Colloids and Surfaces B: Biointerfaces 118 (2014) 226–233
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Effect of ZrO2 additions on the crystallization, mechanical and biological properties of MgO–CaO–SiO2 –P2 O5 –CaF2 bioactive glass-ceramics H.C. Li a,b , D.G. Wang a,b,∗ , X.G. Meng a,b , C.Z. Chen a,b,∗ a Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials, Ministry of Education, Shandong University, Shandong, Ji’nan 250061, People’s Republic of China b School of Materials Science and engineering, Shandong University, Shandong, Ji’nan 250061, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 12 January 2014 Received in revised form 28 March 2014 Accepted 31 March 2014 Available online 13 April 2014 Keywords: ZrO2 Glass-ceramic Mechanical properties Bioactivity Biocompatibility
a b s t r a c t A series of ZrO2 doped MgO–CaO–SiO2 –P2 O5 –CaF2 bioactive glass-ceramics were obtained by sintering method. The crystallization behavior, phase composition, morphology and structure of glass-ceramics were characterized. The bending strength, elastic modulus, fracture toughness, micro-hardness and thermal expansion coefficient (TEC) of glass-ceramics were investigated. The in vitro bioactivity and cytotoxicity tests were used to evaluate the bioactivity and biocompatibility of glass-ceramics. The sedimentation mechanism and growth process of apatites on sample surface were discussed. The results showed that the mainly crystalline phases of glass-ceramics were Ca5 (PO4)3 F (fluorapatite) and -CaSiO3 (-wollastonite). m-ZrO2 (monoclinic zirconia) declined the crystallization temperatures of glasses. t-ZrO2 (tetragonal zirconia) increased the crystallization temperature of Ca5 (PO4 )3 F and declined the crystallization temperature of -CaSiO3 . t-ZrO2 greatly increased the fracture toughness, bending strength and micro-hardness of glass-ceramics. The nanometer apatites were induced on the surface of glassceramic after soaking 28 days in SBF (simulated body fluid), indicating the glass-ceramic has good bioactivity. The in vitro cytotoxicity test demonstrated the glass-ceramic has no toxicity to cell. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Some compositions of glass-ceramics are known as bioactive ceramics and can promote bone regeneration [1–3]. Their surface can form a bioactive apatite layer, which is similar to the mineral phase in bone and provides strong interfacial bonding between implant and biological tissues [4,5]. A/W (MgO–CaO–SiO2 –P2 O5 –CaF2 ) glass-ceramic composes of crystalline apatite and wollastonite. Due to the presence of apatite and wollastonite, A/W glass-ceramic possesses high bioactivity and mechanical properties in comparison to other glass-ceramics [6,7]. Moreover, it has been reported that the bioactivity index of A/W glass-ceramic is higher than that of dense sintered HA (hydroxyapatite), which means A/W glass-ceramic requires less time for bone bonding compared to HA [7,8].
∗ Corresponding authors at: Jing Shi Road # 17923, Jinan 250061, Shandong, People’s Republic of China. Tel.: +86 531 88395991; fax: +86 531 88395991. E-mail addresses: [emailprotected] (H.C. Li), [emailprotected] (D.G. Wang), [emailprotected] (X.G. Meng), [emailprotected] (C.Z. Chen). http://dx.doi.org/10.1016/j.colsurfb.2014.03.055 0927-7765/© 2014 Elsevier B.V. All rights reserved.
A/W glass-ceramic has been considered as a highly desirable implant material, because its desirable bioactivity and mechanical properties can be maintained for a long period in body environment [9]. However, its application may be limited because of its relatively low fracture toughness. An effective strategy for improving fracture toughness is to incorporate some toughening agents into A/W glass-ceramic [10]. Zirconia (ZrO2 ), due to its chemical inertness and good mechanical strength, is often used in dental and orthopaedic application [11,12]. It shows a morphological fixation with surrounding tissues and does not release harmful substances when implanted into human body [13]. Especially, ZrO2 , due to its martensitic phase transformation of tetragonal to monoclinic symmetry, is widely used as an effective toughening material [14–17]. Different preparation methods, such as melting method [18], sintering method [19] and sol–gel method [20], have been introduced to prepare ZrO2 doped bioactive glass-ceramic. Sol–gel bioglass has low preparation temperature and high product purity, however, sol–gel method exists some disadvantages, such as uncontrollable rate, hom*ogeneity of gelation, long production term and so on [21]. Melting method requires high temperature and long heat treatment time, which affects the scope of glass composition and bioactivity of glass. The sintering process has
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lower processing temperature, shorter processing time and higher proportion of crystal phases [22], which overcome the limitations of the conventional melting method [23]. Moreover, it is easier to integrate other useful materials into glass during the sintering process. The glass-ceramic with good performance sometimes can also be prepared by this method without nucleating agent [24]. In recent years, some related researches about the effect of additives on the properties of glass-ceramics have been reported. Kamitakahara et al. [25] studied the effect of ZnO content on the chemical durability and apatite-forming ability of A/W glassceramics. Marghussian’s study [26] showed ZrO2 decreases the crystallization rates and improves the bending strength of CaOTiO2 -P2 O5 microporous glass-ceramics. Mollazadeh’ study showed [11] ZrO2 has no significant influence on the mechanical properties of apatite-mullite glass-ceramic. However, there are few studies about the effect of different crystal types of ZrO2 on the crystallization and mechanical properties of A/W glass-ceramics, and few researches discuss the biocompatibility of ZrO2 doped glassceramics. In the present research, ZrO2 has been incorporated into A/W bioactive glass-ceramics using sintering method. The objective of this study is to evaluate the influence of different crystal types of ZrO2 and ZrO2 content on the crystallization behavior and mechanical properties of glass-ceramic, and study the in vitro bioactivity and biocompatibility of glass-ceramics to find a glass-ceramic with outstanding properties for biomedical applications. Furthermore, the sedimentation mechanism and growth process of apatites on sample surface is discussed.
2. Materials and methods 2.1. Preparation of glasses and glass-ceramics The glasses based on the system of A/W (MgO–CaO–SiO2 –P2 O5 –CaF2 ) were prepared using sintering method. Reagent grade SiO2 , Mg(NO3 )2 ·6H2 O, CaCO3 , NH4 H2 PO4 and CaF2 were mixed hom*ogeneously in the required proportion to obtain the composition of 34% SiO2 , 4.6% MgO, 44.7% CaO, 16.2% P2 O5 and 0.5% CaF2 in weight ratio, and the pre-mixed batches were put into a corundum crucible and ball milled for 5 h to obtain powders. Then the mixed powders were melted in a corundum crucible at 1450 ◦ C for 2 h with a heating rate of 5 ◦ C/min. The melted glasses were quenched into distilled water, after drying, the quenched glasses and anhydrous ethanol were put into the prepared corundum jar at the rate of 1:1 in mass followed by 5 h ball milling. After that the glass powders were dried and then sieved to obtain the original glass powders with particle size of less than 37 m. The original glass powders were modified with different amount of monoclinic zirconia (m-ZrO2 , 300 mesh) and 8 wt.% Y2 O3 partially stabilized tetragonal zirconia (t-ZrO2 , 200–325 mesh) powders, and the mixed powders were ball milled for 5 h to mix evenly. Tetragonal zirconia is unstable and can be easily translated into monoclinic zirconia at room temperature. The addition of yttria (Y2 O3 ) can not only stabilize tetragonal zirconia, but also a right amount of yttrium (Y) has protective effects on cells and can improve the biocompatibility of materials. For preparation of the compact glass-ceramic samples, the obtained glass powders, mixed with polyvinyl alcohol in 10:1 weight ratio, were shaped by pressing and 300 MPa cold isostatic pressure. After that the compact samples were sintered in high temperature furnace with a heating rate of 5 ◦ C/min. The suitable heat treatments were determined by differential thermal analysis (DTA) of the base glass powders. There are seven kinds of glass-ceramics with different components. 0-GC refers to A/W glass-ceramic. 2mGC, 5m-GC and 8m-GC refer to A/W glass-ceramic contained 2, 5
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and 8 wt.% m-ZrO2 , respectively. 2t-GC, 5t-GC and 8t-GC refer to A/W glass-ceramic contained 2, 5 and 8 wt.% t-ZrO2 , respectively, where t-ZrO2 refers to 8 wt.% Y2 O3 partially stabilized tetragonal zirconia.
2.2. Characterization The thermal properties of these glasses were examined by differential thermal analysis (DTA, SDT Q600 of TA Instruments) from room temperature to 1200 ◦ C at a rate of 10 ◦ C/min under an air atmosphere. The crystalline phases were identified using a Bruker D8 Advance X-ray diffractometer (XRD). Scans were run at a speed of 4 ◦ C/min with 0.02◦ increment using Cu K␣ radiation produced at 40 kV and 40 mA on sample surface. Microstructure of samples was examined using a JEOL JSM 6380LA scanning electron microscope (SEM). Fourier Transform Infrared Spectroscopy (FTIR) analysis was conducted in a Bruker Optics VERTEX-70 spectrometer using KBr pellets technique. For transmission electron microscope (TEM) observations, the apatites formed on glass-ceramic surface were milled, then dispersed in ethanol and transferred to copper grids. The examinations were performed on JEOL JEM 2100F.
2.3. Mechanical properties The three-point bending method was used to test the bending strength and elasticity modulus of glass-ceramics, using rectangular bars (36 mm × 4 mm × 3 mm) and RGD-5 type electronic tensile machine at a cross-head speed of 0.5 mm/min (span is 30 mm). Single edge notch beam method (SENB) was used to measure the fracture toughness of glass-ceramics (20 mm × 4 mm × 2 mm) using RGD-5 type electronic tensile machine. The glass-ceramic specimens were notched with the width of 0.25 mm and the notch depth of 2 mm at a low speed of 0.05 mm/min. The experimental results were calculated from five testing samples. Micro-hardness measurements were conducted using HV-1000 Huayin digital micro-hardness tester. Eight measurements were performed at different locations of the flat surface of glass-ceramic through the applied load of 500 g for 15 s. The test results were means of the values, not including the minimum and maximum values. The results of performance test were calculated according to the following formulas [27]: f =
E=
KIC =
3PL 2bh2
L3 (P2 − P1 ) × 10−3 4bh3 (Y2 − Y1 ) √ 3PL a 2bh2
1.93 − 3.07
a h
a 2
+ 14.53
h
a 3 a 4
− 25.07
h
+
h
where f is the bending strength (MPa), E the elasticity modulus (GPa), KIC the fracture toughness (MPa ml/2 ), P the fracture load (N), L the sample span (mm), b the sample width (mm), h the sample depth (mm) and a is the notch depth (mm).
2.4. Thermal expansion coefficient (TEC) The TEC was measured by thermal expansion coefficient tester (PCY-III-1000) on squareness specimens of 50 mm × 6 mm × 6 mm from 20 to 600 ◦ C with a heating rate of 5 ◦ C/min. The experimental results were calculated from three testing samples.
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Fig. 1. DTA curves of the various glass powders (10 ◦ C/min).
2.5. In vitro assays in SBF The assessment of in vitro bioactivity were performed by soaking glass-ceramics in SBF at 37 ◦ C, and the ionic composition of SBF is similar to that of human plasma [7]. During immersion, the SBF was drawn in accordance with VSBF /SA = 10 cm, where VSBF is the volume of SBF and SA is the total surface area of the soaked sample. The SBF was contained in sterile polyethylene containers and renewed every other day. After being soaked, samples were rinsed with deionized water and acetone and then dried at room temperature. After that, these sample were determined by XRD, FTIR, SEM and TEM analysis as described above. 2.6. Tests for in vitro cytotoxicity The in vitro cytotoxicity of glass-ceramic was evaluated with methyl thiazolyl tetrazolium (MTT) cell assay, which is a standard test for screening the toxicity of biomaterials and is carried out in accordance with GB/T 16886.5-2003/ISO 10993-5:1999 standard. This method is based on the formazan formation by the metabolically active cells after their exposure to MTT, and fibroblasts L-929 cell was used to detect the cytocompatibility of glass-ceramic [28]. The preparation processes of the test sample extract fluid and the negative control sample extract fluid are as follows: The test sample (5t-GC) and negative control sample (polyethylene particles) were firstly rinsed with deionized water, then dried by filter paper and autoclaved at 121 ◦ C for 30 min. After cooling, the samples were immersed in extraction vehicle that contained 10% fetal bovine serum (FBS) (Lanzhou National Hyclone Bio-Engineering Co., Ltd.) at 37 ◦ C for 24 h, and the ratio of sample mass to extraction volume was 0.2 g/mL. The extraction was performed in sterile, chemically inert, closed containers by using aseptic techniques. The preparation process of the cell suspension is as follows: The L929 cells (Institute of Materia Medica, Chinese Academy of Medical Sciences) were firstly treated with 0.25% trypsin. After that, RPMI1640 medium (Gibco-BRL Company, USA) supplemented with 10% fetal bovine serum (FBS) was added to these cells to prepare monoplast suspension, and the density of cell suspension was adjusted to 104 per milliliter. MTT method: The prepared cell suspension was seeded in culture plate of 96 wells (Bectondickinson Company, USA), and each well contained 100 L cell suspension. The plate was incubated in humidified atmosphere (37 ◦ C, 5% CO2 ) in culture box (Forma3111, Forma Company, USA) for 24 h to allow the cells to attach to the wells. The 96 wells were randomly divided into experimental group
5t-GC, negative control group and positive control group. The test sample extract fluid (100 L), the negative control sample extract fluid (100 L) and 6.3% phenol (100 L) were, respectively, added to the culture wells of experimental group, negative control and positive control group to replace the original culture medium, then incubated the culture wells for 24 h in humidified atmosphere. Afterwards, 10 L of the 0.5% MTT (5 mg/mL) was added to the culture wells. After 4 h incubation, the culture medium of all wells was removed carefully and then 150 L dimethyl sulfoxide (DMSO) was added into each well following by shaking for 10 min to solubilize the formazan crystals. The optical density (OD) of each well was quantified photometrically using a Victor1420 (PerkinElmer Company, USA) at the wavelength of 570 nm. The test was replicated three times. The relative proliferation rate (RGR) was calculated using the following formula: RGR (%) =
ODf ODnc
× 100%
where ODf refers to the mean value of the measured optical density of the 100% extracts of experimental group and ODnc refers to the mean value of the measured optical density of negative control group. 3. Results 3.1. Thermal property analysis The DTA curves of various glass powders are shown in Fig. 1. A small endothermic peak considers as glass transition temperature range, and its minimum corresponds to glass transition temperature (Tg ) [29]. From the DTA curve, the Tg of sample not contained ZrO2 is 693.74 ◦ C, and the temperatures of glasses contained 2 and 5 wt.% m-ZrO2 don’t appear to be much different from the glass not contained m-ZrO2 . The temperature of sample contained 8 wt.% mZrO2 reduces to 639.70 ◦ C. While the Tg has increased by about 33 ◦ C when 8 wt.% t-ZrO2 was added. When 2 or 5 wt.% t-ZrO2 was added, this temperature has increased slightly. The other three exothermic peaks originate from the amorphous-crystalline transformation, and the maximum temperature refers to crystallization peak temperature [29]. Based on these results, m-ZrO2 drops all the crystallization temperatures. 2 wt.% t-ZrO2 increases the first crystallization temperature, declines the second and third crystallization temperatures. With the increase of t-ZrO2 content, the crystallization temperatures of all phases show the same changing trend. When the temperature
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crystal growth. For the glass contained m-ZrO2 , the nucleation temperature is 700 ◦ C, and the three steps crystallization temperature are 815, 936 and 1006 ◦ C, respectively. For the glass contained tZrO2 , these temperatures are 727, 872, 911 and 965 ◦ C, respectively. The crystallization temperatures of the glass contained different content of ZrO2 are basically consistent, so the same heat treatment system has been adopted. In order to study how the glass composition affects the mechanical properties of glass-ceramics, the holding times at the nucleation temperature is 2 h, and the times at the crystallization temperatures are all 4 h. 3.2. Materials characterization
Fig. 2. XRD patterns of the glasses after heat treatment.
is above 1170 ◦ C, the DTA curves all declined sharply, indicating the glasses begin to melt. According to the analysis result of DTA, the suitable heat treatment systems were determined. For the glass not contained ZrO2 , the heat treatment system is heating the glass 2 h at 700 ◦ C for nucleation followed by 4 h at 820, 974 and 1016 ◦ C, respectively, for
The XRD patterns of the glasses after heat treatment is shown in Fig. 2. Ca5 (PO4 )3 F (Fluorapatite, 15-0876) and -CaSiO3 (wollastonite, 42-0550) are founded in the XRD pattern of sample 0-GC. In addition to these two phases, the peaks assigned to m-ZrO2 appear in sample 5m-GC. The Ca5 (PO4 )3 F and -CaSiO3 peaks also appear in sample 5t-GC. In addition, t-ZrO2 and m-ZrO2 are also observed, and the presence of m-ZrO2 is because of the transformation of t-ZrO2 into m-ZrO2 . 3.3. Mechanical properties The results of mechanical properties of the glass-ceramics are shown in Fig. 3. When t-ZrO2 was added, the fracture toughness value has increased by about 61.96–81.52%, and the bending strength has increased by about 20.53–24.86%. t-ZrO2 increases
Fig. 3. Mechanical properties of the glass-ceramics (where X in X ± SD refers to the mean value, and SD refers to the standard deviation, N = 5. For micro-hardness, N = 8 and X does not include the minimum and maximum values).
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Fig. 4. XRD patterns of glass-ceramic 5t-GC before and after soaking in SBF solution. Fig. 5. FTIR spectra of glass-ceramic 5t-GC before and after soaking in SBF solution.
micro-hardness to 130.95–146.53 GPa from 114.19 GPa. While mZrO2 does not play a positive role to improve these mechanical properties. m-ZrO2 and t-ZrO2 have no obvious influence on elasticity modulus. The elastic moduli of materials are all in the range from 17.47 to 21.5 GPa, which are close to that of the human bone. From this figure, the sample 5t-GC holds good integrated performance. 3.4. Thermal expansion coefficient (TEC) The TECs of glass-ceramics contained different t-ZrO2 content from 20 to 600 ◦ C were measured. The TECs of sample 0-GC, 2tGC, 5t-GC and 8t-GC are 9.70 × 10−6 , 9.71 × 10−6 , 9.83 × 10−6 and 10.3 × 10−6 ◦ C−1 , respectively. The difference of TEC between the coating material and substrate should be less than 1.7 × 10−6 ◦ C−1 , large difference will result the high stresses and lead to the coatings fracture [16,30]. The TECs of these glass-ceramics are all close to that of titanium alloy (Ti6Al4V), so these glass-ceramics are essential to the bioactive coatings on Ti6Al4V substrate. 3.5. In vitro assays in SBF Fig. 4 shows the XRD patterns of glass-ceramic 5t-GC before and after different soaking time in SBF. After soaking in SBF for 7 days, the peak intensity of matrix phases -CaSiO3 and ZrO2 declines significantly, and there is no significant change in the characteristic peaks of Ca5 (PO4 )3 F. Some new peaks of HA (hydroxyapatite: Ca10 (PO4 )6 (OH)2 ) are obviously found but not sharp. The presence of matrix phases Ca5 (PO4 )3 F will hide some HA peaks, because the lattice parameters of HA are very close to that of fluorapatite. With the increasing of immersion time, the matrix phases almost completely disappear, and some peaks of HA and CHA (carbonate hydroxyapatite: Ca10 (PO4 )3 (CO3 )3 (OH)2 ) are observed, which indicates there is a newly formed apatite layer on sample surface. The dispersity of diffraction peak is higher than that before soaking, which shows the crystallinity of the formed apatites is lower, and the apatites have the characteristic of amorphous state. Fig. 5 shows the FTIR spectra of glass-ceramic 5t-GC before and after soaking in SBF. The FTIR spectra of sample before soaking shows that one absorption band at 1039 cm−1 is assigned to the asymmetric stretching vibration Si–O–Si(s, asym), and the peak at 470 cm−1 is the overlap peaks of rocking vibration Si–O(r) and P–O(b). Two non-bridging oxygen bonds (Si–O–NBO) are found
at 950 and 900 cm−1 . The double peaks at 572 and 609 cm−1 are assigned to the bending vibration P–O(b) related to the presence of crystalline phosphate in the glasses [31]. Besides above groups, the band around 1041 cm−1 assigned to P–O(s) is also observed. After soaking in SBF for 2 day, the stretching vibration peaks at 1644 and 3450 cm−1 denoting –OH are observed. After soaking for 7 days, the vibration band corresponding to the carbonate group C–O(b) around at 1465 cm−1 is found. With the extension of soaking time, the peaks of O–H and C–O(b) become stronger and the peak of Si–O–NBO becomes very weaker. After 28 days, the asymmetric stretching vibration Si–O(s) at 1039 cm−1 disappears, which indicates the sample surface is covered by a newly formed apatite. In addition, the C–O(b) around at 873 cm−1 appears. The vibrational peaks of Si–O(s) and Si–O–NBO disappear or weaken and the new vibration peaks of O–H and C–O emerge or strengthen, indicating that the glass-ceramic experiences a dissolution-precipitation in immersion period [32,33]. The morphologies of glass-ceramic 5t-GC before and after different assay periods in SBF were investigated by SEM. As shown in Fig. 6a, the glass-ceramic surface before soaking is smooth. A small amount of spherical grain appears, and no precipitate can be founded. After soaking 2 days, the glass-ceramic surface is covered with a newly discontinuous layer and a large portion of initial glass is still observed. When soaked for 7 days, the newly apatite layer becomes compact. After 14 days, the glass-ceramic surface is fully covered by the apatite layer. With the increasing of soaking time, the surface of the glass-ceramic is more compact and covered with the uniform size of particles. The sedimentary materials are fine spherical particles and the particle size is about 200 nm. Nanoapatite with the characteristics of nanometer materials and apatite itself has been widely used as reconstructive and prosthetic material for osseous tissue, owing to its excellent biocompatibility and tissue bioactivity [34,35]. The microstructure of apatite formed on glass-ceramic 5t-GC surface after soaking 28 days in SBF was investigated by TEM. The image obtained in Fig. 6g clearly shows the plate-like shape of the crystals, and the corresponding selected area diffraction (SAD) pattern of these crystals is assigned to apatite. (1¯ 1 0) and (1 0 1) in ¯ diffraction pattern in Fig. 6h belong to the crystal zone axis (1 1¯ 1). Fig. 6i shows the high-resolution TEM image of (3 0 1) crystal plane of hydroxyapatite, and this crystal plane belongs to the crystal zone axis [0 1 0].
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Fig. 6. SEM morphologies of the sample 5t-GC before and after different assay periods in SBF solution (a) 0d; (b) 2d; (c) 7d; (d) 14d; (e) 21d; (f) 28d, and microstructure (g), the corresponding selected area diffraction pattern (h) and high-resolution TEM image of (3 0 1) crystal plane of hydroxyapatite (i).
3.6. Tests for in vitro cytotoxicity Fig. 7 shows the invert microscopy images of the cells grown of experimental group 5t-GC (a), negative control group (b) and
positive control group (c). It can be seen that cells spread obviously and form visible lamellipodia, and these cells have good activity of adherence and proliferation in Fig. 7(a) and (b). These two groups of cells grow well and the cells are normal in appearance. As is
Fig. 7. Microscopy images of the L-929 cells grown of experimental group 5t-GC (a), negative control group (b) and positive control group (c).
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shown in Fig. 7(c), all cells are round, loosely attached, and the death of most cells has occurred. The quantitative evaluation of the in vitro cytotoxicity test is given by calculating the relative proliferation rate (RGR). The RGR of L-929 fibroblast cells in experimental group 5t-GC, negative control group and positive control group are 93.1%, 99.1% and 6.2%, respectively. The RGR of L-929 fibroblast cells in experimental group 5t-GC is close to the result of the negative control group, indicating the glass-ceramic has no poisonous effect on L-929 cell satisfactory.
4. Discussion To investigate the sintering behaviors of glasses, the glasses were studied with DTA analysis techniques. Various factors, such as the composition of basic glass, the structure and content of additives and so on, affect sintering behaviors of glass [25,11]. The glass transition temperature is mainly affected by the connection strength of glass network structure. In this paper, 2 and 5 wt.% mZrO2 has little effect on the sintering behaviors of glasses, and 8 wt.% m-ZrO2 reduces the transition and crystallization temperatures. These results are agreed with the results reported in the literature [36], which suggests that the effect of m-ZrO2 on the internal structure of glass are related to ZrO2 content. A small amount of m-ZrO2 has filling effect in glass structure. The glass network will be weakened and the structure continuity will be cut off with the increase of m-ZrO2 content, which will lower the transition and crystallization temperatures. While t-ZrO2 increases the transition temperature and the first crystallization temperature, and declines the second and third crystallization temperatures. The reason may be that t-ZrO2 participates the formation of glass [37]. t-ZrO2 raises the glass viscosity by reducing the numbers of non-bridging oxygen, and the glass viscosity relates to the mobility of the structural elements in glass [27]. With the increase of t-ZrO2 content, the glass network is gradually enhanced, which increases the transition and crystallization temperatures. In addition, Y2 O3 exists in glass powders to stabilize t-ZrO2 . Some papers [38,39] suggested that the vacancy defect exists in the structure of Y2 O3 , and other ions in glass may enter the defect, which make the glass network structure rearrange [11]. While t-ZrO2 declines the second and third crystallization temperatures, which reason is likely to be that the precipitation of the first phase changes the composition of the basic glass, which has an impact on the crystallization of other phases [40]. In addition, ZrO2 will appear with the increasing of temperature, which can promote the heterogeneous nucleation and crystal growth of other phases [40]. Three crystallization peaks appear on the DTA curve, which means three phases should be generated. While only two phases Ca5 (PO4 )3 F and -CaSiO3 are detected in XRD patterns after heat treatment, which seems contrary to the DTA results. Combining with the phase diagram of CaO-SiO2 system, ␣-CaSiO3 will be generated when the temperature is higher than 1125 ◦ C. m-ZrO2 and t-ZrO2 all decrease the third crystallization temperatures. Other compositions in glass may also affect the crystallization temperature. The third crystallization temperature should be the crystallization temperature of ␣-CaSiO3 and the reason why ␣-CaSiO3 does not appear in XRD pattern is that ␣-CaSiO3 will transform into low-temperature phase -CaSiO3 during cooling. The addition of t-ZrO2 significantly improves the mechanical properties, especially, the fracture toughness is 61.96–81.52% higher than that of the sample not containing ZrO2 . Such considerable enhancement in toughness is primarily owing to the phase transformation of ZrO2 from t-ZrO2 into m-ZrO2 . The volume expansion occurring in the transformed particles causes stress and then induces microcracks in glass-ceramic. These transformation can absorb or dissipate the energy of these cracks, hence
increasing the toughness of glass-ceramic [41]. When m-ZrO2 was added, the compressive strength reduces slightly and is lower than the strength of glass-ceramic containing no ZrO2 . Various factors such as morphology, crystalline phases, amounts of crystalline phase, size of crystalline particles, porosity distribution and residual strains control the mechanical strength of glass-ceramic [42–45]. The presence of more than one crystalline phase in glassceramic can also lead to a complex mechanical behavior [42,46]. Typically, multi-phased glass-ceramics are prone to microcracking because of the internal thermal stresses caused by the mismatch between density, elastic modulus and thermal expansion coefficient of glass-ceramics and that of crystal phases, which reduces the structural integrity of glass-ceramics and results in low mechanical strengths [11,47]. The surface modifications of glass-ceramic 5t-GC after soaking in SBF were detected by XRD, FTIR, SEM and TEM. The HA and CHA phases are detected after soaking in SBF in XRD patterns (Fig. 4). The disappearance of Si–O(s) at 1039 cm−1 in FTIR spectra (Fig. 5) indicates that the apatite layer is formed on sample surface. The appearance of O–H and C–O absorption bands in FTIR spectra after soaking in SBF confirms the newly formed apatite layer contains HA and CHA. The FTIR spectra analysis results are agreed with the results obtained by XRD patterns. HA is generally accepted to be the prototype for the mineral phase of bone tissues [48]. CO3 2− , is present in these biological apatites tending to increase its solubility and change in the crystal morphology in comparison with pure HA [48]. CHA is strong in flexure and has been reported to have the potential of enhanced bioactive response when compared to noncarbonated HA [49,50]. CO3 2− ions can exist in the structure of HA in two sites. In the A site, CO3 2− ions replace OH− , and the peaks of CO3 2− appear at 880, 1450–1455 and 1540–1550 cm−1 . In the B site, CO3 2− ions replace PO4 3− , and the peaks of CO3 2− appear at 860–870, 1410–1420 and 1455–1470 cm−1 [51]. Some researches [51,52] have shown that HA existed in human bone is acicular crystal in the form of slight crystallization and non-stoichiometric crystal. It contains the group of CO3 2− , and CO3 2− ion is located in the two anionic sites of the structure. In this paper, the C–O peaks denoting CO3 2− appear at 1465 and 873 cm−1 , which shows CO3 2− is assigned to the B site.
Fig. 8. The diagram of chemical reactions between bioactive glass-ceramic and SBF solution (A: SBF solution, B: original glass-ceramic surface, C: glass-ceramic).
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The formation of apatite on glass-ceramic surface is a process of heterogeneous nucleation and growth, and the process involves several reactions [53,54]. There are Ca5 (PO4 )3 F, -CaSiO3 and residual glass phase in glass-ceramic. Ca5 (PO4 )3 F is known to have a much lower dissolution rate than HA in body fluid [55]. Ca5 (PO4 )3 F is supersaturated in SBF, so it does not dissolve in SBF [56]. Due to the stability of the crystal structure, the dissolution of -CaSiO3 is weak. Residual glass phase contains more network ions, and its structure is more loose, which leads to relatively high solubility of the residual glass phase. The lattice parameters between HA, CHA, and fluorapatite are very close, these kinds of apatites can form a continuous solid solution [56]. Fluorapatite can induce the formation of HA [57]. Fig. 8 is the diagram of chemical reactions between bioactive glass-ceramic and SBF solution. Firstly, alkaline earth ions (Ca2+ and Mg2+ ) locating on non-bridging oxygen bonds (Si–O–Ca–O–Si and Si–O–Mg–O–Si) have an rapid ion exchange with H+ or H3 O+ ions in SBF solution. The soluble Si–O–Si in glass-ceramic will be decomposed, and Si–O–H will be generated on glass-ceramic surface. Then the ion exchange induces the formation of SiO2 -rich layer on glass-ceramic surface. SiO2 -rich layer provides favorable conditions for the nucleation of apatite. The reactions are described as follows: Si–O–Ca(Mg)–O–Si
+ 2H3 O+
→ 2 Si–OH + Ca2+ (Mg2+ ) + 2H2 O
Si–O–Si
+ H2 O → 2 Si–OH
Si–OH + OH–Si
→
Si–O–Si
+ H2 O
After that, Ca2+ and PO4 3− are migrated onto SiO2 -rich layer to form the amorphous calcium phosphate compound. The amorphous CaO-P2 O5 layer is unstable and easily combines with OH− and CO3 2− to grow up into crystalline apatite.
5. Conclusion ZrO2 doped MgO–CaO–SiO2 –P2 O5 –CaF2 bioactive glassceramics have been successfully synthesized using sintering method. The mainly crystalline phases of glass-ceramics are Ca5 (PO4)3 F and -CaSiO3 . m-ZrO2 declines the crystallization temperature of glasses, while t-ZrO2 increases the crystallization temperature of Ca5 (PO4 )3 F and declines the crystallization temperature of -CaSiO3 . m-ZrO2 plays a small role in improving the fracture toughness of glass-ceramics. t-ZrO2 significantly increases the fracture toughness, bending strength and micro-hardness of glass-ceramics. The bending strength and fracture toughness of glass-ceramics 5t-GC achieve the maximum value, which increased 24.86% and 81.52% than that of glass-ceramics 0-GC, respectively. The appearance of nano-apatite on the surface of 5tGC glass-ceramic indicates the glass-ceramic has good bioactivity. The in vitro cytotoxicity test demonstrates the glass-ceramic has no toxicity to cell. From the elastic modulus and linear expansion coefficient points of view, the glass-ceramic is one kind of ideal human bone repair materials, and essential to the bioactive coatings on Ti6Al4V substrate.
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