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Yb3+ co-doped bioactive glasses with up-conversion luminescence prepared by containerless processing
Ceramics International 42 (2016) 13168–13175
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Er3 þ /Yb3 þ co-doped bioactive glasses with up-conversion luminescence prepared by containerless processing Qin Li a, Min Xing a, Zhi Chen b, Xiaoya Wang a, Cancan Zhao a, Jianrong Qiu b, Jianding Yu a,n, Jiang Chang a,n a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China b State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 March 2016 Received in revised form 10 May 2016 Accepted 17 May 2016 Available online 20 May 2016
Er3 þ /Yb3 þ co-doped bioactive glasses were prepared via containerless processing in an aerodynamic levitation furnace. The as-prepared glasses were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) equipped with energy dispersive X-Ray spectroscopy (EDX). The up-conversion luminescence of as-prepared glasses was measured using an Omni- 3007 spectrometer. Furthermore, the in vitro bioactivity was evaluated by soaking the materials in simulated body fluid, and the biocompatibility was evaluated in MC3T3-E1 cell culture. The results show that containerless processing is a unique method to prepare homogeneous rare earth doped bioactive glasses. The obtained Er3 þ /Yb3 þ co-doped glasses show green and red up-conversion luminescence at the excitation of 980 nm laser. The XRD analysis confirmed that calcium silicate powders, as starting materials, were completely transformed from the original multi-crystalline phase (CS-P) into the amorphous-glassy phase (CS-G, EYS, LCS) via containerless processing. The SEM observation combined with EDX and FTIR analyses showed that the as-prepared glasses were bioactive. The cell proliferation assay also revealed that the as-prepared glasses were biocompatible and nontoxic to MC3T3-E1 cells. This study suggests that the luminescent bioactive glasses prepared by containerless processing could be used for studying biodegradation of bone implantation materials. & 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: Up-conversion luminescence Bioactive glasses Bioactivity Containerless processing
1. Introduction Bioceramics are widely used for repairing bone defects, and the degradation rate of the bone grafting materials could significantly affect the bone regeneration [1,2]. Moreover, the degradation products are closely related to the material biocompatibility. Therefore, it is of great importance to study the degradation rate and distribution of the degradation products of bioceramics. The radioactive isotope labeling has been applied to trace the degradation of calcium phosphate bioceramics [3], but this method needs strict experimental condition. Hence, the development of new method to label bioceramics for studying the degradation of bone grafting materials after implantation as well as the in vivo distribution of degradation products is meaningful for elucidating the mechanisms of bioceramics/tissue interaction and their clinical n
Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (J. Yu), [email protected] (J. Chang). http://dx.doi.org/10.1016/j.ceramint.2016.05.108 0272-8842/& 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
application. As a traditional silicate compound, calcium silicate is widely studied and used as cement component, refractory materials, anticorrosive and heat insulating coatings [4–7]. In recent years, many studies have shown that calcium silicate ceramics have excellent bioactivity, biocompatibility and biodegradability. Furthermore, calcium silicate ceramics have been found to stimulate bone regeneration after implanting in bone defects, which suggests that this kind of ceramics have great potential as new bone grafting materials [8]. Up-conversion luminescent materials can transform the infrared invisible light into visible light [9]. Rare earth element doped up-conversion luminescent materials have many advantages [10– 18], including higher chemical stability, lower potential toxicity, and longer fluorescence lifetime. In particular, due to the nearinfrared emitting property, the emitting light of these materials has larger optical penetration depth, lower generation of autofluorescence and almost no damage in biological tissue [19–22]. Because of these advantages, the rare earth element doped up-
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Fig. 1. Schematic illustration of the preparation of the glass spheres, (a) adding green samples in an aluminum nozzle; (b) floating the sample with the help of an air flow; (c) melting the sample with laser; (d) rapid-cooling the melt. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
conversion luminescent materials are expected to be used for luminescent bio-analytical assays and luminescent imaging, and might be a new generation of fluorescence bio-labeling materials instead of down-conversion luminescent materials [23]. Up-conversion luminescent materials are usually composed of host materials and doped rare earth ions. Fluoride, oxide, sulfur compounds, fluoride oxide and halide are the most common host materials. Among them NaYF4 exhibits the highest up-conversion luminescence efficiency [24]. For example when Er3 þ , Yb3 þ are co-doped into NaYF4 matrix, Er3 þ works as activator and Yb3 þ as sensitization agent. Oxide host materials have also attracted much attention due to many advantages including simple preparation procedure, uncomplicated working condition and stability [25– 27]. Nowadays many researchers have been investigating up-conversion luminescent materials for biological labeling and drug delivery applications [24,28]. However, most of the studies are mainly focused on the preparation of nanoparticles [29,30]. Less attention is paid on applications as implantable biomaterials, and to the best of our knowledge, there is no report on synthesis and evaluation of up-conversion luminescent calcium silicate glasses as implantable biomaterials. Considering the clinical requirement for the application of bioglasses in human body, one of the most important advantages of the containerless processing for preparing bioglasses is that the purity of the prepared glasses could be well controlled as comparing with the traditional crucible technology for preparing glasses, since no crucible is used which avoids the contamination of glasses during the melting process. In addition, the suppression of heterogeneous nucleation and change of heat transport, achievement of super cooled liquids and rapid solidification, and complete elimination of the internal stress created due to the interaction between samples and the crucible wall are other advantages of the containerless processing method over the traditional technique for preparing glasses [31,32]. Therefore, containerless processing enables developing new type of high performance materials with high purity and different phase composition such as metastable and amorphous phases, and rapid solidification of the materials and study of the undercooled possible [32–34]. Most notably, containerless processing has distinct advantages in developing new functional glasses. Melting under containerless condition can result in the formation of spherical shaped materials automatically by surface tension, which will be transformed into glass ball with ideal spherical surface after cooling down. Levitation is achieved by external applied force such as air in flow on samples to counteract the pull of gravity. In general, materials with high melting point such as calcium silicate are relatively easy to crystallize during the solidification process, so the calcium silicate glass can only be formed when it is under rapid cooling [35]. Therefore, the containerless processing is suitable to fabricate calcium silicate glasses, and the aim of the present study was the preparation and evaluation of up-
conversion luminescent calcium silicate glasses as potential implantable biomaterials. The luminescent property and bioactivity of the prepared materials were investigated.
2. Experimental procedure 2.1. Materials The starting materials were CaSiO3 ceramic powders, Er2O3 (99.99%), Yb2O3 (99.99%), La2O3 (99.99%), TiO2 (99.99%) and ZrO2 (99.9%). The CaSiO3 powders were purchased from Kunshan Huaqiao New Materials Limited (China). And all other chemicals were obtained from China National Medicine Shanghai Chemical Reagent Corporation. 2.2. Preparation of Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses Er3 þ /Yb3 þ co-doped CaSiO3 glasses were prepared by containerless processing in an aerodynamic levitation (ADL) furnace (Fig. 1). Two different approaches were used for preparation of luminescent calcium silicate glasses. The LTZ is known as a good host material for glasses with good luminescent, so we first tried to prepare luminescent bioglasses by direct addition of LTZ luminescent glasses in calcium silicate composition. But, this process requires synthesis of LTZ luminescent glasses first, which is known as a very good luminescent glasses but requires multi-step calcination to fabricate. Besides, for clinical applications, bone graft materials with simple composition are always preferred due to the quality control requirement. Therefore, we also tried another simple way by direct mixing Er3 þ /Yb3 þ component with calcium silicate powders, which only requires one step melting. 2.2.1. Preparation of Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses by doping using LTZ: Er3 þ /Yb3 þ -glasses Firstly Er3 þ /Yb3 þ co-doped La2O3–TiO2–ZrO2(LTZ) glass was prepared in the stoichiometric ratio of (La1 x yErxYby) (Ti0.95Zr0.05)2.25O6 (x ¼ 0.02, y¼0.16) using high purity Er2O3 (99.99%), Yb2O3 (99.99%), La2O3 (99.99%), TiO2(99.99%) and ZrO2(99.99%) powders as raw materials [36]. They were weighed and thoroughly mixed in an agate mortar. Then, the mixture was pressed into green disks and sintered at 1100 °C for 10 h. Secondly, the as prepared disc was placed on an aluminum nozzle (Fig. 1.(a)), then floated by air (Fig. 1.(b)), heated and melted by 100 W CO2 laser (Fig. 1.(c)). The LTZ-glass sphere was solidified via a deep undercooling process at a rate of ca.300 K per second (Fig. 1.(d)). Finally, the LTZ-glass spheres were crushed into pieces and grounded into powder in an agate mortar. The as-prepared glasses were then used as luminescent material for preparation of luminescent calcium silicate glasses. Then, the as-prepared LTZ:Er3 þ /Yb3 þ -glass powders(L) were
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Table 1. Nominal composition of Er3 þ /Yb3 þ co-doped Ca-Si-glass. Sample
Er2O3 (wt%)
Yb2O3 (wt%)
CaSiO3 (wt%)
EYS-1 EYS-2
1 2
9 18
90 80
weighted and mixed in a ratio of 30%, and 50% with CaSiO3 ceramic powders, and named in LCS-30 and LCS-50 respectively; the mixture was then pressed into green disks and melted by containerless processing technique in an ADL furnace. Finally, the LTZ: Er3 þ /Yb3 þ glass doped bioglass spheres were crushed into pieces and grounded into powder by an agate mortar for further analysis. 2.2.2. Preparation of Er3 þ /Yb3 þ direct co-doped luminescent calcium silicate glasses Instead of pre-preparation of luminescent LTZ:Er3 þ /Yb3 þ glasses described in the first approach, Er2O3, and Yb2O3 were directly mixed with CaSiO3 to prepare Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses. Briefly, high purity Er2O3, Yb2O3 powders (99.99%) were weighed and thoroughly mixed in an agate mortar according to the stoichiometric ratio of LTZ: Er3 þ / Yb3 þ glass, and then mixed with different amount of CaSiO3 powders. The ratio of 3 components was listed in Table 1 and the corresponding samples were described as EYS-1, EYS-2 respectively. The difference of two approaches was only that the host material La2O3-TiO2-ZrO2 was substituted by CaSiO3 in the second approach. The mixture was then pressed into green disks and melted by containerless processing in an ADL furnace. The as prepared glass spheres were then crushed and grounded in powders. 2.3. Characterization of Er3 þ /Yb3 þ co-doped calcium silicate glasses X-ray diffraction (XRD) patterns of starting materials such as CaSiO3 powders and as prepared samples were examined on a Rigaku Ultima IV powder diffractometer with Cu-Ka (k ¼0.150465 nm) radiation to identify the crystal and amorphous phase. The morphology of the glass powders before and after soaking in simulated body fluid (SBF) solutions were observed using a scanning electron microscope (SEM; JSE-6700F, JEOL, Japan). In addition, EDX analysis of as prepared glass powders after soaking in SBF was performed. The up-conversion luminescence spectra were measured on Omni-λ3007; Zolix, Beijing China under the excitation of 980 nm laser. All the measurements were
Fig. 3. X-ray diffraction of samples: CS-P, CS-G, EYS-1 and LCS-30.
performed at room temperature. 2.4. Evaluation of bioactivity of Er3 þ /Yb3 þ co-doped calcium silicate glasses For evaluation of the bioactivity of as prepared glasses, the samples were soaked into simulated body fluid (SBF) and then the hydroxyapatite formation on the materials was analyzed with a FTIR spectrophotometer (IR-Vis 8500, Shanghai). Briefly, samples were placed in polystyrene bottles containing SBF with ion concentrations nearly equal to human blood plasma, which was prepared according to the procedure described by Kokubo and Takadama [37]. The bottle with SBF solution and Er3 þ /Yb3 þ codoped CaSiO3 glass powders was incubated at 37.0 °C. After soaking for 14 days, the samples were removed from SBF, rinsed with deionized water, and dried at 60 °C overnight. The surface morphology and structure of the specimens before and after soaking in the SBF solution were characterized by XRD and SEM measurements. 2.5. Cell proliferation assay For evaluating the cytocompatibility of LCS-30, LCS-50, EYS-1, EYS-2, the effects of the material extracts diluted at ratios from 1 to 1/64 on MC3T3-E1 on cell proliferation was evaluated, and cells cultured in α-minimum essential medium (α-MEM) was used
Fig. 2. The up-conversion emission spectra of Er3 þ /Yb3 þ co-doped bioactive glasses (a) LCS-30,-50; (b) EYS-1,-2.
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Fig. 4. SEM images of the as prepared glasses before soaking in SBF: (a) EYS-1, (b) LCS-30; and SEM images after soaking in SBF for 14days: (c) EYS-1, (e) EYS-2; (d) LCS30and (f) LCS-50 respectively.
as control. The newborn mouse calvaria-derived MC3T3-E1 subclone 14 pre-osteoblastic cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and cultured in α-MEM (Gibco), supplemented with 10% (v/v) FBS (HyClone), 1% penicillinstreptomycin (Gibco) at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity. MC3T3-E1 cells were seeded at the density of 2 103 cells per well on 96-well plates. After 24 h, the culture medium was replaced by the diluted LCS-30, LCS-50, EYS-1, EYS-2 extracts at different dilution ratios from 1 to 1/64. Then, the cells were cultured for 7 days. To assess cell proliferation, a Cell
Counting Kit (CCK) 8 assay (Beyotime) was performed by adding 10 μL CCK-8 to each well at 1, 3 and 7 days and incubating for 1 h at 37 °C. The absorbance of the reaction product was read at a wavelength of 450 nm on a microplate reader (ELX800, BIO-TEK, USA).
3. Results and discussion 3.1. The up-conversion luminescence properties of Er3 þ /Yb3 þ co-
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Fig. 5. EDX analysis of (a) EYS-2 and (b) LCS-50 after soaking in SBF for 14 days.
Fig. 6. FTIR spectra of as prepared glasses before (a) and after (b) soaking in SBF for 14 days. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
doped calcium silicate glasses The up-conversion luminescence properties of Er3 þ /Yb3 þ codoped calcium silicate glasses by doping either with LTZ: Er3 þ / Yb3 þ -glasses or with direct addition of Er2O3, Yb2O3 into calcium silicate were evaluated and compared. The up-conversion emission spectra of Er3 þ /Yb3 þ co-doped and LTZ:Er3 þ /Yb3 þ -glass doped Ca-Si-glass powders with different composition are shown in Fig. 2. It is clear to see from Fig. 2 (a) that three emission bands centered at 525, 547, 656 nm at the excitation of 980 nm laser are visible for LTZ:Er3 þ /Yb3 þ -glass doped Ca-Si-glass powders. Previous studies have shown that
single Er3 þ doped glasses exhibit small absorption cross-section causing weak upconversion luminescence. Therefore, Yb3 þ ion is used to enhance the pumping absorption and quantum efficiency of the Er3 þ ions [38,39]. The host material is also an important factor to enhance the up- conversion luminescence and to reduce the multiphonon relaxation. La2O3-TiO2-ZrO2 glass [31] is different from other oxide glasses because of its low phonon energy, and the selection of La-Ti-Zr host matrix for Er3 þ ion doping make a highly efficient up-conversion luminescence possible. However, it is difficult to fabricate La2O3-TiO2-ZrO2 glasses via conventional methods. With the help of containerless processing, we have successfully prepared La2O3-TiO2-ZrO2:Er3 þ /Yb3 þ -glasses with
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Fig. 7. Cell proliferation of as prepared glasses in different concentration: EYS-1,-2; LCS-30,-50.
luminescent property [36]. The trivalent erbium ion (Er 3 þ ) acted as activiation ion, and ytterbium ion (Yb3 þ ) as the sensitizer. In the present study, one approach is to use La2O3-TiO2-ZrO2:Er3 þ / Yb3 þ -glasses as luminescent component to combine with calcium silicate, and the results confirmed that the LTZ: Er3 þ /Yb3 þ -glass doped Ca-Si-glasses are also luminescent. However, this approach required multi-step processes. Therefore, we also tested a second method, in which we directly mixed Er3 þ /Yb3 þ component with calcium silicate powders to fabricate Er3 þ /Yb3 þ co-doped calcium silicate glasses. From Fig. 2(b) we can see that the obtained composite glasses are also luminescent and showed similar emission spectra as the LTZ: Er3 þ /Yb3 þ -glass doped Ca-Si-glasses, and the emission intensity also increased with the increase of the Er3 þ / Yb3 þ co-doping amount. More interesting is that if we compare the luminescent spectra of glasses obtained from two different approaches, we can see that LTZ: Er3 þ /Yb3 þ -glasses (LCS) showed a stronger emission at 540 nm and a weaker emission at 680 nm, while Er2O3, Yb2O3 directly doped glasses (EYS) showed an equal intensity of emission at both wavelength, suggesting an enhanced emission in the red light area as compared to LTZ: Er3 þ /Yb3 þ -glasses. A possible explanation is that addition of CaO-SiO2 will result in an increase of local phonon energy of matrix around Er3 þ , increasing the multiphonon relaxation probability of Er3 þ ions at upper levels. The advantage of this enhanced red emission is that, when used for biological applications, the tissue penetration ability of red light is higher than the green light [40], so the detection sensitivity of the light emission from EYS might be higher than that of LCS, although this needs to be confirmed by in vivo study in the future. 3.2. Phase analysis The XRD patterns of CaSiO3 powder (CS-P), CaSiO3 glass (CS-G), EYS-1 and LCS-30 are shown in Fig. 3. It is clear to see that the starting material CS-P was distinct pure crystal phase, and was transformed to amorphous phase via containerless processing. The Er3 þ /Yb3 þ co-doped samples prepared by two different approaches via containerless processing exhibited also amorphous phase without obvious crystal peaks.
3.3. Bioactivity of Er3 þ /Yb3 þ co-doped CaSiO3 glass powders in SBF The ability to induce apatite formation in simulated body fluid is an important characteristic of bioactive glasses, and the formation of apatite layer between orthopedic implants and bone tissue is important for stabilization of the implants. Therefore, it is critical to evaluate the Er3 þ /Yb3 þ co-doped bioactive glasses, and see if they still have the same activity to induce apatite formation in SBF as the pure bioactive glasses. The surface morphology of as-prepared-glass has been investigated by SEM. As shown in Fig. 4, the as-prepared glasses before soaking in SBF had a smooth surface without any crystalline phase, whereas the samples after soaking for 14 days in SBF showed a rough surface. It is clear to see that the surface of asprepared glass particles was covered by a more (Fig. 4(c),(e),(f)) or less (Fig. 4(d)) dense mineral layer compared with the images before soaking(Fig. 4(a), (b)). As shown in Fig. 5 Ca and P both were observed either in both samples EYS-2 and LCS-50. And the ratio of Ca/P was 1.3 and 1.7 respectively which indicated that calcium phosphate was formed on surface of the glass particles after soaking in SBF for 14 days, in which the Ca/P ratio of LCS-50 was more close to that of the apatite (1.67). Therefore it could be explained that the as prepared glasses induced the formation of hydroxyapatite deposition in SBF. FTIR analyses of as prepared glasses before and after soaking in SBF were carried out and the corresponding spectra are shown in Fig. 6(a), (b). Different colors exhibit different kind of as prepared glass powders, such as red for EYS-1, blue for EYS-2, purple for LCS-30 and green for LCS-50. The spectra of Fig. 6(a) shows that there is only one peak instead of two characteristic peaks of PO4 around 560 cm 1 for samples before soaking in SBF. The Fig. 6(b) shows the spectra of samples after soaking in SBF for 14 days, which reveals some new peaks as compared with that of samples before soaking in SBF, and two characteristic peaks around 560 cm 1 are for hydroxyapatite. FTIR analysis combined with SEM observation and EDX analysis indicates that the Er3 þ /Yb3 þ co-doping did not affect the activity of bioactive glasses in SBF, and the as-prepared Er3 þ /Yb3 þ codoped glass powders still have bioactivity and can induce apatite mineralization in SBF as compared with pure calcium silicate
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glasses. 3.4. Cell proliferation Fig. 7 shows the proliferation of MC3T3-E1 cells cultured in the presence of ionic extracts of as-prepared glasses. The high concentration extracts showed minor inhibition of cell proliferation as compared to the control. In contrast, the lower concentration of the extracts exhibited stimulation of cell proliferation. This is consistent with previous studies on silicate bioceramics and glasses [41–43]. In general, the Er3 þ /Yb3 þ co-doped calcium silicate glasses showed similar biocompatibility and bioactivity as undoped silicate materials. This result suggests that the Er3 þ /Yb3 þ co-doping in certain concentration range did not affect the biocompatibility of the materials.
4. Conclusions In this study, Er3 þ /Yb3 þ co-doped calcium silicate glasses with up-conversion luminescence property were prepared via containerless processing in an aerodynamic levitation furnace. The obtained glasses prepared by doping calcium silicate either with LTZ:Er3 þ /Yb3 þ -glasses or directly with Er2O3 and Yb2O3 exhibit visible up-conversion luminescence. The XRD and SEM analysis confirmed that calcium silicate ceramic powders were completely transformed from the original multi-crystalline phase (CS-P) into the glassy phase (CS-G, EYS-1, LCS-30) via containerless fabrication process. SEM observations combined with FTIR analysis confirmed that the as-prepared glasses are still bioactive in SBF, and the cell proliferation assay indicated that the as- prepared glasses are also biocompatible and bioactive in stimulating cell proliferation as same as pure calcium silicate materials. Therefore, this study suggests that the addition of up-conversional luminescent components in bioglasses is an effective way to synthesize luminescent bioglasses, which might be used for studying biodegradation of bioglasses.
Acknowledgements This study was supported by a fund from the Natural Science Foundation of China (No. 81190132).
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Er3 þ /Yb3 þ co-doped bioactive glasses with up-conversion luminescence prepared by containerless processing Qin Li a, Min Xing a, Zhi Chen b, Xiaoya Wang a, Cancan Zhao a, Jianrong Qiu b, Jianding Yu a,n, Jiang Chang a,n a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China b State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 March 2016 Received in revised form 10 May 2016 Accepted 17 May 2016 Available online 20 May 2016
Er3 þ /Yb3 þ co-doped bioactive glasses were prepared via containerless processing in an aerodynamic levitation furnace. The as-prepared glasses were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) equipped with energy dispersive X-Ray spectroscopy (EDX). The up-conversion luminescence of as-prepared glasses was measured using an Omni- 3007 spectrometer. Furthermore, the in vitro bioactivity was evaluated by soaking the materials in simulated body fluid, and the biocompatibility was evaluated in MC3T3-E1 cell culture. The results show that containerless processing is a unique method to prepare homogeneous rare earth doped bioactive glasses. The obtained Er3 þ /Yb3 þ co-doped glasses show green and red up-conversion luminescence at the excitation of 980 nm laser. The XRD analysis confirmed that calcium silicate powders, as starting materials, were completely transformed from the original multi-crystalline phase (CS-P) into the amorphous-glassy phase (CS-G, EYS, LCS) via containerless processing. The SEM observation combined with EDX and FTIR analyses showed that the as-prepared glasses were bioactive. The cell proliferation assay also revealed that the as-prepared glasses were biocompatible and nontoxic to MC3T3-E1 cells. This study suggests that the luminescent bioactive glasses prepared by containerless processing could be used for studying biodegradation of bone implantation materials. & 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: Up-conversion luminescence Bioactive glasses Bioactivity Containerless processing
1. Introduction Bioceramics are widely used for repairing bone defects, and the degradation rate of the bone grafting materials could significantly affect the bone regeneration [1,2]. Moreover, the degradation products are closely related to the material biocompatibility. Therefore, it is of great importance to study the degradation rate and distribution of the degradation products of bioceramics. The radioactive isotope labeling has been applied to trace the degradation of calcium phosphate bioceramics [3], but this method needs strict experimental condition. Hence, the development of new method to label bioceramics for studying the degradation of bone grafting materials after implantation as well as the in vivo distribution of degradation products is meaningful for elucidating the mechanisms of bioceramics/tissue interaction and their clinical n
Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (J. Yu), [email protected] (J. Chang). http://dx.doi.org/10.1016/j.ceramint.2016.05.108 0272-8842/& 2016 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
application. As a traditional silicate compound, calcium silicate is widely studied and used as cement component, refractory materials, anticorrosive and heat insulating coatings [4–7]. In recent years, many studies have shown that calcium silicate ceramics have excellent bioactivity, biocompatibility and biodegradability. Furthermore, calcium silicate ceramics have been found to stimulate bone regeneration after implanting in bone defects, which suggests that this kind of ceramics have great potential as new bone grafting materials [8]. Up-conversion luminescent materials can transform the infrared invisible light into visible light [9]. Rare earth element doped up-conversion luminescent materials have many advantages [10– 18], including higher chemical stability, lower potential toxicity, and longer fluorescence lifetime. In particular, due to the nearinfrared emitting property, the emitting light of these materials has larger optical penetration depth, lower generation of autofluorescence and almost no damage in biological tissue [19–22]. Because of these advantages, the rare earth element doped up-
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Fig. 1. Schematic illustration of the preparation of the glass spheres, (a) adding green samples in an aluminum nozzle; (b) floating the sample with the help of an air flow; (c) melting the sample with laser; (d) rapid-cooling the melt. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
conversion luminescent materials are expected to be used for luminescent bio-analytical assays and luminescent imaging, and might be a new generation of fluorescence bio-labeling materials instead of down-conversion luminescent materials [23]. Up-conversion luminescent materials are usually composed of host materials and doped rare earth ions. Fluoride, oxide, sulfur compounds, fluoride oxide and halide are the most common host materials. Among them NaYF4 exhibits the highest up-conversion luminescence efficiency [24]. For example when Er3 þ , Yb3 þ are co-doped into NaYF4 matrix, Er3 þ works as activator and Yb3 þ as sensitization agent. Oxide host materials have also attracted much attention due to many advantages including simple preparation procedure, uncomplicated working condition and stability [25– 27]. Nowadays many researchers have been investigating up-conversion luminescent materials for biological labeling and drug delivery applications [24,28]. However, most of the studies are mainly focused on the preparation of nanoparticles [29,30]. Less attention is paid on applications as implantable biomaterials, and to the best of our knowledge, there is no report on synthesis and evaluation of up-conversion luminescent calcium silicate glasses as implantable biomaterials. Considering the clinical requirement for the application of bioglasses in human body, one of the most important advantages of the containerless processing for preparing bioglasses is that the purity of the prepared glasses could be well controlled as comparing with the traditional crucible technology for preparing glasses, since no crucible is used which avoids the contamination of glasses during the melting process. In addition, the suppression of heterogeneous nucleation and change of heat transport, achievement of super cooled liquids and rapid solidification, and complete elimination of the internal stress created due to the interaction between samples and the crucible wall are other advantages of the containerless processing method over the traditional technique for preparing glasses [31,32]. Therefore, containerless processing enables developing new type of high performance materials with high purity and different phase composition such as metastable and amorphous phases, and rapid solidification of the materials and study of the undercooled possible [32–34]. Most notably, containerless processing has distinct advantages in developing new functional glasses. Melting under containerless condition can result in the formation of spherical shaped materials automatically by surface tension, which will be transformed into glass ball with ideal spherical surface after cooling down. Levitation is achieved by external applied force such as air in flow on samples to counteract the pull of gravity. In general, materials with high melting point such as calcium silicate are relatively easy to crystallize during the solidification process, so the calcium silicate glass can only be formed when it is under rapid cooling [35]. Therefore, the containerless processing is suitable to fabricate calcium silicate glasses, and the aim of the present study was the preparation and evaluation of up-
conversion luminescent calcium silicate glasses as potential implantable biomaterials. The luminescent property and bioactivity of the prepared materials were investigated.
2. Experimental procedure 2.1. Materials The starting materials were CaSiO3 ceramic powders, Er2O3 (99.99%), Yb2O3 (99.99%), La2O3 (99.99%), TiO2 (99.99%) and ZrO2 (99.9%). The CaSiO3 powders were purchased from Kunshan Huaqiao New Materials Limited (China). And all other chemicals were obtained from China National Medicine Shanghai Chemical Reagent Corporation. 2.2. Preparation of Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses Er3 þ /Yb3 þ co-doped CaSiO3 glasses were prepared by containerless processing in an aerodynamic levitation (ADL) furnace (Fig. 1). Two different approaches were used for preparation of luminescent calcium silicate glasses. The LTZ is known as a good host material for glasses with good luminescent, so we first tried to prepare luminescent bioglasses by direct addition of LTZ luminescent glasses in calcium silicate composition. But, this process requires synthesis of LTZ luminescent glasses first, which is known as a very good luminescent glasses but requires multi-step calcination to fabricate. Besides, for clinical applications, bone graft materials with simple composition are always preferred due to the quality control requirement. Therefore, we also tried another simple way by direct mixing Er3 þ /Yb3 þ component with calcium silicate powders, which only requires one step melting. 2.2.1. Preparation of Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses by doping using LTZ: Er3 þ /Yb3 þ -glasses Firstly Er3 þ /Yb3 þ co-doped La2O3–TiO2–ZrO2(LTZ) glass was prepared in the stoichiometric ratio of (La1 x yErxYby) (Ti0.95Zr0.05)2.25O6 (x ¼ 0.02, y¼0.16) using high purity Er2O3 (99.99%), Yb2O3 (99.99%), La2O3 (99.99%), TiO2(99.99%) and ZrO2(99.99%) powders as raw materials [36]. They were weighed and thoroughly mixed in an agate mortar. Then, the mixture was pressed into green disks and sintered at 1100 °C for 10 h. Secondly, the as prepared disc was placed on an aluminum nozzle (Fig. 1.(a)), then floated by air (Fig. 1.(b)), heated and melted by 100 W CO2 laser (Fig. 1.(c)). The LTZ-glass sphere was solidified via a deep undercooling process at a rate of ca.300 K per second (Fig. 1.(d)). Finally, the LTZ-glass spheres were crushed into pieces and grounded into powder in an agate mortar. The as-prepared glasses were then used as luminescent material for preparation of luminescent calcium silicate glasses. Then, the as-prepared LTZ:Er3 þ /Yb3 þ -glass powders(L) were
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Table 1. Nominal composition of Er3 þ /Yb3 þ co-doped Ca-Si-glass. Sample
Er2O3 (wt%)
Yb2O3 (wt%)
CaSiO3 (wt%)
EYS-1 EYS-2
1 2
9 18
90 80
weighted and mixed in a ratio of 30%, and 50% with CaSiO3 ceramic powders, and named in LCS-30 and LCS-50 respectively; the mixture was then pressed into green disks and melted by containerless processing technique in an ADL furnace. Finally, the LTZ: Er3 þ /Yb3 þ glass doped bioglass spheres were crushed into pieces and grounded into powder by an agate mortar for further analysis. 2.2.2. Preparation of Er3 þ /Yb3 þ direct co-doped luminescent calcium silicate glasses Instead of pre-preparation of luminescent LTZ:Er3 þ /Yb3 þ glasses described in the first approach, Er2O3, and Yb2O3 were directly mixed with CaSiO3 to prepare Er3 þ /Yb3 þ co-doped luminescent calcium silicate glasses. Briefly, high purity Er2O3, Yb2O3 powders (99.99%) were weighed and thoroughly mixed in an agate mortar according to the stoichiometric ratio of LTZ: Er3 þ / Yb3 þ glass, and then mixed with different amount of CaSiO3 powders. The ratio of 3 components was listed in Table 1 and the corresponding samples were described as EYS-1, EYS-2 respectively. The difference of two approaches was only that the host material La2O3-TiO2-ZrO2 was substituted by CaSiO3 in the second approach. The mixture was then pressed into green disks and melted by containerless processing in an ADL furnace. The as prepared glass spheres were then crushed and grounded in powders. 2.3. Characterization of Er3 þ /Yb3 þ co-doped calcium silicate glasses X-ray diffraction (XRD) patterns of starting materials such as CaSiO3 powders and as prepared samples were examined on a Rigaku Ultima IV powder diffractometer with Cu-Ka (k ¼0.150465 nm) radiation to identify the crystal and amorphous phase. The morphology of the glass powders before and after soaking in simulated body fluid (SBF) solutions were observed using a scanning electron microscope (SEM; JSE-6700F, JEOL, Japan). In addition, EDX analysis of as prepared glass powders after soaking in SBF was performed. The up-conversion luminescence spectra were measured on Omni-λ3007; Zolix, Beijing China under the excitation of 980 nm laser. All the measurements were
Fig. 3. X-ray diffraction of samples: CS-P, CS-G, EYS-1 and LCS-30.
performed at room temperature. 2.4. Evaluation of bioactivity of Er3 þ /Yb3 þ co-doped calcium silicate glasses For evaluation of the bioactivity of as prepared glasses, the samples were soaked into simulated body fluid (SBF) and then the hydroxyapatite formation on the materials was analyzed with a FTIR spectrophotometer (IR-Vis 8500, Shanghai). Briefly, samples were placed in polystyrene bottles containing SBF with ion concentrations nearly equal to human blood plasma, which was prepared according to the procedure described by Kokubo and Takadama [37]. The bottle with SBF solution and Er3 þ /Yb3 þ codoped CaSiO3 glass powders was incubated at 37.0 °C. After soaking for 14 days, the samples were removed from SBF, rinsed with deionized water, and dried at 60 °C overnight. The surface morphology and structure of the specimens before and after soaking in the SBF solution were characterized by XRD and SEM measurements. 2.5. Cell proliferation assay For evaluating the cytocompatibility of LCS-30, LCS-50, EYS-1, EYS-2, the effects of the material extracts diluted at ratios from 1 to 1/64 on MC3T3-E1 on cell proliferation was evaluated, and cells cultured in α-minimum essential medium (α-MEM) was used
Fig. 2. The up-conversion emission spectra of Er3 þ /Yb3 þ co-doped bioactive glasses (a) LCS-30,-50; (b) EYS-1,-2.
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Fig. 4. SEM images of the as prepared glasses before soaking in SBF: (a) EYS-1, (b) LCS-30; and SEM images after soaking in SBF for 14days: (c) EYS-1, (e) EYS-2; (d) LCS30and (f) LCS-50 respectively.
as control. The newborn mouse calvaria-derived MC3T3-E1 subclone 14 pre-osteoblastic cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and cultured in α-MEM (Gibco), supplemented with 10% (v/v) FBS (HyClone), 1% penicillinstreptomycin (Gibco) at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity. MC3T3-E1 cells were seeded at the density of 2 103 cells per well on 96-well plates. After 24 h, the culture medium was replaced by the diluted LCS-30, LCS-50, EYS-1, EYS-2 extracts at different dilution ratios from 1 to 1/64. Then, the cells were cultured for 7 days. To assess cell proliferation, a Cell
Counting Kit (CCK) 8 assay (Beyotime) was performed by adding 10 μL CCK-8 to each well at 1, 3 and 7 days and incubating for 1 h at 37 °C. The absorbance of the reaction product was read at a wavelength of 450 nm on a microplate reader (ELX800, BIO-TEK, USA).
3. Results and discussion 3.1. The up-conversion luminescence properties of Er3 þ /Yb3 þ co-
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Fig. 5. EDX analysis of (a) EYS-2 and (b) LCS-50 after soaking in SBF for 14 days.
Fig. 6. FTIR spectra of as prepared glasses before (a) and after (b) soaking in SBF for 14 days. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
doped calcium silicate glasses The up-conversion luminescence properties of Er3 þ /Yb3 þ codoped calcium silicate glasses by doping either with LTZ: Er3 þ / Yb3 þ -glasses or with direct addition of Er2O3, Yb2O3 into calcium silicate were evaluated and compared. The up-conversion emission spectra of Er3 þ /Yb3 þ co-doped and LTZ:Er3 þ /Yb3 þ -glass doped Ca-Si-glass powders with different composition are shown in Fig. 2. It is clear to see from Fig. 2 (a) that three emission bands centered at 525, 547, 656 nm at the excitation of 980 nm laser are visible for LTZ:Er3 þ /Yb3 þ -glass doped Ca-Si-glass powders. Previous studies have shown that
single Er3 þ doped glasses exhibit small absorption cross-section causing weak upconversion luminescence. Therefore, Yb3 þ ion is used to enhance the pumping absorption and quantum efficiency of the Er3 þ ions [38,39]. The host material is also an important factor to enhance the up- conversion luminescence and to reduce the multiphonon relaxation. La2O3-TiO2-ZrO2 glass [31] is different from other oxide glasses because of its low phonon energy, and the selection of La-Ti-Zr host matrix for Er3 þ ion doping make a highly efficient up-conversion luminescence possible. However, it is difficult to fabricate La2O3-TiO2-ZrO2 glasses via conventional methods. With the help of containerless processing, we have successfully prepared La2O3-TiO2-ZrO2:Er3 þ /Yb3 þ -glasses with
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Fig. 7. Cell proliferation of as prepared glasses in different concentration: EYS-1,-2; LCS-30,-50.
luminescent property [36]. The trivalent erbium ion (Er 3 þ ) acted as activiation ion, and ytterbium ion (Yb3 þ ) as the sensitizer. In the present study, one approach is to use La2O3-TiO2-ZrO2:Er3 þ / Yb3 þ -glasses as luminescent component to combine with calcium silicate, and the results confirmed that the LTZ: Er3 þ /Yb3 þ -glass doped Ca-Si-glasses are also luminescent. However, this approach required multi-step processes. Therefore, we also tested a second method, in which we directly mixed Er3 þ /Yb3 þ component with calcium silicate powders to fabricate Er3 þ /Yb3 þ co-doped calcium silicate glasses. From Fig. 2(b) we can see that the obtained composite glasses are also luminescent and showed similar emission spectra as the LTZ: Er3 þ /Yb3 þ -glass doped Ca-Si-glasses, and the emission intensity also increased with the increase of the Er3 þ / Yb3 þ co-doping amount. More interesting is that if we compare the luminescent spectra of glasses obtained from two different approaches, we can see that LTZ: Er3 þ /Yb3 þ -glasses (LCS) showed a stronger emission at 540 nm and a weaker emission at 680 nm, while Er2O3, Yb2O3 directly doped glasses (EYS) showed an equal intensity of emission at both wavelength, suggesting an enhanced emission in the red light area as compared to LTZ: Er3 þ /Yb3 þ -glasses. A possible explanation is that addition of CaO-SiO2 will result in an increase of local phonon energy of matrix around Er3 þ , increasing the multiphonon relaxation probability of Er3 þ ions at upper levels. The advantage of this enhanced red emission is that, when used for biological applications, the tissue penetration ability of red light is higher than the green light [40], so the detection sensitivity of the light emission from EYS might be higher than that of LCS, although this needs to be confirmed by in vivo study in the future. 3.2. Phase analysis The XRD patterns of CaSiO3 powder (CS-P), CaSiO3 glass (CS-G), EYS-1 and LCS-30 are shown in Fig. 3. It is clear to see that the starting material CS-P was distinct pure crystal phase, and was transformed to amorphous phase via containerless processing. The Er3 þ /Yb3 þ co-doped samples prepared by two different approaches via containerless processing exhibited also amorphous phase without obvious crystal peaks.
3.3. Bioactivity of Er3 þ /Yb3 þ co-doped CaSiO3 glass powders in SBF The ability to induce apatite formation in simulated body fluid is an important characteristic of bioactive glasses, and the formation of apatite layer between orthopedic implants and bone tissue is important for stabilization of the implants. Therefore, it is critical to evaluate the Er3 þ /Yb3 þ co-doped bioactive glasses, and see if they still have the same activity to induce apatite formation in SBF as the pure bioactive glasses. The surface morphology of as-prepared-glass has been investigated by SEM. As shown in Fig. 4, the as-prepared glasses before soaking in SBF had a smooth surface without any crystalline phase, whereas the samples after soaking for 14 days in SBF showed a rough surface. It is clear to see that the surface of asprepared glass particles was covered by a more (Fig. 4(c),(e),(f)) or less (Fig. 4(d)) dense mineral layer compared with the images before soaking(Fig. 4(a), (b)). As shown in Fig. 5 Ca and P both were observed either in both samples EYS-2 and LCS-50. And the ratio of Ca/P was 1.3 and 1.7 respectively which indicated that calcium phosphate was formed on surface of the glass particles after soaking in SBF for 14 days, in which the Ca/P ratio of LCS-50 was more close to that of the apatite (1.67). Therefore it could be explained that the as prepared glasses induced the formation of hydroxyapatite deposition in SBF. FTIR analyses of as prepared glasses before and after soaking in SBF were carried out and the corresponding spectra are shown in Fig. 6(a), (b). Different colors exhibit different kind of as prepared glass powders, such as red for EYS-1, blue for EYS-2, purple for LCS-30 and green for LCS-50. The spectra of Fig. 6(a) shows that there is only one peak instead of two characteristic peaks of PO4 around 560 cm 1 for samples before soaking in SBF. The Fig. 6(b) shows the spectra of samples after soaking in SBF for 14 days, which reveals some new peaks as compared with that of samples before soaking in SBF, and two characteristic peaks around 560 cm 1 are for hydroxyapatite. FTIR analysis combined with SEM observation and EDX analysis indicates that the Er3 þ /Yb3 þ co-doping did not affect the activity of bioactive glasses in SBF, and the as-prepared Er3 þ /Yb3 þ codoped glass powders still have bioactivity and can induce apatite mineralization in SBF as compared with pure calcium silicate
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glasses. 3.4. Cell proliferation Fig. 7 shows the proliferation of MC3T3-E1 cells cultured in the presence of ionic extracts of as-prepared glasses. The high concentration extracts showed minor inhibition of cell proliferation as compared to the control. In contrast, the lower concentration of the extracts exhibited stimulation of cell proliferation. This is consistent with previous studies on silicate bioceramics and glasses [41–43]. In general, the Er3 þ /Yb3 þ co-doped calcium silicate glasses showed similar biocompatibility and bioactivity as undoped silicate materials. This result suggests that the Er3 þ /Yb3 þ co-doping in certain concentration range did not affect the biocompatibility of the materials.
4. Conclusions In this study, Er3 þ /Yb3 þ co-doped calcium silicate glasses with up-conversion luminescence property were prepared via containerless processing in an aerodynamic levitation furnace. The obtained glasses prepared by doping calcium silicate either with LTZ:Er3 þ /Yb3 þ -glasses or directly with Er2O3 and Yb2O3 exhibit visible up-conversion luminescence. The XRD and SEM analysis confirmed that calcium silicate ceramic powders were completely transformed from the original multi-crystalline phase (CS-P) into the glassy phase (CS-G, EYS-1, LCS-30) via containerless fabrication process. SEM observations combined with FTIR analysis confirmed that the as-prepared glasses are still bioactive in SBF, and the cell proliferation assay indicated that the as- prepared glasses are also biocompatible and bioactive in stimulating cell proliferation as same as pure calcium silicate materials. Therefore, this study suggests that the addition of up-conversional luminescent components in bioglasses is an effective way to synthesize luminescent bioglasses, which might be used for studying biodegradation of bioglasses.
Acknowledgements This study was supported by a fund from the Natural Science Foundation of China (No. 81190132).
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