3D Bioprinting of Vessel-like Structures with Multilevel Fluidic

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3D Bioprinting of Vessel-like Structures with Multi-level Fluidic Channels Qing Gao, Zhenjie Liu, Zhiwei Lin, Jingjiang Qiu, Yu Liu, An Liu, Yidong Wang, Meixiang Xiang, Bing Chen, Jianzhong Fu, and Yong He ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00643 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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ACS Biomaterials Science & Engineering

3D Bioprinting of Vessel-like Structures with Multi-level Fluidic Channels Qing Gao1,2, Zhenjie Liu3, Zhiwei Lin1, Jingjiang Qiu2, Yu Liu2, An Liu4, Yidong Wang5, Meixiang Xiang5, Bing Chen3, Jianzhong Fu*1, and Yong He*2 (1State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China 2 Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province , School of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China 3 Department of Vascular Surgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China 4 Department of Orthopaedic Surgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China 5 Department of Cardiology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China * Correspondence to: Yong He; e-mail: [email protected] or Jianzhong Fu; e-mail:[email protected])

Abstract In this study, 3D hydrogel-based vascular structures with multi-level fluidic channels (macro-channel for mechanical stimulation and micro-channel for nutrient delivery and chemical stimulation) were fabricated by extrusion-based three-dimensional (3D) bioprinting, which could be integrated into organ-on-chip devices that would better simulate the microenvironment of blood vessels. In this approach, partially cross-linked hollow alginate filaments loading fibroblasts and smooth muscle cells were extruded through a coaxial nozzle and then printed along a rotated rod template, and endothelial cells were seeded into the inner wall. Due to the fusion of adjacent hollow filaments, two-level fluidic channels, including a macro-channel in the middle formed from the cylindrical template and a micro-channel around the wall resulted from the hollow filaments were formed. By this method, different shapes of vessel-like structures of millimeter diameter were printed. The structures printed using 4% alginate exhibited ultimate strength of 0.184MPa, and L929 mouse fibroblasts encapsulated in the structures showed over 90% survival within one week. As a proof of concept, an envisioned load system of both mechanical and chemical stimulation was demonstrated. In addition, a vascular circulation flow system, a cerebral artery surgery simulator, and a cell co-culture model were fabricated to demonstrate potential tissue engineering applications of these printed structures.

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Keywords 3D bioprinting; Vascularization; Multi-level fluidic channels; 3D cell culture; Tissue engineering 1 Introduction Cardiovascular diseases are serious illnesses among the elderly and often exhibit complex pathogenesis related to vascular performance degradation. This degradation can be caused by abnormal chemical or mechanical stimulation such as occurs with hyperlipidemia, hypertension, hyperglycemia, and blood disorders.1-3 The development of an in vitro model to study the pathogenesis is urgently needed. Over the past few decades, two-dimensional cell culture models have been developed,4-6 however, these models are unable to recapitulate the three-dimensional environment and exhibit important differences from actual biological systems. As a novel analytical tool, organ-on-chip technology has attracted increasing interests,7-9 since Ingber et al. elevated the concept in 2010.10 Some vessel-on-chip models have already been built based on this concept to simulate the microenvironment of blood vessels.11-15 However, only several vascular cells were cultured in these chips. If a tissue engineering vessel could be built that would enable integrated chemical and mechanical stimulation for these organ-on-chip devices, these approaches would be much more informative. Therefore, it is crucial to develop an improved method to fabricate vessel-like structures that allows the integration chemical and mechanical inputs. 3D bioprinting (cell printing or organ printing) has emerged as an exciting new technique in tissue engineering.16-18 This method has been applied for the fabrication of 3D tissues and organs including vessel-like structures, where layer-by-layer freeform fabrication is used to deposit a mixture of cells and biologically compatible hydrogel. For vascular structures, many bioprinting


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methods have been tested, including indirect bioprinting of perfused vasculature fluidic channels19-27 and direct bioprinting of a vessel-like structure.28-42 In the indirect approach, Chen et al.19 reported the printing of 3D filament networks of carbohydrate glass at temperatures above100 °C. Lewis et al.20,

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developed an approach to

rapidly create a vessel-like channel with a perfused open lumen inside cell-laden inks using F127 as the sacrificial template. Dai et al.22,

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tested the printing of a cell-gelatin mixture onto

collagen layers, and then liquefied the gelatin to form a dynamically perfused vasculature inside a thick hydrogel scaffold. Khademhosseini et al.24,

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used agarose template fibers as the

sacrificial layer to fabricate perfused micro-channels inside photo-crosslinkable hydrogel constructs. With an alternate strategy, Forgacs et al.26 introduced a self-assembly approach to use layer-by-layer printing of multicellular spheroids or cylinders on agarose rods as a sacrificial template. In our previous work, another indirect method for the concurrent printing of scaffold and micro-channels was used in which high strength cell-laden hydrogel 3D structures with built-in micro-channels can be fabricated using hollow alginate filaments by controlling the crosslinking time.27 These abovementioned indirect bioprinting approaches have successfully produced vascular channels with lumen surfaces that permitted nutrient delivery, suggesting the potential for chemical loading. However, since the structures printed in these methods were channels that were embedded in thick hydrogel scaffolds, they were unable to be directly used for mechanical loading. Another approach to fabricate a vascular network is to directly bioprint a vessel-like structure. Various bioprinting methods could be used, including inkjet-based printing,28-30 light-based printing,31, 32 or extrusion-based printing.33-42 Nakamura et al.28 developed an inkjet-based 3D bioprinter in which 3D structures including vessel-like tubes were fabricated by the fusion of


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sodium alginate droplets. Similarly, Huang et al.29, 30 used a Z-shape platform-assisted inkjet printing system to fabricate 3D zigzag-shaped cellular tubes and vessel-like structures with bifurcations. This inkjet-based printing method allowed the direct and accurate printing of cellladen alginate droplets with a simple setup. In this system, the cell viability of the printed structures was high, but the mechanical strength was much lower than what is needed for mechanical loading. Light-based printing such as digital mask projection printing and laserinduced forward transfer were also used to fabricate tissue architectures including vessel-like structures. Chen et al.31 introduced a dynamic optical projection stereolithography platform that allows the solidification of photosensitive liquid prepolymer upon activation by UV or other light sources. They used this technology to demonstrate the fabrication of a complex vascular structure. In an alternate strategy, Huang et al.32 used a matrix-assisted pulsed-laser evaporation direct-write to fabricate long alginate tubes. Light-based printing approaches enable the generation of accurate multi-scale 3D structures, but this method requires expensive instruments and results in a poor cell survival rate. Extrusion-based printing is a rapidly growing bioprinting technology that can fabricate more complex hydrogel structures.33 Ozbolat et al.34 and Gelinsky et al.35 presented a coaxial nozzle-assisted extrusion platform to fabricate vessel-like alginate micro-channels. Others used microfluidic chips to fabricate multi-compartmental and multi-scale hollow filaments.36-40 Recently, supporting materials (such as Bingham fluid) were used in extrusion-based printing to fabricate complex hydrogel structures. Angelini et al.41 used carbopol and Feinberg et al.42 used a gelatin slurry support bath as supporting materials to produce complex three-dimensional (3D) biological structures including branched tubular networks. Extrusion-based printing provides increased flexibility in directly printing tubes, but there are few applications.


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The concept of “organ weaving” was presented by Schubert et al.

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in which woven threads

and sheets could be used for artificial organ development. This is an attractive option to fabricate tissue structures, particularly vessel-like tubes. In this method, cell-laden filaments or sheets were weaved around a cylindrical rod and the vessel-like structure were obtained after removal of the template. Takeuchi et al.37 used a microfluidic device with double-coaxial laminar flows to fabricate metre-long cell-laden microfibers, and a vessel-like helical tube could be fabricated by weaving the microfibers over a glass rod. Sher et al.

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presented an approach to fabricate non-

liquefied and liquefied 3D spiral constructs using a perfusion-based layer-by-layer technique. Similarly, cell-laden alginate filaments were wrapped over a glass rod to form a vessel-like structure. The study presented here was inspired by the concept of “organ weaving”. Using coaxial nozzle-assisted extrusion-based printing technology, hollow alginate filaments were used as the building blocks and were printed along a rotated rod to fabricate vessel-like structures. Due to the progressive crosslinking reaction, hollow alginate filaments can undergo self-fusion to result in the formation of multi-level fluidic channels. Multi-layers of cells are deposited in a controlled manner and the resulting printed structures have sufficient strength to meet the requirements of mechanical loading. Additionally, multi-level fluidic channels inside the structures can be used for chemical stimulation. These structures allow the novel designs of blood vessel structures and have potential uses in many diverse tissue engineering applications. 2 Materials and methods 2.1 Material preparation


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Sodium alginate (Na-Alg) powder (Sigma-Aldrich, Shanghai, China) was dissolved in deionized water at 2-4% (w/v). Calcium chloride (CaCl2) powder (Sigma-Aldrich, Shanghai, China) was dissolved in deionized water at 4% (w/v). Collagen (type I, rat tail, Xinyou Biological Technology Co., Ltd., HangZhou, China) was used to treat the inner wall of the vessel-like structure to promote cell adhesion. Collagen solution of 1mg/ml was prepared in 0.006 mol/L acetic acid solution. Red, blue, and yellow dye solutions were injected into the fabricated channels for perfusion testing. To pour a model of the brain, silicone part A and part B (Dongguan Hongfeng Silicone Materials Co., LTD) were mixed at a 1:1 (w:w) ratio and stirred for 2 minutes. To fabricate a skull model, a typical FDM 3D printer (D-Force 400, Trianglelab Co., Ltd., Jiangsu, China) was used and PLA filament (PLA 1.75, Alkht Co., Ltd., Beijing, China) was used as the printing material. 2.2 Cell culture L929 mouse fibroblasts were cultured in MEM (Tangpu Biological Technology Co., Ltd., HangZhou, China) with 10% fetal bovine serum (Tangpu Biological Technology Co., Ltd., HangZhou, China), 1% penicillin (100 units/mL), and streptomycin (100 µg/mL) (Tangpu Biological Technology Co., Ltd., HangZhou, China). MOVAS mouse smooth muscle cells were cultured in DMEM (Tangpu Biological Technology Co., Ltd., HangZhou, China) with 10% fetal bovine serum (Tangpu Biological Technology Co., Ltd., HangZhou, China), 1% penicillin (100 units/mL), and streptomycin (100 µg/mL) (Tangpu Biological Technology Co., Ltd., HangZhou, China). HUVECs were cultured in M199 (Tangpu Biological Technology Co., Ltd., HangZhou, China) with 10% fetal bovine serum (Tangpu Biological Technology Co., Ltd., HangZhou, China), 1% penicillin (100 units/mL), streptomycin (100 µg/mL) (Tangpu Biological Technology Co., Ltd., HangZhou, China), and 1% endothelial growth factors (ECGS, Tangpu


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Biological Technology Co., Ltd., HangZhou, China ). All cells were incubated at 37 °C in 5 % CO2 in polystyrene tissue culture flasks. The culture media was changed every two days and cells were passaged by trypsin-EDTA (Tangpu Biological Technology Co., Ltd., HangZhou, China) dissociation at 90% confluence every four days. 2.3 Cell-laden alginate solution preparation In this study, L929 mouse fibroblasts and MOVAS mouse smooth muscle cells were used to prepare the cell-laden alginate solution. Culture flasks with 90% cells confluence were washed with PBS (Tangpu Biological Technology Co., Ltd., HangZhou, China), and incubated with 0.25% Trypsin-EDTA(Tangpu Biological Technology Co., Ltd., HangZhou, China) for 3-4 min at 37 °C in 5% CO2 to detach the cells from the culture flasks. Next, the cell suspension was centrifuged at 1,000 rpm for 5 min at room temperature, the supernatant was discarded, and cells were resuspended in cell culture medium to a concentration of 4×106 cells/ml. Finally, the cell suspension was mixed at a volume ratio of 1:1 with 4% sodium alginate solution and placed in a magnetic stirrer for 5min at 100 rpm at 37 °C in 5% CO2, resulting in a 2% alginate solution with a cell density of 2×106 cells/ml. 2.4 Fabricating vessel-like structures Using our approach, several steps were performed to bioprint the vessel-like structure with multi-level fluidic channels (Figure 1). In this process, a coaxial nozzle-assisted bioprinting system consisting of two coaxial nozzles was used (Figure S1), as described in detail in our previous work.27 The two coaxial nozzles had an outer needle with an inner diameter of 1600µm and an outer diameter of 2000µm, and an inner needle with an inner diameter of 510µm and an outer diameter of 810µm. Fibroblast-laden Na-Alg solution and smooth muscle cell-laden Na-


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Alg solution were separetely dispensed through the outer tube of the two coaxial nozzles and CaCl2 solution was dispensed through the inner tube. The flow rates of the Na-Alg solution and CaCl2 solution were both 1ml/min. At the exit of the coaxial nozzle, a hollow alginate filament was formed by the reaction of sodium alginate and calcium chloride (Figure 1a). A partially crosslinked filament with gelled alginate inside and ungelled alginate outside was obtained by controlling the concentration and flow rate of the two solutions (Figure 1b). Next, the hollow alginate filament was twined over a rod (Figure 1c). Since the partially crosslinked hollow filament could fuse to an adjacent filament, a 3D alginate structure with a spiral shape could be obtained. In this step, we constrained the coaxial nozzle to horizontal movement and maintained rotational movement of the rod to control adjacent hollow filament deposition in the precise location required for fusion. To obtain a structure similar to blood vessels, a layer of smooth muscle cell-laden structure was first printed (Figure 1c). Next, a layer of fibroblast-laden structure was printed upon the first layer (Figure 1d). When the two-layer structure was completely printed, the rod was carefully removed and the vessel-like structure was transferred to CaCl2 solution to allow complete crosslinking (Figure 1e). Then collagen solution was ejected to the macro-channel to form a layer which could promote endothelial cell adhesion, after one hour, the structure was washed with PBS three times to remove residual collagen (Figure 1f). Finally, endothelial cells were uniformly seeded into the macro-channel according to the method described in Dai’s research23 (Figure 1g). Endothelial cells with a concentration of 6×106 cells/ml were ejected into the macro-channel through an L-shaped connecting tube. The structure was turned over every 20 min to ensure cells uniformly adhere to the wall. As a result, a 3D hydrogel-based vessel-like structure containing three kinds of vascular cells was fabricated with


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multi-level fluidic channels, including a macro-channel in the middle and two micro-channels around the wall (Figure 1f).

Figure 1. Fabrication process of 3D alginate vessel-like structures with multi-scale fluidic channels. a) Formation of hollow alginate filaments. b) Formation of partially crosslinked filaments. c) Printing a layer of smooth muscle cells-laden structure over a rod (inset on the right: cross-section of the selected area). d) Printing a layer of fibroblasts-laden structure upon the first layer. (inset on the right: cross-section of the selected area). e) Removal of the rod and immersion of the structure in CaCl2 solution. f) Ejecting collagen solution. g) Seeding endothelial cells into the inner wall of the structure.

2.5 Mechanical Testing A tensile test was performed to determine the effect of alginate concentration and culture time on the mechanical properties of the vessel-like structures using a DMA Q800 (TA Znstruments, USA) in the controlled mode for the stress/strain test, in which a ramp force with a slope of 0.5N/min was applied to the sample. Here, single-layer structures with 20 mm length and 4 mm diameter served as the samples, and they were flattened between clamps before testing. The stress and strain were determined from the load data and displacement, respectively, by normalizing to sample length, inner diameter, and outer diameter. Three samples were used in


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each group, and all data were expressed as mean ± standard deviation (SD). The Student t-test was used to analyze the statistical significance of data pairs. For analysis of multiple groups of data, the one-way ANOVA test was used. A value of p < 0.05 was considered significant difference and p-value larger than 0.05 (p > 0.05) was taken as indication of no significant difference. 2.6 Cell Viability Analysis The cell viability of the fabricated single-layer structures was tested after 1, 4, and 7 days of culture by a cell LIVE/DEAD assay. In this experiment, nine samples with 40 mm length and 4mm diameter were printed and three samples were concurrently used in a cell viability analysis, respectively on day 1, day 4, and day 7. For the LIVE/DEAD assay, first, cell-laden alginate structures were washed with PBS three times before staining. Next, we performed staining using LIVE⁄DEAD assay reagents (KeyGEN BioTECH Co., Ltd., NanJing, China) according to the manufacturer’s instructions. Calcein AM and PI were diluted with PBS at a concentration of 2 µM and 8 µM respectively. The cell-laden alginate structures were stained by incubation with the Calcein AM/PI mixture for 30-45 min in the dark, and then were washed with PBS to remove residual regents. Finally, a confocal fluorescence microscope (ZEISS LSM780) was used to image the cell-laden structures by acquiring two images of each frame, red and green for live and dead cells, respectively. The cell viability was calculated as the number of green stained cells/number of total cells × 100%. 2.7 Cell Characterization To observe cell distribution in the printed alginate structure, three kinds of vascular cells (fibroblasts, smooth muscle cells, and endothelial cells) were stained using cell tracker


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fluorescent probes (red, green, and orange). First, the fluorescent probes were diluted in DMSO to a final working concentration of 10 µM. Then the cells were harvested by centrifugation and aspirating the supernatant, and were resuspended in cell tracker working solution. After incubating 30 min at 37 °C in 5% CO2, cells were centrifuged and the cell tracker working solution was removed. Finally, Cell-laden alginate solution and endothelial cells suspension were preparation as described in section 2.3 and 2.4. After the vessel-like structure fabricated, it was observed under confocal microscopy (ZEISS LSM780). The excitation/emission wavelengths of three stains are Cell Tracker Red (for L929): 577/602 nm, Cell Tracker Green (for MOVAS): 492/517 nm, and Cell Tracker Orange (for HUVEC): 548/576 nm. 3 Results 3.1 Fabrication of alginate vessel-like structures with multi-level channels To investigate the characteristics of structures fabricated with our method, a single layer structure was printed (Figure 2). This simple structure could be fabricated by a bioprinting method that we described previously,27 in which spiraled structures are printed along the vertical direction with a Z-shape platform (Figure 2a), however, it was difficult to fabricate a long structure using this method and the resulting printed structure was of poor quality. In contrast to this vertical method, we next used a horizontal approach. With the coaxial nozzle moving in the X direction and the rod rotating around the A direction, a vessel-like structure was successfully fabricated using calcium chloride solution mixed with red dye (Figure 2b). To deposit the hollow alginate filaments in the precise location required for fusion, the horizontal speed of the coaxial nozzle and the rotational speed of the rod must be synchronized. The detailed calculation process used to obtain this synchronization is shown in Figure S2. In this method, since the structure was


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fabricated in the horizontal direction with the rod serving as a 3D template, the fabricated structure had a smooth surface and uniform diameter, and the structural integrity was good. Since different rods can be used, spiral structures of various sizes and shapes were fabricated that varied based on the size and shape of the rod. Four structures were fabricated with different diameters (2mm, 4mm, 6mm, and 8mm; Figure 2c). Additionally, a grading structure was fabricated by printing alginate filaments onto a rotating rod with decreasing diameter (Figure 2d), an arched structure was fabricated by printing alginate filaments onto a rotating rod, the diameter of which increased first and then decreased (Figure 2e), and a ripple structure was fabricated by printing alginate filaments onto a rotating rod, the diameter of which changed according to a sine curve (Figure 2f). The successful fabrication of these structures shows the versatility of this method to fabricate many types of vessel-like structures.


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Figure 2. Characterization of the fabrication method. a) Alginate vessel-like structure printed in the vertical direction with a Zshape platform. b) Alginate vessel-like structure printed in the horizontal direction with a rod. c) Alginate vessel-like structures with different diameters. d) A printed grading vessel-like structure. e) A printed arched vessel-like structure. f) A printed ripple vessel-like structure.

As described above, a 3D hydrogel-based vessel-like structure was fabricated based on the fusion of hollow alginate filaments around a rod. A macro-channel was formed by the fusion of adjacent filaments after rod removal. Coupled with the micro-channel of the hollow filament, the vessel-like alginate structure therefore had two-scale fluidic channels, the macro-channel in the middle and the micro-channel around the wall. Figure 3a shows the overview of a single-layer structure with a length of 70 mm and a double-layer structure with a length of 60 mm. Figure 3b and c shows a longitudinal section of the single-layer structure under different magnifications. It can be seen that there is a macro-channel with 6 mm diameter and a micro-channel with 832 µm diameter. Figure 3d and e shows a longitudinal section of the double-layer structure under different magnifications. The macro-channel was in the middle and two micro-channels were around the wall. It can be seen from Figure 3b-e that the fusion section between the adjacent hollow filaments was uniform, which was further confirmed in the scanning electron microscopy image (SEM) shown in Figure 3f. We also conducted a perfusion test by using a syringe pump to push different dye solutions into the macro-channel and the micro-channel of the fabricated structure (Figure 3g-i). Figure 3g and h show that a red dye solution was able to enter both the macro-channel and the micro-channel and the red dye was able to diffuse freely in the fabricated alginate structure (Figure S3). In addition, a yellow dye solution and a blue dye solution were separately perfused through the micro-channel of the inner layer and the micro-channel of the outer layer (Figure 3i). The blue and yellow dye solution were able to mix with each other, and


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the color of the liquid in the overlap of the two layers changed to green, which indicated that the fabricated channels can function to deliver nutrients.

Figure 3. Characterization of the structural feature of the fabricated alginate vessel-like structure. a) Overview of a single-layer structure and a double-layer structure (inset: cross section of the structure). b,c) Longitudinal section of the single-layer structure under different magnification. d,e) Longitudinal section of the double-layer structure under different magnification. f) SEM image of the longitudinal section. g) Perfusion of red dye solution through the macro-channel of single-layer structure. h) Perfusion of red dye solution through the micro-channel of single-layer structure. i) Perfusion of yellow and blue dye solution through the inner micro-channel and the outer micro-channel of the double-layer structure.

3.2 Mechanical testing of the vessel-like structures The mechanical behavior of the fabricated vessel-like structures was next investigated, as shown in Figure 4. The representative tensile process is illustrated in Figure 4a. The structure exhibited mostly linear stress–strain behavior. The ultimate strength increased as the


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concentration of the sodium alginate increased (Figure 4b), from 0.049±0.005 MPa in 2% alginate gels to 0.139±0.006 MPa in 3% alginate gels and 0.184±0.008 MPa in 4% alginate gels (p

3D Bioprinting of Vessel-like Structures with Multilevel Fluidic

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