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3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility
Journal Pre-proof 3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility Qing Gao, Chaoqi Xie, Peng Wang, Mingjun Xie, Haibing Li, Anyu Sun, Jianzhong Fu, Yong He PII:
S0928-4931(19)31353-0
DOI:
https://doi.org/10.1016/j.msec.2019.110269
Reference:
MSC 110269
To appear in:
Materials Science & Engineering C
Received Date: 11 April 2019 Revised Date:
8 September 2019
Accepted Date: 30 September 2019
Please cite this article as: Q. Gao, C. Xie, P. Wang, M. Xie, H. Li, A. Sun, J. Fu, Y. He, 3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110269. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
3D Printed Multi-scale Scaffolds with Ultrafine Fibers for Providing Excellent Biocompatibility Qing Gao#1,2, Chaoqi Xie#1,2, Peng Wang#1,2, Mingjun Xie1,2, Haibing Li3, Anyu Sun1, Jianzhong Fu1,2, Yong He*1,2 (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 Paediatric Orthopaedics, The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310027, China * Correspondence to: Yong He; e-mail: [email protected] #
These authors contributed equally to the work)
Abstract: It is a dilemma that both strength and biocompatibility are requirements for an ideal scaffold in tissue engineering. The normal strategy is mixing or coating another material to improve the biocompatibility. Could we solve this dilemma by simply adjusting the scaffold structure? Here, a novel multi-scale scaffold was designed, in which thick fibers provide sufficient strength for mechanical support while the thin fibers provide a cell-favorable microenvironment to facilitate cell adhesion. Moreover, we developed a promising multi-scale direct writing system (MSDWS) for printing the multi-scale scaffolds. By switching the electrostatic field, scaffolds with fiber diameters from 3µm to 600µm were fabricated using one nozzle. Using this method, we proved that PCL scaffolds could also have excellent biocompatibility. BMSCs seeded on the scaffolds readily adhered to the thin fibers and maintained a high proliferation rate. Moreover, the cells bridged across the pores to form a cell sheet and gradually migrated to the thick fibers to cover the entire scaffold. We further combined the scaffolds with hydrogel for 3D cell culture and found that the fibers enhanced the strength and induced cell migration. We believe that the multi-scale scaffolds fabricated by an innovative 3D printing system have great potential for tissue engineering. Keywords: 3D printing; Multi-scale scaffolds; FDM printing; EHD printing; Tissue engineering
1. Introduction Scaffolds have been widely used in tissue engineering because they act as carriers for cell adhesion and produce a cellular microenvironment for tissue regeneration [1-3]. Moreover, the emergence of three-dimensional (3D) printing has allowed for the rapid fabrication of scaffolds with precisely controlled geometric shape and pore distribution [4-6]. In addition to a controllable structure, an ideal scaffold should also have sufficient mechanical strength to provide mechanical support and superior biological performance to provide a cell-favorable microenvironment. However, widely used printing materials, such as polycaprolactone (PCL) and polylactic acid (PLA), have poor bioactivity. The conventional strategy to solve this limitation is either the addition of bioactive ceramics before printing [7, 8], or coating with a hydrogel after printing [9, 10]. Although the biocompatibility of printed scaffolds is improved, the addition of other materials reduces the printability and coating with a hydrogel adds significant cost and complicates the process. More importantly, a change in the material composition of the printed scaffolds will hinder their applications in clinical tissue repair. Therefore, we pose this question: could we solve this bottleneck by simply adjusting the scaffold structure without changing the material composition? Traditional scaffolds with thick fibers (200-600µm) can be fabricated using conventional fused deposition modeling (FDM) 3D printing [11, 12]. Although scaffolds at this scale can provide mechanical support, they cannot provide the cell-favorable microenvironment for cell adhesion, because the fiber diameter is much larger than the scale of the extracellular matrix (ECM) or the cells (10-20µm). Recently, electrohydrodynamic (EHD) printing has emerged as a promising method for the fabrication of high-resolution scaffolds with ultrafine fibers (3-20µm) [13-16]. Because the scale of the cells is close to that of the fibers, the cells readily adhere to the thin fibers and maintain a high proliferation rate [17, 18]. In order to vividly show the effect of the fiber size on the cell adhesion, a cartoon schematic is given, as shown in Figure 1. The behavior of the cells is similar to that of a person climbing a tree: a person slides down when grasping the thick trunk but
climbs up smoothly by wrapping the arms around a thin branch. Similarly, cells behave differently after being seeded on scaffolds with different fiber sizes. Since a cell is close in size to a high-resolution scaffold but far smaller than the size of the traditional scaffold, when a cell is seeded on the thick fiber of a traditional scaffold, it has difficulty “grasping” the thick fiber and cannot attach itself to it. Conversely, when a cell is seeded on the thin fiber of a high-resolution scaffold, the fiber is thin enough so that the cell can “grasp” it, resulting in good attachment to the thin fiber and good subsequent growth. Therefore, high-resolution scaffolds provide a more cell-favorable microenvironment than traditional scaffolds, which has promising applications related to cell attachment, growth, and function. Nevertheless, the high-resolution scaffolds are prone to deformation and collapse due to low mechanical strength, which constrains their applications in tissue repair and regeneration.
Figure 1. A cartoon schematic showing the effect of the scaffold fiber size on the cell adhesion.
In this study, to balance mechanical strength and biocompatibility of 3D printed scaffolds by adjusting the scaffold structure, a multi-scale scaffold was specially designed by combining the merits of conventional FDM printed scaffold and EHD printed high-resolution scaffold. In current study, an integrated system for fabricating multi-scale scaffold was developed. By a switching module to control electric field and extrusion, FMD and EHD modes could convert during printing.
Thus, thick and thin fibers can be easily printed using one device. In this multi-scale composite scaffold, the thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. Although the strategy of combination of EHD printing with FDM printing to fabricate hierarchical scaffolds has been applied [19, 20], these studies only focused on characterization of the EHD printing process, but neither investigated the stability of the hybrid manufacturing process nor verified the improved biocompatibility of the multi-scale scaffolds. Herein, in addition to demonstrating the feasibility of this strategy, we focused on studying the stability and versatility of the multi-scale direct writing system (MSDWS), and systematically investigated the influence of the multi-scale scaffold in regulating cellular behaviors. These studies made the multi-scale direct writing strategy more practical to ensure both mechanical strength and biocompatibility of scaffolds by simply adjusting the structure without changing the material composition.
2. Materials and Methods
2.1 Materials preparation PCL particle (CAPA6800, Perstorp Ltd, UK) used in this study was with the melting temperature of 60℃ and the molecular weight of 80,000 g/mol. 2.2 MSDWS configuration In this study, a multi-scale direct writing system (MSDWS) is developed to printing the multi-scale scaffolds. The system was composed of motorized XYZ stages attached with a nozzle (inner diameter of 350µm) loaded with PCL particles, a heater to melt PCL polymer and adjust the printing temperature, a pneumatic device to extrude PCL melt and adjust the extruding rate, and a high voltage generator for employing electrostatic field to make the extruded PCL melt into thin fibers.
2.3 Morphological analysis of multi-scale scaffolds The morphology of the multi-scale scaffolds was observed with a scanning electron microscopy (JSM-IT100, Japan) after sputter coating the samples with gold for 3 min. 2.4 Mechanical testing In mechanical test, three types of scaffolds with only thin fibers, only thick fibers, and integrated thin and thick fibers were prepared. The scaffolds of thin fibers were printed with a fiber diameter of 10µm and fiber spacing of 200µm, and those of thick fibers were with a fiber diameter of 400µm and fiber spacing of 1400µm. The integrated multi-scale scaffolds consisted of thin and thick fibers of the same parameters above. In tensile test, all scaffolds were printed at a thickness of 200µm and width of 7mm. The initial length was adjust to 5mm. In compressive test, all scaffolds were 7mm*7mm and 1mm thick. And compressive stiffness was measured at 10% strain. For a simple analysis, the sectional area was calculated as width*thickness ignoring the porous in the scaffolds. 2.5 Cell culture Bone marrow stem cells (BMSCs) were cultured in DMEM added with 10% fetal bovine serum, 1% penicillin, and streptomycin. The culture media was changed every other day, and cells were passaged using trypsin-EDTA dissociation every 4 days. Before seeding cells, the scaffolds were first immersed in 75% ethanol under UV light for 1 h, washed three times with PBS, and incubated in 24-well plates containing culture media overnight. When in 2D cell culture, the cell suspensions were directly seeded on the scaffolds (one scaffold in each well of 24-well plates, at 4×104 cells/scaffold in 1 ml DMEM). When in 3D cell culture, 5% (W/V) Gelatin methacryloyl (GelMA, EFL-GM-90, Suzhou Intelligent Manufacturing Research Institute, Suzhou, China) solution containing 0.5%(w/v) lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) was used to resuspend cells at a concentration of 5×106 cells/ml. Then the cell-laden GelMA was dropped into the multi-scale scaffold, this was followed by photo-crosslinking the GelMA to obtain
reinforced structures. All the cell-seeded scaffolds were incubated at 37°C in 5% CO2 and the media was replaced every other day. 2.6 Cell proliferation analysis Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. Different groups of cell-seeding scaffolds were washed three times with PBS. Then 1450 µl DMEM medium and 50 µl CCK-8 were added to each well of 24-well plates, and incubated for 3 h. Finally, the solutions were transferred to a 96-well plate (200 µl per well) to read the OD values at a wavelength of 450 nm. 2.7 Cell viability analysis Cell viability was analyzed using LIVE/DEAD assay reagents (KeyGEN BioTECH Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Different groups of cell-seeding scaffolds were washed three times with PBS. Then a PBS solution containing 6µM Calcein AM and 24 µM propidium iodide (PI) was added to the scaffolds. The scaffolds were incubated for 40 min in the dark, and washed three times with PBS to remove residual regents. Finally, a confocal fluorescence microscope (OLYMPUS FV3000) was used to image the cell-laden structures by acquiring two images in each frame, red and green for live and dead cells, respectively. 2.8 Cell morphological analysis The cytoskeleton was characterized by staining F-actin and nuclei to determine changes in cell morphology. F-actin and nuclei were stained with TRITC phalloidin and DAPI, respectively, according to the manufacturer's instructions. First, the scaffolds were washed in PBS and fixed in 4% paraformaldehyde for 30 min. The scaffolds were then washed with PBS and permeabilized with 0.5% Triton X-100 for 5 min. Then, the samples were washed with PBS and stained with TRITC phalloidin (0.1 µM, YEASEN BioTECH Co., Ltd., Shanghai, China) for 30 min in the dark. The scaffolds were washed in PBS again and stained with DAPI (10 µg/ml; Solarbio, Beijing, China) for 10 min. Finally, the scaffolds were washed in PBS and imaged under a confocal fluorescence microscope. The SEM analysis was performed to determine changes in cell morphology. Briefly,
cell-seeding scaffolds were fixed in 2.5% glutaraldehyde overnight. The scaffolds were washed in PBS and fixed with 1% osmic acid for 1.5 h. Then, the scaffolds were washed with PBS and dehydrated through a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 95% for 15 min each). Finally, the scaffolds were air dried, sputter coated with platinum, and imaged using a fully digital SEM (SU8010, Hitachi, Tokyo, Japan). 2.9 Statistical analysis Data is presented as mean ± standard deviation of independent replicates. Statistical analysis is conducted using ANOVA, and single asterisk (*) indicates significant differences between groups (p < 0.05), double asterisk (**) indicates significant differences between groups (p < 0.01), and NS indicates no significant differences between groups (p > 0.05).
3. Results and Discussion 3.1 Fabrication strategy of the multi-scale scaffolds As shown in Figure 2A, the designed multi-scale scaffold consists of thick fibers (macro-scale) as frame structures and thin fibers (micro-scale) as filling structures. The thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. To fabricate the designed multi-scale scaffolds efficiently and conveniently, a promising multi-scale direct writing system (MSDWS) is developed for printing the user-specific patterned multi-scale scaffolds using a single nozzle. The schematic of the MSDWS is shown in Figure 2B. By switching the electrostatic field, the FDM printing mode and EHD printing mode can be quickly switched. More specifically, when the high voltage generator was turned off, the MSDWS implemented the FDM printing process and thick fibers were printed using air pressure. When the high voltage generator was turned on, the MSDWS implemented the EHD printing process and thin fibers were printed using an electric force. Therefore, by controlled switching of the electrostatic field in real time, a user-specific patterned multi-scale scaffold was
fabricated. Additionally, by adjusting the printing parameters (printing speed, air pressure, and temperature), thick fibers ranging from 200µm to 600µm and thin fibers ranging from 3µm to 20µm can be obtained. Figure 2C and Video S1 (Supporting Information) shows the printing process. It was observed that the printing was stable and smooth, resulting in a scaffold with uniform thick/thin fibers. Herein, two cell culture models were used to investigate the influence of the multi-scale scaffold in regulating cellular behaviors. As shown in Figure 2D, interestingly, the cells bridged across the pores and formed a sheet in the 2D cell culture; the cells migrated from the hydrogel to the thin fibers in the 3D cell culture, which confirmed the excellent compatibility of the multi-scale scaffold.
Figure 2. Design and manufacturing of a multi-scale scaffold and its application for regulating cellular behaviors. (A) Schematic illustration of the design of the multi-scale scaffold; the thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. (B) Schematic illustration of the multi-scale direct writing system (MSDWS) using on/off electrostatic field. (C) Printing process of the multi-scale scaffold. (D) The application of the multi-scale scaffold in regulating cellular behaviors.
3.2 Fabrication and characterization of the multi-scale scaffolds To confirm that the scale of the printed scaffolds ranged from the micro- to the macro-scale in the MSDWS, the effect of the printing parameters on the fiber size was investigated for the FDM and EHD printing, respectively. Since the resolution of FDM printing is limited by the nozzle size, the fiber diameter cannot be very small and is generally at the macro-scale (>100µm). In contrast, the fiber diameter of EHD printing is significantly smaller than the nozzle size and is at the micro-scale (<30µm), because the high electrostatic field causes the extruded polymer to form a Taylor-cone, followed by a thin jet ejecting from the cone. In the MSDWS printing process, the diameter of the printed fibers can be adjusted in the corresponding scale range and is affected by several parameters, including air pressure, temperature, and printing speed, as shown in Figure 3A. The effect of these parameters on thick and thin fiber diameters is shown in Figure 3B(I) and Figure 3C(I). The result indicates that the fiber diameter increased with increasing air pressure or temperature, whereas it
Figure 3. Characterization of the FDM and EHD printing process. (A) Schematic illustration showing the parameters influencing the fiber diameter, including air pressure, temperature, and printing speed. (B) (I) The effect of the printing parameters on thick fiber diameter in FDM printing, and (II) the thick fiber ranging from 180µm to 330µm obtained by adjusting the printing speed. (C) (I) The effect of the printing parameters on the thin fiber diameter in EHD printing, and (II) the thin fiber ranging from 2.48µm to 18.3µm obtained by adjusting the printing speed. (D) EHD printed scaffolds with complex structures, including (I) cobweb-like structure, (II) flower-like structure, and (III) snail-like structure.
decreased with increasing printing speed. This occurred because a higher air pressure directly increased the extrusion quantity and a higher temperature reduced the polymer viscosity to indirectly increase the extrusion quantity, resulting in an increase in the fiber diameter. In addition, the fiber was stretched and became thinner with increasing printing speed. In this study, the printing speed was used to adjust the fiber diameter. As shown in Figure 3B(II) and Figure 3C(II), the thick fiber size ranged from 180µm to 330µm and the thin fiber size ranged from 2.48µm to 18.3µm. The two printing processes should enable fabricating scaffolds with complex structures to provide better robustness and versatility of the MSDWS. FDM printing is a widely used technology and many strategies have been presented to fabricate complex structures [21, 22]. However, as an emerging technology, EHD printing can only print scaffolds with simple geometries such as grid structures [23, 24]. In this study, complex structures can be successfully printed by adjusting the printing speed in real time, such as cobweb-like structure, flower-like structure, and snail-like structure, as shown in Figure 3D. To determine the feasibility and stability of the MSDWS for fabricating multi-scale composite scaffolds, grid scaffolds were printed as an example by depositing a layer of thin fibers and covering them with a layer of thick fibers. Figure 4A shows a grid thick structure filled with thin fibers in a rectangle with 90° orientation. It can be seen that the EHD printed thin fibers adhered firmly to the thick fibers and were uniform with a homogeneous fiber space of 200µm. It has been reported that micro/nano fiber structures with different geometries and orientations in high-resolution scaffolds induce different cellular behaviors [25, 26]. Because the EHD printing is a
controllable high-resolution 3D printing method, we were able to control the shape and orientation of the fiber structures by adjusting the printing path of the substrate. As shown in Figure 4B and Figure 4C, a parallelogram with 45° orientation and an equilateral triangle with 60° orientation were successfully printed to fill the large pores. Additionally, when printing multilayered scaffolds, three types of thin fiber depositions were observed, including drooping fibers, straight fibers, and curving fibers, as shown in Figure S1 (Supporting Information). This occurred because a simple beam formed after the thin fibers were deposited onto the thick fibers and the printing speed and distance between the adjacent thick fibers affected the suspension. Figure 4D shows the effect of the printing speed and distance on the shape of the deposited thin fibers. By matching the printing speed and space, straight fibers were obtained, and they were desired to obtain multi-scale scaffolds with a controllable structure. Using the straight thin fibers, a multilayered multi-scale scaffold was printed, as shown in Figure 4E. The successful fabrication of these scaffolds demonstrates the stability and versatility of the MSDWS to fabricate morphologically controllable multi-scale scaffolds, which may offer potential applications in regulating cellular behaviors.
Figure 4. Fabrication of multi-scale scaffolds with the MSDWS. (A) The printed multi-scale scaffold with thin fiber structures of a rectangle with 90° orientation. (B) The printed multi-scale scaffold with thin fiber structures of a parallelogram with 45° orientation. (C) The printed multi-scale scaffold with thin fiber structures of an equilateral triangle with 60° orientation. (D) The effect of printing speed and distance on the shape of the deposited thin fibers. (E) The printed multilayered multi-scale scaffold.
To demonstrate the mechanical strength of the multi-scale scaffolds, compressive and tensile tests were conducted. Representative tensile and compressive stress-strain curves are shown in Figure 5A and Figure 5C. Notably, there was significant differences in mechanical properties between scaffolds of thin fibers and the others. The strength was mainly provided by thick fibers, so the curves of multi-scale scaffold and thick fiber scaffold were remarkably similar. The measurement of compressive stiffness and tensile modules showed the same trend (Figure 5B and
Figure 5D). Scaffold of thick fibers and multi-scale fibers had close results in both compressive (59.9±13.7KPa and 64.0±5.7KPa, respectively) and tensile (23.2±0.2MPa and 24.0±1.3MPa, respectively) test. However, the results of scaffold of only thin fibers were about two orders of magnitude lower, which were 0.66±0.06KPa and 0.16±0.07Mpa in stiffness and tensile modulus, respectively. It can be concluded that thick fibers significantly increased the mechanical strength of multi-scale scaffolds.
Figure 5. Mechanical characterization of scaffolds with different fibers. A) Representative tensile stress/strain curves of scaffolds with different fibers. B) Tensile modulus of scaffolds with different fibers. C) Representative compressive stress/strain curves of scaffolds with different fibers. D) Compressive stiffness of scaffolds with different fibers.
3.3 Biocompatibility analysis of the multi-scale scaffolds To confirm the enhanced biocompatibility of the multi-scale scaffold and its effect on regulating the cellular behaviors, bone marrow stem cells (BMSCs) were directly seeded on the multi-scale scaffolds. A schematic illustration of the cell growth process is shown in Figure 6A. In order to study the effect of the thin fibers on the biocompatibility of the multi-scale scaffolds, the
BMSCs seeded on the macro-scale scaffolds with pure thick fibers fabricated by FDM printing were regarded as the control group. The cell attachment, proliferation, viability, morphology, and interaction with the fibers were investigated during a 7-day culture period. As shown in Figure 6B, the cells adhered more readily to the multi-scale scaffold than the macro-scale scaffold after 4 h. This is illustrated in Figure 1. Furthermore, a cell proliferation analysis using cell counting kit (CCK)-8 assay was performed on day 1, 3, 5, and 7. As shown in Figure 6C, the proliferation rate of the BMSCs seeded on the multi-scale scaffolds was twice as high as that of the macro-scale scaffolds during the 7-day culture period, thereby further confirming that the addition of thin fibers was conducive to cell proliferation. Next, a cell viability analysis using live/dead assay was performed to further assess the biocompatibility of the multi-scale scaffolds. As shown in Figure S2 (Supporting Information), most of the cells were alive (green) and a few were dead (red). Additionally, cytoskeletal observations by F-actin staining were used to determine the changes in cell morphology to ascertain the cell growth and interaction with the scaffolds. As shown in Figure 6D and Figure S3 (Supporting Information), on day 1, the BMSCs changed from a spherical shape to an elongated shape and formed cellular junctions on the thin fibers. In the following days, interestingly, the BMSCs gradually bridged across the micro-pores and filled them to form a cell sheet on day 7. In contrast, the BMSCs on the macro-scale scaffolds exhibited less spreading and did not bridge across the large pores and did not fill them, as shown in Figure S4(Supporting Information). This occurred because the scale of the BMSCs (>200µm) was larger than that of the pores, resulting in the cells bridging across the pores. Furthermore, after the cells had filled the pores, they migrated to the thick fibers, continued to proliferate, and finally covered all the fibers of the multi-scale scaffold, as shown in Figure 6E and Video S2 (Supporting Information). And diameter difference of several micrometers in thin fibers would have an impact on cell phenotype which has been reported in our former study [27]. BMSCs were induced to oriented growth on scaffold with fiber diameters of ≈5µm and ≈20µm, but cells on homogeneous scaffold didn’t
exhibit the oriented growth, as shown in Figure S5 (Supporting Information).This result further demonstrated that the thin fibers greatly improved the biocompatibility of the printed scaffold.
Figure 6. Biocompatibility analysis of the printed multi-scale scaffolds. (A) Schematic illustration of the BMSCs seeded and cultured on multi-scale scaffolds. (B) Comparison of cell adhesion between multi-scale scaffolds and macro-scale scaffolds. (C) Comparison of cell proliferation between multi-scale scaffolds and macro-scale scaffolds. (D) Cell morphology change and interaction with the multi-scale scaffolds on day 1, 3, 5, and 7. (E) Cells migrating to the thick fibers and covering all the fibers of the multi-scale scaffold.
3.4 Application of the multi-scale scaffolds in hydrogel-based 3D cell culture Recently, hydrogel-based 3D cell cultures have attracted increasing attention in tissue engineering since hydrogels provide a 3D environment, mimicking an ECM and promoting nutrition exchange, which is close to native tissue. However, hydrogels are intrinsically soft and have poor mechanical strength, which limits their application in long-term cell culture. To overcome this challenge, fiber reinforcing methods have emerged as a promising strategy to increase the mechanical strength as much as dozens of times [28, 29]. Therefore, in this study, to assess whether our printed multi-scale scaffolds could also be integrated with hydrogel-based cell culture, we indirectly seeded cells on the multi-scale scaffolds by encapsulating the cells with a hydrogel. Here, a gelatin methacrylate (GelMA) hydrogel was selected to encapsulate the cells because it not only has excellent biological performance (further confirmed in Figure S6 (Supporting Information)) but also easily crosslinks upon light exposure [30, 31]. In the GelMA-based 3D cell culture experiment, the BMSCs were first mixed uniformly with the GelMA solution and then the cell-laden GelMA solution was dropped into the multi-scale scaffold, this was followed by photo-crosslinking the GelMA to obtain structure similar to reinforced concrete. The schematic illustration of the cell growth process is shown in Figure 7A. As shown in Figure 7B, the BMSCs were evenly distributed in the reinforced structures, and the fluorescent images show that almost all of the cells were alive (green) and were spherically shaped on day 1. Subsequently, the BMSCs began to elongate (Figure 7C), and several cells began to migrate to the thin fibers over time (Figure 7D and Figure S7 (Supporting Information)), this can be clearly seen in Video S3 (Supporting Information). This behavior demonstrated that the printed multi-scale scaffolds could also be used in a 3D cell culture and regulated the cellular behaviors in a manner that was different from that of pure hydrogel-based cell culture. In future studies, controllable cell deposition based on these multi-scale scaffolds/GelMA structures may be achievable by integrating 3D bioprinting technology into the MSDWS [32, 33]. Thus, multi-scale and multi-material structures could be
printed in one step, the macro-scale polymer structures would provide mechanical support, the micro-scale polymer structures would provide a fiber-like microenvironment, and the hydrogel would provide a 3D microenvironment.
Figure 7. 3D cell culture based on “reinforced concrete” structure combined with the GelMA hydrogel. (A) Schematic illustration of the BMSCs seeded and cultured on the “reinforced concrete” structure. (B) Fluorescent images of live/dead staining showing the spherically shaped BMSCs on day 1. (C) Fluorescent images of f-actin/nuclei staining showing the stretched BMSCs on day 7. (D) Fluorescent images of f-actin/nuclei staining showing the migration of the BMSCs to the thin fibers.
4. Conclusion In conclusion, we have developed a novel high-resolution MSDWS that enables the fabrication of multi-scale scaffolds using a single nozzle. The multi-scale scaffold consisted of thick fibers (macro-scale) as the frame structures and thin fibers (micro-scale) for filling the frame; the thick fibers were able to provide sufficient strength for mechanical support and the thin fibers provided a cell-favorable microenvironment for cell adhesion. Through a systematic study of the direct writing process, we have confirmed the feasibility and stability of the MSDWS for fabricating multi-scale composite scaffolds. An improved biocompatibility of the multi-scale scaffold and its effect on regulating the cellular behaviors were also demonstrated by a comparison with macro-scale scaffolds. Furthermore, we successfully combined the multi-scaffolds with a hydrogel for 3D cell culture to enhance the strength and induce cell migration. We believe that the MSDWS will become a generalized 3D printing strategy that can be extended to enhance the biocompatibility of other conventional scaffolds by filling them with ultrafine fibers.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
The authors would like to acknowledge the Testing Center in Suzhou Intelligent Manufacturing Research Institute for providing SEM, confocal and mechanical testing. This work was sponsored by the National Nature Science Foundation of China (No.51805474, 51622510, U1609207, 51605429), the Science Fund for Creative Research Groups of National Natural Science Foundation of China (No. 51821093), and China Postdoctoral Science Foundation (Grant No. 2019T120509).
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Highlights： ： (i) A multi-scale scaffold was designed to balance mechanical strength and biocompatibility of 3D printed scaffolds (ii) A promising multi-scale direct writing system (MSDWS) was developed for printing the multi-scale scaffolds. (iii) By filling ultrafine fibers, the biocompatibility of the scaffold was greatly improved.
S0928-4931(19)31353-0
DOI:
https://doi.org/10.1016/j.msec.2019.110269
Reference:
MSC 110269
To appear in:
Materials Science & Engineering C
Received Date: 11 April 2019 Revised Date:
8 September 2019
Accepted Date: 30 September 2019
Please cite this article as: Q. Gao, C. Xie, P. Wang, M. Xie, H. Li, A. Sun, J. Fu, Y. He, 3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110269. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
3D Printed Multi-scale Scaffolds with Ultrafine Fibers for Providing Excellent Biocompatibility Qing Gao#1,2, Chaoqi Xie#1,2, Peng Wang#1,2, Mingjun Xie1,2, Haibing Li3, Anyu Sun1, Jianzhong Fu1,2, Yong He*1,2 (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 Paediatric Orthopaedics, The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310027, China * Correspondence to: Yong He; e-mail: [email protected] #
These authors contributed equally to the work)
Abstract: It is a dilemma that both strength and biocompatibility are requirements for an ideal scaffold in tissue engineering. The normal strategy is mixing or coating another material to improve the biocompatibility. Could we solve this dilemma by simply adjusting the scaffold structure? Here, a novel multi-scale scaffold was designed, in which thick fibers provide sufficient strength for mechanical support while the thin fibers provide a cell-favorable microenvironment to facilitate cell adhesion. Moreover, we developed a promising multi-scale direct writing system (MSDWS) for printing the multi-scale scaffolds. By switching the electrostatic field, scaffolds with fiber diameters from 3µm to 600µm were fabricated using one nozzle. Using this method, we proved that PCL scaffolds could also have excellent biocompatibility. BMSCs seeded on the scaffolds readily adhered to the thin fibers and maintained a high proliferation rate. Moreover, the cells bridged across the pores to form a cell sheet and gradually migrated to the thick fibers to cover the entire scaffold. We further combined the scaffolds with hydrogel for 3D cell culture and found that the fibers enhanced the strength and induced cell migration. We believe that the multi-scale scaffolds fabricated by an innovative 3D printing system have great potential for tissue engineering. Keywords: 3D printing; Multi-scale scaffolds; FDM printing; EHD printing; Tissue engineering
1. Introduction Scaffolds have been widely used in tissue engineering because they act as carriers for cell adhesion and produce a cellular microenvironment for tissue regeneration [1-3]. Moreover, the emergence of three-dimensional (3D) printing has allowed for the rapid fabrication of scaffolds with precisely controlled geometric shape and pore distribution [4-6]. In addition to a controllable structure, an ideal scaffold should also have sufficient mechanical strength to provide mechanical support and superior biological performance to provide a cell-favorable microenvironment. However, widely used printing materials, such as polycaprolactone (PCL) and polylactic acid (PLA), have poor bioactivity. The conventional strategy to solve this limitation is either the addition of bioactive ceramics before printing [7, 8], or coating with a hydrogel after printing [9, 10]. Although the biocompatibility of printed scaffolds is improved, the addition of other materials reduces the printability and coating with a hydrogel adds significant cost and complicates the process. More importantly, a change in the material composition of the printed scaffolds will hinder their applications in clinical tissue repair. Therefore, we pose this question: could we solve this bottleneck by simply adjusting the scaffold structure without changing the material composition? Traditional scaffolds with thick fibers (200-600µm) can be fabricated using conventional fused deposition modeling (FDM) 3D printing [11, 12]. Although scaffolds at this scale can provide mechanical support, they cannot provide the cell-favorable microenvironment for cell adhesion, because the fiber diameter is much larger than the scale of the extracellular matrix (ECM) or the cells (10-20µm). Recently, electrohydrodynamic (EHD) printing has emerged as a promising method for the fabrication of high-resolution scaffolds with ultrafine fibers (3-20µm) [13-16]. Because the scale of the cells is close to that of the fibers, the cells readily adhere to the thin fibers and maintain a high proliferation rate [17, 18]. In order to vividly show the effect of the fiber size on the cell adhesion, a cartoon schematic is given, as shown in Figure 1. The behavior of the cells is similar to that of a person climbing a tree: a person slides down when grasping the thick trunk but
climbs up smoothly by wrapping the arms around a thin branch. Similarly, cells behave differently after being seeded on scaffolds with different fiber sizes. Since a cell is close in size to a high-resolution scaffold but far smaller than the size of the traditional scaffold, when a cell is seeded on the thick fiber of a traditional scaffold, it has difficulty “grasping” the thick fiber and cannot attach itself to it. Conversely, when a cell is seeded on the thin fiber of a high-resolution scaffold, the fiber is thin enough so that the cell can “grasp” it, resulting in good attachment to the thin fiber and good subsequent growth. Therefore, high-resolution scaffolds provide a more cell-favorable microenvironment than traditional scaffolds, which has promising applications related to cell attachment, growth, and function. Nevertheless, the high-resolution scaffolds are prone to deformation and collapse due to low mechanical strength, which constrains their applications in tissue repair and regeneration.
Figure 1. A cartoon schematic showing the effect of the scaffold fiber size on the cell adhesion.
In this study, to balance mechanical strength and biocompatibility of 3D printed scaffolds by adjusting the scaffold structure, a multi-scale scaffold was specially designed by combining the merits of conventional FDM printed scaffold and EHD printed high-resolution scaffold. In current study, an integrated system for fabricating multi-scale scaffold was developed. By a switching module to control electric field and extrusion, FMD and EHD modes could convert during printing.
Thus, thick and thin fibers can be easily printed using one device. In this multi-scale composite scaffold, the thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. Although the strategy of combination of EHD printing with FDM printing to fabricate hierarchical scaffolds has been applied [19, 20], these studies only focused on characterization of the EHD printing process, but neither investigated the stability of the hybrid manufacturing process nor verified the improved biocompatibility of the multi-scale scaffolds. Herein, in addition to demonstrating the feasibility of this strategy, we focused on studying the stability and versatility of the multi-scale direct writing system (MSDWS), and systematically investigated the influence of the multi-scale scaffold in regulating cellular behaviors. These studies made the multi-scale direct writing strategy more practical to ensure both mechanical strength and biocompatibility of scaffolds by simply adjusting the structure without changing the material composition.
2. Materials and Methods
2.1 Materials preparation PCL particle (CAPA6800, Perstorp Ltd, UK) used in this study was with the melting temperature of 60℃ and the molecular weight of 80,000 g/mol. 2.2 MSDWS configuration In this study, a multi-scale direct writing system (MSDWS) is developed to printing the multi-scale scaffolds. The system was composed of motorized XYZ stages attached with a nozzle (inner diameter of 350µm) loaded with PCL particles, a heater to melt PCL polymer and adjust the printing temperature, a pneumatic device to extrude PCL melt and adjust the extruding rate, and a high voltage generator for employing electrostatic field to make the extruded PCL melt into thin fibers.
2.3 Morphological analysis of multi-scale scaffolds The morphology of the multi-scale scaffolds was observed with a scanning electron microscopy (JSM-IT100, Japan) after sputter coating the samples with gold for 3 min. 2.4 Mechanical testing In mechanical test, three types of scaffolds with only thin fibers, only thick fibers, and integrated thin and thick fibers were prepared. The scaffolds of thin fibers were printed with a fiber diameter of 10µm and fiber spacing of 200µm, and those of thick fibers were with a fiber diameter of 400µm and fiber spacing of 1400µm. The integrated multi-scale scaffolds consisted of thin and thick fibers of the same parameters above. In tensile test, all scaffolds were printed at a thickness of 200µm and width of 7mm. The initial length was adjust to 5mm. In compressive test, all scaffolds were 7mm*7mm and 1mm thick. And compressive stiffness was measured at 10% strain. For a simple analysis, the sectional area was calculated as width*thickness ignoring the porous in the scaffolds. 2.5 Cell culture Bone marrow stem cells (BMSCs) were cultured in DMEM added with 10% fetal bovine serum, 1% penicillin, and streptomycin. The culture media was changed every other day, and cells were passaged using trypsin-EDTA dissociation every 4 days. Before seeding cells, the scaffolds were first immersed in 75% ethanol under UV light for 1 h, washed three times with PBS, and incubated in 24-well plates containing culture media overnight. When in 2D cell culture, the cell suspensions were directly seeded on the scaffolds (one scaffold in each well of 24-well plates, at 4×104 cells/scaffold in 1 ml DMEM). When in 3D cell culture, 5% (W/V) Gelatin methacryloyl (GelMA, EFL-GM-90, Suzhou Intelligent Manufacturing Research Institute, Suzhou, China) solution containing 0.5%(w/v) lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) was used to resuspend cells at a concentration of 5×106 cells/ml. Then the cell-laden GelMA was dropped into the multi-scale scaffold, this was followed by photo-crosslinking the GelMA to obtain
reinforced structures. All the cell-seeded scaffolds were incubated at 37°C in 5% CO2 and the media was replaced every other day. 2.6 Cell proliferation analysis Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. Different groups of cell-seeding scaffolds were washed three times with PBS. Then 1450 µl DMEM medium and 50 µl CCK-8 were added to each well of 24-well plates, and incubated for 3 h. Finally, the solutions were transferred to a 96-well plate (200 µl per well) to read the OD values at a wavelength of 450 nm. 2.7 Cell viability analysis Cell viability was analyzed using LIVE/DEAD assay reagents (KeyGEN BioTECH Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Different groups of cell-seeding scaffolds were washed three times with PBS. Then a PBS solution containing 6µM Calcein AM and 24 µM propidium iodide (PI) was added to the scaffolds. The scaffolds were incubated for 40 min in the dark, and washed three times with PBS to remove residual regents. Finally, a confocal fluorescence microscope (OLYMPUS FV3000) was used to image the cell-laden structures by acquiring two images in each frame, red and green for live and dead cells, respectively. 2.8 Cell morphological analysis The cytoskeleton was characterized by staining F-actin and nuclei to determine changes in cell morphology. F-actin and nuclei were stained with TRITC phalloidin and DAPI, respectively, according to the manufacturer's instructions. First, the scaffolds were washed in PBS and fixed in 4% paraformaldehyde for 30 min. The scaffolds were then washed with PBS and permeabilized with 0.5% Triton X-100 for 5 min. Then, the samples were washed with PBS and stained with TRITC phalloidin (0.1 µM, YEASEN BioTECH Co., Ltd., Shanghai, China) for 30 min in the dark. The scaffolds were washed in PBS again and stained with DAPI (10 µg/ml; Solarbio, Beijing, China) for 10 min. Finally, the scaffolds were washed in PBS and imaged under a confocal fluorescence microscope. The SEM analysis was performed to determine changes in cell morphology. Briefly,
cell-seeding scaffolds were fixed in 2.5% glutaraldehyde overnight. The scaffolds were washed in PBS and fixed with 1% osmic acid for 1.5 h. Then, the scaffolds were washed with PBS and dehydrated through a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 95% for 15 min each). Finally, the scaffolds were air dried, sputter coated with platinum, and imaged using a fully digital SEM (SU8010, Hitachi, Tokyo, Japan). 2.9 Statistical analysis Data is presented as mean ± standard deviation of independent replicates. Statistical analysis is conducted using ANOVA, and single asterisk (*) indicates significant differences between groups (p < 0.05), double asterisk (**) indicates significant differences between groups (p < 0.01), and NS indicates no significant differences between groups (p > 0.05).
3. Results and Discussion 3.1 Fabrication strategy of the multi-scale scaffolds As shown in Figure 2A, the designed multi-scale scaffold consists of thick fibers (macro-scale) as frame structures and thin fibers (micro-scale) as filling structures. The thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. To fabricate the designed multi-scale scaffolds efficiently and conveniently, a promising multi-scale direct writing system (MSDWS) is developed for printing the user-specific patterned multi-scale scaffolds using a single nozzle. The schematic of the MSDWS is shown in Figure 2B. By switching the electrostatic field, the FDM printing mode and EHD printing mode can be quickly switched. More specifically, when the high voltage generator was turned off, the MSDWS implemented the FDM printing process and thick fibers were printed using air pressure. When the high voltage generator was turned on, the MSDWS implemented the EHD printing process and thin fibers were printed using an electric force. Therefore, by controlled switching of the electrostatic field in real time, a user-specific patterned multi-scale scaffold was
fabricated. Additionally, by adjusting the printing parameters (printing speed, air pressure, and temperature), thick fibers ranging from 200µm to 600µm and thin fibers ranging from 3µm to 20µm can be obtained. Figure 2C and Video S1 (Supporting Information) shows the printing process. It was observed that the printing was stable and smooth, resulting in a scaffold with uniform thick/thin fibers. Herein, two cell culture models were used to investigate the influence of the multi-scale scaffold in regulating cellular behaviors. As shown in Figure 2D, interestingly, the cells bridged across the pores and formed a sheet in the 2D cell culture; the cells migrated from the hydrogel to the thin fibers in the 3D cell culture, which confirmed the excellent compatibility of the multi-scale scaffold.
Figure 2. Design and manufacturing of a multi-scale scaffold and its application for regulating cellular behaviors. (A) Schematic illustration of the design of the multi-scale scaffold; the thick fibers provide sufficient strength for mechanical support and the thin fibers provide a cell-favorable microenvironment for cell adhesion. (B) Schematic illustration of the multi-scale direct writing system (MSDWS) using on/off electrostatic field. (C) Printing process of the multi-scale scaffold. (D) The application of the multi-scale scaffold in regulating cellular behaviors.
3.2 Fabrication and characterization of the multi-scale scaffolds To confirm that the scale of the printed scaffolds ranged from the micro- to the macro-scale in the MSDWS, the effect of the printing parameters on the fiber size was investigated for the FDM and EHD printing, respectively. Since the resolution of FDM printing is limited by the nozzle size, the fiber diameter cannot be very small and is generally at the macro-scale (>100µm). In contrast, the fiber diameter of EHD printing is significantly smaller than the nozzle size and is at the micro-scale (<30µm), because the high electrostatic field causes the extruded polymer to form a Taylor-cone, followed by a thin jet ejecting from the cone. In the MSDWS printing process, the diameter of the printed fibers can be adjusted in the corresponding scale range and is affected by several parameters, including air pressure, temperature, and printing speed, as shown in Figure 3A. The effect of these parameters on thick and thin fiber diameters is shown in Figure 3B(I) and Figure 3C(I). The result indicates that the fiber diameter increased with increasing air pressure or temperature, whereas it
Figure 3. Characterization of the FDM and EHD printing process. (A) Schematic illustration showing the parameters influencing the fiber diameter, including air pressure, temperature, and printing speed. (B) (I) The effect of the printing parameters on thick fiber diameter in FDM printing, and (II) the thick fiber ranging from 180µm to 330µm obtained by adjusting the printing speed. (C) (I) The effect of the printing parameters on the thin fiber diameter in EHD printing, and (II) the thin fiber ranging from 2.48µm to 18.3µm obtained by adjusting the printing speed. (D) EHD printed scaffolds with complex structures, including (I) cobweb-like structure, (II) flower-like structure, and (III) snail-like structure.
decreased with increasing printing speed. This occurred because a higher air pressure directly increased the extrusion quantity and a higher temperature reduced the polymer viscosity to indirectly increase the extrusion quantity, resulting in an increase in the fiber diameter. In addition, the fiber was stretched and became thinner with increasing printing speed. In this study, the printing speed was used to adjust the fiber diameter. As shown in Figure 3B(II) and Figure 3C(II), the thick fiber size ranged from 180µm to 330µm and the thin fiber size ranged from 2.48µm to 18.3µm. The two printing processes should enable fabricating scaffolds with complex structures to provide better robustness and versatility of the MSDWS. FDM printing is a widely used technology and many strategies have been presented to fabricate complex structures [21, 22]. However, as an emerging technology, EHD printing can only print scaffolds with simple geometries such as grid structures [23, 24]. In this study, complex structures can be successfully printed by adjusting the printing speed in real time, such as cobweb-like structure, flower-like structure, and snail-like structure, as shown in Figure 3D. To determine the feasibility and stability of the MSDWS for fabricating multi-scale composite scaffolds, grid scaffolds were printed as an example by depositing a layer of thin fibers and covering them with a layer of thick fibers. Figure 4A shows a grid thick structure filled with thin fibers in a rectangle with 90° orientation. It can be seen that the EHD printed thin fibers adhered firmly to the thick fibers and were uniform with a homogeneous fiber space of 200µm. It has been reported that micro/nano fiber structures with different geometries and orientations in high-resolution scaffolds induce different cellular behaviors [25, 26]. Because the EHD printing is a
controllable high-resolution 3D printing method, we were able to control the shape and orientation of the fiber structures by adjusting the printing path of the substrate. As shown in Figure 4B and Figure 4C, a parallelogram with 45° orientation and an equilateral triangle with 60° orientation were successfully printed to fill the large pores. Additionally, when printing multilayered scaffolds, three types of thin fiber depositions were observed, including drooping fibers, straight fibers, and curving fibers, as shown in Figure S1 (Supporting Information). This occurred because a simple beam formed after the thin fibers were deposited onto the thick fibers and the printing speed and distance between the adjacent thick fibers affected the suspension. Figure 4D shows the effect of the printing speed and distance on the shape of the deposited thin fibers. By matching the printing speed and space, straight fibers were obtained, and they were desired to obtain multi-scale scaffolds with a controllable structure. Using the straight thin fibers, a multilayered multi-scale scaffold was printed, as shown in Figure 4E. The successful fabrication of these scaffolds demonstrates the stability and versatility of the MSDWS to fabricate morphologically controllable multi-scale scaffolds, which may offer potential applications in regulating cellular behaviors.
Figure 4. Fabrication of multi-scale scaffolds with the MSDWS. (A) The printed multi-scale scaffold with thin fiber structures of a rectangle with 90° orientation. (B) The printed multi-scale scaffold with thin fiber structures of a parallelogram with 45° orientation. (C) The printed multi-scale scaffold with thin fiber structures of an equilateral triangle with 60° orientation. (D) The effect of printing speed and distance on the shape of the deposited thin fibers. (E) The printed multilayered multi-scale scaffold.
To demonstrate the mechanical strength of the multi-scale scaffolds, compressive and tensile tests were conducted. Representative tensile and compressive stress-strain curves are shown in Figure 5A and Figure 5C. Notably, there was significant differences in mechanical properties between scaffolds of thin fibers and the others. The strength was mainly provided by thick fibers, so the curves of multi-scale scaffold and thick fiber scaffold were remarkably similar. The measurement of compressive stiffness and tensile modules showed the same trend (Figure 5B and
Figure 5D). Scaffold of thick fibers and multi-scale fibers had close results in both compressive (59.9±13.7KPa and 64.0±5.7KPa, respectively) and tensile (23.2±0.2MPa and 24.0±1.3MPa, respectively) test. However, the results of scaffold of only thin fibers were about two orders of magnitude lower, which were 0.66±0.06KPa and 0.16±0.07Mpa in stiffness and tensile modulus, respectively. It can be concluded that thick fibers significantly increased the mechanical strength of multi-scale scaffolds.
Figure 5. Mechanical characterization of scaffolds with different fibers. A) Representative tensile stress/strain curves of scaffolds with different fibers. B) Tensile modulus of scaffolds with different fibers. C) Representative compressive stress/strain curves of scaffolds with different fibers. D) Compressive stiffness of scaffolds with different fibers.
3.3 Biocompatibility analysis of the multi-scale scaffolds To confirm the enhanced biocompatibility of the multi-scale scaffold and its effect on regulating the cellular behaviors, bone marrow stem cells (BMSCs) were directly seeded on the multi-scale scaffolds. A schematic illustration of the cell growth process is shown in Figure 6A. In order to study the effect of the thin fibers on the biocompatibility of the multi-scale scaffolds, the
BMSCs seeded on the macro-scale scaffolds with pure thick fibers fabricated by FDM printing were regarded as the control group. The cell attachment, proliferation, viability, morphology, and interaction with the fibers were investigated during a 7-day culture period. As shown in Figure 6B, the cells adhered more readily to the multi-scale scaffold than the macro-scale scaffold after 4 h. This is illustrated in Figure 1. Furthermore, a cell proliferation analysis using cell counting kit (CCK)-8 assay was performed on day 1, 3, 5, and 7. As shown in Figure 6C, the proliferation rate of the BMSCs seeded on the multi-scale scaffolds was twice as high as that of the macro-scale scaffolds during the 7-day culture period, thereby further confirming that the addition of thin fibers was conducive to cell proliferation. Next, a cell viability analysis using live/dead assay was performed to further assess the biocompatibility of the multi-scale scaffolds. As shown in Figure S2 (Supporting Information), most of the cells were alive (green) and a few were dead (red). Additionally, cytoskeletal observations by F-actin staining were used to determine the changes in cell morphology to ascertain the cell growth and interaction with the scaffolds. As shown in Figure 6D and Figure S3 (Supporting Information), on day 1, the BMSCs changed from a spherical shape to an elongated shape and formed cellular junctions on the thin fibers. In the following days, interestingly, the BMSCs gradually bridged across the micro-pores and filled them to form a cell sheet on day 7. In contrast, the BMSCs on the macro-scale scaffolds exhibited less spreading and did not bridge across the large pores and did not fill them, as shown in Figure S4(Supporting Information). This occurred because the scale of the BMSCs (>200µm) was larger than that of the pores, resulting in the cells bridging across the pores. Furthermore, after the cells had filled the pores, they migrated to the thick fibers, continued to proliferate, and finally covered all the fibers of the multi-scale scaffold, as shown in Figure 6E and Video S2 (Supporting Information). And diameter difference of several micrometers in thin fibers would have an impact on cell phenotype which has been reported in our former study [27]. BMSCs were induced to oriented growth on scaffold with fiber diameters of ≈5µm and ≈20µm, but cells on homogeneous scaffold didn’t
exhibit the oriented growth, as shown in Figure S5 (Supporting Information).This result further demonstrated that the thin fibers greatly improved the biocompatibility of the printed scaffold.
Figure 6. Biocompatibility analysis of the printed multi-scale scaffolds. (A) Schematic illustration of the BMSCs seeded and cultured on multi-scale scaffolds. (B) Comparison of cell adhesion between multi-scale scaffolds and macro-scale scaffolds. (C) Comparison of cell proliferation between multi-scale scaffolds and macro-scale scaffolds. (D) Cell morphology change and interaction with the multi-scale scaffolds on day 1, 3, 5, and 7. (E) Cells migrating to the thick fibers and covering all the fibers of the multi-scale scaffold.
3.4 Application of the multi-scale scaffolds in hydrogel-based 3D cell culture Recently, hydrogel-based 3D cell cultures have attracted increasing attention in tissue engineering since hydrogels provide a 3D environment, mimicking an ECM and promoting nutrition exchange, which is close to native tissue. However, hydrogels are intrinsically soft and have poor mechanical strength, which limits their application in long-term cell culture. To overcome this challenge, fiber reinforcing methods have emerged as a promising strategy to increase the mechanical strength as much as dozens of times [28, 29]. Therefore, in this study, to assess whether our printed multi-scale scaffolds could also be integrated with hydrogel-based cell culture, we indirectly seeded cells on the multi-scale scaffolds by encapsulating the cells with a hydrogel. Here, a gelatin methacrylate (GelMA) hydrogel was selected to encapsulate the cells because it not only has excellent biological performance (further confirmed in Figure S6 (Supporting Information)) but also easily crosslinks upon light exposure [30, 31]. In the GelMA-based 3D cell culture experiment, the BMSCs were first mixed uniformly with the GelMA solution and then the cell-laden GelMA solution was dropped into the multi-scale scaffold, this was followed by photo-crosslinking the GelMA to obtain structure similar to reinforced concrete. The schematic illustration of the cell growth process is shown in Figure 7A. As shown in Figure 7B, the BMSCs were evenly distributed in the reinforced structures, and the fluorescent images show that almost all of the cells were alive (green) and were spherically shaped on day 1. Subsequently, the BMSCs began to elongate (Figure 7C), and several cells began to migrate to the thin fibers over time (Figure 7D and Figure S7 (Supporting Information)), this can be clearly seen in Video S3 (Supporting Information). This behavior demonstrated that the printed multi-scale scaffolds could also be used in a 3D cell culture and regulated the cellular behaviors in a manner that was different from that of pure hydrogel-based cell culture. In future studies, controllable cell deposition based on these multi-scale scaffolds/GelMA structures may be achievable by integrating 3D bioprinting technology into the MSDWS [32, 33]. Thus, multi-scale and multi-material structures could be
printed in one step, the macro-scale polymer structures would provide mechanical support, the micro-scale polymer structures would provide a fiber-like microenvironment, and the hydrogel would provide a 3D microenvironment.
Figure 7. 3D cell culture based on “reinforced concrete” structure combined with the GelMA hydrogel. (A) Schematic illustration of the BMSCs seeded and cultured on the “reinforced concrete” structure. (B) Fluorescent images of live/dead staining showing the spherically shaped BMSCs on day 1. (C) Fluorescent images of f-actin/nuclei staining showing the stretched BMSCs on day 7. (D) Fluorescent images of f-actin/nuclei staining showing the migration of the BMSCs to the thin fibers.
4. Conclusion In conclusion, we have developed a novel high-resolution MSDWS that enables the fabrication of multi-scale scaffolds using a single nozzle. The multi-scale scaffold consisted of thick fibers (macro-scale) as the frame structures and thin fibers (micro-scale) for filling the frame; the thick fibers were able to provide sufficient strength for mechanical support and the thin fibers provided a cell-favorable microenvironment for cell adhesion. Through a systematic study of the direct writing process, we have confirmed the feasibility and stability of the MSDWS for fabricating multi-scale composite scaffolds. An improved biocompatibility of the multi-scale scaffold and its effect on regulating the cellular behaviors were also demonstrated by a comparison with macro-scale scaffolds. Furthermore, we successfully combined the multi-scaffolds with a hydrogel for 3D cell culture to enhance the strength and induce cell migration. We believe that the MSDWS will become a generalized 3D printing strategy that can be extended to enhance the biocompatibility of other conventional scaffolds by filling them with ultrafine fibers.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
The authors would like to acknowledge the Testing Center in Suzhou Intelligent Manufacturing Research Institute for providing SEM, confocal and mechanical testing. This work was sponsored by the National Nature Science Foundation of China (No.51805474, 51622510, U1609207, 51605429), the Science Fund for Creative Research Groups of National Natural Science Foundation of China (No. 51821093), and China Postdoctoral Science Foundation (Grant No. 2019T120509).
References
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Highlights： ： (i) A multi-scale scaffold was designed to balance mechanical strength and biocompatibility of 3D printed scaffolds (ii) A promising multi-scale direct writing system (MSDWS) was developed for printing the multi-scale scaffolds. (iii) By filling ultrafine fibers, the biocompatibility of the scaffold was greatly improved.