PVA and PVP nanofibers combined with Helichrysum italicum oil preserve skin cell interactions, elasticity and proliferation

Abstract

Development of electrospun nanofibers with suitable properties to promote wound healing is an advantage in developing non-invasive skin treatments. We showed the potential application of Polyvinyl acetate (PVA) and Polyvinylpyrrolidone (PVP) combined with Helichrysum italicum oil (HO) in wound healing. During this process, Tight junctions (TJs) play a crucial role in maintaining skin integrity. TJs are intercellular junctions composed of a variety of transmembrane proteins, including Occludin (OCLN), observed also in migrating epithelial cells. Changes in OCLN expression affect epidermal permeability, indicating an active role in the healing process. Within this context, we studied the OCLN expression during healing after scratch assay on Keratinocytes (HaCaT), by a confocal microscopic analysis. In addition, we evaluated the effect of treatment after scratch on cell elasticity by Atomic Force Microscopy (AFM) analysis. All results show a positive trend in cell proliferation and viability on HaCaT treated with functionalized nanofibers. These results were confirmed by the expression of genes involved in the early stages of the regenerative process. Understanding the cell mechanisms involved in skin changes during repair process would allow future application of nanomaterials combined with HO in vivo.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-95788-z.

Introduction

Each layer of the skin plays an important role in maintaining the structural integrity and pathophysiological functions of this tissue. The epidermis is the outermost layer and plays a protective role, while the dermis is the middle layer, providing nourishment and mechanical support to the epidermis¹. Commonly, the integrity of the skin is damaged by extrinsic factors, and this requires the activation of the physiological process to repair damaged tissue². Cutaneous wound healing (WH) is a complex process involving proliferation and migration of different cell types and several molecular mediators. These mediators, as pro- and anti-inflammatory cytokines, are crucial in promoting tissue proliferation, re-epithelialization and remodeling, also contributing to inflammation^3,4. In the early stages of wound healing, pro-inflammatory cytokines as IL-1β, IL-8 and different chemokines play a key role in successful wound repair^3,5. These cytokines are produced and released within a specific timeline. Specifically, IL-1β is highly expressed at the beginning of the wound and during the inflammatory phase, but its levels decrease significantly along with healing improvement. On the other hand, IL-8 has been shown to play an integral role in the re-epithelialization phase⁶. It promotes migration and proliferation of keratinocytes, a key event for the formation of a new epithelial layer on the wound⁷. IL-8 contributes to wound closure and restoration of the skin barrier by stimulating the growth of keratinocytes and guiding their movement toward the wound site³. Regeneration of the basal layer leads keratinocytes to proliferate and differentiate vertically, restoring the physiological features of the multilayer epithelial tissue⁸.

The epithelial barrier is the first line of defense against external damages, thanks to bicellular tight junctions (TJs). It has been shown that normal human epidermal keratinocytes can produce a continuous network of TJ proteins during the formation of the permeability barrier⁹. TJs are complex proteins counteracting the movement of microorganisms through the paracellular space, creating physical barriers by tightly connected cells. This type of junctions is responsible for the selective barrier function, i.e. the replacement of organ-specific molecules, and thus plays a key role as regulators of inflammatory processes¹⁰. Additionally, they mediate signal transduction, able to modulate cell proliferation, migration, and differentiation, as well as immune response and homeostasis¹¹. For their wide range of functions and their presence especially in endothelial and epithelial cells, are critically involved in wound healing¹². TJs are composed of different transmembrane proteins, including cytoplasmic actin-binding proteins and adhesive transmembrane proteins, that build up morphologically distinguishable strands and connect neighbouring cells^9,12. The mostly known proteins specifically localized to tight junctions are Occludin (OCLN), claudin (CLDN) and junctional adhesion molecules (JAMs). OCLN plays an important role in cell adhesion and a reduced expression of this protein increases paracellular permeability, compromising the protective function of epithelial barrier¹³.To understand its role during wound repair, the behaviour of OCLN has been studied in vitro, using three-dimensionally reconstructed human skin^12,14.Characterizing the biomechanical properties of the individual skin layers is important for understanding the ageing process¹⁵ and the mechanisms of other dermatological conditions¹⁶.

Cellular elasticity is a parameter that reflects the biophysical aspect of many known and unknown cellular processes. These include intracellular signaling, cytoskeletal activity, changes in cell volume and morphology, and many others¹⁷. Several methods have been developed to measure cellular elasticity using mechanical techniques such as tonometry, indentation, suction, and torsion, which are useful for measuring the various mechanical properties¹⁸. Within such context, Atomic Force Microscopy (AFM) is an extremely sensitive method for high-resolution imaging of any surface, including those of living and fixed cells. This powerful technique is used for characterizing the mechanical, electrical, and magnetic features of samples to be studied, both qualitatively and quantitatively¹⁹. In the field of regenerative medicine, plants have been widely acknowledged since ancient times for their ability to enhance the healing process.

We have already described the regenerative and anti-ageing properties of Helichrysum Italicum oil (HO) combined with polyvinyl alcohol (PVA) and Polyvinylpyrrolidone (PVP) nanofibers²⁰. In this context, we considered the potential advantages of using nanoformulations for the recovery of skin function. Indeed, nanomaterials functionalized with bioactive substances show discrete advantages, such as a high surface-to-volume ratio and nanoscale dimensions²¹. These nanomaterials, PVA and PVP have a controlled and sustained release, which seems to be very effective for long-term healing²¹. Our results showed that these two biodegradable polymers functionalized with HO can promote cellular growth and proliferation and to reduce the healing time^20–22.

In this study, we evaluated the regenerative effect of these nanofibers on keratinocytes (HaCaT) under mechanical stress. In addition, we investigated the ability of the treatment to increase OCLN expression and its effect on cell morphology and elasticity. These findings would open novel strategies in tissue regeneration based on polymer wound dressings.

Materials and methods

Preparation of HO

The essential oil was obtained from young stems by hydrodistillation, following an established protocol²³ . Subsequently GC–MS analysis was carried out for the extraction of essential oil components, as previously described²⁰. The identification of HO was achieved by comparing the GC retention index (RI) on apolar and polar columns with those of authentic samples of various essential oils, and by comparing MS fragmentation patterns and retention index with the Wiley 7 mass computer library, National Institute of Standards and Technology (NIST) or data in literature, as previously described^20,24,25.

Nanofibers electrospinning and combination with HO

PVA e PVA nanofibers were fabricated by an electrospinning method using a Nanospider 1S500U machine (Elmarco s.r.o. Liberec, Czech Republic). The solutions for the electrospinning process contain 10% w/w of PVA (Mowiol 5–88 + 40–88 in 20% v/v ethanol with deionized water) or 12% of PVP (Sigma PVP360) in ethanol (w/v). 1 h before spinning, 1% of oil was added to 1 g of polymer, as previously described²⁰. All samples were studied under a scanning electron microscope (SEM) (Device Vega 3 from Tescan, Brno, CZ Czech Republic) after spinning due to sputtering tiny samples using a thin film of gold (Quorum Q150R S, Laughton, UK). The features of PVA 1% and PVP1% nanofibers were already described²⁰

Cell culture

Human skin keratinocytes (HaCaT) were obtained from ATCC (Manassas, VA, USA) and cultured in a Dulbecco’s modified Eagle’s Medium (DMEM) low-glucose medium (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS Life Technologies), 2 mM l-glutamine (Euroclone, Milano, Italy), 1% of penicillin/streptomycin (Euroclone, Milano, Italy)²⁶, and transferred in an incubator at 37 °C and 5% CO₂²⁷.

Experimental condition

HaCaT were cultured under two experimental conditions. A group of cells was maintained in a basic growing medium after scratch assay, without any treatment, and used as control cells (CTRL). A group of cells was treated after scratch assay with encapsulated PVA or PVP nanofibers, both functionalized with 1% Helichrysum italicum oil (PVA1% and PVP1%).

Scratch assay

HaCaT were seeded at a concentration of 45,000 cells/well in 12 well-plates and incubated until confluence. Once reached the confluence, the culturing medium was removed, and scratch was made in each well using a 200 μl pipette tip. Cells were washed in PBS to remove detached cells before adding PVA1% or PVP1% nanofibers. The scratches of each well were analyzed by optical microscopy (Leica, Nussloch, Ger-many) after 0 and 24 h following the induced damage at two different magnifications (4 × and 10x).

MTT assay: evaluation of cell viability

The water-soluble tetrazolium salt assay (MTT) (Sigma-Aldrich, Saint Louis, MO, USA) was performed to evaluate the metabolic activity. HaCaT were seeded at a concentration of 6000 cells/well in a 96-well plate and treated with PVA1% or PVP1% for 24 h. After culturing in the above-described conditions, the MTT assay (Sigma-Aldrich) was performed in cells that were exposed to scratch assay. Cell viability was detected by a plate reader (570 nm) and expressed in Optical Density units (OD) as to untreated cells (CTRL). All results were expressed as mean ± SD, referring to the control.

BrdU assay: evaluation of cell proliferation

The BrdU assay (#6813, Cell Signaling Technology, Euroclone, Milan Italy) is an immunoassay for the quantification of cell proliferation. HaCaT were seeded at a concentration of 6000 cells/well in 96-well plates and treated with PVA1% or PVP1% for 24 h. Cells cultured in the absence of PVA% and PVP1% samples were used as control. Cell viability was detected by plate reader (450 nm) and were expressed in OD units compared with untreated cells (CTRL). Data were expressed as mean ± SD, referring to the control.

Immunostaining

After culturing in the above-described conditions, cells were fixed with 4% of paraformaldehyde (Sigma Aldrich Chemie GmbH, Germany) for 30 min at room temperature (RT). After fixing, 1 h of permeabilization by 0.1% Triton X-100 (Life Technologies, USA)-PBS at RT was performed. Cells were then washed three times in PBS and incubated for 30 min at RT with 3% Bovine Serum Albumin (BSA)—0.1% Triton X-100 in PBS (Life Technologies, USA). Primary anti-rabbit anti-Occludin antibody (SAB5700784-Sigma-Aldrich, Saint Louis, MO, USA) was incubated overnight at 4 °C. Cells were washed twice in PBS and stained at RT with the fluorescence-conjugated goat anti rabbit IgG secondary antibody (AF594) (Life Technologies, USA) for 1 h in the dark. Nuclei were labelled with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI). All microscopy analyses were performed with a confocal microscope (TCS SP5, Leica, Nussloch, Germany).

Preparation of sample for AFM measurement

HaCaT were seeded on glass slides (12 mm Ø) placed in a 24-well multi-well plate until confluence. A scratch test was performed on the cells attached to the glass slides using a pipette tip (200 μL). After culturing in the above-described conditions, cells were fixed with 4% of paraformaldehyde (Sigma Aldrich Chemie GmbH, Germany) for 30 min at room temperature (RT).

Atomic force microscopy analysis

Force-distance curves were acquired with an AFM head (Smena NT-MDT) mounted on an inverted microscope (Nikon Ti-E). The cantilevers used have a spring constant of 0.062 N/m and have a bead for the indentation of 2500 nm of diameter (Novascan). Force–distance curves were analyzed using the Atomic J software to level and offset the baseline, find the contact point, and subtract the lever bending, thus obtaining a pre-processed force-indentation (F–I) curve. Finally, each F–I curve was fitted to the modified Hertz-Sneddon model using a custom procedure on IGOR PRO 9.0 (Wavemetrics Inc.) [Senigagliesi et al., Biomolecular Concepts 2022], which led to the evaluation of two Young’s modulus values, Y1 and Y2, relative respectively to the outer (indentation < 500 nm) and inner (indentation comprised between 500 and 1500 nm) part of the cell. At least 30 cells were measured for each condition. Being the data distribution not normal, non-parametrical statistical tests (Wilcoxon) were performed to evaluate significance of data.

Gene expression analysis

Total mRNA was isolated after 24 h of treatment from cells cultured inthe above-described condition using RNeasy Mini Kit (Qiagen, 40,724 Hilden, Germany) according to manufacturer protocol. Quantity and purity of RNA were measured by Od 260/280 nm using Nanodrop (Thermo Scientific, Waltham, MA, USA). RNA of each sample (2.5 ng) was reverse-transcribed and amplificated with Power SYBR® Green RNA-to-CT PCR under standard conditions via the Thermal Cycler (Bio-Rad, Hercules, CA, USA)²⁰. Each sample was assessed in triplicate under standard qRT-PCR conditions (48 °C for 30 min, 95 °C for 10 min, then cycled at 95 °C for 15 s and 60 °C for 1 min for 40 cycles) using a CFX Thermal Cycler (Bio-Rad, Hercules, CA, USA) (Applied Biosystems, Foster City, CA, USA). Target Ct values were normalized to Glyceraldehyde-3-Phosphate-Dehidrogenase (GAPDH), considered as a reference gene, while the mRNA levels of HaCaT treated with PVA1% or PVP1% were expressed as fold of change (2^−∆∆Ct) relative to the mRNA levels observed control cells (CTRL). The qRT-PCR analysis was performed for the following genes: Interleukin 1 beta (IL-1β) and Interleukin 8 (IL-8). All primers were previously described⁵.

Statistical analyses

The experiments were performed two times with three technical replicates for each treatment. Two-way analysis-of-variance ANOVA tests with Tukey’s correction and the Wilcoxon signed-rank test were used, assuming a p value < 0.05 as statistically significant. We considered *p < 0.05.

Results

PVA1% and PVP1% stimulate cell viability and cell proliferation after scratch assay

Human skin keratinocytes (HaCaT) cultured for 24 h after scratch with PVA1% or PVP1%, based on previously tested condition (Supplementary Fig. S1) exhibit an increase in viability (Fig. 1, panel a) as compared to cells cultured for 24 h after scratch without any treatment (untreated control CTRL) (Fig. 1, panel a). At the same time, the BrdU assay was performed to analyze proliferation of HaCaT (Fig. 1, panel b) cultured under the condition described above. The results revealed that treatment with PVP1%, after scratch test, significantly increased cell proliferation, as compared to the untreated control (CTRL), (Fig. 1, panel b). On the other hand, when HaCaT were treated with PVA1%, they showed only a faint proliferation rate increase, as compared to the untreated control (CTRL) (Fig. 1, panel b).

Fig. 1.

MTT assay (panel a) and BrdU assay (b) of HaCaT cultured for 24 h in the presence of PVA1% or PVP1%. Cell viability (panel a) and cell proliferation (panel b) of treated cells (PVA1% and PVP1%) are expressed as mean ± SD reffering to the untreated control (CTRL). (*p < 0.05).

PVA1% and PVP1% promote cells migration after scratch

The capability of PVA1% and PVP1% to enhance HaCaT migration was detected by the scratch assay. Figures 2 and 3 show the healing process at two magnifications (4 × and 10 ×, respectively) under an optical microscopy (Leica, Nussloch, Ger-many) and quantified by ImageJ Software (Supplementary Fig. S2). Figures 2 and 3 shows HaCaT treated with PVA1% or PVP1% and the migration of cells after scratch at two different time point (day 0 and 24 h). Figures revealed that both treatments increased the number of migrating cells detectable in the wound site as compared to untreated cells (CTRL).

Fig. 2.

Shows migration of HaCaT after scratch and treatment with PVA1% or PVP1%. Images were taken under inverted light microscope at time of cutting (day 0) and after 24 h of treatment. Treated cells were compared to untreated cells (CTRL). Figure shows HaCaT migration at 4 × magnification.

Fig. 3.

Shows migration and/or proliferation of HaCaT after scratch and treatment with PVA1% or PVP1%. Images were taken under inverted light microscope after 24 h of treatment with PVA1% and PVP1%. Treated cells were compared to untreated cells (CTRL). Figure shows HaCaT migration at 10 × magnification.

PVA1% and PVP1% induce the expression of Occludin (OCLN)

The expression of Occludin was evaluated in HaCaT treated for 24 h with PVA1% or PVP1% after scratch (Fig. 4). PVA1% and PVP1% treatment increased the expression of Occludin at nuclear level, as compared to control untreated cells (CTRL). This effect was clearly evident in all the analyzed cells treated with PVA1%, while in cells cultured in the presence of PVP1%, this increased expression could be detected mainly closed to the scratch area (Fig. 4).

Fig. 4.

Analysis of Occludin expression in HaCaT during wound closure. Immunohistochemical analysis of the expression of Occludin was assessed in HaCaT after scratch and treatment with PVA1% or PVP1% as compared to control cells (CTRL). Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). The figures are representative of different independent experiments. Scale bars: 40 µm.

Cell elasticity after treatment

HaCaT showed a different behavior when treated with PVA 1% or PVP 1%, as compared to control untreated cells (CTRL). In particular, we observed in the external layer (Y1) a slight (yet not statistically significant p = 0.08) decrease in apparent Young’s modulus for PVA 1%, as compared to control, while no variations were observed in PVP 1%. This trend was more evident and statistically significant (p < 0.01) in the internal layer (Y2) where we observed a significantly decrease in apparent young’s modulus for the PVA 1% while no variation was observed in PVP 1% as compared to controls. This seems to indicate that PVA 1% induced a change in the nuclear and perinuclear regions, causing a significant softening of these regions, while in the cytoskeleton and membrane regions the effect is less evident. On the other hand, it seems that PVP 1% did not affect at the biomechanical properties of cells as compared to control, leaving both layers (internal and external) unaltered.

PVA1% and PVP1% modulate inflammatory-related genes

Figure 6 shows the effect of PVA1% and PVP1% on IL-8 and IL-1β gene expression. Gene expression of IL-8 and IL-1β were influenced by both treatments. Figure 6 shows that the mRNA levels of IL-8 (panel a) and IL-1β (panel b) were significantly upregulated after 24 h of treatment with PVP1% or PVA1%, as compared to untreated cells (CTRL).

Fig. 6.

Gene expression of proinflammatory cytokines IL-8 and IL-1β. The expression of Interleukin 8 (IL-8) and Interleukin 1 beta (IL-1β) was evaluated in cells treated with PVA1% or PVP1% for 24 h after scratch assay (Figure, panels (a) and (b) respectively). The mRNA levels for each gene was expressed as fold of change (2^−∆∆Ct) of mRNA levels observed in untreated HaCaT (CTRL) and normalized to (GAPDH). Data are represented as mean ± SD referred to the control (*p ≤ 0.05).

Discussion

In recent years, the use of biomaterials in wound healing applications emerged as promising for wound treatment. Biomaterials are classified as synthetic or natural and can be processed or modified to enhance their wound healing capacity^28,29. Indeed, we have already shown that HO combined with PVA and PVP nanofibers gives them properties as controlled release, biocompatibility and biodegradability, increasing their efficacy especially under stress conditions²⁰. The skin repair process comprises several highly integrated steps, and involves different cell types, including keratinocytes. Wound healing is essential to maintain the functions of the skin integrity, influenced by the presence of TJs³⁰. TJs reside immediately below the stratum corneum and their compromised integrity in epithelial cells at the wound edge indicates activation of the tissue repair process³¹. Indeed, total or partial inhibition of the expression of major TJ proteins has been shown to have an impact on epidermal permeability, leading to abnormal stratum corneum (SC) formation^32,33. From a mechanical aspect, cells can be considered as an active soft material that responds, generates, and transmits forces. Mechanical forces can also be converted into biochemical signals by cells in a process termed mechanotransduction³⁴, or transmitted directly to the nucleus, where they can modulate gene expression³⁴. Basic and clinical studies on the mechanobiology of cells and tissues highlight the importance of mechanical forces in the process of skin regeneration and wound healing³⁵. In this context, we performed a scratch assay to reproduce injury induced by mechanical stress and evaluated cell migration after treatment under inverted light microscope at 4 × and 10 ×  magnification (Figs. 2 and 3) and quantification by ImageJ Software (Supplementary Fig. S2). Keratinocytes were scratched and then treated for 24 h with PVA1% and PVP1% nanofibers, as previously described. We have shown that both treatments have no cytotoxic effect in treated cells (Fig. 1, panel a) and at the same time increases cell proliferation (Fig. 1, panel b). To confirm the promoting effect of treatment in the repairing process, we evaluated the expression of Occludin (OCLN) during wound closure by immunofluorescence analysis (Fig. 4).

It has been shown that, although OCLN knockdown does not affect wound closure or proliferation under normal scratch wound conditions, it promotes effective WH in mechanically scratch wounds¹². Indeed, several studies showed that Occludin inhibition affects the migratory and proliferative capacity of human skin keratinocytes³⁶. Our results showed that PVA1% and PVP1% treatment increased the expression of OCLN at nuclear level. Nuclear expression of OCLN could be explained by its role in cell division process³⁷. This effect was clearly visible of PVA1%, for all cells analyzed (Fig. 4), while for PVP1%, this increased expression was more visible closed to the scratch area (Fig. 4). These results are linked with the data obtained by atomic force microscope (AFM). Boxplots showed the apparent Young’s moduli Y1 (external layer) and Y2 (cell body) of the cells, obtained using AFM force-indentation curve of HaCaT cells (Fig. 5, panel a and b). We observed in Y1 a small decrease in apparent Young’s modulus for PVA 1% as compared to control, while no variation was observed in PVP 1% (Fig. 5, panel b). The trend was more evident and statistically significant in Y2, where we observed a strong decrease in apparent young’s modulus for the PVA 1%, while no variation was observed in PVP 1%, as compared to control (Fig. 5, panel b). The results reveal that PVA 1% induced a change in the nuclear and perinuclear regions, causing a significant softening of these regions. On the other hand, PVP 1% didn’t seem to affect the biomechanical properties as compared to control, leaving both layers (internal and external) unaltered (Fig. 5, panel b). The increased cellular elasticity induced by PVA1% could be ascribed to the structural support supplied by the cytoskeleton, which contributes to the creation of an elastic environment within the cell, especially during cell division³⁸. In addition, we evaluated the gene expression levels of two cytokines strongly involved in the early stages of the repair process. Pro-inflammatory cytokines play an important role in wound healing and tissue repair. Indeed, IL-1β and IL-8 are highly regulated during the inflammatory phase of wound healing³⁹. IL-1β is one of the first cytokines released during the first response to tissue repair⁴⁰. IL-1β signaling stimulates the production of IL-8 in inflammatory cells and their levels increase exponentially during the early stages of wound healing^41–44. Therefore, IL-8 and IL-1β have proliferative and pro-motility capabilities, confirmed by our results after 24 h of treatment. Figure 6 shows an early induction of IL-1β (panel b) and IL-8 (panel a) expression, 24 h after injury. These results suggest that functionalized nanofibers are able to influence the molecular mechanisms responsible for the early stages of healing during skin repair. Overall, we showed that PVA1% and PVP1% functionalized with HO could be successfully applied as products capable of reconstituting damaged tissue due to the physiochemical properties of the extract and the ability of the nanomaterials to gradually release bioactive molecules⁴⁵

Fig. 5.

Panel (a) Representative AFM force-indentation curve of HaCaT cells, where we highlighted in light blue and light pink the layers used for the fit with the two-slope modified Hertz-Sneddon model; panel (b) boxplots showing the apparent Young’s moduli Y1 (external layer) and Y2 (cell body) of the cells, obtained as described in panel (a). The lower and the upper boundaries of the box represent Q1 (25 percentile) and Q3 (75 percentile) of the data, respectively; the horizontal bar inside the box represent the median of the data.

Conclusions

Many cellular processes are regulated and related by changes in the mechanical properties of the cell. The study of cellular parameters as cell stiffness is useful for understanding cellular processes that involve mechanical changes and have been related to different conditions⁴⁵. In this context, the nanofibers already recognized for their biomedical characteristics as biocompatibility and biodegradability, showed a rapid response to mechanical stress and induced changes in the cell elasticity of HaCaT, after only 24 h of treatment. The effectiveness of combined nanofibers during the healing process was confirmed using confocal microscopy, which revealed increased expression of the OCLN protein. It would be appropriate to evaluate the effect of PVA1% and PVP1% at different times, assessing involvement in the various phases of skin repair. Furthermore, this pilot study will allow research on other cell types involved in the regenerative process. These nanodevices combined with bioactive molecules can potentially be used in the medical field for topical application on skin wounds.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, D.S; G.G; S.C. and MM; methodology, D.S; G.G; S.C; D.C. A.P, G.P; P.P, F.M, M.T. and A.B.; software, D.S; S.C and P.P; validation, M.M and E.A.; formal analysis, D.S., G.G., S.C.; D.C and A.B; investigation, D.S., G.G., S.C and M.M; resources, R.S; G.P; A.P and E.A.; data curation, D.S; G.G; S.C; writing—original draft preparation, D.S.; writing—review and editing, S.C, C.V. and M.M; visualization, C.V and M.M.; supervision, M.M.; All authors have read and agreed to the published version of the manuscript.

Data availability

The data of the current study are available inside of the manuscript.

Declarations

Competing interests

The authors declare no competing interests.