Porous Bioactive Nanofibers via Cryogenic Solution Blow Spinning

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Porous Bioactive Nanofibres via Cryogenic Solution Blow Spinning and their Formation into 3D Macroporous Scaffolds Eudes Leonnan Gomes de Medeiros, Ana Letícia Braz, Isaque Jeronimo Porto, Angelika Menner, Alexander Bismarck, Aldo R. Boccaccini, William C Lepry, Showan N Nazhat, Eliton S Medeiros, and Jonny J. Blaker ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00072 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 21, 2016

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ACS Biomaterials Science & Engineering

Porous Bioactive Nanofibers via Cryogenic Solution Blow Spinning and their Formation into 3D Macroporous Scaffolds

Eudes Leonnan G. Medeiros1, Ana Letícia Braz1, Isaque Jerônimo Porto1, Angelika Menner2, Alexander Bismarck2, Aldo R. Boccaccini3, William C. Lepry4, Showan N. Nazhat4, Eliton S. Medeiros*1, Jonny J. Blaker5*

1*

Materials and Biosystems Laboratory (LAMAB), Department of Materials Engineering

(DEMat), Federal University of Paraíba (UFPB), CEP58051-900, João Pessoa- PB, Brazil 2

Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research,

Faculty of Chemistry, University of Vienna, Währingerstr. 42, A-1090 Vienna, Austria 3

Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-

Nuremberg, 91058 Erlangen, Germany 4

Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada

5*

School of Materials, MSS Tower, Manchester University, Manchester, M13 9PL, UK

Corresponding Authors: *[email protected], *[email protected]

Abstract There is increasing focus on the development of bioactive scaffolds for tissues engineering and regenerative medicine that mimic the native nanofibrillar extracellular matrix. Solution blow spinning (SBS) is a rapid, simple technique that produces nanofibers with open fiber networks for enhanced cell infiltration. In this work highly porous bioactive fibers were produced by combining SBS with thermally induced phase separation (TIPS). Fibers composed of poly(D,L-lactide) and dimethyl carbonate were sprayed directly into a cryogenic environment and subsequently ACS Paragon Plus Environment

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lyophilized, rendering them highly porous. The surface areas of porous fibers were an order of magnitude higher in comparison to smooth control fibers of the same diameter (43.5 m2·g-1 for porous fibers produced from 15 m/v % PLA in dimethylcarbonate) and exhibited elongated surface pores. Macroporous scaffolds were produced by spraying water droplets simultaneously with fiber formation, creating a network of fibers and ice micro-spheres, which act as in situ macroporosifiers. Subsequent lyophilization resulted in three-dimensional scaffolds formed of porous nanofibers, with interconnected macropores due to the presence of the ice spheres. Nano-bioactive glass (nBG) was incorporated into for the production of 3D macroporous bioactive, therapeutic ion releasing scaffolds with potential applications in non-load bearing bone tissue engineering. The bioactive characteristics of the fibers were assessed, in vitro, through immersion in simulated body fluid (SBF). The release of soluble silica ions was faster for the porous fibers within the first 24 h, with hydroxyapatite confirmed on the fiber surface within 84 h.

Keywords: Solution blow spinning; Nanofibers; Bioactive composite; Polylactide; Thermally induced phase separation

1. Introduction While electrospun scaffolds are widely used in tissue engineering, they inherently suffer from poor cell infiltration.1,2 Densely arranged fiber architecture and small inter fiber spaces prevent threedimensional integration with host tissue, and present significant challenges to clinical translation. Several approaches have been used to improve the inter fiber spacing of electrospun scaffolds, including the use of in situ porosifiers such as salt3, ice4, dissolvable polymer phases1, as well as photopatterning,5 (recently reviewed2,6). An alternative method to produce nanofibers with a more open pore structure is solution blow spinning (SBS).7-9 SBS produces nanofibers, analogous to those electrospun from polymers dissolved in suitable solvents, yet with a production rate circa 100 times more rapid than electrospinning and without the need for electric fields.7 SBS uses a system of

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concentric nozzles with special geometry through which a polymer solution is blown to form microand nanofibers, using pressurised gas.7,10 Process variables such as polymer injection rate and concentration, gas pressure and temperature, and nozzle geometry are of key importance to produce and control fiber diameter and overall morphology.10 The technique has been used to spray polylactide-co-glycolide (PLGA) nanofibers (precursor at 10 w/v % in acetone) directly onto tissues via a commercially available airbrush and has applications as surgical sealant, surgical hemostat and tissue reconstruction.11 Recently, SBS has been used for the production of cellulosic and ceramic fibers for several structural and functional applications, such as microfiltration,12 solar cells, catalysis, and high temperature structural materials.13-16 Porous nanofibers are of interest to tissue engineering due to their enhanced surface area and surface roughness, with potential for the active substance encapsulation. Several techniques have been developed to render electrospun nanofibers porous based on phase separation within the fibers. Such fibers have been created by electrospinning into cryogenic liquid, resulting in thermally induced phase separation (TIPS) within the fibers.17 Porous fibers have also been generated via electrospinning using highly volatile solvents,18 as well as polymer blends followed by selective dissolution.19 Vapour induced phase separation (VIPS) can also be used to generate porous fibers, using humidity and non-solvent/solvent combinations .20-22 Polymers can be rendered bioactive by introducing therapeutic ion releasing glasses or ceramics such as hydroxyapatite (HA), as reviewed.23,24 Bioactive nanoparticles, including flame sprayed bioglass and aerosol derived amorphous tricalcium phosphate (TCP) have been incorporated into PLGA solvent solutions, and electrospun into fibers, with the surfactant Tween 20 used to aid particle dispersion.25,26 The removal of Tween has been reported to result in the presence of small pores at the fiber surface during in vitro testing.26 A commercial airbrush style set up has been used to spray HA nanoparticle loaded PLGA interconnected fiber membranes using a gravity driven feed cup system, the presence of residual dichloromethane as solvent resulted in the conjoining of fibers and membrane formation.27


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In this work we have developed a method to rapidly produce porous bioactive nanofibers by combining SBS7 and TIPS28. We also address the challenge to produce interconnected macropores within 3D nanofiber networks. Ice microspheres are used as in situ macroporosifiers, which become entangled with the nanofibers during processing. Subsequent compression of these ice/nanofiber entanglements and lyophilization, results in bioactive interconnected macroporous scaffolds, composed of porous composite nanofibers.

2. Materials and Methods 2.1 Materials Amorphous poly(D,L-lacide) (PLA 4060D, PDLLA, of Mw 94 kg·mol-1) was obtained from Jamplast Inc. (MO, USA). Dimethyl carbonate (DMC) was used as solvent (Sigma-Aldrich, UK). DMC has a melting point of 3 °C and is facile to freeze-dry. Flame sprayed nano-bioactive glass (nBG) particles29 of the same composition as 45S5 Bioglass®, with a size range 20-50 nm diameter were kindly donated by Prof. Wendelin Stark, ETH Zurich, Switzerland.

2.2 Preparation of Polymer/Composite Solutions for SBS PDLLA was dissolved in DMC at a polymer to solvent ratio of 20 m/v % and serially diluted to 15, 12, 10, 8 and 4 m/v % polymer solutions (equivalent to 16.0, 12.0, 9.6, 8 and 3.2 v/v %, respectively). nBG particles were added to the 15 m/v % polymer solution to produce composites at 5 and 10 wt % loadings in PDLLA (nominally 5nBG and 10nBG, respectively). Nanoparticle agglomerates were broken up using a pestle and mortar, mixed into the polymer solutions and ultrasonicated for 40 min to aid dispersion immediately prior to spinning. The tendency for particle sedimentation was reduced by selecting the more viscous 15 m/v% polymer solution, mixing in a 5 ml syringe by drawing in the mixture multiple (circa 3) times and spun immediately. Thermogravimetric analysis confirmed the target loading in the lyophilized fibres (SI).

2.3 Smooth and Porous Fiber Production via SBS/Cryogenic SBS ACS Paragon Plus Environment

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Porous fibers were prepared by spraying polymer/composite precursor solutions via the SBS head into a bath of liquid nitrogen (Figure 1a), with a working distance (10 cm) selected to limit solvent evaporation ahead of freezing and TIPS.17,28 The solvent phase was then subsequently removed by lyophilization to yield highly porous fibers. Smooth fibers were produced from the same precursor solutions via conventional SBS (Figure 1b), with a working distance sufficient to entirely evaporate the solvent (25 cm) to a rotating collector (spinning at 600 rpm). Polymer/composite solution injection rates were initially varied between 100-200 µL·min-1, and later fixed to 120 µL·min-1, delivered via a precision syringe driver (N-300 New Era Pump Systems, Inc., NY, USA) to the inner coax of the SBS head, surrounded by the high-pressure air sheath.7 The nozzle configuration is identical to that described in the original SBS paper by Medeiros et al.7 Dry compressed air was delivered to the SBS head using a compressor (model: AZ100 40VPF, AirZap, São Paulo, Brazil), equipped with an inline desiccator. Air pressure was initially varied between 10-40 psi (69-276 kPa), and later fixed to 20 psi (138 kPa), as this resulted in a stable stream of fibers (at 15 m/v % concentration, defined above) and prevented excessive liquid nitrogen loss. During cryogenic-SBS the level of liquid nitrogen in the centrifugal bath was maintained to ensure the working distance remained within 2 cm of the target 10 cm. A thermostatic probe was use to determine this level. The temperature at the tip of the SBS head, where fiber formation occurs was measured at 13.3 °C, suitably above the melting point of DMC. Frozen fiber membranes were collected from the liquid nitrogen surface, placed into pre-cooled 50 ml polypropylene FalconTM tubes, then transferred to lyophilization flasks (pre-cooled to -80 °C) and freeze-dried (using a Liofilizador Enterprise II, Terroni Ltd, São Paulo, Brazil).

2.4 Bioactive macroporous scaffold production via in situ ice microsphere/cryogenic fiber spraying Macroporous structures were generated by simultaneously spraying water and either neat polymer, or bioactive particle containing PDLLA fibers via separate SBS heads (Figure 2a), both directed to the same point in the cryogenic bath. The sprayed water droplets froze proximal to the surface of the ACS Paragon Plus Environment

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liquid nitrogen surface, forming an entanglement of ice spheres and frozen fibers. Different water injection rates and air pressures applied to the water SBS head allowed for the volume fraction of the ice spheres (eventual in situ macro-porosifiers) to be controlled. The size of the ice microspheres and their volume fraction relative to the fibers was adjusted by altering the air pressure applied to the water head (between 2-10 psi) (14-69 kPa) and water flux between 340-440 µL·min-1 (polymer solution flux fixed at 120 µL·min-1) in an effort to generate scaffolds with 74-79% volume of ice spheres (macropores) in the scaffolds. The entangled ice sphere/fiber networks were collected and lyophilized (as described in 2.3), resulting in cotton wool-like constructs (Figure 3b), or compressed in pre-cooled metal moulds and lyophilized to form macroporous scaffolds (Figure 3c). The macroporous scaffolds are themselves formed of bioactive porous nanofibers, the macroporous network being a negative 3-D fingerprint of where the ice spheres once were. The amount of macroporosity due to the ice in the entangled fiber network was determined by measuring the difference between injected volume and residual water remaining in the centrifugal collector, relative to the total volume of ice and frozen fibers. Cylindrical scaffolds were sliced using sharp doubleedged razor blades for subsequent analysis.

2.5 Fiber and scaffold physical characterisation Fiber morphology was assessed using scanning electron microscopy (SEM, FEI Quanta 200 FEG, UK). Samples were gold sputter coated and observed using an accelerating voltage of 15-20 kV and spot size of 1-3 to prevent sample deformation. Fiber diameter was determined using the software ImageJ (Version 1.48, NIH, USA), with 100 measurements by sample type. BET surface area measurements of the fibers were made using a surface area and porosity analyser (TriStar II, Micromeritics, Neupurkendorf, A). Prior to the gas adsorption experiments, impurities were removed via a ‘degassing’ step using a FlowPrep 060 (Micromeritics, Neupurkendorf, A). Approximately 0.2 mg (about 0.1 cm3) of each sample was placed inside a glass sample chambers and purged with nitrogen at ambient temperature overnight. Afterwards, the nitrogen adsorption isotherms were measured at 77 K. Apparent water-in-air contact angles were measured on the fiber surfaces using a ACS Paragon Plus Environment

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DSA 100 goniometer (Krüss, Germany). Fiber membranes were carefully adhered to glass microscope slides using double-sided tape, water droplets of 20 µL were added via a motorized syringe pump, 6 drops per sample type were analysed. The bioactive nanoparticle loading in the PDLLA fibers was confirmed using thermogravimetric analysis (TGA), where a heating regime room temperature to 550 °C at a rate of 5 °C·min-1 was applied in an air atmosphere. Starting sample mass was 15 ± 2 mg.

2.6 In vitro apatite formation and ion release Membranes of the porous bioactive glass filled fibers were immersed in simulated body fluid (SBF), 30

according a previously reported protocol31 at a fiber weight to SBF volume of 0.5 mg·mL-1. Fiber

membranes were kept submersed in 5 mL volumes of SBF, by trapping them in cuts ends of disposable Pasteur pipettes, interference fitted in 15 mL Falcon™ tubes. Three samples per time point (2, 12, 36, 84 and 180 h) were incubated individually in SBF, and maintained at 37 °C under tangential agitation of 175 rpm using an orbital shaker (Tecnal TE424, SP, Brazil). At each time point, aliquots of SBF were taken, and the fibers rinsed three times with deionised H2O and then dried in a vacuum desiccator. The concentration of soluble silica in SBF was quantified using inductively coupled plasma–optical emission spectrophotometry (ICP-OES, Thermo Scientific iCAP 6500, USA). Aliquots of the dissolved ions were stored in a 15 ml falcon tube to which 4 m/v % nitric acid (Fisher Scientific, Canada) was added. Values were calibrated against certified NIST standards serially diluted with deionized water. The formation of HA on the fibres assessed via X-ray diffraction (XRD), SEM and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. XRD analysis was conducted using a Bruker D8 Discover theta-theta diffractometer with a LynxEye detector in Bragg-Brentano geometry employing Cu Kα radiation (Kα1=1.54060 Å, Kα2=1.54439 Å, Kα ratio) 0.2 mm incident slits and 2.5° receiving Soller slits. The sample was located on a silicon low background holder and a data collection from 5 to 85° coupled 2theta/theta at 0.02° step 10 s/step was undertaken. ATR-FTIR was conducted on control and vacuum dried


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fibers previously immersed simulated body fluids, using a Nicolet 5700 FTIR Spectrometer (Thermo Electron Corporation, USA), resolution was set to 4 cm-1 and four repeat scans were performed.

3. Results and Discussion 3.1 Porous fibers via cryogenic-SBS Solutions at 15 m/v % polymer to solvent ratio formed a stable spray of fibers (at flux 120 µL·min-1, air pressure 20 psi (139 kPa), whilst 20 m/v % solutions could form fibers, the nozzle blocked frequently due to increased viscosity and solvent evaporation. Polymer solutions at < 4 m/v % concentration resulted exclusively in droplet spray. Fiber formation was more prominent in > 10 m/v % solutions (Figures 1c, and 2). The working distance of 10 cm (SBS nozzle to liquid nitrogen surface) was deemed suitable as exposure of the frozen fibers to room temperature resulted in their dissolution, ergo sufficient solvent was present to cause phase separation in the fibers upon exposure to liquid nitrogen. The fibers were observed to float on the surface of the liquid nitrogen, and form a continuous network of non-interconnected fibers, the rapid freezing prevented the fibers conjoining. Lyophilization resulted in cotton-wool-like constructs (Figure 2b). The structure of these fibers differs from those produced by solvent extraction, which can damage the porous structure and cause fibers to conjoin.19 SEM images of the lyophilized membranes produced here demonstrated porous fibers, characterized by elongated surface pores (along the fibre axis) (Figure 2a-b, Figure S1). It is likely that in addition to TIPS, VIPS concomitantly also contributed to pore formation.20-22 However, it was observed that fibre removal from liquid nitrogen prior to lyophilization, to ambient conditions resulted in rapid fibre dissolution. The diameter of the porous fibers decreased concomitantly with lower polymer concentration (Figure 1c), from 412 ± 157 nm (15 m/v %) to 99 ± 20 nm (8 m/v %). The size of the elongated surface pores related to the fiber diameter, the 12 m/v % fibers having surface pore major and minor axes of 82 ± 15 nm and 29 ± 4 nm for 236 ± 16 µm (dia.), and 65 ± 13 nm and 21 ± 2 nm for 151 ± 3 µm (dia.), both, respectively. The surface area of the fibers was approximately an order of magnitude higher in comparison to the smooth fibers, (Figure 1c), and


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increased with decreasing polymer concentration. The surface area of these fibers is far greater than porous poly(acrylonitrile) fibers prepared by electrospinning a solution (at 7 m/v % in dimethylformamide) into liquid nitrogen followed by drying in vacuo.17 McCann et al., reported specific surface areas of 1 µm diameter fibers to be 9.497 m2·g-1,17 in line with the results in the current work, whereby the porous 15 m/v % fibers have a specific surface area of 43.45 m2·g-1. Highly porous spheres were obtained on lyophilization of the frozen spray droplets obtained from the 4 m/v % polymer solution (Figure 1d, and Figure S1), the spheres have a mean diameter of 19.9 ± 17 µm, the maximum observed size was 75 µm and the smallest ~ 500 nm. An increasing prevalence of porous spheres entangled in nanofiber networks was evident at polymer concentrations lower than 10 m/v % (Figure 2c). The surface area of the fibers at concentrations < 10 m/v % is likely to be higher than reported here due to the presence of the porous microspheres. Porous fiber membranes exhibited higher apparent water contact angles for all concentrations compared to smooth fiber membranes (Figure S2), to 140 ± 9° from 132 ± 3° for fibers at 12 m/v % concentration, substantially higher than neat PDLLA films measured at 78 ± 3°. This is consistent with porous poly(vinylidene fluoride) (PVDF) fibers were produced via cryogenic electrospinning, giving apparent water-in-air contact angles of 135°, compared to smooth fibers at 130°, and 110° for a film of PVDF.17 The presence of pores (air) within the nanofibers and between the fibers acts to cause Cassie-Baxter effects.32 The 15 m/v % polymer solution was selected for incorporation of bioactive glass nanoparticles due to its spinnability and viscosity, which resulted in less particle sedimentation. TGA revealed successful bioactive glass incorporation within the porous fibers at the desired loadings (Figure S3).

3.2 Macroporous scaffolds formed of bioactive/porous fibers Loose (cotton-wool-like) macroporous scaffolds could be formed without any compaction of the ice sphere/entangled frozen fiber network (Figure 3b). Water injected at a flux of 340 µL·min-1 and sprayed using an air pressure of 3 psi (20.7 kPa) provided ice spheres in the range 30 µm - 2 mm (based on SEM/image analysis of the macropores in the lyophilized scaffold; histograms shown in


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Figure S4). Pore throats (interconnects) ~200 µm in diameter in the porous nanofibre network were observed. The macroporosity due to the ice spheres was back calculated to 67.3 ± 3.0 %, however the total porosity of the scaffold shown in Figure 3c was 97.5 ± 1.1% (obtained by simple mass/volume measurement, detailed in the supplementary information). This high porosity was due to the presence of porous/hollow fibers along with inter-fiber voids. The macroporosities due to the ice microspheres were calculated for scaffolds with target macroporosities of 77%, and 79% at 65.8 ± 2%, and 66.2 ± 2%, respectively. These lower values are due to the ice sedimentation from the fiber/ice entanglement at higher water spray fluxes. The scaffold shown in Figure 3c was produced using water at a flux of 340 µL·min-1 and air pressure of 10 psi (690 kPa) that resulted in smaller macropore sizes, 102 ± 15 µm and interconnects of 35 ± 11 µm (comparison of macrosphere size as function of water flux is shown in Figure S4). During compaction of the frozen fibers/ice spheres the ice spheres may conjoin and neck under pressure resulting in interconnections. The structures formed differ

from

those

of

Hild

et

al.,

they

dissolved

PLGA

at

0.083

g·mL-1

(tetrahydrofuran:dimethylformamide ratio of 1:3) with 3 wt % Tween (to the PLGA weight), nBG loadings up to 30 wt %. The dispersion was fed at 50 mL·min-1 and electrospun fibers were collected on a cylinder filled with dry ice to encourage ice crystals deposition, with a working distance of 15 cm, and then dried under vacuum at room temperature.25 The technique developed in the current work allows control over the volume of macropores relative to fibers by independent adjustment to of both polymer solution and water flux, as well as some control over macropore size by adjusting the air pressure, unlike the methods previously described which relied on control over humidity to deposit ice crystals on the collector.4,25 Ice particles (spheres) can be sprayed in situ, rather than generating them separately via ultrasonic nebulizers33 or expensive capillary needles (Eppendorf Femtotips)31 and then mixing sieved microspheres with polymer solutions in a separate steps, and requiring solvent exchange at controlled temperatures and lyophilization to produce the scaffolds.30 Ice microparticles produced via capillary tubes at 80-95 wt % with respect to the PLA have been produced, however the surface area of their scaffolds at the highest loading was 0.574 m2·g-1, far below the constituent fibres porous nanofibers produced here.34 ACS Paragon Plus Environment

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Dimethyl carbonate as solvent could not be used due to its melting point at of 3 °C, which would not enable the ice particles to be mixed in directly.33 In the current work these issues were avoided as the nanofibres and water spheres freeze together and form an entangled membrane on the surface of the liquid nitrogen. Both dimethyl carbonate and water are removed in the same freeze-drying step, resulting in porous nanofiber macroporous scaffolds. Macroporous electrospun scaffolds have been created using a low-temperature rotational fiber collector filled with dry ice.4 By controlling humidity the simultaneous deposition of fibers (PLGA and poly(ester-urethane) in chloroform) and ice crystal codeposition from condensing humidity, which acted as a pore template.4 Whereas in this work, well formed ice microspheres formed an interconnected network with the nanofibers directly on the surface of the liquid nitrogen.

3.3 In vitro apatite formation and ion release in SBF All composite samples exhibited a rapid release of soluble silica within the first 36 h (Figure 4a), followed by stabilization, correlating to the nBG loadings in the fibers. ATR-FTIR spectra for the bioactive glass containing fiber samples exhibited the characteristic double bands at 559 and 600 cm1

, associated with the P-O bond from the calcium orthophosphate after 84 h incubation in SBF

(Figure 4b).35 XRD and SEM indicate CaP formation (Figure 4c, and Figure S5 at 3.5 days), at later time point, 7.5 days immersion typical HA is evident (Figure 4d, and Figure S6), XRD shows this is predominantly HA, however other CaP phases may be present at concentrations below the detection limit of XRD (Figure S7). These results are consistent for the in vitro reactivity of nBG particles. 36

4. Conclusions The cryogenic SBS technique provides a simple and rapid route to produce porous nanofibers with surface areas an order of magnitude higher than smooth fibers of the same diameter. The use of multiple-nozzles enables ice microspheres to be sprayed in situ to fiber formation, acting as in situ porosifiers, and lyophilization result in 3D interconnected macroporous, nanofibrillar scaffolds of controlled porosity. The porous fibers are also rendered bioactive by inclusion of bioactive glass ACS Paragon Plus Environment

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nanoparticles in the precursor spinning solutions. The release of soluble silica ions is faster for the porous fibers in comparison to smooth fibers in the first 24 h of exposure to simulated body fluids and the development of hydroxyapatite is evident within 84 h. The ability to control the flux and air sheath pressures of the polymer solution and water (ice), enables control over eventual scaffold macroporosity.

5. References (1)

Wang, K.; Xu, M.; Zhu, M.; Su, H.; Wang, H.; Kong, D.; Wang, L. Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration. J. Biomed. Mater. Res., Part A 2013, 101, 3474-3481.

(2)

Zhong, A.; Zhang, Y.; Lim, T. K. Fabrication of Large Pores in Electrospun Nanofibrous Scaffolds for Cellular Infiltration: A Review. Tissue Eng., Part B 2012, 18, 77-87.

(3)

Kim, T. G.; Chung, H. J.; Park, T. G. Macroporous and nanofibrous hyaluronic acid/collagen hybrid scaffold fabricated by concurrent electrospinning and deposition/leaching of salt particles. Acta Biomater. 2008, 4, 1611-1619.

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Simonet, M.; Schneider, O. D.; Neuenschwander, P.; Stark, W. J. Ultraporous 3D polymer meshes by low-temperature electrospinning: Use of ice crystals as a removable void template. Polym. Eng. Sci. 2007, 47, 2020-2026.

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Sundararaghavan, H. G.; Metter, R. B.; Burdick J. A. Electrospun fibrous scaffolds with multiscale and photopatterned porosity. Macromol. Biosci., 2010, 10, 265-270.

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Sun, B.; Long, Y. Z.; Zhang, H. D.; Lia, M. M.; Duvaile, J. L.; Jiang, X. Y.; Yin, H. L. Advances in three-dimensional nanofibrous macrostructures via electrospinning. Prog. Polym. Sci. 2014, 39, 862-890.

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Medeiros, E. S.; Glenn, G. M.; Klamczynski, A. P.; Orts, W. J.; Mattoso, L. H. C. Solution blow spinning: A new method to produce micro- and nanofibers from polymer solutions. J. Appl. Polym. Sci. 2009, 113, 2322-2330.

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Tutak, W.; Sarkar, S.; Lin-Gibson, S.; Farooque, T. M.; Jyotsnendu, G.; Wang, D.; Kohn, J.; ACS Paragon Plus Environment

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Acknowledgements JB acknowledges support from CAPES and CNPq (Brazil), grant numbers PVE-18523129, and PVE 304898/2014-7, respectively. SNN, JB, and ESM acknowledge joint financial support from DFAIT (CBJRP 2013-14 CA-1 McGill University) and CAPES. Bioactive glass nanoparticles were kindly provided by Dr. Dirk Mohn and Prof. Wendelin Stark, Functional Materials Laboratory, ETH Zurich, Zurich, Switzerland. The Authors acknowledge Dr. Giovanna Machado, CETENE, Recife, for assistance with FEG-SEM.


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Supporting Information Available The following file is available free of charge, Cryogenic_SBS_SI, comprising: Further experimental detail for TGA; determination of macroporosity due to ice microspheres, additional images of porous fibers and high magnification of porous spheres, produced from 15, 10 and 4 m/v % PDLLA in DMS solutions (Figure S1a-c); results of apparent contact angle on smooth and porous fibres as function of starting polymer concentration (Figure S2); TGA results to confirm nanobioactive glass particle loading in (Figure S3); histograms showing macropore diameters as function of water flux and SBS air pressure (Figure S4); SEM image showing HA deposition on 10 nBG fibres after 3.5 days in SBF (Figure S5); SEM images showing typical HA morphology on the10 nBG fibres after 7.5 days in SBF at low and medium magnification (Figure S6), and XRD showing predominantly HA on this scaffold after 7.5 days in SBF.

Table of contents graphic:

Figure Captions Figure 1. Schematics of cryogenic- and conventional SBS (A and B, respectively); comparison of fiber diameter and surface area as a function of polymer concentration and porous/smooth fibers (C and D, respectively).


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Figure 2. SEM images of porous fibers/spheres produced via cryogenic SBS (A-D). Reducing solvent concentration results in smaller diameter fibers and at concentration 8 m/v % porous beads are observed in the fibre network. Porous spheres were produced at 4 m/v % (D). Figure 3. Schematic of the cryogenic SBS process with in situ spraying of ice microspheres, and subsequent compression into scaffolds with interconnected pores (A); cotton-wool-like structure produced without compression (B); scaffolds with large interconnected pores (C) (scaffold diameter is 16 mm, sectioned and opened, right image), and scaffold produced using small ice microspheres (D). Figure 4. Release profile of silicon in simulated body fluid (SBF) as a function of immersion time, for periods up to 7.5 days (A); ATR-FTIR spectra showing characteristic peaks associated with calcium orthophosphate (bands 600 cm-1 and 559 cm-1 associated with P-O-P bending mode found in HA, and P-O bond formation from calcium orthophosphate) after 84 h 3.5 days in SBF (B); XRD diffractographs showing characteristics peaks for HA after 84 h 3.5 days in SBF, compared to control fibers without and without bioactive glass prior to immersion (C), corresponding indices for HA labelled from left to right are (002), (210) and (211); and SEM image showing the typical morphology of HA on 10 nBG fibers after 7.5 days immersion in SBF (D).


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Figures:

Figure 1.


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Figure 2.


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Figure 3.


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Porous fibres loaded with bioactive glass 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15


Cotton-wool like Porous nanofibres

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1 μm


2 μm

Macroporous scaffolds: Porous nanofibres with ice as in situ porosifiers

Porous Bioactive Nanofibers via Cryogenic Solution Blow Spinning

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