Three-Dimensional Bioprinting of Oppositely Charged Hydrogels with

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Applications of Polymer, Composite, and Coating Materials

Three Dimensional Bioprinting of Oppositely Charged Hydrogels with Super Strong Interface Bonding Huijun Li, Yu Jun Tan, Sijun Liu, and Lin Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19730 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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ACS Applied Materials & Interfaces

Three-Dimensional Bioprinting of Oppositely Charged Hydrogels with Super Strong Interface Bonding Huijun Li, Yu Jun Tan, Sijun Liu, Lin Li* Singapore Center for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore ABSTRACT A novel strategy to improve the adhesion between printed layers of 3D printed constructs is developed by exploiting the interaction between two oppositely charged hydrogels. Three anionic hydrogels (alginate, xanthan, κ-Carrageenan (Kca)) and three cationic hydrogels (chitosan, gelatin, gelatin methacrylate (GelMA)) are chosen in order to find the optimal combination of two oppositely charged hydrogels for the best 3D printability with strong interface bonding. Rheological properties and printability of the hydrogels, as well as structural integrity of printed constructs in cell culture medium, are studied as functions of polymer concentration and the combination of hydrogels. Kca2 (2wt% Kca hydrogel) and GelMA10 (10wt% GelMA hydrogel) are found to be the best combination of oppositely charged hydrogels for 3D printing. The interfacial bonding between a Kca layer and a GelMA layer is proven to be significantly higher than that of the bilayered Kca or bilayered GelMA due to the formation of polyelectrolyte complexes between the oppositely charged hydrogels. A good cell viability of > 96% is obtained for the 3D bioprinted Kca-GelMA construct. This novel strategy has a great potential for 3D bioprinting of layered constructs with a strong interface bonding. Keywords: 3D bioprinting, hydrogels, interface, gelatin, κ-Carrageenan

*

Corresponding author. E-mail address: [email protected] (L. Li)

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1. INTRODUCTION The recent progress in three dimensional (3D) printing technologies promises the great potential of the fabrication of complex artificial tissues or organs with accurate geometries.1, 2 There are two approaches in using 3D printing technologies in tissue engineering.3 The first approach is to print an acellular scaffold first and then seed it with living cells after printing.4 The second method is by 3D bioprinting of cell-laden hydrogels to obtain constructs with living cells.5, 6 The hydrogels for the extrusion-based bioprinting should be printed into a 3D structure without collapsing, and also be a cell-containing medium to maintain the viability of cells during the printing process. During printing, the hydrogel should have a shear-thinning property;7 after printing, the hydrogel should be highly thixotropic,7-9 have a good shape fidelity,10 be biocompatible and have a good structure integrity in the cell culture medium.11 Importantly, the interfacial strength between the hydrogel layers should be sufficiently strong to prevent delamination during and after printing.9 Thus, a challenge lies in developing suitable bioinks and smart strategies for the 3D bio-fabrication.12 Hydrogels are commonly used to prepare bioinks, as they are soft and hydrophilic as well as possess similarities to biological tissues.7, 13 In terms of ionic charge, hydrogels can be neutral (e.g. dextran), anionic (e.g. alginate (Alg), xanthan(Xan), κ-Carrageenan (Kca)) and cationic (e.g. chitosan (Chi), gelatin (Gel), gelatin methacrylate (GelMA)).14,15 Natural hydrogels, such as alginate,8, 16-18 gelatin,19,20 collagen,21,22 and chitosan,23,24 which show good biocompatibility with nontoxic degradation products, have received great attention in the field of biomedical engineering. However, these natural hydrogels still have limitations for their broad applications because they are weak by nature. Most of the studies focus on improving the mechanical performance of hydrogels. One of the common ways to improve the mechanical strength of the natural hydrogels is by increasing the crosslink density

25

or the concentration 26of a hydrogel.

An example is the preparation of an Alg hydrogel with a high concentration (~10 wt%), which could produce a highly viscous Alg hydrogel. Nonetheless, the printed structure using the Alg hydrogel can only achieve a limited height (~0.8 mm) due to the poor stacking ability,26 as also reported in our previous study.8,

9

Various strategies have been reported for developing

hydrogels with good mechanical strengths. For example, the double network hydrogels can sustain large deformation and force without failure.27,28 However, the double network hydrogels often contain a chemical network that is made by UV curing or a chemical process which poses a


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potential risk for utilizing these hydrogels in biomedical fields, as their degradation product is likely to be toxic.29, 30 As 3D printing is a layer-by-layer printing process, there are often layer defects or weak interface adhesion in a 3D-printed layered structure.9,31,32 As a new approach, alternate printing of two kinds of hydrogels might overcome the drawbacks of printing one hydrogel alone. Furthermore, alternate printing of two oppositely charged ionic hydrogels is expected to result in a strong interface adhesion between layers, however such method has not been reported in the literature. In this study, we aim to develop a promising approach that is capable of printing a 3D construct with strong interfacial bonding by utilizing the ionic interaction between two oppositely charged hydrogels. Alg, Xan, Kca and Chi, Gel, GelMA are chosen as the representatives of anionic and cationic hydrogels, respectively. Specific properties, including rheological properties of the prepared hydrogels, shape fidelity of a printed structure, structure integrity of a printed construct in the cell culture medium, are investigated. These properties help us to find the best concentration of each hydrogel and the best combinations for bioprinting. Eventually, Kca2 (2 wt%) and GelMA10 (10 wt%) are found to be the best combination for alternative 3D printing into a desired 3D construct named as Kca2-GelMA10. The interfacial adhesion between the Kca2 and GelMA10 layers was examined. Additionally, the biocompatibility of Kca2-GelMA10 as a pair of bioink was also investigated. 2. Experimental Section 2.1 Preparation of hydrogels Sodium alginate (Alg) (with guluronic acid block content of 50-60%, Sigma-Aldrich, Singapore) was dissolved in the Dulbecco’s phosphate buffered saline (DPBS) solution. The mixture was magnetically stirred overnight at room temperature to obtain a homogeneous hydrogel. The prepared Alg hydrogels were named Alg14, Alg16, Alg18, and Alg20, corresponding to the Alg concentrations of 14%, 16%,18%, and 20% (wt/wt) in the DPBS solutions respectively. Xanthan gum (Xan) (from aerobic fermentation, Sigma-Aldrich, Singapore) hydrogels were prepared by gradually adding Xan powder into a DPBS solution while simultaneously stirring the mixture. The hydrogels Xan4 (4 wt% Xan), Xan5 (5 wt% Xan), Xan6 (6 wt% Xan), and Xan7 (7 wt% Xan) were prepared. 3 ACS Paragon Plus Environment


κ-Carrageenan (Kca) (MW 3.0×105g/mol, Sigma-Aldrich, Singapore) powder was gradually added to the hot DPBS (~80 oC) solution. Meanwhile, the mixture was thoroughly stirred to obtain a homogeneous hydrogel. The hydrogels Kca1 (1 wt% Kca), Kca1.5 (1.5 wt% Kca), Kca2 (2 wt% Kca), and Kca2.5 (2.5 wt% Kca) were prepared. Chitosan (Chi) (medium molecular weight, Sigma-Aldrich, Singapore) powder was gradually added in the DPBS, where the pH of the solution was previously adjusted to 3 by adding acetic acid dropwise. Next, the mixture was stirred at room temperature until the Chi powder was evenly dissolved. The hydrogels Chi4 (4 wt% Chi), Chi5 (5 wt% Chi), Chi6 (6 wt% Chi), and Chi7 (7 wt% Chi) were prepared. Gelatin (Gel) powder (type A from porcine skin, Sigma-Aldrich, Singapore) was gradually adding into DPBS at about 50 oC. The mixture was thoroughly stirred until the gelatin powder was evenly dissolved. The hydrogels Gel6 (6 wt% Gel), Gel7 (7 wt% Gel), Gel8 (8 wt% Gel), and Gel9 (9 wt% Gel) were prepared. The synthesis of gelatin methacrylate (GelMA) was carried out according to the literatures.33, 34 10 g of gelatin was dissolved into a 100 mL DPBS solution and stirred until fully dissolved. 0.7 mL of methacrylic anhydride (MA) (Sigma-Aldrich, Singapore) was added into gelatin solution while stirring. The reaction proceeded at 50 oC for 3~4 hours. The mixture was dialyzed at 37 oC against deionized (DI) water using a 12-14 kDa membrane tubing for one week. Then the solution was moved to 50 mL tubes and freeze dried for another one week. The dry GelMA was stored at -40 oC until further use. GelMA with different weights was dissolved in DPBS to obtain GelMA hydrogels with different concentrations. GelMA8 (8 wt% GelMA), GelMA9 (9wt% GelMA), GelMA10 (10 wt% GelMA), and GelMA11 (11wt% GelMA) were prepared. The photo initiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (SigmaAldrich, Singapore) was added in the hydrogel with a photo initiator: GelMA solution ratio of 0.02 g: 9 mL. 2.2 Rheological measurement The rheological properties of the hydrogels with various concentrations were measured using a rotational rheometer (DHR, TA Instruments, USA) with a 40 mm parallel plate and a 0.55 mm measurement gap. Two rheological tests at 26 oC (the working temperature of the 3D printer) were adopted: (1) steady-state flow tests in a range of shear rate 0.5-500 s-1; (2)


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recovery tests under a calculated shear rate simulating the extrusion process for 3D printing as described in 2.2.1. 2.2.1 Determination of shear rate In an extrusion nozzle, the shear rate exerted on a hydrogel at a radial position,35 r (0 < r < R) could be estimated by a deduced equation based on our previous study: 8, 9 ߛሶ = ൥ ௡

ቀ

௏ோ మ

௡

൩ .‫ݎ‬

ሺయ೙శభሻൗ ೙ ೙൰ ቁ൬ோ య೙శభ

(1)

where R is the inner radius of the nozzle, V is the flow rate of the extruded hydrogel in the nozzle, n is the power-law index of the hydrogel. The inner diameter (i.d.) of the nozzle used in this study was 25GA (0.25 mm). The details of obtaining the flow rate of each sample were described in our previous work.9 2.2.2 Characterization of thixotropic property The rheological properties of each hydrogel before (step I), during (step II), and after (step III) the extrusion process were simulated and observed. Step I simulates the initial state of a hydrogel before printing where a shear rate of 0.1 s-1 was applied and held for 60 seconds. At step II, a shear rate, which was calculated based on equation (1), was applied on the hydrogels for 10 seconds. This step simulates the state of a sheared hydrogel during the extrusion process. At step III, the shear rate was decreased to 0.1 s-1 again and held for another 60 seconds to simulate the final condition of the hydrogel after extrusion. 2.3 Evaluation of the printability of each hydrogel 2.3.1 Determination of the best concentration of each hydrogel The smooth surface and constant width of a printed filament, which resulted in regular edges and corners in the printed 3D construct, are all indications of a good printability. Moreover, the printed 3D shapes should be consistent with the pre-designed pattern. Thus, the printability of a hydrogel can be investigated from the shape fidelity of a printed construct.



2.3.2 Determination of the best hydrogels for printing The construct printed with an anionic hydrogel then a cationic hydrogel alternately, is named as anionic-cationic, i.e. Alg18-Gel5. In this study, a bioprinter (BioFactory bioprinter machine, RegenHU) was used to print constructs. A 3D printing route was generated from the 3D software (BioCAD) on the bioprinter to control the continuous deposition of computerdesigned patterns of hydrogels. Here, 20-layered grids in a 0 °/90 ° pattern were printed out. The printing pressure utilized for each hydrogel was listed in Table S1 (Supporting Information). The optimal pressure for extruding each hydrogel was obtained when a continuous filament could be deposited with a uniform filament diameter. The i.d. of the nozzle used was 25 GA (0.25 mm). The structures printed using each cationic or anionic hydrogel solely were also demonstrated as a control. The working temperature for the bioprinter was ~26 oC. 2.4 Measurement of interfacial bonding strength 2.4.1 Evaluation of interaction between two opposite charged hydrogels A simple experiment was performed to investigate the interaction between an anionic and a cationic hydrogel. GelMA10 hydrogel (stained with orange-colored food dye) and Kca2 hydrogel (stained with blue-colored food dye) were cut into small pieces, respectively. Subsequently, pieces of hydrogels were placed next to each other alternatively, i.e. GelMA10Kca2. The same types of hydrogels pieces, i.e. GelMA10-GelMA10, and Kca2-Kca2 were also investigated as a control. 2.4.2 Quantitative study of interfacial bonding strength The interfacial bonding strength between two hydrogel layers was investigated through lapshear tests using an Instron machine (Instron 5569; U.K.) with a 10 N load cell. 2 mm thick hydrogel sheets were cast, each mimicking one layer in a bioprinted construct. Three types of samples were fabricated, namely 2-layered Kca2, 2-layered GelMA10, and 2-layered Kca2GelMA10. In particular, the 2-layered Kca2-GelMA10 was prepared by placing one Kca2 hydrogel sheet immediately onto a GelMA10 hydrogel sheet to produce a 4 mm thick sample. The maximum shear stress was defined as the ultimate shear stress (USS), which was recorded from the stress-time curve.9


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2.5 Structural integrity of the printed constructs in 37 oC DPBS The structure integrity of the printed Kca2-GelMA10 construct was observed for 30 days after soaking it in DPBS in an incubator (37 oC). The constructs solely printed using Kca2 or GelMA10 served as a control. The blend hydrogel (a mixture of Kca2 and GelMA10 in a volume ratio of 1:1) was cast and investigated in DPBS. Each structure has ten layers. The hydrogels were stained using a food dye for ease of visualization. 2.6 3D bioprinting of Kca2-GelMA10 hydrogel constructs 2.6.1 Bioprinting Mouse myoblasts cells C2C12 were cultured in the cell culture media of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic, incubated under 5% CO2 at 37 °C.36 The cells were detached and counted before being loaded into a syringe for bioprinting. The cell suspension was added to the GelMA10 hydrogel, resulting in a final cell concentration of ~3×105 cells/mL in the hydrogel. Two syringes were utilized for bioprinting. Syringe 1 was loaded with the Kca2 hydrogel, while Syringe 2 was filled with the cell-laden GelMA10 hydrogel. The 3D cell-laden construct was printed layer-by-layer by extruding the Kca2 from syringe 1, then followed by printing the cellladen GelMA10 hydrogel from syringe 2. The freshly bioprinted Kca2-GelMA10 constructs were UV cured for 10 seconds in a UV flood (Shuttered UV system, Epoxy and equipment technology Pte Ltd). Then the constructs were cultured in an incubator at 37 oC for 5 days. Figure 1 illustrates the procedure for bioprinting of a Kca2-GelMA10 hydrogel construct.



Figure 1. Schematic illustration of the bioprinting procedure. The 3D cell-laden construct is printed layer-by-layer by printing the Kca hydrogel from syringe 1, then followed by printing the cell-laden GelMA hydrogel from syringe 2. 2.6.2 Cell viability of the bioprinted Kca2-GelMA10 hydrogel construct The bioprinted constructs were cultured in the medium in an incubator (37 oC; 5% CO2) for up to 5 days. The cell culture medium was changed every 2 days. The viability of cells in the constructs was examined using a live/dead assay (Molecular Probes) via an inverted fluorescent microscope (Zeiss Axio Vert. A1). The constructs were incubated in a DPBS solution containing 5 μmol/L propidium iodide and 2 μmol/L calcein acetoxymethyl ester for 15 minutes before fluorescence imaging. C2C12 cells were also cultured on tissue culture polystyrene (TCPS) as a control. 2.7 Statistical analysis All results were presented as the mean ± standard deviation (S.D.), and compared statistically by means of one-way ANOVA. Differences were statistically significant when p ≤ 0.05. 3. RESULTS AND DISCUSSION


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3.1 Rheological evaluation 3.1.1 Determination of shear thinning and shear rate The results of the steady-state flow test for the alginate hydrogel (Figure 2A(i)) indicate that the alginate hydrogel demonstrates a shear thinning behavior that the viscosity of hydrogel decreases with increasing shear rate. The shear thinning properties for alginate hydrogels were also observed in our previous study.8, 9 Meanwhile, it is also easy to find that the Xan, Kca, Chi, Gel, and GelMA hydrogels with various concentration (Figures 2A(ii)-(iii),2B(i)-(iii)) all demonstrate a shear-thinning property. As is well-known, a highly viscous hydrogel with good printability for an extrusion-based printer should be shear thinning.7 Thus, all the tested hydrogel samples have one of the essential properties desired for the successful printing.



Figure 2. Shear viscosity as a function of shear rate. A) Anionic hydrogels: Alg (i), Xan (ii), and Kca (iii). B) Cationic hydrogels: Chi (i), Gel (ii), and GelMA (iii). The procedure for obtaining the flow rate, and the power-law index (n) were described in our previous work.8, 9 The results are listed in Table S1 (Supporting Information). It is well known that, for a shear-thinning fluid, the value of n should be smaller than 1.35 All of the tested hydrogels show the values of n smaller than 1 (Table S1, Supporting Information), again revealing that they are shear-thinning hydrogels. Based on eq 1, almost all the tested samples (except Kca 2.5) were estimated to be sheared under a maximum shear rate of ~100 s-1 during printing (Table S1, Supporting Information). For a hydrogel used for printing, the relative reduction in viscosity depends on shear rate. Thus, estimating of the range of shear rate exerted 10 ACS Paragon Plus Environment

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on a hydrogel is fundamentally important, especially for further simulating the behaviors of a hydrogel during the extrusion process. 3.1.2 Characterization of thixotropic property The thixotropic properties of anionic hydrogels (Figure 3A) and cationic hydrogels (Figure 3B) were investigated. At step II, all the hydrogels were tested under a shear rate of 100 s-1, which simulated the condition for the hydrogels to bear the shear force during the extrusion process. After that, moving to step III, each hydrogel recovered its viscosity to a comparable value of its initial viscosity (step I). All the tested hydrogels exhibited a thixotropic property. The reason for the changing of viscosity is because the cross-links or entanglements between polymer chains were broken by shearing, which resulted in a decrease in the viscosity of hydrogels (step II). After removing the high shear rate, the hydrogel could rebuild the broken cross-links after a period of rest (step III).



Figure 3. Rheological measurements to simulate the shear thinning and recovery behaviors of different hydrogels with various concentrations: step I, at a shear rate of 0.1 s-1; step II, at a shear rate of 100 s-1; step III, at a shear rate of 0.1 s-1. A) Anionic hydrogels: Alg (i), Xan (ii), and Kca (iii). B) Cationic hydrogels: Chi (i), Gel (ii), and GelMA (iii).


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3.2 Evaluation of the printability of hydrogels 3.2.1 Determination of the best concentration of each hydrogel In this section, the shape fidelity of each hydrogel was investigated to find the best concentration for printing. For one-layer of grids in a 0°/90° pattern (used in this study), regular grids and square holes should be consistent with the designed pattern and dimensions if the hydrogel used has a good printability. Ouyang et al.,37 reported that the extruded filaments would form a pattern with circular-like holes when the hydrogel is in an under-gelation state. But when the hydrogel is in an over-gelation state, the extruded filament is irregular or even shows fracture with a rough surface. Thus, each polymer with different concentrations should be under one of the gelation states: under-gelation, proper-gelation, or over-gelation. The hydrogel in a proper-gelation state is suitable for printing. Thus, to find the best concentration of each polymer is essential before using this polymer for printing. The printability ( Pr ) of a hydrogel can be defined according to the printed square shape:37

Pr =

L2 4C 16A

π

(2)

=

where L is the perimeter of one grid in the printed pattern and A is the area of the grid. C is the circularity of an enclosed area, is defined as C = 4π2A . L

L and A of one grid in the printed

construct can be computed using the ImageJ software. It is known that a circle and a square have a circularity value of 1 and π/4, respectively. Thus, for a hydrogel with ideal printability, the interconnected channels of the printed constructs would demonstrate square shape so that the value of P r should be close to 1. Pr 1, the hydrogel is in an over-gelation condition. Based on the criteria, the best concentration of each polymer can be obtained. Figure 4 illustrates the Pr of each hydrogel with various concentrations. For Alg hydrogel (Figure 4A(i)), irregular shape and obvious spreading are easily observed when the Alg hydrogel with comparatively low concentrations (Alg14 and Alg16). Although Alg20 has the highest concentration of Alg, it exhibits a similar Pr with Alg18. As the Alg powder is difficult to dissolve in DPBS when the concentration of Alg reaches 20 %, 18wt % is chosen as the best concentration for Alg hydrogel for printing although its Pr is not ideal ( Pr 96% up to day 2. Moreover, C2C12 elongated and built their own 3D network after culturing for 5 days. Based on the above findings, we believe that this novel method will open a new door for 3D bioprinting of layered constructs with a super strong interface bonding.

Supporting Information Determination of the shear rate exerted on a hydrogel; Evaluation of interaction between Kca2 and Gel8; 1H NMR characterization verified the formation of different functional groups of GelMA; Quantitative study of interfacial bonding strength between Kca2 and Gel8; Structural integrity of Kca2-Gel8 hydrogels in 37 oC DI water; Structural integrity of Kca2/GelMA10 blend hydrogels in 37 oC DPBS; A movie showing the extraordinary adhesion properties between GelMA10 and Kca2. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Lin Li: 0000-0002-9840-8367 ACKNOWLEDGMENTS


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This work was supported by the Academic Research Fund Tier 1 (RG112/17) from the Ministry of Education, Singapore.

REFERENCES 1. 2.

3. 4. 5.

6.

7.

8. 9.

10. 11. 12.

13. 14.

15.

Murphy, S. V.; Atala, A. 3D Bioprinting of Tissues and Organs. Nat. Biotechnol. 2014, 32, 773-785. Liu, W.; Zhang, Y. S.; Heinrich, M. A.; De Ferrari, F.; Jang, H. L.; Bakht, S. M.; Alvarez, M. M.; Yang, J.; Li, Y. C.; Trujillo‐de Santiago, G., Miri, A. M.; Zhu, K.; Khoshakhlagh, P.; Prakash, G.; Cheng, H.; Guan, X.; Zhong, Z.; Ju, J.; Zhu, G. H.; Jin, X.; Shin, S. R.; Dokmeci, M. R.; Khademhosseini, A. Rapid Continuous Multimaterial Extrusion Bioprinting. Adv. Mater. (Weinheim, Ger.) 2017, 29, No. 1604630. Wu, Z.; Su, X.; Xu, Y.; Kong, B.; Sun, W.; Mi, S. Bioprinting Three-Dimensional Cell-Laden Tissue Constructs with Controllable Degradation. Sci. Rep. 2016, 6, No. 24474. Hollister, S.J. Porous Scaffold Design for Tissue Engineering. Nat. Mater. 2005, 4, 518524. Mannoor, M. S.; Jiang, Z.; James, T.; Kong, Y. L.; Malatesta, K. A.; Soboyejo, W. O.; Verma, N.; Gracias, D. H.; McAlpine, M. C. 3D Printed Bionic Ears. Nano Lett. 2013, 13, 26342639. Tan, Y. J.; Tan, X.; Yeong, W. Y.; Tor, S. B. Hybrid Microscaffold-Based 3D Bioprinting of Multi-Cellular Constructs with High Compressive Strength: A New Biofabrication Strategy. Sci. Rep. 2016, 6, No. 39140. Malda, J.; Visser, J.; Melchels, F. P.; Jüngst, T.; Hennink, W. E.; Dhert, W. J. A.; Groll, J.; Hutmacher, D. W. 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Adv. Mater. (Weinheim, Ger.) 2013, 25, 5011-5028. Li, H.; Liu, S.; Li, L. Rheological Study on 3D Printability of Alginate Hydrogel and Effect of Graphene Oxide. Int. J. Bioprinting 2016, 2, 54-66. Li, H.; Tan, Y. J.; Leong, K. F.; Li, L. 3D Bioprinting of Highly Thixotropic Alginate/Methylcellulose Hydrogel with Strong Interface Bonding. ACS Appl. Mater. Interfaces 2017, 9, 20086-20097. He, Y.; Yang, F.; Zhao, H.; Gao, Q.; Xia, B.; Fu, J. Research on the Printability of Hydrogels in 3D Bioprinting. Sci. Rep. 2016, 6, No. 29977. Hospodiuk, M.; Dey, M.; Sosnoski, D.; Ozbolat, I. T. The Bioink: A Comprehensive Review on Bioprintable Materials. Biotechnol. Adv. 2017, 35, 217-239. Ouyang, L.; Highley, C. B.; Rodell, C. B.; Sun, W.; Burdick, J. A. 3D Printing of ShearThinning Hyaluronic Acid Hydrogels with Secondary Cross-Linking. ACS Biomater. Sci. Eng. 2016, 2, 1743-1751. Chimene, D.; Lennox, K. K.; Kaunas, R. R.; Gaharwar, A. K. Advanced Bioinks for 3D Printing: A Materials Science Perspective. Ann. Biomed. Eng. 2016, 44, 2090-2102. Samal, S. K.; Dash, M.; Van Vlierberghe, S.; Kaplan, D. L.; Chiellini, E.; Van Blitterswijk, C.; Moroni, L.; Dubruel, P. Cationic Polymers and Their Therapeutic Potential. Chem. Soc. Rev. 2012, 41, 7147-7194. Souguir, Z.; About-Jaudet, E.; Picton, L.; Le Cerf, D. Anionic Polysaccharide Hydrogels with Charges Provided by the Polysaccharide or the Crosslinking Agent. Drug Delivery Lett. 2012, 2, 240-250. 27 ACS Paragon Plus Environment


16.

17. 18.

19.

20.

21.

22.

23.

24. 25.

26.

27. 28. 29. 30. 31.

32.

Jia, J.; Richards, D. J.; Pollard, S.; Tan, Y.; Rodriguez, J.; Visconti, R. P.; Trusk, T. C.; Yost, M. J.; Yao, H.; Markwald, R. R.; Mei, Y. Engineering Alginate as Bioink for Bioprinting. Acta Biomater. 2014, 10, 4323-4331. Zehnder, T.; Sarker, B.; Boccaccini, A. R.; Detsch, R. Evaluation of an Alginate–Gelatine Crosslinked Hydrogel for Bioplotting. Biofabrication 2015, 7, No. 025001. Leppiniemi, J.; Lahtinen, P.; Paajanen, A.; Mahlberg, R.; Metsa-Kortelainen, S.; Pinornaa, T.; Pajari, H.; Vikholm-Lundin, I.; Pursula, P.; Hytonen, V. P. 3D-Printable Bioactivated Nanocellulose-Alginate Hydrogels. ACS Appl. Mater. Interfaces 2017, 9, 21959-21970. Shi, W.; Sun, M.; Hu, X.; Ren, B.; Cheng, J.; Li, C.; Duan, X.; Fu, X.; Zhang, J.; Chen, H.; Ao, Y. Structurally and Functionally Optimized Silk-Fibroin–Gelatin Scaffold Using 3D Printing to Repair Cartilage Injury In Vitro and In Vivo. Adv. Mater. (Weinheim, Ger.) 2017, 29, No. 1701089. Duan, B.; Hockaday, L. A.; Kang, K. H.; Butcher, J. T. 3D Bioprinting of Heterogeneous Aortic Valve Conduits with Alginate/Gelatin Hydrogels. J. Biomed. Mater. Res., Part A 2013, 101A, 1255–1264. Rhee, S.; Puetzer, J. L.; Mason, B. N.; Reinhart-King, C. A.; Bonassar, L. J. 3D Bioprinting of Spatially Heterogeneous Collagen Constructs for Cartilage Tissue Engineering. ACS Biomater. Sci. Eng. 2016, 2, 1800-1805. Park, J. Y.; Choi, J.-C.; Shim, J.-H.; Lee, J.-S.; Park, H.; Kim, S. W.; Doh, J.; Cho, D.-W. A Comparative Study on Collagen Type I and Hyaluronic Acid Dependent Cell Behavior for Osteochondral Tissue Bioprinting. Biofabrication 2014, 6, No. 035004. Xu, Y.; Xia, D.; Han, J.; Yuan, S.; Lin, H.; Zhao, C. Design and Fabrication of Porous Chitosan Scaffolds with Tunable Structures and Mechanical Properties. Carbohydr. Polym. 2017, 177, 210-216. Ng, W. L.; Yeong, W. Y.; Naing, M. W. Polyelectrolyte Gelatin-Chitosan Hydrogel 0ptimized for 3D Bioprinting in Skin Tissue Engineering. Int. J. Bioprinting 2016, 2, 53-62. Jang, J.; Seol, Y. -J.; Kim, H. J.; Kundu, J.; Kim, S, W.; Cho, D.-W. Effects of Alginate Hydrogel Cross-Linking Density on Mechanical and Biological Behaviors for Tissue Engineering. J. Mech. Behav. Biomed. Mater. 2014, 37, 69-77. Fedorovich, N. E.; Schuurman, W.; Wijnberg, H. M.; Prins, H. J.; van Weeren, P. R.; Malda, J.; Alblas, J.; Dhert, W. J. Biofabrication of Osteochondral Tissue Equivalents by Printing Topologically Defined, Cell-Laden Hydrogel Scaffolds. Tissue Eng., Part C 2012, 18, 33-44. Gong, J. P.; Katsuyama, Y.; Kurokawa, T.; Osada, Y. Double-Network Hydrogels with Extremely High Mechanical Strength. Adv. Mater. (Weinheim, Ger.) 2003, 15, 1155-1158. Liu, S.; Li, L. Ultrastretchable and Self-Healing Doubl-Network Hydrogel for 3D Printing and Strain Sensor. ACS Appl. Mater. Interfaces 2017, 9, 26429-26437. Luo, K.; Yang, Y.; Shao, Z. Physically Crosslinked Biocompatible Silk-Fibroin-Based Hydrogels with High Mechanical Performance. Adv. Funct. Mater. 2016, 26, 872-880. Donderwinkel, I.; van Hest, J. C. M.; Cameron, N. R. Bio-Inks for 3D Bioprinting: Recent Advances and Future Prospects. Polym. Chem. 2017, 8, 4451-4471. Gleghorn, J. P.; Lee, C. S.; Cabodi, M.; Stroock, A. D.; Bonassar, L. J. Adhesive Properties of Laminated Alginate Gels for Tissue Engineering of Layered Structures. J. Biomed. Mater. Res., Part A. 2008, 85, 611-618. Lee, C. S.; Gleghorn, J. P.; Choi, N. W.; Cabodi, M.; Stroock, A. D.; Bonassar, L. J. Integration of Layered Chondrocyte-Seeded Alginate Hydrogel Scaffolds. Biomaterials 2007, 28, 2987-2993.


Page 28 of 31

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33.

34.

35. 36.

37. 38.

39.

40. 41. 42.

Nichol, J. W.; Koshy, S. T.; Bae, H.; Hwang, C. M.; Yamanlar, S.; Khademhosseini, A. CellLaden Microengineered Gelatin Methacrylate Hydrogels. Biomaterials 2010, 31, 55365544. Van Den Bulcke, A. I.; Bogdanov, B.; De Rooze, N.; Schacht, E. H.; Cornelissen, M.; Berghmans, H. Structural and Rheological Properties of Methacrylamide Modified Gelatin Hydrogels. Biomacromolecules 2000, 1, 31-38. Chhabra, R. P.; Richardson, J. F. Non-Newtonian Flow and Applied Rheology: Engineering Applications, 2nd ed.; Butterworth-Heinemann: Boston, 2008. Tan, Y. J.; Leong, K. F.; An, J.; Chian, K. S.; Tan, X.; Yeong, W. Y. Fabrication and in Vitro Analysis of Tubular Scaffolds by Melt-Drawing for Esophageal Tissue Engineering. Mater. Lett. 2015, 159, 424-427. Ouyang, L., Yao, R.; Zhao, Y.; Sun, W. Effect of Bioink Properties on Printability and Cell Viability for 3D Bioplotting of Embryonic Stem Cells. Biofabrication 2016, 8, No. 035020. Voron’ko, N. G.; Derkach, S. R.; Vovk, M. A.; Tolstoy, P. M. Formation of κ-Carrageenan– Gelatin Polyelectrolyte Complexes Studied by 1H NMR, UV Spectroscopy and Kinematic Viscosity Measurements. Carbohydr. Polym. 2016, 151, 1152-1161. Voron’ko, N. G.; Derkach, S. R.; Vovk, M. A.; Tolstoy, P. M. Complexation of κCarrageenan with Gelatin in the Aqueous Phase Analysed by 1 H NMR Kinetics and Relaxation. Carbohydr. Polym. 2017, 169, 117-126. Zhou, M.; Lee, B. H.; Tan, L. P. A Dual Crosslinking Strategy to Tailor Rheological Properties of Gelatin Methacryloyl. Int. J. Bioprinting 2017, 3, 130-137. Tabriz, A. G.; Hermida, M. A.; Leslie, N. R.; Shu, W. Three-Dimensional Bioprinting of Complex Cell Laden Alginate Hydrogel Structures. Biofabrication 2015, 7, No. 045012. Billiet, T.; Gevaert, E.; De Schryver, T.; Cornelissen, M.; Dubruel, P. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials 2014,35, 49-62.




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