Nanocomposite Membrane Scaffolds for Cell Function Maintaining for Biomedical Purposes

Nanocomposite multilayered membrane coatings have been widely used experimentally to enhance biomedical materials surfaces. By the selection of reliable components, such systems are functionalized to be adjusted to specific purposes. As metal nanoparticles can reduce bacterial cell adhesion, the idea of using gold and silver nanoparticles of unique antimicrobial properties within membrane structure is outstandingly interesting considering dressings facilitating wound healing. The study was aimed to explore the interface between eukaryotic cells and wound dressing materials containing various nanoelements. The proposed systems are based on polyethyleneimine and hydroxyapatite thin layers incorporating metallic nanoparticles (silver or gold). To examine the structure of designed materials scanning electron and transmission electron microscopies were applied. Moreover, Fourier-transform infrared and energy-dispersive X-ray spectroscopies were used. Additionally, water contact angles of the designed membranes and their transport properties were estimated. The functioning of human fibroblasts was examined via flow cytometry to assess the biocompatibility of developed shells in the aspect of their cytotoxicity. The results indicated that designed nanocomposite membrane scaffolds support eukaryotic cells’ functioning, confirming that the elaborated systems might be recommended as wound healing materials.


Introduction
Wound healing belongs to the complex multi-phase processes of dynamic characteristics. The tissue's efficient regeneration depends on the number of factors among which the pathological circumstances, environmental conditions and dressing material can be enumerated [1][2][3][4]. For those reasons, it should not be surprised that the appropriate fitting of wound dressing material to the wound increases therapy effectiveness, facilitates healing and reduces the risk of potential infection for both acute and chronic wound types. Furthermore, the application of personalized wound dressing can accelerate treatment [5]. Nevertheless, it is expected that each material aimed at biomedical usage fulfills a list of restrictive requirements. Thereby, comprehensive studies must be conducted to obtain the material matching the appropriate biological and physicochemical characteristics.
The number of natural and synthetic components involving metallic NPs have been tested for their usefulness and cooperation with the cells [6][7][8][9][10][11][12][13]. However, the concept of applying metal nanoparticles as a part of the wound dressing seems to be incredibly engaging, considering the increase of multidrug resistance in microorganisms. Undoubtedly, the development of antibiotics, antivirals and antimalarials are some of modern the polyelectrolyte membrane's surface occurs by adhesion. The process in which the carrier's ionic interactions with a high density of positive charges and numerous negative charges of cell membranes play the leading role [46]. We perform several experiments using microscopic and spectroscopic techniques to testify the constructed membranes. Furthermore, we evaluated coatings' transport characteristics. Additionally, to assess the biocompatibility of developed membrane platforms in the aspect of their cytotoxicity, we assessed the function of cells in vitro. The human dermal fibroblasts were used as model cells for immobilization within the designed nano-coatings during these studies.

Membrane Preparation
The membrane scaffolds were prepared based on polyethyleneimine solution at a 1 mg/mL concentration in PBS (PEI). To obtain the membranes: 1. polyethyleneimine with AuNPs composite (PEI-Au)-20 ppm AuNPs solution in PBS was added to PEI at a 1:1 ratio and subsequently stirred for 4 h, room temperature. 2.
polyethyleneimine with AgNPs composite (PEI-Ag)-20 ppm AgNPS solution in PBS was added to PEI at a 1:1 ratio and subsequently stirred for 4 h, room temperature. 3.
the bilayer of polyethyleneimine incorporating AuNPs and hydroxyapatite (PEI-Au|HAP)-the HAP layer of 0.3% hydroxyapatite solution in PBS was deposited on a PEI-Au membrane layer. 4.
the bilayer of polyethyleneimine incorporating AgNPs and hydroxyapatite (PEI-Ag|HAP)-the HAP layer of 0.3% hydroxyapatite solution in PBS was deposited on a PEI-Ag membrane layer. 5.
built of the hydroxyapatite mixed with polyethyleneimine incorporating AuNPs (PEI-Au-HAP)-the polyethyleneimine with AuNPs solution (PEI-Au) was added to 0.3% hydroxyapatite solution in PBS at a 1:1 ratio and subsequently stirred for 4 min, room temperature. 6.
built of the hydroxyapatite mixed with polyethyleneimine incorporating AgNPs (PEI-Ag-HAP)-the polyethyleneimine with AgNPs solution (PEI-Ag) was added to 0.3% hydroxyapatite solution in PBS at a 1:1 ratio and subsequently stirred for 4 min, room temperature.
Finally, the concentration of nanoparticles in the membranes involving AgNPs or AuNPs was 10 ppm. HAP concentration was 0.15% in PEI-Ag-HAP and PEI-Au-HAP membranes.
The membrane layers were deposited on the support-sterile coverslips. The membrane was poured onto the coverslips, then allowed to dry overnight at room temperature. Next, the coverslips were washed twice with deionized water and, after drying, transferred to the culture wells (put at the bottom of a culture well). The procedure of membrane deposition was present in Figure 1.
Finally, the concentration of nanoparticles in the membranes involving AgNPs or AuNPs was 10 ppm. HAP concentration was 0.15% in PEI-Ag-HAP and PEI-Au-HAP membranes.
The membrane layers were deposited on the support-sterile coverslips. The membrane was poured onto the coverslips, then allowed to dry overnight at room temperature. Next, the coverslips were washed twice with deionized water and, after drying, transferred to the culture wells (put at the bottom of a culture well). The procedure of membrane deposition was present in Figure 1.  (1). Then, the membrane dries overnight at room temperature (2). The coverslips were washed twice with deionized water (3) and transferred to the culture wells (4).

Wettability and Work of Adhesion Evaluation
The wettability of polyelectrolyte membranes was studied in a surface energy analyzer Phoenix 150 (Surface Electro-Optics Haas, EU). Before the analysis, the membrane layers were deposited on the glass coverslips; then, the slips were washed and dried consecutively at room temperature for 2 h. We applied deionized water as a probe for surface-free energy evaluation. After the water droplet forming on the studied membrane surface, the contact angle was monitored by a magnifying camera using dedicated software-SEO Software-IMAGE XP. The images were analyzed automatically by software algorithm analysis using a wave function. The measurements were performed at room temperature.

Assessment of the Transport Properties of the Membranes
The alginate cores prepared of 1.5% alginate solution in 0.1 M NaCl coated with the evaluated membranes were used to estimate the membrane transport properties. The alginate cores served as a control. Diffusive permeability was evaluated using a thermodynamic description of diffusive mass transport across a homogenous membrane (Fick's law) and a two-compartment model [47]. Dextrans of molecular weight of 70 kDa and 150 kDa was used as the model particle in these studies.

Cell Culture
Human Dermal Fibroblast (HDF) cells were maintained under standard culture conditions (5% CO2, 37 °C) in Fibroblast Growth Medium. Cells were grown to approximately 80% confluency. The medium was then removed from the culture bottles, whereas the cells were washed with PBS without calcium and magnesium ions and trypsinized with trypsin with EDTA.
The HDF cells were placed on tested membranes at a density of 3 × 10 3 cells per well (1.6 × 10 3 /cm 2 ). As a negative control, the cells cultured in the presence of support without deposited membranes were applied. Cell viability was assessed after 1, 3 and 7 days of culture in a cytochemical reaction with propidium iodide in a flow cytometer. Then, the membrane dries overnight at room temperature (2). The coverslips were washed twice with deionized water (3) and transferred to the culture wells (4).

Wettability and Work of Adhesion Evaluation
The wettability of polyelectrolyte membranes was studied in a surface energy analyzer Phoenix 150 (Surface Electro-Optics Haas, EU). Before the analysis, the membrane layers were deposited on the glass coverslips; then, the slips were washed and dried consecutively at room temperature for 2 h. We applied deionized water as a probe for surface-free energy evaluation. After the water droplet forming on the studied membrane surface, the contact angle was monitored by a magnifying camera using dedicated software-SEO Software-IMAGE XP. The images were analyzed automatically by software algorithm analysis using a wave function. The measurements were performed at room temperature.

Assessment of the Transport Properties of the Membranes
The alginate cores prepared of 1.5% alginate solution in 0.1 M NaCl coated with the evaluated membranes were used to estimate the membrane transport properties. The alginate cores served as a control. Diffusive permeability was evaluated using a thermodynamic description of diffusive mass transport across a homogenous membrane (Fick's law) and a two-compartment model [47]. Dextrans of molecular weight of 70 kDa and 150 kDa was used as the model particle in these studies.

Cell Culture
Human Dermal Fibroblast (HDF) cells were maintained under standard culture conditions (5% CO 2 , 37 • C) in Fibroblast Growth Medium. Cells were grown to approximately 80% confluency. The medium was then removed from the culture bottles, whereas the cells were washed with PBS without calcium and magnesium ions and trypsinized with trypsin with EDTA.
The HDF cells were placed on tested membranes at a density of 3 × 10 3 cells per well (1.6 × 10 3 /cm 2 ). As a negative control, the cells cultured in the presence of support without deposited membranes were applied. Cell viability was assessed after 1, 3 and 7 days of culture in a cytochemical reaction with propidium iodide in a flow cytometer.

Fluorescent Staining
After 1, 3 and 7 days of culture of cells immobilized within scaffolds, the samples were fixed with 4% paraformaldehyde. After that, the cell membranes were permeabilized using TRITON X100 detergent to provide the dye's penetration into the individual cells. Then, the fluorescent staining was performed by (1) the addition of fluorochrome-conjugated phalloidin, staining F-actin; (2) the addition of DAPI solution. Phalloidin directly binding to filamentous actin (F-actin) is a toxin isolated from the fungus Phylum Amanita (Amanita phalloides) typically found in significant quantities in fibroblasts. DAPI is an explicitly staining DNA fluorochrome, which allows the emittance of blue light's cell nuclei under UV light.

Flow Cytometry
The eukaryotic cells' presence was assessed using a Canto II flow cytometer (Becton Dickinson Immunocytochemistry Systems, Franklin Lake, NY, USA). The obtained results were processed by the FACS Diva software system (Becton Dickinson, Franklin Lake, NY, USA). Evaluated objects were separated from other events based on the light scattering characteristics.

Scanning Electron Microscopy Evaluation
The systems of cells immobilized within membrane shells after 1, 3 and 7 days of culture were fixed with 2.5% glutaraldehyde. Then, so prepared samples were rinsed several times with water and placed in 75% ethanol for 15 min. The procedure was repeated. The next stage was a 15 min incubation of samples in 99.8% ethanol. The samples were then dried and placed on microscope tables.
Scanning Electron Microscopy (SEM) examinations of the samples were performed using TM-1000 (Hitachi, Tokyo, Japan) scanning electron microscope.

Transmission Electron Microscopy Evaluation
We perform Transmission Electron Microscopy (TEM) analysis of the designed membranes on the FEI Talos F200X transmission microscope at 200 kV (The Thermo Scientific, Waltham, MA, USA). The measurements were conducted in TEM and STEM mode using high-angle annular dark-field imaging. The energy-dispersive X-ray spectroscopy (EDX) on a Brucker BD4 instrument was used to detect the metallic nanoparticles and their mapping. Wherein TEM samples were deposited on a copper grid coated with a carbon holey film.

Atomic Forces Microscopy Evaluation
To examine the surface morphology of the prepared samples, Atomic Forces Microscopy (AFM) was used.
To evaluate the interaction force between the polyelectrolyte layers, we applied the force spectroscopy technique. The silicon cantilever with the SQube (a borosilicate glass colloidal particle sphere of a 10 µm diameter) was used for surface forces acquisition. Before each experiment, we determined a cantilever spring constant value through the ThermalTune method. Forces were measured after 5 s of interaction, wherein the maximum load force was set up to 20 nN. The measurements were performed under in situ conditions (0.15 M NaCl).
To determine the work of adhesion, integration of force-distance dependencies was employed. The calculations were made following the formula: W ad = F ad dz; where F ad refers to adhesion force and z corresponds to the distance of the sphere from a surface.
Atomic Forces Microscopy examinations of the samples were performed using Multi-Mode 8 (Bruker, Billerica, MA, USA) atomic forces microscope. The force-distant curves were acquired in Nanoscope 7.30 software and then analyzed with Origin 8.50 (OriginLab).

Fourier-Transform Infrared Spectroscopy Analysis
The membranes were analyzed using infrared Fourier-transform infrared spectroscopy (FTIR) using a Digilab Excalibur FTS 3000 Mx spectrometer (Bio-Rad, Hercules, CA, USA) equipped with a reusable ZnSe crystal re-amplifier and a transmission adapter.

Statistical Analysis
The mean values and standard deviations and the significance of difference were calculated in the Statistica 7.1 software. Values where p < 0.05 were assumed to be significant.

Water Contact Angle Measurements
The material analysis in terms of wettability (hydrophilicity or hydrophobicity) assesses its suitability for biomedical applications. We tested membranes (1) based on polyethyleneimine incorporating silver or gold nanoparticles (PEI-Ag or PEI-Au); (2) built of hydroxyapatite mixed with polyethyleneimine containing silver or gold nanoparticles (PEI-Ag-HAP or PEI-Au-HAP); (3) a bilayer of hydroxyapatite and polyethyleneimine with silver or gold nanoparticles (PEI-Ag|HAP or PEI-Au|HAP). Obtained results allow stating that there were no significant differences in the contact angles for the membranes involving Au and incorporating Ag and HAP (PEI-Au|HAP; PEI-Ag-HAP). All membranes are hydrophilic ( Figure 2a). Similar conclusions can be drawn from the work of adhesion values ( Figure 2b). Surface energy at the level of 20-30 nm/m is called critical energy, i.e., it is characterized by athrombogenicity and has been defined as a hypothetical biocompatibility zone understood as a zone of minimal cell adhesion. Materials with surface energy above 40 nm/m promote cell adhesion [48]. Obtained values of the surface energy of all constructed membranes (PEI-Ag, PEI-Au, PEI-Ag-HAP, PEI-Au-HAP, PEI-Ag|HAP, PEI-Au|HAP) remained in the range of the value of biomaterials willingly populated by cells ( Figure 3) [48].

Assessment of the Transport Properties of the Membranes
The control group and PEI-Ag, adsorbed Dextran 70 (Dex 70), allowing for about 20% release related to the initial concentration value of 0.05 mg/mL. On the other hand, PEI-Ag|HAP provides for the release of about 50% as related to the initial concentration value of 0.05 mg/mL (Figure 4a). This is reflected in keeping permeability (P) at a consistent level (Figure 4b). It can be stated that PEI-Ag functioning as an independent coating-as a monolayer or in bilayers adsorbed Dex 70. It was observed that only PEI-Ag-HAP allows for a 90% release.

Assessment of the Transport Properties of the Membranes
The control group and PEI-Ag, adsorbed Dextran 70 (Dex 70), allowing for about 20% release related to the initial concentration value of 0.05 mg/mL. On the other hand, PEI-Ag|HAP provides for the release of about 50% as related to the initial concentration value of 0.05 mg/mL (Figure 4a). This is reflected in keeping permeability (P) at a consistent level (Figure 4b). It can be stated that PEI-Ag functioning as an independent coating-as a monolayer or in bilayers adsorbed Dex 70. It was observed that only PEI-Ag-HAP allows for a 90% release.
Analyzing the membranes involving AuNPs, it was noticed that the control group and PEI-Au-HAP adsorbed Dex 70, allowing for about 25% release as related to the initial concentration value of 0.08 mg/mL for 30 min. On the contrary, PEI-Au|HAP and PEI-Au allowed for the release of about 94% of the initial concentration value (0.08 mg/mL) ( Figure 4c). All indicated that PEI-Au functioning in independent coating (monolayer) or in bilayers did not allow for adsorption of more than 6% of Dex70's initial concentration. It was reflected in keeping P at a consistent level (Figure 4d).
The discrepancies between the Dex70 permeability for the membranes encompassing the PEI-Au or PEI-Ag as a layer might be caused by differences in dextran's affinity towards applied nanoparticles. The higher permeability for PEI-Au may occur due to the higher affinity of dextrans towards AuNPs. Some authors reported that dextrans are used as stabilizers of colloidal AuNPs solution [49].
The control group and all membranes involving Ag exhibited Dex150 release up to 10% (Figure 5a).   Analyzing the membranes involving AuNPs, it was noticed that the control group and PEI-Au-HAP adsorbed Dex 70, allowing for about 25% release as related to the initial concentration value of 0.08 mg/mL for 30 min. On the contrary, PEI-Au|HAP and PEI-Au allowed for the release of about 94% of the initial concentration value (0.08 mg/mL) (Figure 4c). All indicated that PEI-Au functioning in independent coating (monolayer) or in bilayers did not allow for adsorption of more than 6% of Dex70's initial concentration. It was reflected in keeping P at a consistent level (Figure 4d).
The discrepancies between the Dex70 permeability for the membranes encompassing the PEI-Au or PEI-Ag as a layer might be caused by differences in dextran's affinity towards applied nanoparticles. The higher permeability for PEI-Au may occur due to the higher affinity of dextrans towards AuNPs. Some authors reported that dextrans are used as stabilizers of colloidal AuNPs solution [49].
The control group and all membranes involving Ag exhibited Dex150 release up to 10% (Figure 5a).
The control group and PEI-Au, PEI-Au-HAP and PEI-Au|HAP allowed the release meanly about 5%, related to the initial concentration. PEI-Au|HAP provided the release of about 10% (Figure 5b). The results indicated the membranes incorporating AgNPs or AuNPs cut-off value at the 150 kDa level. The obtained values are reflected in the P course (Figure 5c,d).

Fourier Transform Infrared Spectroscopy
We assessed Fourier transform infrared spectroscopy (FTIR) signals for polyethyleneimine (PEI), as well as hydroxyapatite (HAP) membranes. Moreover, we analyzed the bilayer build of polyethyleneimine and hydroxyapatite (PEI|HAP). The spectrum was recorded in the range of 4000-400 cm −1 . The characteristic picks were detected at 3375 cm −1 exhibiting N-H stretching vibrations in PEI, 1366 cm −1 exhibiting C-CH 3 presence and 570 cm −1 from n4 symmetric P-O stretching vibration of the PO 4 3− . Moreover, the charac-  Figure S1).

Transmission Electron Microscopy Investigations
We analyzed surfaces of designed polyelectrolyte membranes using Transmission Electron Microscopy (TEM). Figure 6a,b presents photographs showing surfaces of the films built of polyethyleneimine with silver or gold nanoparticles incorporated within their structure (PEI-Ag or PEI-Au).
The single, immobilized silver nanoparticles can be observed within the polyelectrolyte layers; they are deposited evenly in membrane material, creating a uniform structure. Conversely, gold nanoparticles formed separate centers positioned within the polyelectrolyte. Figure 6c,d presents the exemplary photographs of surfaces of the coatings with HAP share. The first image shows the bilayer-built of the polyethyleneimine incorporating AgNPs and hydroxyapatite (PEI-Ag|HAP), whereas the second picture presents the membrane constructed with one layer, consisted of hydroxyapatite mixed with polyethyleneimine incorporating AgNPs (PEI-Ag-HAP). HAP presence does not disturb to distinguish the single silver particles incorporating the polyelectrolyte membrane layer structure.
Nanomaterials 2021, 11, 9 of 17 The control group and PEI-Au, PEI-Au-HAP and PEI-Au|HAP allowed the release meanly about 5%, related to the initial concentration. PEI-Au|HAP provided the release of about 10% (Figure 5b). The results indicated the membranes incorporating AgNPs or AuNPs cut-off value at the 150 kDa level. The obtained values are reflected in the P course (Figure 5c,d).

Fourier Transform Infrared Spectroscopy
We assessed Fourier transform infrared spectroscopy (FTIR) signals for polyethyleneimine (PEI), as well as hydroxyapatite (HAP) membranes. Moreover, we analyzed the bilayer build of polyethyleneimine and hydroxyapatite (PEI|HAP). The spectrum was recorded in the range of 4000-400 cm −1 . The characteristic picks were detected at 3375 cm −1 exhibiting N-H stretching vibrations in PEI, 1366 cm −1 exhibiting C-CH3 presence and 570 cm −1 from n4 symmetric P-O stretching vibration of the PO4 3− . Moreover, the characteristic PO4 3− absorption band was observed at 1631 cm −1 . After HAP deposition, the pick 3375 cm −1 was dislocated to 3325 cm −1 in membrane PEI|HAP (Supplementary Material: Figure S1).

Transmission Electron Microscopy Investigations
We analyzed surfaces of designed polyelectrolyte membranes using Transmission Electron Microscopy (TEM) (Figure 6 a,b). presents photographs showing surfaces of the films built of polyethyleneimine with silver or gold nanoparticles incorporated within their structure (PEI-Ag or PEI-Au).  Since the gold nanoparticles view is overlapped with HAP on TEM's photographs, we could not confirm their occurrence in membranes with AuNPs using this technique.
Thus, to prove gold nanoparticles' presence in the layers, we applied a high-resolution transmission electron microscopy (HRTEM) coupled with energy dispersive X-ray analysis (EDX). Moreover, we analyzed the nanoelements at the atomic scale by electron diffraction (Figure 7a). The energy-dispersive X-ray mapping technique, which uses a scanning transmission electron microscope (STEM), provides simultaneous nanoparticle visualization together with the corresponding Au distribution map tracing. Figure 7b shows EDX spectra of PEI-Au-HAP membrane: the gold nanoparticles are visible as bright spots. On the studied surfaces, we have also observed the presence of the other elements like Na (being a part of the stabilizer of Au), P (as a substrate of HAP), Cu and C (not presented here (Figure 7c).
share. The first image shows the bilayer-built of the polyethyleneimine incorporating AgNPs and hydroxyapatite (PEI-Ag|HAP), whereas the second picture presents the membrane constructed with one layer, consisted of hydroxyapatite mixed with polyethyleneimine incorporating AgNPs (PEI-Ag-HAP). HAP presence does not disturb to distinguish the single silver particles incorporating the polyelectrolyte membrane layer structure.
Since the gold nanoparticles view is overlapped with HAP on TEM's photographs, we could not confirm their occurrence in membranes with AuNPs using this technique. Thus, to prove gold nanoparticles' presence in the layers, we applied a high-resolution transmission electron microscopy (HRTEM) coupled with energy dispersive X-ray analysis (EDX). Moreover, we analyzed the nanoelements at the atomic scale by electron diffraction (Figure 7a). The energy-dispersive X-ray mapping technique, which uses a scanning transmission electron microscope (STEM), provides simultaneous nanoparticle visualization together with the corresponding Au distribution map tracing. Figure 7b shows EDX spectra of PEI-Au-HAP membrane: the gold nanoparticles are visible as bright spots. On the studied surfaces, we have also observed the presence of the other elements like Na (being a part of the stabilizer of Au), P (as a substrate of HAP), Cu and C (not presented here (Figure 7c). EDX spectra of PEI-Au-HAP membrane confirm the presence of the atoms mentioned above (Supplementary Material: Figure S2).
We visualized the presence of gold nanoparticles absorbed on hydroxyapatite by scanning transmission electron microscopy. The appropriate photographs in different magnifications are shown in Figure 8a-c. It can be noted that the STEM images indicated that the maximal size of HAP objects is even about ten times higher than the size of elements stated by the manufacturer (<50 nm), which might be caused by its aggregation.
Every membrane was analyzed in three samples/repetitions. The pictures were taken in different sites of the sample (at eight sites). The concentration of nanoparticles in the membranes involving AgNPs or AuNPs was 10 ppm. EDX spectra of PEI-Au-HAP membrane confirm the presence of the atoms mentioned above (Supplementary Material: Figure S2).
We visualized the presence of gold nanoparticles absorbed on hydroxyapatite by scanning transmission electron microscopy. The appropriate photographs in different magnifications are shown in Figure 8a-c. It can be noted that the STEM images indicated that the maximal size of HAP objects is even about ten times higher than the size of elements stated by the manufacturer (<50 nm), which might be caused by its aggregation.  Figure 9 shows the visualization of the morphological structure of the designed layers. The samples of PEI, PEI-Ag, PEI-Au were deposited on the gold mica substrate. On the other hand, Figure 10 presents the values of the work of adhesion between HAP and PEI-Ag or PEI-Au layers. Every membrane was analyzed in three samples/repetitions. The pictures were taken in different sites of the sample (at eight sites). The concentration of nanoparticles in the membranes involving AgNPs or AuNPs was 10 ppm. Figure 9 shows the visualization of the morphological structure of the designed layers. The samples of PEI, PEI-Ag, PEI-Au were deposited on the gold mica substrate. On the other hand, Figure 10 presents the values of the work of adhesion between HAP and PEI-Ag or PEI-Au layers. 3.1.5. Atomic Forces Microscopy Evaluation Figure 9 shows the visualization of the morphological structure of the designed layers. The samples of PEI, PEI-Ag, PEI-Au were deposited on the gold mica substrate. On the other hand, Figure 10 presents the values of the work of adhesion between HAP and PEI-Ag or PEI-Au layers.    Figure 11 presents the percentage of viable cells immobilized within the membranes involving AuNPs or AgNPs.

Flow Cytometry Analysis
After 7-day culture, no significant difference between PEI-Au and PEI-Au-HAP membranes was observed. The discrepancies in the viability of cells cultured within membranes involving AuNPs did not exceed 8%. There were meanly 95% cells alive.
Similarly, no significant difference between PEI-Ag and PEI-Ag-HAP and PEI-Ag|HAP were observed. However, the viability of cells immobilized within PEI-Ag|HAP was significantly higher than the membranes incorporating AuNPs and compared with PEI-Ag-HAP. Nevertheless, the discrepancies did not exceed about 20%.
Finally, there was no significant difference between the viability of cells immobilized within the membranes built of PEI incorporating AgNPs, functioning as an independent layer in monolayer or bilayer coating.
Gold nanoparticles are generally designed for intracorporeal activity due to the low cytotoxicity towards the organism observed by many authors [25,50,51]. On the other hand, silver nanoparticles are proposed mainly for external usage (e.g., dressings, medical devices, water treatment, decontamination), possibly-in low concentrations-internally, e.g., in implants [50][51][52]. However, when examining the effect of AgNPs and AuNPs on human fibroblast cells, no significant differences in cytotoxicity were found at the NPs concentrations used.
To sum up, it can be stated that applied membranes with metallic NPs did not exert a cytotoxic effect on the HDF cells. Figure 10. The work of adhesion between the HAP and PEI-Ag or HAP and PEI-Au layers. Figure 11 presents the percentage of viable cells immobilized within the membranes involving AuNPs or AgNPs.

Microscopic Evaluation of Immobilized Cells
We performed a scanning electron microscopy (SEM) examination to assess the morphology of the designed scaffolds' systems with immobilized cells. The exemplary images of such analysis are shown in Figure 12. The cells were grown directly on a slide (without membranes) served as a control. After 7 -day culture, no significant difference between PEI-Au and PEI-Au-HAP membranes was observed. The discrepancies in the viability of cells cultured within membranes involving AuNPs did not exceed 8%. There were meanly 95% cells alive.
Similarly, no significant difference between PEI-Ag and PEI-Ag-HAP and PEI-Ag|HAP were observed. However, the viability of cells immobilized within PEI-Ag|HAP was significantly higher than the membranes incorporating AuNPs and compared with PEI-Ag-HAP. Nevertheless, the discrepancies did not exceed about 20%.
Finally, there was no significant difference between the viability of cells immobilized within the membranes built of PEI incorporating AgNPs, functioning as an independent layer in monolayer or bilayer coating.
Gold nanoparticles are generally designed for intracorporeal activity due to the low cytotoxicity towards the organism observed by many authors [25,50,51]. On the other hand, silver nanoparticles are proposed mainly for external usage (e.g., dressings, medical devices, water treatment, decontamination), possibly-in low concentrations-internally, e.g., in implants [50][51][52]. However, when examining the effect of AgNPs and AuNPs on human fibroblast cells, no significant differences in cytotoxicity were found at the NPs concentrations used.
To sum up, it can be stated that applied membranes with metallic NPs did not exert a cytotoxic effect on the HDF cells.

Microscopic Evaluation of Immobilized Cells
We performed a scanning electron microscopy (SEM) examination to assess the morphology of the designed scaffolds' systems with immobilized cells. The exemplary images of such analysis are shown in Figure 12. The cells were grown directly on a slide (without membranes) served as a control. After seven days of the culture, we observed numerous cells on the surface of films built of the hydroxyapatite mixed with polyethyleneimine incorporating metallic nanoparticles (PEI-Au-HAP and PEI-Ag-HAP). However, cell morphology differed between the membranes. The spindle-shaped cells of fibroblastoid features were present on the PEI-Au-HAP layer's surface; the cells' morphology was comparable with the control. On the other hand, on the PEI-Ag-HAP membrane's surface, the spherical shape After seven days of the culture, we observed numerous cells on the surface of films built of the hydroxyapatite mixed with polyethyleneimine incorporating metallic nanoparticles (PEI-Au-HAP and PEI-Ag-HAP). However, cell morphology differed between the membranes. The spindle-shaped cells of fibroblastoid features were present on the PEI-Au-HAP layer's surface; the cells' morphology was comparable with the control. On the other hand, on the PEI-Ag-HAP membrane's surface, the spherical shape cells' presence was confirmed. The form taken by the cells would indicate that the membranes involving AuNPs facilitating cells to form the shape characteristic for the adhesion phase and allowing for higher adhesion of immobilized cells comparing with the membranes containing AgNPs. Though there was no significant difference in adhesion work between the membranes with HAP content: PEI-Au-HAP and PEI-Ag-HAP, the internalization of HAP by cells might appear, leading to the modification of the membrane surface towards the membrane built mainly of PEI-Au and PEI-Ag, respectively. Thus, the higher value of the work of adhesion of the membranes built of PEI-Au may result in a more spindle shape of cells at the interface with the membrane. However, separate studies of the internalization of HAP NPs at the cellular level would be necessary for confirmation.
The obtained results suggest that the membranes involving AuNPs facilitating cells to form the shape characteristic for the adhesion phase allowed for higher adhesion of immobilized cells comparing with the membranes containing AgNPs. Nevertheless, there was no correlation between immobilized cells' shape and the cell viability cytometrically assessed.

Fluorescent Staining
The exemplary images of fluorescence microscopic examination of cells maintained in the presence of designed membranes are shown below, wherein the cells grown directly on a slide (without membranes) served as a control. Figure 13 shows the HDF cells maintained in the absence (control) or the presence of PEI-Ag-HAP or PEI-Au-HAP coatings after seven days of the culture. Cell nuclei are visualized by staining with DAPI blue fluorescent dye; cell cytoskeleton is stained red with F-Actin. respectively. Thus, the higher value of the work of adhesion of the membranes built of PEI-Au may result in a more spindle shape of cells at the interface with the membrane. However, separate studies of the internalization of HAP NPs at the cellular level would be necessary for confirmation. The obtained results suggest that the membranes involving AuNPs facilitating cells to form the shape characteristic for the adhesion phase allowed for higher adhesion of immobilized cells comparing with the membranes containing AgNPs. Nevertheless, there was no correlation between immobilized cells' shape and the cell viability cytometrically assessed.

3..2.3. Fluorescent Staining
The exemplary images of fluorescence microscopic examination of cells maintained in the presence of designed membranes are shown below, wherein the cells grown directly on a slide (without membranes) served as a control. Figure 13 shows the HDF cells maintained in the absence (control) or the presence of PEI-Ag-HAP or PEI-Au-HAP coatings after seven days of the culture. Cell nuclei are visualized by staining with DAPI blue fluorescent dye; cell cytoskeleton is stained red with F-Actin. We observed the cells with different morphology for all days of culture. Additionally, the microscopic fluorescence pictures confirmed the cells of spherical shape on the PEI- We observed the cells with different morphology for all days of culture. Additionally, the microscopic fluorescence pictures confirmed the cells of spherical shape on the PEI-Ag-HAP membrane. The spindle-shaped cells' presence, comparable to the control, was visible on the PEI-Au-HAP membrane's surface. However, the cells of spherical shape were also noticeable.

Conclusions
We applied the nanocomposite material with metallic nanoparticles (AuNPs or Ag-NPs) to ensure the material's bacteriostatic properties and simultaneously maintain its biocompatibility. Some differences were observed between the membranes involving Ag-NPs or AuNPs concerning their properties and morphology at membrane-immobilized cells interface. The different values of adhesion work were observed between HAP and layers incorporating AgNPs or AuNPs. The cells cultured on the shells involving AgNPs and AuNPs differ in morphology. The more spindle form taken by the cells immobilized on the membranes PEI-Au-HAP involving AuNPs comparing with membrane PEI-Ag-HAP involving AgNPs implies the influence of a higher adhesion work in case PEI-Au-HAP membrane. Such a phenomenon may arise in case of internalization of HAP by immobilized cells what needs further studies.
Nevertheless, the presented data indicated that the designed materials were biocompatible in the aspect of cytotoxicity. The AuNPs or AgNPs involvement at an applied concentration in the scaffold material does not exert a cytotoxic effect on HDF cells.
However, application of AgNPs or AuNPs at concentration 10 ppm incorporated in membranes did not exert a cytotoxic effect on HDF cells, the cytotoxicity of NPs towards biological material still needs further complex examinations in general and in a complex with selected materials that can find biomedical application, in particular, in bandages.
The bacteriostatic membrane with a cut-off at IgG level may be regarded as an element of bandage systems. The designed scaffolding layered shells, especially ensuring physiological shape forming by HDF cells, can be recommended as an appropriate element to sustain eukaryotic cell function in bandage applications.