Strontium-Substituted Submicrometer Bioactive Glasses Modulate

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Strontium-substituted sub-micron bioactive glasses modulate macrophage responses for improved bone regeneration Wen Zhang, Fujian Zhao, Deqiu Huang, Xiaoling Fu, Xian Li, and Xiaofeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10378 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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ACS Applied Materials & Interfaces

Strontium-substituted sub-micron bioactive glasses modulate macrophage responses for improved bone regeneration Wen Zhang,†,‡,§ Fujian Zhao, †,‡,§ Deqiu Huang,∥ Xiaoling Fu, †,‡,§ Xian Li, †,‡,§ and Xiaofeng Chen*,†,‡,§

† Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China ‡ National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China § Key Laboratory of Biomedical Materials and Engineering, Ministry of Education, South China University of Technology, Guangzhou 510006, China ∥ College of Biophotonics, South China Normal University, Guangzhou 510631, China

KEYWORDS: strontium-substituted sub-micron bioactive glass; macrophage; osteogenesis; osteoclastogenesis; bone regeneration

ACS Paragon Plus Environment

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ABSTRACT Host immune response induced by foreign bone biomaterials plays an important role in determining their fate after implantation. Hence, it is well worth designing advanced bone substitute materials with beneficial immunomodulatory properties to modulate the host–material interactions. Bioactive glasses (BG), with excellent osteoconductivity and osteoinductivity, are regarded as important biomaterials in the field of bone regeneration. In order to explore a novel BG-based osteoimmunomodulatory implant with the capacity of potentially enhancing bone regeneration, it is a possible way to regulate the local immune microenvironment through manipulating macrophage polarization. In this study, strontium-substituted sub-micron bioactive glass (Sr-SBG) was prepared as an osteoimmunomodulatory bone repair material. To investigate whether the incorporation of Sr into SBG could synergistically improve osteogenesis by altering macrophage response, we systematically evaluated the interaction between Sr-SBG and macrophage during the process of bone regeneration by in vitro biological evaluation and in vivo histological assessment. It was found that the Sr-SBG modulate proper inflammatory status, leading to enhanced osteogenesis of mouse mesenchymal stem cells (mMSCs) and suppressed osteoclastogenesis of RAW 264.7 cells compared to SBG without strontium substitution. In vivo study confirmed that Sr-SBG initiated a less severe immune response, and had an improved effect on bone regeneration than SBG, which corresponded with the in vitro evaluation. In conclusion, these findings suggested that Sr-SBG could be a promising immunomodulatory bone repair material designed for improved bone regeneration.


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TEXT 1. INTRODUCTION As a third-generation biomaterial, bioactive glass (BG) has osteoconductive and osteoinductive properties. BG can induce osteoblast differentiation, thereby making it a suitable material for bone regeneration.1-7 When exposed to biological fluid, ions dissolution from BG (e.g. Si, Ca, P) can be released quickly, resulting in a layer of hydroxycarbonate apatite (HCA) formation on the surface of BG which is the cause of strong bond formation between BG and the host tissue.8 Ionic dissolution products from BG can activate osteogenesis-related genes, including osteogenic-related transcription factors, cell cycle regulators, extracellular matrix components and growth factors.9-12 To evaluate the effect of BG on osteogenesis in vitro, osteoblast cells and mesenchymal stem cells (MSC) are commonly used.1-2 However, with the deepening understanding of bone remolding, it is found that the skeletal and immune system are closely related.13-14 Immune cells are proved to play a crucial role in maintaining the balance of bone resorption and reconstruction under both physiological and pathological conditions.15 Bone biomaterials, as a foreign body, can affect the host immune response significantly, and can elicit detrimental or beneficial outcome in bone repair.16-18 The immune response may cause rejection to implanted materials, and may also play a crucial role for effective bone regeneration. In this regard, designing bone biomaterials with the capability of regulating immune microenvironment will result in more efficient bone regeneration.19 Considering the important role of immune cells in the process of bone regeneration, new evaluation system with bone cells, immune cells, and bone substitute materials involved should be established to help developing optimized bone biomaterials.20-24


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Among all the immune cells, macrophages have high plasticity and have multiple effects on bone healing, which can be regarded as model cells for evaluation of immune response and prime targets for modulation of immune phase.18 Generally, macrophages are divided into two phenotypes, classically activated inflammatory macrophages (M1) and alternatively activated inflammatory macrophages (M2). Macrophages polarize toward different phenotypes in response to their microenvironment. For example, macrophages secret pro-inflammatory mediators such as tumor necrosis factor-α (TNFα) and produce inducible nitric oxide synthase (iNOS) to fight infection when exposed to danger signals. While in a wound healing environment, they express anti-inflammatory cytokines, such as interleukin 10 (IL10) and arginase1 (Arg1) to facilitate tissue repair.25-26 Therefore, it is a feasible way of regulating the immune response for improved osteogenesis through modifying the composition of bone biomaterials and testing the interaction between these biomaterials and macrophage switch pattern. To improve the bioactivity of bone biomaterials, bioactive ions are usually incorporated to manipulate the composition of biomaterials.23,

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For instance, strontium (Sr) substituted

bioactive glasses have got the capacity to enhance osteogenesis and inhibit osteoclastogenesis.2833

The potential mechanism appears to be that inflammatory role of nuclear factor kappa-light-

chain-enhancer of activated B cells (NFĸB) has been antagonized, which suggests that strontium have the anti-inflammatory effect.34-36 In previous studies, when assessing the effect of strontium substituted biomaterials on osteogenesis in vitro, conventional approach has been focused on osteoblast lineage cells and MSCs.37-41 However, there have been more and more research suggesting that bone materials have profound impact on immune responses, and this effect could determine the in vivo fate of bone materials.24, 42 To get a full understanding of the mechanism of strontium incorporated biomaterials in bone regeneration, it is not enough to only study the


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interaction between strontium and osteoblast lineage cells or osteoclast cells, the immune cells should also be involved in. However, there are few studies about the interaction between Sr incorporating bioactive glasses and host immune response, and the effect of immune microenvironment trigged by Sr substituted bioactive glasses on osteogenesis and osteoclastogenesis. In this work, we prepared a potential bone biomaterial Sr-substituted sub-micron bioactive glass (Sr-SBG), and hypothesized that it could modulate proper immune response, in combination with its effects of enhancing osteogenesis and inhibiting osteoclastogenesis, to achieve improved bone tissue regeneration. To verify this assumption, systematical studies were carried out by in vitro biological assessment and in vivo histological evaluation to have a better understanding of the interaction between Sr-SBG and immune response, and the effects of immune microenvironment induced by Sr-SBG on osteoclastogenesis and osteogenesis. (i) Firstly, we characterized the macrophage phenotype switch by examining the expression of surface markers, inflammatory-related gene expression, as well as secretion of inflammatory cytokines. (ii) Then the effects of Sr-SBG on osteogenesis and osteoclastogenesis were evaluated by in vitro experiments when macrophages were factored in. (iii) Finally, an in vivo study were performed to test whether the results of in vitro experiments with macrophages involved in the assessment system were consistent with in vivo findings, which would further verify the possibility of designing more effective bone biomaterials through regulating immune response.

2. EXPERIMENTAL SECTION 2.1. Synthesis and characterization of sub-micron bioactive glasses (SBG) and Srsubstituted sub-micron bioactive glasses (Sr-SBG). SBG and Sr-SBG were synthesized by


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alkali-catalyzed sol-gel methed as previous descried.43-44 Dodecylamine (DDA) was used as both template and catalyst agent. The composition of SBG was 60% SiO2, 36% CaO and 4% P2O5 (mol/mol), while the composition of Sr-SBG was 60% SiO2, 30% CaO, 6% SrO and 4% P2O5. Briefly, DDA with certain amount was added to the mixture of 80 mL ethanol and 25 mL distilled water under stirring. After its complete dissolution, 16 mL tetraethylorthosilicate (TEOS) were added to the above solutions and stirred for 1h. Subsequently, triethylphosphate (TEP) and calcium nitrate tetrahydrate (CN) or strontium nitrate (SN) were added in order in the proportions at 30 minute intervals while magnetically stirring at 40 °C. The resulting mixture was stirred vigorously for 3 h until a white precipitate was formed. The synthesized products were centrifuged, washed three times with distilled water and ethanol, then freeze-dried for 24 h. The final products of SBG and Sr-SBG spheres were obtained by sintering in air at 650 °C for 3 h to remove residual DDA and organic components. The morphology of SBG and Sr-SBG were characterized by scanning electron microscopy (SEM, DSM 982-Gemini, Zeiss, Germany). The hydrodynamic diameter and zeta potentials of SBG and Sr-SBG were examined by Zetasizer Nano (Zetasizer Nano ZS, Malvern Instruments, UK). The elemental compositions of SBG and Sr-SBG were tested by X-ray photoelectron spectroscopy (XPS, Kratos Axis Ulra DLD, UK) and Energy-dispersive X-ray spectroscopy (EDS). The rate of degradation of SBG and Sr-SBG in simulated body fluid (SBF) was evaluated by soaking 10mg of SBG and Sr-SBG in 10 mL SBF. The samples were maintained at 37 ℃ in shaking incubator with the speed set on 120 rpm. At 1, 3, 5, and 7 days, 5 ml of the supernatant extracted from the samples after centrifugation was used for the ion release of Si, Ca, Sr, and P determination by inductively coupled plasma-atomic emission spectrometry technique (ICP-AES, PS1000-AT, Leeman, USA).


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2.2. Preparation of SBG and Sr-SBG extracts and assessment of the ion concentraion of SBG and Sr-SBG extracts in DMEM. SBG and Sr-SBG particles were sterilized by autoclave, and material extracts were prepared by adding sterilized SBG and Sr-SBG into DMEM at a weight to liquid ratio of 1 mg/mL. The samples were maintained in a shaker at 37 °C with the speed set at 120 rpm. After 24 h of incubation, the material extracts were collected after centrifugation and sterilizing the supernatants using a 0.22 µm filter membrane (Millipore). Concentrations of Si, Ca, P and Sr ion were analyzed by ICP-AES. 10% fetal bovine serum (FBS, Gibco), and 1% (v/v) penicillin/streptomycin (Gibco) were added into the materials extracts to prepare complete cell culture medium. 2.3. Cell culture. mMSCs and RAW 264.7 cells, obtained from American Type Culture Collection (ATCC) were used in in vitro study. RAW 264.7 cells and mMSCs were incubated in a humidified CO2 incubator at 37 °C and cultured in DMEM supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin. When the cells reached approximately 90% confluence, RAW 264.7 cells were passaged by gently scraping the cells off, while mMSCs were passaged by trypsinization. Cells used for osteogenic differentiation and osteocalstogenesis experiments were from the third to sixth passages. 2.4. In vitro osteogenesis of SBG and Sr-SBG extracts. mMSCs with a seeding density of 1.5×105 cells/well were plated in 24-well plates. After 24h of attachment, the culture medium was replaced by osteogenic inducing medium with 50 ng/mL ascorbic acid, 10 mM βglycerophosphate, and 10 nM dexamethasone containing material extracts. The media were refreshed every 3 days. Alkaline phosphatase (ALP) staining and ALP activity assay, alizarin red-S staining, mRNA expressions of osteogenesis-related genes and ALP protein expression by


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western blot were applied to investigate the osteogenic differentiation potential of mMSCs stimulated by SBG and Sr-SBG extracts. 2.4.1. ALP activity of mMSCs. The ALP activity was evaluated qualitatively and quantitatively using commercially available kits after mMSCs were incubated with osteogenic inducing medium containing SBG or Sr-SBG extracts for 7 and 14 days. Qualitative assay of ALP was performed by using BCIP/NBT ALP Color Development Kit (Beyotime) after cells were washed with PBS for three times, fixed with 10% formaldehyde. In the quantitative assay, cells were lysed for 1h on ice after rinsed with PBS. Then, the lysates were transferred to a centrifuge tube and centrifuged for 10 min at 4 °C (16,000 g). The supernatant was collected for further assay using ALP assay Kit (Beyotime) following the manufacturer's instruction. The total protein was measured by Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, USA). The ALP activity was normalized to the total content of intracellular protein. 2.4.2. Alizarin red-S assay of mMSCs. To determine the extracellular matrix mineralization of mMSCs stimulated by SBG or Sr-SBG extracts, cells were cultured in SBG or Sr-SBG extracts supplemented with osteogenic inducing component. Alizarin red-S assay was performed at day 14. Briefly, after fixation with paraformaldehyde (4% in PBS) for 30 min, cells were washed three times with ultrapure water and were then submerged to a solution of 0.5% Alizarin Red S (PH 4.2, Alfa Aesar, USA) for 5 min at room temperature (RT). Uncombined dyes were totally removed by rinsing with ultrapure water. Images of alizarin red S staining were taken by an inverted microscope (Eclipsc Ti-U, Nikon, Japan). Quantitative analysis of Alizarin Red S staining was performed by eluting the bound stain with 10% cetylpyridinium chloride in 10 mM Na2HPO4 (PH 7.0) for 1 h. The absorbance of the resulting solution at 562 nm was determined by the microplate reader (Thermo3001, USA).


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2.4.3. Osteogenesis-related gene and ALP protein expression in mMSCs. After incubated with osteogenic inducing medium containing SBG or Sr-SBG extracts for 7 and 14 days, the relative expression level of genes related to osteogenesis (ALP,OCN,OPN,COL1, RUNX2) were analyzed and the results were normalized to the expression of house-keeping gene GAPDH. Total RNA was isolated from the treated mMSCs by applying HiPure Total RNA Micro Kit (Magen). The isolated RNA was then reverse transcribed into cDNA by using the Reverse Transcription Reagents Kit (Takara) according to the manufacturer's instructions. The Real-Time qPCR was performed by using Maxima SYBR Green/ROX qPCR (Thermo Scientific) and was conducted on Quantstudio 6 Flex (Life technologies). The gene expressions were calculated by the 2-∆∆Ct method. The primer sequences and genes studied in this section were presented in Supporting information Table S3. For western blot analysis, mMSCs were treated with SBG or Sr-SBG extracts for 14 days. Then, cells were collected and lysed by RIPA (Beyotime) lysis buffer. Concentration of the whole protein were determined by BCA protein assay kit (Thermo Scientific). 15 µg proteins from each group were separated on SDS-PAGE gels, and were then transferred to PVDF membranes. The membranes were blocked by 5% dried nonfat milk for 45 min at RT, incubated with rabbit polyclonal anti-ALP antibody (Abcam) and rabbit polyclonal anti-β-actin antibody (Abcam) overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h. Targeted proteins were detected with a chemiluminescent reagents (ECL-plus, Beyotime). Quantitative densitometric analysis of the image were carried out using Image J software, with β-actin as a loading control. 2.5. Osteoclastogenesis of SBG and Sr-SBG extracts. 2.5.1. Tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity assay. RAW 264.7 cells were seeded at a


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density of 3,000 cells per well in 96-well plates. After attached for 24 h, cells were then cocultured with SBG or Sr-SBG extracts for 5 days in the presence of 50 ng/mL recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL, R&D). For evaluation of osteoclastogenesis, the cells were stained with TRAP by using the TRAP kit (387-A, Sigma– Aldrich) after fixation with 4% formaldehyde according to the manufacturer's protocol. Quantitative detection of TRAP activity were conducted by fixing cells with 4 % formalin and 95 % ethanol for 15 min and 3 min, respectively. Next, each well was mixed with the working solution involved 10 mM sodium tratrate and p-nitrophenylphosphate in 10 mM citrate buffer (pH 4.6). After incubation for 1 h, 5 M NaOH were added to stop the reaction and the absorbance at 405 nm was measured. 2.5.2. Osteoclastogensis related gene expression. RAW 264.7 cells were seeded at a density of 1×105 cells/well in 24-well plates. After treated with SBG or Sr-SBG extracts containing 50 ng/mL RANKL for 24 and 72 h, total RNA from RAW 264.7 cells were isolated as described in section 2.4.3. The gene expression related to osteoclastogenesis (NFATc1, c-Fos, TRAP, cathepsin K, and MMP-9) were detected by RT-qPCR. The primers for target genes were all listed in supporting information Table S3. 2.5.3. Osteoclastogensis related Immunofluorescence Analysis. RAW 264.7 cells were seeded in 96-well plate and cultured in 50 ng/mL RANKL with or without SBG or Sr-SBG extracts for 24 h. After blocked in bovine serum albumin (BSA) for 1 hour at RT, cells were incubated with primary antibody of NFATc1 (1:100 dilution; Santa Cruz Biotechnology) overnight at 4 °C. After rinsed with Tris-buffered saline, cells were incubated with Cy3-conjugated donkey antigoat immunoglobulin G (1:200 dilution; CWBIO, China) for 1 hour at RT. Then cell nuclei was stained with DAPI (Beyotime). Images were captured by inverted fluorescence microscope.


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2.6. Macrophage polarization stimulated by SBG and Sr-SBG extracts. 2.6.1. Flow cytometry analysis of surface markers expression of macrophage. Flow cytometry analysis was applied to detect the surface markers of M1 (CD11c) and M2 (CD206) of macrophages. RAW 264.7 cells were seeded in 6-well plates and cultured in DMEM containing SBG or Sr-SBG extracts for 5 days. Then, cells were scraped off and centrifuged at 1000 rpm for 5 min. After resuspension and blocking with 1% BSA/PBS, cells were incubated with primary CD11c antibody (1:1000 dilution; BioLegend) and CD206 antibody (1:1000 dilution; Santa Cruz Biotechnology) on ice. 30 min later, cells were incubated with DyLightTM 649 goat anti-hamster (Armenian) IgG (BioLegend) and Alexa Fluor 594 rabbit-anti-goat secondary antibodies (Life tehcnologies) for 30 min. At last, the detection was performed on a guava® easyCyte HT system (Merk Millipore). Data obtained were analyzed by GuavaSoft 2.5. 2.6.2. Inflammatory cytokines expression and gene expression. RAW cells were plated in 6well plates at a density of 1.5×105 cells/well. The medium was removed and changed with SBG or Sr-SBG extracts when cells reached 80% confluence. 3 days later, the culture medium was taken out and centrifuged at 4 °C. Supernatants were extracted for enzyme-linked immunosorbent assay (ELISA) detection of IL1β, IL6 and IL10 content using mouse ELISA kits (RayBiotech) following the manufacturer's protocol. The following macrophage-conditioned materials experiments were performed by using the mixture of the above supernatants and DMEM with the ratio of 1:2. Total RNA was isolated from each group and gene expression analysis was conducted as described in Section 2.4.3. RT-qPCR primers were listed in Supporting information Table S4. 2.7. Osteogenesis and Osteoclastogenesis of macrophage-conditioned SBG and Sr-SBG extracts. 2.7.1. Osteogenesis of macrophage-conditioned SBG and Sr-SBG extracts. ALP


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activity, Alizarin red S assay, osteogenesis-related gene and ALP protein expression of mMSCs in response to macrophage-conditioned SBG and Sr-SBG extracts were evaluated as described in Section 2.4. 2.7.2. Osteoclastogenesis of macrophage-conditioned SBG and Sr-SBG extracts. TRAP activity, immunofluorescence assay and osteoclastogenesis related gene expression of RANKL induced RAW 264.7 cells stimulated by macrophage-conditioned SBG and Sr-SBG extracts were examined as described in Section 2.5. 2.8. In vivo evaluation of SBG and Sr-SBG. 2.8.1. Surgical procedure and treatment. Eight week old Balb/c mice and ten week old Wistar rats obtained from Laboratory Animal Center, South China Medical College were used in this study. The experimental protocols were approved by the Institutional Animal Care. Balb/c mice were anesthetized using 10% chloral hydrate. On the flank region of the mice, an incision of 1 cm was made. Then, a subcutaneous pocket from each side of the skin was created. 20 mg of SBG and Sr-SBG powders mixed with phosphate buffer were embedded subcutaneously to investigate the inflammatory reaction. One week later, all the mice were euthanized. The implanted substances were dissected from the mice. All harvested samples were lysed by RIPA lysis buffer for ELISA detection of IL6, IL1β and IL10. Rat femoral condyle defect model was performed by anesthetizing Wistar rats with 10% chloral hydrate, and a bone defect of 3 mm diameter × 3 mm deep was created on the femoral condyles by electric trephine drill. 40mg of SBG and Sr-SBG particles mixed with phosphate buffer were used to fill the bone defects. The wounds were sutured and prophylactic antibiotic was administered to avoid infections. All the rats were sacrificed after four weeks. The femoral condyles were harvested and fixed in 10% formaldehyde.


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2.8.2. ELISA assay of the subcutaneously embedded SBG and Sr-SBG. All of the harvested subcutaneously embedded samples were transferred into a 2 mL tube with RIPA lysis. Tissue lysis was performed by using a electric tissue grinder. Then, the supernatants were collected by centrifuging protein lysis at 14,000 g for 15 min. The total protein content was measured using BCA assay, and IL 1β, IL6, and IL10 mouse ELISA assay (RayBiotech) were performed as described in Section 2.6.2. 2.8.3. Histological analysis. Femoral bone samples were decalcified by immersing samples into decalcifying fluid for 4 weeks. Then, 5 µm-thick sections were cut from the paraffinembedded tissues to conduct the histological evaluation. Hematoxylin and eosin (H&E) staining and Masson's Trichrome staining were performed with sections from each sample. 2.8.4. Immunohistochemistry. Immunohistochemistry was conducted on deparaffined and rehydrated slides with primary antibodies: M1 marker inducible Nitric oxide synthases (NOS2, Santa Cruz Biotechnology), M2 markers CD206 (Santa Cruz Biotechnology) and Arginase I (Santa Cruz Biotechnology). The stained slides were imaged by a light microscope (Axioskop 40 FL, Zeiss, Germany). 2.9. Statistical Analysis. All values were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS Statistics version 22 (IBM, USA). Data were analyzed with one-way ANOVA with LSD post test. A p-value < 0.05 was considered as statistically significant.

3. RESULTS 3.1. Characterization and ion concentration determination of SBG and Sr-SBG extracts. SBG and Sr-SBG were successfully prepared by the modified sol-gel method. The surface


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morphology of SBG and Sr-SBG spheres detected by SEM were shown in Figure 1. SBG and SrSBG all had uniform particle size of approximately 400~500 nm. The particle distribution of SBG and Sr-SBG determined by dynamic light scattering was accorded with the SEM results (Figure S1), which showed the average sizes of SBG and Sr-SBG were 401.0 nm and 534.6 nm, respectively. The elemental compositions were recorded by XPS and EDS. Figure S1 and Figure S2 showed the composition of SBG and Sr-SBG which indicated the successful incorporation of strontium into SBG. As XPS and EDS characterization could only obtain qualitative and semiquantitative detection of element composition, the actual element contents of SBG and Sr-SBG were examined by ICP-AES. The ICP analysis showed that the Sr content of SBG and Sr-SBG was 0% and 2.71%, respectively (Table S1). Zeta potentials of SBG and Sr-SBG were measured to be -21.3 mV and -29.4 mV, respectively (Table S1). The results of degradability of SBG and Sr-SBG in SBF (Figure S1) revealed that Si ions were sustained released from both SBG and SrSBG group. The concentration of Ca ion and P ion showed similar trend of reduction in SBG and Sr-SBG group, which was ascribed to the consumption of HA formation. The release of Sr ions in 7 days was increased steadily in Sr-SBG group. Ion concentration of SBG and Sr-SBG extracts examined by ICP-AES were shown in Table S2. There was a slight difference in Si and P ions concentration between SBG and Sr-SBG extracts. The concentration of Sr ion in Sr-SBG extracts was 6.227 mg/L, whereas the Ca ion concentration in SBG and Sr-SBG extracts were 53.01 mg/L and 49.25 mg/L, respectively.

Figure 1. SEM images of SBG and Sr-SBG.


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3.2. Effect of SBG and Sr-SBG extracts on osteogenesis. ALP is widely used as a maker of osteogenic phenotypes. The ALP staining and ALP activity detection were performed using mMSC samples collected on day 7 and 14. As shown in Figure S4 and Figure 2A, the ALP activity level of Sr-SBG group was obviously increased compared with the control group and SBG group. The results of Alizarin Red S staining and ALP activity had similar trends (Figure 2C and 2D). More significant mineralization nodules were displayed in mMSCs incubated with Sr-SBG extracts than that with SBG extracts. Osteogenesis-related gene expression such as ALP, OPN and OCN were upregulated in Sr-SBG group and SBG group in comparasion with control group on day 7 and 14 (Fig. 2B), wheras Sr-SBG group had an enhanced effect of osteogenesis compared with SBG group (P

Strontium-Substituted Submicrometer Bioactive Glasses Modulate

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