Nanoscale Resolution 3D Printing with Pin-Modified Electrified Inkjets

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Biological and Medical Applications of Materials and Interfaces

Nanoscale resolution 3D printing with pin-modified electrified inkjets for tailorable nano/macro hybrid constructs for tissue engineering Jeong In Kim, and Cheol Sang Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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ACS Applied Materials & Interfaces

Nanoscale resolution 3D printing with pin-modified electrified inkjets for tailorable nano/macro hybrid constructs for tissue engineering Jeong In Kim1, Cheol Sang Kim1,2* 1

Department of Bionanosystem Engineering, Graduate School, Chonbuk National University,

Jeonju 561-756, Republic of Korea 2

Division of Mechanical Design Engineering, College of Engineering, Chonbuk National

University, Jeonju 561-756, Republic of Korea

*Corresponding author: Tel.: +82-63-270-4284; fax: +82-63-270-2460. E-mail: [email protected] (C. S. Kim)

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ABSTRACT The cells respond to their microenvironment which is a comparable size to the cells. The macroscale features of three dimensional (3D) printing struts (PS) typically result in whole cell contact guidance (CCG). In contrast, at the nanoscale, where features are similar size to receptors of the cells, the response of cells is more complex. The cell-nanotopography interaction involves nanoscale adhesion localized structures which include cell adhesionrelated particles that change in response to clustering of integrin. For this reason, it is necessary to develop a technique for manufacturing a tailorable nano/macro hybrid constructs capable of freely controlling the cellular activity. In this study, a hierarchical 3D nano to microscale hybrid structure was fabricated by combinational processing of 3D printing and electrified ink jets spinning via pin motions. This method overcomes the disadvantages of conventional 3D printing, providing a novel combinatory technique of the fabrication of 3D hybrid constructs with excellent cell proliferation. Through a pin-modified electrified inkjets spinning, we have successfully fabricated customizable nano-microscale hybrid constructs in fibrous or mesh form, which can control cell fate. We have conducted this study of celltopography interactions from the fabrication approach to accelerate the development of nextgeneration 3D scaffolds.


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Keywords: 3D printing; pin motion electrospinning; 3D scaffold; biomedical technology; tissue engineering Introduction Cells respond to nanoscale features, with alterations in cell behavior, proliferation, and differentiation mediated by changes in cell adhesion

1-3

. Furthermore, the interaction of

integrin (in the focal adhesion) with a 3D and nanotopographical features changes how the cells attach to surface of the 3D scaffold and influence cell behavior through alterations in both cell morphology and biochemistry 4. Harnessing topography and controlling a diameter at nano to macro scale of a 3D scaffold to influence cell fate can be crucial to sustain an environment capable of better cell growth

5-7

. For these reasons, the 3D tissue engineered

scaffolds need to mimic both mechanical properties and biological function of the extracellular matrix (ECM) in order to induce tissue regeneration

8-10

. It also requires high

diffusivity in 3D scaffolds to promote mass transport of nutrients, oxygen, and proteins 11. A modern technique for the production of 3D scaffold that meets the requirements is 3D printing

12-13

. 3D printing is a computer-aided layer by layer fabrication method that can

reconstruct 3D model of the defective region14. Not only has this technology notably improved the quality of the structure of scaffold, but it also has the advantage of possibility of a variety of material selection15. 3D printing technique has obvious benefits such as the elimination of solvent removal processing or the possibility of various material selections, but they have limitations like large pore size for use as a regeneration scaffold due to the limited print resolution

11, 16

. To overcome this problem, research is underway to combine the

technology with conventional technologies such as electrospinning (ES)

11, 17

. Recently, in

this way, 3D printing technology has been combined with ES technique to make a scaffold that implements the benefits of various materials in one structure. ES technique has been considered as useful method to fabricate fibrous and highly porous scaffolds that have ability



to produce ECM-like structure

18-20

. This technique allows fabricating non-woven matrix

containing nano to micro-sized fibers with desired mechanical and physical features 21. Also, it has been reported that the diameter and pore size of electrospun fibers affect important aspects of cell behavior, including attachment, migration, proliferation, and differentiation 19, 22-23

. Furthermore, a method of functionalizing electrospun fibrous matrixes with active

compounds, such as ECM protein collagen, cadherin, and laminin, etc., have made it possible to regulate cell attachment and growth

24

. However, conventional ES generally fabricates

tightly packed and dense meshes that allow cells to proliferate only on the two dimensional (2D) surface, without permitting full infiltration of the cells in 3D 16. The techniques for the production of highly porous and stable fibrous structures are needed for further study of cell cultures in 3D

25

. Although many methods have been proposed, they have not resulted in

satisfactory cell penetration into the 3D electrospun fibrous scaffolds. Also, the 3D electrospun fibrous scaffolds fabricated by conventional ES techniques have disadvantages in that they cannot maintain their shape well in a liquid state such as cell media or in vivo. For this reason, studies have been conducted combining other technologies such as 3D printing to develop a 3D scaffold that overcomes the drawback of existing ES techniques11, 16, 26. The combination of ES technique and 3D printing was firstly described in an effort to make bioengineered scaffolds, where nano to macro scale constructs are combined 26. Moroni et al., Kim et al. and Park et al. described a fused manufacturing process (FMP) which was interrupted between each layer to deposit electrified ink jet fibrous structure27-30. The resultant scaffolds contained large sized pores, while the electrified ink jet fibers provided suitable constructs for better cell adhesion, migration, and proliferation; effectively increasing the surface area to volume ratio available for the penetration of the cells to adhere to30. In addition, Yang et al. have reported the direct electrospinning writing for producing 3D


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structures consisting of microfibers and macro-scale mesh16. The width of the electrospun fibrous membrane in the 3D matrix was reasonably controllable, that resulted in enhanced cell adhesion, infiltration, and proliferation. The FMP trend in the scaffold fabrication methods has come about because of the complexity of the native tissue which many researchers are aiming to replace with tissue engineering. In the field of materials science, a variety of applicable biomaterials have been identified, ranging from natural to synthetic polymers that are inexpensive, highly soluble, biocompatible, and the polymers have unique chemical structures providing specific function. Native polymers have excellent biocompatibility, but poor mechanical properties. Thus, when fabricating functionalized electrospun nanofibers having improved characteristics, they need to be prepared in combination with synthetic polymers. Among these synthetic polymers, polycaprolactone (PCL) is a hydrophobic and semi-crystalline thermoplastic polyester and has been approved by the Food and Drug Administration (FDA) in the field of human health. PCL has been extensively studied as a bioinspired material capable of targeting selective cellular responses via controlled intracellular absorption pathway due to prolonged degradation and faster absorption. Also, compared to other aliphatic polyesters, the excellent viscoelastic and rheological characteristics make it easy to fabricate and manipulate PCL in an electrospinning process and 3D printing. Here, we explored a fabrication method of nanoscale resolution 3D printing with pinmodified electrified inkjets for tailorable nano/macro hybrid PCL constructs. We aimed to develop a hierarchical 3D hybrid structure consisting of highly ordered or patterned nanomicrofibers and macro structs with good biological function. Through the pin-modified electrified inkjets spinning, we have successfully fabricated tailorable nano to microscale hybrid constructs in mesh or fibrous form, which could control the cell fate. The electrospun



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fibers were fabricated and interlayered to overcome the drawback of the low resolution of the existing 3D printing and to increase initial cell adhesion, proliferation, and the area to volume ratio. The developed novel hybrid constructs showed adequate physical properties and significantly enhanced responses of the MC3T3-E1 cells compared to the conventional 3D printed scaffold. Results and Discussion The macro to microscale features of the scaffold could result in whole cell contact guidance. However, the nano-sized surface features are several orders of magnitude below that of the cell size, the response could be more sophisticated and complex4. At the nanoscale, where surface features are a comparable size to the cell receptors involved with cell adhesion such as integrin, talin, and vinculin, it might be possible to control the cell response or fate. Cell adhesion molecules (CAMs) are proteins located at the cytoplasmic face involved in binding with the extracellular matrix (ECM) in the cell adhesion process as shown in Figure 1. The focal adhesion portion comprises a signaling layer consisting of the cytoplasmic tails of the integrin, paxillin, and focal adhesion kinase (FAK); an intermediate stratum related to the transduction of forces including vinculin and talin; and actin regulatory surfaces containing zyxin and vasodilator-cstimulated phosphoprotein (VASP) (Figure 1)

4, 31

. Because the

cellular adhesion to nanoscale surface features involve nanoscale adhesion localized structures, nanotopography, which mimics the complicated ECM structure of the scaffold could be an important factor for cell activity. Fabrication of 3D hybrid constructs with pin motioned electrospun nanofibers (3D HCPMNF)


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The illustration and the digital photos of the combinational fabrication processing consisted of a 3D printing technique coupled to the pin motion electrospinning (PMES) were shown in Figure 2. The fabrication method adopted here is composed of an ES technique in which the electric field is specially controlled by using a moving pin collector while performing 3D printing (Figure 2), thereby making it possible to fabricate a nano to macroscale scaffold at one time. Depending on the number, location, and distance of the metallic pins, electrospun fibers were collected in different forms as shown in Figure 3a. Also, when pins are separated by more than 3.2cm, the electrospun fibers between the pins were not connected to each other (Figure 3a). The end of the pins was connected to opposite charge of a needle tip and the residual charges of the opposite polarity on the electrospun fibers get attracted toward the metallic pins during electrospinning. Recent studies have demonstrated that use of pin-shaped electrodes with small radius (R) values resulted in high charge accumulation and the convergence of electric field lines toward the edge of the pins. The charge density (σ) is given by σ = Q/4πR2 (Q, charge; R, radius) which was also given by John et al 32. This process demonstrated produces a hierarchical 3D hybrid structure consisting of nano-microscale fibers and macroscale meshes due to the motion pin collector that created a high charge accumulation, which gathered the fibers effectively. The design is unique compared to the geometry obtained using an immovable pin collector with point electrodes. As shown in Figure 3b, any morphology could be designed and fabricated with nanofibrous mesh form or controllable patterned nanofibers by adjusting the position and number of pins. After the fabrication of the designed nanofibers, the pin with fibers enters the inside of the collector (as presented in Figure 3c) and the fibers are collected on the collector or 3D printed meshes. The supporting information video also shows the



combined manufacturing process consisting of 3D printing and PMES as the animated version of Figure 2. In this study, the morphology, dynamic water wetting ability, and protein adsorption of the samples were characterized, and in-vitro studies were performed for the evaluation of the cell viability of the scaffold by culture MC3T3-E1 cells. The biocompatible and biodegradable polymer, PCL struts were printed in 3D form and PCL solution was electrospun to obtain 3D hybrid structure consisting of nano-microfibers and macro meshes. Morphology of 3D HC-PMNF and FFT analysis This study focused on the design of tailorable nano to macro hybrid constructs which influence cellular activity. The hybrid construct (3D HC-PMNFs) were designed by employing a 3D printing and pin-modified electrified ink jet spinning. This scaffold was comprised of an electrospun fibrous layer and 3D printed meshes. The schematic illustrating generation of the 3D hybrid constructs with pin motioned electrospun nanofibers (3D HCPMNF) is shown in Figure 4a. When two pins come out in a straight line, the nanofibrous thread consisting of microfiber was generated. However, if more than four pins come out, the inner part of the pins was coated with aligned nanofibers (Figure 4b). Also, it was confirmed that randomly oriented nanofibers were collected when a 3D printed mesh was electrospun without a pin (Figure 4b). This fabrication method can overcome the low resolution of the 3D printing by controlling the nanofibers with motioned pin collectors. In addition, it is possible to coat only the desired site with nanofibers or a nanofibrous thread consisting of microfiber, which has a great advantage in that the infiltration and alignment of the cells could be locally controlled. Figure 5a represents the SEM images of the 3D PS and 3D hybrid constructs with fibers fabricated by conventional electrospinning (HC-CNF) or pin motioned electrospinning (HC-PMNF). When pin-motion electrospinning is performed on a 3D PS with two pins (3DPMNF-2P), the electrospun fibers are fabricated in the form of aligned and stack together, but


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with four pins (3D-PMNF-4P), the neatly oriented electrospun fibers are ordered and cover the entire 3D PS as shown in Figure 5a. However, only randomly oriented fibers were observed in 3D HC-CNF. The anisotropy of the electrospun fibers was assessed through the fast Fourier transform (FFT) analysis method33. The plots of FFT function have been used to identify the geometric composition of various images. It is possible to extract the information of the SEM images and use the information to assign an objective numerical value to the anisotropy of electrospun fibers by the image processing with FFT function33-34. From the FFT result, the effect of the convergence of the electric field line toward the edge of the pins was investigated. The aligned fibers generate elliptically and ordered distributed gray pixels; in contrast, randomly oriented fibers generate symmetrically and circularly distributed pixels in the output image (Figure 5b)

34-35

. The plot between 0 and 360° was drawn by the pixel

intensities summed along each degree and the height of the peak in the plot reflected the alignment level of the electrospun fibrous samples (Figure 5c). Characterization of 3D PS and 3D HC-PMNF Hydrophilicity of the scaffold is crucial for the facilitation of biological cell activities, including cell adhesion, migration, and proliferation

36

. A water contact angle tests were

performed to evaluate the hydrophilicity of the 3D PS, 3D HC-CNF, and 3D HC-PMNF. The dynamic water contact angles of 3D PS, 3D HC-CNF, and 3D HC-PMNF after placement of a red dyed water droplet (30 µl) onto the sample surfaces as shown in Figure 6a. The 3D HCCNF and 3D HC-PMNFs showed more rapid wetting ability compared to the 3D PS. The water contact angles for the 3D PS, 3D HC-CNF, 3D HC-PMNF-2P, and 3D HC-PMNF-4P were 98 ± 3°, 62 ± 4°, 81 ± 4°, and 74 ± 4°, respectively. In addition, a much wider water filtration and distribution was shown on the 3D HC-CNF and 3D HC-PMNF because of the interlayered nano-microscale fibers. The volume of spread wetted water on the 3D HC-



PMNF-2P (13.82 mm3) was less than the 3D HC-PMNF-4P (21.95 mm3) because the red dyed water drop wetted differently on the electrospun fibrous mesh and fibers. The high capacity of water promotes cell proliferation, growth, and enhanced nutrient transport16, 37. Every sample was soaked in water for 12 hours and differences in the weight were determined before and after soaking. The water absorption capacity of the 3D HC-CNF and 3D HC-PMNFs was prominently higher than the 3D PS because of the presence of fibrous mesh or fibers (Figure 6b). Furthermore, protein absorption can be a crucial factor for the better cell adhesion, migration, and proliferation10, 16. As shown in Figure 6c, the 3D HCCNF and 3D HC-PMNFs showed a higher protein absorption capacity compared to 3D PS. The 3D HC-PMNF (four pins) showed the highest protein absorption capacity, likely due to the greater surface-area-to-volume ratio of covered aligned oriented fibers on the 3D HCPMNF. Porosity and pore size Porosity and pore size of biomaterial scaffolds play a critical role in tissue formation in vitro and in vivo. Each 3D sample was composed of a printed PCL meshes and electrospun fibers with a diameter of 220 ± 20 µm and 7.0 ± 1.5µm, respectively as shown in Table 1. The 3D hybrid construct (3D HC-CNF and 3D HC-PMNF) included an additional fibrous mesh or fibrous layer generated using PMES. Randomly oriented electrospun fibers completely cover the 3D HC-CNF. However, the 3D HC-PMNF-2P included an aligned fibrous mesh and 3D HC-PMNF-4P was uniformly and broadly covered with aligned fibers. As shown in Table 1, the electrospun fibers covered on the 3D HC-CNF had a fiber diameter and pore size of 7.0 ± 0.4 µm and 11.0 ± 2.4 µm, respectively. The 3D HC-PMNFs have two different styles of pores (nano-microscale pores generated by electrospun fibers and macro scale pores generated by 3D printed meshes). For these reasons, the nano to microscale pores


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can promote cell adhesion, migration, and proliferation; also, the macro-sized pores can effectively enhance cellular infiltration and transport of oxygen and nutrient. Thus, the proposed nanoscale resolution 3D printing with pin-modified electrified inkjets for tailorable nano/macro hybrid constructs could overcome the drawback of the conventional 3D printing, in which cultured cells can only result in whole cell contact guidance (CCG) 4. In addition, the developed 3D HC-PMNF scaffold can result in more complex cell responses at the nanolevel, which is similar in size to the receptors of cells. Cell morphology, viability, and infiltration Integrin, one of the cell receptors involved in cell adhesion, interacts with the 3D condition and nanotopographical features to alter the way cell attach to the surface of a scaffold and to influence cell behavior through changes in cell biochemistry and morphology 4, 38-39

. Control of the diameter at nano-to-macroscale and harnessing surface patterns of a

scaffold to affect cell fate can be important in maintaining an environment capable of better cell proliferation

7, 40

. The proposed fabrication method is combination process of an

electrospinning in which the electric field is controlled by using motion pins as a collector while performing 3D printing, thereby making it possible to fabricate a nano to macroscale scaffold. The surface features of the 3D HC-PMNF might provide a supportive condition for better cellular behavior because the integrin clustering results in the formation of focal adhesion with integrin interspacing of the nanoscale feature as shown in Figure 7a 5. The 3D PS and 3D HC-PMNF were prepared for 3D cell culture and the MC3T3-E1 cells (preosteoblast) were seeded at a density of 2×104cells in 100 µl of culture medium and incubated 30 minutes for uploading the seeded cells on the samples. The morphology of MC3T3-E1 cells cultured on the samples was examined using SEM and confocal laser scanning microscope. As shown in Figure 7b, the MC3T3-E1 cells cultured on 3D PS migrated a gap



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larger than the size of the cell to move from a strut to another strut. In contrast, the cells on the 3D HC-PMNF showed that nanoscale fibers act as connection mediators and migrate a short distance the cells were attached to a large area. The cellular adhesion on the macro to microscale is made from a lot of integrins on the nanoscale

40-41

. When the cells are

considered as a tent, the larger the tent, the larger pegs (the clustering of integrins) is needed and the tension of the rope should be maintained appropriately to cope with the deformation of the rope 4. Confocal laser scanning images also show the nanoscale fiber-cell and micro to macroscale strut-cell interactions on differently coated 3D PS depending on the number of moving pins. The aligned nanofibers in a larger area in the case of more than four pins were evenly coated in plate form, which also affected the alignment of the cells (Figure 8a). There is much evidence that the cytoskeleton is associated with nanotopography (and other material related) signaling transduction events and the change of cytoskeletal morphology are required for indirect alteration in biochemical mechanotransduction

38

. Figure 8b shows an enlarged

image of the cells on the 3D PS and 3D HC-PMNF. The MC3T3-E1 cells on the fibers were more spindle shape, elongated, and oriented. Also, from the cell orientation graph, the degree of cell alignment induced by anisotropic electrospun fibrous scaffolds was confirmed. Alignment of bone cell or bone marrow stem cell (BMSC) and ECM is closely related to the anisotropic mechanical properties of bone. In addition, alteration in cytoskeletal tension along the topography of the scaffold will change cells’ morphology, nucleus, gene transduction, and chromosomal arrangement

38, 42-43

. For these reasons, an intact scaffold which promotes the

proliferation, mineralization, and differentiation of osteoblasts or BMSCs in the desired direction promise in the generation of biomimetic bone tissue 40. Nakayama et al. fabricated highly aligned nanofibrous scaffold to enhance the angiogenic capacity of endothelial cells (ECs) using nanotopographical cues from aligned nanofibers in the setting of tissue ischemia 39

. Xing et al. reported that the aligned nanofibrous ECM scaffold induces a significantly


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lower immune response compared to its randomly oriented counterpart detected by the inflammatory cytokines secreted by macrophages 8. There is a wealth of evidence that the orientation of neuronal cells, bone cells, and stem cells etc. has a positive effect on tissue regeneration. The 3D HC-PMNF manufacturing method developed through this study would hold great potential in engineering organized tissues. The cytotoxicity of the 3D PS and 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS) were measured using the cell counting kit-8 after 12h, 24h, 48h, 72h, and 120h of cell culture. After culturing the MC3T3-E1 cells, at desired points in time, the CCK-8 solution (60µl) was added into the wells. Every sample with the CCK-8 solution was incubated for 4 hours in an incubator with 5% CO2 at 37 °C. The measurement of the absorbance of each sample (100µl) was checked at 450 nm using a microplate reader (Tecan, Austria). The results were presented as the mean ± standard error of the mean. The cell growth on the 3D HC-PMNF tends to be faster and vigorous compared to the 3D PS as shown in Figure 9a. In addition, the number of MC3T3E1 cells was determined using a Quant-iTTM PicoGreenTM dsDNA Assay Kit (Life Technologies, USA). The fluorescence of Picogreen with a nucleic acid strain was evaluated to quantitate the dsDNA in a sample. For the measurement of the DNA content, the samples with the MC3T3-E1 cells were harvested after 24h, 48h, 72h, and 120h of cell culture. As a result, the DNA content on the 3D HC-PMNFs was higher than that on the 3D HC-CNF (Figure 9b). The amount of aligned fibrous material deposited varies depending on the number of motion pins. As the amount of aligned fibers deposited by PMES increased, the number of cells increased. The MC3T3-E1 cell number in terms of DNA content on the 3D PS was significantly lower than that on the other samples on which fibrous layers were deposited by PMES or conventional ES as shown in Figure 9b. The MC3T3-E1 cells on 3D HC-PMNF were observed to be more elongated cytoskeleton and the cell length was longer compare to the cells on 3D PS (Figure 9c). Cellular infiltration into the 3D HC-PMNF was



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assessed using an image processing program of confocal Z stacking with the DAPI and actin green stained cells. Each individual stack was subjected to spectral color coding steps using a function of Z stacking to gather the images with color-coded MC3T3-E1 cells. The MC3T3E1 cells cultured with 3D PS and 3D HC-PMNF appeared to be well attached to the surface of the fibers and struts, and z staking results confirmed that the cells were uniformly distributed and infiltrated from top to the bottom of the 3D scaffold (Figure 9d). In-vitro Immunohistochemical examinations In the immunohistochemical study, the distribution and localization of cell adhesion molecules markers, e.g. F-actin and Integrin, were compared between the 3D PS and 3D HCPMNF. Integrins mediate cell-cell interaction through cell adhesion molecules and they interact with various proteins like receptors, growth factors, and proteolytic enzymes. Also, the formation of granulation tissue and re-epithelialization are dependent on the function of integrins44-46. Actin is a globular multifunctional protein that forms microfilaments and is found in eukaryotic cells at a concentration of 100 µM or more43,

47-48

. Linear polymer

microfilaments, called F-actin, are essential for important cell functions such as cell mobility, contraction, and cell division31, 47. For these reasons, examination of the distribution of cell adhesion molecule markers in immunohistochemical studies is an important factor in evaluating cell behavior, activity, and proliferation in the scaffold. In this study, we investigated the 3D HC-PMNF has advantageous properties for tissue engineering, including a high surface area to volume ratio for cell attachment, migration, proliferation, and the possibility to functionalize the polymer chains with cell-adhesive peptides and natural components of the ECM for the enhanced bioactivity49-50. In the SEM and confocal images, it was demonstrated that the cultured MC3T3-E1 cells aligned parallel to the fibers on the 3D HC-PMNF, while the MC3T3-E1 cells spread in all directions on the 3D PS. In addition, in


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immunohistochemical staining results, on the 3D HC-PMNF, the cells on ordered nanomicrofibers adopted an elongated osteoblastic phenotype, with the cells aligning parallel to alignment of the fibers and the higher and denser F-actin (red) and integrin (red) expression on 3D HC-PMNF was observed in immunohistochemical examinations as shown in figure 10 a-d. Focal adhesion has been studied as the initiation point of the interaction of cell to the surface of scaffolds. The use of physical or topographical cues to control cell adhesion would change biochemical signals that would affect the phenotype of the cell4. The biochemical signals conversion derived from alteration in intracellular tension to phenotypic effects could be explained by an indirect mechanotransduction4. Collagen which has a triple helical structure plays a dominant role in maintaining the structural integrity of ECM and the biological functions51-52. The ideal goal of tissue engineering is to restore the vivid remodeling process of native ECM and the structural integrity, especially restoring the delicate networks of collagen fibrils under which normal physiological regeneration occurs37, 39, 53. To date, 28 types of collagen have been identified; I, II, III, and V are the major types which make up the essential part of collagen in cartilage, bone, tendon, muscle, and skin54. Among the various types of collagen, collagen I is the most abundant collagen type in the human body that forms large and eosinophilic fibers55-57. In the immunohistochemistry studies, the MC3T3-E1 cells on the 3D HC-PMNF adopted an elongated and ordered parallel to the alignment of the fibers and the slightly denser collagen I (green) expression was observed (figure 10 e and f). Most of the ECM is made out of aligned collagen fibrils, this has led to many studies on the effect of the scaffold with ordered topography on the various cell types58-61. The MC3T3-E1 cells on the 3D HC-PMNF expressed higher levels of cell adhesion molecules markers and increased the deposition of



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collagen. This experimental results gave us an idea of how the cells would behave in contact with the 3D HC-PMNF before proceeding to the in-vivo experiment. In vivo study The MC3T3-E1 cell loaded 3D PS and 3D HC-PMNF samples were implanted under subcutaneous region of 10 nude mice. The mice were sacrificed at week 3 for the investigation of the biocompatibility and inflammatory response of the 3D PS and 3D HCPMNF. The 3D samples were processed for histological examination with H&E and collagen I (Figure 11). The H&E staining images showed cell distribution and well-defined construct. The 3D HC-PMNF implanted group showed the denser cell distribution versus the 3D PS group in H&E staining result as shown in Figure 11. For the acceleration of tissue healing, the formation and maturation of the granular tissue could be a significant factor. The group with 3D HC-PMNF showed a much faster formation of the granular tissue compare to the control group (3D PS) as shown in the H&E stained cross-sectional scaffold with integrated tissue for the histopathological analysis as shown in Figure 11 a-b. In addition, the collagen arrangement was investigated using a collagen I staining for the evaluation of the maturity of collagen. Compared with the 3D PS group, the 3D HC-PMNF group showed ordered collagen parallel to fiber alignment and slightly higher expression of collagen I. The newly formed collagen fibrils appeared in both 3D PS and 3D HC-PMNF groups but the arrangement and maturity of collagen were significantly different in the groups (Fig. 11 c and d). As expected, the 3D HC-PMNF group showed enhanced biocompatibility in vivo.

Also,

there was no significant inflammatory response between the 3D HC-PMNF group and their surrounding tissues during the in vivo experiment.

Conclusion


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In conclusion, we have developed a hierarchical 3D hybrid constructs consisting of macro-sized struts with nano to microscale fibers were fabricated by combinational processing of 3D printing and electrospinning applying a moving pin as a collector. The focus of this work is to overcome the limitation of the conventional method of 3D printing technique and develop a new manufacturing paradigm for producing a scaffold with adjustable pore size, diameter, and design. Macro to microscale feature of a similar size to cell itself resulted in whole CCG, however, at the nanoscale, where the feature is a comparable size to the individual cell receptor, it is possible to manage the cell response and target receptor-driven pathways. In addition, the topography of the scaffold also has a great influence on cell attachment, activity, and differentiation, etc., because of the integrin-ligand binding result in contraction of the adhered cytoskeleton through some biochemical pathways. Therefore, the nanoscale fibrous scaffolds collected on the moving pins of the technique developed in this study influence the expression of integrins, the contraction, and alignment of the cytoskeleton. This scalable fabrication approach has a number of possible biomedical microdevice technology and biological applications in the fabrication of a tissue-engineered scaffold.

Experimental Section Materials For the development of hierarchical electrospun nano-micro fibrous scaffold, PCL pellets (Mw = 90,000g mol-1) were purchased from Sigma-Aldrich, Korea. PCL filament for 3D printing was obtained from Glo-one (Sejung, South Korea). 5g PCL pellets were dissolved in a solvent of 45g 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) with continuous magnetic stirring



for making 10 wt% PCL solutions. HFIP was purchased from Sigma-Aldrich, Korea. 12ml syringes and twenty-gauge syringe needles were obtained from NORM-JECT®. All of the cell culture reagents, such as Alpha Minimum Essential Medium, trypsin, fetal bovine serum (FBS), and streptomycin/penicillin were purchased from Hyclone (Logan, UT, USA). Fabrication of the 3D printed struct (3D PS) The control 3D constructs comprised of perpendicular macro scale PCL meshes were fabricated via a 3D printer (Cubicon 3DP-110FAC). The heat bed, replaceable extruder with a nozzle, three-axis stage, and heat circulation controller were performed with the software (Cubicreator v3.0). The PCL filaments (1.75mm) were melted at a temperature of 180°C in a heating dispenser and the struts were extruded on the plate for fabrication the 3D constructs consisting of macro-sized meshes. The PCL filament was constantly pressurized at 250 ± 10 kPa and the level of configuration layer of this 3D printer is 100-300 microns with 0.4mm nozzle size. The diameter of printed PCL wall is around 220 µm and this micro-sized PCL structure was deposited in a layer-by-layer manner. Fabrication of the 3D HC-PMNF The 3D hybrid constructs (HC) consisting of nano-microfibers and macro meshes were fabricated via combinational processing: a 3D printing and pin-modified electrospinning (PMES). Firstly, the PCL filament was extruded on the plate after melting in a heating dispenser and the printed scaffold comprised of perpendicular struts was fabricated. And then, the pins come out of the plate holes, exist the sides of the printed constructs, and induce the electrospun fibers with the desired patterns to collect during electrospinning. The pins from the plate act as collectors. Depending on the number and the distance of pins, nano to microscale fibrous constructs of various designs are fabricated. To develop a nanoscale resolution 3D printing with pin-modified electrified inkjets for tailorable nano/macro hybrid


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constructs, we have made and put a number of electrically connected pins below the collector hole to match the number of the pins to fabricate the scaffold. Because there are many pins below the collector holes, pins can be inserted at desired positions, such as the periphery or inside of the 3D printed strut. After the pin is moved upwards and the electrospinning begins, nano-sized fibers or meshes are fabricated. The pins collect the desired amount of electrospun fibers and then go back into the holes of collector. The PCL solution in a 12ml syringe was extruded through a movement of twenty-gauge needle at a flow rate of 0.4ml/h by a syringe pump. PMES was carried out with a high DC voltage (15kV) at a temperature of 25°C, a humidity of 50%, and a needle-to-collector distance of 10cm. Finally, the hierarchical 3D hybrid structure consisting of macro meshes and nano-microfibers was fabricated by repeating 3D printing and PMES. The hierarchical 3D hybrid constructs were dried in a vacuum oven for a day at 40˚C. Characterization of the 3D PS and 3D HC-PMNF The morphology of the 3D PS and 3D HC-PMNF was examined using scanning electron microscopy (SEM, Hitachi S-7400, Hitachi, Japan) and field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). The samples were dried completely using the vacuum oven at 40°C for 48 hours before the observation. Porosity (%) of the samples was measured using this equation, [1-M/ (ρV)] *100, where M is the mass of the sample (V is the scaffold volume, and ρ is the density of PCL). The pore size of the samples was measured using SEM images (with a magnification of 1000×) taken from ten portions of each sample. The SEM images were processed using a software program of Image J to calculate the average pore size. The water absorption abilities of the 3D samples were calculated as (%) = (W12h – W0)/ W0 * 100, where W0 is the scaffold weight before soaking and W12h is the scaffold weight after 12 hours soaking in distilled water. A bicinchoninic acid



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(BCA) kit (Pierce Kit; Thermo Scientific, Waltham, MA, USA) was used for the measurement of the protein absorption ability. The 3D PS and 3D HC-PMNF were placed in 24-well

plate

with

minimum

essential

medium,

supplemented

with

1%

penicillin/streptomycin and 10% fetal bovine serum (FBS). Every sample was incubated with 5% CO2 at 37 °C, washed with PBS, and lysed with 0.1% Triton X-100. A 25 µl of lysate aliquot was added to 200 µl of BCA working reagent. The absorbance at 562nm was determined using a plate reader after the mixture was incubated for 30 minutes with 5% CO2 at 37°C (Samples incubated in serum-free medium were used as blanks). The alignment of the electrospun fibers was obtained using FFT analysis method and further analyzed using an image J software program. The degree angle of the fiber alignment of the SEM image was converted to the output image with patterned grayscale pixels. Cell viability assay The 3D PS and 3D HC-PMNF were prepared for 3D cell culture. And the prepared samples were sterilized under UV radiation for 48 hours. The MC3T3-E1 cells (preosteoblast) were seeded on the samples in 96-well plate at a density of 2×104cells in 100 µl of culture medium, and incubated 30 minutes for uploading the seeded cells on the samples. After 30 minutes, 200 µl of the culture media was added to each well with samples. For culturing the MC3T3-E1 cells, α-MEM high glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin was used and every sample was then transferred to an incubator with 5% CO2 at 37 °C. All the samples were replenished with new culture medium every 12 hours for 1day. To avoid loss of cells, 96-well plate was used for 1 day and the samples were transferred to 48-well plate and the culture medium of 600 µl was added to each sample. The samples with cultured MC3T3-E1 cells were washed one time with the culture medium for removal the non-adherent cells. The cytotoxicity of the 3D PS and 3D


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HC-PMNF were measured using the cell counting kit-8 after 12h, 24h, 48h, 72h, and 120h of cell culture. After culturing the MC3T3-E1 cells, at desired points in time, the CCK-8 solution (60µl) was added into the wells. Every sample with the CCK-8 solution was incubated for 4 hours in an incubator with 5% CO2 at 37 °C. The measurement of the absorbance of each sample (100µl) was checked at 450 nm using a microplate reader (Tecan, Austria). The results were presented as the mean ± standard error of the mean. DNA assay The measurement of MC3T3-E1 cells numbers or proliferation in terms of DNA content was evaluated using a Quant-iTTM PicoGreenTM dsDNA Assay Kit (Life Technologies, USA). This DNA assay can measure the fluorescence of Picogreen that is a nucleic acid strain for the quantitation of the double-stranded DNA (dsDNA) in a solution. Before the incubation, the 10µl of lysate was mixed with 190µl of Pico Green in TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.5). When incubated with the MC3T3-E1 cells and mixtures, they should be protected from light. The fluorescence was measured with a SYNERGY Mx spectrophotometer (BioTekR, USA) with emission wavelength at 520 nm and excitation wavelength at 480 nm. The scaffolds with the MC3T3-E1 cells were harvested after 24h, 48h, 72h, and 120h for the evaluation of the construct cellularity, that was assessed by determining the DNA content. The amount of DNA content of the MC3T3-E1 cells on each scaffold was expressed in ng/cm2 and this assay was repeated using a dilution of the sample for the confirmation of the quantitation results. SEM and fluorescent imaging of cell morphology The morphology of MC3T3-E1 cells cultured on the samples was examined using SEM and confocal laser scanning microscope. The MC3T3-E1 cells were seeded on the samples in



96-well plate at a density of 2×104cells and incubated for 1, 3 and 7 days. All the samples were replenished with new culture medium every 12 hours for 1day and the samples were transferred to 48-well plate and the culture medium of 600 µl was added to each sample. After that, all the samples were replenished with new media every 2 days. The media was aspirated from each well at desired points in time and adherent MC3T3-E1 cells were rinsed with Phosphate Buffer Solution (PBS) twice. The MC3T3-E1 cells on the samples were fixed with 2.5% glutaraldehyde solution and dehydrated through a graded series of ethanol solution (10, 30, 50, 70, 90, and 100%). Every sample was dried for 15 hours in a clean bench and the morphological studies were carried out using SEM. Furthermore, for the fluorescence images, the MC3T3-E1 cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes and were permeabilized in 0.2% Triton™ X-100 in PBS for 2 minutes. All the samples were stained with Actin-green 488 and 4’,6-diamidino-2-phenylindole (DAPI) solution in each sample in a 48-well plate after blocking with 1% human serum albumin (HSA)/PBS dilution for 30 min. The morphology and fluorescence image of the MC3T3-E1 cells were observed using a confocal laser-scanning microscopy. The cell infiltration and penetration studies of DAPI stained MC3T3-E1 cells on the 3D PS and 3D HC-PMNF were obtained using a Z-stacking program. Individual color coded sliced images of the DAPI stained cells, a function of confocal Z-depth, were collected. Every Z-stacking slice images were collected at 0.3µm intervals using 405 nm excitation lasers and the multiple Z-stacking images were subjected to spectral coding, compressed to make a composite image. In-vitro Immunohistochemical examinations The expressions of F-actin, integrin β1, and collagen I are induced during tissue regeneration when the cells are extensively fusing and plays a key role in regulating cell adhesion, migration, and proliferation in vitro, three processes common during tissue repair.


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The relative quantity of F-actin, integrin β1, and collagen I in the MC3T3-E1 cells can be important to the properties of the 3D PS and 3D HC-PMNF for tissue healing. The cultured MC3T3-E1 cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. All the samples were incubated for 10 min with PBS containing 1M Quenching solution and 0.1% Triton X-100 was added to each sample for 15 min for permeabilization of the MC3T3E1 cells. After washing in PBS three times for 5 min for nonspecific blocking, protein blocking solution (DAKO) was added for 12 min under the dark condition. All the samples were incubated with anti-integrin β1 (1:300, abcam) and anti-collagen I (1:100, Santa Crux Biotechnology, USA) as primary antibodies for 90min at room temperature. Alexa Fluor 594conjugated AffiniPure Donkey Anti-Rabbit IgG (1:250, Santa Crux Biotechnology, USA) and Alexa Fluor 594-conjugated AffiniPure Rabbit Anti-Goat IgG-HRP (1:200, Santa Crux Biotechnology, USA) were used as secondary antibodies for integrin β1 and collagen I detection. Then, the mounting medium with DAPI (Santa Cruz Biotechnology, USA) was used for the staining cell nucleus. In addition, for the F-actin staining, 4% paraformaldehydefixed specimens were permeabilized in 0.2% Triton™ X-100 in PBS for 2 minutes, incubated in 1% human serum albumin (HAS)/PBS dilution to block non-specific labelling, and stained with a 1.2 % BSA solution containing rhodamine phalloidin (1:45) for 1h at room temperature. All the samples mounted with mounting medium with DAPI (Santa Crux Biotechnology, USA) and every immunofluorescence and fluorescence images were obtained by confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany).

Animal care and surgical procedure All animal procedures were performed as follows: firstly, ten 6 weeks old nude mice were anesthetized intramuscularly at 0.06 cc with a mixture of Zoletil 50 (30 mg/kg, Virbac, France) and Domitor (1 mg/kg, Orion, Finland). Next, the cell loaded 3D PS and 3D HC-



PMNF samples were implanted under subcutaneous region of nude mice. After the surgery, the mice, singly housed after surgery, were kept warm on a heating pad and caged and monitored once daily. Lastly, the mice were sacrificed for further examination at 3 weeks of the post-surgery and the implanted 3D samples were collected in 4 % formalin solution (Sigma Aldrich Korea, South Korea) for histological analysis. The samples were made into paraffin blocks, sectioned into 6-8µm, and deparaffinized using Xylene. Every collected sample was stained with Hematoxylin and Eosin (H&E, Sigma-Aldrich, USA) and immunohistochemistrty analysis. Histological analysis The 3D samples with the surrounding tissue were collected and fixed in 4 % formalin solution for the further histological analysis. Every sample was made into paraffin-embedded blocks for the section with thickness of 6-8 µm using a microtome. The center of 3D PS and 3D HC-PMNF with surrounding tissue were sectioned for the staining with H&E after deparaffinization with Xylene. For the histological analysis, cell distribution, granular tissue formation, and arrangement of collagen fibrils were observed with H&E stained 3D samples under a microscope. For immunohistochemistry analysis, the deparaffinized samples were treated with 0.3% hydrogen peroxide (Showa, Japan) in a distilled water for 10 min. The samples were blocked with blocking solution (Ultra

Tek HRP, ScyTek,

USA) for 10 min after washing. The sectioned

3D samples were treated with a primary antibody: collagen I (1:100, Santa Cruz, USA) for 1 h at room temperature. Then, Rabbit Anti-Goat IgG HRP (1:200, Santa cruz, USA) was used as a secondary antibody for the detection of collagen I. After the detection, streptoavidinperoxidase was added to each sample for 30 min under a dark room condition. The peroxidase activity was detected using AEC Substrate Chromogen (Dako, USA) and washed with PBS three times. The sectioned samples were mounted and examined under a


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microscopy (Nikon, Japan). All animal experiment procedures were performed with the approval of the Chonbuk National University Animal Care Committee, Jeonju, Korea. Statistical analysis Data are presented as a mean ± standard error of the mean and analyzed by one-way ANOVA. A p < 0.05 was taken as statistically significant.

Author Information Corresponding Author *E-mail : [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgement This research was supported by grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) by Ministry of Education, Science and



Technology (Project no. 2016R1A2A2A07005160), and the program for fostering nextgeneration researchers in engineering of National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT (2017H1D8A2030449).

Figure 1. Schematic illustration of the nanoscale structure of FA molecular architecture


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Figure 2. (a) Schematic illustration of the fabrication process (3D printing + PMES) for the 3D HC-PMNFs and 3D HC-PMNF (b) Digital photos of the fabrication process of the PMES and (c) 3D HC-PMNF fabricated by PMES and 3D printing



Figure 3. (a) Schematic illustration of the fabrication process for the 3D HC-PMNFs; and the optical images of electrospun PCL fibers collected on each of the moving pin collectors. Illustration of electrospun PCL fibers on motion pin collectors deposited differently depending on distance of the pins. (b) The digital photographs of a various shapes of PMNFs (c) The digital photographs of motion pins (1mm in diameter and 20mm height) with electrospun fibers fabricated using by PMES method


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Figure 4. (a) Schematic illustrating generation of the 3D HC-PMNF (b) Aligned or randomly oriented fibrous membrane or mesh covering the layers of 3D printed scaffold horizontally or vertically, respectively. (Depending on the number of pins, only the desired portion can be covered in the desired design)



Figure 5. (a) SEM images of a top view and a cross section of the 3D PS, 3D HC-CNF, and 3D HC-PMNF (two or four pins arranged diagonally at the end of the 3D PS). Highmagnification SEM images were shown together. 3D PS and motion pins were presented as a blue line and red dots respectively. (b) FFT output images for the electrospun fibers on 3D HC-CNF and 3D HC-PMNFs (c) Pixel intensity plots against the angle of acquisition for 3D HC-CNF and 3D HC-PMNFs


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Figure 6. (a) Dynamic water uptake capacity of the 3D PS, 3D HC-CNF, and 3D HC-PMNF (b) Water uptake capacity of the 3D PS, 3D HC-CNF, and 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS) (c) Protein adsorption of the 3D PS, 3D HCCNF, and 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS) after 1, 6, 12, and 24 hours. 3D PS and motion pins were presented as a blue line and red dots respectively.



Table 1. Porosity and pore size of the 3D PS, 3D HC-CNF, and 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS). 3D PS and motion pins were presented as a blue line and red dots respectively.


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Figure 7. (a) Illustration of cells attached to 3D PS (macroscale) and 3D HC-PMNF (nano to macroscale) (b) SEM images of the MC3T3-E1 cells on 3D PS and 3D HC-PMNFs after 3 day of culture



Figure 8. (a) Confocal microscopy images of MC3T3-E1 cells on 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS) after 3 day of culture. Actin Green 488 (green) was applied for actin filament and DAPI (blue) for staining nuclei (b) Enlarged images of the MC3T3-E1 cells on 3D PS and 3D HC-PMNF (The cells on the 3D HC-PMNF were more spindle shape, elongated, and oriented.) (c) Schematic diagram of the orientation angle of the MC3T3-E1 cells on the 3D PS and 3D HC-PMNF, it is included angles of the stretched direction of the cells


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Figure 9. (a) CCK assay result of MC3T3-E1 cells on 3D PS and 3D HC-PMNFs (two or four pins arranged diagonally at the end of the 3D PS) after 12, 24, 48, 72 and 120 hours of cell culture. The data is reported as the mean ± standard deviation (n = 5, p

Nanoscale Resolution 3D Printing with Pin-Modified Electrified Inkjets

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