Composite Ferroelectric Membranes based on Vinylidene Fluoride-Tetrafluoroethylene Copolymer and Polyvinylpyrrolidone for Wound Healing: A Pilot Study

Herein, we report results of the study of the composite ferroelectric scaffolds based on vinylidene fluoride-tetrafluoroethylene copolymer (VDF-TeFE) and polyvinylpyrrolidone (PVP) produced by electrospinning and their application as a wound-healing material. The physicochemical properties of ferroelectric composite polymer scaffolds depending on the content of PVP (in the range from 0 to 50 wt %) including morphology, composition and crystalline structure were studied. The cytotoxicity of materials and the proliferative activity of cells during their cultivation on the surface of formed scaffolds are reported. It has been found that the optimal PVP content in the VDF-TeFE composite scaffolds is 15 wt%. On a model of a full-thickness contaminated wound in vivo, it was shown that piezoelectric scaffolds based on VDF-TeFE copolymer containing 15 wt% PVP provide better wound healing results in comparison with standard gauze dressings impregnated with a solution of an antibacterial agent.

Wound healing is a long-term multi-stage process which may be inhibited by external environment [2]. Modern concepts of the pathophysiology of the wound healing state several basic requirements for wound dressings including: physical protection of the wound from external injury; inhibition of bacterial invasion; wound exudate absorption; maintaining the physiological temperature and humidity; gas and liquid transport; mechanical flexibility; easy removal without adhesion to the wound and biocompatibility [3]. These requirements are proven to prevent tissue dehydration and cell death, improve intercellular interaction and angiogenesis and the wound healing process. Despite the variety of modern materials used for wound healing (foams, hydrogels, sprays, etc.) [4], electrospun polymer non-wowen membranes [5][6][7] meet all the necessary requirements [3] being the most promising dressing material.
Piezoelectric properties of collagen [8], the most common fibrillar protein that forms dermis, triggered investigations directed towards devices and materials capable of accelerating tissue regeneration processes through electrical stimulation [9]. Closer attention received ferroelectric and piezoelectric polymer materials and their application in reconstructive surgery [10]. Considerable interest in these is due to the possibility of electrical stimulation of tissue regeneration processes via membrane mechanical deformations from cell, tissues or organs [11].
This approach does not require external source of electrical energy, batteries or electrodes, excluding the possibility of accumulation of electrolysis products in tissues [12].
Due to the higher electronegativity of fluorine (F) comparing to carbon (C) and hydrogen (H) in polyvinylidene fluoride (PVDF) and its copolymers with tetra-(TeFE) and trifluoroethylene (TrFE), a certain conformation of the macromolecule results in a dipole moment in polymer chain directed perpendicular to its axis. PVDF the most electrically active piezoelectric polymer [13].
Despite the obvious advantages, such membranes suffer from the high hydrophobicity, which limits the absorption of wound exudate and low encapsulation efficiency due to its the high chemical resistance [18]. The problem can be solved by the development of composite membranes based on fluoropolymer piezoelectrics.
In this work, we report results of our study of the composite membranes based on vinylidene fluoride-tetrafluoroethylene copolymer (VDF-TeFE) and polyvinylpyrrolidone (PVP) produced by electrospinning and their application as a wound healing material.
Five types of spinning solutions were prepared with a mass ratio of VDF-TeFE/PVP polymers of 100/0, 95/5, 85/15, 75/25, 50/50 %. The total concentration of polymers in solution was 5 wt.% for all samples. Polymers were dissolved in a sealed glass reactor at a temperature of 40 °C with the constant stirring until a homogeneous transparent solution was obtained. The resulting solution was cooled to the room temperature.
The viscosity of the spinning solutions was measured using SV-10 viscometer (AND, Japan). The conductivity of the spinning solutions was measured using an InoLab Cond 7319 conductometer with a TetraCon 325 measuring cell (WTW, Germany). The viscosity and conductivity measurements of the spinning solutions were carried out at a temperature of 24°C.
A commercially available NANON 01A installation (MECC Co., Japan), equipped with a cylindrical assembly collector with a diameter of 50 mm and a length of 200 mm, was used for the formation of nonwoven materials. The distance between the dope injector (22G needle) and the assembly manifold was 150 mm. The voltage at the injector is 25 kW. The flow rate of the dope solution was 1.8 mL/hour, assembly speed was 50 rpm.

Scanning Electron Microscopy (SEM)
The morphology of the samples was investigated by SEM using a JCM-6000 instrument (JEOL, Japan). Prior to the investigation, samples were coated with a thin gold layer by the magnetron sputtering system (SC7640, Quorum Technologies Ltd., UK). The fiber diameter was determined from SEM images captured in five fields of view using ImageJ 1.38 software (National Institutes of Health, USA). The average diameter was determined from at least 120 fibres.

Energy-dispersive spectroscopy (EDS)
Chemical composition of the membranes was analyzed using energy-dispersive spectroscopy (EDS) (JEOL JED 2300, Japan). The semiquantitative chemical composition of the membrane was calculated by the method of three corrections: for the average atomic number, absorption, and fluorescence.

Gas Chromatography (GC)
The study of the residual DMF content in the membranes was carried using Kristall 5000 chromatograph with a flame-ionization detector (Khromatek, Russia) equipped with a ZB-5MS column (30 m×0.25 mm×0.25 μm). To determine the DMF content 100 ± 5 mg of each sample were dissolved in 10.0 ± 0.1 ml of acetone. The following parameters were used: the volume of the injected sample -1 μl, the evaporator temperature -222 ° C; detector temperature -250 °С; thermostat temperature -90 ° C; inlet pressure -100 kPa; flow rate -10 mL/min.

Study of the structure of VDF-TeFE copolymer in composite membranes
Ferroelectric properties of PVDF and its copolymers are determined by the conformation of macromolecules and as a result by its crystalline structure. There are three main polymorphs (α, β, γ). α-phase is characterized by a monoclinic lattice in which chain (TGTG − ) conformation have opposite dipole moments, so in general it is nonpolar. γ-phase contains a weakly polar cell with a chain (T3GT3G -) conformation. β-phase is the most electroactive and is characterized by orthorhombic lattice with polar cell in which the chain has a planar zigzag (TTТ) conformation [19]. The presence of polymorphic conformations and crystal structures typical of the paraelectric and ferroelectric phases allows determining their presence, using techniques such as XRD and FITR [20] .
Investigations were carried out in the spectral range of 500-2000 cm -1 with a resolution of 2 cm -1 .

X-ray diffraction analysis (XRD)
The crystal structure copolymer VDF-TeFE in composite membranes was investigated using X-ray diffraction analysis (XRD) (Shimadzu 6000, Japan). The samples were exposed to a monochromatic Cu Kα (1.54056 Å) radiation.

Investigation of biomedical properties of membranes Adhesion, viability and proliferative activity of cells
The study of the interaction of the obtained membranes with cells in vitro was carried out using human dermal fibroblasts. For that 19 mm diameter sterile samples were placed into the well of 24-well plate and fixed with 0.6% agarose solution (Helicon, USA). For each well 1 mL of cell suspension containing 10 5 cells, DMEM (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (Gibco), 1% HEPES buffer (Gibco), 1% L-Glutamine -Penicillin -Streptomycin solution (Gibco) and 0.4% amphotericin B (Gibco) was added. The cells were cultured for 6 days using a CO2 incubator (Sanyo, Japan) in an atmosphere containing 5% CO2 at temperature of 37°C and a humidity of 95%.
The absolute number of cells per 1 mm 2 of the surface and the relative content of dead cells was evaluated using fluorescence microscopy using Axio Observer Z1 inverted microscope (Carl Zeiss, Germany). Cells were stained with ethidium bromide (Sigma Aldrich, USA) 0.03 mg / mL Studies of cell adhesion and proliferative activity were performed on 5 samples of each of the studied groups in triplicate using 10 randomly selected fields of view for each group. Cells cultured without a material were used as a control.

Composite membranes in vivo full-thickness contaminated wound healing
The wound healing activity of membranes was studied in 20 adult Wistar rats (body weight 180-200 g). An infected full-thickness contaminated wounds were formed. Rats were anesthetized and a rectangular excision area (20 × 20 mm) was cut on each animal. The edges of the wounds and underlying muscles were crushed with a Kocher's forceps. After that, microbial suspension containing 10 6 CFU of Staphylococcus aureus was applied topically to the wound area. The surface of the wound was covered with plastic wrap for 72 hours to form an acute inflammation. Animals were divided into two groups of 10 animals in each group. For the animals of the first group, a gauze bandage soaked in an aqueous solution of chlorhexidine (Kemerovo pharmaceutical factory, Russia) was applied to the wound surface. For animals of the second group the wound surface was covered with a VDF-TeFE membranes containing 15% PVP. The dressings were changed on 3, 5 and 7 days of the experiment.
Macroscopic photographs (digital camera EOS 250D, Canon, Japan) were used to evaluate healing activity of membranes. For histological analyses, the tissue samples were harvested from the region of interest. Samples were fixed in formalin and processed for histopathological observation. Prepared 5 μm-thick sections section of tissues were stained with hematoxylin and eosin and studied using transmission light microscopy (Axiosсop40, Carl Zeiss).
The study was carried out in accordance with the principles of humane treatment of laboratory animals described in [21]. Prior to investigation all samples were sterilized in ethylene oxide atmosphere using a gas sterilizer AN4000 (Andersen Sterilisers, UK).

Statistics
The data were analyzed with Origin 9.0 (OriginLab, USA) software using the one-way ANOVA with Tukey's correction. The difference was considered significant at a significance level of p<0.05.

Results and Discussion
The morphology of the formed VDF-TeFE membranes and composite membranes with PVP content 50 % is shown in Figure 1. Regardless of the PVP content in the spinning solution, all the membranes are formed by cylindrical fibers of regular shape, randomly intertwining with each other. However, an increase in the PVP content in spinning solutions leads to a decrease of the fiber diameter from 1.45 ± 0.36 to 1.03 ± 0.26 µm due to a decrease in conductivity and dynamic viscosity of the solutions ( Table   1.) The observed decrease in the dynamic viscosity is attributed to a lower content of the polymer with higher molecular weight in the mixture. Moreover, the solution conductivity drop from 2.84±0.02 to 1.50±0.04 μS/cm, accompanied by a decrease of the spinning solution viscosity, is probably induced by intermolecular interaction leading to the formation of complexes between the polymers and the solvent.  An increase of the PVP concentration in spinning solutions leads to an increase in the content of oxygen (O) and nitrogen (N) in the formed membranes (Table 2). These elements are presented in the composition of both PVP and DMF. At the same time, an increase of the PVP amount leads to higher DMF content in the samples (Table 2). Probably, the observed increase in the DMF content in the formed composite samples, as well as a decrease in the conductivity of spinning solutions is evidence of the formation of complex between PVP and DMF [22].
FTIR spectra of the studied membranes VDF-TeFE scaffolds with different PVP content are shown in Figure 3. tensile forces from the electric field in process of the membranes formation [31] and the alignment effect of TeFE segments on the PVDF macromolecule [32]. With an increase of the PVP content, the half-width of the β-phase peak increases from 0.889° for pure VDF-TeFE membranes to 1.738° for VDF-TeFE membranes containing 50% PVP. The intensity of the halo reflection at 17.8° also increases, indicating difficulties of the electrically active β-phase crystallization process in VDF-   (Table 3).  The decrease in adhesion, viability and proliferation of fibroblasts with an increase in PVP content may be due to the following reasons. PVP is a water-soluble, hydrophilic polymer. Thus, PVP can diffuse into the culture medium [33]. The amount of PVP released into the culture medium over a certain period of time is determined by the content of PVP in the sample. It is known that PVP is protonated in aqueous solutions and can form complexes with anions, including various biologically active molecules [34]. Thus, an increase of PVP content may lead to an increase in the content of complexes with biologically active anions in the culture medium, changing its chemical composition and reducing the availability of anions for cells. On the other hand, diffused PVP in the culture medium can lead to its accumulation in cells causing "lysosomal storage disease" which inhibits the processes of vital activity of cells causing their death [35]. The third possible reason for a decrease in cell viability on the surface of membranes with a high content of PVP is the process of its leaching from the fibers leading to membrane deformation (supplementary file) preventing cell attachment to the scaffold surface [33]. And finally, the fourth reason could be the formation of toxic complexes between PVP and DMF during the spinning solution preparation, followed by its migration into the culture medium, which is indirectly evidenced by an increase in the concentration of DMF in the formed membranes with an increase in the concentration of PVP (Table 2).
It was shown that VDF-TeFE membranes containing 15 wt% PVP have optimal morphology, chemical, crystal structure, and are sufficiently capable of maintaining the necessary conditions for adhesion and proliferation of fibroblasts on their surface, which makes it possible to use this type of membranes for pilot studies to explore the possibility of their use to restore the skin in the case of contaminated full-thickness wounds.
The contaminated full-thickness wound, wound treated with gauze dressing soaked in chlorhexidine solution, and wound treated with VDF-TeFE membrane containing 15 wt% PVP after 7 days are shown in Figure 5.  Figure 5C).
VDF-TeFE membrane containing 15% PVP shows better effect on skin healing in the case of contaminated full-thickness wound compared to standard gauze dressings impregnated with chlorhexidine. It might be due to following reasons. First, membranes made by electrospinning have interconnected porosity formed by micron-sized fibers resulting in significant free surface [36] and the ability of PVP to bind toxins and water [34]. These properties of the obtained membranes make it possible to absorb a significant amount of exudate released by pathogenic microflora, as well as maintain the required moisture level on the wound surface. The absorbed exudate saturated with pathogens is removed along with the membrane, thus reducing the concentration of pathogens with each subsequent dressing. In addition, PVP released from fibers into the exudate can accumulate in pathogenic cells causing "lysosomal storage disease" provoking their death [37]. Thus, the presence of PVP in fibers is a factor that reduces the concentration of pathogens in the regeneration zone.
Second, it is known that PVDF-based piezoelectric polymer membranes are capable of negatively affecting the Staphylococcus culture when under dynamic load the membrane even in the absence of antibacterial agents [38]. Since in membranes containing 15 wt% of PVP, the predominant conformation of VDF-TeFE macromolecules is the electrically active trans conformation ( Figure 2) and crystallites with characteristic ferroelectric properties predominate in the crystal structure (Figure 3), it can be assumed that decrease in the concentration of pathogens in the wound and improved tissue regeneration is due to the piezoelectric properties of the formed membranes.
Third, it is known that under the influence of external mechanical stimuli piezoelectric membranes can enhance migration, adhesion, and cytokine secretion in NIH3T3 fibroblasts in vitro [39]. The capacity of piezoelectric membranes to generate electrical impulses in response to the mechanical action of the tissue surrounding the implant allows to promote the wound healing regardless of the implantation zone [40].
Thus, the piezoelectric properties of the formed membranes make it possible to enhance the processes of tissue regeneration. "Pathogenetic basis for the development of cardiovascular implants from biocompatible materials using patient-oriented approach, mathematical modeling, tissue engineering, and genomic predictors".