A Spider-Inspired Multi-Component 3D Printing Technique for Next

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A Spider-Inspired Multi-Component 3D Printing Technique for Next-Generation Complex Biofabrication You Zhou, Shenglong Liao, Xinglei Tao, Xiao-Qi Xu, Quan Hong, Di Wu, and Yapei Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00230 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Applied Bio Materials

A Spider-Inspired Multi-Component 3D Printing Technique for Next-Generation Complex Biofabrication You Zhou,a Shenglong Liao,a Xinglei Tao,a Xiao-Qi Xu,a Quan Hong,b Di Wub and Yapei Wanga* a

Department of Chemistry, Renmin University of China, Beijing 100872, China.

b

Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease, Beijing 100853, China Abstract: The shortage of tissue resources is currently a serious challenge that limits the clinical therapy to patients with tissue loss or end-stage organ failure. The booming development of 3D printing offers unprecedented hope for tissue engineering since it can construct cells and biomaterials into a 3D tissue-mimicking object with precise control over size and shape. However, it is still challenging to fabricate artificial living tissues or organs due to the extreme complexity of biological tissues. Herein, we propose a new concept of spider-inspired 3D printing technique (SI-3DP) for continuous multi-component 3D printing based on in-situ gelation at multi-barrel printing nozzle. The printing process allows for rapid construction of 3D architectures composed of different inks in desired position. To present the potential in biomedical applications, the SI-DIP also prints vessel-like hollow hydrogel microfibers and cell-laden hollow fibers, indicating good biocompatibility of this technique. The newly-developed SI-3DP technique is envisioned to promote the development of next-generation complex biofabrication.

Keywords: Spider-inspired, Multi-component, In-situ gelation, 3D printing, Hydrogel 1

ACS Paragon Plus Environment

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Introduction Tissue engineering has been proposed as a new field aiming to treat patients with tissue loss or end-stage organ failure based on the principle of biology and engineering since 1993.1 Enormous kinds of non-active scaffolds and bio-active materials have been designed and manufactured for medical implants or clinical therapy according to the principle guideline of tissue engineering.2-7 However, tissue or organ resource shortage is still the bottleneck that severely limits the clinical therapy.8,9 Traditionally, tissue repair or transplantation can only rely on well-matched tissue or organ from other people or artificial tissue made of biomaterials and cells. In recent years, the booming development of 3D printing technique offers promising hope especially for tissue repairing.10-17 Current research on 3D printing plays an unreplaceable role in the field of tissue engineering, while it is still a challenge to fabricate artificial living tissues or organs due to the extreme complexity of biological tissues of human body.18-20 Most tissues or organs composed of functional cells and extracellular matrix present absolutely non-uniform mechanical properties and bioactivities due to the spatial organization of cells and matrix21 which significantly increase the difficulty for printing a bioactive tissue or organ to satisfy clinical uses. Hence, a multi-component 3D printing technique for complex biofabrication is highly desired while related researches are rarely archived as far as we know.

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The ideal multi-component printing technique should meet the requirements for printing different materials, cells and cell factors in specific positions and building sophisticated hydrogel structures to mimick tissues and organs.22,23 So far, the existing 3D printing technologies used for biomedical application mainly include inkjet-based printing, laser-based printing and extrusion-based printing.16,24-27 Among these technologies, the inkjet-based printing and extrusion-based printing are the effective methods that have realized multi-component printing. The inkjet-based printing methods can deliver various kinds of inks to predefined locations just like the principle of colorful inkjet printer. 24,25 However, the inkjet printing methods requires specific inks that meets the printing demands on pH, surface tension and liquid viscosity, which is the key factor that limits the versatility of inkjet-based 3D printing. On the contrary, the extrusion-based printing approach can adapt to various bio-inks and its modification and operation are much more available for deep development in biological labs.22-23, 27 The extrusion-based 3D printing approach has already been designed into multi-nozzle 3D printing devices by combining multiple printing nozzles into one printing system. However, this approach for multi-component printing relies on frequent switching between printing nozzles for printing different inks in different cartridges, which increase the time cost and affect the printing efficiency. Recently, Ali and coworkers made an excellent breakthrough in multi-component printing based on a multi-barrel printing nozzle while their research is limited on shear-thinning hydrogels.23 Hence, it is still challenge yet necessary to

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develop a more versatile multi-component 3D printing technique that can be compatible with more printing systems. To solve the manufacture difficulties of artificial bio-active tissues or organs by innovation on 3D printing technique, dating back and learning from nature creatures is a wisdom approach. As far as we know, spiders can spin many tiny but ultra-strong fibers made by a complex of many kinds of proteins.28 Just like a printing nozzle, spiders can print a well-designed network to hunt for foods via the webs. As shown in Figure 1a, an interesting point of the spider fiber is that the fiber mechanical properties vary in a large range due to the tunable composition of the spider fibers. Large quantities of researches have revealed that the spiders commonly have 7 kinds of spinnerets that can secret 7 kinds of proteins and related biological components. Herein, inspired by the spider spinnerets, we proposed a new concept of spider-inspired 3D printing technique (SI-3DP) for multi-component 3D printing based on in situ gelation at multi-barrel printing nozzle via common extrusion-based printing mode. As illustrated in Figure 1b, a 7-channel capillary was served as the printing nozzle fixed on the 3D motion stage and connected with 7 individual injection systems. By using some active inks that can fast react with each other at the nozzle, in situ gelation can be achieved thus gels can be printed as the controllable motion of the printing nozzle. The printing process allows for rapid deposition of 3D architectures composed of different inks in the desired position. The SI-3DP technique we reported here avoids the switching time between different printing nozzles and thus the printing process is in a rapid and continuous manner. To present 4


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the potential in biomedical applications, we also printed vessel-like hollow hydrogel microfibers and the cell-laden hollow fibers. This technique is hoped to promote the development of next-generation biomedical fabrication via the concept of multi-component extrusion bioprinting.

Experimental section Materials. Sodium alginate (SA) was purchased from Lyntech, Beijing, China. Gelatin (~250g bloom), acrylamide (AM), glutaraldehyde (GA, 50% aqueous solution) and fluorescein diacetate (FDA) were purchased from Aladdin, Shanghai, China. The N,N’-methylenebisacylamide (97%) (MBAM) and rhodamine B were obtained from Alfa

Aesar.

N-hydroxysuccinimide

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

(NHS)

hydrochloride

(EDC)

and were

supplied by J&K Scientific. Calcium chloride and isopropyl alcohol were provided by Beijing Chemical Reagent Company. Irgacure 2959 was provided by BASF. RPMI 1640 Medium was purchased from HyClone. 0.25% Trypsin-EDTA was obtained from Life Technologies. Penicillin-Streptomycin solution (100X) and fetal bovine serum (FBS) were obtained from Cellgro. Phosphate buffer solution (PBS) was purchased from Biotopped and the propidium iodide (PI) was obtained from Sigma. Device setup. The multi-barrel (2, 3 or 7) glass capillary (World Precision Instruments Inc, Sarasota, FL, USA) was heated with a blast burner and stretched into a tapered shape with a tip diameter of about 200~300 µm. Each channel was connected with Teflon tubing and sealed with epoxy resin. The capillary was then

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fixed on the 3D translation stage (Zolix, Beijing, China) driven by a motion controller (Zolix MC-600, Beijing, China). Syringes were connected with Teflon tube and were set on several individual syringe pumps (LSP02-1B, LongerPump, Baoding, China). 3D Printing of hydrogel. For common alginate-calcium hydrogel, sodium alginate solution with weight percentage of 0.1%, 0.5%, 1.0% and 2.0% and calcium chloride solution with weight percentage of 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10.0% and 20.0% were prepared and served as the inks. Then, the two solutions were loaded in two channels of glass capillary separately with the programmed flow rate of 20 µL/min in each channel during the printing process. For the gelatin-glutaraldehyde hydrogel, the gelatin solution with weight percentage of 10% and glutaraldehyde solution with weight percentage of 3.0%, 5.0% and 7.0% were prepared respectively. These two solutions were loaded into the capillary channels separately and the flow rates were set at 20 µL/min (gelatin channel) and 8 µL/min (glutaraldehyde channel) respectively during the printing process. For the alginate-gelatin complex hydrogel, sodium alginate solution (2 wt.%), calcium chloride solution (2.0 wt.%), gelatin solution (10.0 wt.%) and glutaraldehyde solution (2.0 wt.%) were loaded in glass capillary and the flow rates were set at 20 µL/min, 20 µL/min, 5 µL/min and 5 µL/min, respectively. For the alginate-polyacrylamide double network hydrogel, sodium alginate solution (2 wt.%), acrylamide solution mixed with crosslinker and photoinitiator (80 wt.% AM, 0.36 wt.% MBAM, 1.0 wt.% Irgacure 2959) and calcium chloride solution 6


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(5.0 wt.%) were loaded in glass capillary, respectively. The flow rates of sodium alginate solution were 8 µL/min and the other two channels were set at 10 µL/min. Rheological test of printed hydrogels. The hydrogel sample was printed as a square shape with size about 1 cm and then the hydrogel was cut into an 8 mm disk for rheology test. Rheological properties of the hydrogel were characterized using a rheometer (TA ARES-G2). Specifically, the shear stress-strain test was carried out at different strains ranging from 0.01 to 100 % with the temperature fixed at 25 °C. Then the storage modulus (G’) and loss modulus (G’’) were assessed by applying a constant strain (0.1 %) within the linear viscoelastic region over a frequency range from 100 to 0.01 Hz. The qualitative test of interlayer adhesion of hydrogels. Two kinds of hydrogels, pure alginate hydrogel and alginate-gelatin complex hydrogel were prepared as stated above. Both shapes were 3 cm-sized squares consist of 3 mm-sized meshes. The hydrogels were subsequently put into petri dishes filled with CaCl2 solution and the petri dishes were fixed on vortex and shaken with constant rotation speed of 500 rpm. Compressing and releasing test of hydrogels. Two kinds of hydrogels, pure alginate hydrogel and alginate-polyacrylamide double network hydrogel were prepared as stated above. The samples were printed as the shape of 1 cm-length square. The test was conducted on a pressure gauge (MARK-10, Series 5) at a compressing or releasing rate of 40 mm/min with a maximum force of 5 N. Preparation of Rhodamine-modified sodium alginate.

Firstly,

0.43 mL

ethylenediamine was slowly added into 60 mL ethanol solution dissolving 2.4 g 7



Rhodamine B under vigorous stirring at room temperature. Subsequently, the stirred mixture was heated for 12 h with reflux until it becomes clear. Then the mixture was cooled and solvent was removed under reduced pressure via the rotary evaporation. About 100 mL HCl (1 mol/L) was added into the residue to remove unreacted ethylenediamine. After that, the pH of the solution was adjusted to 9~10 by slowly adding 1 mol/L NaOH. The resulting solution was extracted by dichloromethane to get the organic phase, which was subsequently dried by Na2SO4 and evaporated to get the crude product of amine-modified Rhodamine B. Then, 121 mg above product and 500 mg sodium alginate were dissolved into 25 mL distilled water under mild stirring at room temperature. Then, 15 mg NHS and 47.25 mg EDC were added in to the above solution. After stirred for 12 h at room temperature, the resulting mixture was precipitated in isopropyl alcohol and washed for several times. Finally, the product was dried under vacuum at 60 oC. Printing the hollow hydrogel microfiber. 7-channel capillary was chosen for preparation of hollow alginate microfibers. The alginate solution was loaded in 6 outer channels and calcium chloride was loaded in the middle channel. The flow rate of the each alginate channel ranged from 600~800 µL/min and the flow rate of the CaCl2 solution was 1~5 mL/min. Preparation of cell-laden alginate solution. L929 mouse fibroblasts and rat aortic endothelial cells (RAOEC) were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1.0% penicillin-streptomycin solution. All cells were incubated at 37 oC in 5.0% CO2 in polystyrene petri dish. 8


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The petri dish with 90% cells confluence was washed with PBS and then incubated with 0.25% Trypsin-EDTA for 3~4 min at 37 °C in 5.0% CO2 to digest the cells from the substrate. Next, the cell suspension was centrifuged under 1,000 rpm for 5 min at room temperature. Cell pellet was resuspended in cell culture medium to achieve a concentration of 5.0×106 cells/mL. The cell suspension was mixed with 3.0% sodium alginate solution at a volume ratio of 1:1 and the final solution contains 1.5 wt.% sodium alginate and 2.5×106 cells/mL. Cell viability analysis. The cell viability in the fabricated hollow microfiber structures was measured everyday by a live/dead staining assay. Cell-laden alginate microfibers were washed with PBS three times before staining. FDA and PI were diluted with PBS at a concentration of 5 mg/mL. The cell-laden alginate structures in 1 mL PBS solution were stained with 1 µL FDA solution and 1 µL PI solution for 10 min in the dark. Finally, a confocal laser scanning microscope (CLSM, LEICA TCS-SP5) was used to photograph the stained cells with excitation 530nm / emission 620nm for PI and excitation 498nm / emission 517nm for FDA. The amounts of live or dead cells were counted by Bitplane Imaris and the cell viability was calculated as the percentage of FDA positive cells.

Results and discussion The fundamental principles of the SI-3DP are mainly based on the multi-barrel capillary and the in situ gelation process. As shown in Figure 1b and 1d, a typical 7-barrel capillary tapered with blast burner is served as the printing nozzle that 9



contains seven independent extrusion channels with approximately equal size. Each channel was connected with an individual injection system and the gelation process happens in situ once the inks from different channels converge at the printing nozzle. Instructed by the simple principle, fast reaction between alginate and calcium ion based on the chelation interaction (Figure 1c) was chosen as a typical gelation reaction. As predicted by above design, hydrogels with different pre-designed shapes were successfully printed via the SI-3DP method (Figure 1e-g). Compared with traditional multi-component printing method based on switching the printing nozzles, the SI-3DP method does not spend additional time on changing channels since every channel shares a same printing position. Thus the SI-3DP method we reported here is able to continuously print different types of inks in both individual and simultaneous modes and different kinds of inks can be deposited in the specific position through selectively turning on/off the syringe pump. As shown in Figure 1h, a bi-color rectangular lattice was printed as an example for illustrating the concept of multi-component 3D printing.

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Figure 1. (a) A diagram for demonstrating the spinning mechanism by natural spiders. (b-c) Schematic illustration of spider-inspired 3D printing. (d) Optical photograph of the multi-channel printing nozzle. Inset is a Scanning Electron Microscope image of the printing nozzle. (e-g) Photographs of different shaped and single colored (blue ink) alginate hydrogel printed by SI-3DP. Insets are photos from front viewpoint. (h) Photograph of a bi-color alginate hydrogel printed by two kinds of alginate sodium solutions dissolving red ink and blue ink.

The SI-3DP technique can print hydrogels not only with the different shapes, but 11



also with tunable modules through the control of ink components. By tuning the concentrations of sodium alginate and calcium chloride, hydrogels with the different rheology performance have been printed and the rheology properties are shown in Figure 2a and 2b. When the concentration of sodium alginate is set at 1 wt.%, the storage modulus (G’) of printed hydrogel increases as increasing the concentration of calcium chloride since the increased concentration can lead to more crosslinking points thus enhances the mechanical performance. However, excess calcium chloride may also lead to decreased crosslinking degree of alginate hydrogel and poor interaction between different layers, both of which can cause the decrease of mechanical performance (Figure 2a). As a result, the storage modulus of hydrogel printed with 20 wt.% calcium chloride is lower than that of hydrogel printed with 10 wt.% calcium chloride. Similarly, keeping the concentration of calcium chloride at 10 wt.%, the modules of hydrogel increases as the increase of sodium alginate concentration (Figure 2b). Besides considering the mechanical performance, the printing requirement on concentrations of sodium alginate and calcium chloride has been investigated. A phase graph shown in Figure 2c indicates that hydrogel filaments could be produced at the printing nozzle and built up into the designed model, only when the concentration of sodium alginate and calcium chloride are high enough. In addition to the physical chelation between alginate and calcium ions, the fast covalent crosslinking between gelatin and glutaraldehyde can be also served as the gelation reaction for SI-3DP. The rheology properties of the printed gelatin-glutaraldehyde hydrogel are shown in Figure 2d, indicating higher 12


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concentration of glutaraldehyde leads to better mechanical performance. As stated above, high concentration of calcium chloride would lead to poor interlayer adhesion and cause slips between adjacent layers. Interestingly, it can be successfully solved via simultaneously printing the above two gelation systems. In order to qualitatively compare the interlayer adhesion force between pure alginate hydrogel and alginate-gelatin hydrogel, shaking experiments on two square-lattice hydrogels were carried out simultaneously. As shown in Figure 2e, pure alginate hydrogel was appeared in a mess after shaking for 3 hours while the alginate-gelatin kept original shape after shaking for 24 hours, indicating the enhanced adhesive force in alginate-gelatin complex hydrogel.

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Figure 2. Rheological properties of alginate hydrogels printed at different concentrations of calcium chloride and a certain concentration of sodium alginate (1 wt.%) (a) or at different concentrations of sodium alginate and a certain concentration of calcium chloride (10 wt.%) (b). (c) A phase diagram for demonstrating the printing conditions. Green triangles represent successful printing process and the yellow-gray cycle means a failure of 3D printing. (d) Gelatin-glutaraldehyde covalent hydrogel printed at different concentrations of glutaraldehyde and same concentration of gelatin (10 wt.%). (e) Optical photographs of alginate hydrogel and alginate-gelatin complexed hydrogel before and after a mild shaking (500 rpm) in calcium chloride 14


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solution for different times. Scale bar: 1 cm.

Some human tissues such as Achilles tendon own excellent toughness. To imitate these tough tissues, double network hydrogel is an ideal choice. As a multi-component printing technique, the SI-3DP can easily print double network hydrogels by separating different components in different channels. Commonly, double network is composed with a covalent network and a supramolecular network. Herein, as a representative example, alginate and acrylamide were selected as the basic materials to construct the double network hydrogel and the mechanism is illustrated in Figure 3a. Alginate-calcium network formed in situ at the printing nozzle was served as the supramolecular network. Then the monomer, crosslinker and photoinitiator mixed in the supramolecular network were polymerized under UV irradiation, generating the covalent network. Correspondingly, double network hydrogel was printed via the SI-3DP technique. To prove the successful polymerization of polyacrylamide, compressing and releasing tests were carried out on single network (alginate-calcium hydrogel) and double network hydrogel (alginate-polyacrylamide hydrogel). As shown in Figure 3b, the ionically crosslinked alginate hydrogel cannot recover to original shape after compressed with a pressure gauge since the hydrogel is composed by a supramolecular network that cannot provide sufficient elasticity. On the contrary, the alginate-polyacrylamide hydrogel composed with double network can almost recover to original shape (Supporting Movie S1). Even after compressing and releasing for 5 cycles, the double network hydrogel printed via SI-3DP technique can go back to original state and the stress-strain curve in 5 consecutive cycles are highly 15



overlapped (Figure 3c), indicating good elastic performance of the double network hydrogel. Besides, rheology tests shown in Figure 3d also proved better mechanical performance of double network compared with alginate-calcium hydrogel, indicating successful formation of double network hydrogel by the SI-3DP technique.

Figure 3. (a) Chemical principles of printing double network by photocuring the acrylamide in the presence of crosslinker and photo-initiator in alginate-calcium network. (b) Optical photographs of as-printed alginate hydrogel (single network) and alginate-PAM (double network) at original state, compressed state and relaxed state. 16


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(c) Force-strain curves of alginate-PAM hydrogel during five consecutive compressing and releasing cycles. (d) The comparison between alginate hydrogel and alginate-PAM hydrogel in rheological properties.

Another challenge of current 3D biofabrication lays in insufficient oxygen and nutrition supply for the artificial tissues or organs. Inspired by human body, inside network composed by artificial blood vessel seems to be an ideal solution. The preparation of hollow hydrogel microfiber is one of the fundamental steps for the fabrication of artificial blood vessel. Commonly, most hollow microfibers were fabricated by coaxial extrusion methods.29-33 Herein, as a multi-component 3D printing technique, in addition to tuning the modules and components of the hydrogel, the SI-3DP can also print microfibers with well-designed topological structure such as hollow hydrogel fiber via loading the channels with different inks. As presented in Figure 4a-c, using the 7-barrel capillary as the printing nozzle, hollow alginate microfibers can be printed via loading alginate solution in 6 outer channels and calcium chloride in middle channel. Due to the relative large diffusion velocity of calcium ion and the fast gelation speed between alginate and calcium ion, a hollow layer of alginate-calcium hydrogel forms once the two kinds of liquid are encountered at the nozzle tip and it forms a hollow microfiber as the deep diffusion of calcium ion. Optical microscope image and the CLSM image clearly identified the hollow structure with a thin wall less than 100 µm (Figure 4b-c). As a hollow hydrogel fiber, fluid can be injected into this hollow fiber and flow in it just like blood flowing in the vessel (inset in Figure 4a and Supporting Movie S2). When ink concentration and 17



capillary size are kept as constants, the diameter of the hollow microfiber is only influenced by flow rate of outer phase and inner phase. As shown in Figure 4d and 4e, both of outer diameter and inner diameter increase as the flow rate of calcium chloride increases, because larger flow rate of inner phase can lead to larger hollow cavity. Similarly, larger flow rate of outer phase would lead to larger outer diameters while shows slight effect on inner diameter. Thus the outer diameter increases remarkably while the inner diameter exhibits only slight increase when the flow rate of alginate is increased. Compared with traditional methods that rely on the coaxially-aligned extrusion nozzle, the commercial capillary for SI-3DP provides a more convenient approach to realize the preparation of hollow hydrogel microfibers. Moreover, the multi-channel capillary brings more possibilities such as core-sheath structure, none uniform hollow fiber and so on.

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Figure 4. (a) Schematic illustration of the printing mechanism for hollow Ca-alginate microfiber. Inset: injection experiment in hydrogel tube at pre-injection state, injecting state and post-injection state. (b-c) Optical microscope image (b) and CLSM image (c) of the hollow Ca-alginate microfiber. Red dye: alginate modified with Rhodamine B. Inset is a cross-section CLSM image. (d-e) The outer (d) and inner (e) diameter of hollow alginate microfiber printed at different flow rates of alginate and CaCl2.

Besides simple extrusion the hollow hydrogel microfibers, the SI-3DP technique 19



can also generate 3D construction based on these hollow microfibers. As illustrated in Figure 5a, the hollow alginate microfiber extruded from the nozzle was printed into a 3D cylindrical shape (Figure 5b). To verify the hollow structure inside the 3D object, a similar perfusion experiment was carried out and the results showed that liquid can flow along the hollow microfiber in the 3D object (Figure 5c and Supporting Movie S3). Moreover, SEM images of the 3D object after lyophilization in Figure 5d also clearly presented the hollow structure. The hollow microfibers exhibit advantages in mass exchange inside and outside of the microfiber just like blood vessel. Thus the 3D construction made by these hollow hydrogel microfibers lays a solid foundation for nutrition supply in 3D cell culture. It is believed that the SI-3DP technique provides a new approach for constructing 3D microtubular network with ability of 3D culture of cells or artificial tissues.

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Figure 5. (a-b) Schematic illustration and optical photograph of 3D cylindrical hydrogel made of hollow alginate microfibers. The inset in (b) is a magnified picture of the cylindrical wall. (c) Image of liquid injection into the 3D cylindrical hydrogel. (d) SEM image of longitudinal section of lyophilized 3D cylindrical hydrogel. Inset is a large field of view SEM image.

As stated above, the hollow hydrogel microfiber exhibits similar size and shape as blood vessel thus it may be a proper option for preparing artificial blood vessel. The hollow hydrogel microfiber made by alginate and calcium may provide appropriate microenvironments for cell encapsulation and culture. To verify the possibility for cell culture, the mouse fibroblast cells (L929) dispersed in alginate solution was encapsulated in the shell of the microfibers during gelation process. The hollow microfibers loading L929 cells were cultured in standard culture medium and cell viability were calculated via live/dead staining with FDA/PI. The bright-field and fluorescent microscope images of cell-loaded hollow microfibers at day 1, 3 and 5 are shown in Figure 6a-f and the cell viability data is shown in Figure 6h. At day 1 and day 3, no cell death was found and the structure of the microfiber kept well as same as original state. At day 5, the bright-field and fluorescent images indicate the hollow microfiber exhibit obvious degradation due to the diffusion of calcium ion. As a consequence, some cells would be detached from the hydrogel microfibers, which induces cell apoptosis and might be the main reason for the decrease of cell viability. Although cell death was found at day 5, the cell viability is still over 60%. The above results indicate that the hollow alginate hydrogel microfiber can provide a good cell 21



culture microenvironment while the degradation speed should be finely controlled by other chemical crosslinking methods for long term cell culture.

Figure 6. (a-f) The bright-field (a-c) and fluorescent (d-f) microscope images of cell-laden hollow alginate microfiber at day 1, 3 and 5. Live and dead cells (L929) were stained with green fluorescence (FDA) and red fluorescence (PI), respectively. Scale bar: 200 µm. (g) CLSM image of hollow alginate microfiber loading GFP-labeled rat aortic endothelial cells. Inset is a cross section CLSM image. (h) Statistic of cell (L929) viability in five days according to the live/dead staining.

Conclusion 22


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In this work, we proposed a spider-inspired multi-component 3D printing technique that can continuously print hydrogels without any process of switching nozzles. Based on a model gelation reaction between alginate and calcium ion, a series of hydrogels with tunable shapes, modules and topological structures have been printed by the SI-3DP technique, demonstrating that the SI-3DP can be served as a versatile platform for multi-component 3D printing of hydrogels. Moreover, cells dispersed in the printing inks can be printed and loaded in the hollow alginate microfibers. The study of cell culture in the hollow hydrogel convinced the biocompatibility of this printing technique. The perfect combination between multi-component 3D printing and extrusion of vessel-like hollow hydrogel fiber indicates that the SI-3DP is an excellent candidate for next-generation 3D bioprinting. We believe that this multi-component printing strategy is bound to attract broad research interests and exhibit infinite potential in fields of biofabrication and tissue engineering.

Associated content Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:. Movie S1: Compressing and releasing process of single and double network hydrogels. Movie S2: Injecting liquid into hollow alginate filament.

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Movie S3: Injecting liquid into hollow alginate filament of 3D cylinder.

Author information Corresponding Author: [email protected] (Yapei Wang) ORCID Yapei Wang: 0000-0001-5420-0364

Acknowledgement Y. Zhou and S. Liao contributed equally to this work. Shuqiang Wang and Ruiting Li were acknowledged for their help and training on cell culture and staining. Qinyuan Gui was acknowledged for his help on hydrogel compressing measurement. This work is financially supported by the National Natural Science Foundation of China (21674127, 21422407, and 51373197)

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