Europium-Containing Mesoporous Bioactive Glass Scaffolds for

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Europium (Eu)-containing mesoporous bioactive glass scaffolds for stimulating in vitro and in vivo osteogenesis Chengtie Wu, Lunguo Xia, Pingping Han, Lixia Mao, Jiacheng Wang, Dong Zhai, Bing Fang, Jiang Chang, and Yin Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03100 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 25, 2016

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ACS Applied Materials & Interfaces

Europium (Eu)-containing mesoporous bioactive glass scaffolds for stimulating in vitro and in vivo osteogenesis

Chengtie Wu1*, Lunguo Xia2, Pingping Han3, Lixia Mao2, Jiacheng Wang,1 Dong Zhai1, Bing Fang2*, Jiang Chang1*, Yin Xiao3

1. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China. 2. Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Science, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, China 3. Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia.

*

Corresponding authors:

Email: [email protected] (C. Wu) Tel.: +86-21-52412249; fax: +86-21-52413903. Email: [email protected] (B. Fang) Tel: +86-21-23271699; Fax: +86-21-63136856. Email: [email protected] (J. Chang) Tel.: +86-21-52412804; fax: +86-21-52413903. C.W, L.X and P.H contribute equally.

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Abstract Bone tissue engineering offers a possible strategy for regenerating large bone defects, in which how to design beneficial scaffolds for accelerating bone formation remains significant challenging. Europium (Eu), as one of important rare earth elements, has been used as solid-state lighting materials. However, there is few report whether Eu can be used for labeling bone tissue engineering scaffolds, and their biological effect on bone cells and bone tissue regeneration is unknown. In this study, we incorporated Eu into mesoporous bioactive glass (Eu-MBG) scaffolds by an in situ co-template method in order to achieve a bifunctional biomaterial with biolabeling and bone regeneration. The prepared Eu-MBG scaffolds have highly interconnective large pores (300 ~ 500 µm), high specific surface area (140 ~ 290 m2/g) and well-ordered mesopores (5 nm) as well as uniformly distributed Eu elements. The incorporation of 2 ~ 5 mol % Eu towards MBG scaffolds with luminescent property. The in vitro degradation of Eu-MBG scaffolds has functional effect on the change of luminescence intensity. In addition, Eu-MBG can be used for labeling bone marrow stromal cells (BMSCs) in vitro and still presents distinct luminescence signal in deep bone tissues in vivo to label new bone tissue via release of Eu ions. Furthermore, the incorporation of different contents of Eu (1, 2 and 5 mol %) into MBG scaffolds significantly enhances the osteogenic gene expression of BMSCs in scaffolds. The Eu and Si-containing ionic products released from Eu-MBG scaffolds distinctly promote the osteogenic differentiation of BMSCs. Critical sized femur defects in ovariectomised (OVX) rats are created to simulate an osteoporotic phenotype. The results show that Eu-MBG scaffolds significantly stimulate new bone formation in osteoporotic bone defects when compared to MBG scaffolds alone and Eu element may involve in the acceleration of bone regeneration in OVX rats. Our study for the first time reports that the incorporation of earth element Eu into bioscaffolds has ability to accelerate bone regeneration in vivo and thus the prepared Eu-MBG scaffolds possess bifunctional properties with biolabeling and bone regeneration.

Keywords: Eu-containing mesoporous bioactive glass; Scaffolds; Bone tissue engineering; Biolabeling; Bone regeneration. ACS Paragon Plus Environment

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1. Introduction The treatment of bone defects, particularly large bone defects resulting from infections, trauma, tumours or genetic malformations, maintains a major challenge for clinicians1-2. To overcome these problems, bioactive porous scaffolds have been extensively studied to regenerate the lost bone3. In the past several years, different kinds of bioactive scaffolds have been studied for bone regeneration applications, including biopolymers, biometals, bioactive glass/ceramics, and their composites4-12, in which the enhancement of the bone-forming ability of bioactive scaffolds for promoting new bone formation and accelerating bone healing is still one of the most challenging issues. Besides the bone regeneration, the study of the degradation mechanism of biomaterial scaffolds is one of hot topics for bone tissue engineering research13-14, in which tracking the distribution and accumulation of degradation products from the scaffolds within tissues and organs throughout the whole scaffold degradation period is another challenging issue15-16. For these reasons, it is of great interest to obtain bifunctional scaffolds with both biolabeling property and osteogenic activity. Among the different kinds of biolabeling techniques, radiolabeling is one of the earliest used methods which have the capacity for long-term quantitative tracing of biomaterials in vivo. Previously, Wennerberg et al. applied radionuclide calcium (45Ca) to the in situ labeling of a nanometer-sized hydroxyapatite layer on the surface of titanium and employed autoradiography to qualitatively analyze the changes in the radioactivity of the implants17. Klein et al. incorporated

45

Ca into β-tricalcium phosphate (β-TCP) and studied the

degradation and metabolism process in vivo18. The main advantage of radiolabeling method is that it will not have obvious effect on the implanted biomaterials. However, it is not easy to



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analyze the obtained samples by conventional analysis methods, such as TEM and SEM. In addition, radiolabeling method has potential risk for environmental pollution19. In recent years, luminescence imaging has become a significant tool for biomedical applications, especially in the region of near-infrared (NIR) spectrum20. Fluorescence labeling has been used for analysis of DNA biomolecular labeling of animal tumor cells and bone imaging of large animals due to its distinct advantages, such as high sensitivity, simplicity and safety20. Organic dyes are always applied to fluorescence labeling, but many fluorophores of organic molecules show short half-life, photobleaching, low fluorescence quantum yield and in vivo instability, which limits their applications in vivo20-22. Rare earth elements have been used for preparation of luminescent materials. Among the rare earth elements, Europium (Eu), with 4f-4f intra orbital electronic transitions which span both the visible and near-infrared ranges, leads to long lifetimes of the excited states, and allows the use of time-resolved detection, a definitive asset for bioassays and biological luminescence imaging20, 23-25. Patra et al. showed that the in vivo toxicity of Eu is dose-dependent and low dose of Eu has no toxicity26. In addition, they found that Eu possesses significant pro-angiogenic activities27. Up to now, Eu has been mainly used for labeling nanomaterials and tracking the drug delivery by monitoring the change of luminescence of nanomaterials during the process of drug release28-31. Inspired by the Eu-labeled drug delivery system, we supposed that Eu might be used for labeling tissue engineering scaffolds and monitoring their degradation process since the scaffolds would be degraded by releasing ionic products, and the change of ionic compositions in scaffolds (including the inherent ions and Eu ions) during degradation could directly influence the luminescence change of scaffolds. A recent study showed that Eu2O3


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could improve the mechanical strength, but inhibit apatite mineralization of glass ceramic scaffolds32. To our knowledge, there is no systematic study about the effect of Eu in biomaterials on in vitro and in vivo osteogenesis, and the relationship of luminescence change and scaffold degradation is completely unknown. Besides the biolabeling function of Eu, Eu ions have the ability to bind to bone mineral hydroxyapatite in vitro33-35. Although there are some studies about the in vitro and in vivo toxicity of Eu ions26, 29, the biological role of Eu ions is unknown except for a preliminary of pro-angiogenic activity of Eu27. In particular, there is no systematic study about the effect of Eu ions from bioactive materials on the proliferation and differentiation of tissue cells as well as the in vivo bone regeneration, especially osteoporotic bone regeneration. In the past few years, mesoporous bioactive glass (MBG) scaffolds have shown potential application for bone regeneration due to their unique features, such as excellent bioactivity and drug-delivery property36-41. Therefore, the aim of this study is to prepare luminescent Eu-containing MBG scaffolds and investigate their luminescence property, the relationship of luminescence change and scaffold degradation, cell labeling and the effect of Eu in the scaffolds on the proliferation and osteogenic differentiation of BMSCs as well as the in vivo luminescence characteristics and bone regeneration in osteoporotic bone defects.

2. Materials and Methods 2.1 Preparation of Eu-MBG scaffolds Porous europium-loaded mesopore-bioactive glass (Eu-MBG) scaffolds were prepared by incorporating Eu (0, 1, 2 and 5 mol %) into MBG to replace parts of silicon (Si) using



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co-templates

of

polyurethane

sponges

and

non-ionic

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block

polymer

P123

(EO20-PO70-EO20). Polyurethane sponges are used to produce large pores (pore size: several hundred micrometers) and P123 is used to create mesoporous structures (mesopore size: several nanometers). The co-template method was applied as described in our previous publication42. To prepare MBG scaffolds containing of 5% mol Eu, 12 g of P123 (Mw = 5800, Aldrich), 18.85 g of tetraethyl orthosilicate (TEOS, 98%), 4.2 g of Ca(NO3)2·4H2O, 2.7 g of Eu(NO3)3, 2.19 g of triethyl phosphate (TEP, 99.8%) and 3 g of 0.5 M HCl were dissolved in 180 g of ethanol (molar ratio Eu/Si/Ca/P = 5/75/15/5, named 5Eu-MBG) and stirred at room temperature for 1 day. The polyurethane sponges were completely cleaned using ddH2O, dried and entirely immersed into the solution for 10 min, then moved to a petri dish to evaporate at room temperature for 12 h. This procedure was repeated for 3 times. To obtain 5Eu-MBG scaffolds, the samples were calcined at 650 °C for 5 h once they were completely dry. MBG scaffolds without Eu (molar ratio Eu/Si/Ca/P = 0/80/15/5, named: MBG), with 1% mol Eu (molar ratio Eu/Si/Ca/P = 1/79/15/5, named: 1Eu-MBG) and 2% mol Eu (molar ratio Eu/Si/Ca/P = 2/78/15/5, named: 2Eu-MBG) were prepared by the same method.

2.2 Characterization of Eu-MBG scaffolds The large-pore structure, element distribution, mesoporous microstructure, specific surface area, mesopore size distribution/volume and phase composition of the prepared Eu-MBG scaffolds were characterized by scanning electron microscopy (SEM), EDS element mapping, transmission

electron

microscopy

(TEM),

Brunauer-Emmett-Teller

(BET),

Barret-Joyner-Halenda (BJH), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy


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(XPS). The large-pore porosity of the prepared scaffolds was measured using Archimedes’ principle43.

2.3 The luminescence property of Eu-MBG scaffolds 2.3.1 The effect of Eu contents and in vitro degradation of scaffolds on luminescence property To investigate the effect of Eu contents on the luminescence property of Eu-MBG scaffolds, the prepared Eu-MBG (MBG, 1Eu-MBG, 2Eu-MBG and 5Eu-MBG) scaffolds were ground to powders with the particle size lower than 40 µm. The luminescence emission spectra were obtained on a Hitachi F-4600 spectrofluorimeter under excitation of 392 nm. To measure the degradation of scaffolds, all scaffolds were weighed, and soaked in Tris-HCl solution (pH 7.4) at 37 °C for 1, 3, 7, 21 and 35 days. The ratio of the solution volume to the scaffold mass was 200 mL/g. The concentrations of released Eu, Si and Ca ions in the Tris-HCl were determined by inductive coupled plasma atomic emission spectrometry (ICP-AES) (Varian 715ES). Since Tris-HCl solution does not have Si ions and MBG scaffolds mainly contain Si element, the weight loss of the scaffolds was calculated according to the released Si ions. To investigate the effect of scaffold degradation on the luminescence property, the degraded 5Eu-MBG scaffolds were ground to powders, the luminescence intensity was tested by spectrofluorimeter. 2.3.2 The effect of Eu-MBG on the labelling of cells To investigate the effect of Eu-MBG on the labeling of cells, bone marrow stromal cells (BMSCs) were isolated and cultured according to our previous publications44. The use of BMSCs in this study was approved by the Human Ethics Committee of Queensland ACS Paragon Plus Environment


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University of Technology, following informed consent taken from all the participants. 5Eu-MBG particles with the average size of 10 µm were used for cell labeling. 103 cells were added in each well, and 5000, 500, 250 and 125 µg/mL of 5Eu-MBG particles in Dulbecco’s Modified Eagle Medium (DMEM) were used for cell culture. After cultured for 24 h, the Eu-MBG labeled cells were carefully cleaned by PBS, and then observed by confocal microscope (Leica DM6000CS). The Eu contents uptaken by cells were measured by ICP-AES. MBG particles without Eu were used for the control materials and glass slides were used for blank control. 2.3.3 The in vivo luminescence property of Eu-MBG scaffolds To investigate the in vivo luminescence property of Eu-scaffolds, the scaffolds were implanted in the femur defects of ovariectomy (OVX) rats. The detail procedure was presented in the section of In vivo reconstruction of femoral defects of OVX rats by using Eu-MBG. After implanted for 4 weeks, the femurs were fixed in 4% phosphate-buffered formalin solution. Then, the samples were dehydrated by using ascending concentrations of alcohols from 70% to 100%, and then embedded in polymethylmethacrylate (PMMA). Generally, three longitudinal sections for each sample were prepared as described in our previous study45. The in vivo luminescence of Eu-MBG scaffolds in bone defects was observed via the prepared sections by using confocal laser scanning microscope (CLSM, Leica, Germany) with the excitation/emission wavelengths of 610 nm. The elements distribution of Si, Ca, P and Eu ions in the bone defects (including scaffolds and new bone tissues) was further observed by EDS mapping analysis.


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2.4 The effect of Eu contents in Eu-MBG scaffolds on bone-related gene expression of BMSCs The RT-qPCR analysis for alkaline phosphatase (ALP), type I collagen (Col I) and runt-related transcription factor 2 (Runx 2) was performed after culturing BMSCs in the scaffolds.

Total RNA was extracted from the cells in Eu-MBG scaffolds using Trizol®

Reagent (Ambion®, Life Technologies Pty Ltd., Australia). The following procedure was conducted according to our previous publication9. The whole experimental samples were carried out for three independent experiments. The mean cycle threshold (Ct) value of each target gene in experiment was normalized against Ct value of house-keeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). And then the relative expression was computed using the following formula: 2-(normalized

average Cts)

×104. All experiments were

conducted in triplicate.

2.5 The effects of ionic products from 5Eu-MBG scaffolds on cell proliferation, ALP activity and bone-related gene expression of BMSCs In order to investigate the effect of Eu-containing ionic products from 5Eu-MBG scaffolds on the cell proliferation, ALP activity and bone-related gene expression of BMSCs, the dissolution extracts were prepared by soaking 5Eu-MBG scaffolds in serum-free DMEM at the concentration of 200 mg/mL according to International Standard Organization (ISO/EN) 10993-546. The mixture was centrifuged after incubation at 37 ºC for 24 h, and the supernatant was collected and then sterilized using a 0.2 µm filter. To evaluate the ionic concentrations of extracts on the biological response, serial dilutions of extracts were further prepared by using



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serum-free DMEM. The concentrations of Eu, Si and Ca ions in the graded extracts were determined by ICP-AES. For investigating the effect of Eu-containing ionic products from 5Eu-MBG scaffolds on the cell proliferation, BMSCs were cultured with the original and diluted 5Eu-MBG extracts in 96-well plates at the initial seeding density of 103 cells/well. After cultured for 1, 3 and 7 days, MTT assays were performed in duplicate. After 7 and 14 days, the relative ALP activity of BMSCs was determined according to previous protocols44. The following procedure was carried out according to the previous publication47. RT-qPCR was performed on bone-related makers of alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), type I collagen (Col I) and osterix (Osx) on the ABI Prism 7300 Sequence Detection System. The testing protocol is same with that used in the section of The effect of Eu contents in Eu-MBG scaffolds on bone-related gene expression of BMSCs.

2.6 In vivo reconstruction of femoral defects of OVX rats by using Eu-MBG The animal ethics was approved by the Animal Research Committee of the Ninth People’s Hospital (Shanghai, China). The ovariectomy (OVX) rat models have been established as described in our previous studies 45. Eighteen OVX rats were randomly allocated into 4 and 8 week time points. At each time points, the OVX rats were anaesthetized by intraperitoneal injection of pentobarbital (Nembutal 3.5 mg/100 g). Two monocortical plug bone defects (3.5 mm diameter × 5 mm deep) were created in the distal region of each femur diaphysis by using a 3.5 mm diameter trephine bur (Fine Science Tools, USA) 48. Finally, 18 critical-sized femur


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defects in 9 rats were randomly filled with MBG, 2Eu-MBG and 5Eu-MBG particles (n = 6 for each group), respectively. At 4 and 8 weeks after the operation, all the rats in each group were sacrificed by overdose of pentobarbital. The femurs were fixed in 4% phosphate-buffered formalin solution. Micro-CT analysis: The samples implanted for 8 weeks were examined by a microcomputed tomography (micro-CT) system (µCT-100, Scanco Medical AG, Switzerland) as described in our previous study49. In addition, the volume of the new formed bone (BV/TV) and the trabecular thickness (Tb. Th) in bone defects were computed by using the auxiliary histomorphometric software (Scanco Medical AG, Switzerland). Histological and histomorphometric analysis: The samples after implanted for 4 and 8 weeks were stained with Van Gieson's (V-G) picro fuchsin for histological observation. The area of newly formed bone was quantified at three randomly selected sections from the serial sections collected from each sample, using Image Pro 5.0 and reported as a percentage of the whole bone defect area, respectively.

2.7 Statistical analysis All the measurements were presented as means ± standard deviation (SD) and were analysed using SPSS software (SPSS Inc., Chicago, Il, USA) with One Way ANOVA analysis. A value of P < 0.05 was considered to be statistically significant.

3. Results 3.1. Characterization of Eu-MBG scaffolds



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Figure 1 shows that the Eu-MBG (MBG and 5Eu-MBG) scaffolds have a highly porous structure with a large-pore size of 300 to 500 µm and the incorporation of 5Eu into MBG still maintain interconnective large pore structure. Element mapping analysis demonstrates that the elements Eu, Si, Ca and P are uniformly distributed in the pore walls of 5Eu-MBG scaffolds (Figure 1c-f). EDS analysis exhibits that Eu contents in the scaffolds can be effectively modulated (Figure S1). Both 1Eu-MBG and 5Eu-MBG scaffolds have a uniform and well-ordered mesoporous channel structure (mesopore size: around 5 nm) in the inner of scaffold walls (Figure 2a, b). N2 adsorption–desorption analysis for the four Eu-MBG scaffolds exhibits a typical IV isotherm pattern (Figure 2c). The mesopore pore distribution of Eu-MBG scaffolds is in the range of 4-5 nm (Figure 2d). Table 1 shows that, after incorporating Eu into MBG scaffolds, the specific surface area, mesopore volume and mesopore size of MBG scaffolds decrease, while the prepared Eu-MBG scaffolds still maintain high mesopore volume ( > 0.16 cm3/g) and surface area ( > 139 m2/g). The average mesopore size of Eu-MBG scaffolds is in the range of 4.1-5.0 nm. The four Eu-MBG scaffolds with different Eu contents (0, 1, 2 and 5 mol %) have no evident crystal peaks and their diffraction patterns have a wide SiO2 peak with low intensity, indicating that the prepared scaffolds are amorphous (Figure 2e). XPS analysis further shows that there is an obvious Eu3d5 peak in the 5Eu-MBG, but pure MBG has not (Figure 2f).

3.2. The luminescence property of Eu-MBG scaffolds The photoluminescence emission intensity of Eu-MBG scaffolds as a function of Eu contents is shown in Figure 3. It clearly indicates that the photoluminescence intensity (5D0-7F2 of Eu3+


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@ 615 nm) increases with the increase of Eu contents. Pure MBG and 1Eu-MBG scaffolds have no obvious photoluminescence (Figure 3a). The ionic release of Eu, Si, and Ca and the weight loss of scaffolds in Tris-HCl are demonstrated in Figure S2. Generally, the amount of released Eu and Si ions increases with the increase of Eu contents in MBG scaffolds (Figure S2a, b). There is no significant difference for the amount of released Ca ions among four groups (Figure S2c). The weight loss for the MBG scaffolds with the incorporation of 2 mol % and 5 mol % Eu increases after soaking in Tris-HCl as compared to scaffolds without Eu (Figure S2d). The change of photoluminescence intensity for Eu-MBG scaffolds as a function of in vitro degradation is shown in Figure 3b. With the increase of soaking time, the photoluminescence intensity (5D0-7F2 of Eu3+ @ 615 nm) of scaffolds decreases (Fig. 3b). 5Eu-MBG significantly enhances the luminescence intensity of cells (Figure 4a-d) as compared to MBG without Eu (Figure 4e) and blank control (Figure 4f). The luminescence intensity of cells increases with the increase of 5Eu-MBG particle concentrations. The cells cultured with 5000 µg/mL of 5Eu-MBG particles displays the strongest luminescence and the nucleus can be clearly observed after labeled with 5Eu-MBG (Figure 4a). Correspondingly, with the increase of 5Eu-MBG concentrations in culture medium, the Eu contents uptaken by cells distinctly increase (Figure 4g). Further in vivo luminescence signals of MBG, 2Eu-MBG and 5Eu-MBG scaffolds after implanted in bone femur defects of OVX rats for 4 weeks are shown in Figure 5. The luminescence intensity of 5Eu-MBG and 2Eu-MBG scaffolds in the bone defects is obviously higher than that of MBG scaffolds without Eu (Figure 5a-c). SEM and histological staining



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shows that scaffolds degrade with the newly formed bone around materials (Figure 5d-f). After implanted for 4 weeks, the scaffolds degraded obviously (Figure 5d) and new bone formed on the boundaries of scaffolds (Figure 5e, f). Further element mapping analysis suggests that the Si element mainly distribute in the undegraded scaffolds (Figure 5g), and Ca, P and Eu ions mainly distributed in the newly formed bone tissues after 4 weeks of implantation (Figure 5h-k). Eu ions released from scaffolds labels the new bone formation and scaffold degradation (Figure 5j). In the new bone area, Ca and P elements are quite obvious (Figure 5h- k).

3.3 The effect of Eu contents in Eu-MBG scaffolds on bone-related gene expression of BMSCs RT-qPCR analysis reveals that the incorporation of Eu into MBG scaffolds enhances the osteogenic differentiation of BMSCs after cultured for 7 days. The osteogenic gene expression (ALP, COL1 and Runx 2) of BMSCs in 5Eu-MBG scaffolds is significantly enhanced compared to that of MBG scaffolds without Eu (Figure 6a-c). In addition, both 1Eu-MBG and 2Eu-MBG scaffolds also increase ALP and Runx 2 gene expression as compared to MBG group (Figure 6).

3.4. The effects of ionic products from 5Eu-MBG scaffolds on cell proliferation, ALP activity and bone-related gene expression of BMSCs To make an investigation on the effect of ionic products from 5Eu-MBG extracts on the proliferation, ALP activity and bone-related gene expression of BMSCs, cells are cultured in different concentrations of the extracts for different time period. The MTT assay obviously


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shows that the proliferation of BMSCs increases in a time dependent manner in each group. The cell proliferation of BMSCs cultured with 12.5 ~ 100 mg/mL 5Eu-MBG extracts is significantly higher than that for blank group at day 7 (P < 0.05) (Figure 7a). The ionic products from 5Eu-MBG extracts at both 25 and 6.25 mg/mL enhance the ALP activity of BMSCs, compared to blank control without 5Eu-MBG extracts (Figure 7b). However, 100 mg/mL of 5Eu-MBG extract inhibits ALP activity of hBMSCs compared to blank group (Figure 7b). RT-qPCR data demonstrates that 5Eu-MBG extracts significantly promote expression of most bone-related genes within the concentrations range of 100 and 6.15 mg/mL after culturing for 7 days (Figure 8). The relative gene expression is significantly enhanced for BMSCs with OPN ( ~ 3 fold), COLI ( ~ 2 fold), OSX ( ~ 1.4 fold) and BSP ( ~ 1.6 fold) by 5Eu-MBG extracts at the concentration of 100 mg/mL (Fig. 8a-d). 6.25 mg/mL of 5Eu-MBG extracts also increases bone-related gene OPN ( ~ 2.3 fold), COLI ( ~ 2 fold), OSX ( ~ 1.7 fold) and OCN ( ~ 1.6 fold) expression for BMSCs (Figure 8).

3.5. In vivo reconstruction of femoral defects of OVX rats by using Eu-MBG The 3D micro-CT images show that much more new bone tissue formation is observed in 5Eu-MBG and 2Eu-MBG groups as compared to control MBG group especially for 5Eu-MBG group at week 8 (Figure 9a). Furthermore, the morphometrical analysis shows that the significantly greater BV/TV value (47.71 ± 4.01% and 36.69 ± 4.82%) is detected for 5Eu-MBG and 2Eu-MBG groups as compared with MBG group (26.81 ± 4.41 %), respectively, while there is also significant difference between 5Eu-MBG and 2Eu-MBG



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groups (P < 0.05) (Figure 9b). Moreover, 5Eu-MBG and 2Eu-MBG groups possess the higher Tb. Th value (0.125 ± 0.013 mm and 0.102 ± 0.015 mm) as compared with MBG group (0.079 ± 0.014 mm), while 5Eu-MBG group achieves the highest value (P < 0.05) (Figure 9c).

For histological analysis, the undecalcified samples are stained with Van Gieson's picro fuchsine. It is shown that the higher percentages of new bone area are observed in 5Eu-MBG (27.15 ± 4.31% and 46.53 ± 3.44%) and 2Eu-MBG (21.73 ± 2.59% and 36.58 ± 3.19%) groups as compared to MBG group (13.36 ± 1.76% and 25.80 ± 3.48%) at weeks 4 and 8, respectively, while 5Eu-MBG group has the highest new bone area (P < 0.05) (Figure 10).

4. Discussion In this study, we have successfully prepared Eu containing MBG scaffolds with highly interconnective large pores (300 ~ 500 µm), high specific surface area (140 ~ 290 m2/g) as well as well-ordered and uniform mesopores (5 nm) by incorporating different contents of Eu ions into the scaffolds. Eu ions are uniformly distributed in the pore walls of scaffolds and can be released with a sustained release profile. Although there are some studies about the incorporation of Eu into solid-state lighting materials50 and drug-carrier nanomaterials28-31, 51-52

, there are few reports about the luminescence and biolabeling property as well as their

effect on cell proliferation, differentiation and in vivo bone regeneration for Eu ions-incorporated bioactive tissue engineering scaffolds. Therefore, the main innovation of this study is that we found that the Eu-labeled scaffolds possess distinct luminescence property for labeling the in vitro degradation of scaffolds, tissue cells and in vivo deep bone ACS Paragon Plus Environment

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tissues, and at the same time, Eu-labeled scaffolds possess activity to enhance bone regeneration in osteoporotic bone defects. These results suggest that Eu-MBG scaffolds are a bifunctional biomaterial with the properties of biolabeling and accelerating bone regeneration.

4.1. Characterization and luminescence property of Eu-MBG scaffolds One of interesting results is that the incorporation of Eu into MBG scaffolds offers them distinct luminescence property, which mainly depends on the contents of Eu. There is no obvious emission peaks when the Eu contents are lower than 1 mol %. When Eu contents increase to 2 mol %, the two main characteristic peaks from 5D0-7F1 (590 nm) and 5D0-7F2 (615 nm) are dominant. The results suggest that the incorporation of Eu into MBG scaffold system does not change the inherent luminescent property of Eu elements. Previous studies showed that the luminescent property of solid-state materials might be related to their chemical states and structures53-55. In this study, XPS analysis reveals that there is an obvious peak for Eu3d5 in the Eu-MBG pattern, suggesting that Eu is mainly in the form of Eu3+. The chemical state of Eu3+ endows it inherent luminescent property. In addition, Eu ions can be released with a sustained release with the degradation of Eu-MBG scaffolds in Tris-HCl solution. Due to the release of Eu ions from scaffolds, it is interesting to find that scaffold degradation in vitro has a functional effect on the change of luminescence intensity. The results suggest that the degradation of MBG scaffolds can be effectively monitored by testing the luminescence change of scaffolds. It is known to all that the degradation of tissue engineering scaffolds plays a crucial role for new tissue ingrowths56-58. The present available methods for scaffold degradation analysis, such as direct mechanical



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measurements and histology, are unsuitable for the continuous monitoring of the same sample in vivo as they destroy scaffolds, cells and tissue matrix57. However, how to monitor the in situ degradation process of scaffolds still remains significant challenging. Our study shows that the tissue cells can be effectively labeled with distinct luminescence by co-culturing with 5Eu-MBG particles, one of important material forms after scaffold degradation. In addition, the luminescence intensity of the labelled tissue cells depends on the concentrations of 5Eu-MBG particles in the culture medium. However, pure MBG particles and blank control could not produce distinct luminescence to label the cells, indicating the importance of Eu ions released from 5Eu-MBG scaffolds for labeling the tissue cells. Therefore, our study suggests that Eu labeling may provide a non-invasive strategy to track the degradation of scaffolds. Further in vivo study shows that the luminescence intensity of 5Eu-MBG and 2Eu-MBG scaffolds in the bone defects is obviously higher than that of MBG scaffolds without Eu, indicating that Eu-MBG scaffolds still maintain good luminescence property even in the in vivo tissue defects. In addition, it is found that Eu ions can be found in the newly formed bone tissue, which could be used for labeling new bone tissue and scaffold degradation in vivo.

4.2. Stimulating osteogenic differentiation and bone regeneration of Eu-MBG scaffolds As we mentioned, although there are some studies about the cytotoxicity of Eu ions29, the biological role of Eu ions is unclear. In particular, the effect of Eu ions from bioactive scaffolds on the proliferation and osteogenic differentiation of bone-forming cells as well as in vivo bone regeneration is completely unknown. The other important result of this study is


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that the incorporation of Eu in MBG scaffolds significantly enhances the osteogenic differentiation of BMSCs via activating their bone-related gene expression (e.g. ALP, Col I and Runx 2) by culturing BMSCs with both scaffolds and their extracts (ionic products). ALP is well-known as an early marker for osteoblastic differentiation. Col I is a major matrix component of periodontal tissues and an early marker of osteogenic differentiation associated with the formation of extracellular matrix59. And Runx 2 is a master regulator in the commitment of osteogenesis60. The results illustrate that Eu ions released from the bioactive MBG scaffolds probably play a key role in enhancing osteogenic differentiation of BMSCs. To further explain the biological role of Eu ions in the MBG scaffolds, we conducted additional studies to investigate what effect of Eu-containing ionic products released from 5Eu-MBG scaffolds on the proliferation as well as osteogenic differentiation of BMSCs. It is found that the Eu and Si-containing ionic products released from 5Eu-MBG scaffolds also significantly promote the cell proliferation and osteogenic differentiation of BMSCs, indicating that Eu and Si ions may have important effect on the induction of osteogenic differentiation of BMSCs. Therefore, the incorporation of Eu into bioactive MBG scaffolds appears to be a feasible means for enhancing the biological response of BMSCs towards osteogenesis in bone tissue engineering applications. Previously, Eu ions have been reported to bind to bone mineral hydroxyapatite in vitro33-35. In combination of previous studies, it is speculated that Eu-containing bioactive MBG scaffolds might be a promising biomaterial for bone regeneration. To confirm the speculation, Eu-MBG scaffolds were further implanted in the osteoporotic bone defects, and it is interesting to find that Eu-MBG scaffolds distinctly stimulated the



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regeneration of new bone as compared to pure MBG scaffolds. As is well known, osteoporosis is one of the most pervasive bone disorders all over the world characterized by reduction in bone mass, low bone mineral density and poor bone strength which will lead to skeletal fragility and susceptibility to fractures61. Although the clinical application of bioactive scaffolds combined with therapeutic agents is still developing, this study illustrates the successful delivery of low-cost therapeutic Eu ions for the treatment of osseous defects, especially for therapy of osteoporosis. Although the mechanism for Eu-MBG scaffolds stimulating regeneration of osteoporotic bone defects is not quite clear, it is confirmed that the released Eu ions have pro-angiogenic activities according to previous study27, which may play a key role for bone regeneration. In addition, our study has shown that Eu-MBG could stimulate the osteogenic differentiation of BMSCs in vitro, which is of great importance for promoting in vivo bone regeneration. To our knowledge, this is the first report that the incorporation of rare earth element Eu in biomaterials can stimulate the regeneration of bone defects, which opens new direction that rare earth elements will be used for tissue engineering in combination of their specific luminescence property.

5. Conclusions Eu-MBG scaffolds with large pores (300-500 µm) and well-ordered mesopores (5 nm) were prepared by in situ incorporation of Eu ions, which showed dual effects on the biolabeling and bone regeneration. The prepared Eu-MBG scaffolds possess distinct luminescence property for in vitro labeling of scaffolds and tissue cells. The in vivo luminescence signal of Eu-MBG scaffolds is obvious in bone tissue defects to label new bone tissue by release of Eu ions.


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Eu-MBG scaffolds have distinctly improved bone forming ability by activating osteogenic differentiation of BMSCs and regenerating osteoporotic bone defects via the incorporation of Eu ions into bioscaffolds. This study suggests that Eu-MBG scaffolds may be a bifunctional biomaterial for biolebeling and bone regeneration.

Supporting Information. Additional Figures including EDS analysis for Eu-MBG scaffolds and the ionic release of Eu-MBG scaffolds after soaked in Tris-HCl buffer solution are available in supporting information.

Acknowledgements The authors acknowledge the support of the Recruitment Program of Global Young Talent, China (Dr Wu), the National High Technology Research and Development Program of China (863 Program, SS2015AA020302), Natural Science Foundation of China (Grant 31370963 and 81190132), Program of Shanghai Outstanding Academic Leaders (15XD1503900) and Key Research Program of Chinese Academy of Sciences (Grant KGZD-EW-T06).

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Osteoblast-related Gene Expression and Bone-to-Implant Contact in vivo. Int. J. Oral Maxillofac Implants 2009, 24 (2), 205-215.

Table 1. The specific surface area, pore volume and pore size of Eu-MBG scaffolds. Scaffolds

Surface area

Pore volume 3

Average mesopore size

2

(m /g)

(cm /g)

(nm)

MBG

290.0

0.30

5.0

1Eu-MBG

222.5

0.23

4.1

2Eu-MBG

203.7

0.23

4.6

5Eu-MBG

139.3

0.16

4.5


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(a)

(b)

(c)

(d)

(e)

(f)

Figure 1. SEM analysis for MBG (a) and 5Eu-MBG scaffolds (b). The incorporation of Eu into MBG scaffolds still maintain interconnective large pore structure. The large-pore size is in the range of around 300-500 µm. Element mapping analysis for the prepared 5Eu-MBG scaffolds, indicating that the elements of Eu (c), Si (d), Ca (e) and P (f) are uniformly distributed in the pore walls of the scaffolds.


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(b)

(a)

(d)

(c)

(e)

(f)

Figure 2. TEM for 1Eu-MBG (a) and 5Eu-MBG (b) scaffolds with well-ordered mesopore channel structure (mesopore size is around 5 nm). Nitrogen adsorption-desorption isotherm (c), mesopore pore size distribution (d), XRD (e) and XPS (f) of Eu-MBG scaffolds.



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(a)

(b)

Figure 3. Emission spectra of Eu-MBG scaffolds with different Eu contents at the excitation of 392 nm (a). The luminescence intensity of at 610 nm increases with the increase of Eu contents. The photoluminescence emission intensity of 5Eu-MBG scaffolds as a function of in vitro scaffold degradation (b). The luminescence intensity decreases with the increase of scaffold degradation.


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(a)

(b)

(c)

(d)

(e)

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Figure 4. The confocal images of cells cultured with different concentrations of 5Eu-MBG particles ((a) 5000, (b) 500, (c) 250 and (d) 125 µg/mL), MBG particles ((e) 5000 µg/mL) and blank control (f). The Eu contents uptaken by cells after cultured with different concentrations of 5Eu-MBG particles (f). 5Eu-MBG significantly enhances the luminescence intensity of cells (a, b, c and d) as compared to MBG without Eu (e) and blank control (f). The luminescence intensity of cells increases with the increase of 5Eu-MBG particle concentrations. The scale bar is 25 µm. The Eu concentrations uptaken by cells increase with the increase Eu-MBG concentrations (g). ACS Paragon Plus Environment


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Figure 5. The in vivo luminescence signals of MBG (a), 2Eu-MBG (b) and 5Eu-MBG (c) scaffolds after implanted in bone femur defects of OVX rats for 4 weeks. The luminescence intensity of 5Eu-MBG and 2Eu-MBG scaffolds in the bone defects was obviously higher than that of MBG scaffolds without Eu. The scale bar is 100 µm. SEM (d), histological images (e, f) and element mapping analysis (g, h, i, j, k) for the scaffold degradation and new bone formation. After implanted for 4 weeks, the scaffolds degraded obviously (d) and new bone formed on the boundaries of scaffolds (e, f). Yellow arrows point to new bone tissue. Element ACS Paragon Plus Environment

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analysises suggest that Eu released from scaffolds labels the new bone formation and scaffold degradation (j). In the new bone area, Ca and P elements are quite obvious (h, i and k).

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Figure 6. The osteogenic gene expression (ALP (a); Col I (b); Runx2 (c)) of BMSCs cultured in different Eu-MBG scaffolds for 7 days. The incorporation of Eu into MBG scaffolds significantly enhances the osteogenic gene expression.



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Europium-Containing Mesoporous Bioactive Glass Scaffolds for

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