New Dimensions of Electrospun Nanofiber Material Designs for Biotechnological Uses

Abstract

Electrospinning technology has garnered wide attention over the past few decades in various biomedical applications, including drug delivery, cell therapy, and tissue engineering. This technology can create nanofibers with tunable fiber diameter and functionality. However, the 2D membrane nature of the nanofibers, low porosity, and rigidity of electrospun fibers lower their efficacy in tissue repair and regeneration. Recently, new avenues have been explored to resolve the challenges associated with 2D electrospun nanofiber membranes. This review discusses the recent trends in creating different electrospun nanofiber microstructures from the 2D nanofiber membrane using various post-processing methods and their application in biotechnological uses.

Emerging applications of electrospun nanofiber scaffolds

Amongst various scaffolds used to repair tissue, nanofiber scaffolds have gained much attention due to their extracellular matrix (ECM) (see Glossary) mimetic architectures [1]. Generally, nanofibers are made using phase separation, electrospinning, melt-blowing, bicomponent spinning, force spinning, flash-spinning, template synthesis, or self-assembly [2]. Electrospinning has surpassed other methods due to its versatility in fiber compositions, precise therapeutic loading, fibrous morphology and size, fiber orientation, simple and low-cost laboratory experimental setup, and large-scale production rate [3]. Electrospun fibers have gained significant importance in various biotechnological applications due to their unique properties and versatility [4]. For example, in tissue repair and regeneration, electrospun fibers are frequently used to build scaffolds that resemble the ECM of living tissues. The high surface area and interconnected porosity of electrospun fibers facilitate cell attachment and proliferation along with better mechanical properties. Electrospun nanofibers have a tendency to mimic the structure and characteristics of particular tissues, including skin, bone, cartilage, blood vessels, and nerves [5, 6].

Cell-laden electrospun fibers can act as artificial organs and implants [1]. Electrospun scaffolds, for instance, can facilitate the development of synthetic organs, like the liver and kidney, while electrospun vascular grafts can serve as replacements for damaged or blocked blood vessels [7, 8]. Furthermore, the electrospun fibers’ high surface area and porous structure facilitate the loading of therapeutic drugs and growth factors that allow the controlled release of factors which makes them an ideal candidate for therapeutic delivery systems [9–12]. In this case, the drug release kinetics can be optimized for different applications by altering fiber properties like surface chemistry and degradation rate [13, 14]. Moreover, functionalized electrospun fibers that contain biomolecules like enzymes or antibodies render the possibility of sensing particular analytes and show a great potential application in biosensors. Effective analyte capture and signal transduction are made possible by the fibers’ large surface area and high sensitivity [15]. These biosensors are used in environmental monitoring, food safety, and medical diagnostics [16–18]. The biocompatibility and tissue-mimicking properties of the fibers make them an excellent choice for all these applications.

However, the traditional electrospinning technique produces 2D electrospun nanofiber membranes from synthetic or naturally derived polymer materials. Although these 2D electrospun membranes have shown promising results in tissue engineering, they still lack the ability to mimic the 3D milieu of the target tissue, are poorly adaptive to site shape adaptability, and have poor cell infiltration and mechanical strength [19, 20]. To improve the microstructure design and cell-scaffold interaction, researchers have used several different methods to make various new forms of nanofiber microstructures from 2D electrospun nanofiber mats [21–24].

This review outlines new dimensions of electrospun nanofiber scaffolds in versatile biological applications. We highlight the recent progress in the translation of 2D electrospun nanofiber into the development of 3D nanofibrous structures using various techniques. Subsequently, we discuss electrospun 3D scaffolds in regenerative medicine and remark on the challenges associated with their tunable characteristics and limitations. Overall, electrospun fiber’s significance in biotechnological uses lies in its ability to provide a biomimetic platform with tailored properties for tissue engineering, drug delivery, wound healing, biosensing, filtration, and the development of artificial organs.

Extracellular mimetic 3D microstructures from 2D nanofibers

The electrospun 2D nanofiber membrane cannot mimic the 3D extracellular milieu of the cells or organs. Relatively low porosities and small pore diameters in 2D nanofiber mats hinder cell infiltration [25–27]. To circumvent these drawbacks, many efforts have been devoted to making porous 3D nanofiber scaffolds with various post-processing methods like mechanically stabbing, 3D weaving technique, sacrificial templating, crystallization, and combining melt electro writing with electrospinning which is discussed elsewhere [25]. Among them, the expansion of the membrane attracted lots of attention because it provides control over shape, porosity, and fiber alignment within scaffolds [17, 18]. In this method, the flat mats convert into 3D structures by direct expansion of the membrane by submerging it in foaming aqueous solutions such as NaBH₄ (Figure 1A (i)) or by gas foaming, with subcritical CO₂ (Figure 1A (ii)). On the other hand, cutting the 2D nanofiber mat into short segments as injectable nanofibers (Figure 1B (i)), injectable nanofibrous microspheres (NMs) (Figure 1B (ii)), and aerogels with tunable porosity (Figure 1B (iii) has been widely employed for various applications, including cell and therapeutic delivery, bone repair, cartilage repair, and wound healing [25–27]. Furthermore, short fibers have been exclusively used for fabricating hybrid scaffolds (Figure 1B (iv)).

Figure 1:

Schematic representation of different strategies used for translation of 2D electrospun nanofiber into 3D nanofibrous scaffold. (A). 3D expansion technology namely (i) NaBH₄ method which converts 2D nanofiber mat into 3D structure with two different directions such as vertically and radially aligned, (ii). 3D expansion using CO_2. (B). 3D nanofibrous scaffold formulation from fragmented short nanofibers, (i). Injectable nanofibers, (ii). Nanofibrous microspheres fabricated using electrospraying technique, (iii). Aerogels, and (iv). Hybrid bioink, nanofiber reinforced hydrogels for 3D printing applications.

3D Expansion Methods

Expansion by NaBH₄

2D nanofiber mats can be expanded to form 3D micro and macro structures by submersion in a solution of NaBH₄ [19, 20, 28–30]. In 2015, Joshi and colleagues, [19] reported a post-electrospinning method to expand 2D nanofiber membranes of different polymers to low-density 3D nanofiber scaffolds. At the same time, Xie’s research team widely explored and modified this technique to create various 3D electrospun nanofiber microstructures for tissue engineering applications [20]. Most of their work belongs to clinically relevant electrospun poly(ε-caprolactone) (PCL) nanofiber mats using a gas foaming technique followed by freeze-drying. They explained the versatility of this process to generate confined and controlled expansions of the 3D nanofiber scaffolds with a highly ordered architecture.

Inspired by solids of revolution, Xie’s research team [25–27] transformed 2D mats into 3D objects with complex predesigned shapes. For example, Chen and colleagues reported a rectangular nanofiber mat and used thermal treatment to fix one side of it [23]. They obtained different 3D shapes including cones, spheres, and hollow spheres. The authors evaluated the potential of the 3D scaffolds for neural tissue construct formation, by cultivating rat neural progenitor cells on cylindrical structures composed of radially aligned nanofibers. Figure 2A shows some of the different shapes that can be obtained by expansion. Aligned nano topographic cues have a significant impact on controlling the shape and activity of cells, as well as directing the deposition of ECM [28–31]. It is critical to use the constructs with ordered structures for tissue repair. Gelatin coating on 3D expanded scaffolds makes them super elastic, compressible, and shape recoverable [29]. Instead of gelatin, Chen and colleagues used gelatin methacryloyl (GelMA) coated, for minimally invasive delivery of cells [30, 31]. Stem cells seeded 3D expanded nanofiber scaffolds formed 3D tissue constructs for cell therapy that can be compressed and transported to diseased tissue while maintaining the viability of the seeded cells [28]. Figure 2B and Figure 2C, show the fluorescence images of a 3D radially aligned nanofiber scaffold and an illustration of the 3D scaffold for wound healing [30].

Figure 2.

(A) 2D nanofiber mats transform in a variety of shapes including circular cones, spheres, and hollow spheres [28]. (B) Schematic illustration of the implantation of (radially aligned nanofibers (RAS) and vertically aligned nanofibers (VAS) to critical-sized cranial bone defects in rats with cross views, Micro-CT images of the RAS, VAS groups after 8 weeks of operation [33]. (C)The fluorescence images of 3D RAS with dual gradations in rhodamine 6G and FITC-BSA contents [32]. (D) Schematic illustration of the 3D scaffold with gradations in the bFGF growth factor showing guidance and acceleration of cell migration. Inset shows an in vitro (8 mm) wound model (8 mm GelMA hydrogel with human dermal fibroblasts seeded on the surrounding area [32]. (E) Schematic illustration showing the migration route of BMSCs from the surroundings to the center of RAS and VAS [33]. Image reproduced with permission from [30, 32, 35]

The introduction of structural and compositional gradients within 3D hierarchical assemblies created gradient pores which were achieved by varying the contents of surfactants such as Pluronic F-127 in each layer of 2D mat [32, 33]. These findings suggest that nanofiber scaffolds with higher density introduce a hypoxic environment and promote BMSCs chondrogenic differentiation better than porous scaffolds [32, 33]. The same research group used these 3D radially and vertically aligned nanofiber scaffolds for various wound healing models [29, 33–35]. Another group developed a 3D layered nanofiber sponge using chitosan and polyvinyl alcohol to regenerate skin tissue for smaller scars [32]. However, less control was applicable to the shape and size of the scaffold. Using this novel technique, 3D scaffolds can now be tailored to specific dimensions, depths, and configurations, allowing them to function as personalized constructs that precisely match diabetic ulcers. After seeding with BMSCs, they enhance the formation of granulation tissue, promoting angiogenesis, facilitating collagen deposition, and switching the immune responses to the pro-regenerative direction [33]. The pore size and porosity of the cylindrical scaffolds can be easily adjusted by modifying the initial membrane thickness. These scaffolds show significant promise in enhancing bone repair and regeneration by expediting the movement of keratinocytes and fibroblasts. This acceleration fosters the process of re-epithelialization and the creation of granulation tissue [36]. Figure 2D and Figure 2E show a schematic illustration of the radially and vertically aligned scaffolds implanted to critical-sized cranial bone defects in vivo and the migration behavior of BMSCs from the surrounding area to the center [36].

Synthetic polymers including PCL, poly(vinylene difluoride) (PVDF), and nylon have been widely employed to engineer various 3D gas-foamed scaffolds. However, a group of scientists used this technology to expand poly(l-lactide-co-ε-caprolactone)/silk fibroin (PLCL/SF) and hyaluronic acid-crosslinked fiber nanofibers [37]. The 3D scaffolds exhibited high porosity, great water absorption capacity, and excellent mechanical stability [37]. In another research, the authors cross-linked PLCL/SF scaffolds with chondroitin sulfate (CS) to obtain more enhancement in mechanical and biological properties. The cross-linked 3D scaffolds exhibited more mature cartilage-like tissue formation which resulted in a good repair outcome and less expression level of pro-inflammatory cytokines [38]. Nanofiber sheets expanded with NaBH₄ have also been shown to enhance the growth and regeneration of peripheral nerves. For example, Rao and colleagues, [39] used 3D PLA/silk fibroin sponge scaffolds implanted into chitosan nerve guiding conduits to regenerate Schwann cells in rat sciatic nerve defects. The scaffolds show the formation of more nerve fibers, a larger radius, and thicker myelin sheaths compared to the hollow conduits. Overall, expanded nanofiber scaffolds offer a wide range of applications in tissue regeneration and wound healing.

Expansion by Subcritical CO₂

Despite the advancement of gas foaming in an aqueous solution, this method has some drawbacks, including multiple time-consuming steps, an aqueous solution suitable only for hydrophobic polymers, the need for freeze-drying, and the possibility of reactions with NaBH₄ as a strong reducing agent that may impair the bioactivities of loaded components [25]. Expansion of electrospun nanofibers using subcritical CO₂ takes advantage of CO₂’s phase transitions to control the shape of electrospun mats selectively. Jiang and colleagues, reported the first subcritical CO₂ fluid-induced 3D transformation of electrospun nanofiber membranes. To enhance cellular penetration and blood vessel formation, nanofiber membranes can be micro-punched under cryogenic conditions [40]. The same team visualized the experiments later by cutting the mats into an extruded surface when CO₂ is converted from liquid form to gas [41]. Inspired by the above-mentioned technology in 2019, Meng and colleagues, expanded a 2D PCL mat using CO₂ solubility differences in ethanol and water [42]. The nanofibers wetted with CO₂-saturated ethanol were highly expanded in the water bath due to CO₂ gas escaping which can be applied to expand hydrophobic electrospun nanofibers such as PCL and PLA. The fluffy scaffolds had 95.3 % porosity, compared to 2D PCL.

Recently Chen and colleagues, [43] studied the immune response of granulocyte-macrophage colony stimulating factor (GM-CSF) using hierarchically 3D nanofiber scaffolds that expanded by CO₂ method. The researchers loaded the scaffolds with GM-CSF to achieve immunoregulation and tissue regeneration at the same time. The locally released GM-CSF showed a significant role in cell migration and ECM deposition. Furthermore, increased levels of human cytotoxic T cells (CD3+/CD8+), as well as M1 macrophages (CD68/CCR7), are detected in RAS loaded with GM-CSF, both within the scaffolds themselves and in the neighboring tissues, which resulted in acceleration of cell migration from surrounding tissues boosting wound healing. Despite the advantages of expanded 3D nanofibers construct in tissue regeneration they lack mechanical properties when it comes to hard tissue repair. Several methods could possibly enhance its strength by reinforcing with synthetic materials or developing composite scaffold with core and shell structure.

3D Nanofiber Aerogels

Despite their advantages, traditional 2D nanofibers or 2D mesh have some drawbacks associated with their practical biomedical use including liquid absorption ratio, weak mechanical properties, and porosity. When rigid structures are desirable to use, short nanofibers are employed as foundational units to construct porous, 3D nanofiber scaffolds called nanofiber aerogels. Generally, two main approaches have been developed for making nanofiber aerogels that have been discussed in detail elsewhere [25]. The first approach includes generating nanofibers through electrospinning, creating short nanofibers, and dispersing them, forming templates or molds, and ultimately freezing/freeze-drying, and cross-linking the scaffolds [44–46]. On the contrary, the alternative method distinguishes itself in the stage involving the dispersion and templating of short nanofibers, achieved by assembling short nanofiber aggregates through a thermally induced self-assembly [47–49]. Furthermore, the technique of anisotropic/directional freezing can be employed at various temperatures on thoroughly mixed short nanofibers to create oriented porous arrangements exhibiting diverse pore dimensions. However, chemically or thermally crosslinking or subjecting nanofiber-based aerogels to pyrolysis will greatly bolster their mechanical stability over extended durations [44].

Electrospun nanofiber aerogels are promising for cell implantation due to their macrostructure and microstructure with variable pore size and have many bioengineering applications when used with sacrificial or permanent templates to create different morphologies [50–52]. They can be used as a bone regeneration material with the ability to carry miRNAs to promote osteogenic differentiation [53]. Aerogels composed of nanofibers can also be loaded with different biochemical agents such as metal oxides, to provide cell interactions. [54] For instance, Mao and colleagues, created a PLA/gelatin/ZnO aerogel to be used for wound healing [54]. Their aerogel serves as a wound dressing scaffold, creating a physical barrier to prevent infection and enabling the exchange of gases while absorbing fluids released by the wound. Figure 3A, Figure 3B, and Figure 3C show the preparation of PLA/gelatin/ZnO aerogel scaffolds with different ZnO content and good antibacterial and wound healing properties of scaffolds.

Figure 3.

(A) The steps for fabrication of polylactic acid/gelatin/ZnO aerogel scaffolds and TEM images of single polylactic acid/gelatin/ZnO nanofiber with different ZnO concentrations[54]. (B) Schematic illustration of the fabrication processes of nanofiber aerogels with and without macrochannels; with the corresponding SEM images of horizontal and longitudinal cross sections of 3D polycaprolactone/gelatin nanofiber aerogels [55]. (C) Mechanism of antibacterial properties in Polylactic acid/gel/ZnO nanofiber aerogel scaffolds [54]. (D) Digital photos of infectious wounds show better healing for aerogels with ZnO after 14 days [54]. (E) H&E staining of 3D polycaprolactone/gelatin nanofiber aerogels with and without patterned macrochannels implanted in rats for 2 and 4 weeks [55]. (F) Schematics illustration of cell distributions showing significantly improved cellular infiltration on aerogels with patterned macrochannels [55]. Image reproduced with permission from [54, 55]

When it comes to wound healing application, the cellular infiltration rates and host tissue integration are the key factors that the scaffold should maintain. Recently, researchers introduced a novel approach for creating macrochannels and microchannels inside the 3D scaffolds to enhance the cell infiltration rate [27, 55]. Incorporated sacrificial templates into the PCL/gelatin and PCL/gelatin/GelMA nanofiber aerogels, allow them to be fine-tuned to improve morphology and cell adhesion [55]. Isotropic freezing led to partially aligned microchannels while anisotropic freezing led to honeycomb-like channels. Figure 3D, Figure 3E, and Figure 3F illustrate the preparation and mechanism of bone formation on these scaffolds [55]. The same team used aerogels with macrochannels for fast healing of diabetic wounds [27]. To improve the mechanical properties of aerogel, non-sacrificial scaffolds have also been used which gradually form cartilage-like tissues, maturing with time [56]. Poly(l-lactide)/gelatin (0.5–1.5 wt%) nanofibers around a PCL tubular scaffold created tracheal implants that closely mimic the mechanical properties of native trachea [56]. The scaffolds were comparable to the native trachea possessing a stable mechanical property. With favorable features such as strength, elasticity, and biocompatibility, these scaffolds offer the potential for wound healing in different anatomical areas, including the trachea.

Injectable nanofiber designs

Technological advancements in tissue engineering and the simplicity of clinical implementation have increased the minimally invasive therapies (MIT) over the past two decades [10]. Specifically, injectable hydrogels and microgels have been widely employed for therapeutic delivery, bone repair, and wound healing [25, 57]. Since electrospun nanofiber scaffolds are neither injectable nor very flexible, they have received less attention as potential MIT. To overcome these limitations, scientists have been testing various post-processing techniques to create novel injectable electrospun nanofiber materials. Recent advancements in the injectable forms of electrospun nanofiber design like short nanofiber and nanofiber microsphere with tunable porosity, have enabled a new avenue of the electrospun nanofibers in MIT.

Short nanofibers

MIT offers simplified surgical procedures and minimal secondary damage using an injectable material matrix, which favors the defect shape adaptation upon injection and multiplicity of therapeutic agents [58]. To overcome the constraints on the use of traditional electrospun, material scientists developed injectable short fiber fillers using various cutting methods. The first report on the preparation of short nanofiber is based on the UV cutting method, which is limited by the use of UV cross-linkable polymers [59]. Later, other methods were introduced, including shear stress liquid flow [60], mechanical cutting [61], electric spark-induced cutting method [62], and tuning polymer molecular weight and solvents [63]. Short nanofibers demonstrated diverged tissue engineering applications among them fiber-oriented ECM alignment became popular. Burdick’s team reported a preparation of a short NorHA nanofiber scaffold which directed ECM alignment parallel to fiber orientation (Figure 4A) [60]. Short nanofibers have the potential to deliver biological molecules/drugs to a target site through injection in addition to providing shape adaptability [64]. This injectable short nanofiber system proved its function in vaccine delivery when Moore and colleagues, prepared different lengths of ribbon-like microconfetti from electrospun acetalated dextran loaded with Resiquimod. It stimulated cytokine inflammatory response in human dendritic cells without inducing cytotoxicity thereupon providing a new platform for vaccine delivery [12]. Similarly, Shi and colleagues, prepared dual drug-loaded injectable short nanofibers (DOX@LDH/a-TOS/PLGA) for tumor drug delivery. These pH-sensitive short nanofibers slowly release DOX from the short fibers and function against drug-resistant tumors [11]. Overall, short nanofibers present as therapeutic delivery vehicles however these short nanofibers necessitate secondary material for injection. The addition of shear thinning material enhances the injectability of short nanofibers nevertheless challenges associated with composite material.

Figure 4.

New forms of 2D electrospun fiber mat into 3D structures such as (A) Short nanofiber (prepared using fluid shear flow method, provides an advantage of fiber direction-oriented ECM secretion) [60]. (B) Nanofiber microspheres (fabricated using electrospraying technique with short nanofiber solution, interconnected porous microsphere favors cell migration and improves cell adhesion and proliferation) [26]. (C) Lamellar scaffold a heterogeneous layered structure renders a stability [82]. (D) Hybrid 3D scaffolds such as hydrogel and nanofiber blend enhance cell attachment motifs in encapsulated cells [93]. (E) Hybrid bioink utilized for 3D bioprinting and fibers exhibiting alignment corresponding to printing direction and cells succeeding the similar direction which shown using F-actin (red) staining [99]. Image reproduced with permission from [26, 60, 82, 93, 99]

Nanofiber microspheres

Short nanofibers provide better wound structure adaptation and mobility with numerous coating strategies [11, 64]. However, short nanofibers are unable to create ECM microstructure for cell migration in tissue repair. Further developments in this area explored the possibility of engineering NMs as an ECM-mimicking scaffold with high biocompatibility and adaptability of tissue structure [65]. Miniature size and injection stability of NMs present as an injectable scaffold and drug carrier in MIT. Various methods have been employed in the fabrication of NMs for example self-assembly, electrospraying (dripping), and co-axial electrospraying. Ma and colleagues, [66] reported the use of self-assembled PLLA-based NMs for chondrocyte culturing. However, this self-assembly manufacturing strategy was limited to functional groups (−OH, −NH₂, and −COOH) [67]. To circumvent the drawbacks of the self-assembly approach in the development of NMs, Xie and colleagues, reported a method that combined electrospraying of electrospun short nanofibers prepared using mechanical cutting (Figure 4B). NMs with controlled size were engineered by varying the voltage and fiber solution speed, and porous NMs were developed using a co-axial system. Further, biological molecules were loaded onto NMs to promote their predetermined function. Such as BMP-2 and VEGF analog peptides were loaded to NMs to promote osteogenic differentiation of BMSCs and aided in the bone regeneration [10]. Recently, Mo and colleagues, fabricated injectable fibrous PLA/gelatin microspheres with metal phenolic compounds for osteoarthritis treatment. These strontium and tannic acid (TA) loaded microspheres exhibited sustained release of TA and it positively affected the cell behavior thus secretion of cartilage matrix [68]. These outcomes present the diversity and compatibility of NMs for drug loading and coatings, indicating NMs’ potential in treating multiplex tissue damages and showing broad prospects as an injectable platform. Nonporous NMs significantly enhanced surface-limited cell behavior, while porous NMs promoted improved cell proliferation due to sufficient exchange of oxygen and nutrients through open pores [69, 70]. The coaxial electrostatic spray method mediated by air bubbles demonstrated the fabrication of porous NMs and, pore density and size can engineered by voltage and airflow velocity [26]. In vivo experiments on these porous NMs showed significantly enhanced cellular infiltration and neovascularization [26]. While NMs generally aid in host cell infiltration and ECM formation, there are still concerns regarding these microspheres load bearing capacity. Additionally, these NMs act as MIT most suitable for soft tissue regeneration. Integrating them with stimuli-responsive materials will improve their applicability in tissue repair. Further, incorporating cell-laden NMs into a continuous phase of the hydrogel scaffold will upgrade the biological activity of the construct.

Hybrid scaffolds

Hybrid scaffolds are a combination of hydrogel and nanofiber fragments which provide synergistic effects of both components during tissue repair [71]. Hydrogel holds an advantage of a highly hydrated 3D porous network which facilitates diffusion of nutrients and metabolic wastes as well as cell proliferation, differentiation, and migration, most importantly they simulate native tissue microenvironment [72, 73]. However, the inferior biomechanical property of hydrogel lacks to support damaged tissue, and techniques to enhance its strength are still perceived to fail. Isotropic hydrogel system strives to form a heterogeneous and stable 3D structure hindering the construction of multilevel structures and topological design [74, 75]. To address these issues, electrospun nanofibers were added to enhance scaffold stiffness and the degree of stiffness can be tuned by fiber density. Researchers able to design anisotropic composite structures using nanofibers and concentration gradient of spatial heterogeneity allow gradient drug delivery [76]. Common integration strategies include lamellar and short nanofiber homogenous structures (Table 1). The lamellar structure is the simplest layered structure of electrospun nanofiber and hydrogel plates to obtain a composite scaffold and mechanical and biochemical properties can be regulated through stacking sequences. Among them, remarkable progress has been made in vascular and neurological treatment by curling laminated hydrogels into tubular scaffolds and incorporation of electrospun fiber mat into the system which provided mechanical strength by reinforcement strategy [8, 77–80]. Further, Dual-scale scaffolds were developed using PCL by simultaneous 3D printing and rotational electrospinning. These scaffolds demonstrated enhanced cell attachment and osteogenesis due to nanofibrous structure and microporous architecture [81]. With further advancements drug-loaded electrospun nanofibers were prepared in combination with hydrogel to act as a drug-eluting scaffold for on-demand drug delivery (Figure 4C) [82]. Native tissue mimicking short nanofibers integrated hydrogel molded into 3D printed scaffold demonstrated enhanced chondrogenesis and mechanical property in vivo [83]. However, these stacked scaffolds suffer from delamination, to overcome those researchers developed a homogeneous structure of electrospun nanofiber and hydrogel scaffold with enhanced mechanical properties, self-adaptable to complex and irregular wounds [84–87]. These homogenous composite scaffolds showed potential use in soft and hard tissue engineering [88–92]. Fibers in the hydrogel system demonstrated fiber density-mediated cell behavior such as cell spreading regulated by the YAP mechanosensing pathway in fibroblast encapsulated fiber-reinforced hydrogel composite [93] (Figure 4D). Similarly, this can be utilized to study the cell migration during cancer metastasis [94] and also act as a superparamagnetic scaffold with nanoparticles to fiber alignment which in turn affects the cell migration [74]. Injectable hydrogel incorporated with polyacrylonitrile and reduced graphene oxide nanofibers were employed as a scaffold and biosensor for pressure ulcers which mimic the native microenvironment and simultaneously monitor the wound pressure [95]. Electrospun fiber provides the possibility of drug release strategies of composite scaffolds by customizing fiber distribution, diameter, porosity, cross-linking degree, and orientation degree [82, 96].

Table 1.

Lamellar and homogenous hybrid scaffolds and their application in the biomedical uses

Scaffold component		Techniques involved	Results	Application	Reference
Hydrogel	Fiber sheet
Lamellar structured scaffold
Fibrinogen-thrombin	PEUU	Grafting, UV crosslinking	High tensile strength and suture retention	Vascular graft	[77]
Gelatin-chitosan	SF-PU	Freeze drying	Improved biocompatibility and cell mobility with mechanical integrity	Vascular scaffold	[7]
GelMa	PCL	Photo-polymerization	Tunable mechanical properties and supported the different stages of skeletal muscle	Skeletal muscle regeneration	[78]
Gelatin+Chlorotetracyline hydrochloride	PEOT/PBT + Diclofenac	Sandwiched	Optothermal guided dual drug release system and biocompatible	Drug delivery for soft tissue tumor	[96]
PNIPAAm/AuNR	PLLA-RhB	Embedding, NIR stimulation	Photothermal and mechanical shrinkage responsiveness prolonged drug release	Drug delivery	[82]
Alginate-Alginate sulfide-(Kartogenin NP, PLGA as a carrier)	PCL-Gelatin	Freeze drying	Interconnected pores, improved mechanical features, sustained release of Kartogenin	Kartogenin delivery for tissue regeneration	[79]
PLEOF-HA	PLA	UV crosslinking	Increased elastic modulus, osteogenic differentiation	Bone tissue engineering	[80]
PAM	Silk yarn, TPU	UV crosslinking	High elasticity, fast endothelialization	Triple-layered vascular graft	[8]
Homogenous structure scaffold
PEGDAC	PCL	Coagulation bath	Fluidity, dose dependent increase in compression modulus, chondrogenic potential	Cartilage repair	[85]
Chitosan	Bioactive fiber glass	Freeze drying	Shape recovery property, elasticity, and biomineralization	Osteoporotic Bone Regeneration	[86]
Selfassembling peptides	PLA-DNF	pH driven self-assembly	Electrospun nanofibers provided a more stable, continuous delivery	Growth factor delivery	[126]
Hyaluronic acid	PCL		Inward migration of hASCs due to pores, angiogenesis, in vivo macrophage infiltration	Soft tissue construction	[87]
Thiolated hyaluronic acid	PCL	Injectable composite	Reduced injury size and more astrocytes and axons	Spinal cord repair	[90]
HA modified with PEG dithiol	PCL grafted with PAA	Injectable	Improved stability, EC maker expression and neovascularization	Soft tissue engineering	[89]
Methacrylated HA	PCL nanofiber		Increased compressive modulus, bone specific marker expression	Bone tissue engineering	[88]

3D bioprinting is gained popularity in tissue engineering and commonly soft hydrogel was used with better cell proliferation and migration however the ink viscosity was challenging to achieve [97]. In this regard, hybrid inks gained attention as incorporating nanofibers improves the mechanical strength of 3D scaffold [98] and also includes drug/GF loading capacity. The most significant advantage of 3D printing is to create an anisotropic structure from isotropic fiber-reinforced hydrogel bioinks. This can mimic the extracellular matrix of various tissues and regulate cell behavior according to the specificity of regional structure. Recently, Burdick’s team explored a new avenue in 3D bioprinting using a blend bioink consisting of NorHA electrospun fiber fragments and GelMa hydrogel [99]. 3D bio-printed scaffolds demonstrated cell actin orientation in the direction of printed filaments (Figure 4E). Further investigations have been conducted to demonstrate the fiber distribution in the effect of printing variables [100]. Further, it can be used to push the anisotropic expansion behavior into different regions of the material and offers broad application on soft active materials.

Platforms for effective sampling and sensing

The accurate collection of biological samples is essential for effective analysis, intervention, and treatment in the early stages of diseases [101, 102]. Population-based endoscopic screening and biopsy analysis not only prolong patient suffering but also time and finance liable [103, 104]. In recent years, minimally invasive techniques have received extensive attention, for example, the cell-collecting device cytosponge is mostly used for patient ingestion [105, 106]. However, they resulted in bleeding due to higher friction against the esophagus during the removal of the device, and inadvertent ingestion due to a broken rope. To overcome these problems, John and colleagues developed a 3D PCL nanofiber sponges coated with gelatin and an ex vivo swine esophagus test showed that the device can collect cells and tissue without harming the lining of the esophagus and stomach [107]. During COVID times, sample collection was made using cotton swabs. However effective biological sampling was challenging due to the false positive cases. A group of researchers created a PCL nanofiber swab for collecting SARS-CoV-2 samples and nanofiber swabs presented hierarchical structures which resulted in the extraction of biological samples efficiently [108]. In addition to obtaining cells and tissues from the lesion site, single-cell capture is also one of the important strategies for effective sampling [109]. In view of improving the single-cell capture recently, nanofibers were employed with functional moieties to reduce nonspecific adhesion of blood cells [110, 111]. Capture and nondestructive release of circulating tumor cells (CTCs) were achieved by incorporation of ZnO nanofibers into the device for efficient sampling owing to dense nanofibers improved cell-binding sites and cell-material matrix contact time [112]. Electrospun nanofibers could also be used as wearable health monitoring devices. Microstructure and porous nanofibers combined with conductive ink and flexible substrate can increase the detection limit and compliance of wearable electrical sensors [16, 17, 113–118]. The grafted nanofibers can be sensitive to temperature and help piezoelectric sensors detect body temperature through time [119–121]. Multilayer, core-shell electrospun nanofibers capture more electrons and enhance the sensor response for the diabetes detection [122–124]. Also, studies have shown that the addition of electrospun nanofibers has improved breathability, stretchability, response time, and detection range of wearable sensors, but its cost, production capacity, and corrosion resistance need to be further improved [125]. Future research and development will primarily focus on these open questions. Recent advancements in the use of short nanofibers in hydrogel system for biosensing increased the availability of nanofibers and sensitivity of sensors in biosensor applications in health care.

Concluding remarks and future perspectives

The new dimensions of a 2D electrospun fiber mat into a 3D microstructure have biotechnological multiple applications (Figure 5). Stepping into new dimensions of electrospun nanofiber mats will enhance their use in real-time biotechnological uses including wound healing, cartilage repair, bone regeneration, cell delivery, and therapeutic delivery due to their enhanced porosity, cell infiltration, and mimicking native tissue microstructure. Incorporating electrospun nanofibers enhances bulk scaffold physical and biochemical characteristics. However, several gaps have been identified in terms of scaffold fabrication and underlying mechanisms to improve the functioning of the scaffold. Although 3D expanded scaffolds can support cell migration and angiogenesis, the mechanical stability of these scaffolds in hard tissue replacements is questionable. Hence, further advancements in these 3D expansion techniques will focus on improving the mechanical strength of the structure while retaining its biological characteristics (see Outstanding Questions).

Figure 5:

Summary of the various 3D electrospun nanofiber microstructure designs from 2D nanofiber membrane for various biotechnological uses. 2D nanofibers can be expanded to different shapes and forms including Aerogel, 3D printing ink, radially or vertically aligned scaffolds and microspheres. as illustrated in the middle circle. Theo outer part of the graph illustrate potential applications for variety of forms including applications in tissue regeneration and repair.

Outstanding questions.

• Can expansion approaches develop 3D scaffolds that provide comparable mechanical strength to the native tissue loads?

• What degree of cell differentiation or drug delivery can be accomplished using short nanofibers in composite structures?

• Can nanofibers exactly mimic the native tissue or organ environment in synthetic scaffolds? How can the similarity be measured?

• How can we validate the clinical relevance of these nanofiber-based scaffolds to demonstrate their accuracy and predictivity in terms of tissue repair and drug or cell delivery?

Hybrid inks show promising results despite their printability issues, so new opportunities will create more solutions for effective ink formulation with fiber incorporated. Despite the benefits, it is uncertain that what could be the degree of cell differentiation or drug delivery can be accomplished using short nanofibers in composite structures, necessitating advanced validating methods. The unique properties of electrospun nanofibers increase their usage in biosensor applications, but the sensitivity of the electrospun nanofibers decreases after post-processing. Future avenues will improve the performance of electrospun nanofibers in biosensors by designing surface chemistry, and biomimetic topography will further enhance their availability. The opportunity to translate 2D fiber mats into 3D structures will connect nanofiber architecture and hybrid scaffold performance, which will expand their use in biological applications.

Highlights.

• Electrospun scaffolds are confined to 2D structures.

• Various strategies have been employed to translate 2D electrospun mats into functional 3D nanofiber scaffolds, including expansion techniques, nanofibrous microsphere technology, and hybrid constructs.

• The expansion method allows the direct expansion of 2D mat into 3D structures, and control over the direction of expansion plays a major role in cell adhesion and migration, which in turn affects tissue regeneration.

• In combination with the electrospraying technique, 3D nanofibrous microspheres enhance the efficacy of minimally invasive therapies using injectable microspheres for diabetic wound healing.

• Recently, hybrid scaffolds/bioinks have demonstrated an advantage over pristine structures due to their improved mechanical strength and cell-biomaterial interactions.

Glossary

2D nanofiber membrane

a polymeric mat that contains layers of nano scale diameter fibers stacked together.

Aerogel

an open-celled, dry, mesoporous, solid foam with a porosity (non-solid volume) of at least 50% and is made up of a network of interconnected nanostructures.

Circulating tumor cells (CTCs)

tumor cells that extravasate from the primary tumor or metastasis loci and intravasate into the blood circulation and detecting CTC has a tremendous potential in cancer metastasis.

Cytosponge

A small capsule attached to a string and upon swallowing allows sample collection. In this review, the mentioned cytosponges are electrospun nanofiber scaffolds that provide more surface area for cell adhesion/sampling.

Electric spark-induced cutting

a method to cut metal surfaces where an electric spark cuts fibers into small pieces using a high-voltage electrode, similar to electrical discharge machining.

Extracellular matrix (ECM)

a non-cellular portion of a tissue composed of an intricate network of macromolecules.

Electrospraying

a voltage-driven process like electrospinning, but it produces polymeric spherical particles which are collected in the liquid nitrogen reservoir.

Gas foaming

a method of creating a porous 3D matrix without use of any solvents thus resulting in the expansion of a 2D electrospun sheet into a 3D scaffold.

Mechanical cutting

a technique used for the generation of short nanofibers from a whole electrospun mat using a cryosectioning/freeze-cutting method which eliminates the damage to nanofiber.

Minimally invasive therapy (MIT)

a term that encompasses a variety of techniques to treat damaged tissue with less harm or without additional secondary surgery.

Nanofiber microsphere

a polymeric sphere that contains a nanofibrous structure and is made from a solution of electrospun short fibers.

Shear stress fluid flow method

an approach where an electrospun fiber mat is segmented and mixed with a solution to extrude through a needle, which creates a fragmented fiber such as short fibers.

Short nanofiber

a fragmented electrospun fiber mat that resembles individual small threads and can be produced by various techniques.