Binary Doping of Strontium and Copper Enhancing Osteogenesis and

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Binary Doping of Strontium and Copper Enhancing Osteogenesis and Angiogenesis of Bioactive Glass Nanofibers While Suppressing Osteoclast Activity Lin Weng, Sunil Kumar Boda, Matthew Teusink, Franklin D Shuler, Xiaoran Li, and Jingwei Xie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06521 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 5, 2017

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ACS Applied Materials & Interfaces Revised ms # am-2017-065217

Binary Doping of Strontium and Copper Enhancing Osteogenesis and Angiogenesis of Bioactive Glass Nanofibers While Suppressing Osteoclast Activity

Lin Weng†, Sunil Kumar Boda†, Matthew J. Teusink‡, Franklin D. Shuler¶, Xiaoran Li◊,*, Jingwei Xie†,* †

Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program,

College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States ‡

Department of Orthopedic Surgery and Rehabilitation, University of Nebraska Medical Center,

Omaha, Nebraska 68198, United States ¶

Department of Orthopaedic Surgery, Joan C. Edwards School of Medicine, Marshall

University, Huntington, WV, 25755 United States ◊

Key Laboratory for Nano-Bio Interface Research, Division of Nanobiomedicine, Suzhou

Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, China

ABSTRACT: Electrospun bioactive glass fibers show great potential as scaffolds for bone tissue engineering due to their architectural biomimicry of the bone extracellular matrix and their composition capable of providing soluble bioactive cues for bone regeneration and remodeling. Trace elements can be doped to further promote osteogenesis and angiogenesis during bone regeneration. Cationic substitution of strontium for calcium in bioactive glass positively enhances osteoblast phenotype, while suppressing osteoclast activity. Further, the addition of copper spontaneously improves the vascularization during neobone formation. The objective of this study was to fabricate and characterize electrospun bioactive glass fibers doped with strontium and copper and evaluate their potential for bone repair/regeneration in vitro. Different ratios of strontium and copper were doped in electrospun bioactive glass fibers. The released strontium and copper from doped fibers could reach effective concentrations within 40 h and last for 4 weeks. These bioactive glass fibers demonstrate their bioactivity by promoting osteoblastic and endothelial cell activity and inhibiting the formation of osteoclasts or bone resorbing cells.

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Additionally, in vitro cell culture of different cell types in the presence of extraction solutions of the electrospun bioactive glass fibers showed that the dopants achieved their individual goals without causing significant cytotoxicity. Altogether, this novel class of bioactive glass fibers hold great promise for bone regeneration. KEYWORDS: bioactive glass fibers, strontium, copper, electrospinning, bone tissue regeneration, vascularization

1. INTRODUCTION The treatment of large bone defects remains a major clinical and socio-economical problem due to trauma, tumor removal or age related bone diseases such as osteoporosis or bone thinning. In the United States alone, 1.3 million people undergo bone graft surgeries each year for skeletal defects of the load bearing bones resulting from either accidents or age-related bone disease.1 Particularly, osteoporotic bone fracture is a major epidemic in post-menopausal women due to the decrease in estrogen secretion levels, that is responsible for maintaining bone mineral density.2 Currently, autologous bone graft is still the gold standard despite its limited resources, associated done site morbidity and size mismatch.3 Allografts and xenografts have issues such as being weakly osteoinductive, immunogenic response, and potential risk of infection and disease transmission.4 Also, currently there are no synthetic 3D bioactive materials that can resorb at a programmed rate identical to rate of bone ingrowth as well as mimic the architecture and composition of the bone extracellular matrix (ECM). Therefore, there is an urgent need to develop synthetic bone grafts that can overcome the limitations of bone autografts and allografts. Bioactive glasses belong to a class of well-known synthetic bone substitute materials. The first bioactive glass of the composition 45SiO2-24.5Na2O-24.5CaO-6P2O5 (all components in wt. %) (45S5 Bioglass®), was developed by Hench et al. 5 in the late 1960s and it has been in clinical use for orthopaedic and dental applications since 1985.6 However, the bioactive glass materials produced previously were either in the bulk or granular form or fiber-type melt-derived glass with diameters of hundreds to tens of micrometers. Although bioactive by virtue of leaching of ions essential for bone formation, these materials have a lesser resemblance to the architecture of bone ECM. To recapitulate the 3D architecture and nanofibrillar structure of bone ECM, several researchers have fabricated bioactive glass nanofibers using electrospinning and demonstrated their bioactivity by the biomineralization of hydroxyapatite crystals in simulated body fluids.7 2 ACS Paragon Plus Environment

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However, an optimal composition of electrospun bioactive glass nanofibers for bone regeneration has not been established yet. Some other works have also illustrated how biomineralization of glass fibers in the presence of proteins of interest can be exploited for the controlled delivery of therapeutic molecules for bone regeneration.8 While several protein-based medications such as bone morphogenetic protein (BMP-2) have been used for the treatment of bone disorders via repair and regeneration, these protein-based therapeutics can cause significant side effects such as ectopic bone formation, osteoclast-mediated bone resorption, inappropriate adipogenesis, and unwanted immunogenic responses in the host.9 An alternative to protein-based bone therapeutics is the addition of different elements to bioactive glass, that can enhance their dissolution and bioactivity. For instance, researchers have elucidated the efficacy of Si based bioactive glass in enhancing metabolic process, and formation and calcification of bone tissue.10 Further, the addition of Mg, Ca, or Sr has been reported to promote osteoblast proliferation and differentiation while reducing osteoclast activities.11-13 Also, trace amounts of Zn and Cu have been for implicated in eliciting anti-inflammatory and angiogenic properties.14, 15 Among the dopant elements, Sr can easily substitute for Ca in many graft materials by virtue of their chemical similarity. Also, Sr can aid in bone homeostasis by stimulation of osteoblast differentiation and bone formation, as well as inhibition of osteoclastogenesis and bone resorption.16 In the light of the therapeutic effect of Sr on bone health, strontium ranelate (PROTELOS®) has been approved in Europe for the treatment of severe postmenopausal osteoporosis in women with an increased risk for bone fracture.17 Inspired by such results, many efforts have been devoted to incorporate Sr into various calcium phosphates and mesoporous bioactive glasses to achieve long-term release of Sr2+ for bone repair/regeneration.18-20 In a parallel to osteoinductive effect of strontium, copper in addition to its antibacterial effect has been demonstrated to stimulate the proliferation of endothelial cells in a dose dependent manner in vitro, and promote wound healing in rats by up-regulating VEGF expression.21, 22 The amount of oxygen required for cell survival is limited to a diffusion distance between 150 and 200 µm from the supplying blood vessels,23 making a porous interconnected scaffold architecture critical for the successful angiogenesis and vascularization of the neobone. Summarizing, an ideal bone grafting material should be able to enhance osteogenesis and angiogenesis, while reduce bone resorption. The addition of Sr to bone grafts can promote osteogenesis and decrease bone resorption, while the addition of Cu can promote angiogenesis. In the current study, our objective 3 ACS Paragon Plus Environment


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is to fabricate bioactive glass nanofibers co-doped with Sr and Cu in trace quantities and evaluate the effects of released ions released on the viability and cellular functionality of relevant cell types.

2. EXPERIMENTAL SECTION 2.1. Fabrication of Sr and Cu doped electrospun bioactive glass fibers. The electrospinning in the present work was performed using a conventional electrospinning setup except that the mandrel collector was placed over the spinneret against gravity for quality control (Figure 1A). The solution for electrospinning was prepared by mixing two precursor solutions (Table 1).24 One was that of the silica sol mixture for the bioactive glass and the other was that of a polymer meant to be added to adjust the rheology of the silica sol for electrospinning. The doped bioactive glass composition is as follows: 1.340 g of tetraethyl orthosilicate (TEOS; Sigma-Aldrich), 0.116 g of triethyl phosphate (TEP; Sigma-Aldrich), 0.148 g of Ca(NO3)2·4H2O (Sigma-Aldrich), 0.132g of Sr(NO3)2(Sigma-Aldrich), 0.200/0.400 mL of 0.0053 g ml-1 CuCl2 (Sigma-Aldrich) aqueous solution (0.5% Cu or 1% Cu doped bioactive glass electrospun fibers), 0.10 mL of HCl (Sigma-Aldrich) solution (1.00 mol L–1), 4.00 mL of ethanol (Decon labs, Inc.), and 3.00 mL of deionized (D.I.) water. The second solution was that of poly(vinylpyrrolidone) (PVP) prepared by dissolving 1.65 g of PVP (Sigma-Aldrich, Mol.wt.1,300,000) in 10.0 mL of absolute ethanol. In order to adjust the solution viscosity prior to electrospinning, the two precursor solutions were mixed in 1:1 ratio by stirring for 2 h at 4 ˚C. The electrospinning parameters were optimized to obtain non-beaded fibers and the typical feeding rate for the final solution mixture was 0.60 ml h–1. The fabricated fibers were collected on a slow rotating mandrel to obtain random fibers. In order to ensure the randomness of the glass fibers and their easy removal from the collector, the mandrel was pre-coated by a thin memory layer of electrospun polycaprolactone (PCL; Sigma-Aldrich, Mol. wt. 80,000) fibers. The bioactive glass fibers were obtained by a post-processing heat treatment, wherein the organic polymers PCL and PVP in electrospun fibers were removed by sintering the collected fibers at 600 ˚C for 5 h in a muffle furnace in ambient atmosphere. Overall, four compositions of the bioactive glass fibers were fabricated in this study. They have been designated as follows – (i) Undoped or Ca bioactive glass fibers; (ii) 50% Sr doped bioactive glass fibers, where 50 mol % of Ca is replaced by Sr; (iii) 1% Cu and 50% Sr doped bioactive glass fibers, where 1 mol % Cu and 50 mol % Sr replace 4 ACS Paragon Plus Environment

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Ca; and (iv) 0.5% Cu and 50% Sr doped bioactive glass nanofibers, where 0.5 mole % Cu and 50 mol % Sr replace Ca.

2.2. Morphology and elemental composition of Sr and Cu doped electrospun bioactive glass fibers. The surface morphology and diameters of bioactive glass electrospun fibers was characterized by field emission scanning electron microscopy (FE-SEM; Hitachi S4700). To avoid charging of the insulating electrospun fibers under the electron beam, the composite samples were fixed on a metallic stub with double-sided conductive carbon tape and coated with chromium for 240 s using a sputter coater. The SEM images were acquired at low accelerating voltage of 5 kV in the secondary electron mode. The fiber diameters were quantified from the SEM images by measuring twenty individual fibers using the Image J software. The elemental composition of the glass fibers was characterized by energy dispersive spectroscopy (EDX) mapping, line scan and point probing at different locations on the fiber mats.

2.3 Biomineralization and release kinetics of ions from bioactive glass fibers. The bioactivity of the doped glass fibers was determined by immersing them in simulated body fluid (SBF, pH 7.4) solution followed by characterization of formed minerals. The SBF for the biomineralization test was prepared following earlier studies.25 The fibers were sampled at a specific time, washed by D.I. water thrice, each at 5 min intervals, and then dried before examination under FE-SEM. For the cumulative ion release kinetics, a pre-cut fiber mat that weighed around 3.0 mg was sterilized and immersed in 3.00 mL of Dulbecco’s modified eagle’s medium (DMEM) in a 15 mL conical centrifuge tube. At each pre-defined time points, 3.00 mL of supernatant DMEM was collected, followed by rinsing the fibers with 2.00 mL D.I water, which was also collected. Subsequently, 3.00 mL of fresh DMEM was replenished to the conical centrifuge tubes containing the glass fibers. The collected DMEM supernatant was filtered and either stored at 4 ˚C for future cell culture studies or taken for inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The concentrations of Sr, Cu, Si and Ca released into the cell culture medium were determined using ICP-MS (Agilent 7500 cx). The test was run by using argon gas for generating the plasma. Each sample was recorded four times and the data presented are a mean value of four replicates. 5 ACS Paragon Plus Environment


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2.4. In vitro cellular response. The effect of the ions released from doped bioactive glass fibers on osteogenesis, osteoclast activity and angiogenesis was examined in vitro. The study on the osteogenesis of human bone marrow stromal osteoprogenitor cells (hBMSCs) included one control group, four electrospun bioactive glass fiber groups (0.5 mol % Cu, 1 mol % Cu, 50 mol % Sr, and Ca electrospun bioactive glass fiber groups, and one free Sr2+ group as positive control. For evaluating the angiogenic effect of Cu on HUVECs, the two Cu2+ groups (0.5 and 1 mol% Cu) were tested against the undoped bioactive fibers as controls. Table 2 lists the sample designations of the extraction solutions from the differently doped bioactive glass fibers. The extraction solutions of electrospun bioactive glass fibers were prepared by immersing the corresponding glass nanofibers into the cell culture medium for different time periods, before being filtered and stored at 4 ºC for the cell culture tests. The duration of the bioactive glass fiber extractions was determined by the in vitro release profiles. For example, during the osteogenesis of ADSCs, the culture medium was changed every 72 h with the extractions at 36 h, 108 h, 180 h and 252 h, which can simulate the ion concentrations from bioactive glass nanofibers during the in vitro release. The positive control groups (Sr2+ and Cu2+ for osteogenesis and angiogenesis respectively) were prepared by adding filtered Sr(NO3)2·4H2O and/or CuCl2·2H2O solutions at final concentrations of 1 mM and 0.01/0.001 mM into the culture medium, respectively.

2.4.1. Cell lines and their culture. RAW264.7 macrophage cells were obtained from the ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco/BRL). ADSCs were obtained from Lonza and cultured in 50% DMEM/50% F12k medium supplemented with 10% FBS. Human vascular endothelial cells (HUVECs) were obtained from Lonza and cultured in EMP-2®. hBMSCs were obtained from ATCC and cultured in the low glucose DMEM supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 °C and 5% CO2. 2.4.2.

Osteogenesis

lipopolysaccharide

and

angiogenesis

(Escherichia

coli,

supplementing

serotype

055:

B5),

agents.

L-Ascorbic

dexamethasone,

and

acid, β-

glycerophosphate disodium salt hydrate were supplied by Sigma. Human VEGF Quantikine® Elisa was purchased from R&D SYSTEMS, and Alizarin Red S (ARS) solution was obtained from EMD Millipore Corp. 6 ACS Paragon Plus Environment

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2.4. 3. Cell viability assay. RAW264.7 cells were plated at a density of 5×103 cells/well in 96-well plates and treated with the designated extraction solutions for 48 h and 96 h. HUVECs, ADSCs and hBMSCs were plated at a density of 3×103 cells/well in 96-well plates and exposed to dopant ions from bioactive glass fiber extraction solutions for 48 h and 96 h (24 h and 48 h for HUVECs). For the study, the plated cells were allowed to attach to the polystyrene well bottoms for 6 h, followed by replacement of the culture media with the designated extraction culture medium. After the predetermined exposure durations, the cells were washed thrice with PBS and (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide or MTT (5 mg ml-1) was added to the culture medium at a volume ratio of 1:9, and further incubated for 4 h. Subsequently, the culture media were removed and the formazan crystals were solubilized in 200 µL of dimethyl sulphoxide (DMSO). The absorbance of the dissolved formazan crystals was measured at 490 nm using a microplate reader (Dynatech MR-7000; Dynatech Laboratories).

2.4.4. TRAP staining of RAW264.7 cells. Tartrate-resistant acid phosphatase (TRAP) is a metalloenzyme highly expressed in activated macrophages and osteoclast cells. This acid phosphatase can be detected by naphthol AS-Bi phosphoric acid in conjunction with diazonium salts. For evaluating the anti-osteoclast potential of doped bioglass fibers, RAW264.7 cells were plated at a density of 2×104 cells/well in 24-well plates for 6 h and then incubated with DMEM supplemented with lipopolysaccharide (LPS) at a final concentration of 100 ng ml-1 for 3 days to stimulate RAW264.7 into macrophage-like cells. Then, the culture medium was replaced with the designated glass fiber extraction media containing 10% FBS and LPS (100 ng ml-1). The extraction media was replenished every three days for cell culture. TRAP staining was performed on days 2, 4 and 6 after the induction of macrophage phenotype, following the protocol (Sigma, Procedure No. 387). After staining, the 24-well plates were photographed by a Leica digital MC120 camera. To avoid bias, all the wells were photographed starting from the same position and surveyed over by the same pattern. The TRAP-positive cells with three or more nuclei (multinucleate cells) were counted from multiple image frames and statistically analyzed. 2.4.5. ALP activity of ADSCs. ADSCs were plated at a density of 5×104 cells/well in 6-well plates for 6 h, followed by treatment with designated extraction culture media supplemented with 7 ACS Paragon Plus Environment


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0.05 mM of ascorbic acid, 100 nM of dexamethasone, and 10 mM of β-glycerophosphate disodium. The extraction culture media was replenished every three days. The alkaline phosphatase (ALP) activity of ADSCs was assayed on day 5 and 10 following the protocol of ALP assay kit (ab83369, Abcam). The alkaline phosphatase was extracted by lysing the cells using a high-speed centrifuge (15000 rpm, 15 min, 4 ̊C). The cell lysate was reacted with pnitrophenyl phosphate (pNPP), which turned yellow upon dephosphorylation to p-nitrophenol. The absorbance was measured at a wavelength of 405 nm using a microplate reader.

2.4.6. Alizarin Red staining of ADSCs. The mineralization of ADSCs was detected by the formation of calcium deposits detected by Alizarin Red S (ARS) staining. For assessing the potential of doped bioglass extracts in induction of CaP mineralization, ADSCs were plated at a density of 5×103 cells/well in 24-well plates for 6 h, followed by treatment with designated extraction media supplemented with 0.05 mM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate disodium. The extraction culture media was replenished every 3 days. The alizarin red staining procedure was performed on day 14. Post-staining of the culture wells, a minimum of four images were taken for each well at fixed positions without overlapping. Subsequently, the images were analyzed by the Image J software, in which the reddish area was surveyed and compared with each other.

2.4.7. Vascularization of HUVECs. The angiogenic potential of copper doped bioactive glass fibers were characterized by the formation of tubes by HUVECs. Sub-confluent HUVECs were harvested and suspended in designated extraction media containing Endothelial Cell Growth Medium-2® (Lonzo). The cells were seeded into growth factor-reduced matrigel coated 96 well plates at a seeding density of 7×104 cells/well. Tube formation was examined at 16 h under a microscope and photographed at 5× magnification. These photographs were subsequently analyzed using the Image J software.

2.4.8. VEGF assay of hBMSCs. The secretion of vascular endothelial growth factor (VEGF) protein is a characteristic feature of angiogenesis. For the VEGF assay, hBMSCs were plated at a density of 2×104 cells/well in 24-well plates for 6 h, followed by treatment with designated extraction media. The culture media was changed on day 3. The VEGF ELISA was performed 8 ACS Paragon Plus Environment

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on days 3 and 7 following the protocol of Human VEGF Elisa kit (R&D Systems). Briefly, the cell culture medium was collected and centrifuged at 4 ˚C, 1000 rpm for 15 min.

The

supernatant collected was diluted 10 times by PBS. The absorbance of the supernatant was measured at wavelength of 450 nm by a microplate reader.

2.4.9. Statistical analysis. Data are presented as mean ± standard deviation (SD) of triplicates from three individual experiments. Statistical significance was determined by performing analysis of variance (ANOVA) with Tukey test and with a significance accepted at p-value < 0.05.

3. RESULTS AND DISCUSSION 3.1. Fabrication and characterization of Cu or/and Sr doped bioactive glass fibers. In order to improve osteoinduction and vascularization for neobone formation, different compositions of bioactive glass fibers were fabricated by electrospinning followed by thermal treatment of the as spun fibers. In all the cases the fiber diameters of the glass fibers after thermal treatment were smaller than that of the as spun PVP-glass precursors implying a decrease in fiber diameter due to the loss of organic polymer framework during calcination. The SEM images present the nanofibrous morphology of the synthesized bioactive glass fibers with the four different compositions (Figure 1). The substitution of Ca with Sr in the bioactive glass fibers resulted in a significant increase in the fiber diameters from 229 ± 39 nm to 397 ± 67 nm, 420 ± 89 nm, or 457 ± 66 nm (Figure 1, B-E). A plausible explanation is the higher limiting equivalent ionic conductance of Ca2+ compared to Sr2+, which could lead to higher electrical current during the electrospinning process of undoped glass fibers and eventually a lower diameter for the electrospun nanofibers.26 Interestingly, the co-doping of copper and strontium in the glass sol mixture led to further increase in the fiber diameters, despite the low dopant concentration of copper compared to strontium. These findings indicated the nature of the dopants or the substituent ions played some role in governing fiber diameters. The elemental composition of the different bioactive glass nanofibers was determined by energy dispersive x-ray spectroscopy (EDX). Figure S1 (in the supporting information) shows the elemental mapping images of the glass nanofibers. The data from the elemental mapping scans are in agreement with the starting sol-gel precursors of the different glass compositions and the 9 ACS Paragon Plus Environment


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density of the signals detected are proportional to the density of fibers in the areas scanned. More importantly, all the expected elements (Si, O, P, Ca, Sr) were distinctly detected including trace amounts of Cu. Figure S2 corresponds to line scans across the glass nanofibers of different compositions. A semi-quantitative information can be ascertained from the line scans with the following order of elemental abundance: O > Si > Ca ≥ Sr > P > Cu. Figure S3 are point probe EDX spectra recorded from different regions of the glass nanofibers. All the expected elements were detected and quantified from their characteristic peaks, the results of which are listed in Table 3. The abundance of the detected elements is in good agreement with the starting compositions of the sol-gel mixtures used for fabricating the different bioactive glass nanofibers. Further, the composition of commercial 45S5 bioglass has been shown in Table 3 for comparison. A close observation of Table 3 will reveal the closeness of the silicon content of the sol-gel derived fibers fabricated in the present study with the commercial melt-quenched 45S5 bioglass. To ascertain the amorphous nature of the bioactive glass nanofibers after sintering at 600 ºC for 5 hours, X-ray diffraction (XRD) of the crushed fibers was performed with a powder XRD machine. Figure S4 shows the XRD patterns recorded in the 2θ (degrees) range of 10-90 º for the different compositions of the bioactive glass nanofibers. The broad amorphous hump can be observed in all the samples indicating the absence of any crystalline phases or glass crystallization that can influence bioactivity.

3.2. Biomineralization and release kinetics of ions from bioactive glass fibers. To examine the biomineralization or apatite-forming ability of the fabricated bioactive glass nanofibers, the fibers with different compositions were immersed in SBF and incubated at 37 oC. We used SBF, the standard buffer concentration that mimics the concentration of ions in the blood plasma for biomineralization as SBF is the golden standard for evaluating the apatiteforming ability of implant materials in vitro, and predicting the bioactivity of implants in vivo.27 We observed that all four bioactive glass nanofibers were mineralized over time though their mineralization rates varied (Figure 2). On day 1, all fiber samples showed no evident deposition of minerals on their surfaces (Figure 2, A1-D1)). A few minerals deposited were observed on all the fibers on day 3 (Figure 2, A2-D2). The pristine undoped bioactive glass nanofibers were partially covered with apatite, while the doped glass fibers were sparsely mineralized in the order 10 ACS Paragon Plus Environment

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50% Sr < (0.5% Cu + 50% Sr) < (1 %Cu + 50% Sr). The retarded biomineralization in the case of Cu-doped glass fibers can be rationalized from the lewis acid character of Cu2+ and their binding affinity to phosphates, thus reducing apatite formation.28 On day 7, the pristine bioactive glass nanofibers (without Sr and/or Cu doping) were completely covered by flaky hydroxyapatite (Figure 2A3), while the doped fibers were partially covered with minerals (Figure 2, B3-D3). All the fiber samples were fully covered with apatite minerals after 15 days of incubation in SBF (Figure 2, A4-D4). Our previous work demonstrated that bioactive glass hollow tubes formed rod-like minerals in water.24 However, the solid bioactive glass fibers developed in this study exhibited no such behavior (Figure S5). The mineralization of bioactive glass in SBF includes a series of reactions proposed by Hench and co-workers.29 According to them, the dynamic balance of release and absorption of Ca2+ from SBF onto the electronegative Si-OH surface groups created by hydrolysis of silicate network, subsequently attracts PO43− from SBF to form apatite nucleation sites. According to the classic apatite formation mechanism,30 the apatite formation on bioactive glass nanofibers are affected by multiple factors such as the surface polarity, availability of nucleation sites, and the supersaturation of Ca2+. The released Sr2+ into solution may interfere with HAp formation by competitively binding to PO43- compared to Ca2+. Further, previous reports suggested the smoother surfaces of Sr doped bioactive glasses and electrospun polymer nanofibers mineralized with Sr2+ doped HAp make them less preferred nucleating sites for apatite formation.31,

32

The released Cu from the doped bioactive glass fibers further reduced

HAp formation, by interaction with CO32- and OH- in SBF. The tendency of Cu2+ to precipitate at alkaline pH can disrupt the biomineralization process and reduce HAp formation.33 The release kinetics of therapeutic ions from the doped glass nanofibers into cell culture medium were studied by ICP-MS. At pre-determined time points, aliquots of the extraction solutions were analyzed over a time duration of one month. The ion concentration measurements showed that the cumulative release profile of Si, Cu and Sr in the dissolution medium displayed an initial logarithmic increase followed by a steady release profiles as can be observed from the plots of ion concentrations versus time (Figure 3). The release profiles of Si, Sr and Cu indicated much higher releasing rates of the bioactive glass fibers compared to their bulk counterparts. Such accelerated release kinetics can be attributed to the large surface area to volume ratio of bioactive glass nanofibers compared to mesoporous bioglasses.19, 34, 35 Among them, Si exhibited 11 ACS Paragon Plus Environment


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a sustained and controlled release profile over one month (720 h) period. On the other hand, both Cu and Sr showed a rapid burst release in the first 120 h, followed by steady release kinetics. Nevertheless, a first order ion release kinetics was observed in all cases. The accelerated release of the dopants could be due to the higher solubility of Sr2+ and Cu2+ compared to Si as well as the possible chelation between Cu2+ and amino acids present in DMEM.36 On the contrary, Ca exhibited a different release pattern compared to Si, Cu, and Sr. The absolute concentration of Ca surged in the first 4 h and then dropped to the basal Ca2+ concentration in DMEM. After 40 h, Ca concentration dropped to a level far below that of the Ca2+ present in DMEM, which indicated the depletion of Ca2+ from DMEM. The depletion in extracellular calcium could be attributed to the mineralization of hydroxycarbonate apatite (HCA) on bioactive glass nanofibers in DMEM (Figure S6). The drop in Ca2+ in DMEM became stabilized after 120 h, which could be explained by the steady state of calcium dynamics i.e., the rate of apatite nucleation and crystal growth equaled the release of calcium from the crystallized hydroxyapatite. The release kinetics of phosphate are not expected to vary significantly with time as shown in earlier studies on a similar series of strontium-doped bioactive glass of the type SiO2-P2O5-Na2O-CaO.19 The dissolution and release kinetics of doped bioactive glasses have a dependence on the ionic radius of the dopant in a way that the dopants with smaller ionic radius (such as Li) stabilizes the silicate framework of the bioactive glass thus decreasing ion release, while dopants with larger ionic radius (such as K) destabilize the tetrahedral silicate framework and increase glass dissolution and ion release kinetics.37 In this light, both Sr2+ and Cu2+ with larger ionic radii than Si4+ are envisaged to enhance the dissolution and ion release from doped bioactive glass nanofibers as compared to their pristine counterpart. In the context of glass based biomaterials, it may be worthwhile to compare the bioactivity of sol-gel glasses with commercial melt-quenched 45S5 bioactive glass. Such a comparative analysis was made by Hench and co-workers.38 Particularly, the melt-derived 45S5 glass was compared with sol-gel based 58S bioactive glass powders. Physicochemical characterization of the two types of glass powders revealed low porosity in the melt-quenched glasses in comparison to highly mesoporous texture of the sol-gel glass.38 This promoted higher dissolution and surface apatite layer formation as well as better resorption of the sol-gel glass in vivo.34, 38 Furthermore, melt-quenched glasses suffer from crystallization during sintering which reduces their bioactivity unlike the sol-gel derived glasses.39 In a noteworthy work, the tunable release of a model protein, 12 ACS Paragon Plus Environment

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bovine serum albumin (BSA) from glass nanofibers was demonstrated by regulating the hydrolysis of the silicate framework in sol-gel derived bioactive glass nanofibers by manipulating the ratio of the silicate precursor (TEOS) and water.8 Thus, it is possible to modulate bioactivity by manipulating the dissolution and ion release kinetics from sol-gel derived bioglasses, which make them preferable choice of bioglasses compared to melt-derived ones. Therefore, in the present study, we have focused on sol-gel based bioactive glass nanofibers and their doping to confer specific bioactivity.

3.3. Cell viability. The pristine and doped bioactive glass nanofibers were assessed for their biocompatibility by exposing the glass fiber leachates to multiple cell types. In particular, we examined the potential cytotoxicity of the Cu or Sr doped bioactive glass nanofibers using MTT assay by incubating RW264.7, HUVECs, ADSCs and hBMSCs with the designated extraction culture media. Previous reports indicate a dose and exposure duration dependent cytotoxicity of Cu released from intrauterine contraceptive devices (IUCDs) on mouse fibroblasts in vitro.40 Similarly, we observed that the release of Cu2+ elicited a decline in the cell proliferation of all the treated cell types. Figure 4 shows that Cu2+ released from Cu doped bioactive glass nanofibers or the addition of soluble Cu2+ as a metal salt had a detrimental effect on the proliferation of multiple cells including HUVECs and ADSCs. Specifically, the cytotoxic effects of Cu were distinctly observed in HUVECs. At 48 h, the viability of HUVECs treated with 0.5% Cu doped bioactive glass nanofiber extraction media dropped to 76%, showing statistically significant decline compared to the control group. Therefore, the exposure of Cu-doped glass fiber leachates to HUVECs was not performed beyond 48 h unlike all the other cases, where the cell types were more tolerant towards Cu2+. The generation of reactive oxygen species (ROS) is one of the major mechanisms by which copper elicits cytotoxicity in cell monolayers in vitro.41 At elevated copper concentrations, the spike in ROS production can even lead to mutations and DNA damage by interaction with nuclear chromatin as well as peroxidation of other biomolecules such as proteins and lipids in cell membranes.42 In order to avoid such detrimental effects, we limited the doping concentrations of Cu in the bioactive glass nanofibers to 0.5 and 1.0 mole %. On the other hand, we observed that Sr, Ca, and Si alone as metal salts or in combination from their corresponding bioactive glass nanofibers had little impact on the growth and proliferation of the treated cells (Figure 4). Also, previous work from our research group showed that the 13 ACS Paragon Plus Environment


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proliferation of mouse preosteoblastic MC3T3-E1 cells was similar for hollow as well as solid glass nanofibers, while mineralization occurred on both the inner and outer surfaces of the hollow fibers in simulated body fluid (SBF).24 Overall, our findings were in line with the previous reports in which the biocompatibility and non-cytotoxicity of these elements (Sr, Ca, Si) in doped calcium phosphates and silicates have been verified and established.43-45 However, it is important to note that strontium can cause osteomalacia or bone softening in patients with renal failure or chronic kidney disease (CKD),46 which is also another critical ailment in aged people.

3.4. In vitro cell response. Apart from evaluating the cell viability, the effect of the dopants in the bioactive glass nanofibers on the functional behavior of cells was determined using biochemical assays, ELISA and cell morphological analysis. As pre-meditated, the glass nanofibers were doped with Sr to enhance osteogenic and anti-osteoclastogenic activity were tested against ADSCs and RAW264.7 cells, respectively. Similarly, the purposeful addition of Cu intended to promote angiogenesis was evaluated in hBMSCs and HUVECs. The osteogenic and angiogenic differentiation of ADSCs and hBMSCs as well as vascularization of progenitor HUVECs were examined after prolonged exposure to the designated extraction media from the doped glass nanofibers. The following sub-sections present the results and discuss the effect of the doped glass nanofiber leachates on the functionality of the cells in question.

3.4.1. Strontium mediated osteogenesis and anti-osteoclastogenesis. Strontium has been demonstrated to play a key role in inhibiting osteoclastogenesis or the bone resorption activity by inducing apoptosis in osteoclast cells. Therefore, the effect of strontium released from bioactive glass nanofibers on the differentiation and maturation of RAW264.7 macrophage cells into multinucleated osteoclasts was investigated. To examine the influence of Sr2+ on the osteoclast activity, we performed the TRAP assay, a marker for osteoclast differentiation and bone resorbing activity. In particular, we compared and quantified the number of TRAP positive macrophages derived from RAW264.7 cells treated by lipopolysaccharide (LPS) and the extracted solution from Sr-doped bioactive glass nanofibers.47 Figure 5 shows that all the groups containing either released Sr2+ or free soluble Sr2+ (as a metal salt) showed an inhibition on the osteoclast activity, while the undoped bioactive glass electrospun fibers showed no significant 14 ACS Paragon Plus Environment

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difference from the control group (cell culture medium). On day 2, the 50% Sr doped bioactive glass fibers maximally inhibited osteoclast activity, while the glass fibers co-doped Cu and Sr were more effective on day 4. A plausible explanation for the antagonistic effect of Cu on osteoclast activity could arise from its role in strengthening osteoblast activity via collagen crosslinking by lysyl oxidase, a copper-dependent enzyme.48 Overall, our results are in line with previous reports, which demonstrated the reduction in osteoclast formation by strontium ions or strontium ranelate.16, 19 Furthermore, few studies have elucidated the inhibitory effect of Sr2+ on bone resorption by the quantification of erosion pits created by osteoclast cells on CaP coated surfaces.19 The molecular basis for such an anti-osteoclastogenic effect elicited by strontium is centered around the receptor activator NF-κB ligand (RANKL) signaling pathway. Strontium has been implicated in promoting the binding of osteoprotegerin (OPG) to RANKL and prevent it from binding to RANK.49 Thus, Sr2+ is a key player in bone remodeling and homeostasis by its direct influence on the RANK/RANKL/OPG system. However, a moderately high concentration of Sr2+ is necessary for eliciting such anti-osteoclastogenic effects. In a previous study, the low concentration (of the order of 200 ppb) of Sr2+ released from β-tricalcium phosphate (Sr-doped β-TCP) based bioceramics could not deter the osteoclastogenesis of RAW 264.7 mononuclear cells into mature osteoclasts.50 On the contrary, the Sr2+ levels in the extraction media from the doped bioactive glass nanofibers in the current study are sufficiently high (of the order of 0.3-0.4 mM) to significantly hinder osteoclast activity. The osteoinductive effect of strontium on osteoprogenitor cells or mesenchymal stem cells has been more profoundly reported in literature as compared to the inhibition of osteoclast differentiation. To examine the effect of doped bioactive glass nanofibers on the osteogenesis, we cultured ADSCs with osteogenic supplemented media containing bioactive glass nanofibers extraction solutions for 15 days. We evaluated ALP activity and also performed ARS staining to detect the extracellular calcium deposition of the differentiating ADSCs. In particular, we quantified ALP (a hydrolase enzyme responsible for removing phosphate groups) activity of ADSCs after incubation with osteogenic media in the presence of bioactive glass nanofibers extracted solutions for 5 days and 10 days. We observed enhanced enzymatic activity of ALP in ADSCs treated with the Sr2+ containing extraction media for the all groups (Figure 6A), among which the addition of Sr2+ almost tripled the ALP activity. Meanwhile, Cu and Sr co-doped bioactive glass fibers doubled the ALP activity on days 5. However, the enhancing effect was 15 ACS Paragon Plus Environment


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diminished on days 10. This temporal trend of an initial increase followed by a subsequent drop in ALP, an early stage osteogenic marker in characteristic of the ALP dynamics that occur during bone formation.51 The temporal changes in ALP activity can be rationalized from the functional role of ALP, which converts pyrophosphate into inorganic phosphate in the bone and sets the stage for mineralization via the calcium deposition by the maturing osteoblasts. The calcium mineral deposits formed by the differentiating ADSCs were detected by ARS staining. Figure 6B and Figure 6C show that the Cu and Sr doped fiber groups significantly enhanced mineralization, which could be attributed to the effect of various ions including Sr, Cu, Ca, and Si. All the groups containing Sr2+ showed a distinct effect, including the free Sr2+ ion group. In addition, electrospun bioactive glass fibers co-doped with Cu and Sr had an enhancing effect on ARS staining, in particular for 0.5% Cu-doped bioactive glass fibers. As mentioned earlier, Cu is implicated in bone anabolism through its strengthening role via crosslinking of bone collagen by lysyl oxidase, a copper dependent enzyme.48 Similarly, the mechanism of the anabolic effect of strontium on bone formation was elucidated to involve the Ras/MAPK signaling pathway in that strontium increased the phosphorylation of mitogen-activated protein kinase (MAPK) as well as activated the Rat Sarcoma viral oncogene (Ras), which is an important upstream regulator of extracellular signal regulated kinase (ERK1/2) and p38, that in turn control the downstream transcription of Runx2,52 an early bone marker intranuclear protein. In addition to osteogenic effects, strontium released from bioactive glasses have also been reported to bring about antibacterial effects against oral pathogens such as P. ginigivalis, presenting them as excellent dental restorative materials.53 Altogether, strontium doped bioactive glass nanofibers suppressed osteoclastogenesis and promoted osteogenesis indicating its potential for use in bone and dental tissue engineering. However, the brittle nature of bioactive glass fibers makes them unsuitable for tissue engineering of load bearing bones. To address this shortcoming, nanocomposites of collagen with sol-gel derived bioactive glass nanofibers and mesoporous bioactive glass nanoparticles were fabricated to form stiff hydrogels, while promoting osteogenesis of human osteoblastic cells and mesenchymal stem cells (MSCs) from rats, respectively.54, 55

3.4.2. Copper induced angiogenesis and vasculogenesis. The successful vascularization or sprouting of blood vessels in the neobone is the key to bone regeneration. While mesoporous 16 ACS Paragon Plus Environment

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bioactive glasses (MBG) are excellent osteoinductive biomaterials by enhanced release of bioactive ions, the mesopores in the glass scaffolds are too small to support blood capillaries in the developing bone. One strategy to overcome this shortcoming is to engineer the pore size to macropores and another method is the release of pro-angiogenic factors essential for vascularization.56 We resorted to the latter method by doping with copper to confer angiogenic potential to bioactive glass nanofibers. It is well known that HUVECs can form blood vessels and capillaries when cultured at a high density

57

and Cu2+ promotes angiogenesis.21,

58-60

To

evaluate the effect of copper released from bioactive glass nanofibers on vascularization, we measured the length of tubes and number of nodes formed by HUVECs as reported in published literature.61 Figure 7(C1-C6) presents the typical images showing the formation of tubes and nodes/ branches/ meshes formed by HUVECs upon treatment with the designated extraction media from the various groups. The statistics of tube length and nodal density were presented in Figure 7A, indicating that Cu-doped bioactive glass nanofibers and free Cu2+ significantly increased the number of nodes/ branches and tube length formed by HUVECs cultured on Matrigel compared to the control and undoped bioactive glass nanofibers. These results confirmed that the released Cu2+ from doped bioactive glass nanofibers has a positive effect on the vascularization. Vascular endothelial growth factor (VEGF) is one of the most potent signaling proteins produced by cells that stimulates vasculogenesis and angiogenesis.62 The functional role of VEGF lies in the restoration of the oxygen supply to ischemic tissues when blood circulation is inadequate such as in hypoxic conditions.63 We also quantified the VEGF secretion from hBMSCs after treatment with designated extraction media. Figure 7B shows that Cu-doped bioactive glass nanofibers promoted the secretion of VEGF from hBMSCs into the culture medium over time. On day 6, the amount of secreted VEGF from Cu-doped bioactive glass nanofibers were about 1.5 times higher than those of control and bioactive glass nanofibers without Cu doping. These results confirmed the expression of angiogenic factors induced by copper and differentiation of hBMSCs into vascular endothelial cells. In both HUVECs and hBMSCs, strontium did not influence angiogenesis/ vasculogenesis unlike the anabolic and catabolic effect of copper on ADSCs and RAW 264.7 cells, respectively. The mechanism of copper induced angiogenesis was discerned recently. Both, copper and hypoxia can trigger hypoxia-inducible factor-1 (HIF-1) element, a transcription factor that controls the expression of 17 ACS Paragon Plus Environment


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VEGF.64 However, excess copper can hinder the formation of HIF-1 transcriptional complex and consequent VEGF production.65 On the other hand, copper has also been reported to be a limiting factor for tumor growth due to its ability to promote blood vasculature and proliferation of cancer cells within solid tumors.66 Therefore, the concentration of bioavailable copper is critical for the function it performs in biological systems. In this light, we demonstrate the controlled release of copper from doped bioactive glass fibers in therapeutically relevant quantities, which is a pre-requisite for the clinical application of copper based biomaterials in promoting vascularization of neobone. The supplementation of copper may also be potent in treating angiogenic pathologic conditions such as ischemia, cardiac hypertrophy and diabetic wounds.67 The reported effective concentration of Cu2+ for stimulating in vitro vascularization of HUVECs was around 100 µM.68 Wu et al.

69

studied the use of Cu doped bioactive glasses to

stimulate HUVECs, where they reported that 14.2 µg/ml of Cu2+ was the critical concentration that induced a positive angiogenic effect. However, Kong et al.

59

reported that 1.5 µg/ml Cu2+

rendered by Cu doped CaSiO3 was optimal, and a higher concentration inhibited the proliferation of HUVECs. In their study, Cu and Si synergistically promoted the expression of angiogenic growth factors, and the effective copper level was not necessary to be as high as free Cu2+ ion concentration. Summarizing, we tailored the ratio of copper and strontium in doped bioactive glass nanofibers, which effectively induced osteogenesis and inhibited osteoclastogenesis as well as encouraged angiogenesis and vascularization without any signatures of cytotoxicity. Our study illustrated that the incorporation of elements with complementary functions into bioactive glass nanofibers could pave the way for the design of multifunctional biomaterials.

4. CONCLUSIONS In summary, we have demonstrated the fabrication of Sr and/or Cu doped bioactive glass nanofibers by electrospinning followed by a post-processing heat treatment to remove the polymer. The Sr-doped glass nanofibers exhibited accelerated apatite crystal formation on their surfaces when immersed in SBF, as compared to the Cu-doped counterparts. The ion release kinetics show the sustained release of Cu2+, Sr2+, and Si4+ over an experimental period of 30 days. The ion release profiles of the dopants in the bioactive glass nanofibers can be finely tuned in terms of the Cu and Sr content. Importantly, Sr dopant significantly enhanced osteogenesis 18 ACS Paragon Plus Environment

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and suppressed osteoclastogenesis, and meanwhile, Cu dopant promoted the angiogenesis as testified by in vitro cell culture studies using multiple cell lines. In order to improve the mechanical property and cellular infiltration into the nanofibers, we will generate 3D hybrid scaffolds composed of doped bioactive glass nanofibers and polymeric nanofibers for bone regeneration in our future study. Such hybrid scaffolds with multifunctional elements performing complementary functions is a promising direction towards the development of tissue replacements.

ASSOCIATED CONTENT Supporting Information Elemental mapping, line scans and point probe EDX analysis of bioactive glass nanofibers in Figure S1, S2 and S3, respectively; XRD patterns of bioactive glass fibers in Figure S4; SEM image of Ca bioactive glass fiber after 15 days of immersion in water as Figure S5; SEM images of glass fibers after ion extraction in DMEM for 30 days as Figure S6.

AUTHOR INFORMATION Corresponding authors. *

E-mail addresses: [email protected] (X. Li), [email protected] (J. Xie)

ACKNOWLEDGMENTS This work was supported partially from startup funds from University of Nebraska Medical Center (UNMC) and UNMC Regenerative Medicine Program pilot project grant number 371209-2004-007.

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Figure 1. (A): Schematic illustrating the experimental setup. (B-E): SEM images of different bioactive glass electrospun fibers. B: undoped bioactive glass nanofibers; C: 50% Sr doped bioactive glass nanofibers; D: 0.5% Cu and50% Sr co-doped bioactive glass nanofibers; E: 1% Cu and 50% Sr co-doped bioactive glass nanofibers. Scale bar = 2 µm.



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Figure 2. SEM images of the biomineralization of different bioactive glass nanofibers in SBF at 37 ˚C for different times. A1-A4: undoped bioactive glass nanofibers; B1-B4: 50% Sr doped bioactive glass nanofibers; C1-C4: 0.5% Cu and 50% Sr co-doped bioactive glass nanofibers; D1-D4: 1% Cu and 50% Sr co-doped bioactive glass nanofibers. Scale bar = 4 µm.


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Figure 3. Cumulative release of various ions and the change of Ca2+ concentration from Cu and Sr co-doped bioactive glass nanofibers in DMEM at 37 ˚C over time. (● 1% Cu and 50% Sr doped bioactive glass nanofibers; ■ 0.5% Cu and 50% Sr doped bioactive glass nanofibers). Values represent means ± SD.



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Figure 4. MTT assay for different cells cultured with designated culture media at different times. S1/C1- Cell culture medium (negative control), S2/C2 – Extraction medium from 1% Cu and 50% Sr doped bioactive glass nanofibers, S3/C3 – Extraction medium from 0.5% Cu and 50% Sr doped bioactive glass nanofibers, S4/C4 – Extraction medium from 50% Sr doped bioactive glass nanofibers, S5 – 1 mM Sr2+ and C5 (2/3) – 0.01/ 0.001 mM Cu2+ (positive control), S6 – Extraction medium from undoped/ Ca bioactive glass nanofibers. Values represent means ± SD. *p

Binary Doping of Strontium and Copper Enhancing Osteogenesis and

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