A Versatile Method for Fabricating Tissue Engineering Scaffolds with a

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A Versatile Method for Fabricating Tissue Engineering Scaffolds with a Three-Dimensional Channel for Pre-vasculature Networks Shuai Li, Yuanyuan Liu, Lijun Liu, and Qingxi Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07725 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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ACS Applied Materials & Interfaces

A Versatile Method for Fabricating Tissue Engineering Scaffolds with a Three-Dimensional Channel for Pre-vasculature Networks Shuai Li,1 Yuan-Yuan Liu,1,2* Li-Jun Liu,1 Qing-Xi Hu1,2* 1

Rapid Manufacturing Engineering Center, Shanghai University, Shanghai 200444, People’s

Republic of China 2

Shanghai Key Laboratory of Intelligent Manufacturing and Robotics, Shanghai University,

Shanghai 200072, People’s Republic of China AUTHOR ADDRESS. 99 Shangda Road, Baoshan District, Shanghai, China. KEYWORDS. 3D Printing; Sacrificial Template; 3D Channel; Pre-vasculature; Interface Compensation.

ABSTRACT. Despite considerable advances in tissue engineering over the last two decades, solutions to some crucial problems remain elusive. Vascularization is one of the most important factors that greatly influence the function of scaffolds. Many research studies have focused on the construction of a vascular-like network with pre-vascularization structure. Sacrificial

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materials are widely used to build perfusable vascular-like architectures, but most of these fabricated scaffolds only have a 2D plane-connected network. The fabrication of threedimensional perfusable branched networks remains an urgent issue. In this work, we developed a novel sacrificial molding technique for fabricating biocompatible scaffolds with a threedimensional perfusable branched network. Here, 3D-printed polyvinyl alcohol (PVA) filament was used as the sacrificial material. The fused PVA was deposited on the surface of a cylinder to create the 3D branched solid network. Gelatin was used to embed the solid network. Then, the PVA mold was dissolved after curing the hydrogel. The obtained architecture shows good perfusability. Cell experiment results indicated that human umbilical vein endothelial cells (HUVECs) successfully attached to the surface of the branched channel and maintained high viability after a few days in culture. In order to prevent deformation of the channel, paraffin was coated on the surface of the printed structure, and hydroxyapatite (HA) was added to gelatin. In conclusion, we demonstrate a novel strategy towards the engineering of pre-vasculature thick tissues through the integration of the fused PVA filament deposit. This approach has great potential in solving the issue of three-dimensional perfusable branched networks and opens the way to clinical applications.

1.

INTRODUCTION

Tissue engineering, with its ultimate goal of planting artificial tissues or even organs to replace a lesion or an implant site inside the human body, provides varied solutions for tissue or organ transplantation.1,2 Great successes have been achieved in this field; a wide range of biomaterial scaffolds, such as skin, bone, cartilage, and nerve,3-6 have been made for clinical use. Despite


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considerable progress, some technological barriers still need to be overcome;7 one of them is the construction of vascular-like structures that contain a three-dimensional perfusable branched network, especially for complex tissues and organs.8 These networks are needed to deliver necessary nutrients and oxygen to the tissue and organ systems to avoid necrosis in the body and are critical to the rise and survival of large-scale multicellular organisms.9-11 For this reason, the pre-vasculature architecture inside engineered tissue constructs is crucial to effectively transfer the in vitro fabrication of large tissues to clinical practice. Several researchers have investigated different methods to build scaffolds with perfusable branched networks.12-14 One approach is to apply a sacrificial template to the biomanufacturing process, which is based on the dissolution of the sacrificial material in a bulk hydrogel, thus forming a scaffold with a branched connected network. Hydrogels such as sodium alginate and agarose have been used as sacrificial substrate to create branched networks inside synthetic materials,15 PDMS,16 and fibrous scaffolds.17 Sucrose, which can be dissolved in water, has been used to create cylindrical microfluidic channels in lab-on-a-chip.18 Aqueous bio-ink has also been employed in the fabrication of branched vascular-like structures. Moreover, the replication of plant leaf venation is another approach to fabricating nature-inspired artificial microvascular networks for perfusable tissue constructs.19-21 The biomaterial Pluronic F127, which can be easily printed and removed under mild conditions, was also employed in fabricating the embedded vasculature and building the heterogeneous cell-laden tissue constructs.22 With the development of 3D bioprinting, cell-laden hydrogels and polymers have become widely used to print three-dimensional branched vascular systems.23 The microtissues and tissue spheroids are living materials with certain biological properties. A fugitive organic ink has been printed and encapsulated in a thermally treated or photocurable resin and subsequently removed


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by liquefaction to yield a microvascular network consisting of uniform microchannels interconnected in three dimensions.24 In addition to this, photocrosslinkable hydrogel constructs can be utilized to encapsulate printed sacrificial template fibers.15 Printing cell-laden hydrogel drops in a high-density fluid environment was another choice to build a three-dimensional construct that can mimic a vascular bifurcation.25,26 Similarly, printing sodium alginate in a liquid cross-linking agent can be applied to create cellular structures.27,28 Based on these different methods for building a perfusable branched network, it can be seen that both the sacrificial molding technique and 3D printing approach have drawbacks that will definitely hamper their clinical application. For instance, most proposed sacrificial templates focus on a planar perfusable branched network; the 3D network could not be easily fabricated via these mentioned methods. The structures of tissues and organs are complex; vascular systems within tissues and organs are not distributed only in a plane.11 Healthy adult vasculature presents as a hierarchical vascular structure with different branched networks.29-31 The shape of vasculature in the human body is close to that of the leaf skeleton.32 Thus, two basic features, three-dimensional and branched structures, should be addressed in fabricating the prevascularized construct. Although 3D bioprinting has opened our eyes to printing threedimensional, cell-laden constructs, the printed networks were easily accessible Y-shaped structures, and some complicated structures were embedded in thermally treated materials33 or photocurable resin.20 Thus, this kind of method still lacks flexibility in creating heterogeneous vasculature; the effect of material and treatment on cell viability should also not be ignored. Albeit the emergence of hollow hydrogel fibers can solve the problem of biocompatibility and non-toxicity,34,35 the uniform hollow channels’ diameter limits the scalability of the engineered scaffolds. Therefore, there is still the need to develop a novel approach to produce pre-


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vasculature architectures within sophisticated three-dimensional connected network scaffolds for the production of 3D constructs with different scales for specific tissue engineering applications. In this work, we demonstrate a novel approach combining printing and sacrificial molding techniques that overcomes these limitations, enabling the fabrication of biocompatible scaffolds with an embedded three-dimensional perfusable branched connected network. This approach is based on printing water-soluble poly (vinyl alcohol) (PVA) filaments on a cylinder; the printed solid structure itself has three-dimensional structure according to the structural characteristics of the cylinder. Three branched unit and a complicated structure were built in this work to meet the two basic issues for fabricating the pre-vascularized construct. Then, the solid network was embedded in gelatin, the product of partial hydrolysis of collagen. We added microbial transglutaminase (mTG) to keep the shape of the gelatin bulk. After cross-linking, the PVA structure can be easily dissolved in water, and a three-dimensional perfusable connected network can be obtained. The presented approach is shown to be flexible for the fabrication of 3D networks with different sizes and structures, which is the origin of the channels, allowing the engineering of complicated vascular geometries in inhomogeneous scaffolds. Moreover, the fabricated perfusable scaffolds were successfully attached with HUVECs, and cell viability was maintained at high levels after culturing, thus demonstrating the non-toxicity of the materials and fabrication process. Finally, hydrophobic paraffin was coated on the surface of the printed structure, and HA was added to gelatin to avoid interaction in the interface of the sacrificial template and gelatin and reduce the deformation of the branched network.

2.

MATERIALS AND METHODS


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2.1. MATERIALS Commercially available filaments of the water-soluble polymer, poly (vinyl alcohol) (PVA, 3dHi-Tech, Shenzhen, China) were used to print the sacrificial mold. Enzymatically crosslinkable gelatin solution was prepared as previously described.36,37 Gelatin (Type A from porcine skin, 300 blooms, Sigma Aldrich, St. Louis, MO) was fully dissolved in deionized water in a 60 ºC water bath to prepare a 15 wt% pre-polymer solution. Microbial transglutaminase (mTG, activity of approximately 100 U/g, Hengsheng BioTech CO., LTD, Shenzhen, China) was added when the temperature of the gelatin solution reached 30 ºC and mixed thoroughly to obtain a gelatin/mTG mixed solution. The final weight ratio of gelatin to mTG in the mixed solution was 10:1.

2.2.

SACRIFICIAL TEMPLATE FABRICATION

To fabricate a hydrogel scaffold within a three-dimensional perfusable connected network, a fused-deposit extrusion 3D filament printing system was implemented in this study. This system was used to fabricate complex 3D mold constructs such as tree branches. As shown in Figure 1, the mold printing system consists of two key subsystems. The first is a computer-controlled fused printhead mounted to a solo motorized linear Z stage. The printhead can be controlled to move up and down for future studies on different receiving platforms that have complex structures. The other crucial part of this system, which is also the novelty of this study, is the receiving platform, which is not a traditional panel but a cylinder with two degrees of freedom (moving left and right, or rotating following a software program); the diameter of the


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receiving cylinder can be changed accordingly. In 3D printing, a 2D plane is typically used as the receiving platform. The 2D plane can be considered a cylinder whose radius is infinite. Thus, the cylinder was chosen as the receiving platform in our study. Motion-control programs were developed in-house using G-code commands based on the geometry of the receiving cylinder surface and the designed 3D solid structure. The PVA filament, which was purchased for immediate use, was heated using the fused printhead’s material-feeding mechanism and a heating module for deposition. The orifice diameter of this printhead is 200 µm.

Figure 1. Schematic for the printing of the three-dimensional solid sacrificial template.

2.3.

FABRICATION OF 3D PERFUSABLE CONNECTED NETWORK


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After the complex mold construct was printed, it was taken from the cylinder and placed into a container containing freshly prepared mixture of mTG and gelatin, as shown in Figure 2. It was kept at room temperature for 15 min and then at 37 ºC for 6 h to fully cross-link the hydrogel. It was then removed from the hydrogel container and trimmed with microsurgery scissors to expose the head and tail parts of the embedded PVA structure. Finally, it was placed in a container with PBS, generally for 1 h, to fully dissolve the inner PVA.

Figure 2. Fabrication of the 3D connected network. (a) Printed solid template was placed into a container. (b) Fresh mixture of gelatin and mTG was poured into the same container. (c) Container was kept at 37 ºC to cross-link gelatin. (d) After cross-linking, the redundant part of the construct was cut to reveal the head part of the sacrificial template. (e) PBS was applied to dissolve the sacrificial template. (f) A 3D connected network in gelatin was obtained.

2.4.

CELL VIABILITY OF THE BRANCHED NETWORKS


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HUVECs from Zhongqiaoxinzhou Biotech CO., Ltd, Shanghai, China, were routinely cultured in RPMI 1640 culture medium with 10% fetal bovine serum, 0.30 mg mL-1 L-glutamine, 4.766 mg mL-1 HEPES, 0.85 mg mL-1 NaHCO3, 1% penicillin (10000 units/mL), and 10000 µg/mL streptomycin. The cells were maintained in a humidified incubator at 37 ºC in 5% CO2 in a standard petri dish (Corning, NY, USA), passaged every 3 days, with the media being refreshed every day. The branched network construct was placed into a 70 ºC water bath for 30 min to inactivate the residual mTG before the cell experiment. After that, the hydrogel matrix was immersed in ethanol (75%) for 12 h under UV radiation, and then the network in gelatin was washed with phosphate buffer solution (PBS) three times. Finally, the construct was immersed in fresh RPMI 1640 medium for 6 h before injection. After trypsinization, a cell suspension with a density of 5 × 106 cells/mL was prepared in RPMI 1640 medium. Then, 10 mL cell suspensions were slowly perfused into the branched network until the whole network was covered. After 4 h of cell attachment, the cell-laden constructs were statically cultured, with the medium changed every day in a CO2 incubator. A Live-Dead Cell Staining Kit (Biovision, Inc., San Francisco, USA) was used to assess the cell viability of the attached HUVECs. Green fluorescent dye LiveDyeTM was used to stain live cells, while the dead cells were stained by the red fluorescent dye propidium iodide (PI). The cellular morphology and Live-Dead fluorescence were observed under an inverted fluorescent microscope (Eclipse Ti-U, Nikon Instruments Inc., Japan).

2.5.

COMPENSATION FOR INTERFACE DEFORMATION

The sacrificial template designed with the three-dimensional construct may experience certain process-induced deformation mainly due to the dissolution of PVA in gelatin. To compensate for


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the deformation from the interface of these two materials, liquid paraffin was used to prevent the dissolution of PVA in gelatin. After the sacrificial template was printed, it was placed into liquid paraffin, and the paraffin was coated on the surface of printed sacrificial template. Then, the template was put into an oven at 37 ºC to dry the coated paraffin. For the cell experiment, the coated could thoroughly removed by melting at 60 ºC38 when the branched network construct was placed into a 70 ºC water bath. Although the dissolution of PVA in gelatin can be avoided by coating paraffin onto the surface of the sacrificial template, PVA swelling still existed in the fabrication process. Therefore, hydroxyapatite (HA) was added into gelatin to prevent swelling of the sacrificial template.

3. 3.1.

RESULTS AND DISCUSSION FABRICATED SACRIFICIAL TEMPLATE

The method used to fabricate the sacrificial template is presented in Figure 1.


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Figure 3. Different shapes of sacrificial templates were designed and printed using the fused printhead and receiving cylinder. (a, b, c) One-to-two, one-to-three, and one-to-four structures, respectively. (d) A complex structure. The black dot represents the redundant path. Scale bar is 3 mm.

A fused printhead and commercially available water-soluble PVA filaments were used to print the sacrificial template. The fused PVA was deposited on an aluminium alloy cylinder with 16 mm diameter. Masking tape for 3D printing was attached to the receiving cylinder to increase the friction between the fused PVA and the receiving surface. First, different CAD models of 3D solid structures were designed as shown in Figure 3. In Figure 3a, 3b, and 3c, different branched networks were envisioned. In Figure 3d, a widely used 2D symmetrical connected network was changed to 3D. Through the successive printing of such structures, different combinations of branched features may be fabricated. Under command from the G-code program, all these designed structures can be printed with our homemade 3D bio-printer (Rapid Manufacturing Engineering Center, Shanghai University, Shanghai, China). Finally, the redundant path, marked with black dots, was cut to obtain the sacrificial template. 3.2. FABRICATED 3D PERFUSABLE CONNECTED NETWORK After fabrication of the sacrificial templates, they were immersed into a freshly prepared mixture of mTG and gelatin. Following the processing mode in section 2.3, different hydrogel scaffolds with connected channels can be obtained. Then, orange acrylic paint was injected into the channel to verify the perfusion of these channels. Figure 4 shows the front and side views of these connected networks; the injected paint fills the channel without any gaps inside the


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hydrogel. Structurally speaking, channel shapes that come from the dissolution of the sacrificial template are well maintained. This is mainly due to the strength of the printed 3D structures.

Figure 4. Perfusion of the fabricated connected networks. (a) Front view, (b) side view. Scale bar is 3 mm.

3.3. CELL VIABILITY OF THE BRANCHED NETWORK The branched network was built as pre-vasculature to facilitate the transportation of oxygen, nutrients, etc. needed to maintain the viability of cells at the in vitro stage and provide conditions for the formation of vascularization.


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(a)

(d)

(b)

(e)

(c)

(f)

Figure 5. Cell morphology and fluorescent images of the linear channel: (a, d) after 4 h attachment, (b, e) cultured for 2 days, and (c, f) cultured for 4 days.


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In order to investigate the biocompatibility of the branched network in gelatin, HUVECs were seeded into the channel and statically cultured inside the gelatin constructs. The one-to-two channel in Figure 4 was used for the cell experiment. Figure 5a shows the microscopic morphology of endothelial cells inside the linear part after 4 h attachment. It is obvious that the seeded endothelial cells could attach to the surface of the branched channel. Almost all cells could grow into a homogenous and flat appearance, which is the original shape of the HUVECs.39 However, the cell distribution in this part was not so uniform, and the absence of cells was mainly found in the center part of the channel. This may be caused by the uniformity of the cell suspension. Thus, uniformity should be improved to avoid this phenomenon in further research. Moreover, the absence of cells may cause by the flow of cell suspension inside the channel during the 4 h attachment period. After 2 days of culturing, the amount of cells increased, and the cells grew into the blank part where cells had not previously attached, as shown in Figure 5b. On the third observation day, HUVECs are distributed in the linear part (Figure 5c) uniformly, as well as the branched channel (Supporting Information, Figure 1a) after culturing for 4 days. Figures 5d–5f show the corresponding fluorescent images of the cellattached channel. Although cell distribution in the channel was not very uniform at the initial stage, cells maintained high viability both in linear part and branched part (Supporting Information, Figure 1b) and were distributed uniformly after culturing for 4 days. These results indicate that the materials and the fabrication process were non-toxic for the growth of cells and the engineered 3D channel is favorable to cellular attachment, spreading, and growth.


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3.4. COMPENSATION FOR INTERFACE DEFORMATION A 3D Y-shaped construct (Figure 6a) that has a square cross-section was designed to verify that the coating of paraffin and the mixed HA (HA:mTG is 1:1 by weight) can prevent the dissolution and swelling of PVA in gelatin. The width of the designed square cross-section is 2.0 mm, as shown in Figure 6b. Three branched structures were printed on a platform using the same printhead (Figure 1). The first printed Y-shaped constructs were encapsulated in gelatin only. Some burrs appeared on the surface of the printed Y-shaped construct in Figure 6c. This phenomenon can be clearly seen in the microscopic image (Figure 7a). The printed construct color turned white after it was placed in gelatin for a while. This is an obvious signal of PVA dissolution.

(b)

(a)

(c)

(d)

(e)

Figure 6. Paraffin coating and HA addition were done to reduce the degree of deformation of the connected channel. (a) Designed Y-shaped structure. (b) Printed Y-shaped structure. (c) Putting the Y-shaped structure into gelatin. (d) Putting the paraffin-coated, Y-shaped structure into gelatin. (e) The coated Y-shape structure within the mixture of gelatin and HA.


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The second printed structure was placed into gelatin after coating a layer of paraffin (Figure 6d) using the method described in section 2.5. The burr phenomenon disappeared and the channel surface was smooth (Figure 7b), but the swelling of PVA still existed in this condition. The last structure, coated with a layer of paraffin, was placed into a mixture of gelatin and HA, and its shape was maintained well after the same period compared with the other two structures, as shown in Figure 6e and 7c.

(a)

(b)

(c )

Figure 7. Microscopic image of the channel (a) after the dissolution of the Y-shaped structure within gelatin, (b) after dissolution of the coated Y-shaped structure within gelatin, and (c) after dissolution of the coated Y-shaped structure within gelatin and HA. In order to quantitatively investigate the effect of paraffin and HA on the formation of the perfusable connected network, the width of the channel in gelatin was measured. As shown in Figure 8, the size of the first sample is 1.0 mm wider than the designed width. For the second one, the coating of paraffin on PVA only slightly decreased the dissolution. Although the addition of HA in gelatin also results in increased channel width, the dimension in this condition was close to 2.0 mm. In fact, the sacrificial material PVA showed thermal deformation behavior,40 and the swelling phenomenon of PVA filaments in water is not due to heat.41,42 During the cross-linking of gelatin and mTG, the optimal cross-linking temperature of 37 ºC which can be seen as the


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thermal source that caused the deformation. Moreover, the embedded PVA template would swell when dissolved in PBS. In our study, the combination of hydrophobic paraffin and HA effectively reduced the dissolution and swelling of the sacrificial template in gelatin. The dissolution of PVA in gelatin, caused by the interaction in the interface, was effectively prevented. The scale of the HA particles far exceeded the diameter of water molecules. Thus, HA can partly prevent the flow of water molecules and the dissolution of PVA. Therefore, the burr around the sacrificial template could be avoided. The addition of HA also prevented the thermal deformation and swelling phenomenon, probably by increasing the hydrogel strength, so that the swelling of PVA could not overcome the structural force. The coated paraffin and the existence of HA particles prevented the water molecules in gelatin from penetrating into and dissolving PVA.

Figure 8. Measuring the width of the connected channel.

4.

CONCLUSIONS AND FUTURE WORK


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In this study, the potential applicability of printing sacrificial templates on a cylinder has been explored towards the realization of the three-dimensional perfusable branched network, which can be used for human tissue and organ implantation in the future. The sacrificial template printing system was composed of two key subsystems: a motorized Z stage attached to a computer-controlled fused printhead, and a receiving cylinder that can rotate and move along the center axis. The obtained 3D solid sacrificial template was embedded into a gelatin and mTG mixture. After cross-linking, the sacrificial structure can be dissolved in PBS to form the perfusable branched channel in the hydrogel construct. HUVECs have been successfully injected and attached to the surface of the branched channel. The morphology of cells was almost the same as the original after a few days of culture. Live/Dead staining results showed high viability of cells, which proves that the fabrication process and materials were nontoxic to cell growth. Due to the dissolution of PVA in water, the dimension and shape of the channel inside the hydrogel may be deformed. Thus, paraffin was coated on the surface of the printed solid structure to prevent shape deformation, and the addition of HA in gelatin was used to prevent the dimensional deformation. Finally, the channel dimensions approached the dimensions of the sacrificial template. Future work should aim to further the ability to fabricate 3D pre-vasculature networks, including the capability to print more complex sacrificial templates to meet the complexity of tissues and organs. In fact, the swelling ratio of the PVA hydrogel system was dependent on the concentration of PVA; higher concentrations result in reduced swelling ratios.43,44 Thus, modifying the PVA filament to improve the moisture absorption to better compensate for process-induced deformation should be an urgent task. Moreover, the endothelialization of cells and vasculature formation should be further investigated.


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ASSOCIATED CONTENT Supporting Information Details of additional fluorescent images (PDF)

AUTHOR INFORMATION Corresponding Authors *Y.-Y.L.: phone, 86-021-66133157-8019; e-mail, [email protected] *Q.-X.H.: phone, 86-021-66133298; fax, 86-021-66133297 ; e-mail, [email protected] Funding Sources National Natural Science Foundation of China, Grant No.51375392 and 51475281. National Science Fund for Young Scholars, Grant No. 51105239. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge funding support from the National Natural Science Foundation of China (Grant No.51375392 and 51475281) and the National Science Fund for Young Scholars (Grant No. 51105239).


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Table of Contents Graphic


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275x167mm (96 x 96 DPI)



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Figure 1. Schematic for the printing of the three-dimensional solid sacrificial template. 209x198mm (96 x 96 DPI)


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Figure 2. Fabrication of the 3D connected network. (a) Printed solid template was placed into a container. (b) Fresh mixture of gelatin and mTG was poured into the same container. (c) Container was kept at 37 ºC to cross-link gelatin. (d) After cross-linking, the redundant part of the construct was cut to reveal the head part of the sacrificial template. (e) PBS was applied to dissolve the sacrificial template. (f) A 3D connected network in gelatin was obtained. 318x165mm (96 x 96 DPI)



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Figure 3. Different shapes of sacrificial templates were designed and printed using the fused printhead and receiving cylinder. (a, b, c) One-to-two, one-to-three, and one-to-four structures, respectively. (d) A complex structure. The black dot represents the redundant path. Scale bar is 3 mm. 331x228mm (96 x 96 DPI)


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Figure 4. Perfusion of the fabricated connected networks. (a) Front view, (b) side view. Scale bar is 3 mm. 221x219mm (96 x 96 DPI)



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Figure 5. Cell morphology and fluorescent images of the linear channel: (a, d) after 4 h attachment, (b, e) cultured for 2 days, and (c, f) cultured for 4 days. 297x419mm (96 x 96 DPI)


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Figure 6. Paraffin coating and HA addition were done to reduce the degree of deformation of the connected channel. (a) Designed Y-shaped structure. (b) Printed Y-shaped structure. (c) Putting the Y-shaped structure into gelatin. (d) Putting the paraffin-coated, Y-shaped structure into gelatin. (e) The coated Y-shape structure within the mixture of gelatin and HA. 345x262mm (96 x 96 DPI)



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Figure 7. Microscopic image of the channel (a) after the dissolution of the Y-shaped structure within gelatin, (b) after dissolution of the coated Y-shaped structure within gelatin, and (c) after dissolution of the coated Y-shaped structure within gelatin and HA. 250x127mm (96 x 96 DPI)


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Figure 8. Measuring the width of the connected channel. 296x209mm (300 x 300 DPI)



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Figure 1. Phase images of cell morphology (a) and fluorescent images (b) inside the branched channel. 419x256mm (96 x 96 DPI)


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