferred to a 96-well plate. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Thermo 3001, Thermo, USA). To investigate alkaline phosphatase (ALP) activity and protein concentration, 5  104 cells were seeded on the samples in the wells and cultured by adding 500 μL of osteogenic induction medium containing 90 vol% H-DMEM with 10 vol% FBS, 10 mM sodium glycerophosphate, 10 nM dexamethasone, and 82 mg/mL vitamin C. After 7, 10 and 14 days of cell culture, cell lysis was achieved with the addition of 11 ACS Paragon Plus Environment


250 μL of lysis buffer (0.1 vol% Triton X-100 in 10 mM Tris–HCl buffer solution, pH = 7.4) and incubation at 4°C for 4 h. To determine the ALP activity, 20 μL of cell lysate was incubated with 200 μL of 5 mM p-nitrophenyl phosphate (PNPP, Sigma-Aldrich, USA) solution at 37°C for 15 min. Subsequently, 200 μL of 1 M NaOH was used to terminate the reaction, and the released p-nitrophenol concentration was determined photometrically at 405 nm. Total protein content was assessed using a BCA Protein Assay Kit (Thermo Scientific, USA). For this purpose, 20 μL of cell lysate was incubated with 160 μL of working reagent at 37°C for 15 min, which was then measured at a wave length of 562 nm using an ELISA reader. ALP activity was calculated as enzyme activity unit per milligram of total protein content.

2.3. Statistical Analysis All collected data are expressed as the mean values and standard deviations. Statistical analyses were performed using Student’s t-test for multiple comparisons among the different groups. A confidence level of p

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