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. 2026 Sep 11;11(37):55910–55920. doi: 10.1021/acsomega.6c05640

Improvement of the Stability of Functionalized PVA Nanofibers for Sensitive Compound Incorporation by Gentle Freeze–Thaw Treatment

Maxim Lisnenko †,‡,*, Sarka Hauzerova †, Katerina Strnadova †, Jana Mullerova †, Renata Prochazkova §, Lubos Behalek ∥, Jan Valtera ⊥, Kristyna Havlickova †, David Lukas †, Vera Jencova †,*
PMCID: PMC13613426  PMID: 42798875

Abstract

Recent advances in tissue engineering focus on enhancing the biological performance of nanofibrous materials that mimic the extracellular matrix. These materials provide mechanical stability and form nanofibrous membranes that support cell adhesion, migration, and growth. Their functionality can be further improved by incorporating bioactive compounds, especially in water-soluble polymer-based nanofibers, enabling controlled release. However, a major limitation is the “burst release” effect, where more than 90% of the embedded substances are released within the first 24 h. The second challenge is the stabilization of these water-soluble systems. The most commonly used chemical crosslinking methods require the addition of chemical agents (crosslinkers) that are potentially toxic and may lead to structural and chemical changes in the nanofibrous materials, while also causing denaturation of the incorporated bioactive compounds. This study presents the development of functionalized nanofibers based on water-soluble poly­(vinyl alcohol) (PVA), produced by an advanced alternating current (AC) electrospinning method, and their subsequent stabilization using a non-toxic Freeze-Thaw (FT) method. This physical stabilization technique avoids chemical crosslinkers and instead induces crystallinity through polymer chain reorganization, reducing solubility while preserving structural integrity. The impact of FT stabilization on the physicochemical properties and solubility of PVA nanofibers was evaluated, along with its effect on the release profile of human platelet lysate (PL), a growth factor-rich additive. The results show that FT treatment effectively stabilizes PVA nanofibers and significantly slows down the release of incorporated PL. Specifically, the solubility of neat PVA nanofibers after 10 FT cycles under thawing at +4 °C decreased by 19.5%, whereas in PVA + PL nanofibers the reduction in PVA solubility under the same conditions reached 12.2%. The release of PL itself slowed by 18.2% after 10 FT cycles. Changes in crystallinity were also observed in the FT-treated materials. This approach offers a promising platform for developing safe, functional nanofibrous materials for chronic wound treatment and skin regeneration.

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1. Introduction

Water-soluble polymers play a crucial role in a wide range of industries, including biomedicine, pharmaceuticals, food processing, and cosmetics. They are particularly valuable in the development of nanofibrous scaffolds due to their ability to form biocompatible, three-dimensional structures that support cell adhesion, proliferation, and tissue regeneration. , These polymers are generally classified as either natural, such as gelatin, chitosan, and xanthan, or synthetic, including poly­(vinyl alcohol) (PVA), poly­(ethylene oxide) (PEO), and polyvinylpyrrolidone (PVP). Among these, PVA is well known for its excellent biocompatibility, non-toxic nature, and favorable mechanical properties, making it widely used in medical and pharmaceutical applications such as soft contact lenses, wound dressings, and drug delivery systems. −

However, a major limitation of water-soluble polymers like PVA is their tendency to rapidly dissolve in aqueous environments. This often results in a burst release of incorporated bioactive agents, with over 90% released within the first 24 h. , To overcome this, stabilization techniquesboth chemical and physicalare employed to enhance the mechanical and chemical resilience of the polymer matrices. , While chemical crosslinking forms strong covalent bonds, it often involves toxic agents like glutaraldehyde that may compromise biocompatibility. , In contrast, physical stabilization methods such as UV irradiation, plasma treatment, and thermal processes avoid harmful chemicals and are more suitable for biomedical applications. However, physical methods generally provide a lower degree of crosslinking and mechanical strength compared to chemical techniques.

This study focuses on physical-thermal stabilization of PVA nanofibers using the Freeze-Thaw (FT) technique, a method originally introduced by Stauffer and Peppas for producing physically crosslinked PVA hydrogels. The FT process involves cycles of freezing at sub-zero temperatures and thawing at above-zero temperatures. During freezing, ice crystals are formed from water, confining PVA microdomains and encouraging crystallization. Each cycle enhances physical crosslinking by forming hydrogen bonds between hydroxyl groups on PVA chains. The effectiveness of this stabilization depends on parameters such as the number of cycles, the applied temperatures, and the exposure times. , Due to its mild conditions and absence of harmful reagents, the FT method represents a highly suitable approach for the stabilization of materials incorporating sensitive bioactive molecules, including proteins and growth factors, thereby preserving their structural integrity and biological activity.

Research by Wang, Yang, and Xu demonstrates that controlled heat treatment significantly improves the water stability and mechanical properties of PVA porous membranes through thermal crosslinking. Similarly, FT treatment has also been shown to reduce solubility and enhance mechanical performance by increasing hydrogen bonding. Adelnia et al. observed that increasing the number of FT cycles led to improved tensile strength and reduced solubility of PVA hydrogels, confirming the role of FT in promoting physical crosslinking through crystalline domain formation. Additionally, the degree of hydrolysis of the PVA used for material preparation significantly affects the resulting solubility. A higher content of hydroxyl groups leads to stronger hydrogen bonding between chains, resulting in higher crystallinity and consequently lower solubility.

Electrospun nanofibrous materials differ substantially from other classes of materials used in tissue engineering, such as hydrogels, films, and microspheres, both in their structural architecture and functional properties. Their high surface-to-volume ratio, interconnected porous network, and morphological resemblance to the extracellular matrix make them highly attractive for biomedical applications. However, these characteristics also render water-soluble nanofibers particularly susceptible to dissolution and loss of structural integrity upon exposure to aqueous media. Consequently, stabilization strategies developed for PVA hydrogels , or microspheres cannot be directly extrapolated to electrospun nanofibrous materials, and their effectiveness must be specifically evaluated for nanofiber-based systems.

As part of presented research, platelet lysate (PL)a biologically active substance rich in sensitive growth factorswas incorporated into PVA nanofibers. The FT-stabilized nanofibers demonstrated reduced dissolution and a slower, more controlled release of PL compared to non-stabilized materials. These findings highlight the potential of FT-stabilized PVA scaffolds for use in regenerative medicine, particularly for the treatment of chronic and complex skin wounds, where gradual and sustained bioactive release is essential.

2. Materials and Methods

2.1. Preparation of Nanofibrous Materials by Alternating Current (AC) Electrospinning

The electrospinning apparatus for alternating current (AC) was assembled at the Faculty of Mechanical Engineering, Technical University of Liberec. The electrospinning process utilized a 10 wt % poly­(vinyl alcohol) (PVA) solution with a molecular weight of 125,000 and a hydrolysis degree of 98%–98.8%, dissolved in distilled water. The PVA was supplied by Merck (Germany). The distilled water was provided by Penta (Czech Republic). Following this, a mixture of 10 wt % PVA and 10 wt % platelet lysate (PL) protein content was used. The PL was prepared as described by Koprivova et al., using a platelet concentrate with a concentration of 738 × 106 PL/mL obtained from the Regional Hospital in Liberec. Briefly, the platelet concentrates were prepared from pooled human buffy coats obtained from four donors, followed by the removal of residual erythrocytes and leukocytes. Unlike the previously described procedure, the InterSol additive solution (Fresenius, Germany) was used during platelet concentrate preparation. All blood donors provided informed consent for the use of their blood-derived products for research purposes, and the study was approved by the hospital’s ethics committee. The platelet lysate was subsequently prepared according to the previously described protocol. The total protein concentration in the platelet lysate was determined using the Bradford assay with BSA as the calibration standard (see the Bradford assay section in the Materials and Methods), yielding a protein concentration of 15.9 mg/mL.

The electrospinning parameters were established based on preliminary optimization experiments performed to identify suitable operating conditions for the AC electrospinning process, while also taking into account the parameters previously reported in our published work. Several parameters, including applied voltage, waveform type, frequency, collector rotational speed, and electrode distance, were varied and evaluated with respect to process stability, jet formation behavior, and the resulting fiber morphology. The final conditions were selected as a compromise between stable continuous electrospinning and uniform fiber deposition. A signal generator (Owon AG 1022; China) coupled with a high-voltage amplifier (TREK 50/12C-H-CE; United States of America) was employed to generate high-amplitude AC potentials with adjustable waveforms and frequencies. In this study, square waveforms at a frequency of 40 Hz and an effective voltage of 50 kV were used to initiate and sustain the electrospinning process. Upon activation of the alternating electric field, polymer jets were formed on the electrode surface, where they underwent rapid solvent evaporation, leading to the creation of nanofibers. The screw pump operated at approximately 550 rpm, ensuring a constant solution flow rate of 14–18 mL/min to the spinneret surface (diameter: 22 mm), while the fibrous plume was continuously collected on a layer of spun-bond polypropylene fabric mounted on a rotating drum serving as an electrically inactive collector. The peripheral speed of the collector was maintained between 30 and 35 m/min, with the distance between the electrodes set to 255 mm. All experiments were conducted at a temperature of 23 °C and a relative humidity of 50%. Further details of the AC electrospinning setup are available in Valtera et al.

2.2. Stabilization of Materials

For material stabilization, the fabricated fibers were subjected to a low-temperature physical-thermal treatment. The stabilization cycles were configured so that the materials were kept for 16 h at −20 °C in a freezer, followed by 8 h periods at temperatures of +4 °C, +22 °C, or +37 °C. The impact of these varying temperatures and the number of stabilization cycles (5 and 10 cycles) was assessed by comparing the results with those of reference materials that had not undergone any stabilization process.

2.3. Morphological Analysis

The morphology of the prepared samples was examined with a scanning electron microscope (SEM), specifically the Vega 3 Tescan (Czech Republic), using magnifications of 1000×, 3000×, and 5000×, and a voltage setting of 15 kV. To prepare the samples, a small section of the material was cut and fixed onto a metal target with double-sided adhesive tape. A 10 nm gold coating was then applied to the samples using a Quorum Q150R S Plus sputter coater from Quorum Technologies (United Kingdom). Fiber diameters were measured via image analysis with ImageJ, developed by Fiji Contributors. A total of 400 fiber diameters per material were randomly measured at a magnification of 5000×. The data were processed to calculate the mean ± standard deviation and were visualized using GraphPad Prism 11.0.1 software from GraphPad Software (United States of America).

2.4. Fourier-transform Infrared Spectroscopy (FTIR)

FTIR (Fourier-transform infrared) spectroscopy was utilized for structural analysis of materials using a Nicolet iZ10 instrument from Thermo Fisher Scientific (United States of America). This technique involves the absorption of electromagnetic radiation at various wavelengths, which interacts with the sample. During the analysis, the samples were positioned on a diamond crystal (using the attenuated total reflection method), and identification of organic compounds occurred in the spectral range of 400–4000 cm-1 at room temperature. Functional groups, such as O–H and C–H bonds in the organic compounds, were detected sequentially from the highest to the lowest wavenumbers. A spectrum of an unstabilized material, serving as a negative control, was included in each set of spectra.

2.5. Differential Scanning Calorimetry (DSC)

DSC was employed to evaluate the thermal behavior of PVA-based materials. Three replicates from each FT condition (weighing 6–10 mg per sample) were placed in individual aluminum pans with a volume of 25 μL, sealed with pressure-tight lids, and analyzed using a DSC 1/700 instrument (Mettler Toledo, Switzerland). The measurements were carried out under a nitrogen atmosphere (flow rate: 50 mL/min) at a heating rate of 10 °C/min over a temperature range from 0 °C to 300 °C. The samples were stored under ambient laboratory conditions (room temperature and relative humidity), as they were not exposed to water during preparation or handling and therefore no additional drying procedure was applied prior to DSC measurements. The thermograms obtained from the first heating cycle were processed and evaluated using the STARe software (Mettler Toledo, Switzerland). The degree of crystallinity was calculated from the melting enthalpy obtained during the first heating cycle, assuming a theoretical melting enthalpy of 138.6 J/g for fully crystalline PVA according to Guirguis and Moselhey. The raw DSC thermograms are provided in the Supporting Information (Figures S1–S4).

2.6. The Dissolving of PVA

For the dissolution analysis of PVA, each material sample (before and after FT) was carefully weighed to 10 ± 0.1 mg. Three replicates of each material were prepared and placed in 1.5 mL microtubes containing 1 mL of PBS (pH 7.4) with 0.02% sodium azide, supplied by Sigma-Aldrich (Germany). The samples were incubated at 37 °C, and aliquots of 200 μL were taken at predetermined intervals (1, 2, 4, 6, 8, 24, and 72 h). After each collection, an equal volume of buffer was added back to the tubes. The collected samples were immediately frozen at −20 °C for subsequent analysis.

The aliquots were analyzed using ultraviolet–visible (UV–Vis) spectroscopy. Prior to measurement with a Spark spectrometer from Tecan (Switzerland), calibration solutions of PVA at known concentrations were prepared (0.05, 0.1, 0.25, 0.5, 1, 2, 3, 4, and 5 mg/mL). Both calibration solutions and aliquots were diluted 10-fold with distilled water. All measurements were performed in triplicate. To form the color complex, the following reagents were added to a 96-well microtiter plate: 37.5 μL of potassium iodide solution, 187.5 μL of 4 wt % boric acid (H3BO3), 45 μL of distilled water, and 5 μL of the sample, resulting in a 10-fold dilution. After thorough mixing, absorbance was measured at 630 nm. The average absorbance of the blank (0 mg/mL solution) was subtracted from each sample measurement, and the PVA concentration in the aliquots was determined using the linear regression equation from the calibration curve. The cumulative mass of dissolved PVA was then calculated from this concentration.

2.7. Protein Release Analysis of Platelet Lysates Using the Bradford Assay

To determine the protein concentration, a calibration curve was created using known concentrations of bovine serum albumin (BSA) dissolved in distilled water (0, 0.25, 0.5, 0.75, 1 mg/mL). For the assay, 96-well microtiter plates were used. A 5 μL sample was added to each well, followed by 250 μL of Bradford reagent, supplied by Merck (Germany). After incubating for 5 min at room temperature, the absorbance of each sample was measured at 595 nm. From these readings, the protein concentration was calculated, and the protein amount per gram of the original nanofibrous material was determined.

2.8. Statistical Analysis

Quantitative evaluation of the obtained data is presented as the mean ± standard deviation. Data normality was assessed using the Shapiro–Wilk test. Statistical comparison of nanofiber diameters obtained from morphological analysis, which showed a non-normal distribution, was performed using the non-parametric Kruskal–Wallis test for multiple comparisons. Statistical evaluation of normally distributed data obtained from the solubility testing of PVA nanofibers and the release of the incorporated active substance PL was performed as follows. First, the individual PVA and PL release profiles obtained under different FT conditions and numbers of FT cycles were quantitatively compared. The profiles were evaluated using the area under the curve (AUC), calculated by the trapezoidal method. The AUC approach was selected because it provides a single quantitative parameter describing the overall dissolution and release behavior over the entire experimental period, allowing direct comparison of complete time-dependent release profiles while avoiding multiple statistical comparisons at individual time points. Subsequently, statistical analysis of the AUC values was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. , All statistical analyses were performed using GraphPad Prism 11.0.1 (GraphPad Software, United States of America). Statistical significance was defined as not significant (ns), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

3. Results and Discussion

The aim of this study was to investigate the effect of freeze-thaw (FT) method on the stability of poly­(vinyl alcohol) (PVA) nanofibers in an aqueous environment. PVA with a high degree of hydrolysis was used to prepare 10 wt % solutions, which were subsequently processed into nanofibrous scaffolds using alternating current (AC) electrospinning. The primary objective was to enhance the water resistance of these nanofibers through physical stabilization by FT treatment. The materials were subjected to repeated FT cycles, in which samples were frozen at −20 °C for 16 h and then thawed for 8 h at +4, +22, and +37 °C. Two different FT cycle counts were tested: 5 and 10. Following the testing of pure PVA nanofibers, the most effective FT conditions were applied to PVA nanofibers containing incorporated platelet lysate (PL), a protein-rich bioactive additive, and their stability and release behavior were subsequently evaluated.

3.1. FT Stabilization of PVA-Based Nanofibrous Materials

3.1.1. Preparation and Morphological Analysis of Non-functionalized PVA Nanofibrous Material

Materials were prepared using AC electrospinning. The production rate for PVA material was 5.6 ± 0.7 g/h, the areal weight of the prepared material was 7.3 ± 0.5 g/m2, and the average fiber diameter was 375 ± 100 nm. Figure displays the prepared PVA material (Figure A) and samples subjected to various stabilization conditions: after 5 FT cycles at a thawing temperature of +4 °C (Figure B) and after 10 FT cycles at +37 °C (Figure C). Following the FT stabilization process, all PVA materials exhibited no significant changes in fiber diameters, as shown in Table and see in (Figure D). In addition to the preservation of fiber dimensions, morphological analysis revealed the presence of bead-like structures within the nanofibrous mats. The formation of these structures can be attributed to several phenomena occurring during electrospinning of aqueous PVA solutions. Due to the relatively high surface tension and low volatility of water compared with many organic solvents, complete solvent evaporation from the polymer jet may not always occur before deposition onto the collector. Consequently, locally retained solvent and polymer solution droplets may reach the deposited nanofibrous layer and partially merge with the forming fibers, resulting in the formation of bead-like structures. It should also be noted that bead formation is a commonly observed phenomenon during electrospinning of PVA-based nanofibers, particularly in aqueous systems. Similar bead-like structures have been observed in our previous studies using PVA nanofibrous systems. ,,, A detailed morphological analysis of the nanofibrous materials exposed to different thawing temperatures and subjected to both 5 and 10 cycles of physical - thermal stabilization via FT revealed no significant variations in fiber diameters. Minor deviations were attributed to the inherent heterogeneity of electrostatically spun nanofibers, which explains the observed variability in fiber diameters.

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Comparison of selected SEM images of PVA materials under different FT conditions (scale bar: 10 μm) with corresponding histograms of fiber diameter distribution; (A/D) PVA material without FT treatment; (B/E) PVA material after 5 FT cycles (−20 °C / +4 °C); (C/F) PVA material after 10 FT cycles (−20 °C / +37 °C); (G) Boxplots showing fiber diameters in the electrospun materials. Statistical analysis: Kruskal–Wallis test (n = 400; ns = not significant).

1. Average Fiber Diameters of FT-Stabilized PVA Materials.
designation material fiber diameter [nm]
0c / +4 PVA 0 cycles 375 ± 100 nm
5c / −20 / +4 PVA 5 cycles 16 h −20 °C/8 h + 4 °C 385 ± 105 nm
5c / −20 / +22 PVA 5 cycles 16 h −20 °C/8 h + 22 °C 375 ± 120 nm
5c / −20 / +37 PVA 5 cycles 16 h −20 °C/8 h + 37 °C 360 ± 125 nm
10c / −20 / +4 PVA 10 cycles 16 h −20 °C/8 h + 4 °C 360 ± 110 nm
10c / −20 / +22 PVA 10 cycles 16 h −20 °C/8 h + 22 °C 365 ± 100 nm
10c / −20 / +37 PVA 10 cycles 16 h −20 °C/8 h + 37 °C 370 ± 100 nm
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3.1.2. PVA Solubility after FT

Stabilization of PVA via FT process is expected to reduce its solubility by promoting the formation of hydrogen bonds between polymer chains. In this study, we investigated how the number of FT cycles and different thawing temperatures affect the solubility of PVA nanofibers. The release of PVA was monitored spectrophotometrically during incubation of the nanofiber samples in PBS at 37 °C, with measurements taken at 1, 2, 4, 6, 8, 24, and 72 h. As shown in Figure , approximately 68.1 ± 2.7% of untreated PVA dissolved within the 72 h. After 5 FT (Figure A) cycles with thawing at +4 °C, the solubility of pure PVA decreased by 15.3% (to 52.8 ± 2.9%), while thawing at +22 °C and +37 °C led to reductions of 4.2% (to 63.9 ± 1.5%) and 5.1% (to 63.0 ± 1.5%), respectively. Increasing the number of FT cycles to 10 (Figure B) further decreased PVA solubility, with a 19.5% reduction (to 48.5 ± 2.2%) observed at +4 °C thawing temperature. Corresponding decreases of 12.1% (to 56.0 ± 2.6%) and 13.3% (to 54.8 ± 3.0%) were observed at +22 °C and +37 °C, respectively. Furthermore, Figure C presents the statistical evaluation of the obtained PVA dissolution curves after 5 and 10 FT cycles. The analysis was performed using the area under the curve (AUC), which is commonly employed to evaluate the rate of substance release from materials. , As shown in Figure C, the 5c/–20/+4 sample exhibited significantly lower cumulative PVA release compared to the other groups, whereas no statistically significant difference was observed between the 5c/–20/+22 and 5c/–20/+37 samples. The highest AUC values were recorded for the 0c/+4 samples, which differed significantly from most FT-treated materials. AUC analysis comparing the effect of the number of FT cycles further showed that, under the FT −20/+4 condition, increasing the number of cycles from 5 to 10 did not result in a significant decrease in solubility. In contrast, for the other two thawing temperatures (+22 °C and +37 °C), the decrease observed after 10 cycles was significant. At the same time, it is evident that the reduction in solubility achieved under the −20/+4 condition after 5 cycles was comparable to that observed after 10 cycles at thawing temperatures of +22 °C and +37 °C. Overall, the FT treatments led to a reduction in PVA release; however, the FT -20/+4 condition appears to be the most effective. Although no statistically significant difference was observed between samples subjected to 5 and 10 FT −20/+4 cycles, a clear trend toward decreased solubility is evident. The observed decrease in PVA solubility after thawing at +4 °C can be related to changes in crystallite development during the FT process. FT cryogelation is governed by liquid–liquid phase separation during freezing, where ice formation induces polymer chain concentration in confined polymer-rich regions, promoting chain alignment and crystallite nucleation. The final crystallite structure depends not only on the freezing conditions but also on the subsequent thawing stage. During slow thawing at temperatures slightly above the melting point of ice, such as +4 °C, polymer chains regain sufficient mobility to undergo secondary crystallization and crystallite reorganization processes. Small and unstable crystallites may partially reorganize and recrystallize into larger and more stable crystalline domains, increasing the density and stability of physical crosslinking points. , These larger crystallites restrict water penetration and decrease the accessibility of hydrophilic PVA chains, which can explain the lower solubility observed for samples thawed at +4 °C. In contrast, faster thawing conditions provide less time for crystallite reorganization and growth, resulting in less pronounced structural reinforcement. Similar effects of FT conditions on crystallite size and crystallinity development have been reported for PVA-based systems. ,

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Plots of cumulative PVA release (expressed as [%] of total sample weight) from PVA nanofibers as a function of the number of FT cycles and thawing temperature; (A) PVA nanofibers stabilized with 5 FT cycles; (B) PVA nanofibers stabilized with 10 FT cycles; (C) Quantitative analysis of PVA release expressed as AUC for samples exposed to different FT conditions and numbers of cycles. Data are presented for n = 3 independent samples per FT condition. Statistical evaluation of differences between individual materials was performed using two-way ANOVA. Differences not indicated in the graph were not statistically significant. Statistical significance is denoted as (*) for p < 0.05, (**) for p < 0.01, (***) for p < 0.001, and (****) for p < 0.0001.

These results suggest that a higher number of FT cycles significantly improves the stability of PVA by reducing its solubility. This finding is consistent with Hassan and Peppas, who reported decreased solubility in PVA hydrogels with an increasing number of freeze–thaw cycles. Interestingly, the strongest reduction in PVA solubility was observed at the lowest thawing temperature of +4 °C, which contrasts with some previous reports suggesting higher thawing temperatures enhance stabilization. This could be related to slower and more controlled thawing at +4 °C, which might allow for more orderly rearrangement and stronger hydrogen bonding between polymer chains. In contrast, faster thawing at higher temperatures (+22 °C and +37 °C) might disrupt this gradual process, resulting in less effective physical crosslinking.

3.1.3. Chemical Structure and Crystallinity of PVA after FT

FTIR spectroscopy was employed to investigate potential chemical and structural changes induced by physical crosslinking via the FT method. This analysis was performed as a complementary technique to provide further insight into the structural behavior of PVA-based nanofibers after FT stabilization. As shown in Figure , no visible changes were observed in the FTIR spectra after stabilization. All samples exhibited the characteristic absorption bands of PVA without noticeable peak shifts or the appearance of additional bands, indicating that the FT treatment did not induce chemical modifications and that the chemical structure of the PVA nanofibers remained unchanged. It should be noted that FTIR analysis primarily provides information on chemical bonding and molecular interactions. Therefore, minor variations in the crystalline fraction or polymer chain organization may not result in detectable changes in the FTIR spectra, particularly when the overall chemical structure remains unchanged.

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Infrared spectra of PVA nanofibers subjected to thermal stabilization by the FT method for 16 h at −20 °C and 8 h at +4, +22, and +37 °C.

Apart from our results, Tavakoli et al. reported an increase in PVA crystallinity after FT treatment in PVA-based films containing tetraphenylethene-cored diboronic acid (TPE-2BA). In their study, FTIR spectra showed changes in the intensity of characteristic PVA bands associated with crystalline domains, particularly at 1142 and 1095 cm–1. The differences between their observations and the results obtained in the present study may originate from variations in polymer characteristics, sample preparation, morphology, and FT treatment conditions. Tavakoli et al. used PVA with a higher molecular weight (145,000 g mol–1) and a higher degree of hydrolysis (>99%) compared with the PVA used in the present study (125,000 g mol–1, 98%–98.8% hydrolysis). Moreover, their samples were prepared as relatively thick PVA films (450 ± 50 μm) by a drop-casting method from aqueous DMSO solutions containing TPE-2BA, whereas the present study investigated electrospun PVA nanofibers prepared from distilled water. In addition, Tavakoli et al. applied multiple short FT cycles (1 h at −20 °C and 1 h at 80 °C), while the present study employed longer stabilization cycles consisting of 16 h at −20 °C followed by 8 h at elevated temperatures (+4, +22, or +37 °C). These differences in polymer structure, sample morphology, and thermal history can significantly affect molecular mobility, chain rearrangement, and crystallization behavior during FT treatment.

A comparable, though less pronounced, trend can also be observed for the prepared PVA nanofibers in Table based on the DSC analysis. A slight increase in crystallinity was evident for all samples subjected to 10 FT cycles. However, all measured crystallinity values ranged between 47.42% and 48.86%, which cannot be considered a significant difference. Thus, it is difficult to draw a clear conclusion regarding the relationship between crystallinity and PVA solubility based solely on DSC results. The greatest reduction in PVA solubility was observed in samples thawed at +4 °C (see Figure ), where crystallinity was only marginally higher compared with other conditions. This phenomenon can be explained by the fact that solubility is influenced not only by crystallinity but also by the organization of the polymer network, the density of hydrogen bonds, and the material’s microstructure. At +4 °C, a more uniform and compact physical crosslinking network may have formed, effectively restricting water penetration. Nevertheless, it is well known that the crystalline phase can be unevenly distributed within the material, which could partially explain the observed variability and larger deviations in some results. ,

2. Crystallinity of PVA Nanofibrous Materials After FT Treatment as a Function of the Number of Cycles and Thawing Temperatures.
conditions FT crystalline content of PVA in nanofibers
0 cycles FT 5 cycles FT 10 cycles FT
–20 °C / +4 °C 47.42 ± 2.38% 47.89 ± 2.60% 48.86 ± 2.32%
–20 °C / +22 °C 47.53 ± 4.13% 48.20 ± 2.80%
–20 °C / +37 °C 47.45 ± 4.44% 48.22 ± 2.05%
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3.2. FT Stabilization of PL - Containing PVA Nanofibers

3.2.1. Preparation and Morphological Analysis of Functionalized PVA Nanofibrous Material

Subsequently, the PVA solution enriched with proteins derived from platelet lysate (PL) was electrospun using AC electrospinning. Based on the measured protein concentrations used for the preparation of the nanofibrous materials, the theoretical protein content in the PL stock solution was 15.9 mg/mL. This a loading efficiency of 1.56%, representing a substantial increase compared with our previous study, which may also have contributed to the differences observed in fiber diameters. The resulting PVA + PL nanofibers exhibited an areal weight of 4.5 ± 0.7 g/m2 and an average fiber diameter of 265 ± 70 nm, as shown in Table and Figure . In comparison, pure PVA nanofibers had an average fiber diameter of 375 ± 100 nm, indicating that the incorporation of platelet lysate resulted in significantly thinner fibers. This effect is likely due to the presence of proteins in the polymer solution, which not only alters its chemical composition but also affects its spinnability. Although the overall production rate decreased to 2.4 ± 0.2 g/h for PVA + PL, the fibers became finer. A similar trend was observed by Gilotra et al., who reported that the incorporation of sericin protein into PVA nanofibers led to a decrease in the average fiber diameter compared to pure PVA fibers.

3. Average Fiber Diameters of FT-Stabilized PVA + PL Materials.
designation material fiber diameter [nm]
0c / +4 PVA + PL 0 cycles 265 ± 70 nm
5c / −20 / +4 PVA + PL 5 cycles 16 h −20 °C/8 h + 4 °C 260 ± 65 nm
10c / −20 / +4 PVA + PL 10 cycles 16 h −20 °C/8 h + 4 °C 255 ± 65 nm
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Comparison of selected SEM images of PVA + PL materials under different FT conditions (scale bar: 10 μm) with corresponding histograms of fiber diameter distribution; (A/D) PVA + PL material without FT treatment; (B/E) PVA + PL material after 5 FT cycles (−20 °C / +4 °C); (C/F) PVA + PL material after 10 FT cycles (−20 °C / +4 °C); (G) Boxplots showing fiber diameters in the electrospun materials. Statistical analysis: Kruskal–Wallis test (n = 400; ns = not significant).

Importantly, morphological analysis of the nanofibrous scaffolds after 5 and 10 FT stabilization cycles revealed no significant changes in fiber diameter, underscoring a key advantage of this physical stabilization method. Specifically, the mean fiber diameter was 260 ± 65 nm after 5 FT cycles and 255 ± 65 nm after 10 cycles, indicating that the FT process preserves the structural integrity of the nanofibers despite repeated exposure to sub-zero temperatures.

The average fiber diameter of pure PVA nanofibers in this study (375 ± 100 nm) is comparable to that reported by Koprivova et al. (380 ± 170 nm) for fibers electrospun using DC electrospinning. However, the fiber diameters of PVA + PL materials differ notably. In this work, PVA + PL nanofibers exhibited an average diameter of 265 ± 70 nm, whereas Koprivova et al. reported substantially larger diameters (370 ± 150 nm) for fibers produced by DC electrospinning using a Nanospider - type device. The exact cause of this discrepancy remains unclear. For example, a study by Holec et al., which compared polyamide fibers produced by AC and DC electrospinning, found no consistent correlation between fiber diameters and the type of electrospinning used. Potential influencing factors may include the type of polymer, solvent system, or polymer concentration. In the study by Koprivova et al., although the same polymer and protein additives were used, PVA was dissolved in an 8:2 mixture of distilled water and ethanol, whereas in the present work, PVA was dissolved exclusively in distilled water. This difference in the solvent system, along with the specific electrospinning parameters, may influence the resulting fiber morphology and overall material properties.

3.2.2. PVA Solubility and PL Release from Functionalized Materials after FT

Proteins and growth factors present in human PL play a crucial role in promoting wound healing. Therefore, reducing their rapid release from nanofibrous scaffolds is essential to ensure a gradual delivery to the wound site. Based on earlier observations of the solubility behavior of non-functionalized PVA nanofibers, the most significant reduction in dissolution, following 5 and 10 FT stabilization cycles, was achieved when the materials were thawed at +4 °C for 8 h. Accordingly, this specific FT condition was selected for further experiments with PVA nanofibers containing human PL. As shown in Figure A, materials containing native proteins followed a similar trend in solubility reduction with increasing FT cycles, comparable to non-functionalized PVA materials. However, the effect of FT stabilization was slightly less pronounced in protein-loaded samples. Approximately 61.1% ± 2.4% of untreated PVA + PL dissolved within the 72 h. After 5 cycles, the solubility decreased by only 5.1% (to 55.9% ± 1.9%), while after 10 cycles, the reduction reached 12.2% (to 48.8% ± 2.1%). The AUC analysis in Figure C showed that increasing the number of FT cycles reduced the cumulative release of PVA, with the 10c/–20/+4 sample exhibiting significantly lower values compared to the control group (0c/+4). No statistically significant difference was observed between the 0c/+4 and 5c/–20/+4 samples, whereas the difference between 5c and 10c was significant.

5.

5

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Plots of cumulative release from functionalized PVA nanofibers as a function of the number of FT cycles; (A) Solubility of PVA (expressed as [%] of total sample weight); (B) Release of PL (expressed as [%] of its initial content); (C) AUC analysis of PVA solubility after FT treatment; (D) AUC analysis of PL release after FT treatment. Statistical differences between materials were analyzed using one-way ANOVA. Data are presented for n = 3 independent samples per FT condition. Differences not indicated in the graph were not statistically significant. Statistical significance is denoted as (*) for p < 0.05, (**) for p < 0.01, and (****) for p < 0.0001.

The slower release of incorporated proteins correlated with the decreased solubility of PVA after physical stabilization, likely due to the formation of additional hydrogen bonds within the polymer structure. The data in Figure B illustrate the protein release profiles from non-stabilized and FT-stabilized materials (5 and 10 cycles). For the untreated PVA + PL nanofibers, the amount of released proteins reached 15.4 mg per gram of material after 3 days of the experiment, which closely corresponds to the theoretical protein content of 15.6 mg per gram of nanofibrous material. Following 5 FT cycles, the protein release was reduced by 8.8% (to 89.8% ± 1.8%), and after 10 cycles, a more substantial reduction of 18.2% (to 80.4% ± 1.6%) was observed. Although FT stabilization reduced the release rate, the majority of incorporated proteins was released within the investigated 72 h period. Therefore, the observed release profile should be regarded as providing prolonged local availability of PL-derived proteins rather than representing a long-term sustained-release system capable of delivering growth factors throughout the entire wound healing process. Nevertheless, even a temporary extension of protein availability may be advantageous in chronic wound applications, where the rapid degradation of exogenously applied growth factors by proteases present in wound exudate remains a major limiting factor. Considering that PVA + PL materials with an areal weight of 4.5 ± 0.7 g/m2 contain approximately 7.8 μg of total protein per 1 cm2 sample, the amount of individual growth factors is expected to be considerably lower, most likely in the range of tens to hundreds of nanograms per sample. As the therapeutic efficacy of growth factors depends not only on the administered dose, but also on the maintenance of biologically relevant local concentrations, estimated to be within the range of 10–100 ng/mL, repeated application of the material may represent a more appropriate strategy than attempting to achieve a single prolonged release event. Such an approach could compensate for the gradual decline in protein bioactivity and help maintain the availability of native PL-derived factors throughout the different phases of wound healing. The AUC analysis of cumulative PL release in Figure D also confirmed significant differences between the evaluated groups and demonstrated a progressive decline with an increasing number of FT cycles. While in pure PVA nanofibers without PL content under FT −20/+4 conditions the number of applied cycles did not lead to a significant reduction in PVA solubility (although a clear trend was apparent), in samples with incorporated proteins an increase in the number of cycles resulted in a significant decrease in PVA solubility as well as in the rate of protein release. A similar reduction in the release rate of an incorporated compound was reported by Lee et al., where the release of bovine serum albumin (BSA) from PVA microspheres was slowed after applying three FT cycles (20 h at −20 °C and 4 h at +22 °C), with cumulative release measured over 60 h. An analogous reduction in the release rate has also been achieved using alternative fabrication strategies. For example, Mares-Bou et al. compared single-component PVA fibers with coaxial fibers consisting of a PVA core and a PLLA (poly­(l-lactic acid)) or PCL (polycaprolactone) shell and found that the presence of the shell significantly slowed the release of BSA. These findings suggest that physical stabilization via FT treatment can produce diffusion-limiting effects on bioactive compounds similar to those obtained through comparatively more complex manufacturing approaches.

3.2.3. Chemical Structure and Crystallinity of PVA in Functionalized Materials after FT

The chemical composition of the prepared materials and the homogeneity of protein distribution within the nanofibrous layers were evaluated using FTIR. The results of the analysis are presented in Figure . Compared to the spectra of pure PVA, the PVA + PL samples showed characteristic amide bond absorption bands typical for proteins, particularly a strong band in the 1600–1690 cm–1 region. This confirms the presence of protein components in the functionalized nanofibers. Moreover, the analysis of PVA + PL samples after FT stabilization revealed a similar signal intensity in this spectral region, indicating that the protein content remained preserved and evenly distributed even after the physical crosslinking process.

6.

6

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Infrared spectra of functionalized PVA + PL nanofibers depending on the number of FT cycles performed.

Results of the crystallinity changes assessed by DSC, presented in Table , show that PVA materials containing proteins exhibit lower crystallinity (approximately 5% lower for material without FT) compared to non-functionalized PVA nanofibers (see Table ). This reduction in crystallinity is likely attributed to the more uniform structural arrangement of polymer chains in pure PVA, whereas the incorporation of the bioactive protein component PL may partially disrupt the organization of the polymer’s macromolecular structure. Nevertheless, similarly to the behavior observed in pure PVA nanofibers, a slight increase in crystallinity with an increasing number of FT cycles can also be observed in PVA nanofibers incorporating PL.

4. Crystallinity of Functionalized PVA + PL Nanofibrous Materials After FT Treatment as a Function of the Number of Cycles and Thawing Temperatures.
conditions FT crystalline content of PVA in nanofibers
0 cycles FT 5 cycles FT 10 cycles FT
–20 °C / +4 °C 42.11 ± 1.55% 45.05 ± 2.15% 45.78 ± 2.18%
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4. Conclusion

This study explored the effects of physical crosslinking on the stability of electrospun PVA nanofibrous materials, with a focus on material solubility, changes in PVA crystallinity, and protein release kinetics. Nanofibers were fabricated using high molecular weight and highly hydrolyzed PVA, both in pure form and functionalized with PL (proteins were incorporated in fibers). Uniform fiber materials were produced via AC electrospinning, and FT cycles were applied as a physical crosslinking method to enhance material stability.

The results of the present study show that the method can significantly reduce the solubility of PVA nanofibrous materials. The treatment performed at −20 °C (freezing) and +4 °C (thawing) proved to be the most effective, with solubility decreasing by 19.5% after 10 cycles. Statistical analysis comparing the effect of the number of FT cycles indicated that, under FT conditions of −20/+4 °C, increasing the number of cycles from 5 to 10 did not lead to a statistically significant reduction in solubility, although a clear trend was observed. The effect of reducing the solubility of PVA nanofibers via FT cycles at thawing temperatures of +22 °C or +37 °C was lower compared to +4 °C, with solubility decreasing by 12.1% and 13.3% after 10 cycles, respectively. Our findings thus correspond to the behavior of PVA in hydrogels, where thawing at lower temperatures (e.g., +4 °C) provides sufficient molecular mobility for further crystallite growth and recrystallization while preserving the polymer-rich domains generated during freezing. These conditions favor the formation of larger and more stable PVA crystallites, which serve as physical crosslinking points and limit water accessibility to polymer chains. In contrast, at thawing temperatures of +22 °C and +37 °C, the reduction in PVA solubility after 10 cycles was already statistically significant when compared with 5 FT cycles. For PVA samples functionalized with PL, the effect of the number of cycles at a thawing temperature of +4 °C was already statistically significant, and after 10 FT cycles the solubility of PVA decreased by 12.2%. Crystallinity increased progressively with the number of FT cycles in both material types. Importantly, FT stabilization did not induce any notable morphological changes in the fiber structure.

Protein release studies also revealed differences between untreated samples and FT-treated samples in the initial protein release (burst release), with untreated samples exhibiting approximately 100% release of incorporated proteins within 72 h. Stabilized samples after 10 FT cycles exhibited a more sustainable release profile, with protein release reduced by up to 18.2% after 72 h. These findings demonstrate that FT crosslinking effectively increases the stability of PVA nanofibers in water and possibly also promotes the formation of hydrogen bonds between PVA and proteins, thereby synergistically slowing down protein release. This approach shows strong potential for applications in regenerative medicine, particularly for the treatment of large and chronic skin wounds, and specifically in the targeted drug delivery of sensitive biologically active substances, where non-aggressive stabilization methods are required.

Supplementary Material

ao6c05640_si_001.pdf (565.4KB, pdf)

Acknowledgments

The authors would like to thank the “Excellent Research in Regenerative Medicine” project, registration number CZ.02.01.01/00/22_008/0004562, of the European Union Programme entitled Johannes Amos Comenius in the call “Excellent Research” for financial support, as well as the project SGS-2025-4519 at the Technical University of Liberec for conducting the research. This work was also supported by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council under project No. NW24-08-00073.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05640.

  • Raw DSC thermograms of the investigated samples, Figures S1–S4 (PDF)

The authors declare no competing financial interest.

References

  1. Kelleher C. M., Vacanti J. P.. Engineering extracellular matrix through nanotechnology. J. R. Soc. Interface. 2010;7(suppl_6):1742–5662. doi: 10.1098/rsif.2010.0345.focus. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Grasso G., Zane D., Foglia S., Dragone R.. Application of Electrospun Water-Soluble Synthetic Polymers for Multifunctional Air Filters and Face Masks. Molecules. 2022;27(24):8753. doi: 10.3390/molecules27248753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kadajji V. G., Betageri G. V.. Water Soluble Polymers for Pharmaceutical Applications. Polymers. 2011;3(4):1972–2009. doi: 10.3390/polym3041972. [DOI] [Google Scholar]
  4. Alves M.-H., Jensen B. E. B., Smith A. A. A., Zelikin A. N.. Poly­(Vinyl Alcohol) Physical Hydrogels: New Vista on a Long Serving Biomaterial. Macromol. Biosci. 2011;11(10):1293–1313. doi: 10.1002/mabi.201100145. [DOI] [PubMed] [Google Scholar]
  5. Halima B. E. N.. Poly­(vinyl alcohol): review of its promising applications and insights into biodegradation. RSC Adv. 2016;6(46):39823–39832. doi: 10.1039/C6RA05742J. [DOI] [Google Scholar]
  6. Phan C.-M., Walther H., Riederer D., Lau C., Lorenz K. O., Subbaraman L. N., Jones L.. Analysis of polyvinyl alcohol release from commercially available daily disposable contact lenses using an in vitro eye model. J. Biomed. Mater. Res., Part B. 2019;107(5):1662–1668. doi: 10.1002/jbm.b.34259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castillo-Henríquez L., Vargas-Zúñiga R., Pacheco-Molina J., Vega-Baudrit J.. Electrospun nanofibers: A nanotechnological approach for drug delivery and dissolution optimization in poorly water-soluble drugs. ADMET and DMPK. 2020;8(4):325–353. doi: 10.5599/admet.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Koprivova B., Lisnenko M., Solarska-Sciuk K., Prochazkova R., Novotny V., Mullerova J., Mikes P., Jencova V.. Large-scale electrospinning of poly (vinylalcohol) nanofibers incorporated with platelet-derived growth factors. eXPRESS Polym. Lett. 2020;14(10):987–1000. doi: 10.3144/expresspolymlett.2020.80. [DOI] [Google Scholar]
  9. Campiglio C. E., Negrini C. N., Silvia F., Draghi L.. Cross-Linking Strategies for Electrospun Gelatin Scaffolds. Materials. 2019;12(15):2476. doi: 10.3390/ma12152476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ehrmann A.. Non-Toxic Crosslinking of Electrospun Gelatin Nanofibers for Tissue Engineering and Biomedicine - A Review. Polymers. 2021;13(12):1973. doi: 10.3390/polym13121973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nataraj D., Reddy R., Reddy N.. Crosslinking electrospun poly (vinyl) alcohol fibers with citric acid to impart aqueous stability for medical applications. Eur. Polym. J. 2020;124:109484. doi: 10.1016/j.eurpolymj.2020.109484. [DOI] [Google Scholar]
  12. Rezaee S., Moghbeli M. R.. Crosslinked Electrospun Poly (Vinyl Alcohol) Nanofibers Coated by Antibacterial Copper Nanoparticles. Iran. J. Chem. Eng. 2014;11(3):45–58. [Google Scholar]; https://www.ijche.com/article_10196.html. (accessed November 2025).
  13. Beishenaliev A., Lim S. S., Tshai K. Y., Khiew P. S., Moh’d Sghayyar H. N., Loh H. S., Hwei-San L. O. H.. Fabrication and preliminary in vitro evaluation of ultraviolet-crosslinked electrospun fish scale gelatin nanofibrous scaffolds. J. Mater. Sci.: Mater. Med. 2019;30(6):62–4838. doi: 10.1007/s10856-019-6264-4. [DOI] [PubMed] [Google Scholar]
  14. Stauffer S. R., Peppast N. A.. Poly­(vinyl alcohol) hydrogels prepared by freezing-thawing cyclic processing. Polymer. 1992;33(18):3932–3936. doi: 10.1016/0032-3861(92)90385-A. [DOI] [Google Scholar]
  15. Miraftab M., Saifullah A. N., Çay A.. Physical stabilisation of electrospun poly­(vinyl alcohol) nanofibres: comparative study on methanol and heat-based crosslinking. J. Mater. Sci. 2015;50(4):1943–1957. doi: 10.1007/s10853-014-8759-1. [DOI] [Google Scholar]
  16. Hasimi A., Stavropoulou A., Papadokostaki K. G., Sanopoulou M.. Transport of water in polyvinyl alcohol films: Effect of thermal treatment and chemical crosslinking. Eur. Polym. J. 2008;44(12):4098–4107. doi: 10.1016/j.eurpolymj.2008.09.011. [DOI] [Google Scholar]
  17. Homer W. J. A., Lisnenko M., Hauzerova S., Heczkova B., Gardner A. C., Kostakova E. K., Topham P. D., Jencova V., Theodosiou E.. Thermally Stabilised Poly­(vinyl alcohol) Nanofibrous Materials Produced by Scalable Electrospinning: Applications in Tissue Engineering. Polymers. 2024;16(14):2079. doi: 10.3390/polym16142079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wang Y.-Li, Yang H., Zhen-Liang XU.. Influence of post-treatments on the properties of porous poly­(vinyl alcohol) membranes. J. Appl. Polym. Sci. 2008;107(3):1423–1429. doi: 10.1002/app.24712. [DOI] [Google Scholar]
  19. Adelnia H., Ensandoost R., Moonshi S. S., Gavgan Nasrollah J., Vasafi E. I., Hang T. T.. Freeze / thawed polyvinyl alcohol hydrogels: Present, past and future. Eur. Polym. J. 2022;164:110974. doi: 10.1016/j.eurpolymj.2021.110974. [DOI] [Google Scholar]
  20. Agudelo D. I. J., Badano J. M., Rintoul I.. Kinetics and thermodynamics of swelling and dissolution of PVA gels obtained by freeze-thaw technique. Mater. Chem. Phys. 2018;216:14–21. doi: 10.1016/j.matchemphys.2018.05.038. [DOI] [Google Scholar]
  21. Akter, F.. , 2016. Principles of Tissue Engineering. In: Tissue Engineering Made Easy. Academic Press, pp 3–16. ISBN 978-0-12-805361-4. Available at: doi: 10.1016/B978-0-12-805361-4.00002-3. [DOI] [Google Scholar]
  22. Hassan C. M., Peppas N. A.. Structure and Morphology of Freeze/Thawed PVA Hydrogels. Macromolecules. 2000;33(7):2472–2479. doi: 10.1021/ma9907587. [DOI] [Google Scholar]
  23. Lee M.K., Bae H., Lee S., Chung N.-O., Lee H., Choi S., Hwang S., Lee J.. Freezing/thawing processing of PVA in the preparation of structured microspheres for protein drug delivery. Macromol. Res. 2011;19(2):130–136. doi: 10.1007/s13233-011-0203-7. [DOI] [Google Scholar]
  24. Low W. M.-T., Hsieh Y.-H., Chu Y.-C., Hsiao J.-T., Shu Y.-T., Lee H.-M., Hsieh M.-F.. Effect of Freeze-Dried Porcine Platelet Lysate on Wound Healing in Rats. Medicina. 2025;61(6):1098. doi: 10.3390/medicina61061098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Blanquer A., Musilkova J., Filova E., Taborska J., Brynda E., Riedel T., Klapstova A., Jencova V., Mullerova J., Kostakova E. K., Prochazkova R., Bacakova L., Bacakova L.. The Effect of a Polyester Nanofibrous Membrane with a Fibrin-Platelet Lysate Coating on Keratinocytes and Endothelial Cells in a Co-Culture System. Nanomaterials. 2021;11(2):457. doi: 10.3390/nano11020457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Homer W. J. A., Lisnenko M., Gardner A. C., Kostakova E. K., Valtera J., Wall I. B., Jencova V., Topham P. D., Theodosiou E.. Assessment of thermally stabilized electrospun poly­(vinyl alcohol) materials as cell permeable membranes for a novel blood salvage device. Biomater. Adv. 2023;144:213197. doi: 10.1016/j.bioadv.2022.213197. [DOI] [PubMed] [Google Scholar]
  27. Valtera J., Kalous T., Pokorny P., Batka O., Bilek M., Chvojka J., Mikes P., Kostakova E. K., Zabka P., Ornstova J., Beran J., Stanishevsky A., Lukas D., Lukas D.. Fabrication of dual-functional composite yarns with a nanofibrous envelope using high throughput AC needleless and collectorless electrospinning. Sci. Rep. 2019;9(1):1801. doi: 10.1038/s41598-019-38557-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Guirguis O. W., Moselhey M. T. H.. Thermal and structural studies of poly­(vinyl alcohol) and hydroxypropyl cellulose blends. Natural Science. 2012;04(01):57–67. doi: 10.4236/ns.2012.41009. [DOI] [Google Scholar]
  29. Havlickova K., Kostakova K. A., Maxim L., Sarka H., Martin S., Stanislava V., Lucie V., Jiri M., David L., Renata P., Jana H., Sarka J., Bohdana H., Vera J.. The Impacts of the Sterilization Method and the Electrospinning Conditions of Nanofibrous Biodegradable Layers on Their Degradation and Hemocompatibility Behavior. Polymers. 2024;16(8):1029. doi: 10.3390/polym16081029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Adams E., Coomans D., Smeyers-Verbeke J., Massart D. L.. Application of linear mixed effects models to the evaluation of dissolution profiles. Int J Pharm. 2001;226(1–2):107–125. doi: 10.1016/S0378-5173(01)00775-X. [DOI] [PubMed] [Google Scholar]
  31. Li C., Zhang K., Lin Q., Huang S., Cheng W., Lei Y., Zhao X., Zhao J.. Major depressive disorder recognition based on electronic handwriting recorded in psychological tasks. BMC Med. 2025;23(1):282. doi: 10.1186/s12916-025-04101-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sivan M., Madheswaran D., Valtera J., Kostakova E. K., Lukas D.. Alternating current electrospinning: The impacts of various high-voltage signal shapes and frequencies on the spinnability and productivity of polycaprolactone nanofibers. Mater. Des. 2022;213:110308. doi: 10.1016/j.matdes.2021.110308. [DOI] [Google Scholar]
  33. Filova E., Blanquer A., Knitlova J., Plencner M., Jencova V., Koprivova B., Lisnenko M., Kostakova E. K., Prochazkova R., Bacakova L., Bacakova L.. The Effect of the Controlled Release of Platelet Lysate from PVA Nanomats on Keratinocytes, Endothelial Cells and Fibroblasts. Nanomaterials. 2021;11(4):995. doi: 10.3390/nano11040995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reneker D. H., Yarin A. L.. Electrospinning jets and polymer nanofibers. Polymer. 2008;49(10):2387–2425. doi: 10.1016/j.polymer.2008.02.002. [DOI] [Google Scholar]
  35. Ricciardi R., Auriemma F., Gaillet C., De Rosa C., Lauprêtre F., Laupretre F.. Investigation of the Crystallinity of Freeze/Thaw Poly­(vinyl alcohol) Hydrogels by Different Techniques. Macromolecules. 2004;37(25):9510–9516. doi: 10.1021/ma048418v. [DOI] [Google Scholar]
  36. Holloway J. L., Lowman A. M., Palmese G. R.. The role of crystallization and phase separation in the formation of physically cross-linked PVA hydrogels. Soft Matter. 2013;9(3):826–833. doi: 10.1039/C2SM26763B. [DOI] [Google Scholar]
  37. Tavakoli J., Gascooke J., Xie Ni, Tang B. Z., Tang Y.. Enlightening Freeze–Thaw Process of Physically Cross-Linked Poly­(vinyl alcohol) Hydrogels by Aggregation-Induced Emission Fluorogens. ACS Appl. Polym. Mater. 2019;1(6):1390–1398. doi: 10.1021/acsapm.9b00173. [DOI] [Google Scholar]
  38. Ting-Yu L. I. U., Chan T.-Yi, Wang K.-S., Tsou H.-M.. Influence of magnetic nanoparticle arrangement in ferrogels for tunable biomolecule diffusion. RSC Adv. 2015;5(109):90098–90102. doi: 10.1039/C5RA17306J. [DOI] [Google Scholar]
  39. Li C., Hou T., She X., Wei X., She F., Gao W., Kong L.. Decomposition properties of PVA/graphene composites during melting-crystallization. Polym. Degrad. Stab. 2015;119:178–189. doi: 10.1016/j.polymdegradstab.2015.05.011. [DOI] [Google Scholar]
  40. Lujuan L. I., Xiaodong X. U., Lei L., Pingan S., Qianqian C., Zhiguang X., Zhengping F., Wang H.. Water governs the mechanical properties of poly­(vinyl alcohol) Polymer. 2021;213:123330. doi: 10.1016/j.polymer.2020.123330. [DOI] [Google Scholar]
  41. Gilotra S., Chouhan D., Bhardwaj N., Nandi S. K., Mandal B. B.. Potential of silk sericin based nanofibrous mats for wound dressing applications. Mater. Sci. Eng. 2018;90:420–432. doi: 10.1016/j.msec.2018.04.077. [DOI] [PubMed] [Google Scholar]
  42. Holec P., Jirkovec R., Kalous T., Batka O., Brozek J., Chvojka J.. The Potential for the Direct and Alternating Current-Driven Electrospinning of Polyamides. Nanomaterials. 2022;12(4):665. doi: 10.3390/nano12040665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Eming S. A., Martin P., Tomic-Canic M.. Wound repair and regeneration: Mechanisms, signaling, and translation. Sci. Transl. Med. 2014;6(265):1946–6242. doi: 10.1126/scitranslmed.3009337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tayalia P., Mooney D. J.. Controlled Growth Factor Delivery for Tissue Engineering. Adv. Mater.:Compos. Carbon, Pap. Symp. 2009;21(32–33):3269–3285. doi: 10.1002/adma.200900241. [DOI] [PubMed] [Google Scholar]
  45. Sofía M.-B., Serrano M.-A., Gómez-Tejedor J. A.. Core–Shell Polyvinyl Alcohol (PVA) Base Electrospinning Microfibers for Drug Delivery. Polymers. 2023;15(6):1554. doi: 10.3390/polym15061554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Jackson M., Mantsch H. H.. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit. Rev. Biochem. Mol. Biol. 1995;30(2):95–120. doi: 10.3109/10409239509085140. [DOI] [PubMed] [Google Scholar]
  47. Li J., Mooney D. J.. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 2016;1(12):16071. doi: 10.1038/natrevmats.2016.71. [DOI] [PMC free article] [PubMed] [Google Scholar]

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