Characterization of Engineered Scaffolds with Spatial Prevascularized

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Characterization of Engineered Scaffolds with Spatial PreVascularized Networks for Bulk Tissue Regeneration Shuai Li, Hai-Guang Zhang, Dong-Dong Li, Jian-Ping Wu, Cheng-Yan Sun, and Qing-Xi Hu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00355 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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ACS Biomaterials Science & Engineering

Characterization

of

Engineered

Scaffolds

with

Spatial

Pre-Vascularized Networks for Bulk Tissue Regeneration Shuai Li1, Hai-Guang Zhang1, 2, 3, *, Dong-Dong Li1, Jian-Ping Wu1, Cheng-Yan Sun1, Qing-Xi Hu1, 2, 3, * 1

Rapid Manufacturing Engineering Center, Shanghai University, Shanghai 200444,

China 2

Shanghai Key Laboratory of Intelligent Manufacturing and Robotics, Shanghai

University, Shanghai 200072, China 3

National Demonstration Center for Experimental Engineering Training Education,

Shanghai University, Shanghai 200444, China

Abstract Despite significant progress in the fabrication of pre-vascularized networks over the past decade, a number of challenges remain. One of the most relevant issues is the lack of three-dimensional structures, which limits the clinical applications of the engineered scaffolds. Another problem is the complexity of pre-vascularized networks in engineered scaffolds, which is still less than that of human tissues, especially in the case of mature and bulk tissues. Thus, there is still the need to develop more flexible methods to better simulate the structure of natural tissues. In this work, we used a versatile sacrificial template method for fabricating bulk scaffolds with spatial pre-vascularized networks. Soft poly(vinylalcohol) (PVA) filaments were used to print the sacrificial template, and the receiving platform was a stepped shaft, allowing the

ACS Paragon Plus Environment


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sacrificial template to have 3D and complex structures. The obtained template was embedded into gelatin and microbial transglutaminase (mTG). The inner PVA template could be extracted from the enzymatic crosslinking system, and an engineered scaffold with spatial pre-vascularized networks was obtained. In vitro experiments demonstrated that the fabrication process is biocompatible with cells.

Keywords: 3D; Complex Structure; Pre-Vascularized Networks; Bulk Scaffolds

1. Introduction Biofabrication, which aims to engineer artificial organs or tissues to replace damaged or diseased ones in vivo, has been a promising research area of tissue engineering, providing effective technological methods to bridge the gap between organ shortages and transplantation needs2-4. Considerable progress has been made in the fabrication of engineered tissues and organs, including nerve5, skin6, 7, ear8, and cartilage9. However, the manufacturing of complex organs and bulk tissues in vitro remains a great challenge in this field. One of the most significant problems in this fabrication process is vascularization. The existence of vascularization in tissues and organs provides delivery channels of oxygen, nutrients, and metabolic products, allowing cells to grow and proliferate successfully10, 11, and prevent necrosis. For this reason, the fabrication of pre-vascularized networks in engineered scaffolds is crucial for tissue regeneration and beneficial for the integration of the engineered scaffolds with the host vasculature12.


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Three-dimensional bioprinting offers a series of methods to fabricate pre-vascularized networks within engineered scaffolds. The first commonly used approach was the printing of hollow fibers. In this method, the coaxial extrusion of sodium alginate and calcium chloride is used to form hollow fibers13-16. After printing according to requirements, an engineered scaffold with pre-vascularized networks is obtained. Building on this method, water-soluble polymer poly(vinyl alcohol) (PVA) can be added into the sodium alginate to create 3D porous scaffolds with regular macro-pores and an artificial vasculature-like system17. Moreover, biocompatible materials with different forming performances, such as chitosan and sodium tripolyphosphate, have been used to replace sodium alginate and calcium chloride18. Although the use of hollow fibers is well established, this method is generally restricted to planar footprints, and the final shape of the channels in the engineered scaffolds is limited. Another 3D bioprinting-based method is the extrusion of hydrogel droplets or bioinks, such as printing cell-laden alginate in a bath of Ca2+ 19, 20, agarose gel in a hydrophobic high-density fluid perfluorotributylamine21, 22, or cell pellets with the support of hydrogel23. The methods have mostly been used for fabrication of artificial arteries, blood vessels, and vascular-like structures. Owing to the technical limitations of additive manufacturing for obtaining spatial pre-vascularized networks within engineered scaffolds, sacrificial materials emerged as a good alternative for fabricating artificial channels in engineered scaffolds. Sodium alginate dissolved in chelating agent ethylenediaminetetraacetic acid (EDTA) has been used as a sacrificial material to engineer interconnected 3D vascular



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networks17. However, the concept of 3D in this research was still restricted to layer-by-layer assembly, and fully 3D structure was not achieved. Fugitive ink Pluronic F127, which can be easily printed and removed under mild conditions, has also been employed to yield the desired microchannel networks within an engineered matrix24, 25. Although the sacrificial template strategy exhibits more flexibility than the printing method, it was reported that cell viability was low when higher concentrations of sodium alginate were used19, and highly concentrated Pluronic F127 had significant cytotoxic effects26,

27

. In order to achieve nanoscale features,

electrospinning nonwoven mats were used as sacrificial fibers to generate 3D nanoscale vascularized composites containing interconnected networks with channels and tubes28. However, the biocompatibility of the fabrication process and materials has not yet been reported. In this paper, we report an optimized approach combining printing and sacrificial molding techniques, which overcomes these limitations, enabling the fabrication of biocompatible scaffolds with embedded spatial pre-vascularized networks. This approach is based on printing soft PVA filaments on a stepped shaft; the printed solid structure itself has a spatial structure according to the structural characteristics of the receiving shaft. Next, the solid network is embedded in a mixture of gelatin and microbial transglutaminase (mTG). After solidifying and extracting the inner PVA template, a spatial channel can be obtained in the hydrogel. The present approach is shown here to be flexible in terms of the fabrication of spatial channels at multiple scales,

allowing

the

engineering

of

complicated

vascular


geometries

in

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inhomogeneous scaffolds. Owing to the elasticity of gelatin hydrogel and the softness of printed PVA templates, the sacrificial template does not have any significant influence on dimension of the spatial channel. Mechanical properties of the scaffolds were investigated, and human umbilical vein endothelial cells (HUVECs) were injected into the spatial channel to demonstrate the biocompatibility of the materials and the fabrication process.

2. Materials and Methods

2.1. Materials and reagents

For the sacrificial template, PVA filament (1.75 mm; 3d Hi-Tech, Shenzhen, China) was used. A solution of gelatin (Type A from porcine skin, 300 blooms; Sigma-Aldrich) and deionized water was mixed on a magnetic stirrer at 60 °C to fully dissolve gelatin particles. Finally, mTG (activity of approximately 100 U/g; Hengsheng BioTech Co., Ltd, Shenzhen, China) was added to the gelatin solution until the temperature cooled to 30 °C and mixed thoroughly.

2.2. Development of sacrificial template printing platform

PVA filament was used to print the sacrificial template and was finally extracted to form a spatial channel in the hydrogel. The structure of the sacrificial part was



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determined during the printing process. For bulk tissue such as massive bone, the distribution of vasculature is complex. The vascular structure in tissues is hierarchical and resembles to tree branches1.Thus, we required a complex, 3D sacrificial template. The sacrificial template printing platform was almost the same as that used in our earlier study29. PVA filament was extruded from a fused printhead, which was mounted on a 3D printer to print the sacrificial template. The only difference was the receiving stepped shaft, which has a more complex spatial structure compared to the cylinder previously used. G-code commands were developed according to the designed sacrificial part to control the movement of the printing system. The fused extrusion system was heated to 180 °C to melt the PVA filament, and extruded from the printhead. The printhead orifice diameter was 0.2mm.

2.3. Design and 3D printing of sacrificial template

The stepped shaft consists of a large-diameter part (LDP), conical part (CP), and small-diameter part (SDP), as shown in Figure 1. Different sacrificial templates were designed to test the printability of our printing system. A Y-like template (Figure 1a) was designed to test the printability from LDP-to-SDP, while a double-channel model (Figure 1b) was devised for both LDP-to-SDP and SDP-to-LDP printability. The dimensions of each branch in the above two models are the same. In addition, a tree branch-like structure was designed, the branches of which have different sizes (D > d), which can better mimic the vasculature system of a tissue or organ.


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Figure 1. Different PVA templates were designed to verify the printability (a) from LDP-to-SDP and (b) from SDP-to-LDP on the stepped shaft, and (c) a tree branch-like multiscale structure.

2.4. Fabrication of the spatial channel in hydrogel

The printed sacrificial template was embedded in enzymatically crosslinking gelatin to obtain the spatial channel. Thus, after printing of the sacrificial template, the redundant part was trimmed as necessary and placed it in container filled with freshly prepared crosslinked gelatin solution. The container was then kept at –80 °C for 3 min to induce the aqueous gelatin to quickly turn to gel. Next, the hydrogel part containing the sacrificial template was cut to expose the head and tail part of the embedded PVA template, and extracted from the hydrogel construct. Then, the hydrogel construct was placed at 37 °C for 6 h to fully crosslink the gelatin, and a spatial channel was obtained within the hydrogel construct. The whole process is shown in Figure 2.



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Figure 2. Fabrication of a spatial channel in hydrogel. A multiscale PVA template was placed in a container (a). A mixture of gelatin and mTG was poured into the container and placed at –80 °C for 3 min (b). The redundant part of the partially crosslinked gelatin was cut, revealing the head part of PVA template (c). The PVA template was extracted from the partially crosslinked gelatin (d). The partially crosslinked gelatin with spatial channel was placed at 37 °C for 6 h for full crosslinking (e).

In order to verify the impact of the extraction process and PVA template on the spatial channel, the PVA template shown in Figure 1c was printed and used to fabricate a hydrogel construct. The widths at points A, B, C, D, E, and F of the printed template (Figure S1) and the corresponding channel were measured and compared. Three separate PVA templates and hydrogel constructs were prepared.

2.5. Mechanical properties

Uniaxial compression tests were conducted to test the compressive mechanical


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properties of the hydrogel construct. A constant compression speed of 0.5 mm/min was applied to each construct using a WDW-1 materials testing machine (Songdun machine equipment Co., Ltd, Shanghai, China) until fracture. The maximum compressive modulus and Young’s modulus were recorded. Three samples were tested.

2.6. Endothelialization of the spatial channel

HUVECs (Zhongqiaoxinzhou Biotech Co., Ltd, Shanghai, China) were cultured in RPMI 1640 culture medium, which consisted of 10% fetal bovine serum, 0.292 mg/mL L-glutamine, 4.766 mg/mL HEPES, 0.85 mg/mL NaHCO3, 1% penicillin (100 units/mL), and streptomycin (100 µg/mL), in a CO2 incubator. The cells were cultured for 3 days and then suspended in fresh medium; the final density of the suspension was 6 × 106 cell/mL. The engineered construct was immersed in 75% ethanol for 12 h under UV light, washed with phosphate-buffered saline (PBS) three times and immersed in freshly prepared medium under UV light for 6 h before use. 10 mL suspension was slowly injected into the spatial channel. The construct was cultured for 30 min to allow cells to attach to the bottom of the channel. The scaffold was rotated by 90°, and another 10 mL of cell suspension was injected into the channel. This process was repeated three times until the whole channel was covered with HUVECs. After adherence for 4 h, the construct was cultured with medium that was changed every day. The cellular morphology images were obtained using an inverse



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fluorescence microscope (Eclipse Ti-U, Nikon Instruments Inc., Japan) with a bright field.

2.7. Cell imaging

A Live/Dead cell staining kit (Biovision, Inc., San Francisco, USA), which is based on a cell-permeable dye (Live-Dye) for staining of live cells and a cell-impermeable dye (propodium iodide (PI)) for staining of dead and dying cells, was used for the visualization of live and dead cells in the spatial channel. Briefly, the cell-attached constructs were removed from the culture medium and gently washed with PBS. The dye solution was injected directly into the spatial channel and incubated for 15 min at 37 °C (as per manufacturer’s instructions). Then, the stain medium was removed and the hydrogel construct was mounted on microscope slides for imaging. An immunofluorescence study was performed to visualize the morphology of the attached cells in the inner surface of the spatial channel. After culturing for 4 days, HUVECs were immunostained with tetramethylrhodamine (TRITC)–phalloidin and 4',6-diamidino-2-phenylindole (DAPI) (Yeason, Shanghai, China) to visualize the cell cytoskeleton and nucleus. The construct was fixed (4% paraformaldehyde), permeabilized (5 min, 0.5% Triton X-100 in PBS). 100 µl TRITC–phalloidin was injected into the channel, followed by culturing in a dark environment for 30 min at room temperature to stain the cell cytoskeleton, after which the channel was washed


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three times with PBS. Next, 100 µl DAPI solution was injected into the channel to stain the cell nucleus for 30 s. A fluorescence microscope was used to observe the morphology of cells.

2.8. MTT assay

Cell

proliferation

was

assessed

using

(3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (MTT) (BS030B; Biosharp, China). HUVECs were seeded in three 24-well plates (No. 3524; Costar) at 6 × 106 cells/ml, and an mTG cross-linked gelatin cube (3mm × 3mm × 3mm) was placed in each well. HUVECs were seeded in another three 24-well plates without the mTG cross-linked gelatin cubes as the control group. All of the 24-well plates were incubated in CO2. The number of metabolically active cells was estimated after culturing for 0, 2, and 4 days by measuring absorbance using a microplate reader (Infinite 200Pro, Tecan Group Ltd., Switzerland) at wavelengths of 490 nm. Each condition was determined using triplicates.

2.9. Statistical analysis

All data are presented as the mean ± standard deviation (SD) and were analyzed using one-way analysis of variance (ANOVA) to determine which populations were significantly different. Differences were considered significant at p< 0.05.



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Results

3.1. Printing the complex 3D PVA template

In this research, additive manufacturing and subtractive manufacturing were combined to fabricate engineered scaffolds with spatial pre-vascularized networks. Spatial PVA templates were printed through our 3D printing system (Rapid Manufacturing Engineering Center, Shanghai University, China). A stepped shaft was used to print ideal models. The cylinder part of the shaft provides spatial structure along the axial direction for the sacrificial templates, while the spatial structure in the radial direction comes from the conical part. Therefore, a fully 3D PVA template can be printed. The first Y-like template (Figure 3a) verified printability from LDP to SDP on the stepped shaft. After cutting the redundant paths, the obtained structure was the same as our designed model. In addition, the printability from SDP to LDP was tested using the second double-channel model, as shown in Figure 3b. In each case, the designed model could be obtained smoothly using our printing system. As is well known, the vasculature in human is multiscale30, 31. Besides the spatial structure, multiscale PVA templates can be achieved with our printing system. Thus, a tree branch-like structure was designed and printed (Figure 3c). The width of the main stem is greater than that of the branches, as can be clearly seen from the side view of the printed tree branch-like structure (Figure 3).


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Figure 3. Printing results of (a) Y-like template, (b) double-channel template, and (c) tree branch-like template on a stepped shaft as the receiving platform. Black dot represents a redundant path. Scale bar is 3 mm.

3.2. Perfusion of hydrogel constructs

After fabricating the hydrogel constructs according the procedure described in section 2.4, orange acrylic paint was injected into the spatial channel to verify the perfusion of the channel. The injected paint filled the spatial channel perfectly in each construct, and no gaps were found (Figure 4). Thus, we could conclude that all the designed shapes were achieved through our printing system and fabrication method.

Figure 4. Perfusion of the spatial channels in hydrogel constructs. (a), (b), and (c)



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correspond to Y-like, double-channel, and tree branch-like structures, respectively. Scale bar is 3 mm.

In our fabrication process, the PVA templates were extracted from the hydrogel constructs, and the resulting channels were observed using fluorescence microscopy. Taking the tree branch-like construct as the test sample, the structure in the linear part and branched part were both intact, as shown in Figure 5. There were no differences between the shape of the spatial channel obtained and that of the designed model. The corresponding multiscale channel is shown in Figure S2. The maximum width of the spatial channel was around 900 µm; the minimum width was below 500 µm.

Figure 5. Morphology of the PVA template at (a) linear part and, (c) branched part; and corresponding channels at (b) linear part and, (d) branched part.


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Figure 6. Investigation of the impact of extracted process on the dimensions of the 3D channel by comparing the printed width and channel width at points A, B, C, D, E, and F of the sacrificial template.

To verify the impact of the extraction process and the PVA template on the spatial channel, the dimensions of the original PVA template and the corresponding channel were measured. Six points were selected as the observation sites (Figure S1). Comparisons of printed widths and actual widths are shown in Figure 6. Although the widths of channel at all six places were greater than those of the corresponding PVA template, the maximum difference was around 70 µm, and the minimum was less than 15 µm.

3.3. Mechanical properties

The compressive characteristics of engineered scaffolds are vital parameters in



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many tissue engineering applications. Thus, we performed compression tests of the fabricated constructs.

Table 1(Unit: MPa)

Compressive

Sample 1

Sample 2

Sample 3

Average Value

2.360

2.194

2.398

2.317 ± 0.108

4.661

4.595

4.610

4.622 ± 0.035

strength Young’s modulus

Samples were recorded as the stress from the test start to the fracture point of the scaffold, and the maximum compressive strength was recorded as 60% strain (almost all the tested samples were fractured at 60% strain). Young’s modulus was recorded as a measure of the rigidity of the scaffolds. The scaffolds had a compressive strength of 2.317 ± 0.108MPa, and the Young’s modulus was 4.622 ± 0.035 MPa before the fracture point (Table 1).

3.4. Morphology of cell adhesion

The main object of fabricating spatial pre-vascularized networks inside hydrogel scaffolds is to facilitate nutrient transport for the injected cells at the initial stage and during the formation of vascular networks. In this study, the most complicated PVA template (Figure 1c) was chosen and extracted from the crosslinked hydrogel


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constructs to fabricate hydrogel scaffolds for in vitro research. HUVECs in suspension were injected into the spatial channel and cultured statically in a CO2 incubator to explore the biocompatibility of the hydrogel scaffolds. Figure 7 shows the microscopic morphology of HUVECs inside the linear part and branched part of the spatial channel after being cultured for 4 days. Original cell morphology after attachment is shown in Figure S3. Although cells were distributed unevenly just after the adhesive, the cells were found to be well spread, and they completely covered the inner surface, even on the side surface of the spatial channel (Figure S4). Moreover, cells maintained normal cellular phenotypes from day 0 to day 4. The live/dead images (Figure 7) showed that HUVECs could maintain high viability; very few dead cells were found (Figure S5). The cross-section of the fabricated channel was a rectangle. The live/dead images of the whole channel are shown in Figure S6. Almost all cells on all four sides of main channel were live (Figure S6a), grooves on the side surface of the channel were owing to layer-to-layer printing of the PVA templates. Figure S6b shows the cell viability of the branched channel, which has smaller dimensions compared with the main channel. In addition, immunostaining of the hydrogel scaffold was carried out to visualize the cell nucleus and cytoskeleton. Corresponding magnification could is shown in Figure S7. Homogeneous and confluent distribution of cells at linear part and branched part can be seen in the DAPI and TRITC–phalloidin images (Figure 7). Figure 8 shows the endothelialized networks on the whole spatial channel. Live/dead and immunostaining images further demonstrated that the cells were uniformly distributed, with a high density of live



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cells inside the spatial channel, and were well attached to the inner surface of the network with a spread-out morphology.

Figure 7. Images of cell adhesive, live and dead cells, cell nucleus, and cytoskeleton at the linear part and branched part.


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Figure 8. 3D fluorescence images of cytoskeleton. (a) and (b) the main channel, (c) and (d) the branch channel. White curves represent the boundaries of the spatial channel.

3.5. MTT assay In order to further understand the cytoactivity of HUVECs and the growth conditions they require, cell proliferation was investigated under the same culturing conditions as used for the cell attachment studies. The MTT assay data (Figure 9) shows that after culturing for 2 days, cell numbers increased by around 40%, in both the experimental groups and the control group. Cell growth in all groups was seen to be sharply improved (by approximately 250%) on day 4. This indicates that cells can maintain their proliferation ability when cultured with the fabricated hydrogel



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constructs.

Figure 9. MTT assays of HUVECs after being cultured for 0, 2, and 4 days.

Discussion

In this paper, a new scalable and reproducible technique for fabricating engineered scaffolds with defined spatial pre-vascularized networks has been presented. The technique is easy to use and involves casting of the biomaterial gelatin within a soft, multiscale, 3D PVA template which defines the geometry of the channels within the scaffold. A stepped shaft was used as the receiving platform, which was different from the traditional planar receiving platform. The PVA template obtained in this way has a 3D structure, owing to the cylindrical surface and circular conical surface of the stepped shaft. Thus, the PVA template has spatial geometry in every axial direction or radial direction. In our previous study, a cylinder was used, which resulted in PVA template that only had spatial geometry in the radial direction. In this work, the complexity of the pre-vascularized networks obtained was increased. After the PVA


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template had been printed, it was embedded in a mixture of gelatin and mTG which could crosslink it, but the template was extracted from the hydrogel construct before total crosslinking occurred. Although our earlier research also used PVA as a sacrificial template to fabricate engineered scaffolds29, that PVA template swelled in the hydrogel and the dimension of the spatial channel increased, owing to the water solubility of PVA. We had investigated the possibility of PVA modification to control the swelling phenomenon in hydrogel. However, modified PVA still swelled in a water environment. Moreover, the printed PVA template used in previous studies only had 3D structure in the axial direction. Therefore, a more effective method was needed to meet the requirements of bulk tissue regeneration. We used gelatin as the scaffold material. Gelatin is low cost, and its biocompatibility has been tested repeatedly as it is the denatured product of collagen32. mTG has also been widely used in tissue engineering as a crosslinking agent for gelatin33-36. Here, we combined the printed 3D PVA template and an enzymatically induced crosslinking mechanism to fabricate engineered scaffolds with spatial pre-vascularized networks for bulk tissue regeneration. Although the PVA template was extracted from the mixture of gelatin and mTG at the initial stage of crosslinking, the perfusion test indicated that the integrity of the channel was maintained (Figure 4). Images of the channel (Figure 5) showed the morphology of the inner surface; an obvious rough appearance was detected. However, the comparison of width of the PVA template and the channel itself revealed that the extraction of the template did not have any significant impact on the dimensions of the channel. Beside this, many



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studies have shown that roughness of the scaffold surface plays an important part in cell attachment and proliferation37-41. Thus, we consider that the rough appearance of the inner surface of pre-vascularized networks has a positive influence on cell attachment. The compressive strength and Young’s modulus of the scaffolds reached 2.3 MPa and 4.6 MPa, respectively. These data have already exceeded those of many previous studies of hydrogel vascularization scaffolds16, 42, 43, which indicate that the fabricated scaffolds have appropriate mechanical properties for tissue regeneration. The longest channel in the scaffolds produced in this study had a length of 60 mm. As the diffusion limit of oxygen in mammalian tissue is 100–200µm, cells within the construct need to be located within that distance of the nearest capillaries44. Therefore, it is a challenge to maintain cell viability within long channels inside thick engineered scaffolds, owing to insufficient oxygen and nutrient levels. In vitro results demonstrated that HUVECs could successfully attach to the inner surface of the pre-vascularized networks and were uniformly distributed throughout the entire channels of the scaffolds after 4 days culturing. Immunostaining images show that luminal endothelialized layers could be formed on the inner surface. MTT assay data, as shown in Figure 9, shows that cells can proliferate in the gelatin environment as fast as when cultured in a petri dish. The homogeneity achieved can be attributed to the following factors. First, the morphology of the spatial channel is conducive to cell attachment and proliferation, and it provides a favorable condition for the even distribution of cells throughout the spatial channel. Second, the designed branched channels in the hydrogel constructs promote cell survival and proliferation, because


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the spatial channel allows sufficient oxygen and nutrient mass transport into the scaffold. Thus, our results indicate that the scaffolds and designed channel can meet mass transportation requirements in a static environment. Although HUVECs could survive and proliferate in this study, more work should be done to investigate optimal conditions. A dynamic culture environment should be created to provide the endothelial cells with in-vivo-like flow conditions, in order to better mimic the shear stress of body fluid. Thus, the development of a bioreactor for vascularization scaffolds is one of the most important considerations for further research. Based on this, functions such vascularization of the scaffolds should be explored in vivo to determine the potential of clinical applications.

Conclusion

We have presented a versatile method to fabricate engineered scaffolds with spatial pre-vascularized networks for bulk tissue regeneration by combining 3D printing on a stepped shaft and enzymatic crosslinking of gelatin. Different PVA templates were designed and smoothly printed on the novel receiving platform, thereby verifying the printability of our system. Scaffolds were obtained after extracting the PVA templates and crosslinking at 37 °C for 6 h. Perfusion tests indicated that the extraction process did not have a significant influence on the dimensions of spatial channels, and that structural integrity could be maintained. The hydrogel scaffolds with spatial channels facilitated the attachment and spreading of



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HUVECs; a high density of cells could attach to the inner surface after 4 days culturing, and were distributed across the 3D channel homogeneously owing to good oxygen and nutrient transport. It is envisioned that this approach might provide a versatile way to achieve more complex 3D pre-vascularized networks within engineered scaffolds, to broaden the applications of vascularization scaffolds in clinical tissue and organ research.

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

AUTHOR INFORMATION Corresponding Author *H.-G.Z.: phone, 86-021-66132941 ; e-mail, [email protected].

*Q.-X.H.: phone, 86-021-66133298; fax, 86-021-66133297 ; e-mail, [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge funding support from the National Natural Science


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For Table of Contents Use Only

Characterization

of

Engineered

Scaffolds

with

Spatial

Pre-Vascularized Networks for Bulk Tissue Regeneration Shuai Li1, Hai-Guang Zhang1, 2, 3, *, Dong-Dong Li1, Jian-Ping Wu1, Cheng-Yan Sun1, Qing-Xi Hu1, 2, 3, * 1

Rapid Manufacturing Engineering Center, Shanghai University, Shanghai 200444,

China 2

Shanghai Key Laboratory of Intelligent Manufacturing and Robotics, Shanghai

University, Shanghai 200072, China 3

National Demonstration Center for Experimental Engineering Training Education,

Shanghai University, Shanghai 200444, China


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Figure 1. Different PVA templates were designed to verify the printability (a) from LDP-to-SDP and (b) from SDP-to-LDP on the stepped shaft, and (c) a tree branch-like multiscale structure. 279x267mm (96 x 96 DPI)



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Figure 2. Fabrication of a spatial channel in hydrogel. A multiscale PVA template was placed in a container (a). A mixture of gelatin and mTG was poured into the container and placed at –80 °C for 3 min (b). The redundant part of the partially crosslinked gelatin was cut, revealing the head part of PVA template (c). The PVA template was extracted from the partially crosslinked gelatin (d). The partially crosslinked gelatin with spatial channel was placed at 37 °C for 6 h for full crosslinking (e). 330x140mm (96 x 96 DPI)


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Figure 3. Printing results of (a) Y-like template, (b) double-channel template, and (c) tree branch-like template on a stepped shaft as the receiving platform. Black dot represents a redundant path. Scale bar is 3 mm. 364x147mm (96 x 96 DPI)



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Figure 4. Perfusion of the spatial channels in hydrogel constructs. (a), (b), and (c) correspond to Y-like, double-channel, and tree branch-like structures, respectively. Scale bar is 3 mm. 411x120mm (96 x 96 DPI)


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Figure 5. Morphology of the PVA template at (a) linear part and, (c) branched part; and corresponding channels at (b) linear part and, (d) branched part. 333x252mm (96 x 96 DPI)



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Figure 6. Investigation of the impact of extracted process on the dimensions of the 3D channel by comparing the printed width and channel width at points A, B, C, D, E, and F of the sacrificial template. 297x209mm (150 x 150 DPI)


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Figure 7. Images of cell adhesive, live and dead cells, cell nucleus, and cytoskeleton at the linear part and branched part. 287x406mm (96 x 96 DPI)



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Figure 8. 3D fluorescence images of cytoskeleton. (a) and (b) the main channel, (c) and (d) the branch channel. White curves represent the boundaries of the spatial channel. 393x295mm (96 x 96 DPI)


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Figure 9. MTT assays of HUVECs after being cultured for 0, 2, and 4 days. 297x209mm (150 x 150 DPI)


Characterization of Engineered Scaffolds with Spatial Prevascularized

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