Visible Light-Induced Hydrogelation of an Alginate ... - ACS Publications

Feb 2, 2018 - Application to Stereolithographic Bioprinting Using a Visible Light. Projector and ... desktop lamp. The hydrogelation process and the r...
4 downloads 28 Views 7MB Size
Download PDF
Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

pubs.acs.org/Biomac

Visible Light-Induced Hydrogelation of an Alginate Derivative and Application to Stereolithographic Bioprinting Using a Visible Light Projector and Acid Red Shinji Sakai,*,† Hidenori Kamei,† Toko Mori,‡,§,∥ Tomoki Hotta,† Hiromi Ohi,† Masaki Nakahata,† and Masahito Taya† †

Division of Chemical Engineering, Department of Materials Science and Engineering, Graduate School of Engineering Science, and SEEDS Program, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan § Shitennoji Senior High School, 1-11-73 Shitennoji, Tennoji, Osaka 543-0051, Japan ∥ JST Global Science Campus, 4-1-8 Honmachi, Kawaguchi, Saitama 332-0013, Japan ‡

S Supporting Information *

ABSTRACT: Visible light-induced hydrogelation is attractive for various biomedical applications. In this study, hydrogels of alginate with phenolic hydroxyl groups (Alg-Ph) were obtained by irradiating a solution containing the polymer, ruthenium II trisbipyridyl chloride ([Ru(bpy)3]2+) and sodium persulfate (SPS), with visible light. The hydrogelation kinetics and the mechanical properties of the resultant hydrogels were tunable by controlling the intensity of the light and the concentrations of [Ru(bpy)3]2+ and SPS. With appropriate concentrations of [Ru(bpy)3]2+ and SPS, the hydrogel could be obtained following approximately 10 s of irradiation using a normal desktop lamp. The hydrogelation process and the resultant hydrogel were cytocompatible; mouse fibroblast cells enclosed in the Alg-Ph hydrogel maintained more than 90% viability for 1 week. The solution containing AlgPh, [Ru(bpy)3]2+ and SPS was useful as a bioink for stereolithographic bioprinting. Cell-laden hydrogel constructs could be printed using the bioprinting system equipped with a visible light projector without a significant decrease in cell viability in the presence of photoabsorbent Acid Red 18. The hydrogel construct including a perfusable helical lumen of 1 mm in diameter could be fabricated using the printing system. These results demonstrate the significant potential of this visible light-induced hydrogelation system and the stereolithographic bioprinting using the hydrogelation system for tissue engineering and regenerative medicine.

1. INTRODUCTION Hydrogels have been widely studied and applied in the biomedical and biopharmaceutical fields.1−3 These hydrogels have been prepared through a variety of approaches. The most common approach is to cross-link polymer molecules in an aqueous solution. In particular, the rapid hydrogelation of aqueous polymer solutions under mild conditions for the encapsulation of cells and tissues has attracted increasing attention for applications such as tissue engineering, regenerative medicine, and drug delivery.3−6 To date, rapid and mild hydrogelation have been achieved through various types of cross-linking approaches such as click chemistry, enzymemediated cross-linking, and Michael-type addition.3 Hydrogelation induced by irradiation using ultraviolet (UV) or visible light is also a well-known approach.3 An advantage of lightinduced hydrogelation is the ease of control of the progress of cross-linking by changing the light exposure time and light intensity. Comparing the use of UV light and visible light, using visible light is more suitable for fabricating cell-laden hydrogels. The adverse effects of UV light irradiation such as inducing chromosomal and genetic instability are well-known.7 In © XXXX American Chemical Society

addition, visible light demonstrates a deeper penetration that is effective for overall hydrogelation efficiency for thick constructs. Recently, the utility and the potential of visible light-induced hydrogelation mediated by ruthenium II trisbipyridyl chloride ([Ru(bpy)3]2+) and sodium persulfate (SPS) were reported.8−10 In this system, methacryloyl groups10 and phenolic hydroxyl (Ph) groups, including those in tyrosine residues,8,9,11 are cross-linked to create hydrogels. The Ph groups have also been cross-linked through horseradish peroxidase-catalyzed reactions. For enzymatic cross-linking, various Polymer-Phs have been developed by chemically incorporating Ph groups into natural and synthetic polymers.12−17 To date, to the best of our knowledge, poly(vinyl alcohol)-Ph (PVA-Ph) is the sole Polymer-Ph that has been applied in [Ru(bpy)3]2+/SPS-mediated hydrogelation.8,9 In addition, detailed studies of the hydrogelation kinetics have not been performed for this hydrogelation system.8,9,11,18 To Received: December 28, 2017 Revised: January 19, 2018

A

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules extend the biomedical application of the [Ru(bpy)3]2+/SPSmediated hydrogelation system, further investigations of different materials, and more detailed studies of the hydrogelation mechanism are required. In the current study, we applied an alginate derivative with chemically incorporated Ph groups (Alg-Ph) in a visible light induced hydrogelation system (Figure 1a,b). Alginate is a

2. EXPERIMENTAL SECTION 2.1. Materials. Sodium alginate with a high guluronic acid content (molar ratio of mannuronic acid to guluronic acid of 0.65) and an average molecular weight of 70 000 Da was obtained from Kimica (Kimica I-1G, Tokyo, Japan). Alg-Ph was obtained by conjugating the alginate and tyramine hydrochloride (Tokyo Chemical Industry, Tokyo, Japan) via the carbodiimide-mediated condensation of the carboxyl groups of alginate and amino groups of tyramine, as previously reported.15 The content of Ph groups in the Alg-Ph was 2.8−3.0 × 10−4 mol-Ph/g-Alg-Ph. Water-soluble carbodiimide (Peptide Institute, Osaka, Japan) and N-hydroxysuccinimide (Wako Pure Chemical Industries) were used for the conjugation. Tris(2,2′bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+), SPS, and Acid Red 18 (Acid Red) were purchased from Sigma-Aldrich (MO, USA), Wako Pure Chemical Industries (Osaka, Japan), and Tokyo Chemical Industry, respectively. The UV−visible absorption spectrum of [Ru(bpy)3]2+ aqueous solution is shown in Figure S2. Mouse fibroblast 10T1/2 cells were obtained from the Riken Cell Bank and cultured in Dulbecco’s modified Eagle’s medium containing 10 v/v% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. 2.2. Hydrogelation Kinetics and Mechanical Properties. Rheological measurements to assess the hydrogelation kinetics and mechanical properties of the hydrogels were performed for phosphatebuffered saline (PBS, pH 7.4) containing 1.0 w/v% Alg-Ph using a rheometer (HAAKE MARS III, Thermo Fisher Scientific, MA, US) equipped with a parallel plate geometry. The top plate was a stainlesssteel plate of a 25 mm radius. The bottom plate was a transparent glass plate to allow for irradiation of the samples. The sample solution (200 μL) was poured on the lower plate, and then the upper plate was set at the distance of 0.1 mm. Time-course studies were conducted at 25 °C using an oscillatory shear strain of 3% and a frequency of 0.3 Hz. Light, with a spectrum shown in Figure S1a and 33 mW/m2 at 452 nm wavelength (33 mW/m2@452) was emitted from a desktop lamp (LTL-CK9N, Ohm Electric, Inc., Figure S1b) to irradiate the samples and study the effect of [Ru(bpy)3]2+ (0.1−3.0 mM) and SPS (0.2−3.0 mM) concentrations on hydrogelation. The effect of light intensity was determined by changing the distance between the light source and the samples. The light intensity at 452 nm was measured using an illuminance meter (CL-70F, Konica Minolta, Tokyo, Japan). Gelation time was defined as the time it took to achieve the intersection point of the transitions of the storage elastic moduli (G′) and loss elastic moduli (G′′).22 2.3. Effect on Cells Encapsulated in the Hydrogels. The effect of encapsulating cells in the Alg-Ph hydrogel through the visible lightinitiated hydrogelation was investigated by measuring the viability of 10T1/2 cells in the gel. The cells were encapsulated in hydrogels (5.0 × 105 cells/mL, 1 mm gel thickness, 4 mm radius) composed of PBS containing 1.0 w/v% Alg-Ph, 3.0 mM [Ru(bpy)3]2+, and 3.0 mM SPS. To evaluate the effect of Acid Red on the cells, cell-laden hydrogels of the same composition were produced but containing 0.06 w/v% Acid Red. The solutions were irradiated with the light (1.2 W/m2@452, Figure S1c) for 10 min. The resultant cell-laden hydrogels were rinsed several times using medium and incubated at 37 °C. After 1, 3, and 7 days of incubation, the cells were stained using Calcein-AM and propidium iodide (PI) to determine cell viability. The statistical significance of the difference in viabilities of the cells enclosed in the specimens prepared with and without using Acid Red, and that of the cells at each time period of incubation was evaluated by an unpaired Student’s t-test. 2.4. Stereolithographic Bioprinting. A commercially available stereolithographic printing system (LittleRP, LittleRP, CA, USA) equipped with a visible light projector (H6510BD, Acer, Taiwan) was used to prepare 3D hydrogel constructs. PBS containing 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+, 3.0 mM SPS, and 0−0.08 w/v% Acid Red (total volume of 12 mL) was poured into a transparent plastic dish and exposed to visible light with an intensity of 0.5−2.3 W/m2@ 452 and a spectra shown in Figure S1c. The thickness and light irradiation time of each hydrogel layer were set at 25−500 μm and 1−

Figure 1. (a) Schematic of Alg-Ph hydrogel formation through crosslinking of Ph groups of Alg-Ph through visible light irradiation in the presence of [Ru(bpy)3]2+ and SPS. (b) Photographs of an Alg-Ph, [Ru(bpy)3]2+ and SPS mixture before and after 15 min of light irradiation using a normal indoor fluorescent lamp. (c) Photograph of a 3D printer used in this study and a schematic showing printing with visible light irradiation from a visible light projector.

naturally occurring polysaccharide and has been widely investigated in biomedical and biopharmaceutical applications.19,20 We demonstrate the potential to obtain hydrogels from Alg-Ph, including controllable hydrogelation times and mechanical properties, depending on the concentrations of [Ru(bpy)3]2+ and SPS, and the intensity of light. In addition, we demonstrate the cytocompatibility of the materials and the hydrogelation process, using a cell-laden Alg-Ph hydrogel. Furthermore, we show the potential of a solution containing Alg-Ph, [Ru(bpy)3]2+, and SPS as a bioink for threedimensional (3D) bioprinting using a stereolithographic bioprinting system composed of a commercially available visible light projector (Figure 1c). The light irradiated from the light sources used in this study contained negligible ultraviolet (UV) light (Figure S1a, c,d). Compared with other popular bioprinting technologies using nozzles to print bioink containing cells, such as inkjet bioprinting and microextrusion bioprinting, this nozzle-free system has a low risk in terms of damaging cells during printing.21 In addition, an attractive feature of stereolithographic bioprinting using projectors is that the printing time depends only on the thickness of the constructs but not on the complexity or the size of each layer.21 B

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules 6 s, respectively. Cell-laden hydrogel was printed from PBS containing 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+, 3.0 mM SPS, 0.06 w/v% Acid Red, and 5.0 × 105 cells/mL by irradiating 1.2 W/m2@452 light for 3 s to each printing layer of 50 μm thickness. The resultant specimens were rinsed using medium and incubated at 37 °C. At 1, 3, and 7 days of incubation, the viability of the encapsulated cells was evaluated through staining using Calcein-AM and PI.

3. RESULTS AND DISCUSSION 3.1. Hydrogelation Kinetics and Mechanical Properties. We first studied the hydrogelation of an Alg-Ph aqueous solution, produced through visible light irradiation, by putting a glass vessel containing 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+ and 1.0 mM SPS under a conventional fluorescent indoor lamp with an intensity of 7 mW/m2@452 (Figure S1d). A hydrogel was obtained after 15 min of irradiation (Figure 1b). This result was surprising because special lamps for curing resins have been used in previous studies using [Ru(bpy)3]2+ and SPS for photoinitiated hydrogelation.8,9 The hydrogelation was also confirmed by the light irradiation-dependent transition of the mechanical properties (Figure 2). The values of G′ and G′′

Figure 3. Effects of concentrations of (a) [Ru(bpy)3]2+ and (b) SPS, and (c) light intensity on the hydrogelation time of PBS containing 1.0 w/v% Alg-Ph, [Ru(bpy)3]2+ and SPS. Data: mean ± SD (n = 5). Light in panels a and b means the light intensity at 452 nm. Light spectra are shown in Figure S1a.

Figure 2. Effect of visible light irradiation on the transitions of G′ and G′′ of PBS containing 1.0 w/v% Alg-Ph, 0.1 mM [Ru(bpy)3]2+ and 1.0 mM SPS. Off: No irradiation; On: light irradiation.

increased only during the period of light irradiation. The tunability of the cross-linking, simply using an on−off light irradiation, is attractive for a variety of applications in which the mechanical properties of hydrogels affect their functionality. The ease in controlling the degree of cross-linking is an advantage of photoinduced hydrogelation compared with the other hydrogelation methods. We next investigated the effects of concentrations of [Ru(bpy)3]2+ and SPS, and the intensity of the light on the hydrogelation kinetics and mechanical properties of the resultant hydrogels. A detailed study of the effects of these factors has not been performed previously.8,9,11,18 As shown in Figure 3, the hydrogelation kinetics strongly depended on these factors: the hydrogelation time of a 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+ and 1.0 mM SPS sample at 33 mW/m2@452 was 36 ± 3 s. The hydrogelation time decreased with increasing concentrations of [Ru(bpy)3]2+ (Figure 3a) and SPS (Figure 3b), and increased with decreasing light intensity (Figure 3c). The shortest hydrogelation time was 13 ± 1 s (Figure 3b). The storage elastic modulus (G′) of the individual samples continued to increase significantly after the hydrogelation, for around 300 to 1200 s (Figure 4). The maximum value of G′ seemed to plateau (ca. 550 Pa) when prolonging the light irradiation time beyond a certain [Ru(bpy)3]2+ concentration (Figure 4a) and light intensity

Figure 4. Effects of concentrations of (a) [Ru(bpy)3]2+ and (b) SPS, and (c) light intensity on G′ of PBS containing 1.0 w/v% Alg-Ph, [Ru(bpy)3]2+ and SPS. Data: mean ± SD (n = 5). Light in panels a and b means the light intensity at 452 nm. Light spectra are shown in Figure S1a.

(Figure 4c). By contrast, the maximum value of G′ strongly depended on SPS concentration (Figure 4b). These results can be understood intuitively considering the reaction induced by visible light irradiation. The cross-linking mechanism using [Ru(bpy)3]2+ and SPS through visible light irradiation is known as following three steps:8 (1) [Ru(bpy)3]2+ is excited to Ru(III) by absorbing photons in the visible light, and then an electron is donated to SPS. (2) The SPS dissociates into radicals and sulfate anions. (3) The radicals propagate through the functional Ph groups resulting in cross-links between Ph groups. In theory, the amount of [Ru(bpy)3]2+ is not reduced, C

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

The round-shaped morphology of the encapsulated cells is not specific for the photoinduced Alg-Ph hydrogel. It has been reported that adherent cells maintained a round shape in the Alg-Ph hydrogels obtained through the horseradish peroxidasecatalyzed hydrogelation without obvious growth due to the lack of moieties for cell adhesion,23,24 as with those in Ca-alginate hydrogels.25 The promising result for cytocompatibility was surprising considering previously reported results:8 Lim et al. reported that almost all mouse fibroblast cells died after being encapsulated in a PVA-Ph hydrogel without using an antioxidant material, gelatin, to scavenge reactive oxygen species resulting from the radicals generated during the photoinduced hydrogelation.8 In another paper by the same authors, approximately 90% viability was achieved in breast adenocarcinoma cells following encapsulation in a hydrogel containing antioxidant gelatin derivative methacryloyl groups, created through the same photoinduced hydrogelation method.10 In the current study, the cell-laden polymer-Ph hydrogels maintained cell viability without additives to protect the cells. This may be attributed to the fact that it was possible to achieve hydrogelation without excess light irradiation, based on the hydrogelation kinetics and mechanical property studies. Because of the existence of varieties of Polymer-Phs developed for horseradish peroxidase-catalyzed hydrogelation,12−16 the results obtained in this study could increase the feasibility of [Ru(bpy)3]2+/SPS-mediated hydrogelation systems for biomedical applications. 3.3. Stereolithographic Bioprinting. Finally, we investigated the potential of a solution containing Alg-Ph, [Ru(bpy)3]2+, and SPS as a bioink for a stereolithographic 3D bioprinting system equipped with a commercially available business projector (Figure 1c). In a preliminary study, we attempted to prepare hydrogel constructs based on the blueprints shown in Figure 6a and using the bioink containing no Acid Red by irradiating 1.2 W/m2@452 light for 3 s to each printing layer of 50 μm thickness. However, the resultant constructs were poorly defined (Figure 6b). We hypothesized that light scattering caused these suboptimal results. Therefore, we added Acid Red to the bioink, which is used as a food dye, as an absorbent of the scattered light. In fact, the absorbance at 452 nm decreased with increasing Acid Red concentration (Figure S3). The addition of 0.06 w/v% Acid Red to the bioink drastically improved its printability. The resulting hydrogel constructs had geometries comparable with those in the blueprints (Figure 6a), as shown in Figure 6c. A further increase in Acid Red to 0.08 w/v% resulted in an impairment of hydrogel construct formation (Figure 6d). This result can be explained by the excess absorption of light by Acid Red. This explanation is supported by the results obtained from the measurements of the hydrogelation time and the light irradiation time-dependent transition of the storage elastic modulus (G′) of the specimens (Figure 7). The hydrogelation time (Figure 7a) and the G′ (Figure 7b) increased and decreased, respectively, with increasing Acid Red concentration. A concern of the use of Acid Red was the cytotoxicity. Thus, we evaluated it by measuring the viability of cells encapsulated in a hydrogel containing 0.06 w/v% Acid Red through 10 min of light irradiation. The viability of the cells at 1 day of encapsulation was 95.8 ± 2.1%. There was no significant difference in the viability even at 7 days of encapsulation (98.8 ± 0.6%, Figure 5c,d,e, p = 0.13). In addition, no significant decrease in the viability was found compared to those encapsulated in Alg-Ph hydrogel construct without using Acid

but the amount of SPS reduces with the progress of the reaction. A notable result of the hydrogelation kinetics study is that the shortest hydrogelation time of around 10 s (Figure 3) was achieved using a conventional desktop lamp (Figure S1b) as a light source. This result means that it is possible to establish cost-effective hydrogelation systems with the lamps used in our daily lives. 3.2. Effect on Cells. We next studied the effect on cells of encapsulation in an Alg-Ph hydrogel. The cytocompatibility of the photoinduced Alg-Ph hydrogel and the hydrogelation process was confirmed from the viability of cells. The cells encapsulated in an Alg-Ph hydrogel created through irradiation at 1.2 W/m2@452 for 10 min of a cell-suspension containing 1.0 w/v% Alg-Ph, 3.0 mM [Ru(bpy)3]2+, and 3.0 mM SPS showed around 95% viability during 7 days of incubation with a round-shaped morphology (Figure 5a,b,e). There was no significant difference between the viabilities at 1 and 7 days of incubation (p > 0.63). No obvious increase in density of encapsulated cells was found during the period (Figure 5a,b).

Figure 5. Fluorescence microphotographs of 10T1/2 cells after (a,c) 1 and (b,d) 7 days of encapsulation in Alg-Ph hydrogels created from PBS containing 1.0 w/v% Alg-Ph, 3.0 mM [Ru(bpy)3]2+, 3.0 mM SPS and 5.0 × 105 cells/mL through 10 min light irradiation. Prepared (a,b) without and (c,d) with 0.06 w/v% Acid Red. Live and dead cells show green and red fluorescence, respectively. (e) Viabilities of the encapsulated 10T1/2 cells in Alg-Ph hydrogels prepared (white column) without and (black column) with 0.06 w/v% Acid Red. Data: means ± SD (n = 3). D

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 6. (a) Blueprint of hydrogels, and (b−k) hydrogels obtained using PBS containing 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+, and 3.0 mM SPS for evaluating the effects of (b−d) Acid Red concentration, (c,e−g) layer thickness, (c,h,i) light intensity, and (c,j,k) light irradiation time of each hydrogel layer. The conditions applied for each hydrogel construct preparation are shown in and under each panel. The light with spectra shown in Figure S1c was irradiated.

not decrease the viability of the encapsulated cells. The viabilities of 10T1/2 cells at 1, 3, and 7 days of bioprinting were 91.3 ± 3.0%, 97.9 ± 0.4% and 99.6 ± 0.4% (n = 3), respectively (Figure S4). As described above, the gradual increase in viabilities from day 1 to day 3 and from day 3 to day 7 (p < 0.04) are likely caused by the decomposition of dead cells over time without a notable increase in new dead cells. These results demonstrate the potential of a solution containing Alg-Ph, [Ru(bpy)3]2+, SPS, and Acid Red as a bioink for stereolithographic bioprinting using a visible light projector. The encapsulated cells maintained a round shape and showed no obvious growth during 7 days of culture (Figure S4) as the same with those encapsulated without being exposed to the bioprinting process (Figure 5c,d). These results indicate that the Alg-Ph hydrogels obtained through the stereolithographic bioprinting are suitable for encapsulating the cells, which do not need spreading and migration for expressing biological functions.26−28 In the operation of the stereolithographic 3D bioprinting system, printing layer thickness, light intensity, and light irradiation time are controllable parameters. Therefore, we next studied the effects of these parameters by changing one of the parameters applied for fabricating the hydrogel construct shown in Figure 6c (50 μm layer thickness, 1.2 W/m2@452 light intensity, 3 s light irradiation for each layer printing). The decrease and increase in layer thickness to 25 and 100 μm, respectively, did not hinder hydrogel construct formation (Figure 6e,f). However, a further increase to 500 μm completely prevented 3D hydrogels formation (Figure 6g). The decrease in printability with increasing the layer thickness can be explained by an increase in light scattering and a hindrance of hydrogel formation resulting from a decrease of

Figure 7. Effect of concentration of Acid Red on (a) hydrogelation time and (b) G′ of PBS containing1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+, and 3.0 mM SPS. Data: mean ± SD (n = 4 or 5). The light with spectra shown in Figure S1a and 33 mW/m2@452 was irradiated.

Red (Figure 5e, p > 0.18). This result demonstrates the possibility of using Acid Red for fabricating cell-laden hydrogel constructs. In addition, stereolithographic bioprinting also did E

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules light intensity. It is known that thinner layer thickness results in 3D constructs with smoother surfaces.29 For the effect of light intensity, both the decrease and increase in light intensity from 1.2 W/m2@452 reduced the printability (Figure 6h,i). The printability reduction was also caused by decreasing the light irradiation time to 1 s, and by increasing the time to 6 s, respectively (Figure 6j,k). A notable result was that circular column- and quadrangular prism-shaped hydrogel constructs did not form in spite of the formation of the hemisphere- and quadrangular pyramid-shaped hydrogel constructs. The impairments of hydrogel constructs formation attributed to the decreases in light intensity and light irradiation time can be understood intuitively by a decrease in the light energy consumed for each layer formation. By contrast, the reason for the impairments induced by the increases of these values is unclear. A possible explanation is that the circular column- and quadrangular prism-shaped hydrogel constructs could not withstand the load attributed to the adhesion strength of the hydrogels to the bottom surface of the ink bath. Hydrophobic interaction between the hydrogel and the bottom surface of the ink bath would increase with increasing the density of crosslinked hydrophobic Ph moieties. It was reported that the hydrophobicity of hydrogel increased with increasing the density of cross-linked Ph moieties.30 The formation of hydrogels with a higher cross-link density of Ph moieties is indicated by the formation of harder hydrogels through the irradiating of higher light energy as shown in Figure 4. The reason for the formation of the hemisphere- and quadrangular pyramid-shaped hydrogel constructs is that the load attributed to the adhesion of the hydrogel to the bottom of the ink bath decreased as the printed layer increases because of the decrease in printing area with increasing printing depth. By contrast, the printing area is constant in the fabrication of the circular column- and quadrangular prism-shaped hydrogel constructs. As a final study, we attempted to print the hydrogel constructs demonstrating the usefulness of the current printing approach using the bioink containing 1.0 w/v% Alg-Ph, 1.0 mM [Ru(bpy)3]2+, 3.0 mM SPS, and 0.06 w/v% Acid Red. The operation parameters of the stereolithographic bioprinting system were set to be 1.2 W/m2@452 light irradiation for 3 s to each printing layer of 50 μm thickness. Figure 8 shows blueprints, a lattice-shaped hydrogel construct (dark orange part) printed on a preprinted thin base hydrogel layer (light orange part) (Figure 8a), and a rectangular prism-shaped hydrogel including a helical lumen with 1 mm in diameter (Figure 8b) based on the blueprints. Individual frames (0.5 mm width) of the lattice-shaped hydrogel were distinguishable until around 0.2 mm distance. The minimum distinguishable distance is comparable to the lattice-shaped hydrogel constructs made from the bioinks containing sodium alginate using microextrusion printing,31−33 and inkjet printing systems.34 For the hydrogel construct including a helical lumen structure, the helical structure was smooth, and it was possible to flow aqueous solution in the lumen by introducing from an upper opening. Development of the methods for fabricating cell-laden 3D constructs having a perfusable vascular-network is a challenging issue in tissue engineering field.35−37 It would be difficult to prepare 3D hydrogel constructs containing a smooth helical lumen of 1 mm diameter using conventional microextrusion printing and inkjet printing systems. The reason is that 3D hydrogel constructs are fabricated by layering hydrogel filaments of 0.1−0.5 mm in diameter and droplets of 0.1 mm in diameter, respectively, in these printing systems.

Figure 8. (a) Blueprint and printed lattice-shaped hydrogel. (b) Blueprint and printed rectangular prism-shaped hydrogel containing a perfusable helical lumen (1 mm in diameter) structure. Blue color in panel b is a blue ink flowed from an upper opening of the lumen. The light with spectra shown in Figure S1c and 1.2 W/m2@452 was irradiated.

4. CONCLUSIONS In the current paper, we have reported the hydrogelation of an Alg-Ph solution containing [Ru(bpy)3]2+ and SPS. Hydrogelation was achieved through irradiation using visible light to induce the cross-linking of Ph groups in Alg-Ph. The hydrogelation and mechanical properties of the resultant hydrogels could be controlled using varied light intensity, and different concentrations of [Ru(bpy)3]2+ and SPS. The shortest time required for the hydrogelation of an Alg-Ph solution through irradiation using a normal desktop lamp was approximately 10 s. The superior cytocompatibility of the Alg-Ph hydrogel was confirmed by more than 90% viability of 10T1/2 cells encapsulated in the hydrogel at 24 h of culture. We applied the visible light-induced Alg-Ph hydrogelation to a stereolithographic bioprinting process using a visible light projector. Hydrogel constructs, in accordance with the blueprints, were obtained by adding Acid Red as an absorbent of the scattered light to the mixture of Alg-Ph, [Ru(bpy)3]2+, and SPS. In addition, cells were encapsulated in the bioprinted Alg-Ph hydrogel without a significant decrease in viability. The hydrogel construct having a perfusable helical lumen of 1 mm in diameter was obtained using the printing system consisting of the bioink and the stereolithographic printer equipped with a F

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

(6) Kondiah, P. J.; Choonara, Y. E.; Kondiah, P. P.; Marimuthu, T.; Kumar, P.; du Toit, L. C.; Pillay, V. A review of injectable polymeric hydrogel systems for application in bone tissue engineering. Molecules 2016, 21 (11), 1580. (7) Dahle, J.; Kvam, E.; Stokke, T. Bystander effects in UV-induced genomic instability: antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation. J. Carcinog. 2005, 4, 11. (8) Lim, K. S.; Ramaswamy, Y.; Roberts, J. J.; Alves, M. H.; PooleWarren, L. A.; Martens, P. J. Promoting cell survival and proliferation in degradable poly(vinyl alcohol)-tyramine hydrogels. Macromol. Biosci. 2015, 15 (10), 1423−1432. (9) Lim, K. S.; Alves, M. H.; Poole-Warren, L. A.; Martens, P. J. Covalent incorporation of non-chemically modified gelatin into degradable PVA-tyramine hydrogels. Biomaterials 2013, 34 (29), 7097−7105. (10) Lim, K. S.; Schon, B. S.; Mekhileri, N. V.; Brown, G. C. J.; Chia, C. M.; Prabakar, S.; Hooper, G. J.; Woodfield, T. B. F. New visiblelight photoinitiating system for improved print fidelity in gelatin-based bioinks. ACS Biomater. Sci. Eng. 2016, 2, 1752−1762. (11) Jeon, E. Y.; Hwang, B. H.; Yang, Y. J.; Kim, B. J.; Choi, B. H.; Jung, G. Y.; Cha, H. J. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking. Biomaterials 2015, 67, 11−19. (12) Jin, R.; Hiemstra, C.; Zhong, Z.; Feijen, J. Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates. Biomaterials 2007, 28, 2791−2800. (13) Park, K. M.; Lee, Y.; Son, J. Y.; Bae, J. W.; Park, K. D. In situ SVVYGLR peptide conjugation into injectable gelatin-poly(ethylene glycol)-tyramine hydrogel via enzyme-mediated reaction for enhancement of endothelial cell activity and neo-vascularization. Bioconjugate Chem. 2012, 23, 2042−2050. (14) Sakai, S.; Yamada, Y.; Zenke, T.; Kawakami, K. Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidasecatalyzed enzymatic reaction. J. Mater. Chem. 2009, 19 (2), 230−235. (15) Sakai, S.; Kawakami, K. Synthesis and characterization of both ionically and enzymatically crosslinkable alginate. Acta Biomater. 2007, 3, 495−501. (16) Kurisawa, M.; Chung, J. E.; Yang, Y. Y.; Gao, S. J.; Uyama, H. Injectable biodegradable hydrogels composed of hyaluronic acidtyramine conjugates for drug delivery and tissue engineering. Chem. Commun. 2005, 34, 4312−4314. (17) Ren, K.; He, C.; Xiao, C.; Li, G.; Chen, X. Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering. Biomaterials 2015, 51, 238−249. (18) Bjork, J. W.; Johnson, S. L.; Tranquillo, R. T. Rutheniumcatalyzed photo cross-linking of fibrin-based engineered tissue. Biomaterials 2011, 32 (10), 2479−2488. (19) Venkatesan, J.; Bhatnagar, I.; Manivasagan, P.; Kang, K. H.; Kim, S. K. Alginate composites for bone tissue engineering: a review. Int. J. Biol. Macromol. 2015, 72, 269−281. (20) Bidarra, S. J.; Barrias, C. C.; Granja, P. L. Injectable alginate hydrogels for cell delivery in tissue engineering. Acta Biomater. 2014, 10 (4), 1646−1662. (21) Wang, Z.; Abdulla, R.; Parker, B.; Samanipour, R.; Ghosh, S.; Kim, K. A simple and high-resolution stereolithography-based 3D bioprinting system using visible light crosslinkable bioinks. Biofabrication 2015, 7 (4), 045009. (22) Djabourov, M.; Leblond, J.; Papon, P. Gelation of aqueous gelatin solutions. II. Rheology of the sol-gel transition. J. Phys. 1988, 49 (2), 333−343. (23) Sakai, S.; Kawakami, K. Both ionically and enzymatically crosslinkable alginate-tyramine conjugate as materials for cell encapsulation. J. Biomed. Mater. Res., Part A 2008, 85 (2), 345−351. (24) Sakai, S.; Hashimoto, I.; Ogushi, Y.; Kawakami, K. Peroxidasecatalyzed cell encapsulation in subsieve-size capsules of alginate with phenol moieties in water-immiscible fluid dissolving H2O2. Biomacromolecules 2007, 8 (8), 2622−2626.

visible light projector. Considering a preceding paper demonstrating the hydrogelation of PVA-Ph through the same visible light-induced cross-linking system,8,9 the findings obtained in the current study demonstrate the versatility, utility, and significant potential of this visible light-induced hydrogelation system and the stereolithographic bioprinting using the hydrogelation system for various biomedical applications. While the great potential, a concern when applying biomedical applications is the safety issue of [Ru(bpy)3]2+, SPS, and Acid Red remaining in resultant hydrogels. In particular, [Ru(bpy)3]2+ and SPS will generate radicals when the hydrogels are exposed to visible light and may induce unwanted effects. To enhance the potential of the current technology, the study on the removal of them from the resultant hydrogels is currently under investigation and will be reported in our future reports.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01827. Intensity spectra of the light used to irradiate samples (Figure S1), UV−visible absorbance spectrum of [Ru(bpy)3]2+ aqueous solution (Figure S2), effect of Acid Red concentration on the absorbance at 452 nm (Figure S3), and fluorescence micrographs of live/dead cells encapsulated in a bioprinted Alg-Ph hydrogel using a stereolithographic bioprinting system at 1, 3, and 7 days after encapsulation (Figure S4) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shinji Sakai: 0000-0002-1041-4798 Funding

Japan Society for the Promotion of Science KAKENHI Grant Numbers 15H04194 and 16H02423. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers 15H04194 and 16H02423, and Global Science Campus, Japan Science and Technology Agency, in 2016.

■

REFERENCES

(1) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2002, 54, 3−12. (2) Vashist, A.; Kaushik, A.; Vashist, A.; Jayant, R. D.; Tomitaka, A.; Ahmad, S.; Gupta, Y. K.; Nair, M. Recent trends on hydrogel based drug delivery systems for infectious diseases. Biomater. Sci. 2016, 4 (11), 1535−1553. (3) Ko, D. Y.; Shinde, U. P.; Yeon, B.; Jeong, B. Recent progress of in situ formed gels for biomedical applications. Prog. Polym. Sci. 2013, 38 (3−4), 672−701. (4) Macaya, D.; Spector, M. Injectable hydrogel materials for spinal cord regeneration: a review. Biomed. Mater. 2012, 7 (1), 012001. (5) Norouzi, M.; Nazari, B.; Miller, D. W. Injectable hydrogel-based drug delivery systems for local cancer therapy. Drug Discovery Today 2016, 21 (11), 1835−1849. G

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules (25) Hunt, N. C.; Smith, A. M.; Gbureck, U.; Shelton, R. M.; Grover, L. M. Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation. Acta Biomater. 2010, 6 (9), 3649−3656. (26) Kim, J.; Sachdev, P.; Sidhu, K. Alginate microcapsule as a 3D platform for the efficient differentiation of human embryonic stem cells to dopamine neurons. Stem Cell Res. 2013, 11 (3), 978−989. (27) Bozza, A.; Coates, E. E.; Incitti, T.; Ferlin, K. M.; Messina, A.; Menna, E.; Bozzi, Y.; Fisher, J. P.; Casarosa, S. Neural differentiation of pluripotent cells in 3D alginate-based cultures. Biomaterials 2014, 35 (16), 4636−4645. (28) van Susante, J. L.; Buma, P.; van Osch, G. J.; Versleyen, D.; van der Kraan, P. M.; van der Berg, W. B.; Homminga, G. N. Culture of chondrocytes in alginate and collagen carrier gels. Acta Orthop. Scand. 1995, 66 (6), 549−556. (29) Pham, D. T.; Gault, R. S. A comparison of rapid prototyping technologies. Int. J. Mach. Tool. Manu. 1998, 38 (10−11), 1257−1287. (30) Ogushi, Y.; Sakai, S.; Kawakami, K. Phenolic hydroxy groups incorporated for the peroxidase-catalyzed gelation of a carboxymethylcellulose support: cellular adhesion and proliferation. Macromol. Biosci. 2009, 9, 262−267. (31) Seidel, J.; Ahlfeld, T.; Adolph, M.; Kummritz, S.; Steingroewer, J.; Krujatz, F.; Bley, T.; Gelinsky, M.; Lode, A. Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds. Biofabrication 2017, 9 (4), 045011. (32) Zhang, Y. S.; Arneri, A.; Bersini, S.; Shin, S. R.; Zhu, K.; GoliMalekabadi, Z.; Aleman, J.; Colosi, C.; Busignani, F.; Dell’Erba, V.; Bishop, C.; Shupe, T.; Demarchi, D.; Moretti, M.; Rasponi, M.; Dokmeci, M. R.; Atala, A.; Khademhosseini, A. Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomaterials 2016, 110, 45−59. (33) Akkineni, A. R.; Ahlfeld, T.; Funk, A.; Waske, A.; Lode, A.; Gelinsky, M. Highly concentrated alginate-gellan gum composites for 3D plotting of complex tissue engineering scaffolds. Polymers 2016, 8 (5), 170. (34) Arai, K.; Tsukamoto, Y.; Yoshida, H.; Sanae, H.; Mir, T. A.; Sakai, S.; Yoshida, T.; Okabe, M.; Nikaido, T.; Taya, M.; Nakamura, M. The development of cell-adhesive hydrogel for 3D printing. Int. J. Bioprinting 2016, 2 (2), 10. (35) Liu, Y.; Sakai, S.; Taya, M. Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules. Heliyon 2016, 2, e00067. (36) Miller, J. S.; Stevens, K. R.; Yang, M. T.; Baker, B. M.; Nguyen, D. H.; Cohen, D. M.; Toro, E.; Chen, A. A.; Galie, P. A.; Yu, X.; Chaturvedi, R.; Bhatia, S. N.; Chen, C. S. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat. Mater. 2012, 11 (9), 768−774. (37) Attalla, R.; Ling, C.; Selvaganapathy, P. Fabrication and characterization of gels with integrated channels using 3D printing with microfluidic nozzle for tissue engineering applications. Biomed. Microdevices 2016, 18, 17.

H

DOI: 10.1021/acs.biomac.7b01827 Biomacromolecules XXXX, XXX, XXX−XXX