Three-dimensional Bioprinting of Cell-laden Constructs Using

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Three-dimensional Bioprinting of Cell-laden Constructs Using Polysaccharide-based Self-healing Hydrogels Sang Woo Kim, Do Yoon Kim, Hyun Ho Roh, Hyun Seung Kim, Jae Won Lee, and Kuen Yong Lee Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Submitted to Biomacromolecules (revised manuscript)

Three-dimensional Bioprinting of Cell-laden Constructs Using Polysaccharide-based Selfhealing Hydrogels Sang Woo Kim,† Do Yoon Kim,† Hyun Ho Roh,† Hyun Seung Kim,† Jae Won Lee,† and Kuen Yong Lee*,†, § † Department § Institute

of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea

of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of

Korea

* To whom correspondence should be addressed: Kuen Yong Lee, Ph.D., Professor Department of Bioengineering, Hanyang University 222 Wangsimni-ro, Seongdong-gu Seoul 04763, Republic of Korea Phone: +82-2-2220-0482 Fax: +82-2-2293-2642 E-mail: [email protected]

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ABSTRACT Development of biomaterial-based bioinks is critical for replacement and/or regeneration of tissues and organs by three-dimensional (3D) printing techniques. However, the number of 3D-printable biomaterials in practical use remains limited despite the rapid development of 3D printing techniques. Controlling the flow properties of bioinks and the mechanical properties of the resultant printed objects are key considerations in the design of biomaterialbased bioinks for practical applications. In this study, a printable hydrogel comprising biocompatible polysaccharides that has potential for cartilage regeneration via tissue engineering approaches was designed. Self-healing hydrogels were prepared from partially oxidized hyaluronate (OHA) and glycol chitosan (GC) in the presence of adipic acid dihydrazide (ADH). The self-healing ability of OHA/GC/ADH hydrogels was attributed to combination of two dynamic bonds in the gels, including imine bonds obtained via Schiff base reaction between OHA and GC, as well as acylhydrazone bonds formed by reaction between OHA and ADH. The OHA/GC/ADH hydrogels did not require any post-gelation or additional cross-linking processes for use in the fabrication of 3D constructs using an extrusion-based 3D printer. The concentrations and molecular weights of the constituent polymers were found to be critical parameters affecting the flow and mechanical properties of the self-healing hydrogels, which showed great potential as bioinks for fabricating cell-laden structures using a 3D printer. The expression of chondrogenic marker genes such as SOX-9 and collagen type II of ATDC5 cells encapsulated in OHA/GC/ADH hydrogel was not significantly affected by the printing process. This self-healing hydrogel system may have the potential in tissue engineering applications, including cartilage regeneration.

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KEYWORDS Bioprinting; self-healing hydrogel; sodium hyaluronate; glycol chitosan; cartilage regeneration

INTRODUCTION Three-dimensional (3D) bioprinting with living cells has recently been proposed as a means to design and fabricate artificial tissues and organs. Furthermore, it presents the ability to construct functional and biological structures for various biomedical applications.1-3 Typical bioprinting strategies include inkjet printing, extrusion printing, and laser-associated bioprinting.4-7 Extrusion-based bioprinting is currently a widely used method owing to its low cost and high compatibility with various bioinks. Hydrogels are often employed in extrusionbased bioprinting8-12 as they can mimic the structures and functions of extracellular matrices (ECMs) in the body.13 However, external factors such as elevated temperatures and/or high shear stresses applied to 3D printer nozzles can lead to cell death and gel fracture during the printing process.2,14 Inspired by the autonomous healing exhibited by living tissues after damage, self-healing hydrogels have been proposed for 3D bioprinting and have been widely developed in recent years.15-17 Self-healing hydrogels are typically fabricated by incorporating species capable of dynamic and reversible covalent bond formation,18 such as that by Schiff-base-forming,19 disulfide-bond-forming,20 and Diels-Alder reactions.21 However, the number of synthetic and natural polymers suitable for the fabrication of self-healing hydrogels is very limited. Polysaccharides are often used in the preparation of self-healing hydrogels and have been 3



successfully applied to the delivery of drugs22,23 and cells24,25 owing to their excellent biocompatibilities and ability to heal under physiological conditions. However, the mechanical properties of these hydrogels are generally poor without additional cross-linking, which is known to significantly influence tissue formation.26,27 Hyaluronate (HA) was chosen as the base polysaccharide for the bioinks in this study because it is a natural biomaterial used in various biomedical applications.28,29 HA is one of the most hydrophilic molecules in nature and it has been widely exploited to fabricate hydrogels.30 In addition, HA can bind to CD44 receptors and regulate the expression of chondrogenic marker genes such as collagen type 2, SOX-9, and aggrecan,31-33 indicating that it is an excellent candidate biomaterial for cartilage regeneration. HA typically requires small molecular chemical cross-linker to generate network structures. However, unreacted crosslinking reagents remaining in the gel can cause side effects in the body, including inflammatory reactions and immune responses.34 Glycol chitosan (GC), a water-soluble form of chitosan, can be utilized as a polymeric cross-linker to form cross-linked networks of partially oxidized hyaluronate (OHA) under physiological conditions.35 However, OHA/GC gels do not exhibit self-healing properties, despite the reversible imine bond formation possible between OHA and GC. Accordingly, we hypothesized that the addition of a species capable of reversible covalent bond formation to OHA/GC hydrogels would impart them with autonomous healing properties due to competitive covalent bond formation throughout the entire network system. A simple mixture of adipic acid dihydrazide (ADH) and GC was added to OHA, allowing the formation of reversible acylhydrazone bonds by condensation between the hydrazide groups of ADH and the carbonyl group of OHA. The viscoelastic properties of OHA/GC/ADH hydrogels were controlled by varying the concentration and molecular weight of the HA as a means to optimize the printability of the gels as bioinks. The 4


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self-healing properties of the OHA/GC/ADH hydrogels along with their biocompatibilities were evaluated. Chondrogenic differentiation of ATDC5 cells in a printed 3D construct was also investigated in vitro to demonstrate the potential application of the hydrogels to cartilage tissue engineering.

EXPERIMENTAL SECTION Materials. Sodium hyaluronate (HA) was purchased from Humedix, Korea (MW = 1,000 kDa) and Lifecore (MW = 2,500 kDa). Glycol chitosan (GC; MW = 50 kDa) was provided by Wako. Adipic acid dihydrazide (ADH) and sodium periodate were purchased from Sigma. Phosphate buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F-12) and penicillin streptomycin (PS) were supplied by Gibco. Formaldehyde was purchased from Junsei, and a Live/Dead viability/cytotoxicity kit® was supplied from Invitrogen. Preparation of Oxidized Hyaluronate. HA (1 g) was dissolved in distilled water (90 mL) by mixing for 24 h. Sodium periodate (0.2673 g) was dissolved in distilled water (10 mL) and added to the HA solution. The oxidation reaction was carried out for 24 h under dark conditions. The solution was then purified by dialysis against deionized water containing sodium chloride for 4 d (molecular weight cut-off = 3,500 g/mol), treated with activated charcoal (Sigma), filtered with a 0.22-m filter, and lyophilized. GC (1 g) was dissolved in 100 mL of distilled water and the same purification process was applied to the solution. Characterization of Oxidized Hyaluronate. Fourier-transform infrared spectroscopy (FT-IR, Nicolet IS50; Thermo) was used to confirm the formation of aldehyde groups in the 5



oxidized hyaluronate (OHA). The degree of oxidation (%) was defined as the number of oxidized units per 100 repeating units in the HA. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) assays were used to determine the number of aldehyde groups in the OHA. Since the aldehyde groups are not able to react with TNBS directly, an excess amount of t-butyl carbazate was added in advance and the amount of unreacted t-butyl carbazate was determined to calculate the degree of oxidation. Hydrogel Fabrication and Characterization. Hydrogels were fabricated from OHA and GC in the presence of ADH. Briefly, OHA was dissolved in DPBS overnight in an amber bottle, and GC and ADH were also dissolved in DPBS overnight. Equal volumes of each solution were then mixed to form the hydrogels. FT-IR spectroscopy was used to confirm imine bond formation in the hydrogels. The viscoelastic properties of the hydrogels were measured by a rotational rheometer (Bohlin Gemini 150; Malvern) equipped with a cone-andplate fixture (20 mm diameter plate, 4° cone angle) at 37 °C (5 Pa, 1 Hz). Characterization of Self-healing Hydrogels. The self-healing properties of the OHA/GC/ADH hydrogels were initially evaluated macroscopically. Briefly, a hydrogel disk (10 mm diameter, 1 mm thick) was cut into pieces using a razor blade. The gel pieces were then reassembled and allowed to self-heal for 10 min at room temperature without additional treatment, and an image of the integrated gel disk was then taken. The self-healing properties of the OHA/GC/ADH hydrogels were also investigated using a rotational viscometer (Bohlin Gemini 150; Malvern) equipped with a cone-and-plate fixture (20 mm diameter plate, 4° cone angle) at 37 °C (5 Pa, 1 Hz). Amplitude sweeps were carried out in the strain range 1–300% to determine the breaking strains of the OHA/GC/ADH hydrogels. Next, the strain was alternated for 2 min from 1% to 300% repeatedly to evaluate the self-healing performances of the gels. The strain value was varied from 400% to 800%. 6


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The recovery rate of the self-healing hydrogel as a function of ADH concentration was determined by measuring the storage shear modulus before (G′0) and after (G′t) strain was applied. The % recovery was calculated using the following equation: % Recovery =

G′t G′0

× 100

3D Printing. A 3D bioprinter (Invivo®, Rokit, Korea) equipped with a pressurecontrolled cartridge was used to print the OHA/GC/ADH hydrogels. The hydrogel was loaded into a disposable syringe and inserted to the cartridge for printing. The printability of the hydrogel was optimized by varying the moving speeds of the platform and nozzle along with the input flow through the fixed nozzle to achieve stable and constant printing of precision gel filaments. Cell Culture. The ATDC5 cell line was purchased from RIKEN cell bank (Tsukuba) and cells were cultured in DMEM/F-12 media containing 10% FBS, 1% PS, 10 μg/mL human transferrin, and 3 × 10-8 M sodium selenite. Bovine insulin (10 μg/mL) was added to the media for chondrogenic differentiation. For 3D culturing of ATDC5 cells, the cells were premixed with an OHA solution ([OHA] = 2 wt%, [ATDC5] = 1 × 107 cells/mL). The OHA solution was then mixed with a GC solution containing ADH ([GC] = 1 wt%, [ADH] = 0.3 wt%) to form the hydrogel. The cell-encapsulating hydrogel was incubated at 37 °C under a 5% CO2 atmosphere using DMEM/F-12 media containing 10% FBS and 1% PS. In Vitro Cell Viability. The viability of ATDC5 cells encapsulated within the OHA/GC/ADH hydrogel was evaluated using a Live/Dead viability/cytotoxicity kit® (Invitrogen) before and after the printing process. Briefly, 20 μL of EthD-1 (Invitrogen) and 5 μL of calcein AM (Invitrogen) were dissolved in 10 mL of DPBS, and 150 μL of reagent was applied to each sample. After 30 min incubation, images were taken using fluorescence 7



microscopy (ECLIPSE TE2000-E; Nikon). The number of live cells and dead cells in the images were counted to evaluate cell viability. In Vitro Chondrogenic Differentiation. Chondrogenic differentiation of ATDC5 cells cultured within the hydrogel was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analyses. The samples were homogenized to retrieve the cells from the hydrogel, and the liberated cells were then treated with RNAiso Plus reagent (Takara) for RNA extraction. The expression of chondrogenic marker genes (SOX-9 and collagen type II) was evaluated using a RT-PCR thermal cycler machine (Takara). The sequences of the primers are as follows: GAPDH, 5′-CCATCACCATCTTCCAGGAGCGA3′, 5′-GGATGACCTTGCCCACAGCCTTG-3′; SOX-9, 5′ATCGGTGAACTGAGCAGCGAC-3′, 5′-GCCTGCTGCTTCGACATCCA-3′; collagen type II (COL2), 5′-AAGAGCGGTGACTACTGGATAG-3′, 5′TGCTGTCTCCATAGCTGAAGT-3′. The gene expression level was also quantified by comparison with that of -actin, the reference gene cycle threshold (CT), using an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems) and SensiMaxTM SYBR (Bioline). The sequences of the primers are as follows: -actin, 5′CCCTGAACCCTAAGGCCAAC-3′; 5′-GCATACAGGGACAGCACAGC-3′; SOX-9, 5′AAGTCGGAGAGCCGAGAGCG-3′, 5′-ACGAAACCGGGGCCACTTGC-3′; collagen type II (COL2), 5′-CACACTGGTAAGTGGGGCAAGACCG-3′, 5′GGATTGTGTTGTTTCAGGGTTCGGG-3′. Statistical Analysis. All data are presented as mean ± standard deviation (n = 4). Statistical analyses were performed using Student’s t-test. **P-values < 0.01 and ***P-values < 0.001 were considered to be statistically significant.

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RESULTS AND DISCUSSION Preparation and Characterization of OHA/GC/ADH Hydrogel. HA was first treated with sodium periodate to obtain partially oxidized hyaluronate (Figure 1a). Sodium periodate causes the formation of reactive aldehyde groups in carbohydrates by the oxidation of hydroxyl groups on adjacent carbon atoms and is often used to generate aldehyde groups in HA.36,37 A peak assigned to the aldehyde group (C=O) of OHA is observed at 1720 cm-1 in the FT-IR spectrum (Figure S1, Supporting information). A mixture of GC and ADH was then added to the OHA solution to form a hydrogel. In the spectrum of the hydrogel, the aldehyde peak (1720 cm-1) disappears and a new peak appears at 1456 cm-1. This peak corresponds to the imine bond (C=N) generated upon Schiff base formation between OHA and GC and the hydrazone bond (C=N-N) formed by the reaction between OHA and ADH (Figure S1, Supporting information). OHA/GC forms a transparent hydrogel in the presence of ADH within seconds under physiological conditions at room temperature (Figure 1b). The schematic illustration for the gelation reaction is shown in Figure S2 (Supporting information) and Figure 1c. Controlling the mechanical stiffness and gelation kinetics of OHA/GC/ADH hydrogels is critical for their application to 3D bioprinting. The degree of oxidation (%), defined as the number of oxidized units per 100 repeating saccharide units in HA, is an important factor affecting the mechanical stiffness of the OHA/GC/ADH hydrogels. The use of highly oxidized OHA improves the mechanical stiffness of OHA/GC hydrogels but leads to the formation of inhomogeneous gels owing to its near-instant gelation behavior.35 Accordingly, the degree of OHA oxidation employed in this study was kept constant at 34%.35 The optimal OHA/GC ratio and total polymer concentration were determined in our previous study to be 9



[OHA]:[GC] = 2:1 and [polymer] = 3 wt%,35 and these conditions were used to form OHA/GC/ADH hydrogels in the present study. The addition of ADH results in a decrease of the mechanical stiffness of the hydrogel compared to that of OHA/GC hydrogel. The decrease is dependent upon ADH concentration (Figure 1d). Since OHA/ADH does not form hydrogel (no GC, [OHA] = 2 wt%, [ADH] = 0.3 wt%), hydrazone bonds may not actively participate in the cross-linked network formation of OHA at this condition. Thus, the addition of ADH to OHA/GC hydrogel is most likely to reduce the total number of active cross-links in the OHA/GC network and decreases the mechanical stiffness of the hydrogel. However, existence of both hydrazone bonds and imine bonds may alter the exchange kinetics of dynamic and reversible bonds in the network38-40 and induce self-healing of the hydrogel. Controlling the molecular weight of the OHA is critical for regulating the mechanical stiffness of the resultant hydrogel. OHA1000 and OHA2500 were prepared from HA with molecular weights of 1,000 kDa and 2,500 kDa, respectively. The gelation begins instantly in both cases, and the complete gelation time is not significantly affected by the molecular weight of the HA (Figure 1e). However, the higher the molecular weight of the HA, the stiffer the hydrogel (G' = 1,362  96 Pa and 2,528  163 Pa for OHA1000/GC/ADH and OHA2500/GC/ADH, respectively). The mechanical stiffness of a hydrogel usually decreases upon encapsulation of cells for tissue regeneration as the cells can inhibit cross-linking. Accordingly, OHA/GC/ADH hydrogels lose some of their mechanical stiffness upon ATDC5 encapsulation ([OHA] = 2 wt%, [GC] = 1 wt%, [ADH] = 0.3 wt%, [ATDC5] = 1 × 107 cells/mL). A much more significant decrease in the G' value is observed upon cell encapsulation for the OHA1000/GC/ADH hydrogel (G' = 283  27 Pa) compared with that for the OHA2500/GC/ADH hydrogel (G' = 620  59 Pa). OHA2500/GC/ADH gel disks retained 10


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more than 80% of their original diameter for 3 weeks when incubated in PBS at 37oC. Hydrogel disks lost their weight during in vitro degradation. About 40% weight loss was found for OHA2500/GC/ADH gel disks after 3 weeks of incubation, and a slight decrease in the degree of swelling of the gel disks was observed (Figure S3, Supporting information).

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Figure 1. (a) Chemical structures of oxidized hyaluronate (OHA), glycol chitosan (GC), and adipic acid dihydrazide (ADH). (b) Photographs of the polymer solutions (OHA and GC+ADH) and the resultant hydrogel. (c) Schematic illustration for the gel structure. (d) Changes in the storage moduli of OHA/GC/ADH gels depending on ADH concentration 12


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([OHA] = 2 wt%, [GC] = 1 wt%). (e) Gelation kinetics of OHA/GC/ADH hydrogels (■, G'; □, G''; [OHA] = 2 wt%; [GC] = 1 wt%; [ADH] = 0.3 wt%).

Self-healing Properties of OHA/GC/ADH Hydrogel. The self-healing ability of the OHA/GC/ADH hydrogel was first assessed by macroscopic self-healing tests. Hydrogel disks were prepared and cut into two pieces using a razor blade, and the pieces were then placed in contact and allowed to reassemble. Completely integrated gel disks were obtained in 10 min at room temperature without additional treatment (Figure 2a). When the gel disks were cut into four pieces and reassembled, the disks completely integrated and no splitting was observed upon shaking (Figure S4, see the video in the Supporting Information). This autonomous healing behavior of the OHA/GC/ADH hydrogel is due to the existence of dynamic and reversible covalent cross-links comprising imine bonds and hydrazone bonds in the hydrogel. When OHA is mixed with GC in the presence of ADH, a covalently cross-linked network is generated upon Schiff base formation between the OHA and GC. Although imine linkages are dynamic and reversible, OHA/GC hydrogel did not exhibit self-healing properties (see the video in the Supporting Information). Self-healing gels consisted of imine bonds have been often reported, including hydrazide-functionalized PEG/aldehyde-functionalized PEG,40 chitosan/dibenzaldehyde-PEG,41 and GC/dibenzaldehyde-PEG.42 However, GC/dibenzaldehyde-PEG gel was not fully recovered (recovery rate = ~38%). Non-selfhealing feature of OHA/GC hydrogel might be attributed to inherent rigidity of the polysaccharide backbone used in our study. N‐carboxyethyl chitosan and oxidized sodium alginate also required ADH to fabricate self-healing gel.43 The addition of ADH to OHA/GC hydrogel enables hydrazone bond formation between 13



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OHA and ADH. Interestingly, combination of two dynamic and reversible bonds (i.e., imine bonds and acylhydrazone bonds) would just change the exchange kinetics in the gel39,44 and enable self-healing of the gel. Hydrolytic degradation of acylhydrazone bonds in the gel may influence the dynamic imine bond exchange and resultant self-healing of the gel. Thus, the OHA/GC/ADH hydrogel maintains its cross-linked network structure through the rapid breaking and reforming of the reversible imine and hydrazone linkages (Figure 2b).

a)

cutting

self-healing < 10 min

1 cm

b)

Figure 2. (a) Photographs showing the self-healing process of OHA2500/GC/ADH hydrogel. 14


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Hydrogel disks were cut into two pieces that were then reassembled and formed completely integrated gel disks in 10 min at room temperature. (b) Schematic description of the selfhealing process by reversible breaking and reforming of imine and hydrazone bonds in the OHA/GC/ADH hydrogel.

Next, the self-healing behavior of the OHA/GC/ADH hydrogel was investigated in more detail using a rotational rheometer. First, an amplitude sweep was performed to determine the strain that breaks the hydrogel network, which was found to be 192% (Figure 3a). The strain sweep revealed that the G′ values are higher than the G″ values at low strain, indicating the solid-like structure of the OHA2500/GC/ADH hydrogel. However, the G′ values decrease with increasing strain, indicating that the gel network is disrupted. From this point, the gel exhibits dominantly fluidic behavior. When high strain (300%) is applied to the OHA2500/GC/ADH hydrogel for 2 min, the G′ values decrease substantially (close to the G″ values), indicating gel breakage. Interestingly, the G′ values recover instantly to their original values when the strain is removed (Figure 3b), and this process can be repeated several times. These results clearly demonstrate the self-healing behavior of the OHA2500/GC/ADH hydrogel. The hydrogel also retains its self-healing ability upon a notable increase in strain (Figure 3c). Next, the effect of ADH concentration on the stiffness recovery rate of the self-healing hydrogel was investigated. The recovery rate increases with ADH content (Figure 3d), and the hydrogel prepared with [ADH] = 0.3 wt% ([OHA] = 2 wt%, [GC] = 1 wt%) recovers most of its initial mechanical stiffness. However, as the addition of ADH results in a decrease in mechanical stiffness (as shown in Figure 1d), the OHA2500/GC hydrogel containing 0.3 wt% 15



ADH was used for all further experiments.

Figure 3. (a) Amplitude sweep carried out from 0 to 250% strain with the OHA2500/GC/ADH hydrogel ([OHA2500] = 2 wt%, [GC] = 1 wt%, [ADH] = 0.3 wt%). (b) The repeated self-healing behavior of the OHA2500/GC/ADH hydrogel was confirmed at 300% strain, (c) and was also observed when strain was increased to 800%. (d) Storage modulus recovery rate for the OHA/GC/ADH gel as a function of ADH concentration.

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3D Printing with the OHA/GC/ADH Hydrogel. The primary concerns in extrusion bioprinting are printability and stability. The stability of a construct after printing can be reinforced by secondary cross-linking.7 However, OHA/GC/ADH hydrogels do not require any post-gelation or additional cross-linking after the fabrication of 3D constructs. The selfhealing hydrogels are capable of recovering integrity and mechanical stiffness following structural damage. Model objects of various shapes and sizes were 3D printed via extrusion bioprinting of the OHA2500/GC/ADH hydrogel (Figure 4). The mechanical properties along with the self-healing ability of the OHA2500/GC/ADH hydrogel allowed stacking of the gel as a bioink, resulting in the fabrication of constructs with different heights and volumes. The self-healing OHA2500/GC/ADH hydrogel can be used for 3D printing without additional cross-linking or reinforcement, making it highly efficient in terms of both minimization of material consumption and selective implementation for specific self-healing applications. The self-healing behavior also allows the fusion of filamentous layers into unitary objects, as confirmed by microscopic observation (Figure 4d). Cells encapsulated within a gel may interfere the cross-linked network formation and reduce the self-healing capability of the gel. Interestingly, self-healing properties of OHA2500/GC/ADH were maintained even upon cell encapsulation (Figure 4d).

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b)

a)

c)

d)

0 min

15 min

30 min

Figure 4. 3D printed OHA2500/GC/ADH hydrogel in (a) donut, (b) disk, and (c) meniscus shapes (scale bar, 1 cm). (d) Microscopic observation of self-healing by the filamentous structure of OHA2500/GC/ADH gel encapsulating chondrocytes ([ATDC5] = 1 × 107 cells/mL; scale bar, 200 m).

In Vitro Chondrogenic Potential of the OHA/GC/ADH Hydrogel. The chondrogenic differentiation of ATDC5 cells encapsulated in 3D printed OHA/GC/ADH hydrogel was investigated. ATDC5 cells were encapsulated into OHA2500/GC/ADH hydrogel ([OHA2500] = 2 wt%, [GC] = 1 wt%, [ADH] = 0.3 wt%), and their viability was first evaluated using a live-and-dead assay method before printing ([cell] = 1 × 107 cells/mL). Cell viability within the 3D printed construct was also assessed after 2 h of in vitro culturing. The ATDC5 cells almost maintain their viability within the hydrogel after the printing process (Figure 5a). In addition, the cells were viable within the gel over time. More than 80% of cells were viable in the gel after one week of incubation (Figure S5, Supporting information). This 19



clearly demonstrates the potential of 3D-printed OHA2500/GC/ADH hydrogel as a scaffold for tissue engineering applications. We next investigated the chondrogenic differentiation of ATDC5 cells encapsulated within 3D printed OHA/GC/ADH hydrogel. The expression of chondrogenic marker genes such as SOX-9 and COL-2 was evaluated by RT-PCR and real-time PCR analyses. Recent studies have demonstrated that the mechanical stiffness of a hydrogel can regulate chondrogenic differentiation,45 and the mechanical stiffness of the OHA/GC/ADH hydrogel was varied by changing the molecular weight of the HA (OHA1000/GC/ADH, G' = 283  27 Pa; OHA2500/GC/ADH, G' = 620  59 Pa). The expression of SOX-9 at day 7 is more prominent for the OHA2500/GC/ADH hydrogel than the OHA1000/GC/ADH hydrogel (Figure 5b). This may be because of the difference in the mechanical stiffness of the two hydrogels. However, the expression level of SOX-9 decreases along with a notable increase in collagen type II (COL-2) gene expression by day 21. SOX-9 is expressed in the early phase of chondrogenic differentiation31 and can directly regulate the expression of type II collagen, which is a major ECM component in cartilage tissue.46,47 This finding was also supported by quantitative analysis using the real-time PCR method (Figure 5c). Again, an increase in the molecular weight of HA leads to an increase in the mechanical stiffness of the resultant OHA/GC/ADH hydrogel and thus enhances chondrogenic differentiation of ATDC5 cells in vitro. We also confirmed that self-healing properties of OHA/GC/ADH hydrogel did not significantly influence chondrogenic differentiation of ATDC5 cells encapsulated in the gel (Figures S6 and S7, Supporting information). Thus, OHA2500/GC/ADH hydrogel can be used as a bioink without any adverse effects to chondrogenic differentiation of ATDC5 cells.

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Before printing

After printing

b) O HA 10 00 O HA 25 00

a)

Cell viability (%)

GAPDH SOX-9 COL-2 Day 7

3

After

Relative gene expression (COL-2) R e la t iv e g e n e e x p r e s s io n ( C o l -l l)

Before

c) Relative (SOX-9) Relativegene gene expression expression (SOX-9)

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7days Day 7 21days Day 21

*** 2

1

0

O HA 10 00 O HA 25 00

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1000k printed

OHA1000

2500k printed

OHA2500

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7 d a7y s Day 21days Day 21

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**

6 5 4 3 2 1 0

1 0 0 0 k p rin te d

OHA1000

2 5 0 0 k p r in te d

OHA2500

Figure 5. (a) Live-and-dead assay results for ATDC5 cells encapsulated in OHA2500/GC/ADH hydrogel before and after printing. (b) Expression of chondrogenic marker genes (SOX-9 and collagen type II). (c) Quantitative analysis of the gene expression levels of ATDC5 cells cultured within the OHA/GC/ADH gel (**P-values < 0.01, ***Pvalues < 0.001).

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CONCLUSIONS We have demonstrated the design and preparation of a printable self-healing hydrogel based on biocompatible polysaccharides that can be applied as a bioink for fabricating cell-laden structures using an extrusion-based 3D printer. The self-healing hydrogel is prepared from OHA, GC, and ADH, and the competitive formation of imine and hydrazone bonds endows it with highly efficient self-healing properties. The viscoelastic properties of the OHA/GC/ADH hydrogel were tailored by controlling the polymer concentration and molecular weight of the HA in order to optimize the hydrogel as a bioink for 3D printing. The resultant 3D constructs showed structural stability, even without the addition of supporting materials or the use of secondary cross-linking for further stabilization. The excellent biocompatibility of the OHA/GC/ADH hydrogel and controlled chondrogenic differentiation of ATDC5 cells cultured within it demonstrate its potential for tissue engineering applications, including cartilage regeneration.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

Infrared spectra, schematic gelation reaction, degradation data, cell viability, and PCR analysis data (PDF). Video clips of non-self-healing and self-healing hydrogels (MP4).

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (K.Y.L.)

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2016R1A2A2A 10005086).

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For Table of Contents Use Only

Three-dimensional Bioprinting of Cell-laden Constructs Using Polysaccharide-based Self-healing Hydrogels

Sang Woo Kim, Do Yoon Kim, Hyun Ho Roh, Hyun Seung Kim, Jae Won Lee, and Kuen Yong Lee*

Self-healing hydrogel cut

Assembly

Self-healing 10 min

Hydrogel disks

0 min

3D bioprinting with cells

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15 min

30 min

Three-dimensional Bioprinting of Cell-laden Constructs Using

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