Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with

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Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting Ali Nadernezhad, Ozum S. Caliskan, Fuat Topuz, Ferdows Afghah, Batu Erman, and Bahattin Koc ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00665 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Scheme 1. Schematic illustration of bioprinting of agarose bioinks in the (a) absence and (b) presence of Laponite RD. 929x424mm (200 x 200 DPI)

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Figure 2. Dynamic rheological characterization of bioinks containing various concentrations of Laponite RD. (a) Strain amplitude sweep profiles of bioinks with different Laponite RD concentrations at 37 ˚C. (b) Yield shear stress of the bioinks calculated from the cross-over of G’ and G” from the respective strain sweep tests (a) at 37 ˚C. Significant difference is denoted by different letters (p < 0.05) (c) Temperature sweep profiles of nanocomposite bioinks in the range of 55-20 ˚C showing the evolution of agarose networks over G’ by decreasing temperature. Inset shows the narrow range of the respective plot. 314x183mm (300 x 300 DPI)


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Figure 4. Cellular evaluation and cytotoxicity of Laponite RD incorporated nanocomposite hydrogels. (a) Overlay z-stack confocal images of HeLa cells encapsulated in nanocomposite hydrogels for different culture periods. Red fluorescence indicates cells at G1 phase and yellow-green fluorescence belong to cells at S/G2/M phases of cell division cycle; scale bars: 200µm. Metabolic activity of (b) Fucci HeLa and (c) NIH/3T3 cells normalized to the internal control (mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3). (d) The metabolic activity of encapsulated Fucci HeLa cells in Bioprinted bioinks (RD 2%) compared with the cast cell laden samples of the same composition after different post printing incubation periods.



Figure 5. Cell spreading and morphology on nanocomposite hydrogel surfaces. (a-d) Bright field images of fixed 3T3 cells; scale bars: 50 m. (e-h) Confocal images of NIH/3T3 cells stained with fluorescent dyes; scale bars: 30 m. (i-k) high magnification images of spread cells on the surface of nanocomposite hydrogels. Cells tend to spread more with elongated F-actin filaments by increasing Laponite RD concentration; scale bars: 20 m. (l) the quantified spreading area of cells seeded on different bioinks with different Laponite RD content calculated from the microscopy images. 303x160mm (300 x 300 DPI)


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Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting Ali Nadernezhad,†, ‡,§ Ozum S. Caliskan,†, ‡,§ Fuat Topuz, ‡,§ Ferdows Afghah,†, ‡ Batu Erman,∥ and Bahattin Koc†, ‡, * †

Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey

‡

Bioprinting Laboratory, Sabanci University Nanotechnology Research and Application Center

(SUNUM), Istanbul, Turkey ∥ Molecular

Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural

Sciences, Sabanci University, Istanbul, Turkey. KEYWORDS: 3D bioprinting, nanocomposite hydrogel, bioink, agarose, nanosilicate

1 ACS Paragon Plus Environment


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ABSTRACT. Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties, and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.


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1. INTRODUCTION Extrusion based 3D bioprinting is considered as one of the mainstream additive manufacturing strategies for tissue and organ regeneration. 1, 2 Hydrogels as bioinks, are the main cell carrier in the process of bioprinting due to their water-rich biocompatible environment. However, the physico-chemical properties of hydrogel bioinks should be tailored to meet the specific requirements of 3D bioprinting process. 3,

4

This demands screening criteria for selection of

hydrogel bioinks, which includes the biological activity of bioinks as well as mechanical and structural stability throughout the process. 4, 5 3D bioprinting of different natural and synthetic hydrogel bioinks was investigated extensively, among them the synthetic bioinks usually showed high adaptability with the requirements of bioprinting process although they usually lack the biomimicry and bioactivity of natural hydrogels. 6 Natural hydrogels, such as collagens and their derivatives, on the other hand, can mimic the richness of natural extracellular matrix to support cell attachment, migration, proliferation, and differentiation and can facilitate the matrix remodeling.7 However, natural hydrogels usually do not comply with the process of bioprinting in terms of cross-linking conditions, flow properties and mechanical stability. To overcome the major drawbacks of natural hydrogels in adaptation with 3D bioprinting process, innovative approaches have been developed, including the modification of gelation kinetics, viscoelasticity, and mechanical properties.5,

6

These advanced bioinks demonstrated significant tunability to be

adapted to the process of 3D bioprinting, while they possess high degrees of biocompatibility. Agarose as a naturally derived polysaccharide has been mostly used as the support material in 3D bioprinting.8,

9

Although agarose hydrogels show high viscosity and shape fidelity after

gelation, they lack cell binding sites in their backbone and thus have limited bioactivity. Additionally, the thermal cross-linking mechanism of gelation of agarose is highly dependent on



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the polymer concentration: the low concentration of agarose tends to form gel structures below physiological temperature, which results in very low viscosity and no fidelity of extruded bioinks. However, from the perspectives of the bioprinting process, agarose could be considered as an interesting bioink candidate due to the ease of tuning mechanical properties, rapid gelation kinetics, and the potential for further chemical functionalization. On the other hand, highly concentrated agarose solutions rapidly form gels with the intrinsic stiffness of helical fiber bundles formed by noncovalent association of agarose chains in a short temperature span,10 which results in a high imposed shear rate during extrusion and structure breakdown. To overcome similar problems in other natural or synthetic hydrogels, the synergistic tuning of bioactivity and flow properties by the synthesis of nanocomposite hydrogels was introduced.11 Nanoengineered hydrogels revealed a range of new possibilities and unique properties, such as bioactivity, controlled drug release, photoresponsiveness, and electrical conductivity. 11 The addition of a small amount of nanomaterials can alter the physiochemical properties of hydrogels and enable their use for 3D bioprinting.11-13 Among these nanomaterials, Laponite nanosilicates showed to be an effective additive being able to induce noteworthy effects on the flow behavior and bioactivity of hydrogel matrix. Laponite, a synthetic hectorite-type layered nanosilicate, is widely used as a rheology modifier, and recent studies showed its potential in tissue engineering applications. 14-18 Laponite nanosilicates comprise disc shaped nanoparticles, while upon dispersion in water the individual nanodiscs possess negatively charged surfaces with positively charged edges. This heterogeneous distribution of charges throughout nanodiscs results in the formation of a structured fluid due to the ionic interactions between nanoparticles. The resultant thixotropic gel-like structure is


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extremely responsive to shear deformation and shows an unequalled degree of shear thinning with a high degree of recoverability.19

Scheme 1. Schematic illustration of bioprinting of agarose bioinks in the (a) absence and (b) presence of Laponite RD. The promising effects of incorporation of Laponite in different hydrogel matrices suggests its potential in the improvement of flow behavior and bioactivity of agarose hydrogels to meet the criteria for extrusion 3D bioprinting. Recent studies showed that the incorporation of Laponite nanosilicates into polysaccharide hydrogel matrices, such as alginate and kappa-carrageenan, yielded shear thinning nanocomposite hydrogels, which could be processed as bioinks in 3D bioprinting.17, 18 Depending on the type of Laponite nanosilicates used, an increase or decrease in viscosity of nanocomposite blends was observed compared with the initial polysaccharide hydrogels. Based on the information available through the literature reports, we hypothesized that the incorporation of Laponite nanosilicates within agarose hydrogels will improve the bioactivity of the hydrogel, while the flow behavior of agarose can be altered at the same time to adapt to the extrusion 3D bioprinting (Scheme 1). To evaluate these hypotheses, the role of Laponite RD



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nanosilicates on improvement of bioactivity of agarose hydrogels was investigated and a detailed study on the rheological properties of nanocomposite hydrogels was conducted. Moreover, the current study demonstrated that an increasing concentration of Laponite nanosilicates enhanced the cell attachment and morphogenesis in early culture times. 2. MATERIALS AND METHODS 2.1. Materials Laponite nanosilicates (Laponite RD) were a kind gift from BYK-Chemie Gmb. Agarose NuSieve 3:1 was purchased from Lonza. For cell culture experiments, Dulbecco’s modiﬁed Eagle’s medium (DMEM), penicillin/streptomycin (PS) antibiotic mix and fetal bovine serum (FBS) were obtained from Gibco, Life Technologies. Trypsin-EDTA (ethylenediaminetetraacetic acid) (0.025%) solution was purchased from Sigma. PrestoBlue cell viability reagent was purchased from Thermo Scientific. F-actin Staining Kit - Orange Fluorescence - Cytopainter was purchased from Abcam. 2.2. Formulation of Bioinks Nanocomposite hydrogel bioinks were formulated with different concentrations of nanosilicates. Water was used as a medium instead of PBS, since Laponite clays form large aggregates in PBS buffer by means of electrostatic interactions between the surface ions of Laponite and cations in PBS.20 Initially, nanosilicates were fully exfoliated in distilled water by vigorous mixing until a clear solution was formed. Agarose powder was slowly added to the solution while stirring, and the mixtures were left on stirrer for additional 15 min at room temperature before autoclaving. The initial mixtures of gel formulations were autoclaved at low temperature sterilization cycles (115 ˚C for 15 min) to avoid structural degradation of agarose. Established bioink compositions were thoroughly mixed and cooled to 70 ˚C in an oven until further use. Agarose concentration was kept 6 ACS Paragon Plus Environment

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at 3 wt.% for all bioink compositions while the concentration of Laponite RD varied between 1 and 3 wt.%. 2.3. Characterization FTIR spectra were recorded on a Bruker-VERTEX 70 spectrophotometer. The samples were blended with KBr and pressed into transparent pellets. A total of 64 scans were taken per sample with a resolution of 4 cm-1. A serial ethanol dehydration step was performed on the gels and afterwards, the gels were dried in a critical point dryer (Tousimis, Autosamdri-815B, Series C critical point dryer). The inner morphology of the dried gels was explored by scanning electron microscopy (SEM) (Quanta 200 FEG, FEI). The specimens were coated with 5 nm Au with a PECS-682 sputter. The Zeta Potential measurements were performed using a Malvern Nano ZS ZEN3600 (Malvern Instruments Inc., US) by measuring the electrophoretic mobility in water. The electrophoretic mobility is converted to a zeta potential via the Henry equation by the instrument. During the measurements, disposable zeta potential cuvettes were used. All analyses were carried out in triplicate, and the mean data with the standard deviation were reported. All rheological characterizations were performed with a MCR 302 rheometer (Anton Paar, Austria) and a parallel plate geometry (diameter = 25 mm) at a gap of 1 mm. The sample perimeter was covered with a thin layer of low viscosity silicone oil to prevent water evaporation during the measurements. Rotational measurements were performed for the characterization of flow behavior of the formulations. The viscosity of solutions was investigated in the shear rate range of 10 -2 - 102 s-1. Time sweeps at constant shear rate of 10 s-1 were performed to investigate the thixotropic properties of formulations during extrusion. Oscillatory deformation tests were conducted to investigate the structural dynamics of formulated bioinks. Strain-sweep tests were carried out in the strain range of 0.01-500% at a constant angular frequency of 10 rad/s. The recoverability of 7 ACS Paragon Plus Environment


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the formulation during extrusion was investigated by applying sequential low-high oscillatory strains of 1 and 50% at the constant angular frequency of 10 rad/s for 60 s durations. Temperature sweeps were performed from 55 to 20 ˚C with the cooling rate of 3 ˚C/min at the constant strain and angular frequency values of 1% and 10 rad/s, respectively. All the measurements, except for temperature sweeps, were performed at 37 ˚C while the samples were thermally equilibrated prior to testing in situ for at least 10 min. Compression tests were performed using the Instron 5982 universal tensile system (Instron, USA) with a 10 kN static load cell. The samples were prepared by casting the bioink formulations in an aluminum cylindrical mold with diameter and height of 25 and 30 mm, respectively. The samples were incubated in distilled water at 4 ˚C overnight prior testing to achieve the equilibrium swelling conditions. The testing was performed at room temperature while the compression rate was set as 2 mm/min and the compression moduli were determined using the slope of the linear portion of the stress-strain plots (0-4% compressive strain). 2.4. Three Dimensional Bioprinting Process A three axes robotic platform (Poysan Makina, Turkey) equipped with a pneumatic dispensing unit (Nordson EFD Performus V) was used. Bioinks were dispensed through 10 mL heated syringes mounted on the z-axis through a 22-gauge double thread screwed plastic needle (Musashi Engineering, Japan). The motorized x-y-z robotic stages were controlled by MACH3 software (Newfangled Solutions, USA). Rhinoceros 5 (Robert McNeel & Associates) was used for the development of computer aided designs and the tool-paths were generated by using Rhinoscript. 3D printing parameters were set as the following: printer head speed of 3 mm/s and air pressure of 6 psi, while the temperature of the syringe heater set to 37 ˚C. The build platform was at room temperature during the 3D bioprinting experiments. 2.5. Cell Culture and Maintenance 8 ACS Paragon Plus Environment

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Fluorescent ubiquitination-based cell cycle indicator (Fucci)-expressing HeLa cells and NIH3T3 cells were maintained in DMEM supplemented with 10% FBS and 1% PS. The cells were incubated at 37 °C in a humidified chamber supplied with 5% CO 2. The culture medium was replaced every three days, and the cells were passaged at 80-90% conﬂuency by trypsin-EDTA. 2.6. Viability Analysis The cell-laden hydrogel bioinks with a cell density of 5×105 cell/mL were prepared using a spacer with 1 mm thickness. The viability of cell-laden hydrogel constructs was determined by PrestoBlue cell viability reagent at 1, 3, and 7 days after encapsulation. Hydrogel discs having no cell were used as the negative control for the correction of the absorption. Cell-laden and cell-free hydrogel discs were treated with PrestoBlue according to the manufacturer’s protocol. Simply, Presto Blue was added to each well in reagent: medium of 1:9 in the experiment day. The plates were incubated at 37 ˚C for 90 minutes in standard conditions. 100 µL of the Presto Blue containing medium from each well was transferred to 96-well-plate for spectroscopy measurements. All attempts were done in dark starting with the addition of Presto Blue reagent into the wells. Hydrogel discs having no cell were used as negative control for cell-containing hydrogels samples. Cell-laden, Laponite-free agarose hydrogel was used as an internal control and the absorbance values of samples were normalized to that of an internal control. Fucci HeLa cells encapsulated in hydrogel discs were imaged on day 1, 3, and 7 of culture using Carl Zeiss LSM710 confocal microscope. The metabolic activity of bioprinted cell laden bioinks were measured with the same protocol, while the density of encapsulated Fucci HeLa cells were set as 2×10 6 cell/mL. 2.7. Cell Spreading Assay NIH/3T3 cells were seeded on the top of nanocomposite hydrogel discs to assess the spreading of cells over short culture times. The as-prepared hydrogel discs were equilibrated in culture 9 ACS Paragon Plus Environment


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medium at 37 °C for 30 min prior to seeding. Following the equilibration period, all the medium in wells were aspirated, and NIH/3T3 cells (25000 cells/8 µL) were carefully seeded on top surfaces, and the discs were incubated at standard conditions for 1 hour. Afterwards, 300 µL of medium was added to each well and incubated overnight. Bright field microscopy was used to track the attached cells. The cells were fixed prior to imaging with 4% paraformaldehyde for 25 minutes, followed by 1X PBS washing. Nikon Eclipse LV100 POL upright microscope was used for imaging. Cell spreading over the hydrogel samples was quantified by calculation of cell area in microscopy images using ImageJ software. At least 5 different images of each sample were analyzed, and the mean values were reported. F-actin and DAPI staining were used to analyze the cytoskeleton development. Fixed samples were incubated in 1% (w/v) BSA in PBS solution for 5 minutes to decrease unspecific binding to the nanocomposite hydrogels and to reduce the background noise. The samples were washed with PBS and permeabilized with 0.2% Triton X100 solution for 15 minutes, followed by washing with 0.1 M glycine solution and PBS for 5 minutes. F-actin staining was performed according to manufacturer’s protocol, followed by DAPI staining. Stained samples were washed with PBS and imaged with Carl Zeiss LSM710 confocal microscope. 2.8. Statistical Analysis The obtained data were evaluated by two-way analysis of variance followed by Tukey multiple comparison tests (GraphPad Prism 7). Significant differences were assumed at p < 0.05. 3. RESULTS AND DISCUSSION 3.1. Structural and Physical Characterization of Bioinks


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Previous studies have shown that the non-covalent interactions between Laponite and hydrogel matrix result in significant alteration of the physical properties of hydrogel nanocomposites.21 The presence of Laponite in the gel was confirmed by FTIR analysis, where a dominant peak at ~1000 cm-1 and a small peak at ca. 700 cm-1 were observed due to the stretching vibration of Si-O and bending vibration of O-Si-O (Figure 1a). The influence of the incorporated Laponite on the morphology of agarose gels was explored through SEM analysis. Figure S1 shows the SEM photos of the pure and nanocomposite agarose gel containing 2 wt.% Laponite RD. Pure hydrogels revealed the inter-connected nanofibrous matrix, while addition of Laponite induced the collapse of the fibers to some extent, forming thicker fibers. Due to the charged surface of Laponite, they interacted with agarose chains during the network evolution and led to the thickening of agarose nanofibers. The interaction between agarose and Laponite was further explored through examination of the flowability of various agarose solutions with different Laponite content (0-3 wt.%). As the respective solutions were not exposed to any heating-cooling cycles, pure agarose was in solution state, while the addition of small amount of Laponite RD led to significant increase in the viscosity. When the Laponite content reached to 2 wt.%, the solution did not flow at inverted position, suggesting the presence of interactions between Laponite and agarose (Figure S2a, b). On the other hand, Laponite RD could form a gel network at 3 wt.% and no gelation was observed below 2 wt.% (Figure S2c). Moreover, zeta-potential measurements were performed to confirm the interactions between Laponite and agarose. Incorporation of higher agarose content resulted in decrease of the negative surface potential of the Laponite due to adsorption of polymer chains on the Laponite surface (Figure 1b). Similar finding was observed on the mixture of Laponite and PEG, where the zeta-potential increased with higher PEG content. 22



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Figure 1. The interaction between Laponite RD and agarose characterized by various experiments. (a) FTIR spectra of the Laponite RD powder and agarose and nanocomposite gels. (b) Zeta potential values of Laponite RD dispersion (2 wt.%) containing various agarose concentrations (0.5-3 wt.%). (c) The results of compression test on different hydrogel samples containing different Laponite

RD

contents

at

constant

agarose

concentration

of

3

wt.%.

Different

letters/numbers/symbols denote statistically significant difference (p < 0.05). (d) Representative plots of deformation for each formulation in (c) during the uniaxial compression. The presence of strong interaction between Laponite RD nanodiscs and agarose chains was not only related to the sol state, and significant alteration of physical and mechanical properties during 12 ACS Paragon Plus Environment

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and after gelation can be expected. The results of compression testing showed that the toughness of nanocomposite hydrogels significantly increased by increasing the Laponite RD content (Figure 1c, d). This indicates a strong interaction between ionically charged nanodiscs and agarose chains even after gelation, which resulted in the enhancement of energy dissipation during deformation due to secondary interactions between nanodiscs and agarose chains, as well as the elastic contribution of the rigid network of Laponite RD nanodiscs. Similar findings were reported by Chang et al. for the nanocomposite hydrogels based on PEG-nanoclays.23 Although the incorporation of Laponite RD resulted in significant increase in toughness, no significant enhancement of compression moduli between different nanocomposites with Laponite RD contents up to 3 wt.% was observed. This could be attributed to the dependency of the compressive moduli of the hydrogels on the elastic contribution of the condensed double-helix network of agarose chains during deformation, rather than the viscoelastic effects imposed by the physical interactions between nanoparticles and polymer chains. The same trend and behavior were also observed in the previous studies.23-24 These results confirm the occurrence of a strong interaction between agarose chains and Laponite nanodiscs, which could be further utilized in tuning the physical properties of nanocomposite bioinks. 3.2. Bioink Formulations and Their Rheological Behavior Laponite is known to induce thickening or thinning effects as efficient rheology modifiers, and the type of flow modification depends on the structure and composition of Laponite nanosilicates. Figure 2 shows the results of dynamic rheological characterization of bioinks with different Laponite RD contents. The interaction of Laponite RD nanodiscs with each other and with the agarose chains prior to gelation resulted in increased stability and dynamic moduli of nanocomposite bioinks (Figure 2a), characteristic behavior for nanosilicates, which was reported 13 ACS Paragon Plus Environment


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previously.25-27 Agarose forms a weak structure at 37 ˚C, which is far above its gelation temperature (31 ˚C). At this elevated temperature, which is determinant of the bioprinting process, bioinks based on only agarose cannot be printed due to their low shear moduli and instable weak interactions. However, the presence of a thickening rheology modifier like Laponite RD resulted in the enhancement of shear moduli over a large range of shear strains. The stability of LaponiteLaponite and Laponite-agarose interactions was highly dependent on the applied shear stress. The extent of intermolecular interactions, such as hydrogen bonding and the ionic interactions induced by the charged surface of Laponite RD, determined the yield shear stress upon, which the bioinks tend to flow. The yield stress of bioinks at 37 ˚C increased by increasing the Laponite content (Figure 2b), which is referenced as the crossover point of elastic (G’) and viscous (G”) moduli. A significant portion of this increase in yield stress can be associated to the formation of a 3D gel structure by Laponite nanosilicates, which is stabilized by both its viscosity and ionic interactions. Increasing the concentration of Laponite resulted in the formation of transient networks governed by electrostatic interactions between Laponite and the backbone of randomly coiled agarose molecules, while the conformational transition of agarose chains from coil to double helix was not occurred. Further, the linear viscoelastic region (LVR) increases with a higher Laponite content.


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Figure 2. Dynamic rheological characterization of bioinks containing various concentrations of Laponite RD. (a) Strain amplitude sweep profiles of bioinks with different Laponite RD concentrations at 37 ˚C. (b) Yield shear stress of the bioinks calculated from the cross-over of G’ and G” from the respective strain sweep tests (a) at 37 ˚C. Significant difference is denoted by different letters (p < 0.05) (c) Temperature sweep profiles of nanocomposite bioinks in the range of 55-20 ˚C showing the evolution of agarose networks over G’ by decreasing temperature. Inset shows the narrow range of the respective plot. The gel forming property of Laponite RD is due to its inherent ionic interaction and hence being stable at elevated temperatures. This can be confirmed by monitoring the change in dynamic moduli (G’ and G”) of bioinks in a wide temperature range prior forming a gel structure (Figure 2c). Stable elastic modulus (G’) of bioinks containing Laponite RD prior to the thermal gelation of agarose indicates the homogeneity and uniformity of mechanical properties of the pre-gel



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solutions, which is a key factor in process consistency during bioprinting. The gelation temperature of bioinks with different Laponite RD concentrations was not altered significantly by changing the nanosilicate content. However, all the nanocomposite bioinks showed higher gelation temperatures (33 ˚C compared with 31 ˚C), which indicates the interaction of nanosilicates with agarose chains during their conformational transition. The elastic modulus (G’) of agarose hydrogel at 20 ˚C (gel state) was found to be significantly less than the bioink with the lowest nanosilicates content (i.e., 19200 Pa for RD 0.0% compared with 31600 Pa for RD 1.0%), which suggests the existence of strong interactions between Laponite nanosilicates and agarose chains. However, the elastic moduli at 20 ˚C of bioinks containing nanosilicates did not show noteworthy dependency on the concentration of nanosilicates and were almost at the same value. Rheological characterization of nanocomposite bioinks revealed that processing of bioinks at 37 ˚C will not induce the gelation and at the same time, the nanosilicates embedded bioinks retained high enough elastic moduli to withstand reasonable amount of applied shear stress. Flow behavior of nanocomposite bioinks was characterized by rotational rheometry and the corresponding results are presented in Figure 3. The electrostatic interactions between Laponite nanodiscs induced formation of a gel structure that was readily broken down by the applied shear, and strong shear thinning effect was observed (Figure 3a,b). Although the bioinks containing nanosilicates possessed high viscosity at rest (i.e., while no shear is applied), they were able to flow easily by applying relatively small shear forces. The evolution of shear stress during incremental shear rates showed two distinctive regions. At low applied shear rates, an increase in shear stress was observed which can be attributed to the yield behavior of viscous nanocomposite hydrogels. This agrees well with the reported yield stress in Figure 2b. Further increase in deformation resulted in structure breakup, followed by hydrogel thinning. Laponite nanosilicates


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are known to exhibit dependency of restructuration on the sweep rate.28 Since the shear rate sweeps were performed at a logarithmic time scale, the Laponite mesostructure likely had the chance to undergo partial restructuration at intermediate sweep rates. This resulted in observation of a second gradual shear stress buildup during shear rate sweeps. Further increase in shear rate and decrease in sweep rate resulted in second breakup of the structure. By increasing the concentration of Laponite RD nanosilicates the restructuration rate at intermediate shear rates increased and less decrease in shear stress was observed. It should be noted that if the flow behavior of bioinks in the context of bioprinting process is the subject of interest, the shear thinning properties of agarose hydrogels are substantially not comparable with those of nanocomposite hydrogels at 37 ˚C, due to their low viscosity and weak intermolecular interactions. However, by considering RD 1.0% bioink as the reference material, it was observed that nanocomposite hydrogels showed consistent behavior in terms of initial viscosity, yield behavior, structure breakup, and restructuration while being imposed to incremental shear rates. Nanocomposite hydrogels bioinks demonstrated increased thixotropic behavior at constant shear deformation by increasing the nanosilicates concentration (Figure 3c). This has important implications for consideration of Laponite containing hydrogels as bioinks in 3D bioprinting process. Since the nozzle dimensions and applied pneumatic or mechanical pressures are usually set constant during bioprinting, hence the applied shear rate on bioinks during extrusion are defined with a known tolerance based on the design. Progressive drop in viscosity during extrusion will result in the loss of shape fidelity and precision, which will eventually affect the quality of print. Increasing the Laponite concentration resulted in more evident drop in viscosity at early deformation times. However, the percentage reduction in initial viscosity turned to be decreasing by increasing the Laponite RD concentration, although the initial viscosity was demolished to more extents; from 1.7 to 1.4 Pa·s (~ -18%) for



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RD 1.0% bioink compared to the drop of viscosity from 21.6 to 19.5 Pa·s (~ -10%) for RD 3.0% bioink. In this way, weaker gels faced with more viscosity loss percentage during deformation than the stronger gels. On the other hand, different extents of deformation will induce different dynamics of structural breakup and restructuration. Figure 3d shows the effect of cyclic low and high deformations during oscillation on the elastic modulus of nanocomposite hydrogels, which might mimic the process of extrusion during bioprinting of a construct with start/stop intervals. High strain values (i.e., extrusion) resulted in quick breakup of structure and flow of bioink accompanied with gradual decrease of moduli during deformation. This gradual decrease of moduli during high strain deformation was successively minimized by increasing the cycle numbers. However, a more profound increase in moduli during low deformation cycles (i.e., rest times) was observed which can be attributed to the continuous restructuration of Laponite nanosilicates within the hydrogel. This is due to the time dependency of re-orientation of Laponite particles to form a gel structure upon removal of the shear stress. 29 Moreover, a uniformity between the values of G’ during periodic high or low deformation cycles was observed, which indicates well consistency in mechanical properties of the nanocomposite hydrogels, including Laponite nanosilicates. Considering the difference between moduli during low and high deformation cycles, a selection criterion for compatibility of the bioink with bioprinting process can be drawn, in terms of providing a viable environment for encapsulated cells with minimal alteration of induced mechanical forces.30


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Figure 3. Characterization of flow behavior of nanocomposite bioinks for 3D printing. Shear rate sweeps of nanocomposite bioinks with different formulations and evolution of corresponding (a) viscosity () and (b) induced shear stress () during deformation. (c) Steady shear rate deformation of nanosilicates bioinks and their thixotropic behavior. (d) Change in elastic modulus (G’) of the bioinks during cyclic deformation with high (50%) and low (1%) shear strain periods to mimic the 3D bioprinting process. (e) 3D printed nanocomposite hydrogel structures containing 2 wt.% Laponite RD at 37 ˚C with excellent shape fidelity and structural integrity; scale bars: 5 mm. By taking the results of flow characterization and gelation dynamics of nanocomposite bioinks into account, hydrogels with 2 wt.% Laponite RD were chosen for 3D bioprinting (Figure 3e). The implemented strategy in selection of a suitable hydrogel bioink was in such a way that the selected



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bioink required the minimum amount of mechanical force to flow (based on the results of flow behavior characterization) while the dynamics of interactions between nanodiscs and also agarose chains provided enough mechanical stability for the extruded filament to retain its shape (dynamic rheological measurements) (Figure S3, Videos S1-2). The other key factor in selection of a proper bioink from different Laponite RD concentrations was the corresponding biocompatibility of different bioink formulations, which will be discussed in detail in the next section. 3.3. Cell Viability in Presence of Laponite RD Nanosilicates Different Laponite RD concentrations were chosen to study the effect of nanosilicates on viability of Fucci HeLa and NIH-3T3 cells. Design of experiments included three working concentrations of Laponite RD as low, intermediate, and high (1, 2, and 3 wt.%, respectively). Due to the strong autofluorescence of bioinks and extreme affinity of Laponite nanosilicates to adsorb fluorescence dyes, a staining-free approach was chosen for the visualization of encapsulated cells within the bioink formulations. For this purpose, Fucci HeLa cells were used for microscopy studies due to their inherent, cell-cycle progression dependent fluorescence properties. Fucci HeLa cells are genetically modified to give orange emission during G1 phase, and green emission in S/G2/M phases of the division cycle. 31 Having such a property, these cells and cell-cycle progression of the cells can be observed in real-time without the need for additional staining.


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Figure 4. Cellular evaluation and cytotoxicity of Laponite RD incorporated nanocomposite hydrogels. (a) Overlay z-stack confocal images of HeLa cells encapsulated in nanocomposite hydrogels for different culture periods. Red fluorescence indicates cells at G1 phase and yellowgreen fluorescence belong to cells at S/G2/M phases of cell division cycle; scale bars: 200µm. Metabolic activity of (b) Fucci HeLa and (c) NIH/3T3 cells normalized to the internal control (mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3). (d) The metabolic activity of encapsulated Fucci HeLa cells in Bioprinted bioinks (RD 2%) compared with the cast cell laden samples of the same composition after different post printing incubation periods.



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Figure 4 shows the cell-laden nanocomposite hydrogels with the corresponding results of cellular metabolic activity. All the hydrogel bioinks showed homogeneous distribution of encapsulated cells and compared with Laponite-free agarose hydrogels, cells in the nanocomposite hydrogels possessed their characteristic shapes as in 2D culture after early incubation time of 1 day, and they kept their morphology throughout the incubation period (Figure 4a). The autofluorescence property of Laponite RD resulted in generation of strong background radiations in nanocomposite hydrogels, which might be misleading in terms of the number of visible cells compared to the agarose hydrogel. In this way, any quantification on the cell numbers and behavior in nanocomposite samples will likely be inaccurate. The metabolic activity and relative viability of encapsulated Fucci HeLa and NIH/3T3 cells were evaluated at day 1, day 3, and day 7 of incubation (Figure 4b, and 4c). Normalized viability of Fucci HeLa cells in Laponite RD nanocomposite hydrogels were around 100 % in all experimental days. The spectrometry measurements for Laponite RD incorporated hydrogels were in similar range with the internal control, and Fucci HeLa cells did not show significant variations in cell viability for incremental concentrations of Laponite RD in all experimental days. The effect of bioprinting process on cell viability (i.e., the applied shear during extrusion) is of great importance in evaluation of the functionality of the bioinks. Passing through a fine nozzle during extrusion will result in exertion of high shear rates on the cells embedded within the bioinks, and the viscoelasticity of the surrounding polymer matrix plays a great role in stress transfer and shielding during the extrusion. Based on the rheological profiles of different bioinks, RD 2.0% bioink was used for bioprinting experiments with encapsulated Fucci HeLa cells. The results showed that, compared to the cast hydrogel bioinks of the same composition, encapsulated cells did not show any significant decrease in their metabolic activity (Figure 4d).


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Increased metabolic activity was observed in NIH/3T3 cell by increasing the Laponite RD concentration. On the other hand, the overall metabolic activity of encapsulated cells decreased throughout the culture period. There are similar reports available in the literature regarding the influence of Laponite nanosilicates on the metabolic activity of encapsulated cells. 32-34 However, most of the studies reported unchanged or decreased metabolic activity of encapsulated hydrogels throughout the culture period. We speculate that the main difference between these results rise from different experiment designs. In most of the reported studies, metabolic activity of cells cultured 2D in standard culture plates were chosen as the internal control for viability assessments, which is substantially different from the current design of experiments. In this study, agarose cellladen hydrogels with the same density of cells were used as the internal control, to demonstrate a clear view of the effect of presence of Laponite RD nanosilicates within the same culture conditions. In this way, encapsulated cells in the internal control hydrogel experienced the limitations of 3D cultures as in the sample groups. Due to the presence of a matrix, cells cultured in 3D cannot directly connect and communicate with each other as in 2D culture systems, highlighting the role of matrix in controlling cell-cell interactions. Limitations in delivery of nutrients and oxygen through the matrix; decreased proliferation rates; and decreased cell viability in 3D compared to 2D culture was already investigated in the literature.35-37 From this perspective, selection of the internal control group as cells encapsulated within agarose hydrogel seems to be reasonable. 3.4. Cell Spreading Assessment of Nanocomposite Bioinks The adhesion of cells on the material is one of the early signs of bioactivity and cell spreading is the natural outcome of the adhesion. Cell adhesion is one of the first requirements of a proper cell-material interaction. Cell adhesion is required and regulates many important cellular functions, 23 ACS Paragon Plus Environment


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including cell survival, migration, and cell-cell interaction.38 The effect of Laponite RD nanosilicates on cell adhesion in 2D culture was determined by using NIH/3T3 cells seeded on the surfaces of nanocomposite hydrogels. NIH/3T3 cells were chosen for this experiment rather than Fucci HeLa cells, due to ease of tracking the differences in their morphology during culture. NIH/3T3 cells have a fibroblastic morphology with elongated shapes, and Fucci HeLa cells are epithelial-like cells having polygonal shapes. Cells cultured on or within a hydrogel lacking binding sites will remain rounded, lacking the actin fibers. 39 Cells that are seeded on or embedded within bioactive hydrogels that provide binding sites for cells, will demonstrate spread morphology and extended actin fibers. In this way, the visualization of the material’s bioactivity by means of changes in cell morphology and spreading is more distinguishable with fibroblastic type cells rather than epithelial-like cells.

Figure 5. Cell spreading and morphology on nanocomposite hydrogel surfaces. (a-d) Bright field images of fixed 3T3 cells; scale bars: 50 m. (e-h) Confocal images of NIH/3T3 cells stained with fluorescent dyes; scale bars: 30 m. (i-k) high magnification images of spread cells on the 24 ACS Paragon Plus Environment

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surface of nanocomposite hydrogels. Cells tend to spread more with elongated F-actin filaments by increasing Laponite RD concentration; scale bars: 20 m. (l) the quantified spreading area of cells seeded on different bioinks with different Laponite RD content calculated from the microscopy images. It was observed by bright field microscopy that cells were attached on the nanocomposite hydrogels containing Laponite RD, while no attachment of cells took place for the pure agarose hydrogel (Figure 5a to 5d). A trend in the population of the attached cells on the nanocomposite hydrogel was observed, indicating that number of attached cells increased by higher Laponite RD content. Moreover, quantified spreading area of attached cells showed that the cells tend to spread more on the surface by increasing the Laponite RD content (Figure 5). Agarose is considered as an inert hydrogel due to the lack of binding sites within the structure, and no attachment of cells on the agarose hydrogel surface was expected. The observed trend of an increased cell attachment by higher Laponite RD content can be attributed to the cell-nanosilicates interactions due to the charged nature of nanosilicate. It has been shown previously that cell attachment to inert surfaces will enhance upon introducing charged densities.40 Increased Laponite content means the increased distribution of charged entities within the hydrogel, which will eventually contribute in more affinity of cells to spread on the surface. Confocal laser microscopy was used for the detailed visualization of cell spreading through factin and nuclei staining with fluorescent dyes (Figure 5i-k). Although the background signal from Laponite autofluorescence was noticeable, the spreading and extension of cells on the surfaces of nanosilicates was evident. This was in contrast to agarose hydrogels, were no cell attachment was observed. It should be noted that spreading analysis was done after a relatively short incubation period. However, even this short period of incubation was enough for cells to express elongated 25 ACS Paragon Plus Environment


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shapes and to produce high number of f-actin filaments on the Laponite RD incorporated hydrogels, which highlights the significant effect of Laponite RD nanosilicates in the enhancement of the bioactivity of agarose hydrogels. 4. CONLUSIONS Bioactive nanocomposite hydrogels based on agarose and Laponite nanosilicates were developed as strongly shear thinning bioinks for extrusion 3D bioprinting applications. The rheological characterization of bioinks showed that the incorporation of Laponite RD nanosilicates caused dramatic changes in the flow behavior. The interaction between Laponite nanosilicates and agarose chains led to higher elastic moduli (G’) of nanocomposite hydrogels and the gelation temperature. The induced shear thinning behavior of nanocomposite hydrogels together with time dependent restructuration of Laponite RD mesostructure contributed in formation of a highly printable hydrogel system, which can retain its shape after extrusion through a fine nozzle. Moreover, it was shown that the incorporation of Laponite RD nanosilicates significantly improved the bioactivity of nanocomposite hydrogels, by means of increased metabolic activity of encapsulated cells and the ability of cells to extend and change their morphology. Overall, this study reports that nanocomposite hydrogels based on agarose and Laponite nanosilicates possess a great potential to be used as bioactive and 3D printable bioinks for tissue engineering applications. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The scanning electron micrographs of the dried gels, optical photos of Laponite/agarose solutions, and photos and videos of 3d printing of pure and nanocomposite agarose gels. 26 ACS Paragon Plus Environment

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AUTHOR INFORMATION Corresponding Author Prof. Dr. B. Koc ([email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ A. Nadernezhad, O. S. Caliskan and F. Topuz contributed equally to this work. ORCID ID Bahattin Koc: 0000-0001-9073-8516 Fuat Topuz: 0000-0002-9011-4495 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT F. T. thanks the TUBITAK and European Union Marie Curie Co-Fund Brain Circulation Scheme Fellowship (Project No: 116C031). REFERENCES (1)

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TOC Graphic


Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with

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