Three-dimensional Objects Consisting of Hierarchically Assembled Nanofibers with Controlled Alignments for Regenerative Medicine

Abstract

Assembling electrospun nanofibers with controlled alignment into three-dimensional (3D), complex, and pre-designed shapes have proven a difficult task for regenerative medicine. Herein, we report a novel approach inspired by solids of revolution that transforms two-dimensional (2D) nanofiber mats of a controlled thickness into once-inaccessible 3D objects with pre-designed shapes. The 3D objects are highly porous, consisting of layers of aligned nanofibers separated by gaps ranging from several micrometers to several millimeters. Upon compression, the objects are able to recover their original shapes. The porous objects can serve as scaffolds, guiding the organization of cells and producing highly-ordered 3D tissue constructs. Additionally, subcutaneous implantation in rats demonstrates that the 3D objects enable rapid penetration of cell, formation of new blood vessel, and collagen matrix deposition. This new class of 3D, hierarchical nanofiber architectures offers promising advancements in both in vitro engineering of complex 3D tissue constructs/models or organs, and in vivo tissue repair and regeneration.

Graphical Abstract

Complex, three-dimensional (3D) assemblies of nanofibers are ubiquitous in the extracellular matrix (ECM) of most human tissues.¹ Though nanofiber-based scaffolds have been widely used to mimic the architecture of ECM in native tissues, their 3D assembly remains a great challenge, despite the development of various fabrication techniques.^2–5 Recent studies have established the use of Origami or Kirigami, an ancient paper folding and cutting approach, to transform two-dimensional (2D) structures into 3D objects.^6–11 Such an approach is restrained to the rolling, bending, folding, wrinkling, and buckling of 2D structures, limiting the types of 3D objects that are producible. Researchers have also attempted to control the deposition of nanofibers during electrospinning for the production of 3D scaffolds, yet only several simple 3D architectures have been demonstrated including grids, walls, and hollow cylinders.^12–14 In general, direct collection of electrospun nanofibers as 3D objects remains in the early stage of development, facing many technological challenges. Despite its high cost, time consumption, combining 3D printing and melt electrospinning can only produce 3D structures of microscale fibers with limited thicknesses (typically, less than several millimeters).¹⁵

In mathematics, a solid of revolution refers to an object obtained by rotating a plane curve around a straight line (the axis of revolution) on the same plane. Based on the aforementioned concept, a 3D object can be obtained by rotating an area around a predetermined center line (called the axis of rotation). Based on the same concept, people have used the potter’s wheel to produce works of art several thousand years ago.¹⁶ Our recent studies demonstrated an innovative gas-foaming technique for expanding 2D nanofiber mats along the fiber deposition direction, generating 3D scaffolds with well-controlled thickness and porosity.^17–19 Inspired by the concept of solids of revolution, we argue that 2D nanofiber mats can be transformed into 3D objects with complex, pre-deigned shapes if we fix one side of the mat during the expansion process.

To test our hypothesis, we chose poly(ε-caprolactone) (PCL), a biocompatible, biodegradable polymer with several Food Drug Administration (FDA) approved clinical applications. To demonstrate the concept, we fabricated 1-mm thick mats of PCL nanofibers using a rotating mandrel to collect from electrospinning, as described by our previous studies.^17–20 We then cut the mats in liquid nitrogen into 2D objects with various shapes (e.g., rectangle, half circle, arch, and triangle) and used thermal treatment to fix one side of each object. Subsequently, the 2D objects were expanded in an aqueous NaBH₄ solution according to our previously established protocols.^17,18

As illustrated in Figure 1 a–d, the rectangular 2D object can transform into a 3D cylinder. During expansion, we observed the formation of a fan-shaped object at t=30 min, a half cylinder at t=60 min, a three-quarter cylinder at t=90 min, and a cylinder at t=135 min (Figure S1). Therefore, obtaining objects with pre-designed shapes can be achieved by freeze-drying sample at different time points of expansion. Applying negative pressure through a lyophilizer can speed up the expansion process (i.e., shorten the expansion time) following an initial expansion in NaBH₄ aqueous solution (Video S1). Figure 1e shows a photograph of the cylinders derived from 2D rectangular nanofiber mats after freeze-drying. Further characterization of the cylinders using scanning electron microscopy (SEM) revealed that they were comprised of numerous thin nanofiber layers (Figure 1f). The X-Y plane of the cylinder consisted of radially-aligned nanofibers, while the X-Z and Y-Z planes showed a highly porous structure (Figure 1, f–h). Gaps between adjacent cylinder layers ranged from several micrometers to hundreds of micrometers and could be tailored by varying the initial thickness of the 2D nanofiber mats. The layers of nanofibers were ca. 15 μm in thickness. The initial thickness of 2D nanofiber mats determined the porosity and pore size after the transformation to 3D scaffolds.

Figure 1. Schematic illustrating the transformation of 2D nanofiber mats into 3D objects with pre-designed shapes.

(a) A 2D nanofiber mat is produced by electrospinning and collected on a rotating mandrel. (b) The 2D nanofiber mat is cut into a rectangular shape in liquid nitrogen. (c) One side of the rectangular mat is fixed by thermal treatment. (d) The rectangular mat with one side fixed is expanded using a gas-foaming technique to generate a cylinder. (e) Photograph of the resultant cylinders. (f) SEM image showing the X-Y plane made of radially aligned nanofibers and the porous structure of the X-Y plane. (g) SEM image showing the porous structure of the X-Z plane. (h) SEM image showing the porous structure of the Y-Z plane. The fiber alignment is along the X-axis. Arrows indicate the direction of fiber alignment.

Applying the same principle, a triangular, semicircular, and arch-shaped 2D nanofiber mats were successfully transformed into a pre-designed cone, sphere, and hollow sphere, respectively, as demonstrated in Figure 2. Establishing a different direction of fiber alignment in the 3D objects was readily achieved by switching the side subject to thermal fixation (Figure S2). Cylinders comprised Z-axis aligned nanofibers mimic the structures of tissues with anisotropic properties, such as tendon, muscle, and nerve.^21–23 Similarly, other complex shapes, including circular cones, spheres, and hollow spheres, could be fabricated using the same strategy (Figure S2).

Figure 2. Transformation of 2D nanofiber mats in the shapes of triangle, semicircle, and arch, respectively, into a circular cone, sphere, and hollow sphere.

The fiber alignment is along the X-axis direction indicated by the blue arrows.

Tissues and organs with tubular structures are omnipresent throughout the human body, present in the vasculature (e.g., arteries, veins, capillaries), respiratory (e.g., trachea, esophagus), urinary (e.g., ureter, urethra, bladder), and gastrointestinal systems.²⁴ Given their widespread occurrence, tissue engineering of tubular organs is of great importance as a large number of surgeries are performed annually on these organs.²⁵ To this end, we sought to fabricate tubular nanofiber scaffolds (hollow cylinders) based on the cylinders derived from 2D nanofiber mats. Figure 3 a–d, illustrates the fabrication of hollow cylinders. Briefly, we compressed the cylinders into a 2D mat and cut along the fixed side. Then, we re-expanded the compressed and sliced mats, yielding hollow cylinders. Figure 3e shows a photograph of a hollow cylinder. Figure 3f shows the relating SEM image of the cylinder’s cross-section, revealing a highly porous, layered structure with gaps between layers that range from tens of micrometers to hundreds of micrometers (Figure 3g). Similarly, the thickness of each layer was around 15 μm (Figure 3h). By varying the alignment of fibers in the 2D mats, hollow cylinders consisting of aligned fibers in either the radial or longitudinal direction could be readily generated, which could be useful in mimicking the smooth muscle structures in the tubular tissues.

Figure 3. Fabrication of hollow cylinders of nanofibers.

(a-d) Schematic illustrating the fabrication of a hollow cylinder through repeated expansion (red line indicates the fixed side). A typical procedure including the compression of the cylinder into a 2D mat, cutting off a certain portion (marked by the black dash line in c), and re-expansion. (e) Photograph of a hollow cylinder. (f-h) SEM images of a hollow cylinder at different magnifications. The nanofibers were aligned along the longitudinal direction of the tube. The red arrow in (g) indicates the fiber alignment direction.

Previously, we demonstrated the expansion of 2D nanofiber mats into ordered 3D structures using gas bubbles generated by chemical reactions in an aqueous solution.^17,18 Despite the advancement, this method has a number of limitations, including multiple time-consuming steps, the use of an aqueous solution, the necessity of freeze-drying, possible reactions between NaBH₄ and the polymers or encapsulated substances, the possible loss of encapsulated bioactive materials in the fibers, bioactivities of biomacromolecules incorporated in the fibers, and the poor suitability for water-soluble materials. Our recent study demonstrated that many of these issues could be alleviated by switching to a low-temperature, CO₂-based expansion, which better maintains the activity of the encapsulated bioactive materials compared to the previous approach.¹⁹ Therefore, we also aimed to transform 2D nanofiber mats into 3D objects with pre-designed shapes through depressurization of subcritical CO₂ fluid. To demonstrate the concept, a unilaterally fixed 2D rectangular mat of nanofibers was depressurized in subcritical CO₂ fluid, as described in our recent study.¹⁹ Depressurization of subcritical CO₂ at different timepoints successful synthesized the desired 3D shapes (Figure S3). Similar to the expansion in aqueous NaBH₄ solution for 30 min, fan-shaped objects were formed from both one and two depressurizations of subcritical CO₂ (Figure S3, a and b), three depressurizations yielded a three-quarter cylinder (Figure S3c), and four depressurizations yielded a full cylinder (Figure S3d). Our recent work demonstrated that gelatin-coated, expanded nanofiber matrices showed superelastic and shape-recovering properties in air and liquid.²⁶ Based on a similar principle, gelatin-coated nanofiber cylinders almost entirely recovered their original shape (>95%) after first compression (Video S2). Interestingly, the coated nanofiber cylinders recovered more than 75% of their original shapes after the fourth compression (Video S3–S5). By reducing the pressure during re-expansion, 100% shape recovery was readily achieved in water (Video S6). Such a shape-recoverable property allows for the development of nanofiber-based objects with complex shapes for use in the minimally invasive surgery as the compressed 3D nanofiber objects could be delivered using catheters or cannula and then re-expand to their original shapes after delivery.

Previous studies showed that aligned nanofibers provide contact guidance for various types of cells.^27–30 The 3D objects comprised of thin films of aligned nanofibers could aid the organization of seeded and proliferated cells to form highly-ordered tissue constructs. To demonstrate, we seeded green fluorescent protein (GFP)-labeled dermal fibroblasts into transformed cylinders (diameter: 10 mm; height: 1.5 mm) with radially and longitudinally-aligned nanofibers in the X-Y plane, respectively. Figure 4 shows the dermal fibroblasts seeded on the cylinders for 1 day and 3 days, respectively. Due to the limited imaging thickness of optical confocal microscopy, cells 135 and 150 μm from the top surface of the cylinder were imaged and shown in Figure 4. Uniform cell distribution was observed throughout the thickness of the cylinder, with the proliferated cells displaying a radially-aligned pattern in each scanning layer (Figure 4a, Video S7, S8). Cellular alignment emulated the X-Y plane of the nanofiber-based cylinders, with dermal fibroblasts longitudinally aligned in the X-Z cylinder plane (Figure 4b, Video S9, S10). To evaluate the potential for neural tissue construct formation, we further demonstrated the culture of rat neural progenitor cells on the cylinders consisting of radially-aligned nanofibers. Neural precursor cells were evenly distributed throughout the cylinder and able to proliferate and differentiate into neurons, exhibiting an organized structure (Figure S4). The neurites were displayed in a radial fashion emulating the alignment of the nanofiber in the cylinder. Such 3D, highly-ordered neural tissue constructs could be used for the construction of in vitro 3D neural tissue models and repair of nerve injuries.

Figure 4. GFP-labeled dermal fibroblasts cultured on the expanded scaffolds consisting of radially- and longitudinally-aligned PCL nanofibers, respectively.

(a) Distribution of the human dermal fibroblasts in the 1-mm-thick, expanded scaffold of radially-aligned PCL nanofibers after incubation for 1 day and 3 days, respectively. (b) Distribution of the human dermal fibroblasts in the 1-mm-thick, expanded scaffold of longitudinally-aligned PCL nanofibers after incubation for 1 day and 3 days, respectively.

Previous studies reported direct deposition of radially-aligned 2D nanofiber membranes on a special collector consisting of a ring electrode and a centrally located point electrode during electrospinning.^27,31–34 Though this 2D membrane directed cell migration from surrounding areas to a center point, it is restricted to the generation of 2D nanofiber membranes with limited thicknesses. Our current work overcomes this limitation by generating radially-aligned nanofiber scaffolds/devices with increased thicknesses while maintaining ideal porosity. Such nanofiber cylinders have applications for in situ tissue regeneration and wound healing, as the radially-aligned nanofibers can direct and promote cell migration into the scaffolds from surrounding host tissues. In addition, cells migrating from host tissues can penetrate the cylinders through the surrounding side faces and upper and lower surfaces, offering a major advantage when compared to the previously developed 3D expanded nanofiber scaffolds, where cells mainly infiltrated from only the surrounding side faces.^17–19

To further demonstrate the in vivo response, we implanted cylindrical objects of nanofibers (diameter: 10 mm; height: 1.5 mm) subcutaneously in rats as acellular scaffolds. Phenomenally, H&E staining results showed many cells infiltrated and migrated throughout the objects, even after implantation for 1 week, a phenomenon that was rarely seen for electrospun nanofiber-based scaffolds (Figure 5, a and b).¹⁸ Cell penetration into the scaffolds increased with time post-implantation, culminating with tissue formation at 8 weeks (Figure S5). Given the tissue formation results, we can assume fiber alignment helps guide cell infiltration and organization, thus forming new tissues. Gaps between adjacent nanofiber layers, possible cell penetration from all sides (including top and bottom surfaces), and contact guidance rendered by aligned nanofibers all likely contributed to the rapid cell penetration. Further, Masson’s trichrome staining showed corresponding collagen deposition (i.e., ECM production) and new blood vessel formation within the implanted nanofiber cylinders (Figure 5, c and d). Similarly, collagen deposition and blood vessel formation occurred throughout the cylinders after implantation for 1 week. The amount of deposited collagen positively corresponded with time post-implantation (Figures 5 and S5). These results indicate that implantation of such nanofiber scaffolds can rapidly generate new tissues through cell infiltration, ECM deposition, and neovascularization. One possible limitation for this technology is that the fibers become crimped after transformation from 2D mats to 3D scaffolds. It is not clear if crimped structures could affect the applications of 3D scaffolds in guiding/regulating cellular behavior (e.g., cell migration). On one hand, the collagen fibrils in some soft tissues exhibit crimped structures.³⁵ The transformed 3D scaffolds could show better biomimetic property. On the other hand, the crimped structures may not be optimal for providing contact guidance of neurite outgrowth as axons may take longer time to reach the target along the curved nanofibers than the straight ones.

Figure 5. In vivo tests of the expanded scaffolds consisting of radially- and longitudinally-aligned PCL nanofibers by subcutaneous implantation in rats for 1 and 8 weeks.

(a) H&E staining showing cell infiltration in the expanded, radially-aligned PCL nanofiber scaffolds. (b) H&E staining showing cell infiltration in the expanded, longitudinally-aligned PCL nanofiber scaffolds. (c) Collagen deposition (green arrows) and formation of new blood vessels (red arrows) within the expanded, radially-aligned PCL nanofiber scaffolds. (d) Collagen deposition (green arrows) and formation of new blood vessels (red arrows) within the expanded, longitudinally-aligned PCL nanofiber scaffolds. Green dots indicate the boundary between the surrounding tissues and the scaffolds.

In summary, we have demonstrated a novel method of transforming 2D nanofiber mats into 3D objects with pre-designed, complex shapes. The 3D objects contained highly porous, layered structures while simultaneously retaining nanofiber alignment. Such 3D nanofiber objects exhibited shape-recovery properties following compression. Additionally, the 3D objects were guided the organization of seeded cells, generating highly-ordered 3D tissue constructs. Similarly, the 3D objects were able to promote cellular infiltration, ECM deposition, and neovascularization after subcutaneous implantation in rats. Future studies investigating the combined use of the complex 3D nanofiber objects with hydrogels and 3D printing technology for engineering 3D in vitro tissue models and constructs and regenerating a variety of tissues in vivo are underway. Finally, we envision the 3D objects fabricated in this study are highly translatable to other fields, such as energy and environmental science.

Supplementary Material

Supporting Information

Experimental details are included in the Supporting Information.