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

2Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, Faculty of

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

3Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058

Erlangen, Germany

4Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada

5*Bio-/Active Materials Group, School of Materials, MSS Tower, Manchester University, Manchester, M13 9PL, UK

Corresponding Authors: *eliton@ct.ufpb.br, *jonny.blaker@manchester.ac.uk

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 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. 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.

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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 three-dimensional 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 concentric nozzles with special geometry through which a polymer

solution is blown to form micro-and 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

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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

In this work we have developed a method to rapidly produce porous bioactive nanofibers by combining SBS 7 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

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

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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 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

double-edged 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

4

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 cm 3)

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 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 maintained 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)

5

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

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, and Figure S1).

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

6

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 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 32c). 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

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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

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

8

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

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 cm -

1, 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

9

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

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.

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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

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(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.

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

14

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).

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 3.5 days in SBF (B); XRD

diffractographs showing characteristics peaks for HA after 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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