Layer-by-layer 3-dimensional nanofiber tissue scaffold with controlled gap by electrospinning

In recent years, a significant amount of research has been directed toward the development of tissue scaffolds used for tissue engineering owing to their ability to mimic natural surroundings of tissues, which is beneficial to cells growth and differentiation into tissues [1–7]. The three-dimensional (3D) substrates that possess adequate mechanical strength and an appropriate bio-degradation rate without undesirable by-products is known as an ideal tissue scaffolds serves as a template for tissue regeneration. For example, it has been proven to be effective stimulants for chondrogenesis in articular cartilage tissue engineering [8].

Electrospinning is a simple yet versatile technique that allows the fabrication of continuous nanofibers with diameters from micrometers down to a few nanometers [9–13]. Ultrafine nanofibers have been produced with large surface-area-to-volume ratios and superior mechanical properties of stiffness [14] and strength [15]. These outstanding properties of electrospun nanofibers show a great potentiality in the fabrication of bio-scaffolds [16] from a variety of polymers. However, conventional electrospinning process generally produces two-dimensional (2D) mats with randomly arranged structures on the collectors, which may limit their application. Several novel approaches have been considered for the electrospinning of aligned or 3D polymer nanofibers (several examples were shown in figure S1 is available online at stacks.iop.org/MRX/5/025401/mmedia, supporting information) [17–20]. Chase et al demonstrated a simple method for spinning aligned polymer nanofiber sheets by the use of copper wires spaced evenly in the form of a circular drum as a collector [17]. Jiang et al have fabricated well-aligned polymeric micro and nanofibers over large areas. The technique involves a polymeric solution magnetized with small amounts (<0.5 wt%) of magnetic nanoparticles as the substrate and carrying out the spinning process in a magnetic field [18].

To date, only a few examples of tissue cells embedded in 3D architectures were reported in the literature, which may be attributable to their uncontrollable structural morphology by using conventional electrospinning techniques [3, 21–23]. In 2008, Moroni et al developed a novel 3D scaffolds fabricated by combining 3D fiber deposition and electrospinning, which consisted of integrated 3D fiber periodical macrofiber and random electrospun microfiber networks [21]. Among natural polymers, collagen is the most abundant protein in mammals, which is widely distributed in nature. To date, collagen scaffolds have already been used in the medicinal field for drug delivery, soft tissue augmentation, tissue regeneration and tissue engineering [24]. Generally, polycaprolactone (PCL) is also considered to be useful in tissue engineering due to its biocompatibility, biodegrad-ability, structural stability, and mechanical properties. Kannarkat et al reported a 3D fibrous scaffold based on PCL for tissue engineering fabricated through the electrospinning process [3]. The fabricated nanofibrous polymer scaffolds of PCL incorporating magnetic nanoparticles are beneficial to cell growth, proliferation, and differentiation. It has already been reported that collagen/gelatin could attribute to a better cell attachment with its excellent biocompatibility and affinity. An increasing attention has been aroused to the combination of the PCL and collagen composite scaffolds. Bak et al demonstrate collagen nanofibers with low humidity and high ethanol content supported the cell growth [25]. Chong et al reported fibroblast cells were successfully cultivated on both sides of a thin PCL/gelatin nanofiber scaffold [26]. Choi et al reported the combination of fish collagen and PCL scaffolds at a certain ratio (1:9 and 2:8) increased the cell proliferation compared to the PCL's [27].

Many studies have shown that the size and surface texture of electrospinning nanofibrous polymer scaffolds have a pronounced impact on both cell proliferation rate and morphology [28, 29]. Therefore, the development of 3D structures of nanofibers with finely controlled morphology by electrospinning is quite desirable in the field of tissue scaffolds. Here, we show a facile method of fabrication of layer-by-layer three-dimensional PCL nanofiber structures with controlled gap by utilizing booklet collector. The 3D architectures were coated with the collagen based solution before cell culture. We expect that this class of manufacturing will allow the design of a variety of 3D architectures and can serve as building blocks for new metamaterials in the field of tissue scaffolds.

Materials

Preparation of 3D cell scaffold

A solution of 10 wt.% polycaprolactone (PCL) and chloroform was prepared by dissolving 1.0 g of powdered PCL in 9.0 g of chloroform. A solution of 5.0 mg ml⁻¹ collagen was prepared by dissolving 0.05 g of powdered collagen in 10 ml deionized water. Then the ingredients were stirred at 4 °C for 12 h to form the desired transparent and uniform dissolution of the polymer into the solvent.

As shown in figure 1, the spinneret in which the polymer solution is hosted was connected to a positive high voltage power supply of +15 kV. Plastic hollow sheets having side length 1 cm, length hollow 2 mm and thickness 20 ± 2 μm were mounted to the surface of the rotary cylinder used as the collector. The diameter of the cylinder is 10 cm. During electrospinning process, the rotating speed of the cylinder was kept for 2800 rad min⁻¹.

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As shown in figure 2(a), soluble collagen extracted from the body wall of sea cucumber [30]. In figures 2(b)–(e), the obtained single sheet that supported by aligned polymer nanofibers via this electrospinning method was immersed in a solution of 5 mg ml⁻¹ collagen for 1 min, and then dried in a vacuum oven at 40 °C to constant weight. Then these dried nanofibers sheets (30 layers) were superimposed on each other and fixed by a synthetic ethyl α-cyanoacrylate adhesive for gluing on the near surface of the resulting nanofibers multilayer. The resulting nanofibers multilayer was dried in a vacuum oven at 40 °C to constant weight, forming layer-by-layer 3D nanofiber scaffolds.

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Cell studies

Characterization

In this work, we mainly propose a novel methodology of creating layer-by-layer 3D polycaprolactone (PCL) nanofiber structures by utilizing booklet collector with controlled morphology (figures 1 and 2). The representative scanning electron microscopy (SEM) images of the self-bundling 3D scaffold PCL/collagen nanofibers prepared via this directly utilizing booklet collector are shown in figure 3. The layer-to-layer gap distance could be easily controlled by using plastic sheets with different thickness values. We choose HeLa cells as a typical example in the potential application of this created layer-by-layer 3D nanofiber structures as tissue scaffolds in tissue engineering. The experimental results show the fabricate 3D nanofiber structures with controlled gap (approximately 20 μm layer-to-layer distance) is beneficial for the HeLa cervix cancer cells migrate into tissues and induce cells in an ordered arrangement. The LSM images of HeLa cervix cancer cells growth in 3D scaffolds multilayer structures was shown in figure 4. In contrast, most of those reported 3D scaffold prepared by conventional methods generally suffer from disordered structure, which is unfavorable for cells migrate into tissues. Comparison of the non-layer structured 3D nanofibers and fabricated layer-by-layer 3D polycaprolactone (PCL) nanofiber structures on cells growth was carried out (figure S2, supporting information). The LSE results showed cells only grew on the surface of the non-layer structured 3D nanofibers after 24 h culture. A series of experiments were further undertaken to study the effect of the ratio of collagen to fibers on cells proliferation (table S1, supporting information). The different concentration of PSC (1, 2.5, 5, 10 mg ml⁻¹) on cell proliferation effect comparing with the control after 48 h of cell culture was shown in table S1 (supporting information). The results of addition of PSC concentration at 2.5 and 5 mg ml⁻¹ was of extremely significant difference (p < 0.01), and was significant difference (p < 0.05) for 10 mg ml⁻¹, while there is no significant difference for PSC at 1 mg ml⁻¹ (p > 0.05). The collagen ratios to fibers on cells proliferation result indicate the collagen solution at a concentration of 5.0 mg ml⁻¹ is much more favorable for the cells proliferation.

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The generation of two dimensional mats with conventional method limits electrospinning, the electrical charging of polymer liquids, as a mean of nanofiber fabrication. The main difference between our electrospinning arrangement and the conventional electrospinning apparatus is that plastic hollow sheets overlay bonded on the surface of a rotary cylinder and were used as the collector (figure 1). As the rotational speed of the cylinder increased to levels of 2800 rad min⁻¹, the polymer jets that are electrospun from the spinneret could be spontaneously attracted to the collector and fabricated sheet-like structure of aligned PCL nanofibers via this self-bundling and rotating electrospinning method.

As can be seen in the images of the hollow sheet surface of the produced 3D nanofibers (figure 3(c)), it was found clearly that aligned nanofibers can be obtained by electrospinning process. These results indicated that the generated nanofibers sheets were superimposed on each other by utilizing booklet collector with controlled morphology. Generally, the pore size as well as the morphology regularity of the scaffolds has a profound effect on the cell attachment, growth and migration [23]. For example, relatively bigger pores are beneficial to the cell migration within the scaffolds. In addition, the orientation selectivity of the cells could be significantly increased for parallel scaffold compared to that obtained in random scaffold. In this work, we have developed an empirical recipe to ensure the formation of 3D scaffold based on parallels nanofibers sheets with layer-to-layer distance of approximately 20 ± 2 μm, indicating the cells migrate freely into tissues and induce cells in an ordered arrangement. It can be observed in figure 3(d) the adhered cells was spread well over the substrate inside the gap of 3D scaffolds multilayer.

It is worth noting that the initial cell attachment and spreading are crucial factors in developing scaffolds for tissue engineering [23, 31]. To test the ability of 3D nanofiber assembly to boost cells growth as tissue engineering scaffolds, we cultured HeLa cervix cancer cells. The schematics of the cells growth in the 3D cell scaffold are illustrated in figure 4. Chemically dissociated HLCCs were seeded on the constructs where they bound to, and spontaneously aligned with the 3D fibers. It was found clearly that although a small number of cells were grown at the initial stage (figure 4(c)), cell clusters could be formed on 3D scaffolds after 3 days of culture (figure 4(d)), which demonstrated by the blue color in most of the volume. However, the cells only growth on the surface of the non-layer structured 3D nanofibers were observed after 24 h culture under the same conditions (figure S2, supporting information). This is presumably due to the limited diameter of the nanofiber compared to the cells, which prevents the adhered cells spread into the body of the non-layer structured 3D nanofibers.

The collagen/gelatin has been applied in the field of the cell culture and tissue engineering with its low viscosity and high solubility, the excellent biocompatibility and affinity that could probably enhance the cell adhesion and proliferation [25–27]. Jain et al illustrated the tumor cells can alive well in the pure collagen hydrogel and demonstrated the tumor cell migration distance was statistically significant on the aligned nanofiber scaffold compared with the smooth film [32].

The results in this study indicate the fabricated 3D PCL/collagen scaffolds by utilizing booklet collector might play an important role for cell attachment, growth and migration. It could be a useful model of 3D scaffolds for cell culture and may have a wide applicability for tissue engineering in the future.

To summarize, we have proposed a novel technique for creating 3D structures of nanofibers with finely controlled morphology. The size and morphologies of artificial 3D nanofibrous materials can be well-controlled by utilizing a booklet collector with controlled morphology. The experimental results show that the layer gap can be controlled in microns so that the cells can move to the inner structure of such a 3D tissue scaffold. The fabrication of 3D nanofibrous materials with controlled gap may also have far reaching impacts in many other important domains, such as material separation diaphragms, energy storage systems, and catalyst materials, etc.

This work was funded by the Science Technology Department of Zhejiang Province (No. 2012C37042), Scientific Research Fund of Zhejiang Provincial Education Department (No. Y201224739).