Nanofiber with hydrogel

Hydrogel-nanofiber composite

Hydrogel is one of the most commonly used 3D scaffolds for tissue engineering. Nevertheless, incorporation of nanofibers into hydrogel has been shown to improve the mechanical properties of the hydrogel [Kai et al 2012; Visser et al 2015; Liu et al 2014] and the presence of nanofibers may also potentially improve or influence cell activity in the resultant composite [Yang et al 2011]. Since hydrogel is often prepared from a solution state, constructed nanofiber membranes can be unraveled (by manual tearing or cutting of the membrane) and mixed with the solution [Sakai et al 2008]. Another method is to incorporate layers of nanofibers in hydrogel in a layer by layer assembly [Yang et al 2011, Xu et al 2013]. By having electrospinning and electrospraying of hydrogel simultaneously, a hybrid nanofiber and hyrdogel 3D structure can be constructed [Ekaputra et al 2008]. Other researchers have resorted to chopping electrospun nanofibrous membrane or yarn for incorporation into hydrogel blocks [Kai et al 2012; Liu et al 2014]. Using a single electrospun PCL layer, Ghafari et al (2017) was able to form mechanically stable hybrid structure with natural urinary bladder extracellular matrix (ECM). The decellularized rat bladder tissue used in this model was in the form of a gel which lacks mechanical stability and integrity. In this hybrid structure, the gel was first injected into a cylinder. Next, the electrospun membrane was placed on the gel before a second gel layer was added on top of the membrane. High porosity of the electrospun membrane probably helped to ensure integration with the gel.

Chopping electrospun fibers into shorter strands will allow better distribution of the fibers throughout the hydrogel if they can be separated into individual strands. Rivet et al (2015) fabricate individual short strand nanofibers by collecting aligned fibers on a substrate with a thin film of polyvinyl alcohol (PVA) as release agent. The substrate with the deposited aligned fibers was cut into short segments of about 1 mm width, perpendicular to the fiber orientation. The PVA was subsequently removed from the fibers by dissolving in water. The dispersed chopped fibers were finally mixed in hydrogel.

Gel may be used in combination with electrospun fibers to give a more transparent scaffold. For thicker electrospun membrane, spaces may be created on it to increase its optical transparency in a hybrid structure. To enhance the transparency of electrospun aligned nanofibers, Kong et al (2017) used laser to create perforations of diameters in the range of 100 to 200 µm at intervals of 50 to 100 µm on the membrane. This perforated electrospun poly (lactic-co-glycolide) (PLGA) membrane was sandwiched between collagen gels and compressed to give a hybrid construct. With the perforation, the optical transmittance was 15 fold higher than hybrid construct with non-perforated membranes. This hybrid construct was found to exhibit an optical transmittance of 63% and this was increased to 72% after 7 days of immersion in PBS solution.

The presence of electrospun nanofibers in hydrogel regardless of form has been shown to improve its mechanical properties. Anderson et al (2008) used electrospun polycaprolactone as scaffolding to support the vertical stability of standing swollen hydrogel that surrounds microfabricated sensor hairs. Focusing ring was used to concentrate electrospun polycaprolactone fibers such that it rest vertically on the sensor hairs before the hydrogel was added over it. With the fiber scaffolding, the hydrogel capula can be extended up to 3 times that of unsupported hydrogel and this raises its aspect ratio. Liu et al (2014) incorporated chopped electrospun yarn made of poly(L-lactide-co-ε-caprolactone) (P(LLA-CL) with length of about 1 mm and mixed with the collagen to give a weight ratio of 1:1. The storage modulus of the collagen hydrogel with chopped nanofibrous yarns was found to be much greater than pure collagen hydrogel. Kai et al (2012) cut electrospun poly(ε-caprolactone) (PCL)/ gelatin nanofibrous sheets into small pieces and adding these to gelatin solution at a weight ratio of 25 or 50 mg/ml. Both poly(ε-caprolactone) (PCL)/ gelatin nanofibers made from blended system or core-shell system were shown to significantly improve compressive strength, degradation rate and swelling ratio over pure gelatin hydrogel.

Electrospun membrane without further modifications or processing has also been encapsulated within hydrogel to form a composite structure. Since hydrogels are generally water-based, the electrospun membrane should be hydrophilic to allow full integration of the hydrogel solution within the pores of the membranes. Eslami et al (2014) showed that with electrospun polycaprolactone (PCL), the resultant membrane was too hydrophobic to absorb the hydrogel. With the addition of poly(glycerol sebacate) (PGS) to PCL, the resultant electrospun membrane was sufficiently hydrophilic for hydrogel absorption. Culturing of cells on the composite seem to suggest that they were able to penetrate into the fibrous layer while cells cultured on electrospun membrane alone were restricted to the membrane surface [Eslami et al 2014]. In this case, swelling of the hydrogel component within the composite may open the pores between the fibers such that cells were able to infiltrate into the structure. Visser et al (2015) used melt-electrospun 3D printed scaffold for incorporation with hydrogel. The resultant stiffness of the gel/scaffold increased up to 54-fold compared to either gel or scaffold alone. The stiffness and elasticity of the gel/scaffold composite was found to approach that of articular cartilage. Hydrogel may function as a binder in a hydrogel-electrospun fiber combination. Apart from stacking layers of electrospun membrane, a single long membrane may be rolled into a spiral cylinder and fixed using a hydrogel. Kosowska et al (2020) demonstrated this technique using electrospun polylactide/hydroxyapatite (PLA/HA) fiber membrane and chitosan/graphene (CS/GO) hydrogel in the construction of a hierarchically organized bone scaffold. Electrospinning was first carried out using a blend of PLA and HA to form a fibrous membrane. A CS/GO suspension was spread over the membrane before rolling into a cylinder. The construct was frozen before immersing in gelling solution to form the final scaffold. The steps of coating the membrane and freezing the construct is important to maintain a gap between the overlapping membrane. This gap meant that cells will be able to infiltrate into the depth of the scaffold.

An advantage of using hydrogel in combination with electrospun 3D scaffold is in its cell seeding. Cells may be encapsulated in hydrogel and injected into a loosely packed 3D scaffold. Formica et al (2016) used cryoelectrospinning of polyε-caprolactone to create a loosely packed network of fibers. In this process, the fibers are deposited on a -78 °C mandrel where ice crystals formed over the deposited fibers forms a barrier for incoming fibers. This created a very loose network of electrospun fibers which allows cell carrying hydrogel to be injected into it. This creates a composite scaffold with a uniform distribution of seeded cells.

Concurrent electrospinning of nanofibers and electrospraying of hydrogel [Ekaputra et al 2008]. Click image to enlarge

A limitation of hydrogel is that they generally do not influence direction of cell growth. Electrospun scaffold in contrast is able to orientate cells through contact guidance when the fibers are aligned. Given that a 3D environment is easy to form using hydrogel, using contact guidance property of aligned nanofibers within hydrogel may be used to achieve desirable cell behavior. McMurtrey (2014) showed that the combination of hyaluronic acid hydrogel with laminin coated electrospun aligned polycaprolactone fibers were able to encourage the longest neurite extension from SH-SY5Y cell compared to aligned fibers alone and hyaluronic acid hydrogel alone.

a) 3D hydrogel scaffold showing neuronal cells clustering in globules. b) Neurons can be seen on the aligned nanofiber scaffold embedded within the hydrogel. [McMurtrey 2014 J Neural Eng 2014; 11: 066009. This work is licensed under a Creative Commons Attribution 3.0 Unported License.]

An advantage of combining hydrogel with electrospinning is that cells can be easily encapsulated within the hydrogel before combining with the electrospun fibers. This may benefit clinical application where immediate incorporation of cells into the scaffold is required. Trypsinized cells may take a couple of hours before they adhered onto a surface. Loading the scaffold with hydrogel and cells eliminates the need to wait for the cells to adhere to the scaffold. Xu et al (2013) used a combination of electrospinning and printing of cells incorporated hydrogel in a layer by layer fashion to create a 3D scaffold. The construct comprises of electrospun polycaprolactone fibers and chondrocytes loaded into a collagen/fibrin hydrogel. While in vitro studies suggest that chondrocytes activity are depressed at the core of the cartilage construct compared to the outer perimeters, in vivo studies using a subcutaneous immunodeficient mouse model showed presence of Type II collagen and GAG throughout the scaffold. This demonstrates the viability of hydrogel/electrospun fibers/cell construct in tissue repair. Similarly, Keller et al (2015) constructed an electrospun nanofibers/hydrogel hybrid structure for cartilage regeneration. Human mesenchymal stem cells (hMSCs) and human chondrocytes were introduced into alginate/hyaluronic acid hydrogel in the form of micro-tissue and this was placed under a nanofibrous membrane layer loaded with BMP-7. The nanofibrous membrane was used for subchondral bone repair while the hydrogel is for cartilage repair.

A hydrogel/electrospun membrane combination may also be used to mimic native tissue environment. The skin is made out of a thin epidermis layer and a thicker dermis layer. Pan et al (2014) attempts to reconstruct the structure using electrospun Poly(ε-caprolactone-co-lactide)/Poloxamer(PLCL/Poloxamer) as the upper layer and a hydrogel composed of 10% dextran and 20% gelatin as the lower layer. The upper electrospun layer is designed to provide mechanical support while the lower, more porous hydrogel layer is designed to facilitate cell infiltration and proliferation. Liu et al (2023) constructed a skin substitute using multi-layers of electrospun polycaprolactone (PCL) fibers and sprayed alginate hydrogel powder by alternating electrospinning and spraying. The multi-layered structure was wetted and cross-linked so that the alginate formed a stable matrix with the PCL fibrous layers to form a fiber hydrogel interpenetrated network (FHIPN). Amino-terminated hyperbranched polyamide (ATHBP) with antibacterial properties was added to the FHIPN by dipping the FHIPN in ATHBP solution to form functionalized fiber-hydrogel interpenetrating network (FFHIPN). The amino groups on ATHBP would bind to the carboxyl groups of sodium alginate through Coulomb interaction. In this setup, the PCL fibrous layers would provide the necessary elasticity and mechanical strength. On a wound injury, the alginate hydrogel would absorb the wound exudate and swell to create a more open FHIPN structure which encourages cell infiltration and at the same time provides a wet environment for wound healing. Yao et al (2018) constructed an aligned fibrin hydrogel (AFG) composite comprising of aligned electrospun poly(ethylene oxide) (PEO) and fibrin hydrogel for the purpose of spinal cord injury repair. The presence of electrospun fibers in the hydrogel may mimic the mechanical properties of native nerve tissue. Aligned electrospun fibers may potentially guide neurite extension and aid recovery. In their animal study using a rat model, the AFG group showed significantly faster recovery compared to random electrospun fibers with fibrin hydrogel. In the research of fibrosis where tissue scarring is caused by activated myofibroblast (MF), it has been shown that conventional 2D hydrogel alone may not be able to elicit appropriate myofibroblast differentiation [Matera et al 2020]. The addition of fibers to form a multi-component hydrogel matrix is better able to imitate the 3D fibrous structure of interstitial tissue regions. Matera et al (2020) used a functionalized a biocompatible and protein-resistant polysaccharide, dextran, with pendant vinyl sulfone groups amenable to peptide conjugation (DexVS) for electrospinning into fibers and constructing a hydrogel. Electrospun DexVS fibers were added into the DeVS hydrogel precursors prior to gelation. MF differentiation as measured by α-smooth muscle actin (α-SMA) was found to be absent in transforming growth factor-β1 (TGF-β1) supplemented conditions that lacked fibrous architecture and other markers suggested that higher fiber density drives fibrotic phenotype and gene expression in the absence of a stiff hydrogel environment. Tests using primary human dermal fibroblasts and mammary fibroblasts showed that higher fiber density promoted proliferation in dermal fibroblast while mammary fibroblasts underwent MF differentiation. Higher fiber density also prompted greater hydrogel contraction compared to hydrogels with no or low fiber density. Greater collagen production was also found in hydrogels with higher fiber density. This study showed a clear influence of fiber density in hydrogel on MF differentiation and phenotype in 3D environment.

Hydrogel and electrospun fiber combination may also be used to replicate more complex organ structure. While it may be relatively easy to replicate the shape of a complex organ using hydrogel, having the full surface of the hydrogel replica coated with electrospun fibers may be challenging. During electrospinning, fibers tend to straddle across any gaps on the collector instead of filling the recessed area. Song et al (2021) demonstrated the feasibility of uniformly coating the surface of a flexible hydrogel with electrospun fibers by flattening the hydrogel structure before fiber deposition. Song et al (2021) selected a mixture of alginate and gelatin to form the shape of an ear. Flattening of the alginate/gelatin hydrogel collector enables fiber to be deposited within the complex geometries of helix and antihelix of the ear. The thickness of the deposited fibers across the surface of the flattened hydrogel ear template was found to be uniform in contrast to the unflattened, original 3D ear shaped hydrogel collector where no fibers were deposited in the indented part.

Schematized process of conformal fabrication of an electrospun nanofiber mat on a 3D ear cartilage-shaped hydrogel collector. a The 3D printing process for the 3D ear cartilage-shaped template. b The PDMS negative mold replicated by the 3D ear-cartilage-shaped template. c The alginate-gelatin hydrogel collector with the shape of the ear cartilage. d Conformal fabrication of an electrospun nanofiber mat with the flattened 3D ear cartilage-shaped hydrogel collector [Song et al 2021].

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Content [3D nanofibrous structures]