Origami meets electrospinning: a new strategy for 3D nanofiber scaffolds
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Inspired by the constitution of things in the natural world, three-dimensional (3D) nanofiber scaffold/cells complex was constructed via the combination of electrospinning technology and origami techniques. The nanofiber boxes prepared by origami provided a limited space for the layer-by-layer nanofiber films, and the human fetal osteoblasts (hFOBs) seeded on the both sides of the nanofiber films were expected to facilitate the bonding of the adjacent nanofiber films through the secretion of extracellular matrix. Specifically, the hFOBs presented 3D distribution in the nanofiber scaffold, and they can stretch across the gaps between the adjacent nanofiber films, forming the cell layers and filling the whole 3D nanofiber scaffold. Eventually, a 3D block composed of electrospun nanofiber scaffold and cells was obtained, which possesses potential applications in bone tissue engineering. Interestingly, we also created 3D nanofiber structures that range from simple forms to intricate architectures via origami, indicating that the combination of electrospinning technology and origami techniques is a feasible method for the 3D construction of tissue engineering scaffolds.
KeywordsElectrospinning Origami 3D construction Nanofiber scaffold/cells complex
Electrospinning, as a universal and cost-effective technology, has aroused worldwide attention and has been explored for extensive applications including photovoltaic devices , actuators [2, 3], filtration, catalyst supports , dielectric separators [5, 6, 7, 8], drug delivery [9, 10, 11, 12], composite reinforcements [13, 14, 15]. In recent years, electrospinning has been widely adopted in tissue engineering because it can continuously produce nano- to microscale fibers that simulate the structure of extracellular matrix (ECM) [16, 17, 18, 19]. Although electrospun nanofibers possess many merits including large surface area-to-volume ratio, high specific surface and multiple designs of surface modification [20, 21, 22], traditional electrospinning only can prepare two-dimensional (2D) membranes due to the technical limitations. To overcome the inherent nature of the electrospinning process and obtained three-dimensional (3D) structures, two main kinds of methodologies have been developed to address this issue . The first one relies on the precise manipulation of the electrospinning process via either continuous electrospinning or multilayer electrospinning [11, 24, 25], introducing 3D template to replace the 2D planar collector [26, 27, 28] or adjusting parameters (such as solution concentration, electric field strength and relative humidity) to realize the self-assembly of nanofibers [29, 30, 31]. 3D nanofiber structure with a thickness of several hundred micrometers could be obtained by continuous electrospinning time, but this was time-consuming and the thickness of the 3D structure was still limited . Most researchers designed collectors with micro-/nanoscale layouts to fabricated patterned nanofibers, and they focused on the configuration in microscale, rather than the 3D architecture in macroscale [32, 33, 34, 35]. Although 3D cotton-like nanofiber structures were prepared by adjusting electrospinning parameters, the mechanism of the nanofiber assembly was uncertain and the mechanical properties of the 3D structures were poor. Instead of direct control over electrospinning process, the second one realized 3D construction through post-treatment of electrospun nanofibers, such as overlaying, folding and curling [36, 37, 38]. Post-treatment is relatively simple, but the 3D nanofiber structures often have large distances between adjacent fiber surfaces, and in this case, cells only adhere and spread on the 2D surfaces rather than forming a bridge between adjacent surfaces. Thus, the 3D nanofiber structures fabricated by post-treatment cannot be directly used for tissue engineering. To improve this methodology, we examined closely the processes of folding and unfolding, that is, origami , and proposed a facile and effective method for 3D construction of electrospun nanofibers by combining the origami and electrospinning.
As a traditional paper art, origami can produce elegant and intricate 3D objects from planar sheets through a series of folding techniques . Since people recognized that nature achieved complex architectures ranging from proteins  to plants  via the utilization of controlled folding and unfolding sequence, the method of origami assembling has attracted significant scientific and technological interest. So far, the novel origami assembly has been used to fabricate DNA-based objects [42, 43], which are nanoscale. In addition, nano- to mesoscale structures also have been obtained via origami, such as 3D metal objects [44, 45, 46, 47] and silicon solar cells , that are lithographically patterned and spontaneously folded via surface tension effects [49, 50]. Therefore, how to exert the ability to assemble electrospun structures of arbitrary 3D architecture and functionality with the ease and versatility of origami is significant.
Materials and methods
The PCL pellets (molecular weight 80,000), nHA powders (particle size less than 200 nm) and Docusate sodium salt (AOT, BioUltra) were purchased from Sigma-Aldrich (USA). Dichloromethane (DCM, AR) and dimethylformamide (DMF, AR) were purchased from Aladdin (China). All chemical reagents were used as received.
Preparation of PCL/nHA composite nanofibers
A certain quality of nHA (0, 0.156, 0.350 and 0.600 g) and AOT (0.005 g) were simultaneously added to a 10-mL mixture of DCM and DMF (volume ratio = 1:1) and underwent ultrasonic dispersion for 60 min. During ultrasonication, an anionic surfactant AOT was added to promote the complete dispersion of nHA in DCM/DMF and lower the surface tension of the mixed solution . Cold water was then added discontinuously to the ultrasonic cleaner (SB-5200DT, Ningbo Scientz Biotechnology, China) to ensure a constant temperature. After that, the PCL (1.4 g) was added to the homogeneous suspension of nHA/(DCM + DMF) and completely dissolved by continuous magnetic stirring overnight at room temperature (RT) following the pretreatment of stirring (60 min) and ultrasonication (30 min). According to the initial quality of nHA, a series of PCL/nHA compounds with nHA concentrations of 0, 10, 20 and 30 wt% were produced, which were the mass ratios of nHA and the total solute (nHA + PCL) in the final mixtures. Before electrospinning, for each trial, the PCL/nHA mixture was ultrasonicated for 30 min.
The electrospinning was conducted using a conventional procedure as described in our previous work . Typically, the mixture was filled in a 10-mL plastic syringe equipped with a 24-gauge stainless steel blunt-tipped needle (inner diameter: 0.30 mm), and then, electrospinning was performed under a series of constant parameters: flow rate, 1 mL/h; distance between the blunt needle tip and the collector, 12 cm; supplied positive voltage, 16.2 kV; temperature, 37 °C ± 3; and relative humidity, 45% ± 3. All nanofibers were collected with an aluminum foil wrapped on a grounded rotary drum (diameter = 100 mm). The rotating speed of the rotary drum was 200 rpm, and the collecting time was 13 min (for morphological characterization) or 4.5 h (for the other physicochemical characterization and 3D construction via origami). To obtain samples with suitable size and thickness for mechanical characterization and origami, the spinneret moved along the lateral scanning frame during electrospinning (moving distance, 54 mm; and moving speed, 27 mm/s). The collected nanofiber films were subsequently dried for at least 2 days under vacuum to remove any residual solvents. The electrospinning process was conducted using a commercial electrospinning machine (FM1108-Electrospinning System, Beijing Future Material Sci-tech, China).
To construct 3D nanofiber scaffolds with massive structure, we assembled the electrospun films via origami, and the specific process is as follows (Fig. 1): (1) The PCL/nHA composite nanofiber films were cut into small squares (1.0 cm × 1.0 cm) using a commercial punch; (2) In addition, the nanofiber films were also cut into a plurality of rectangles [width of 2.7 cm (1.0 cm × 2 + 0.7 cm) and length of 3.0 cm (1.0 cm × 3)], which were used for the origami construction of nanofiber boxes (the inset in Fig. 1, without cover, length of 1.0 cm, width of 0.7 cm and height of 1.0 cm); (3) The normal cultured hFOBs were seeded on one side of the nanofiber film (1) with an appropriate concentration (5 × 104/cm2), adhered and cultured for 1 day, a duration that ensure cells adhere to nanofibers well with a suitable density; (4) Turning the nanofiber film (3), seeding hFOBs on the other side of the nanofiber film with the same concentration and culturing for 1 day; (5) When hFOBs adhered well on the both sides of the nanofiber film, the nanofiber films (4) were overlaid layer by layer and inserted into the nanofiber boxes (2). During the process of overlaying, the structural integrity of the nanofiber films should be ensured; (6) Repeating the operation of overlaying and inserting (5) until the nanofiber boxes were filled with nanofiber films; (7) The 3D structures (6) were transferred into a humidified incubator with 5% CO2 (37 °C) and then cultured in vitro. During the process of the culture, the cell culture medium was refreshed every three days; (8) When endpoints (14 and 30 days) were reached for assays, the 3D structures were removed and detected via various analysis methods. In this process, we expect the hFOBs will proliferate and migrate through the nanofiber films, which are confined in the nanofiber boxes. Most importantly, the cells adhered on the both sides of the nanofiber films also secrete ECM, which can bond the adjacent nanofiber films, resulting in the formation of 3D scaffold/cells complex.
Morphologies of the composite nanofibers and the 3D scaffolds
The micromorphologies of the nanofibers and the 3D scaffolds were observed by a field emission scanning electron microscope (FESEM, Merlin, Zeiss, Germany) with an accelerating voltage of 5 kV. Prior to SEM imaging, the samples were sputtered with platinum for 120 s by a high-vacuum sputter coater ion sputter (Q150T, Quorum, UK). Moreover, the macromorphologies of the nanofiber boxes and the 3D scaffold/cells complex were characterized via a digital single-lens reflex camera (DSLR, Nikon, Japan).
Mechanical properties of the composite nanofiber films
The mechanical properties of the nanofiber films were determined using a universal material testing machine (5967, Instron, USA) at a crosshead speed of 5 mm/min with a 250-N load cell (the maximum) in an ambient environment. All samples were sectioned into dumb-bell shapes with a test rectangular dimension of 13 × 5 mm (length × width) and a thickness of about 0.08 mm, measured using a digital screw micrometer. At least five samples were tested for each type of nanofiber films, and the mean and standard deviation were calculated.
Surface components of the nanofiber
Powder X-ray diffraction patterns of PCL, nHA and PCL/nHA composite nanofibers were recorded on a X-ray diffractometer (XRD, D8 ADVANCE, Bruker, Germany) with Cu Kα (λ = 0.15418 nm) incident radiation. The XRD data were collected between 5° and 90° in intervals of 0.02° and a scan rate of 2°/min.
Thermal properties of the scaffold
A differential scanning calorimeter (DSC, Pyris Diamond DSC, PerkinElmer, USA) was used in a temperature range of − 40 to 100 °C at a heating rate of 5 °C/min under a nitrogen atmosphere to evaluate the thermal properties of the scaffolds. To eliminate the thermal history, all specimens underwent two heat cycles from − 40 to 100 °C. Dried samples with a weight of approximately 10 mg were loaded in an aluminum crucible. The thermal stability of the scaffolds was determined using a simultaneous thermal analyzer (STA, STA449 C, Netzsch, Germany) under a nitrogen atmosphere. The temperature range used was 30–1000 °C, with unchanged sample weight and heating rate.
Human fetal osteoblasts (hFOBs, ATCC, USA) were propagated in conventional growth medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM/F12 (1:1), Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin stock solution (Beyotime Institute of Biotechnology, China). Studies on cell behaviors were performed with hFOBs within four to five passages, which were digested and collected by the addition of 0.25% trypsin–EDTA solution (Gibco, USA). In all experiments, the cell culture medium was refreshed every 3 days. The PCL/nHA-20 wt% composite nanofiber films which have been sectioned were introduced into 6-well TCP plates (Jet Bio Filtration, China) and sterilized with an irradiation of 15 kGy. After immersing in the cell culture medium for 3 days to remove any residual, the hFOBs were seeded on the nanofiber films as introduced in the chapter of 2.2 (the construction of 3D nanofiber scaffolds with massive structure, Fig. 1).
Assembling of 3D scaffold/cells complex
To analyze the cell behaviors on the 3D nanofiber scaffolds and the assembling of 3D scaffold/cells complex, the staining of cell skeleton/nucleus was performed. The hFOBs were seeded onto the nanofiber scaffolds and cultured for 14 and 30 days. Then, the scaffold/cells samples were transferred to a new TCP plate and washed twice with DPBS. The samples were fixed with 4% neutral formaldehyde (ACS, Aladdin) at 37 °C for 40 min and then immersed in 0.1% Triton X-100 (Biochemical, Aladdin) for 7 min to increase permeability. Finally, the fixed cells were stained with Cell Navigator TM F-Actin Labeling Kit (AAT Bioquest Inc, USA) and DAPI (Beyotime Institute of Biotechnology, China) working solution at 37 °C for 70 and 9 min, respectively. All the stained cells were rinsed thrice and observed by a laser scanning confocal microscope (LSCM, Leica, Germany).
Results and discussion
Morphologies of the composite nanofibers
Mechanical properties of the composite nanofiber films
In addition to the biocompatibility, biodegradability and interconnected pores, the ideal scaffold should also have mechanical stability. The combination of chemical and mechanical properties of scaffold plays an important role in cell proliferation and tissue formation, and it is also a key technique for the successful construction of nanofiber scaffold. The mechanical properties of electrospun nanofiber scaffold include elastic modulus, tensile strength and elongation, which are reflected in the stress–strain curves. Specifically, they correspond to the linear slope of the initial elastic region, the highest point of the coordinate, and the abscissa value corresponding to the highest point of the coordinate, respectively. As shown in Fig. 3b, all the nanofiber scaffolds have similar stress–strain curves, showing the initial elastic region and the final fracture failure. The elastic modulus and tensile strength of PCL/nHA composite nanofiber scaffolds increased significantly with the increase in nHA content. On the contrary, the elongation at break decreased dramatically. When the nHA content increased to 30 wt%, the elastic modulus and tensile strength of the composite nanofiber scaffold decreased, even lower than those of the pure PCL nanofiber scaffold. Interestingly, the stress–strain curve of the PCL/nHA-30 wt% composite nanofiber scaffold showed the double yielding. This may be due to the content of nHA particles is too high, and the nHA aggregates with large scale will break the entanglement of PCL polymer chain, which reduces the mechanical properties of the composite nanofibers, whereas the friction and the slide between the inorganic particle aggregates and organic polymer chain lead to the double yielding after the fracture. In other words, with the appropriate incorporation of the nHA, the elastic modulus and tensile strength of PCL/nHA increased, while the elongation decreased.
For the composite nanofiber scaffolds, the increase in tensile strength may be attributed to the effective integration of nHA and PCL, which is inspired by the organic/inorganic composite nanostructure of natural tissues (such as bone). Higher elastic modulus of elasticity and lower elongation at break can be explained by the fact that the nHA particles endow the polymer nanofibers more hardness and less plasticity during the deformation process, which is the necessary form of the inorganic phase filling. It is well known that nHA can promote the interaction between materials and osteoblasts, improving the bone conductivity. However, the brittleness of nHA has limited its applications, and it is only used in unloaded and nonbearing applications or as the coating material for metal implants [54, 55]. Although PCL is a synthetic polymer material with superior extension performance, it cannot be used in hard tissue engineering because of its poor mechanical properties . Therefore, the combination of the advantages of these two components in composite nanofibers may synergistically overcome their mechanical defects, providing satisfactory nanofiber scaffolds for the application of bone tissue engineering.
Surface components of the nanofibers
Thermal properties of the scaffolds
DSC results of PCL/nHA composite nanofiber scaffolds
nHA concentration (wt%)
Morphologies of the 3D nanofiber scaffolds
Assembling the scaffold/cells complex
After culturing for 30 days, the nanofiber films in the 3D nanofiber box which was prepared by origami were taken out, and the results are shown in Fig. 7b and Video S1. It is clear that the fibrous films were no longer individuals and separate sheets, but a block of nanofiber films. When the nanofiber block was picked by clamping the local part, it would not be scattered and maintained integrity, even with shake. That is, without the use of other bonding process, the nanofiber films limited in a specific space (the nanofiber box prepared by origami) realized the filling of gaps and the bonding of the adjacent nanofiber films through the secretion of ECM, resulting in the formation of the 3D nanofiber scaffold/cells complex.
Macromorphologies of 3D nanofiber structures prepared by origami
The 3D nanofiber scaffold/cells complex was obtained through the combination of origami craft and electrospinning technology. Considering the applications of the 3D nanofiber scaffold, the bioactive nHA was incorporated into the biocompatible PCL, and we eventually selected PCL/nHA-20 wt% composite nanofibers to construct 3D scaffolds after investigating the properties of the PCL/nHA composite nanofibers with different nHA concentrations. With the structural design, the layered nanofiber scaffold/cells complex with 3D structure was constructed, and the gap between the layers was about 10 μm. The hFOBs cultured on the both sides of the nanofiber films stretched across the gaps and presented 3D distribution. The ECM secreted by cells realized the gap filling and film bonding between the adjacent nanofiber films, eventually forming a 3D nanofiber scaffold/cells complex. In addition, the origami performance of the electrospun nanofiber was verified by the construction of 3D structures with simple and intricate shapes. These 3D origamis underwent repeated folding and unfolding operations which can maintain the structures and shapes well. All these results indicate the operability of this method—the combination of origami and electrospinning, and it provides a new strategy for the construction of tissue engineering scaffold.
The verification of the formation of the 3D nanofiber scaffold/cells complex (Video S1).
This study was financially supported by grants from the National Natural Science Foundation of China (51232002, 51502095, 31771027), the Guangdong Natural Science Funds for Distinguished Young Scholar (2016A030306018) and the Guangdong Natural Science Funds (2017B090911008).
Supplementary material 2 (MOV 15823 kb)
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