3D Printing of Silk Particle-Reinforced Chitosan Hydrogel Structures

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Tissue Engineering and Regenerative Medicine

3D printing of silk particle-reinforced chitosan hydrogel structures and their properties Jun Zhang, Benjamin James Allardyce, Rangam Rajkhowa, Yan Zhao, Rodney J. Dilley, Sharon Redmond, Xun-Gai Wang, and Xin Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00804 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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ACS Biomaterials Science & Engineering

3D printing of silk particle-reinforced chitosan hydrogel structures and their properties Jun Zhanga, Benjamin J. Allardycea*, Rangam Rajkhowaa, Yan Zhaob, Rodney J. Dilleyc, Sharon L. Redmondc, Xungai Wanga, Xin Liua* a

Deakin University, Institute for Frontier Materials, Geelong, 75 Pigdons Road, Waurn

Ponds VIC 3216, Australia b

College of Textile and Clothing Engineering, Soochow University, Suzhou, 215123, China

c

Ear Science Institute Australia, 8 Verdun Street, Nedlands, WA 6009, Australia

* Corresponding authors: Dr. Xin Liu, and Dr Benjamin J. Allardyce Email: [email protected]; [email protected]

Abstract: Hydrogel bioprinting is a major area of focus in the field of tissue engineering. However, 3D printed hydrogel scaffolds often suffer from low printing accuracy and poor mechanical properties due to their soft nature and tendency to shrink. This makes it challenging to process them into structural materials. In this study, natural chitosan hydrogel scaffolds were, for the first time, reinforced with milled silk particles and fabricated by 3D printing. Compared with pure chitosan scaffolds, the addition of silk particles resulted in up to a 5-fold increase in compressive modulus as well as significantly better printing accuracy and improved scaffold stability. The chitosan/silk inks flowed well during printing; loading of up to 300% silk (w/w) resulted in only minor changes in the rheological properties of the ink. Particle loading also enabled tuning of the surface roughness of the scaffolds and improved scaffolds’ biodegradability. The printed composite hydrogel scaffolds showed no cytotoxicity and supported adherence and growth of human fibroblast cells. Keywords: 3D Bioprinting, Silk particles, Hydrogel scaffold, Chitosan, Reinforcement

1. Introduction 3D printing for biomedical application aims to produce complex shapes that can be used to guide tissue regeneration.1 Printed hydrogels, which are highly hydrated polymer networks, Page 1

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are favoured materials for cell scaffolds, because they mimic the extracellular matrix (ECM) by supporting cell attachment, proliferation and differentiation.2 Due to their low stiffness, hydrogel scaffolds are perfect candidates for soft tissue regeneration. To produce a 3D printed hydrogel scaffold, bioinks must possess high enough viscosity to allow printing into solid filaments while remaining fluid enough to flow during printing. Rapid gelation after extrusion is also desirable since it helps to maintain the shape of printed filament.3 In addition, to produce robust hydrogel scaffolds, the printed filaments should be strong enough to selfsupport while offering good adherence between adjacent layers.4 Compared with commonly used synthetic biopolymers such as poly-l-lactide5 , polyurethane6 or polyethylene glycol, naturally-derived materials can interact with cells and biological systems better since they deliver biological cues to cells and surrounding tissues.7-8 Chitosan, which is the second most abundant natural biopolymer after cellulose, is a polysaccharide produced from exhaustive deacetylation of chitin, a structural element in the exoskeleton of crustaceans and insects.9-10 It has received considerable attention as a potential scaffold material due to its well documented cytocompatibility and biodegradability.11-13 Furthermore, chitosan can be easily dissolved in dilute acidic solution and its viscosity can be easily tuned by adjusting the concentration. Chitosan remains in solution up to a pH of approximately 6.2; above this pH it rapidly precipitates to form a hydrogel. This transition results from the neutralization of amino groups within chitosan and the subsequent removal of inter-chain electrostatic forces, which subsequently enables the formation of extensive hydrogen bonding and hydrophobic interactions between adjacent chains.14 However, despite the excellent printability and the ability to form pH-induced hydrogels, the low stiffness and compressive strength of chitosan, due to high water content, makes it unstable and prone to collapse.15-16 Shrinkage is also common in hydrogel materials, representing the biggest problem for maintaining shape fidelity after printing.17 One proposed solution to these problems is the introduction of a filler material. In polymer composite studies, the addition of rigid particles to polymers or other matrices can produce a number of desirable effects, such as increases in stiffness, creep resistance and fracture toughness.18 Thus, the use of composites has been suggested as a way of improving hydrogel shape fidelity.19 Milled silk particles have been used in the past as a filler to reinforce scaffolds since silk has excellent mechanical properties and biocompatibility.20-21 Our group previously used milled silk particles to achieve a 40-fold increase in the compressive modulus of Page 2


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porogen leached composite scaffolds.22 A similar study using alkali-hydrolysed short silk fibres also resulted in high strength silk-based scaffolds suitable for use in bone repair.23 The aim of this study was therefore to develop composite chitosan/silk particle scaffolds that retain the printability of chitosan while improving post-printing shape fidelity and scaffold mechanical properties. Chitosan/silk particle (CS/SP) composite scaffolds were fabricated using a rapid and economic extrusion-based 3D printing procedure. The processing conditions, including material and printing parameters were optimised and CS/SP inks with different silk particle loading were evaluated for their rheological properties. The effect of silk particles on printing accuracy, surface roughness and scaffold compressive modulus were quantified and the reinforcing performance was validated using established theoretical models. Finally, cytocompatibility and cell interactions were evaluated using human fibroblasts to support its potential application for soft tissue repair.

2. Materials and methods 2.1 Materials Materials and media were used as received, including milled silk powder (average particle size of 5 µm, Smiss Natural, China), chitosan powder (medium molecular weight, Sigma Aldrich, St. Louis, MO, USA), acetic acid and ethanol (analytical grade, Chem-Supply, Australia), sodium hydroxide (Chem-Supply), two-part silicone elastomer (ACEO, Germany), Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, MA, USA), fetal bovine serum (FBS, Bovogen, Australia), penicillin (10,000 U/mL) and streptomycin (10,000 g/mL, Aldrich), phosphate buffered saline (PBS, 10 mM, pH 7.4, Aldrich), MTS-kit bioassay (Promega Corporation), triton X-100 (Aldrich),

4,6-diamidino-2-phenylindole (DAPI,

Molecular Probes), and Phalloidin Alexa568 (Molecular Probes). Human skin fibroblasts (passage number > 20)24 were used from stock cultures.25 Cells were incubated in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin and 1% (v/v) streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

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2.2 Particle characterisation The morphology of the silk particles was observed by gold coating (5nm) a small sample of the powder before being imaged under a scanning electron microscope (Zeiss Supra 55VP, Germany) with an accelerating voltage of 5 kV and a working distance of 6–8 mm. The average silk particle size and size distribution was quantified using a digital camera-based particle analyser (CAMSIZER P4, Retsch Technology, Germany).

2.3 Rheological characterisation of CS/SP printing ink The rheological properties of chitosan solutions (concentration ranging from 3% to 5% w/v) with different silk particle loading (0%, 50%, 100% and 300% of the weight of chitosan) were measured using a HR-3 Rheometer (TA Instruments, New Castle, DE, USA). A cone and plate geometry (CP 4/40, cone diameter of 40 mm with 4° angle) was used for flow tests and a parallel plate geometry (sandblasted to reduce slip effects, cone diameter of 40 mm) was used for oscillation tests. The environmental cuff was used in all the experiments and care was taken to avoid under or overfilling the geometry, adapting the procedure used previously after slight modifications26. To evaluate flow behaviour, the response of CS/SP inks to shear was examined in continuous flow experiments over a shear rate range of 0.1 to 1000 s−1 by selecting 10 points in each order of magnitude across the logarithmic scale. To evaluate the thermo-responsive behaviour of CS/SP inks, their viscosity was measured across a temperature range of 0˚C to 60˚C at a fixed shear rate of 100 s−1, which was determined to be the shear rate experienced during extrusion as suggested by previous literature3. To understand the fluid behaviour of CS/SP inks, the dynamic elastic modulus (G’) and viscous modulus (G”) were measured using the oscillatory mode of the rheometer. The linear viscoelastic region of the materials was first determined using amplitude and frequency sweep measurements. These measurements formed the basis for a gelation kinetics study. In the amplitude sweep measurements, the materials were tested in a strain range of 10-1 to 104 % while keeping the frequency constant at 1 Hz; for frequency sweep measurements, materials were tested from 0.1 to 100 Hz while keeping the strain constant at 5%. For gelation kinetics study, the target strain value of 5% and frequency value of 1 Hz were selected as they represented one-third of the linear region of the curve. These values were Page 4


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selected to ensure that the measurements were conducted within the linear viscoelastic region and that the G’ and G” were independent of the strain amplitude and frequency. To evaluate differences in gelling rates after addition of the coagulant, the gelation kinetics of the solutions were studied as a function of time in oscillation mode. The coagulant, a mixture of 12% (w/v) NaOH in a 7/3 water/ethanol mixture (3 ml), was pipetted onto the CS/SP solutions during the amplitude sweep test and changes in gel strength were recorded by the rheometer in real time. Each test was carried out in triplicate.

2.4 Preparation of printing ink CS/SP inks were prepared by adding different amounts (0 to 300%, w/w) of silk particles to chitosan solution. Typically, the required amount of silk powder was weighed and suspended in deionized water. This suspension was ultra-sonicated for at least 1h to ensure homogeneity. Acetic acid was added to the particle suspension (2% v/v final concentration) to aid chitosan dissolution before chitosan was dissolved with mechanical stirring for at least 3 h at room temperature (RT). The resulting paste was then centrifuged at 5,752 × g for 15 min to remove bubbles.

2.5 Scaffold printing The scaffold printing process is summarised in Fig. 1. The prepared CS/SP inks were transferred into polyethylene injection cartridges and mounted on a 4th generation 3D Bioplotter™ (EnvisionTEC GmbH, Germany) for printing. A lattice CAD model file (15×15×3 mm) was loaded into the Bioplotter software. The scaffolds were extruded layerby-layer through a 580 µm diameter nozzle using a pressure of 0.4 to 0.7 bar, printing speeds of 10-20 mm s-1 and 1mm inter-filament distance. Each layer was solidified after printing by manually pipetting coagulant solution (12% (w/v) NaOH in a 7/3 water/ethanol mixture) onto the printed filaments. The scaffold architecture was constructed by plotting filaments with 0 and 90° angle steps between each layer (for surface, dimension and mechanical characterisations). Another dense membrane structure was also prepared by narrowing the inter-filament distance to 800 µm and solidified after 3-layer printing (for cell culture and stability tests). After printing, all samples were rinsed 3 times with deionised water before Page 5



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being submerged in deionised water at RT with water changes every day until the pH became neutral.

Fig. 1. Work flow of the CS/SP scaffold printing process.

2.6 Scaffold characterisation To evaluate printing accuracy using the different inks, a porous cube structure with a side length of 15 mm, height of 3 mm, and the inner structure of 1 mm inter-filament distance was designed and printed. The dimensions of the printed scaffolds were measured using a Vernier caliper and compared to that of the CAD model to determine printing accuracy using a method suggested by a previous study.27 The printed distance Di (mm, length, width and thickness) was compared to the designed distance D (Dlength = Dwidth =15mm, Dthickness =3mm) using the equation (1) below, with an average of 5 samples for each measurement. Printing accuarcy % 1

| |

100

(1)

The surface morphology of the as-printed CS/SP hydrogel scaffolds was observed using an Olympus DP71 optical microscope under bright field. The surface areal roughness (Sa) of the scaffolds was determined using an Olympus LEXT OLS4100 laser scanning digital microscope (Olympus, UK). To measure the compressive properties of the printed structure without interference of the pore architecture, a porous cube structure with a side length of 15 mm, height of 3 mm, and the same inner structure of 1 mm inter-filament distance was designed and printed. Compression tests were performed using an Instron 5967 mechanical Page 6


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testing machine with a 5 N load cell (Instron, Norwood, MA, USA): scaffolds were tested using a wet compression bath (Fig. 2). Compressive strength was tested using a linear strain rate from 0−50% of the initial length at an extension rate of 0.5 mm/min. In order to approximate biological conditions, tests were conducted at 37°C in PBS (0.01M, pH 7.4) solution. The compressive modulus of each sample was determined by linear fitting (R2 of 0.94 to 0.99) of the stress−strain curve in the strain region between 0 and 10%. All samples were tested in triplicate.

Fig. 2. Testing rig setup for wet compression test.

2.7 Cell culture on CS/SP constructs Cytotoxicity A 3-day cytotoxicity test was performed based on ISO10993 (Biological evaluation of medical devices -- Part 5: Tests for in vitro cytotoxicity) with minor modification. Briefly, samples were sterilized by immersion in 70% ethanol for 30 min, then in fresh 70% ethanol for a further 2 h, followed by 3 brief rinses in ethanol. Samples were then washed with PBS and equilibrated in cell culture medium for 72 h to extract any chemical leachates released by the scaffolds. Dimethyl sulfoxide (DMSO) at 5% v/v in medium was used as a cytotoxic Page 7



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(negative) control while cell culture media was used as the positive control for the assay. Human fibroblasts28 were then seeded at a density of 1 × 104 cells/ml into standard 24 well plates (Nunclon Delta for adherent cells) containing the media extracted from the samples as well as the DMSO and culture media control samples. The fibroblasts were maintained under standard culture conditions. After 3 days incubation, viable cell numbers grown in each sample extract were determined by MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethox-yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,

inner

salt,

CellTiter

96®

AQueous One Solution, Promega, Madison, WI, USA). Specifically, culturing mediums were removed and samples were rinsed with PBS first. As colour-development solution, 200 µL of DMEM media and 40 µL of MTS solution were added into the wells. The samples were then incubated at 37˚C for 1 h, after which 100 µL of solution was withdrawn from each well. The absorbance of these aliquots was measured at 490 nm on a microplate reader. The average reading of 6 replicates was calculated for each experiment and the experiment was repeated 3 times in total. Cell proliferation To determine cell proliferation, 1 × 104 human fibroblasts were seeded on the scaffolds (3 replicates for each scaffold) with tissue culture plastic (TCP) as control. At 1, 3 and 7 days post cell seeding, the culture scaffolds were rinsed with PBS to remove non-adhering cells before being transferred to new wells. The proliferation of the cells on each specimen was determined using a standard MTS assay (as described for the cytotoxicity study above). Cell attachment Confocal laser microscopy (Carl Zeiss, LSM 700, Germany) imaging was used to visualize cell morphology on the scaffolds. After 3-days culture of 1 105 fibroblasts cell seeding density on each sample, each specimen was rinsed three times with PBS, then fixed with 4% paraformaldehyde for 30 min at 4 °C. The cells were then permeabilised using 0.1% Triton X-100 (Sigma, USA) for 10 min. After washing 3 times with PBS, the cytoskeleton and nucleus of cells were stained with 25 µg/mL rhodamine-conjugated phalloidin (Invitrogen, USA) and 10 µg/mL 4’,6’-diamidino-2-phenylindole hydrochloride (DAPI, Invitrogen, USA) for 30 min and 5 min, respectively. Subsequently, the cells were visualized using laser scanning confocal microscopy.

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2.8 Scaffold stability The printed hydrogel scaffolds incubated for 2 weeks in DMEM media (control group); scaffolds were removed and weighed at different time points during 2 weeks to determine scaffold stability in media. Repeated stability study of hydrogel scaffolds in DMEM media with 1×105 cells / well (experimental group) was also conducted to determine if the cells contributed to degradation at all. Three replicates of each sample were measured for each group and media were changed every 3 days. Representative images of scaffolds in cellcontaining media were also captured at days 7 and 14.

2.9 Statistical analysis All data were obtained at least in triplicate and all values were reported as mean and standard deviation (SD). Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey's test using Origin 8.0 software (Origin Lab Inc., USA). The statistical difference between two sets of data was considered significant when p < 0.05.

3. Results and discussion 3.1 Material development and shear rate calculation The aim of this study was to develop a 3D printed scaffold from natural material reinforced with silk particles. During extrusion and solidification of the ink, three criteria were considered critical for the printing process. First, the extrusion of the printing ink out of the micro-nozzle should be continuous, without blockage. Second, the ink should exhibit shear thinning so that it demonstrates solid-like behaviour (G’>G”) when loaded in the canister, then transition to a liquid-like state (G”>G’) while under shear during printing before solidifying again after printing. Third, the timing of ink solidification after extrusion is important. Rapid solidification helps to maintain the cylindrical filament shape, however, it can result in poor adhesion between layers. Slower solidification is better for adhesion between layers, but results in poor printing resolution due to spreading of the printed filament. Thus, to better understand the extrusion process for printing ink development, the shear rates of different inks in the nozzle upon extrusion were calculated prior to printing. Page 9



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For Newtonian fluids, according to Mooney analysis,29 the shear rate of a flowing liquid can be calculated as

γ,

!"

(2)

where Q is the volumetric flow rate (mm3/s) of the liquid being extruded and r is the radius of the nozzle (mm). In the printing process, pressure is adjusted to maintain the flow rate as close to printing speed as possible. Therefore, the volumetric flow rate can be calculated using the printing speed V (mm/s), where A is the area of the nozzle:

Q A. V '( ) . *

(3)

For non-Newtonian fluids, according to Power-law model,30 the equation can be corrected as

+

,-./ -

γ,

(4)

where n refers to flow index (non-Newtonian index). When n=1, the fluid is Newtonian; when n>1, the fluid exhibits shear thickening; when n0.05) compared to the positive control (fresh culture media), suggesting that none of the scaffolds produced cytotoxic leachates (Fig. 8A). DMSO, as the negative control, showed significantly lower cell viability (p0.05), indicating no significant cytotoxicity of all printed scaffolds. The results of the proliferation assay (Fig. 8B) indicated that all printed scaffolds supported cell growth over the 7-day experiment. Growth at early times was lower on scaffolds than on culture plastic but by 7 d all were statistically similar (P>0.05) to the culture plastic control.

Fig. 8. A. Cytotoxicity of leachate from the printed CS/SP hydrogel scaffolds with varying silk particle content. DMSO and cell culture medium were used as negative and positive controls respectively. B. MTS assay results for human fibroblast proliferation on printed CS/SP hydrogel scaffolds with varying silk particle content at various time points. Cells were grown on tissue culture plastic (TCP) as positive controls. (All results are presented as the mean ± standard deviation (SD). Statistical significance was defined as *p < 0.05, **p < 0.01 and ***p < 0.001 using one-way analysis of variance (ANOVA));

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3.7 Cell attachment When testing fibroblast adhesion (Fig. 9), the seeded cells adhered in small clumped colonies; some cells appeared more spread out on the surface of CS scaffolds (Fig. 9A, E). With increasing silk loading, the fibroblasts appeared to separate from each other and show less clumping (Fig. 9B-D and F-H). This behaviour agrees with previous studies discussing the physiochemical features on the surface affecting cell adhesion and proliferation.46-48 Surface properties including roughness and stiffness can play a role in cellular behaviour. It was suggested that stiffer substrates tended to direct cells to show spread and flattened morphology,47 while less spreading and cell clumps could be observed on soft substrates (

3D Printing of Silk Particle-Reinforced Chitosan Hydrogel Structures

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