Interwoven Aligned Conductive Nanofiber Yarn/Hydrogel Composite Scaffolds for Engineered 3D Cardiac Anisotropy Yaobin Wu,†,# Ling Wang,†,# Baolin Guo,*,† and Peter X Ma†,‡,§,∥,⊥ †
Frontier Institute of Science and Technology, and State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049, China ‡ Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States § Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States ∥ Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor, Michigan 48109, United States ⊥ Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *
ABSTRACT: Mimicking the anisotropic cardiac structure and guiding 3D cellular orientation play a critical role in designing scaffolds for cardiac tissue regeneration. Significant advances have been achieved to control cellular alignment and elongation, but it remains an ongoing challenge for engineering 3D cardiac anisotropy using these approaches. Here, we present a 3D hybrid scaffold based on aligned conductive nanofiber yarns network (NFYs-NET, composition: polycaprolactone, silk fibroin, and carbon nanotubes) within a hydrogel shell for mimicking the native cardiac tissue structure, and further demonstrate their great potential for engineering 3D cardiac anisotropy for cardiac tissue engineering. The NFYs-NET structures are shown to control cellular orientation and enhance cardiomyocytes (CMs) maturation. 3D hybrid scaffolds were then fabricated by encapsulating NFYs-NET layers within hydrogel shell, and these 3D scaffolds performed the ability to promote aligned and elongated CMs maturation on each layer and individually control cellular orientation on different layers in a 3D environment. Furthermore, endothelialized myocardium was constructed by using this hybrid strategy via the coculture of CMs on NFYs-NET layer and endothelial cells within hydrogel shell. Therefore, these 3D hybrid scaffolds, containing NFYs-NET layer inducing cellular orientation, maturation, and anisotropy and hydrogel shell providing a suitable 3D environment for endothelialization, has great potential in engineering 3D cardiac anisotropy. KEYWORDS: nanofiber yarns network, hydrogel shell, cardiomyocyte, 3D cellular alignment, cardiac anisotropy, endothelialization cells packing together.17,18 Therefore, to effectively mimic the complex 3D anisotropic structure and replicate the biological function of native cardiac tissue, developing well-defined scaffolds that can guide cardiac cells orientation and organization within a 3D environment would be highly beneficial for cardiac tissue engineering. Different approaches have so far been explored to generate engineered scaffolds for 3D cardiac tissues.7,9,12,19 For instance, a series of 3D scaffolds containing structural patterns and
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ardiac failure has become a critical international health issue,1 and myocardial infarction (MI) or heart attack is one of the major causes of death associated with cardiovascular disease.2−4 Tissue engineering strategies have been demonstrated as a potential approach for cardiac tissues regeneration5−7 and as an alternative technique for the investigation of cardiovascular diseases by building engineered cardiac tissue models in vitro.8−10 Although a range of tissue engineering approaches have been successfully used for cardiac tissue regeneration applications,11−13 developing a suitable artificial scaffold for engineered functional cardiac tissue remains remarkably challenging.14−16 Heart muscles are richly vascularized and dense quasi-lamellar tissues in which multiple layers made of extracellular matrix (ECM) and highly oriented © 2017 American Chemical Society
Received: February 15, 2017 Accepted: June 1, 2017 Published: June 7, 2017 5646
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Figure 1. Designing a scaffold mimicking native cardiac tissue structure and NFYs-NET scaffolds preparation. (a) Native cardiac tissue exhibits an interwoven structure and scaffold with network structure would be beneficial for cardiac tissue regeneration. (b) Schematic illustration of the PCL/SF/CNT NFYs prepared by a developed wet−dry electrospinning and (c) the NFYs with smaller diameter shows the better alignment performance of nanofibers. (d) Scheme of the NFYs-NET scaffolds fabrication by a weaving technique using surgical suturing threads as warp and aligned NFYs as the weft (i); the SEM images of NFYs (ii); and the optical (iii) and SEM images (iv) of NFYs-NET scaffold illustrated the interwoven microstructure. (e) The NFYs-NET scaffolds were prepared by NFYs with different diameters including 50, 100, and 200 μm. The top views (i−vi) and 3D views (vii−ix) of fluorescent images of NFYs-NET scaffolds, which were prepared by NFYs stained with Nile red dye.
layer assembly techniques that have been introduced to prepare 3D cardiac tissue constructs consisting of multiple cell layers,24,31−35 developing a 3D multiple scaffold that can individually control the cellular orientation in each layer within a 3D environment remains challenging. Aligned nanofibers prepared by electrospinning technique have been increasingly investigated in cardiac tissue engineering,36,37 because their aligned structures can mimic the anisotropic structural organization of native cardiac tissue38 and their nano- or microenvironment and excellent mechanical properties provide the guidance cues to enhance CMs maturation.39 However, insufficient cell migration into the dense nanofibrous structures and the limited thickness of scaffolds are great barriers for developing a well-defined scaffold that can control CMs orientation within a 3D environment and further mimic the highly organized anisotropic structure of myocardium. Contrastively, in our previous study, composite scaffolds by incorporating aligned nanofiber yarns (NFYs) within hydrogel have been investigated to enhance 3D cellular alignment and elongation for skeletal muscle tissue regeneration.40 However, unlike the simple aligned structure of skeletal muscle tissue, the myocardium shows the complex 3D
various microstructures such as accordion-like honeycombs have been fabricated to control CMs orientation and organization within 3D environment.20−24 However, the nonuniform cell seeding and distribution inside these 3D scaffolds showed the limitation of large-scale cardiac tissues regeneration. Contrastively, hydrogel scaffolds present a 3D environment similar to native tissues and cells can be easily encapsulated within hydrogel scaffolds homogeneously, which showed great potential for engineering 3D cardiac tissues.12,25 The external stimuli, such as mechanical stimulation or electrical stimulation, has been applied to control the 3D cellular alignment and elongation within hydrogel scaffolds.26−30 Nonetheless, these complex processes of controlling cellular organization limited their clinical applications. More importantly, mimicking cellular multilayers structure plays a critical role for functional cardiac tissue, but preparing engineered cardiac tissues with multiple layers within hydrogels is difficult. Especially, for the multilayers structure of native cardiac tissues, the cellular alignment is uniform in each layer with a gradual transition of alignment between layers, which contributes to the unique biomechanical behavior of cardiac tissue.25 Although there are some approaches, such as layer-by5647
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ACS Nano anisotropic architecture composed of multiple layers of fibrous networks ECM and highly orientated cells together. Especially, each cellular layer performed the uniform alignment morphology, while a slight shift of alignment between layers was observed from endocardium to the epicardium.41,42 Such 3D anisotropic architecture of cardiac muscle ensures a circular contraction and leads to a strong pump function. Unfortunately, there have been no reports on developing a scaffold that can individually control the orientation of cardiac cells on each layer within a 3D environment. To address these challenges, we hypothesize that incorporating multiple aligned nanofibers layers within hydrogel would be a creative approach to develop a 3D scaffold that can control multilayer cellular orientation within a 3D environment. Here, we present a 3D hybrid scaffold by encapsulating conductive nanofiber yarns network (NFYs-NET) within hydrogel shell to engineer anisotropic and endothelialized cardiac constructs by controlling cellular orientation and organization of cardiomyocyte within a 3D multilayer environment. To mimic the complex interwoven structure of native cardiac tissue and control cellular alignment and elongation (Figure 1a), we prepared the interwoven conductive aligned NFYs-NET structures via a weaving technique, and the CMs orientation and organization on NFYs-NET were investigated. Furthermore, a series of 3D hybrid scaffolds were fabricated by encapsulating NFYs-NET layers within a photocurable methacrylated gelatin (GelMA) hydrogel shell after photocross-linking. CMs were seeded within these NFYs-NET/Gel scaffolds to investigate 3D cellular alignment and elongation and cardiac tissue function. Furthermore, we also developed an endothelialized myocardial tissue construct by the coculture of CMs and endothelial cells (ECs) within this scaffold, where aligned CMs were cultured on NFYs-NET layers while ECs formed a homogeneous network within hydrogel shell. We expect that these 3D hybrid scaffolds consist of NFYs-NET layers that can induce aligned and elongated CMs maturation, and the GelMA hydrogel shell that can provide a suitable 3D environment for mechanical protection and endothelialization via the coculture of ECs, will have a great potential for the practical application for cardiac tissue regeneration.
the regeneration of cardiac tissue due to their conductivity and nanostructures.52−55 Therefore, to ensure the composition of NFYs would be beneficial for cardiac cell adhesion, viability, and maturation, in this study we chose PCL, SF, and CNT as the components to prepare aligned NFYs. We hypothesize that such conductive NFYs would be highly beneficial for mimicking the mechanical and electrophysiological features of native cardiac tissue. As illustrated in Figure 1b, the random PCL/SF/ CNT electrospun nanofibrous web was formed on the surface of water/ethanol solution during the electrospinning, and then such nanofibrous web was drawn and lifted off the liquid surface by a rotating receptor to form the continuous NFYs. The diameter of NFYs and the orientation of nanofibers were both regulated by the rotation rate of receptor, that is, the higher drawing speed of the receptor led to the smaller diameter of NFYs with the better alignment performance of nanofibers (Figure 1c and Figure S1). For instance, the percentages of nanofibers aligned within ±10° in NFYs with diameters of 50 μm (NFY-50) and 100 μm (NFY-100) were as high as 89 ± 3% and 87 ± 6%, respectively, while the percentage in NFYs with diameter of 200 μm (NFY-200) decreased significantly to 70 ± 6% (Figure S1i). However, there was no significant difference of the nanofiber diameter in these NFYs with different diameters to be observed (Figure S1a−f and h), which indicated that the electrospinning situations rather than the receptor speed contributed to the nanofiber diameter within NFYs. Moreover, the representative TEM image of PCL/SF/CNTs nanofibers showed that CNTs were embedded within nanofiber and well aligned along the fiber axis (Figure S2a). In addition, these CNTs tended to be connected in fiber to form a conductive network. These data confirmed that the random CNTs were well dispersed in electrospun solution and then ultimately performed the parallel orientation within nanofiber axis due to the uniaxial direction of the fluid jet during the electrospinning process. Weaving technique containing warps and wefts has been regarded as a promising approach to create the scaffolds with interwoven structures in various tissue engineering fields, such as cartilage, osteochondral, and heart valve tissue engineering applications.56−59 The NFYs-NET scaffolds with interwoven structure were fabricated via a weaving method, which contained biodegradable surgical suturing threads as warp and aligned NFYs as the weft (Figure 1d). In weaving, surgical suturing threads were prestretched across a 3D-printed model with battlements pattern to form a warp structure and the NFYs weft thread was woven across the warp threads in the perpendicular direction to obtain the interwoven network scaffold. The biodegradable surgical suturing thread was chosen as the weft yarn due to its good biocompatibility and strong enough mechanical properties to provide a stable warp structure. For a clear demonstration of the effect of NFYs structure on CMs morphology and behavior, here we prepared three NFYs-NET scaffolds based on NFYs with diameter of 50, 100, and 200 μm, and the scaffold samples were named as NFYs-NET-50, NFYs-NET-100, and NFYs-NET-200. The optical and SEM images revealed that a couple of parallel aligned NFYs across surgical suturing threads with the orthogonal orientation and the interwoven microstructure was performed very well (Figure 1d (iii, iv) and Figure S3). In addition, both the top views and 3D views of fluorescent images of NFYs-NET scaffolds (NFYs were stained with Nile red dye) further illustrated such interwoven structure (Figure 1e). The conductivities of these NFYs-NET samples (ranging
RESULTS AND DISCUSSION The fabrication of NFYs-NET scaffolds was performed by a weaving technique via using aligned nanofiber yarns. The first step is to prepare the aligned nanofiber yarns with different diameters by a developed wet−dry electrospinning process similar to our previous work described.40 In our previous study, poly(caprolactone) (PCL), silk fibroin (SF), and polyaniline (PANI) were used to prepare aligned NFYs for enhancing myoblast alignment, elongation, and differentiation, because the composition of PCL and SF shows the suitable mechanical properties and a good biocompatibility and blending conductive PANI and its derivatives in biomaterials could promote myotube formation.40,43−48 To ensure the conductivity of PANI and its derivatives, acids, such as hydrochloric acid or camphorsulfonic acid, were necessary to dope PANI. However, these acids would have a negative effect on cell viability when they were released from PANI over time and also decreased the conductivity of PANI along with the dedoping process.44,49−51 Contrastively, carbon nanotubes (CNTs) perform the good and stable conductivity without doping with any acid, and many previous studies have demonstrated that biomaterials based on CNTs showed the positive effect on 5648
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Figure 2. Cardiomyocytes culture on NFYs-NET scaffolds prepared by NFYs with different diameters. (a) Scheme of cardiomyocytes seeded and cultured on NFYs-NET scaffolds. (b) Relative cells viability percentages of cardiomyocytes on NFYs-NET scaffolds, which was evaluated by live/dead assay. (c) The top views and 3D views of fluorescent images of cardiomyocytes on NFYs-NET scaffolds by staining with F-actin (green) and DPAI (blue) after 5 days of cultivation. Scale bar: 300 μm. (d) Cell elongation was estimated by nucleus aspect ratio (max/min Feret diameter) and cell orientation was evaluated by the angle between the long axis of the cells and the direction of aligned NFYs. The statistical analysis shows that the nucleus aspect ratios (e) and the percentage of cells aligned within ±10° (f) on NFYs-NET scaffolds were significantly higher than that on a 2D control group (*P < 0.05).
from 6.5 × 10−5 to 8.1 × 10−5 S/m) increased with the increase of NFYs diameter from 50 to 200 μm (Figure S2b), which was mainly because the NFYs with larger diameter would more easily form a better conductive network to promote the electrical conductivity. The mechanical properties of these NFYs-NET samples in NFYs weft direction and in sutures warp direction were both investigated, respectively. As shown in Figure S4, all of NFYs-NET samples showed the higher tensile strain in NFYs weft direction (ranging from 140% to 170%) compared with that in sutures warp direction (only about 10%) due to the good elasticity of aligned NFYs. However, the Young’s modulus of NFYs-NET samples in sutures warp direction (about 110 MPa) were significantly higher than those in NFYs weft direction (about 20 MPa) due to the tough and stiff properties of surgical suturing threads. These results showed that these interwoven NFYs-NET scaffolds with aligned NFYs as the weft and sutures as warp performed notable anisotropic mechanical characteristics and suitable conductivities, which would be beneficial for cardiac tissue engineering application. To test the effect of these interwoven architectural cues on cardiac cells morphology and functionality, cardiomyocytes with a high cell density (1.0 × 106 cells/mL) were seeded on
the NFYs-NET scaffolds prepared by NFYs with different diameters as illustrated in Figure 2a, and cells cultured on the glass-bottomed Petri dish (35 mm) were regarded as the 2D control group. Cells viability, alignment, and elongation morphologies, and their contractile behavior were investigated during a period of 8 days of culture. To assess the biocompatibility of these NFYs-NET scaffolds, cell viability was evaluated by using live/dead viability assay after cell cultivation on scaffolds for 2 days. The data indicated that about 80% cells were alive during the 2 days of incubation, and there was no significant difference of cell viability to be observed when cells were cultured on NFYs-NET scaffolds prepared by NFYs with different diameters and on 2D control group (Figure 2b). Furthermore, the cellular distribution and morphology on scaffolds were measured by fluorescent images via F-actin staining (Figure 2c). The top views and 3D views of fluorescent staining images showed that aligned CMs distributed uniformly and fully filled on the peripheral surface of NFYs-NET scaffolds after 5 days of cultivation (Figure 2c and Movie S1). Cells on all of these NFYs-NET samples with different NFYs diameters performed the alignment and elongation behavior due to the anisotropy of the aligned NFYs, and the higher magnification images showed the 5649
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Figure 3. Phenotype of cardiomyocytes on NFYs-NET scaffolds prepared by NFYs with different diameters. Immunofluorescence staining of sarcomeric α-actinin (green), nuclei (blue), and CX43 (red) revealed that cardiac tissues (8 days of cultivation) on NFYs-NET scaffolds (a−c) and on 2D control group (d) were phenotypically different. Immunostaining of α-actinin showed the well-organized sarcomeric α-actinin and sarcomere alignment on NFYs-NET scaffolds, and immunostaining of CX43 exhibited the better interconnected sarcomeric structure with robust intercellular junctions on NFYs-NET scaffolds than on 2D. Scale bar: 30 μm. Quantified analysis of the relative percentage of area coverage by α-actinin (e) and CX43 (f) on NFYs-NET scaffolds and on the 2D, respectively (*P < 0.05). (g) Scheme of cardiomyocytes on the gap of NYFs and the surface of NFYs-NET. (h) The beating behavior of cardiomyocytes in different zones on NFYs-NET scaffolds after 5 days of cultivation compared with cells on 2D control group. (i) Beating frequency (BPM) of cardiomyocytes on the surface of NFYs-NET scaffolds prepared by NFYs with different diameters and 2D control group after 5 days of cultivation.
formation of the interconnection network of cells between the adjacent NFYs within scaffolds (Figure S5). Contrastively, cells on 2D only showed the normal random morphology. Quantitative analysis of cellular elongation on NFYs-NET scaffolds was estimated by the nucleus aspect ratio (Figure 2d), and the statistical data showed that the nucleus aspect ratios on NFYs-NET samples were significantly higher than that on 2D environment (Figure 2e). Furthermore, the nucleus aspect ratios on NFYs-NET scaffolds with the NFYs diameter of 50 and 100 μm were as high as 2.7 ± 0.7 and 2.4 ± 0.6, while the aspect ratio decreased to 1.7 ± 0.3 when the NFYs diameter increased to 200 μm, which was mainly due to the larger diameter of NFYs and lower nanofibers alignment index of NFYs-NET-200 compared with NFYs-NET-50 and NFYs-
NET-100. On the other hand, the quantitative analysis of cells alignment behavior was evaluated by the orientation of cells though measuring the angle between long axis of the cells and the direction of aligned NFYs in scaffolds (Figure 2c). The histograms of cellular angles distribution displayed that cells on NFYs-NET scaffolds exhibited significantly higher alignment morphology than on 2D control group (Figure 2f and Figure S6). Particularly, both the NFYs-NET-50 and NFYs-NET-100 performed as high as 88 ± 4% and 79 ± 5% of cell aligned within ±10° orientation, respectively, while cells alignment decreased to 69 ± 3% when cells on NFYs-NET-200. Contrastively, only about 14 ± 3% of cells were within ±10° orientation on 2D environment. These data demonstrated the good biocompatibility of aligned interwoven PCL/SF/CNT 5650
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Figure 4. Fabrication of 1-layer 3D NFYs-NET/Gel hybrid scaffolds and cardiomyocytes cultivation within these 3D scaffolds. (a) The schematic fabrication process of 1-layer 3D scaffold via encapsulating single NFYs-NET layer within GelMA hydrogel shell after photo-crosslinking. The gross image (b), optical image (c), and the 3D view of confocal image (d) of 1-layer 3D scaffolds. (e) Scheme of cardiomyocytes seeded and cultured within 1-layer 3D scaffold and the fluorescent image of cardiomyocytes via F-actin staining (f). The confocal images of cardiomyocytes via F-actin/DAPI staining (green/blue) showed the cellular alignment and elongation within 1-layer 3D scaffold (g,h) while the random morphology of cells within 3D GelMA hydrogel (i,j). The 3D confocal images of α-actinin/CX43/DAPI staining (green/red/ blue) indicated that cells within 1-layer 3D scaffold (k) showed more orientated sarcomeric α-actinin and higher expression of CX43 compared with cells within 3D GelMA hydrogel (l). The quantitative analysis of cellular orientation distribution demonstrated cellular alignment along with the direction of NFYs within 1-layer scaffold (m,n), in contrast to the random morphologies within GelMA hydrogel (o,p). Quantified analysis of the relative percentage of area coverage by α-actinin (q) and CX43 (r) and sarcomere length (s) within 1-layer 3D scaffold and in GelMA hydrogel, respectively (*P < 0.05).
NFYs-NET scaffolds, the fluorescent images and the histograms of cellular angle distribution showed that both CMs on scaffolds degraded for 6 and 12 h still performed the aligned and elongated morphologies after 5 days of cultivation (Figure S8). Quantitative analysis of cellular elongation showed that nucleus aspect ratios of CMs on scaffolds degraded for 6 and 12 h were still significantly higher compared with 2D control group (Figure S8f). These data indicated that NFYs prepared by PCL, SF, and CNTs performed a good biodegradation behavior in vitro, and also demonstrated that the ability of NFYs-NET scaffolds to induce cellular alignment and elongation even during degradation period, which further suggested their great potential as an excellent candidate for cardiac tissue engineering applications in vivo. Immunofluorescence staining results further demonstrated that CMs on NFYs-NET scaffolds expressed a mature
NFYs-NET scaffolds and their ability to guide CMs to form an elongated and aligned interconnection network on their surface. Furthermore, the morphology changes of these NFYs-NET scaffolds were evaluated during lipase enzyme degradation over time, because lipase enzyme was widely used to investigate the degradation of ester groups in polyesters including PCL.60−62 After degradation in lipase solution for 6, 12, and 24 h, the obvious morphological changes on micrometer-scale were observed by the SEM images (Figure S7). The NFYs-NET scaffolds remained an intact interwoven structure while some aligned NFYs were disrupted by the erosion of lipase enzyme after degradation for 6 h (Figure S7a,d). Contrastively, the interwoven structure was partially destroyed after degradation for 12 h (Figure S7b,e), and even completely destructed after degradation for 24 h (Figure S7c,f). Furthermore, when CMs were cultured on these degraded 5651
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assembled only by these NFYs-NET is complex and difficult. In our previous study, we developed a series of core−shell scaffolds prepared by aligned NFYs and photo-cross-linked hydrogel for skeletal muscle regeneration, and such scaffolds have been demonstrated that the aligned NFYs core can guide the myoblasts alignment and differentiation, and the hydrogel shell can provide a suitable 3D environment for nutrition exchange and mechanical protection.40 Therefore, in this study, we supposed that NFYs-NET encapsulated within hydrogel shell would not only prevent the damage of NFYs-NET microstructure but also protect cardiac cells arrangement and function within a 3D environment. To ensure the viability and biofunction of CMs within a 3D environment, we selected methacrylated gelatin (GelMA), a good biocompatible hydrogel and an excellent candidate material for cardiac tissue engineering,66−68 to prepare the hydrogel shell via photo-cross-linking. Moreover, we also anticipated that 3D scaffolds containing multiple layers would be easily fabricated by embedding stacked NFYs-NET layers within GelMA hydrogel shell to further mimic multiple cellular layers of native cardiac tissue. To test the operability of NFYs-NET/Gel scaffolds and investigate positive effect of such 3D scaffolds on cellular alignment in 3D environment, the scaffold containing single NFYs-NET layer within hydrogel shell was first fabricated. The schematic process of 1-layer 3D scaffold fabrication was shown in Figure 4a. A single NFYs-NET was fixed in the 3D-printed frame model and then placed into a microsheet PDMS pattern to act as one layer core. The GelMA solution was dropped into the PDMS pattern and then the hydrogel shell with 1 mm thickness was formed after photo-cross-linking. The representative gross image and optical microscope image showed that a single NFYs-NET-50 layer was placed well within the GelMA hydrogel sheet (Figure 4b,c). Furthermore, to distinguish NFYs-NET layer and hydrogel shell, the aligned NFYs were stained red by Nile red dye and the hydrogel shell was stained by FITC dye. The fluorescence images clearly showed the individual interwoven NFYs-NET layer and hydrogel shell (Figure S11), and the 3D view of confocal images further confirmed that such NFYsNET was localized within the hydrogel shell very well (Figure 4d). In addition, for the SEM images of the scaffolds after lyophilization, the nanofibrous structure of NFYs, and the interwoven network of NFYs-NET, and the multiporous microstructure of GelMA hydrogel shell were all observed from the cross section of scaffolds (Figure S12). Furthermore, the structure stability of these NFYs-NET/Gel 3D scaffolds was evaluated under the dynamic mechanical test by using a rheometer. As shown in Figure S13, the interwoven network structures of NFYs-NET scaffolds were easily disrupted after dynamic mechanical test for 1 min. Contrastively, NFYs-NET/ Gel 3D scaffolds remained an intact interwoven network after the same dynamic mechanical test, because of the mechanical protection by hydrogel shell. These data suggested that the 3D hydrogel shell provided a mechanical protection for NFYsNET, and further ensured the structure stability of such 3D hybrid scaffolds in practical use. For culturing cells within NFYs-NET/Gel 3D scaffolds, the CMs were first seeded on the NFYs-NET surface and cultured for 48 h to allow cells adhesion and organization (Figure 4e,f). The cell-laden NFYs-NET was encapsulated by GelMA hydrogel shell after photo-cross-linking, and further cultured in medium for another 6 days. The morphology and maturation phenotype of CMs within 3D scaffolds were evaluated by Factin staining and immunofluorescence staining, respectively,
phenotype, which was evaluated via the expression of cardiac markers sarcomeric α-actinin and gap junction protein connexin-43 (CX43) (Figure 3a−d and Figure S9). The immunofluorescence staining of sarcomeric α-actinin, a crucial marker for strong contractility and maturation of CMs,63 showed that CMs on NFYs-NET scaffolds expressed wellorganized sarcomeric α-actinin and performed the alignment of sarcomeres along with the major direction of NFYs. However, such cross striated structures were elusive and cells did not exhibit any preferred orientation when CMs were cultured on 2D environment. Additionally, the coverage areas of sarcomeric α-actinin in NFYs-NFY groups were significantly higher than that in 2D group (Figure 3e). On the other hand, CMs exhibited higher expression and uniform distribution of CX43 on aligned NFYs-NFY scaffolds compared with on random 2D surface (Figure 3f and Figure S9), which indicated enhanced cell−cell interactions and improved contractile properties. CX43 has been demonstrated as a kind of critical gap junction protein that allows the passage of ions and solutes between cells, which contributed to the cell−cell communication and the regulation of electrical and mechanical junctions between adjacent cardiomyocytes.64,65 Therefore, it is envisioned that the well-developed and interconnected sarcomeres and the uniform distributed networks of gap junctions on the surface of NFYs-NET scaffolds improved the overall cell−cell interactions, which would enhance the spontaneous and synchronization activity of adjacent CMs. The beating behavior of CMs on these NFYs-NET scaffolds was also investigated to further evaluate the CMs function. First, we seeded CMs on the surface of a single NFY-50 (diameter 50 μm), and CMs exhibited spontaneously synchronous beating after 5 days of culture and the CM beating rate (BPM) showed 57 ± 3 beats/min (Figure S10 and Movie S2). Furthermore, when CMs were cultured on NFYsNET-50 scaffold, cells within the gaps of adjacent NFYs and on the surface of NFYs-NET both performed the spontaneously synchronous beating behavior similar to ones on single NFY (Figure 3g,h, Figure S10 and Movie S3−4). Contrastively, contractions of CMs on 2D control group were weaker and less synchronized compared with that on single NFY or on NFYsNET, and the CM beating rate was also lower than NFYs groups (Figure 3h, Figure S10 and Movie S5). In addition, CMs on NFYs-NET-100 and NFYs-NET-200 scaffolds also showed the similar spontaneously synchronous beating behavior and beating rate with cells on NFYs-NET-50 scaffolds (Figure 3i and Movie S6−7). Taken together, the immunofluorescence staining results and the beating behavior analysis suggested that the formation of the aligned CMs network on NFYs-NET scaffolds enhanced the cellular maturation and protein expression, which contributed to the spontaneously synchronous beating. CMs cultured on the surfaces of NFYs-NET scaffolds showed the aligned and elongated morphologies and performed the maturation phenotype and spontaneously synchronous beating ability. These data suggested that such NFYs-NET scaffolds showed the great potential for cardiac tissue regeneration. However, the uniform interwoven network structures of these scaffolds were easily disrupted when using in the complex practical environment. Furthermore, to be of clinical relevance, engineered cardiac tissue scaffolds were required to not only induce single oriented cardiac layer regeneration but also enhance the organization of multiple cardiac layers, while the preparation of multilayer scaffolds 5652
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Figure 5. Fabrication of 2-layer 3D NFYs-NET/Gel hybrid scaffolds and cardiomyocytes cultivation within these 3D scaffolds. (a) Myocardium showing a gradual transition of aligned cell layers from endocardium to epicardium. (b) Schematics of multiple layers of NFYsNETs assembled with the gradual transition of orientation. (c) The schematic process of 2-layer 3D scaffold fabrication via encapsulating two layers of NFYs-NET with orthogonal orientation within GelMA hydrogel shell after photo-cross-linking. The gross image (d) and 3D view of confocal image (e) of 2-layer 3D scaffolds, where NFYs-NET was stained with red and hydrogel was stained with green, respectively. (f) The scheme of culturing cells on two NFYs-NET layers within hydrogel shell. Fluorescent images of cardiomyocytes via F-actin (green) staining on the NFYs-NET layer with horizontal direction (g) and vertical direction (h) within 2-layer 3D scaffold. The top view (i) and 3D view (j) of confocal images of cells via F-actin/DAPI staining. The quantitative analysis of cellular orientation distribution demonstrated that cells were aligned on each layer while were perpendicular on different layers (k,l).
GelMA hydrogel performed the nucleus aspect ratio (1.2 ± 4) and alignment index (13%) similar to cells on 2D environment (Figure 4o,p and Figure S15c,d). Although cells within NFYsNET/Gel scaffolds prepared by NFYs with larger diameter (100 and 200 μm) showed the slightly lower nucleus aspect ratio and alignment index than that in NFYs-NET-50/Gel scaffolds, they still exhibited the much better elongation and alignment performances compared with 3D Gel control group (Figure S15). Furthermore, immunofluorescence staining images showed that cells within NFYs-NET/Gel scaffolds showed the clearly orientated sarcomeric α-actinin and higher expression of CX43 protein compared to cells in 3D GelMA hydrogel (Figure 4k,l and Figure S16). The coverage areas of sarcomeric α-actinin and CX43 in NFYs-NFY groups were significantly higher than that in 3D gel groups (Figure 4q,r).
and cells encapsulated within 3D GelMA hydrogel (3D Gel) were used as a control group (Figure 4g−l and Figure S14). From the top view and 3D view of F-actin staining images, it shows that CMs were fully filled on the peripheral surface of NFYs-NET (Figure 4g,h), and the cellular alignment and elongation were both observed within these 3D scaffolds compared with the random morphology of cells within 3D GelMA gel (Figure 4i,j and Figure S14). Compared with the CMs on NFYs-NET, the nucleus aspect ratio and cellular alignment index were both slightly decreased when cells in NFYs-NET/Gel scaffolds. However, the quantitative analysis results showed that the nucleus aspect ratio and the percentage of cells aligned within ±10° orientation of NFYs-NET-50/Gel group were as high as 2.3 ± 4 and about 82%, respectively (Figure 4m,n and Figure S15c,d). Contrastively, cells within 5653
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Figure 6. Construction of endothelialized myocardium based on the coculture of cardiomyocytes and endothelial cells (ECs) within 1-layer 3D scaffolds. (a) Schematics showing the coculture procedure that CMs were cultured on the NFYs-NET layer while green fluorescent protein-positive endothelial cells (GFP-ECs) were encapsulated within hydrogel shell. The fluorescent images of GFP-ECs (green) (b) and CMs (red) (c) and their merge image (d). (e) The 3D view of confocal images of GFP-ECs and CMs within scaffolds. The quantitative analysis of cellular orientation distribution of GFP-ECs (f) and CMs (g). (h,i) The fluorescence intensity analysis for the cross section of this cell-laden scaffold showed the distribution of ECs in hydrogel and CMs on NFYs-NET.
Importantly, the cardiomyocytes in NFYs-NET/Gel scaffolds exhibited significantly longer sarcomeres than that in 3D Gel (Figure 4s). These results demonstrated that such 3D scaffolds also performed the ability to enhance CMs maturation and improve cell−cell coupling similar to cells on NFYs-NET without hydrogel shell. These results indicated that such NFYsNET/Gel scaffolds not only induced 3D cellular alignment and elongation but also provided a suitable 3D environment for nutrition exchange and mechanical protection. For native cardiac tissue, the myocardium shows the uniform aligned cardiac cells in each layer, while it performs a gradual transition of alignment between cell layers (Figure 5a). Therefore, designing a scaffold that can individually control cellular orientation and organization in different cell layers would be more beneficial while still an ongoing challenge. We supposed that these NFYs-NET/Gel scaffolds would overcome this issue, because cells alignment and elongation can be easily controlled by the NFYs-NET layer and hydrogel shell provides a 3D environment to assemble cell-laden NFYs-NETs via layerby-layer (Figure 5b). To confirm this hypothesis, we designed and prepared the 2-layer 3D scaffold containing two layers of NFYs-NET with the orthogonal orientation to prove the ability to guide multiple layers of cellular alignment and elongation with different orientation between layers in 3D environment.
The 2-layer 3D scaffold was prepared by a similar process to that for 1-layer 3D scaffold’s fabrication (Figure 5c). Two NFYs-NET-50 layers with orthogonal orientation were encapsulated well within GelMA hydrogel shell, as shown in the optical gross image and fluorescence merge images (Figure 5d and Figure S17a−c). Furthermore, these two layers of NFYs-NET with orthogonal orientation were clearly distinguished as observed in the fluorescence images with different focus distance (Figure S17d−g), and the 3D view of confocal images further illustrated the two perpendicular stacked layers within 3D hydrogel shell (Figure 5e). Analogously, after culturing cells within 2-layer 3D scaffolds for 6 days (Figure 5f), the fluorescence images showed that CMs were fully filled on the two NFYs-NET layers with horizontal direction (Figure 5g) and vertical direction (Figure 5h) within 2-layer 3D scaffold, respectively. From the top view (Figure 5i) and 3D view (Figure 5j) of confocal images, the aligned and elongated CMs with orthogonal orientation were further confirmed on the two NFYs-NET layers. In addition, the quantitative analysis indicated the polar distribution of cellular orientation along with the two perpendicular NFYs-NET layers (Figure 5k,l). According to these promising results, we supposed that the more complex 3D scaffolds containing multiple layers, such as three, four, and even more layers of NFYs-NET, were easily 5654
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weaving method based on aligned conductive nanofiber yarns as the weft and surgical suturing threads as warp. Cardiomyocytes (CMs) were seeded and cultured on these NFYs-NETs, and the cultivation results demonstrated the good biocompatibility of these NFYs-NETs and their ability to guide cellular alignment and elongation and enhance CMs maturation and function. Furthermore, the 3D hybrid scaffolds were fabricated by encapsulating one layer or two layers of NFYsNET within a hydrogel shell after photo-cross-linking, and such 3D scaffolds performed the ability to not only promote aligned and elongated CMs maturation on each layer but also individually control the cellular orientation on different layers within a 3D environment. Endothelialized myocardium was also constructed by using this hybrid strategy based on the coculture of CMs seeding on the NFYs-NET layer and ECs encapsulating within hydrogel shell, demonstrating the potential for the development of integrated cardiovascular organoids. These results indicate that NFYs-NET layer mimicking the interwoven structure of native cardiac tissue have the ability to induce cell alignment, elongation functional maturation, and anisotropy of CMs between different layers, and the GelMA hydrogel shell provides a suitable 3D environment for mechanical protection and endothelialization via the coculture of ECs, which suggests that these 3D hybrid scaffolds performed a great potential application for cardiac tissue engineering.
developed by using the same fabrication process. Furthermore, these scaffolds exhibited the ability to not only induce CMs alignment and elongation on each layer but also control the cellular orientation with different layers within a 3D environment, suggesting their great potential for engineering 3D multilayer cardiac anisotropy. Vascularization plays an important role in developing 3D engineered cardiac tissue for long-term survival and function, while fabrication of integrated cardiovascular organoids is still a great challenge due to the hierarchical structure of the native myocardium.7,69 Previous studies have been demonstrated that the addition of endothelial cells into cardiac myocytes performed a positive effect on the spontaneous vascularization during the in vitro or in vivo cultivation and then improved cardiac tissue structure and function.7 Although various strategies have been recently developed to prepared engineered endothelialized myocardial tissues via the coculture of ECs and CMs,70−72 the incorporation of confluent endothelium layers and aligned cardiac tissue layers within a suitable 3D environment was still limited due to their hierarchical structures. In this study, we proposed that our NFYs-NET/ Gel hybrid scaffolds were suitable to produce integrated endothelialized myocardium structures by coculture of ECs and CMs within the 3D scaffolds, and therefore enhanced nutrition transformation in a 3D environment and promoted 3D cardiac constructs for the long-term survival. For the coculture, CMs were seeded and cultured on single layer of NFYs-NET for 2 days following the same process of cultivation cells within 1-layer 3D scaffold, and then GelMA solution encapsulating ECs was added to the CMs-laden NFYs-NET to form a ECs-laden hydrogel shell after photo-cross-linking (Figure 6a). To distinguish ECs and CMs in 3D environment, the green fluorescent protein (GFP)-positive endothelial cells (GFP-ECs) were used in this study and the CMs were stained by red via immunofluorescence staining of sarcomeric α-actinin, and both cells morphologies and distributions within scaffolds were investigated after coculture for 5 days. GFP-ECs (green) were found to be homogeneously distributed and exhibited network formation and CMs (red) exhibited the aligned and elongated morphology, as observed in their fluorescent images and alignment index analysis (Figure 6b−e and f,g). Specially, the 3D view of fluorescent images further showed that such homogeneous ECs networks were distributed within 3D hydrogel shell and the CMs were fully filled on the NFYsNET layer, respectively (Figure 6e). In addition, the fluorescence intensity analysis for the cross section of this cell-laden scaffold also showed the good distribution of ECs in hydrogel and ECs on NFYs-NET (Figure 6h,i). These data suggest that using this hybrid strategy based on NFYs-NET/ Gel scaffolds can easily develop the endothelialized myocardium structure containing the aligned and elongated cardiac tissue on NFYs-NET layers within hydrogel shell encapsulating homogeneous ECs networks, therefore performed the great potential for developing the integrated cardiovascular organoids for clinical cardiac regeneration applications.
METHODS Synthesis and Preparation of Polymers. Regenerated silk fibroin (SF) were prepared following our previous study.40 Briefly, degummed silk (Buke Pharmaceutical Co.) was dissolved in CaCl2/ H2O/ethanol mixture solution and then dialyzed (Mw = 3500 Da) in distilled water for 3 days, and the resulting solution was filtered and lyophilized to obtain the regenerated SF sponges and maintained at −80 °C. GelMA was synthesized following a procedure as described previously.67 Gelatin from cold water fish (Sigma-Aldrich) was added into DPBS at 50 °C and stirred until fully dissolved to obtain 10 wt% solution. Methacrylic anhydride was then added into gelatin solution under stirring and allowed to react for 3 h at 50 °C. The resulting mixture was diluted with DPBS (5× dilution) to stop the reaction, and then dialyzed (Mw = 5000 Da) against distilled water for 10 days at 40 °C to remove unreacted reagents. The solution was lyophilized for 3 days to obtain a white porous GelMA foam and maintained at −80 °C. Preparation of Aligned PCL/SF/CNT Nanofiber Yarns (NFYs). CNT (Nanjing XFNANO Materials Tech Co.) was added in HFIP and then sonicated for 1 h (VCX 400, 80 w, 2 s on and 1 s off) in an ice−water bath to obtain a homogeneous dispersion of CNTs. PCL (Mn = 80 kDa, Sigma-Aldrich) and SF were dissolved in the HFIP at room temperature for 24 h, and the selected amount of sonicated CNT suspension was added in to PCL/SF solution and further stirred for 24 h to prepare a PCL/SF/CNT homogeneous suspension. The final concentrations of each materials were as follows: PCL 120 mg/ mL, SF 30 mg/mL, and CNT 1.5 mg/mL. The aligned PCL/SF/CNT nanofiber yarns were prepared by an enhanced wet−dry electrospinning process following our previous study.40 In brief, the PCL/SF/ CNT solution was added into a 10 mL syringe with a 21 G hypodermic needle, and then electrospun at a volume flow rate of 1 mL/h with the voltage of 12 kV. The nanofibrous random web was first received on the surface of a distilled water/ethanol (V/V 8:2) bath, and then drawn with a rotating receptor and lifted off the surface of solution to obtain the continuous nanofiber yarns (Figure 1b). The diameter of NFYs was controlled by the rotating rate of the receptor ranging from 20 to 100 mm/min. All of these aligned NFYs were dried under vacuum at room temperature for 2 days to remove solvent and residue, and their micromorphology was analyzed by a scanning electron microscope (SEM) (Quanta FEG 250, FEI) at an accelerating voltage of 5 kV. The CNTs distribution within electrospun nanofibers
CONCLUSIONS In summary, we developed a simple and efficient strategy to prepare a 3D hybrid scaffold based on NFYs-NET layers within a hydrogel shell for mimicking the native cardiac tissue structure, and demonstrated their great potential for engineering anisotropic 3D cardiac constructs. The NFYs-NETs with anisotropic interwoven structure were prepared by a developed 5655
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ACS Nano prepared by electrospinning the PCL/SF/CNTs solutions onto copper grids directly for about 10 s was examined using transmission electron microscope (TEM, Hitachi H-700H). Fabrication of NFYs-NET Scaffold. The NFYs-NET scaffolds were prepared based on a weaving technique via aligned PCL/SF/ CNT nanofiber yarns as weft and the surgical suturing threads (size: 6−0) as warp, and the schematic diagram in Figure 1d(i) illustrated the waving process. Briefly, the surgical suturing threads were winded and fixed through a 3D-printed model (Length × Width × Height: 18 mm × 12 mm × 3 mm) with battlements pattern to provide a stable warp structure, and then NFYs with different diameter (50, 100, and 200 μm) as weft threads were passed through the warp threads to achieve the interwoven network structure. Fabrication of NFYs-NET/Gel 3D Scaffold. To fabricate 1-layer NFYs-NET/Gel 3D scaffold, the NFYs-NET was peeled off from the 3D-printed model and then placed into a PDMS (Sylagrad 184, Dow Corning) microsheet pattern (18 mm × 12 mm × 1 mm), and then GelMA solution (5% w/v in DPBS) was added into the PDMS microsheet pattern and then photopolymerized at 365 nm UV light at ∼12 mW/cm2 for 60 s with the presence of Irgacure 2959 (SigmaAldrich) as photoinitiator (0.05% w/v) to obtain the 3D hydrogel sheet. Similarly, for the 2-layer NFYs-NET/Gel 3D scaffold fabrication, two layers of NFYs-NETs with the orthogonal direction were placed into a PDMS microsheet pattern (18 mm × 18 mm × 1 mm), and the GelMA hydrogel shell was then prepared by UV irradiation following the same fabrication process of 1-layer 3D scaffold. In order to label NFYs-NET and the 3D hydrogel sheets, Nile red (Sigma-Aldrich) was mixed with the PCL/SF/CNT electrospun solution before electrospinning and the FITC (Sigma-Aldrich) was mixed with GelMA solution before photo-cross-linking. The microstructure of NFYs-NETs and l-layer or 2-layer 3D scaffolds were characterized by using a fluorescence microscope (IX53, Olympus) and a confocal laser microscope (FV1200, Olympus). The micromorphology of these samples was analyzed by a SEM (Quanta FEG 250, FEI) after freeze-drying. Characterizations. The conductivities of NFYs-NET scaffolds prepared by NFYs with different diameters were measured by the standard Van Der Pauw DC four-probe method, and the mechanical properties of these NFYs-NET samples in NFYs weft direction and in sutures warp direction were investigated by using a MTS Criterion tester. In vitro enzymatic degradation tests of NFYs-NET samples were performed by using lipase enzyme from Thermomyces lanuginosus at an activity of 10000 U/mL in phosphate buffer solution (PBS, pH 7.4). The structure stability of NFYs-NET scaffolds and NFYs-NET/Gel 3D scaffolds were evaluated under the dynamic mechanical test by using a rheometer (TA Instruments) at a strain of 50% and a shear frequency of 5 Hz. These details are available in SI Materials and Methods. Cardiomyocytes Isolation and Culture. Cardiomyocytes (CMs) were isolated from the hearts of 2-day-old Sprague−Dawley rats following an established protocol according to the guide for the care and use of laboratory animals, established by the committee on animal research at Xi’an Jiaotong University. The details are available in SI Materials and Methods. Cardiomyocytes Culture on NFYs-NET. Cardiomyocytes were seeded and cultured on NFYs-NET samples with different NFYs diameter (50, 100, and 200 μm) as described in the following process. First, the NFYs-NET samples were placed in PDMS mold and then sterilized by UV light for 2 h. Before seeding with cardiomyocytes, sterile aligned NFYs-NET samples were incubated with cell culture medium at 37 °C for 2 h. Cell suspension (0.3 mL, 1.0 × 106 cells/ mL) was dropped onto the NFYs-NET and then incubated for 6 h. After most of cells were adhered onto the surface of NFYs-NET, another 1 mL of culture medium was added and cultivation continued for 8 days. The medium was changed every day. Cardiomyocytes Culture within NFYs-NET/Gel Scaffolds. The cultivation of cardiomyocytes within NFYs-NET/Gel scaffolds was performed by a process similar to cells culture on NFYs-NET. Briefly, to culture cells within 1-layer 3D scaffold, cardiomyocytes were seeded and then cultured on NFYs-NET for 48 h as the process previously
described, and the cell-laden NFYs-NET was coated with the GelMA hydrogel (prepared by culture medium) shell after photo-cross-linking by UV irradiation at 12 mW/cm2 for 30 s. Similarly, for preparing cellladen 2-layer 3D scaffold, cardiomyocytes were seeded on two layers NFYs-NET samples and cultured for 48 h, respectively. Such two cellladen NETs−NETs with orthogonal direction were stacked together, and then encapsulated by GelMA hydrogel shell after photo-crosslinking following the same UV irradiation with 1-layer 3D scaffold preparation. The cell-laden 3D scaffolds were then transferred into a Petri dish and further cultured for another 6 days. The medium was changed every day. Cell Characterization on NFYs-NET or within NFYs-NET/Gel Scaffolds. A live/dead viability kit (Molecular Probes) was used according to the manufacture’s process to investigate cell viability. The fluorescence images were taken by using an inverted fluorescence microscope (IX53, Olympus), and the green color resulted from staining with calcein AM indicating living cells and red color resulted from staining with ethidium homodimer-1 indicating dead cells. The ImageJ software was used to analyze the number of live/dead cells from 10 randomly selected areas of 3 samples for each group. Immunofluorescence measurement of cardiomyocytes was performed after 8 days of cultivation. In brief, cultured cells were rinsed twice gently with DPBS and then fixed with 2.5% glutaraldehyde for 15 min at room temperature. After fixation, cells were rinsed twice gently with DPBS and then treated with 0.3% Triton X-100 for 30 min. The samples were treated with FITC-phalloidin for 90 min and counterstained with DAPI. In addition, cardiomyocytes constructs were stained with two different kinds of primary antibodies including sarcomeric α-actinin and connexin-43 (CX43) at 1:200 dilution in blocking buffer at 4 °C overnight. Then, Alexa Flour-488 conjugated secondary antibody (Molecular Probes) for sarcomeric α-actinin and Alexa Flour-594 conjugated secondary antibody for CX43 were added and incubated for 1.5 h at room temperature. After being washed with DPBS, cell nuclei were counterstained with DAPI for 5 min. The fluorescence images of these stained samples were taken by a confocal laser microscope (FV1200, Olympus). The elongation and orientation of CMs on NFYs-NET samples or 3D scaffolds were analyzed by using ImageJ software based on F-actin fluorescence images following our previous study.40 Briefly, the cell elongation was quantitatively measured by nucleus aspect ratio and the orientation of cells was determined by measuring the angle between long axis of the cells and the direction of aligned NFYs to generate alignment histograms. The quantified analysis of the relative percentage of area coverage by αactinin and CX43 and the statistics of sarcomere length of CMs were evaluated by using ImageJ software. In addition, cell cultivation on the glass-bottomed Petri dish (35 mm) was used as 2D control group, and cells within GelMA hydrogel (5% w/v) were regarded as 3D control group, respectively. To visualize the beating behavior on NFYs-NET, CMs were stained with 5 μm CellTracker Green (Molecular Probes) for 45 min in the incubator. The green fluorescence labeled CMs were seeded and cultured on NFYs-NET for 5 days. The beating video of CMs was recorded by the inverted microscope equipped with a CCD camera. The video sequences were digitized at a rate of 25 frames per second. The beating signal patterns of CMs on NFYs-NET were obtained using a custom written MATLAB program.53 Coculture of CMs and Endothelial Cells (ECs) within 3D Scaffolds. The coculture of CMs and green fluorescent protein (GFP)-positive endothelial cells (GFP-ECs) (Biovector Science Lab, Inc.) within 1-layer 3D was performed with a process similar to the cultivation of CMs within 3D scaffolds. First, CMs were seeded and then cultured on NFYs-NET for 48 h as described in the previous process, and then GelMA solution (5% w/v) containing with GFPECs (1.0 × 106 cells/mL) was dropped onto the CMs-laden NFYsNET within the PDMS mold. The 3D hydrogel shell encapsulating GFP-ECs and CMs-laden NFYs-NET was formed after photo-crosslinking by UV irradiation at 12 mW/cm2 for 30 s. The coculture 3D scaffolds were then transferred into a Petri dish and further cultured for another 5 days within the mixture cell medium (CMs medium: GFP-ECs medium = 1:1 v/v). The medium was changed every day. The CMs and GFP-ECs cellular morphologies and distributions were 5656
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investigated by a confocal laser microscope (FV1200, Olympus) after immunofluorescence staining CMs via sarcomeric α-actinin as red. Statistic. Experiments were run in triplicate for each sample, and results are presented as mean ± standard deviation. Quantitative data and measurement of cell aspect ratio and orientation were obtained using the ImageJ software, and the results are from at least 5 images of 3 independent locations of each sample (cell number >200). Statistical differences were obtained through analysis of variance followed by Tukey’s significant difference post hoc test. A significance level of 0.05 was applied to determine significant differences.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01062. The materials and methods description; micromorphologies of aligned NFYs, NFYs-NET, and NFYs-NET/Gel 3D scaffolds; cells behavior on NFYs-NET and within NFYs-NET/Gel 3D scaffolds; SEM images; confocal fluorescent images; immunostaining staining images; and cellular orientation and elongation analysis (PDF) The 3D side view of aligned CMs fully filled on NFYsNET surface (AVI) The beating behavior of CMs on aligned NFYs and NFYs-NET prepared by different diameters (AVI) The beating behavior of CMs within the gap of adjacent NFYs (diameter 50 μm) after 5 days of cultivation (AVI) The beating behavior of CMs on the surface of NFYsNET (diameter 50 μm) after 5 days of cultivation (AVI) The beating behavior of CMs on 2D control after 5 days of cultivation (AVI) The beating behavior of CMs on the surface of NFYsNET (diameter 100 μm) after 5 days of cultivation (AVI) The beating behavior of CMs on the surface of NFYsNET (diameter 200 μm) after 5 days of cultivation (AVI)
AUTHOR INFORMATION Corresponding Author
*Tel.:+86-29-83395361; Fax: +86-29-83395131; E-mail:
[email protected]. ORCID
Baolin Guo: 0000-0001-6756-1441 Author Contributions #
Y.W. and L.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS The National Natural Science Foundation of China (grant number: 51673155) and “The Fundamental Research Funds for the Central Universities”, and Xi’an Jiaotong University are acknowledged for financial support of this work. REFERENCES (1) Alwan, A. Global Status Report on Noncommunicable Diseases 2010; World Health Organization, 2011. (2) Sutton, M. G. S. J.; Sharpe, N. Left Ventricular Remodeling after Myocardial Infarction Pathophysiology and Therapy. Circulation 2000, 101, 2981−2988. (3) Kajstura, J.; Gurusamy, N.; Ogórek, B.; Goichberg, P.; ClavoRondon, C.; Hosoda, T.; D’Amario, D.; Bardelli, S.; Beltrami, A. P.; 5657
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DOI: 10.1021/acsnano.7b01062 ACS Nano 2017, 11, 5646−5659
