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DOI: 10.1002/marc.201900019

Communication

Rheological Characterization of Biomaterials Directs Additive

Manufacturing of Strontium-Substituted Bioactive Glass/Polycaprolactone

Microfibers a

Naomi C Paxton, Jiongyu Ren, Madison J Ainsworth, Anu K Solanki, Julian R Jones, Mark C

Allenby, Molly M Stevens, Maria A Woodruff*

–––––––––

N.C. Paxton, Dr J. Ren, M.J. Ainsworth, Dr M.C. Allenby, Prof M.A. Woodruff

Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology

(QUT), 60 Musk Ave, Kelvin Grove QLD 4059, Australia

E-mail: mia.woodruff@qut.edu.au

Dr A.K. Solanki, Prof J.R. Jones, Prof M.M. Stevens

Department of Materials, Department of Bioengineering and Institute of Biomedical

Engineering, Imperial College London, London SW7 2BP, United Kingdom

–––––––––

Additive manufacturing via melt electrowriting (MEW) can create ordered microfiber scaffolds

relevant for bone tissue engineering, however there remains limitations in the adoption of new

printing materials, especially in MEW of biomaterials. For example, while promising

composite formulations of polycaprolactone with strontium-substituted bioactive glass have

been processed into large or disordered fibres, to our knowledge, biologically-relevant

concentrations (>10wt%) have never been printed into ordered microfibers using MEW. In this

study, we used rheological characterization in combination with a predictive mathematical

model to optimize biomaterial formulations and MEW conditions required to extrude various

PCL and PCL/SrBG biomaterials to create ordered scaffolds. Previously, MEW printing of

PCL/SrBG composites with 33wt% glass required unachievable extrusion pressures. We

modified the composite formulation using an evaporable solvent to reduce viscosity 100-fold

a Supporting Information is available online from the Wiley Online Library or from the author.

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to fall within the predicted MEW pressure, temperature, and voltage tolerances, which enabled

printing. This study reports the first fabrication of reproducible, ordered high-content bioactive

glass microfiber scaffolds by applying predictive modeling.

FIGURE FOR ToC_ABSTRACT

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1. Introduction

Melt electrowriting (MEW) has been established as a versatile additive manufacturing

technique for biofabrication, tissue engineering and regenerative medicine research.1,2 MEW is

a specialized electrospinning platform which creates micro- and nano-scale fibers usually

collected as mats3 with high levels of control over fiber size,4,5 porosity,6 and fiber laydown

patterns.7,8 A high potential difference between the nozzle and collector plate instigates the

formation of a ‘Taylor cone’ at the base of the nozzle through complex interactions between

the polymer and strong electric field, drawing out micron-scale fibers. By controlling the

extrusion velocity and collector plate movements, complex scaffold geometries can be

fabricated layer-by-layer using this technique.1

Numerous polymers have been fabricated into scaffolds using MEW, with polycaprolactone

(PCL) being the most commonly reported polymer used for MEW optimization4,9–11 for bone,12

cartilage,13–15 periosteum,16 endosteum17 and cardiac tissue engineering.18 PCL has tailorable

biodegradability19–22 and a melting point of approximately 60°C, ideal for extrusion-based 3D

printing.23 However, PCL is hydrophobic, which limits cell attachment.24 By combining PCL

with other materials such as strontium substituted bioactive glass (SrBG), designer composites

which attempt to mimic the composition of natural bone are formulated to possess more suitable

biological and mechanical properties.23,25,26 SrBG was previously reported by incorporating the

highly bioactive components of Sr into bioactive glass formulations by substituting Sr in for

Ca.27,28 The resultant material offers exceptional bioactive properties, increasing alkaline

phosphatase activity in cells and promoting the proliferation of osteoblasts whilst inhibiting

osteoclast differentiation to induce rapid bone formation.29–31 This material has been optimised

for printability via incorporation with PCL to overcome the brittleness of SrBG alone.32

However in our experience, the bulk rheological properties of PCL/SrBG composites prevent

their extrusion via MEW into ordered scaffolds at SrBG concentrations above 10wt%.33–35 This

has motivated research to understand both the material properties governing printability11 and

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to provide a systematic framework to fabricate MEW scaffolds using novel, complex and

composite biomaterials.

The limited availability of functional biomaterials which can be processed and fabricated into

ordered micron-fiber structures using MEW has hindered the development of potential new

tissue substitutes. Since rheological behavior affects material extrusion rate,36,37 the aim of this

article is to systematically apply a predictive framework for characterizing the influence of PCL

and PCL/SrBG composite rheology on MEW extrusion behavior. This formulation framework

directs the first MEW extrusion of high bioglass-content micron-fiber scaffolds, and can be

applied to formulate and print future biomaterials.

2. Experimental Section

2.1. Materials

Poly(ɛ-caprolactone)Capa 6430 and Capa 6500 were supplied by Perstorp (Perstorp, Sweden).

PCL Capa 6430 (𝑀𝑤 = 37,000 𝐷𝑎) is commonly used for MEW.6,10,38 PCL Capa 6500 (𝑀𝑤 =

50,000 𝐷𝑎 was selected for incorporation into a composite with SrBG with a composition of

46.13 SiO2, 2.60 P2O5, 24.35 Na2O, 20.18 SrO, 6.73 CaO (mole %) processed into particles as

previously described.[28,37]

2.2. PCL/SrBG Composite Preparation

SrBG particles were ground for 8 h using a micronizing mill (McCrone, Westmont IL, USA)

according to previously reported methods32 and the particle size distribution was measured

using a laser particle size analyzer (Mastersizer 3000, Malvern, UK) (Supporting Information

1). Following grinding, the particles were freeze dried for 3 days. For composite preparation,

10% w/v PCL solution was prepared by dissolving 2 g PCL Capa 6500 pellets into 20 mL

chloroform (MERCK, Millipore, Australia). Under constant stirring, 1 g SrBG particles were

added to the solution and stirred for 8 h for a final concentration of 33wt% SrBG in PCL (2:1

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ratio of PCL to SrBG, optimized previously32). In order to fully dissolve the components, the

suspension was repeatedly ultra-sonicated during stirring. Finally, the solution was precipitated

into 200 mL absolute ethanol (MERCK, Millipore, Australia) and the precipitate was dried in

air at room temperature for 30 min.

2.3. Material Characterization

2.3.1. Thermogravimetric Analysis

Thermogravimetric Analysis (TGA) was performed under nitrogen on a Q500 TGA V6.5 Build

196 from TA Instruments to characterize material degradation behavior. Approximately 30 mg

of each sample was weighed and loaded onto a platinum measurement pan and subjected to a

temperature ramp of 10 °C/min from 20-800 °C.

2.3.2. Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) was conducted on a Q100 DSC V9.6 Build 290 from

TA Instruments under a constant nitrogen sample purge flow of 50 mL/min. The samples were

cycled between 20 °C and 200 °C at 10 °C/min and the melting point was determined at the

lowest peak of the second heating step.

2.3.3. Rheometry

Material viscoelastic properties were measured after heating to potential MEW operating

temperatures with a Physica MCR302 rheometer (Anton Paar, Graz, Austria) using a plate-plate

measurement system (PP25, SN44936, 0.5 mm plate gap). Rotational measurements were

conducted to mimic the extrusion process in which the polymer chains are naturally aligned

during extrusion. Shear viscosity measurements were performed between shear rates of 0.001

and 10 s-1 at 80 °C, 100 °C and 120 °C. Temperature sweeps were also conducted from 70-

150 °C at 1 s-1 shear rate.

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2.4. MEW Printing

Samples were loaded into 2 mL syringes (B. Braun, Australia) and melted in a heated jacket for

30 min within a custom built MEW device. Air pressure was applied to the top of the syringe

controlled by an air pressure regulator and motorized collector plates (Velmex, Bloomfield NY)

were controlled by Repetier Host software (Hot-World GmbH & Co. KG, Willich, Germany).

A 10 mm tip-to-collector plate distance was used for all scaffolds. Gcode governing the MEW

machine collector plate was written for 3 x 3 cm square crosshatch scaffolds with 1 mm fiber

spacing.

2.5. Scaffold Imaging

Scaffolds were imaged by light microscopy (Zeiss Axio Imager A2, Germany and Nikon Stereo

Microscope SMZ25, Tokyo, Japan). For scanning electron microscopy (SEM) and energy

dispersive spectrometry (EDS), samples were gold sputter-coated and imaged using a TESCAN

MIRA3 SEM (Brno, Czech Republic) with a back scattered electron detector for EDS imaging

contrast.

3. Results and Discussion

3.1. Material Characterization

The TGA results indicate that PCL degradation occurs above 250 °C for all studied materials,

therefore only lower temperatures are relevant for printing (Figure 1a). Furthermore, bioactive

glass does not melt within the operating limits of the instrument. Therefore the degradation

profile of the PCL/SrBG 33wt% composite plateaus to approximately 33% of the total sample

weight, comprised of residual SrBG. The melting temperatures of the two PCL polymers and

PCL/SrBG 33wt% were characterized by DSC, whereby PCL Capa 6500 demonstrated a

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melting point of 56°C, and its SrBG composite was shown to melt at 61 °C (Figure 1b).

Rheological measurements were performed between 70-150 °C to maintain the polymer in a

molten state, whilst not inducing degradation (Figure 1c, d).

From the temperature sweep, PCL decreases in viscosity with increasing temperature (Figure

1c). At 80 °C, 100 °C and 120 °C, very weak shear thinning of the PCL samples (near-

Newtonian behavior) was observed which increased once SrBG particles were added. These

shear-viscosity profiles were quantified using a Power Law regression of the form:

𝜂 = 𝐾�̇�𝑛−1 (1)

Where 𝜂 is the viscosity, �̇� is the shear rate, and n and K are coefficients fitted to each curve.

Figure 1. (a) Thermal degradation of samples during TGA heating ramp to 800 °C. (b) DSC

results show the melting point during 2nd heating cycle. (c) Temperature sweep rheometry

measurements performed at 1 s-1 shear rate characterize the decrease in viscosity of PCL as the

temperature increases. (d) Shear viscosity rheometry measurements between 0.001-10 s-1

performed at 80 °C, 100 °C and 120 °C allows characterization of the shear thinning properties

of the materials.

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3.2. Mathematical Model to Predict Extrusion Pressure

For MEW, previous publications have identified the importance of maintaining a sufficient

print speed such that the fiber is drawn out in a straight line from the printer nozzle.[1,3] Here,

the optimal lag distance between the nozzle and place at which the fiber touches the collector

plate to optimize fiber deposition was found to be 2.3 mm (Supporting Information 2) and an

average velocity of polymer extrusion from the nozzle was calculated to be 0.239 mm/min using

a previously published non-Newtonian shear rate model.36 Rearranged, this model then predicts

the printing pressure, ∆p, required to maintain this average extrusion velocity �̅� through a

cylindrical nozzle of radius, R and length, L, dependent on a material’s power law extrusion

behavior characterized by Power Law coefficients n and K (Figure 2);

∆𝑝 = (−�̅�

2𝐿𝐾)

1

𝑛(

𝑛

3𝑛+1) 𝑅

𝑛+1

𝑛 (2)

Figure 2. (a) Schematic diagram of the MEW process using an applied pressure (∆p) to

extrude a molten polymer or polymer solution through a grounded nozzle onto a motorized

collector plate. The length and radius of the nozzle are measured according to the inset

diagram. (b) Visualisation of the custom MEW device used in this study where the entire print

head in contained within the indicated holder and collector plate translates in x and y

controlled by computer programming. MEW device image courtesy of Dr Sean Powell,

Queensland University of Technology (QUT).

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This rheological model neglects physical processes surrounding the MEW Taylor cone which

forms at the tip of the extrusion nozzle, in which the shear conditions remain poorly

understood39–41 but appear negligible.42 This model only ensures a standardized flow rate of

material from the tip of the nozzle, after which fibers were readily printable under these

extrusion conditions.

This model identifies the ability to extrude fibers using PCL-only materials, as well as the

inability to extrude osites which require pressures at least 13-fold above what typical MEW

devices allow (0.5 MPa; Table 1). We addressed this by using dissolution of the PCL/SrBG

sample in a chloroform solvent which lowered the solution viscosity and enabled the calculated

printing pressure to fall within MEW tolerances.

Table 1. Power Law coefficients (n, K), printing parameters (∆p), and printed fibers derived for

each material at different temperatures. Voltages were selected to keep the fiber lag distance

consistent (Supporting Information 1). Fibers were produced for two PCL samples.

PCL/SrBG 33wt% composite could not be printed alone in molten phase.

Material Temp

[°C] n K

∆p

[MPa]

Voltage

[kV] Printed Fiber c

PCL Capa

6430

80 0.998 812.1 0.050 a -7.0

100 0.995 453.8 0.029 -5.2

120 0.993 281.9 0.018 -4.2

PCL Capa

6500

80 0.976 5252 0.437 -9.5

100 0.991 2979 0.200 -8.0

120 0.996 1821 0.115 -7.8

PCL/SrBG

33wt%

80 0.767 15,257 73.35 - Unprintable b

100 0.828 7,883 8.264 - Unprintable b

120 0.803 4,373 6.658 - Unprintable b

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a Control extrusion pressure; 0.050 MPa was used to calibrate the predictive model to ensure

consistent flow rate across all other samples and temperatures. b Material could not be extruded in molten phase; pressure required exceeds experimental

pressure limit. c Scale bar = 100 µm

3.3. Optimizing Extrusion of PCL/SrBG Composite

Informed by the predictive model above, three concentrations of PCL/SrBG 33wt% dissolved

in chloroform were characterized using the same shear-viscosity rheology measurement and

theoretical extrusion model. Figure 3a shows the shear-viscosity profiles for 1 g, 0.5 g and

0.25 g PCL/SrBG 33wt% dissolved in 1 mL chloroform, demonstrating that the viscosity was

significantly lower than the raw composite sample and demonstrates shear thinning. Using the

Power Law regression and predictive model, theoretical printing pressures of 0.380 MPa,

0.069 MPa and 0.036 MPa were estimated for the three concentrations respectively, all below

the operational limit of the MEW device and all theoretically printable.

Using these pressures, fabrication of PCL/SrBG 33wt% scaffolds was achieved using 6 kV

applied to the collector plate, grounded nozzle, 8 mm working distance, polymer maintained at

55 °C and extruded in ambient conditions. Comparing the TGA profiles of PCL/SrBG 33wt%

(‘pre-printing’) and PCL/SrBG 33wt% after dissolution in 1 mL chloroform and MEW

extrusion into fibers (‘post-printing’) showed negligible differences, indicating that all the

chloroform evaporated after printing (Figure 3b).43 The ratio of 1 g composite material in 1

mL chloroform was selected to minimize the quantity of chloroform in the solution for more

rapid drying and solidification. Composite scaffolds up to 6 layers high were successfully

printed using the predicted operational parameters (Figure 3c).

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Figure 3. (a) Shear-viscosity profile for three formulations of /PCL/SrBG 33wt% dissolved in

chloroform at 50°C compared to the raw PCL materials at 80°C, each at printing temperature;

(b) TGA results demonstrate negligible difference in the composition of the PCL/SrBG 33wt%

composite bulk versus after dissolving the composite bulk in chloroform, extrusion and

subsequent evaporation of the solvent from the composite; (c) Scaffold fabricated via a hybrid

MEW approach using 1 g PCL/SrBG 33wt% dissolved in 1 mL chloroform, printed at elevated

temperatures; (d) SEM imaging showing the surface morphology and typical diameter of the

fiber; and (e) EDS confirms the presence of SrBG particles (white) evenly distributed in the

composite fibers.

SEM was used to visualize the microfibers and SrBG particle distribution. Additionally, energy

dispersive spectroscopy (EDS) confirmed the homogeneous distribution of SrBG throughout

the PCL fibers. This is highly desirable to enable the sustained release of Sr2+ ions from the

SrBG particles which has been shown to stimulate bone remodeling and scaffold degradation

to assist osteoinduction and integration.29,43,44

4. Conclusions

The methodology presented directs the MEW additive manufacturing of viscous polymer

formulations into ordered microfibers which previously required unachievable extrusion

pressures. This methodology is especially relevant to fabricate biocompatible, micro-

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architecture scaffolds for bone tissue engineering and is applied toward creating bioactive

PCL/SrBG composites. Where MEW of untested materials typically follows ad hoc

experimentation, this model quickly identifies possible MEW material formulations and

printing parameters through rheological properties, applicable to a breadth of materials. Using

this framework, high quality, ordered PCL/SrBG composite scaffolds with 33wt% bioactive

glass content were fabricated, the highest content via MEW reported to date with previously

demonstrated biological relevance. This study therefore offers a new platform for the

fabrication of high ceramic-content polymer scaffolds for bone tissue engineering and a method

to assist the rapid application and screening of new biomaterials for MEW.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Appendix/Nomenclature/Abbreviations

DSC Differential scanning calorimetry

EDS Energy dispersive spectroscopy

MEW Melt electrowriting

PCL Polycaprolactone

SEM Scanning electron microscopy

SrBG Strontium-substituted bioactive glass

TGA Thermogravimetric analysis

Acknowledgements: The authors would like to thank Dr Tim Krappitz, Dr Cynthia Wong, Ms

Elizabeth Graham, Mr Peter Hegarty and Mr Trent Brooks-Richards for their technical

assistance. The data reported was obtained using the resources of the Central Analytical

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Research Facility at the Institute for Future Environments, Queensland University of

Technology, with funding from the Science and Engineering Faculty. The authors also

gratefully acknowledge Perstorp for supplying PCL samples and NCP acknowledges the

support of ARC ITTC in Additive Biomanufacturing and industry partner, Anatomics Pty Ltd.

Keywords: Melt electrospinning writing, additive manufacturing, polycaprolactone, bioactive

glass, tissue engineering

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Melt electrowriting additive manufacturing of composite biomaterials for bone tissue

engineering is explored in this study, examining the influence of viscosity on the ability to

extrude microfibers. A polycaprolactone-bioactive glass composite biomaterial is successfully

printed into bioactive scaffolds for bone tissue engineering.

NC Paxton, J Ren, MJ Ainsworth, AK Solanki, JR Jones, MC Allenby, MM Stevens, MA

Woodruff

Rheological Characterization of Biomaterials Directs Additive Manufacturing of

Strontium-Substituted Bioactive Glass/Polycaprolactone Microfibers

ToC figure

100 µm