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. 2026 Feb 20;11(9):15365–15376. doi: 10.1021/acsomega.5c12907

Electrospun 3D Nanofiber Scaffolds with Adhesion-Enhanced Interfaces for Cell Culture

Seo Hyung Moon †, Jin Haeng Jo †, Sol Ji Park †, Hye Ryeong Jeon †, Yun Jung Yang †,‡,*
PMCID: PMC12980440  PMID: 41835546

Abstract

Creating three-dimensional (3D) fibrous scaffolds has long been a challenging task due to difficulties in controlling fiber assembly and maintaining structural stability. Polystyrene (PS), a synthetic polymer with self-assembling properties, enables the formation of porous 3D architectures via electrostatic fiber repulsion without the need for auxiliary templates. However, despite its suitability for constructing self-standing fibrous structures, the inherent hydrophobicity and bioinert nature of PS limit its applicability in cell-interactive platforms. To overcome these limitations, this study proposes a gravity-defying electrospinning strategy combined with a surface functionalization approach to fabricate bioactive 3D scaffolds. Tannic acid (TA) is employed as a physical cross-linker, and silk sericin (SS), a hydrophilic protein, is applied as a coating layer. The TA-mediated SS coating enhances surface wettability and introduces abundant functional groups, thereby improving cell–scaffold interactions. The engineered PS3D/TA/SS scaffolds exhibit a mesoporous structure with high specific surface area, interconnected porosity, and a fibrillar morphology that closely mimics the extracellular matrix (ECM). Overall, the synergistic integration of TA and SS onto a PS scaffold provides a simple and robust strategy to transform hydrophobic synthetic substrates into bioactive platforms suitable for regenerative medicine.

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1. Introduction

A pore-based three-dimensional (3D) structure provides ample space for cell infiltration, vascularization, and nutrient exchange, while its interconnected porosity facilitates efficient waste removal and intercellular communication. − Despite these advantages, constructing such 3D architectures solely from fibrous materials has remained a considerable challenge. By effectively harnessing electrostatic interactions, however, it becomes possible to fabricate stable fibrous scaffolds that closely replicate the extracellular matrix (ECM)’s hierarchical and porous architecture, thereby promoting functional tissue regeneration.

Interestingly, the electrostatic repulsion mechanism observed in spider ballooning shares similarities with the principles governing electrospinning processes. The ballooning behavior of spiders enables aerial fiber dispersal over hundreds of kilometers, primarily facilitated by naturally occurring electric fields. The process is initiated when the spider elevates its abdomen by tip-toeing, allowing it to sense atmospheric conditions and electric field gradients. , The spider then releases a durable silk fiber, called a dragline, to anchor itself, followed by the extrusion of ballooning silk. These fibers, influenced by electrostatic repulsion and aerodynamic lift, enable spiders to become airborne in a gravity-defying manner. ,

Electrospinning involves applying a strong electric field to create charged polymer jets that repel each other, facilitating the formation of 3D fibrous structures. In this study, polystyrene (PS) was selected as the base material, enabling rapid growth and 3D fiber stacking without auxiliary collecting templates or secondary components. , Under the strong electric field, the negatively charged PS-spun fibers attract spin doping at the Taylor cone, which is positively charged. Because the deposited fiber carries a negative charge, it repels incoming fibers via electrostatic interactions. This electrostatic repulsion induces a stable 3D porous structure at the centimeter scale. The fiber stacks horizontally and resists gravitational collapse. This biomimetic approach overcomes the challenge of forming gravity-defying, self-standing, and spatially expanded 3D scaffolds through PS-driven self-assembly electrospinning without the need for external supports or post-processing steps.

This parallel between the natural spider ballooning phenomenon and electrospinning highlights how similar electrostatic principles can be harnessed in material fabrication to overcome the challenge of creating spatially expanded 3D scaffolds. As a result, these fluffy PS scaffolds exhibit a deeply interconnected porous architecture and fibrous morphology. Such structural features promote efficient cell migration, infiltration, and proliferation, thus enabling robust in vitro 3D cell culture. , The fibrillar network of scaffolds closely mimics the native ECM, facilitating precise tissue remodeling, enhancing intercellular communication, and improving cellular accessibility. These advantages surpass those of the 2D planar fiber mats.

PS has served as fundamental substrate for cell culture platforms due to its ease of manufacture, optical clarity and cost-effectiveness. However, cell adhesion to PS surfaces is inherently limited by their hydrophobic nature, which creates an unfavorable environment for adequate cell adhesion and proliferation. To promote cell anchoring on PS, surface modification with hydrophilic and biologically favorable functional groups, such as amines, hydroxyls, and carbonyls, is required. Although plasma activation and liquid surface treatments have been widely proposed, they require high ionization energy for corrosive substances. These treatments inevitably result in a certain degree of surface damage, leading to a decrease in the mechanical properties and structural integrity of the scaffolds , and limit the penetration of plasma species into 3D porous structures with uncontrolled submicrometer scale of pores. , Therefore, advanced surface-modification strategies using bioactive substances are required.

Silk sericin (SS) is a coating material used to functionalize the PS surfaces. SS is a naturally adhesive and hydrophilic protein in cocoons that covers silk fibroin filaments. SS has emerged as promising biomaterial in biomedical applications owing to its diverse biofunctional properties, including antimicrobial, antioxidant, anti-inflammatory, and wound healing effects. The well-preserved hydroxyl and polar carboxyl groups in hydrothermally extracted SS contribute to its hydrophilicity and reactivity, , making it an effective coating material without the need for additional chemical modifications that provides a suitable environment for cell adhesion, growth, and differentiation. , To achieve a uniform and stable coating of SS on the PS fibers, tannic acid (TA) was employed as a physical cross-linker via noncovalent interactions. TA, a naturally occurring plant polyphenol, contains abundant phenolic hydroxyl groups, which not only adsorb firmly onto the PS surface via π–π stacking but also function as hydrogen bond donors. Conversely, SS can act as a hydrogen bond acceptor due to its numerous carboxyl groups. ,

This study aimed to fabricate 3D scaffolds using electric repulsion that exhibit characteristics similar to spider ballooning, thereby providing both spatial and surface microenvironments suitable for cellular substrates. The PS/TA/SS scaffold exhibited a mesoporous structure and high specific surface area compared with conventional 3D nanoconstructs, mimicking the fibrous architecture of the native ECM to enhance HaCaT cell infiltration and integration. In addition, it demonstrated improved surface wettability, which is conducive to cell attachment. These biomimetic 3D nanoconstructs are promising for cellular cultivation and implantable clinical applications in various biomedical fields.

2. Experimental Section

2.1. Extraction of SS

Raw silk cocoons (Bombyx mori, Uljin Silk Farm, Korea) were cut into small pieces and autoclaved. The cocoon: distilled water ratio (v/v) was 1:30. A pure SS solution was obtained by filtration using Miracloth (EMD Millipore Corp.). The SS solution was frozen at −80 °C and lyophilized (FD8508, IlShinBioBase, Korea).

2.2. 3D Electrospinning of PS

PS (Mw ∼ 280,000, Sigma-Aldrich) was dissolved in a mixture of tetrahydrofuran (THF, J.T. Baker) and dimethylformamide (DMF, Samchun, Korea) in a 1:1 volume ratio. PS solutions with different concentrations ranging from 12% (w/v) to 20% (w/v) were prepared using a 5 mL syringe (BD Company) fitted with a 23 G needle (NanoNC, Korea). The spinning rate was set between 0.01 and 0.1 mL min–1. The working distance between the needle tip and the collector was 10–20 cm. The applied voltage ranged from 12 to 20 kV. All electrospinning parameters were experimentally optimized at 25 °C, with a relative humidity of 20–50%.

2.3. PS3D/TA/SS Scaffold Fabrication

Tannic acid TA (Samchun, Korea) and SS were dissolved in distilled water to concentrations of 0.2% and 1.3% (w/v), respectively. The TA and SS solutions were blended at a volumetric ratio of 1:10. The 3D electrospun PS (PS3D) was dip-coated in each solution (SS, TA, and TA/SS). The samples were denoted as PS3D/SS, PS3D/TA, and PS3D/TA/SS, respectively. All the samples were washed twice with DW and dried in a fume hood for 24 h.

2.4. Scanning Electron Microscope (SEM) Analysis

The samples were sputter-coated (MCM-100, SEC, Korea) with gold (Au) for 3 min. The morphologies of PS3D, PS3D/TA, and PS3D/TA/SS were assessed using a SEM (S-4300SE, HITACHI, Japan) at magnifications of 500× and 1000× at an accelerating voltage of 15 kV. Diameter distributions were analyzed using ImageJ (National Institutes of Health, Bethesda, MD).

2.5. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

The intramolecular interactions of the PS fibrous scaffolds (PS3D, PS3D/SS, PS3D/TA, and PS3D/TA/SS) were analyzed using FT-IR (VERTEX 80 V, Bruker). Before analysis, the nanofibers were dried at 80 °C for 24 h to minimize moisture-related artifacts. The dried specimens were mixed with potassium bromide (KBr) and pressed into disc molds for analysis. Absorption spectra were recorded at a resolution of 4 cm–1 in the wavenumber range of 4000–400 cm–1.

2.6. Rheological Measurement

The dynamic rheological properties of the TA/SS coating solution were analyzed using a rheometer (MCR702e, Anton Paar, Austria) with cone–plate geometry (1°, 50 mm diameter). The viscoelastic moduli (G′ and G″) were measured under frequency sweep mode with a 0.1 to 10 Hz range. The constant strain amplitude was set to 1%. The linear viscoelastic region (LVER) was determined using a strain sweep conducted at 1 Hz over 0.1–100% strain. The viscosity of TA/SS was measured by a shear rate from γ = 0.01 to 300 s–1. All experiments were performed at 25 °C and repeated three times with high accuracy. The results were analyzed using the RheoCompass 1.34 software (Anton Paar).

2.7. Brunauer–Emmett–Teller (BET) Surface Area Analysis

The adsorption isotherms and BET specific surface areas of the PS scaffolds were analyzed using an ASAP 2020 adsorption apparatus (ASAP 2020, Micromeritics). The samples (PS3D and PS3D/TA/SS) underwent degassing at 50 °C for 4 h to remove adsorbed gases. The ASAP 2020 software (Micromeritics) was used to calculate the theoretical surface area, pore size, and pore volume based on the BET and Barrett–Joyner–Halenda (BJH) equations.

2.8. Contact Angle Measurement

The wettability of the PS nanofiber was assessed using a contact angle goniometer (Phoenix 300 touch, Surface Electro-Optics Co., Korea) equipped with Surfaceware 9. A 10 μL water droplet was placed on the PS samples (PS3D, PS3D/TA, and PS3D/TA/SS). The contact angle was determined using ImageJ.

2.9. Cytocompatibility of PS3D/TA/SS

The human keratinocyte cells (HaCaT, RRID: CVCL-0038, Addexbio) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) antibiotic-antimycotic solution (Welgene, Korea). HaCaT cells were routinely tested and confirmed to be free of mycoplasma every three months. Cells were incubated in a humidified incubator at 37 °C and 5% CO2. 5 × 104 cells were suspended in 200 μL of cell culture media (n = 5) and seeded on the PS scaffolds (PS3D, PS3D/TA, and PS3D/TA/SS). After 1 h of cell attachment, the scaffolds were transferred to a new 48-well plate and incubated for a day.

Cell proliferation was assessed at 24, 48, and 72 h using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate absorbance spectrophotometer (EPOCH2, BioTek, Korea). Cell viability was assessed using the Live/Dead Viability/Cytotoxicity kit (Invitrogen) on day 3. Z-stack fluorescence images were obtained using a digital imaging system (CELENA S; Logos Biosystems, Korea) at 10× magnification.

2.10. Mechanical Property Analysis

The tensile properties were evaluated using a universal testing machine (34SC-1, Instron). Rectangular fibrous specimens (5 × 20 mm) were fixed between grips spaced 10 mm apart and tested at a crosshead speed of 5 mm min–1 with a 1 kN load cell.

2.11. Statistical Analysis

Statistical significance was calculated by Student’s t test using the SigmaPlot Software (Systat Software Inc.). A p-value less than 0.05 represented the presence of a significant difference (*p < 0.05, **p < 0.01, and ***p < 0.001).

3. Results and Discussion

3.1. Spider Ballooning-Mimetic Electrospinning for 3D PS Construct Formation

In nature, spiders release nanoscale silk fibers that become electrostatically charged, resulting in mutual repulsion and spatial dispersion, and forming a 3D silk configuration through ballooning. , Similarly, this electrostatic repulsion mechanism was realized in the electrospinning system by applying a high-voltage electric field, where the negatively charged collector served as the preferred deposition site, attracting the positively charged PS jet from the nozzle (Figure (A)). As the fibrous mat accumulated on the surface, it acquired the same negative charge, leading to electrostatic repulsion between the arriving fibers and the existing layer. This repulsion induced a horizontal expansion of the fibrous structure, resulting in self-standing scaffolds produced within minutes and extending over the centimeter scale. The resultant 3D fluffy scaffolds exhibited structural stability under gravity.

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(A) Schematic of spider ballooning and 3D electrospinning process. (B) Representative photographic and SEM images of 3D electrospun PS fibers and their diameter were obtained using different flow rates. Data are expressed as average diameter ± standard deviation (n = 40). Scale bars indicate 10 mm in photographic images and 10 μm in SEM images.

To successfully obtain 3D controllable architectures, the PS solution concentration, working distance, flow rate, and applied voltage must be adjusted. Uniform fibrous structures are essential for cellular scaffold use because they provide a consistent pore size, mechanical support, and spatially interconnected networks, all of which are critical for efficient cell infiltration, adhesion, and proliferation. ,

PS solutions with concentrations of 12, 15, 18, and 20% (w/v) were electrospun using an applied voltage of 18 kV, a tip-to-collector distance of 5 cm, and a flow rate of 0.05 mL h–1. At a concentration of 12% (w/v), the low viscosity of the solution led to reduced jet stability during electrospinning, resulting in a mixture of beads and fibers. As shown in Figure S1, the average fiber diameter increased with polymer concentration, reaching 1427 ± 197, 2497 ± 279, and 2597 ± 236 nm for 15, 18, and 20% (w/v), respectively. A concentration of 15% (w/v) was selected for further electrospinning optimization owing to its consistent fiber morphology and well-formed interconnected fiber networks.

The effects of the applied voltage were examined in the range of 12–20 kV, with a working distance of 15 cm and a flow rate of 0.05 mL min–1. The electrospinning jet was unstable at applied voltages below 15 kV, leading to bead formation and nonuniform fiber morphology (Figure S2). The average fiber diameters were 1099 ± 207, 480 ± 68, and 425 ± 100 nm for the applied voltages of 15, 18, and 20 kV, respectively. A uniform fiber morphology and stable 3D structures were obtained when the applied voltage exceeded 18 kV. At 18 kV or higher, the PS fibers formed 3D structures within seconds. In 20 min electrospinning, the resulting 3D structures grew to heights greater than 10 cm. This observation suggests that the top of the 3D structure shifted closer to the charged nozzle, thereby placing it within a stronger electric field. This enhanced polarization and charge induction promoted fiber deposition onto the growing structure rather than onto the flat collector. Consequently, at higher applied voltages, the deposited mat polarizes more rapidly, accumulates sufficient negative charge, and attracts the positively charged jet more effectively, thereby initiating 3D structure formation. An applied voltage of 18 kV was selected to reduce the initiation time for 3D structure formation and maintain stable electrospinning conditions during further optimizations involving extended working distances or elevated flow rates.

The effect of the working distance on the 3D build-up was studied at 18 kV and 0.05 mL min–1. As the working distance increased, the fiber deposition area expanded, which was attributed to the whipping and bending instabilities of the electrospun jet. Therefore, working distances greater than 20 cm did not result in a stable 3D build-up. However, between 10 and 15 cm, an appropriate 3D structure was observed. The measured average fiber diameters were 798 ± 442 and 491 ± 68 nm, respectively (Figure S3). Increasing the working distance decreased the fiber diameter because a more extended jet travel allowed more solvent evaporation, promoting further thinning and elongation of the fibers. A working distance of 15 cm was selected as the optimal distance to yield well-defined fibers.

Flow rates ranging from 0.01 to 0.1 mL min–1 were evaluated at an applied voltage of 18 kV and a working distance of 15 cm. Among these, 0.03 mL min–1 was the minimum flow rate capable of generating 3D structures. This outcome may be explained by the reduced number of fibers available for static induction and polarization at lower flow rates, resulting in an insufficient accumulation of negatively charged fibers that act as favorable deposition sites for the incoming electrospun jets. At 0.03 and 0.05 mL min–1, the 3D structures showed a lower height than those formed at 0.1 mL min–1 (Figure (B)). Similar to the effects observed at low polymer concentrations, a reduced number of fibers resulted in weaker fiber–fiber repulsion and a limited supply of fibers available to contribute to the buildup of 3D structures. The average fiber diameters were 443 ± 82, 404 ± 59, and 840 ± 218 nm for the flow rates of 0.03, 0.05, and 0.1 mL min–1, respectively. Flow rates of 0.1 mL min–1 produced the thinnest diameters, whereas higher flow rates resulted in a greater volume of solution being ejected from the needle. The increased solution volume necessitates a longer evaporation time, and the jet will require additional stretching to achieve a finer diameter. However, increasing the flow rate while maintaining a constant voltage may yield detrimental effects. This occurs because the number of available charged ions may become insufficient to effectively stretch the solution’s larger volume, potentially resulting in suboptimal fiber formation. Although higher flow rates increased production, a flow rate of 0.05 mL min–1 was selected to achieve delicate control over 3D structure formation and uniform fiber morphology. Altogether, the optimum manufacturing conditions for 3D electrospun PS scaffolds were 15 cm of working distance, 0.05 mL min–1 spinning rate, and 18 kV voltage, with 15% (w/v) spin dope.

3.2. Fabrication of PS3D/TA/SS Scaffold

PS has been used as a fundamental substrate for human and animal cell cultures for over five decades because of its cost-effectiveness, large-scale manufacturing process, nontoxicity, and ease of molding into diverse shapes. , However, cell adhesion to PS surfaces is limited because of their hydrophobic nature, which hinders the formation of a suitable environment for cell growth. To promote cell anchoring on PS-based scaffolds, surface modification is required to enhance hydrophilicity and introduce biologically relevant functional groups, such as amine, hydroxyl, and carbonyl groups. SS was employed as a coating material to functionalize the PS surfaces (Figure (A)). To achieve the uniform deposition of SS onto the PS fibers, TA was used as a physical cross-linker via noncovalent interactions. TA, which contains abundant phenolic hydroxyl groups, firmly adheres to the PS surface through π–π stacking interactions and is an effective hydrogen bond donor. Conversely, SS can act as a hydrogen bond acceptor with numerous carboxyl groups. ,

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(A) Schematic of the SS-coated electrospun PS scaffold fabrication using TA as a cross-linker. (B) Storage modulus as a function of the frequency and (C) effect of shear rate on the apparent viscosity of TA/SS coating solutions. (D) FT-IR spectra of nanofibers (PS3D, PS3D/SS, PS3D/TA, and PS3D/TA/SS) from 2000 to 1000 cm–1 wavelength, and (e) from 4000 to 3000 cm–1 wavelength (red box: hydroxyl group interaction).

An ideal coating solution should achieve complete surface coverage and maintain uniformity and consistency upon deposition to obtain a homogeneous coating layer. Rheological properties are critical for the development of coating materials because they affect the spreadability, homogeneity, and redispersion of substances. Figure (B),(C) show the viscoelastic properties of the TA/SS coating solutions within a linear viscoelastic range. The storage modulus (G′) and loss modulus (G″) are key viscoelastic parameters for TA/SS coating solutions. G′ and G″ moduli were measured at a fixed strain of 1% within the linear viscoelastic region (LVER) (Figure S4), while varying frequency (Figure (B)). The G′ exceeded the G″ across all frequency ranges, indicating that the elastic component of the dispersions dominated over the viscous component. The TA/SS coating solutions exhibited a plateau region with minimal frequency dependence, which indicates quasi-elastic behavior typical of weak gel systems. After 6 h of the coating process, both the storage modulus (G′) and the loss modulus (G″) increased, which was attributed to the formation of additional hydrogen bonds between TA and SS. This increase suggests a transition toward predominantly elastic behavior, reflecting the enhanced ability of the material to resist deformation and maintain its structure. For coating applications, such an elastic dominance implies that the dispersion behaves similarly to a solid rather than a liquid (viscous), supporting the formation of a stable, uniform layer that resists uncontrolled spreading or dripping. This rheological behavior ensures coating integrity, consistency, and functional performance on the substrate surface.

A decrease in viscosity with increasing shear rate, as shown in Figure (C), demonstrates the shear-thinning behavior typical of non-Newtonian fluids. This behavior was attributed to the structural deformation of the network formed in the equilibrium state. As the shear rate increased, the particles aligned in the flow direction, disrupting the TA/SS molecular network and reducing the flow resistance. The shear-thinning behavior of TA/SS is crucial for obtaining a thin coating layer on 3D electrospun PS scaffolds via dip-coating, ensuring that the porous structure of the fibrous scaffold remains intact. After 6 h of coating, a slight increase in the apparent viscosity of TA/SS was observed across the entire shear rate range, indicating the formation of a viscoelastic gel state through physical cross-linking, which allowed a firm coating.

To verify the successful deposition of TA/SS onto the PS 3D fibers, FT-IR analysis was performed (Figure (D),(E)). Firm absorption peaks corresponding to the C–O aromatic ester (1224 and 1365 cm–1) and CO aromatic ester (1622 and 1714 cm–1), which are characteristic of the galloyl group in TA, confirmed the presence of TA. , These peaks were observed in both PS3D/TA and PS3D/TA/SS spectra, indicating that TA was successfully coated on the 3D PS fiber via π–π interactions between PS and TA (Figure (D)). The peaks at 1068, 1506, and 3280 cm–1, corresponding to C–N stretching, N–H bending, and N–H stretching, respectively, were attributed to the amide bonds in SS. Notably, these peaks were observed in the PS3D/TA/SS spectrum, however, were absent in the PS3D/SS spectrum. The PS3D/SS spectrum closely resembled that of the untreated PS3D, which indicates the absence of SS deposition. Both PS3D/TA and PS3D/TA/SS spectra exhibited a broad peak of 3200 to 3550 cm–1, resulting from intramolecular hydrogen bonding and hydroxyl group stretching (Figure (E)). The peak at 3280 cm–1 confirmed the presence of an intermolecular hydrogen bond between TA and SS, which was only observed in the PS3D/TA/SS spectrum, suggesting that direct coating of SS on PS fibrous scaffolds is not feasible without using TA as a cross-linker.

The formation of stable TA-SS interactions on the PS scaffolds was expected to not only to modify surface chemistry but also to influence the macroscopic mechanical properties of the scaffold. To investigate whether the TA-mediated SS coating affects the mechanical performance of the PS scaffold, tensile tests were conducted. As shown in Figure S5 and Table S1, compared to pristine PS3D (152.354 ± 1.682 kPa), the PS3D/TA/SS (178.159 ± 14.622 kPa) scaffold exhibited enhanced tensile strength. This enhancement was attributed to the formation of intermolecular interactions between TA and SS, which contribute to improved structural integrity of the fibrous network.

Additionally, the storage stability of the TA–SS coating was evaluated under ambient conditions. PS3D/TA/SS maintained their coating integrity without visible delamination or degradation after storage at room temperature under typical humidity for up to 6–12 months, as shown in Figure S6. The storage stability of the TA–SS coating supports the potential practical applicability of PS3D/TA/SS as a 3D scaffold for in vitro cell culture systems.

3.3. Surface Wettability of PS3D/TA/SS

Surface wettability is a key parameter that significantly affects the mechanical properties of scaffolds, cell adhesion, and proliferation. It influences cell morphology and protein binding interactions in the culture medium, thereby promoting cell attachment. Surface modification of scaffolds is essential for controlling cellular behavior in various biomedical and biotechnological applications.

Water contact angle measurements were conducted to evaluate the effect of the SS treatment on the surface wettability of the PS scaffolds (Figure (A)). The water contact angle values for untreated PS were 119.7 ± 1.0°, indicating high surface hydrophobicity. This was attributed to the inherent hydrophobicity of PS and to the enhanced hydrophobicity of the fibers during electrospinning. , However, modifications with PS3D/TA and PS3D/TA/SS significantly improved the wettability of the PS surface, with contact angle values of 7.9 ± 2.3° and 39.2 ± 2.7°, respectively. This substantial enhancement in the treated PS is primarily attributed to the surface chemistry/functional groups, as well as surface micro- and nanoscale morphological features.

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(A) Contact angle measurement of nanofiber surface (PS3D, PS3D/TA, and PS3D/TA/SS). (B) SEM images of PS3D, PS3D/TA, and PS3D/TA/SS fiber morphology. All error bars represent the standard deviations (n = 3) with statistical significance (***p < 0.005). Scale bars indicate 20 μm.

The phenolic hydroxyl groups in the TA molecules reduced the hydrophobicity of PS in the PS3D/TA samples, thereby increasing surface wettability. The presence of functional groups, as confirmed by FT-IR (Figure (D)), further validated successful TA treatment. The incorporation of SS altered the surface characteristics and morphology of the electrospun PS fibers due to SS’s soluble, hydrophilic nature. The amine, hydroxyl, carboxyl, and carbonyl groups of amino acids with polar uncharged side chains (such as serine, asparagine, threonine, and glutamine) or charged side chains (such as lysine, arginine, glutamic acid, and aspartic acid) contribute to the increased hydrophilicity of PS scaffolds, thereby lowering their water contact angle. Moreover, the 3D electrospun PS morphology was analyzed using SEM images. Figure (B) shows the smooth surfaces observed for both PS3D/TA and PS3D/TA/SS compared with the rough surface of the uncoated PS. The hydrophilicity of a solid surface can be enhanced by increasing its smoothness. Per the SEM images, the SS coating evenly covered the fiber surface, with some coating penetrating the fibrous network.

These results suggest that the PS3D/TA/SS scaffolds with improved surface wettability promote cell spreading and anchoring and interact with proteins from the culture medium, thereby enhancing cell proliferation. This simple colorless coating strategy enabled the successful formation of hydrophilic PS scaffolds, making them highly suitable for various biomedical applications and biological studies.

3.4. Specific Surface Area of PS3D/TA/SS

The effect of SS treatment on the structure and morphology of the PS scaffolds was analyzed using nitrogen (N2) adsorption–desorption measurements based on BET technology. The detailed specific surface area, pore volume, and average pore diameter of the porous PS scaffolds (PS3D and PS3D/TA/SS) are presented in Table . Figure (A) shows the adsorption–desorption curves in the isotherms of both the PS3D and PS3D/TA/SS scaffolds, indicating H1 hysteresis loops (Figure (C)). They exhibited a sharp increase at relatively high pressures near P/P 0 = 1, corresponding to a Type IV isotherm according to IUPAC (Figure (D)), demonstrating the presence of layered pores and a mesoporous structure. , Moreover, the PS3D and PS3D/TA/SS scaffolds exhibited pore diameters of <20 nm (Table ), consistent with the mesoporous (<100 nm) architecture. Additionally, mesoporous structures enhance cellular responses and biological activity in cell-laden scaffolds by providing a large specific surface area and high pore volume, thereby improving initial cell adhesion, proliferation, and nutrient exchange. ,

1. BET Analysis Results for 3D Electrospun PS Scaffolds.

  BET surface area (m2 g–1) BJH pore volume (cm3 g–1) average pore diameter (Å)
PS3D 40.882 (±4.832) 0.098 (±0.012) 111.768 (±1.863)
PS3D/TA/SS 35.451 (±1.699) 0.112 (±0.019) 146.719 (±25.570)
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BET analysis of 3D PS scaffolds. (A) BET isotherm plots for nitrogen adsorption capacity and (B) calculated BET surface area of PS3D and PS3D/TA/SS. Classification of (C) hysteresis loops and (D) adsorption isotherms. All error bars represent the standard deviations (n = 3) with statistical significance (n.s.: not significant).

The specific surface areas of untreated PS3D and SS-coated PS3D were obtained using BET equations, resulting in 40.882 ± 4.832 and 35.451 ± 1.699 m2 g–1, with no statistically significant difference (Figure (B)). These results demonstrated that SS treatment did not hinder the formation of the pure PS3D area during the coating process. Moreover, both PS3D and PS3D/TA/SS not only had a higher specific surface area than the 2D PS mat (data not shown), but also a 6-fold higher specific surface area than PS3D scaffolds electrospun under the same solvent conditions using a different fabrication method in a previous study.

The cumulative pore volume and average pore diameter of the porous PS scaffolds were calculated using BJH desorption analysis (Table ). The total pore volume, representing the vacant spaces between nanofibers in the 3D network, was 0.098 ± 0.012 and 0.112 ± 0.019 cm3 g–1 for PS3D and PS3D/TA/SS, respectively. The average pore diameter was 111.768 ± 1.863 and 146.719 ± 25.570 Å, respectively. The properties of the porous structures showed no significant difference between PS3D and PS3D/TA/SS. The average pore diameter is related to the fiber diameter in the 3D electrospun scaffolds. − As the PS3D/TA/SS scaffolds were obtained from PS3D via SS coating while maintaining the same fiber diameter, no significant differences in their porous characteristics were observed.

These results demonstrated that nanofibrous PS3D scaffolds can be fabricated with an enhanced BET-specific surface area compared to the conventional method. Moreover, the SS treatment improved the wettability of the scaffolds without hindering their specific surface areas. Given their porous characteristics and high permeability, they are expected to be utilized as cellular scaffolds in 3D environments. Furthermore, these mesoporous scaffolds facilitate cell infiltration throughout the 3D structures, maintain cellular organization, and meet internal mass transfer requirements, which are essential for actively growing cells within porous constructs.

3.5. Biocompatibility of PS3D/TA/SS

Several existing scaffolds for 3D cell cultures, including hydrogels, aerogels, and bioprinted constructs, have inherent limitations. These systems typically involve cell-gel suspensions that, once cross-linked, encapsulate cells within a 3D environment or rely on static seeding through the drop-on method. , However, these approaches often result in cell aggregation, limited penetration, poor migration, and less homogeneous cellular distribution, given the polymeric cross-linked network. , In addition, cells tend to proliferate locally and inconsistently within isolated scaffold regions.

3D nanofibrous scaffolds with high wettability, porosity, and surface-specific area are expected to offer promising alternatives to overcome these challenges by enhancing scaffold infiltration, diffusion, attachment, and cell–cell interactions. − As proof of concept, the human keratinocyte HaCaT cells were employed as the model cell line for cultivation on 3D electrospun PS scaffolds (PS3D, PS3D/TA, and PS3D/TA/SS), owing to their subculturing variations and short culture life. Given the requirement for a 2D to 3D culture transition, HaCaT cells provide a simple and reliable approach for establishing skin equivalent models with potential clinical relevance. Nondegradable PS scaffolds maintained structural stability for up to 3 weeks without shrinkage, correlating with the duration of differentiation and full-thickness skin tissue regeneration. Additionally, the thickness of the 3D electrospun PS scaffolds can be precisely controlled, allowing the fabrication of layer-by-layer architectures with thicknesses ranging from micrometers to several millimeters. Keratinocytes are anchorage-dependent cells that require substrate adhesion for their growth. The 3D electrospun PS scaffolds were designed to address the limitations of cell attachment and migration typically observed in 2D cultures, providing a 3D environment that closely replicates the native ECM’s nanofibrous architecture, thereby supporting HaCaT cell growth and proliferation.

The coated PS (PS3D/TA and PS3D/TA/SS) exhibited enhanced initial attachment to HaCaT cells compared to PS (Figure (A)). PS3D/TA/SS exhibited a 2-fold higher adhesion than PS, followed by PS3D/TA with a 1.5-fold increase. After 2 days of culture, HaCaT cell proliferation stagnated in PS3D and accelerated in both PS3D/TA and PS3D/TA/SS (Figure (B)). On day 3, the OD values of both coated PS3D were significantly higher than those of PS, indicating that the improvement in cell proliferation was attributable to the enhanced wettability of the scaffold surface, as shown in Figure (B). Both the TA and TA/SS treatments improved scaffold wettability, facilitating greater cell support. The resulting higher cell confluence potentially improved subsequent cell proliferation.

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(A) Initial attachment plots according to the CCK-8 assay at day 1 and (B) CCK-8 assay results of HaCaT cells on 3D fibrous scaffolds (PS3D, PS3D/TA, and PS3D/TA/SS) over 3 days. (C) Live/dead staining images of HaCaT cells cultured on 3D fibrous scaffolds at day 3. The data were presented as the mean ± standard deviation (n = 5) with statistical significance (*p < 0.05 and ***p < 0.001). Scale bars indicate 100 μm.

The viability of HaCaT cells on the 3D PS-based scaffolds (PS3D, PS3D/TA, and PS3D/TA/SS) was assessed on day 3 using a Live/Dead assay (Figure (C)). Most cells were viable, as indicated by green staining. In contrast, dead red-stained cells were rarely observed across all tested fibrous meshes (PS3D, PS3D/TA, and PS3D/TA/SS), indicating that 3D electrospun PS matrix exhibited excellent biocompatibility. Moreover, in all PS3D-based matrices, HaCaT cells adhered and spread with a rounded morphology and could infiltrate the internal scaffolds, as evidenced by the z-axis image stack. However, the cells formed small island-like clusters on both PS3D and PS3D/TA surfaces, indicating limited lateral migration over the surface. Conversely, they were well distributed across the entire curved surface of the PS3D/TA/SS fibrous matrix. Homogeneous cell distribution is crucial for replicating specific natural tissues, whereas crowded cells spontaneously aggregate, further aggravating cellular inhomogeneity.

Although the adhesion and proliferation mechanisms of HaCaT cells differ across substrates and remain unclear, surface wettability and functional groups influence cell growth and attachment. Additionally, the presence of polar surface functional groups is a critical factor, as HaCaT cells primarily adhere via carbonyl and carboxyl groups, favoring hydrophilic surfaces for enhanced cell proliferation. This adhesion was further stabilized by van der Waals forces, hydrogen bonding, reinforcing cells, and substrate surface interactions. The PS3D/TA surface was functionalized with hydroxyl groups. Conversely, the PS3D/TA/SS surface was modified with various chargeable functional groups, including amine, hydroxyl, carboxyl, and carbonyl groups derived from the SS amino acids. This surface functionalization significantly enhanced cell migration and proliferation on PS3D/TA/SS compared with PS3D and PS3D/TA. This remarkable performance of PS3D/TA/SS was attributed to the synergistic effect of the scaffolds’ suitable wettability and the biochemical functionalities provided by the coated SS.

In summary, PS-based scaffolds have been the primary substrates for in vitro cell culture for over five decades, owing to their ease of fabrication and cost-effectiveness. However, unmodified PS scaffolds have limited capacity to support cell adhesion and growth due to their hydrophobic surfaces. The SS-coated 3D electrospun PS scaffolds served as a porous, advanced platform for cell culture in a 3D environment, allowing keratinocytes to grow, proliferate, infiltrate, and migrate across them. Moreover, previous studies have reported that SS promotes collagen production without inducing pro-inflammatory responses, suggesting that PS3DTA/SS may provide a favorable regenerative microenvironment for skin tissue regeneration. , Notably, similar to Alvetex, a commercially available porous PS scaffold used in 3D cell culture systems and as a matrix for bioengineered human skin (REPROSKIN), the PS3D/TA/SS exhibits considerable promise as a platform for three-dimensional cell culture, particularly in the context of in vitro biological studies and artificial skin substitutes.

4. Conclusion

This study reports the fabrication of 3D electrospun PS scaffolds, which naturally exhibit similarities to the spider ballooning phenomenon and mimic the fibrous architecture of the native ECM. By distinguishing itself from previously reported 3D electrospinning approaches that rely on external supports, auxiliary collectors, or secondary components, this biomimetic approach enables the formation of spatially expanded, self-standing 3D constructs using only single base material.

To address the hydrophobic nature of PS and improve its bioactivity, a simple biocompatible coating approach was developed using TA as a cross-linker and SS as the bioactive component. TA-mediated SS coating strategy facilitated uniform SS deposition on both the external and internal scaffold surfaces. This coating process facilitates biofunctionalization of 3D fibrous scaffolds while improving surface wettability and physicochemical properties without reducing the specific surface area of the porous architecture.

The resulting PS3D/TA/SS scaffolds displayed cell-favorable surface characteristics, supporting uniform cell distribution, improved adhesion, and enhanced proliferation. These effects were mainly attributed to the multiple chargeable functional groups of SS, which strengthened the cell-scaffold interactions. Moreover, the scaffolds supported keratinocyte infiltration throughout the 3D matrix, thereby facilitating robust tissue ingrowth. These results demonstrate the potential of the biomimetic PS3D/TA/SS scaffolds for in vitro studies and clinical applications in skin tissue engineering. In addition to skin-related applications, the proposed platform offers a broadly applicable framework for 3D cell culture and the development of advanced fibrous biomaterials requiring spatially organized and bioactive scaffold.

Supplementary Material

ao5c12907_si_001.pdf (421.1KB, pdf)

Acknowledgments

This study was supported by the Global Research Development Center (GRDC) Cooperative Hub Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT) (RS-2024-00435183); the National Research Foundation of Korea (NRF) grant, funded by the Korea government (MSIT) (RS-2025-00515679); and the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant, funded by the Korea government (MSIT) (RS-2024-00403941).

Data used is available throughout the manuscript text.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12907.

  • Supporting figures (SEM characterization, mechanical and rheological analysis) (PDF)

S.H.M.: Investigation, validation, visualization, writingoriginal draft, writingreview and editing. J.H.J. and S.J.P.: Investigation. H.R.J.: Visualization. Y.J.Y.: Conceptualization, supervision, funding acquisition, writingoriginal draft, writingreview and editing.

The authors declare no competing financial interest.

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