Bacterial Cellulose-Based Nanocomposites Containing Ceria and Their Use in the Process of Stem Cell Proliferation

A technique for the fabrication of bacterial cellulose-based films with CeO2 nanofiller has been developed. The structural and morphological characteristics of the materials have been studied, their thermal and mechanical properties in dry and swollen states having been determined. The preparation methodology makes it possible to obtain composites with a uniform distribution of nanoparticles. The catalytic effect of ceria, regarding the thermal oxidative destruction of cellulose, has been confirmed by TGA and DTA methods. An increase in CeO2 content led to an increase in the elastic modulus (a 1.27-fold increase caused by the introduction of 5 wt.% of the nanofiller into the polymer) and strength of the films. This effect is explained by the formation of additional links between polymer macro-chains via the nanoparticles’ surface. The materials fabricated were characterized by a limited ability to swell in water. Swelling caused a 20- to 30-fold reduction in the stiffness of the material, the mechanical properties of the films in a swollen state remaining germane to their practical use. The application of the composite films in cell engineering as substrates for the stem cells’ proliferation has been studied. The increase in CeO2 content in the films enhanced the proliferative activity of embryonic mouse stem cells. The cells cultured on the scaffold containing 5 wt.% of ceria demonstrated increased cell survival and migration activity. An analysis of gene expression confirmed improved cultivation conditions on CeO2-containing scaffolds.


Introduction
In the 21st century, there has been growing attention among researchers and chemical engineers in the field of novel polymer materials in so-called natural or "green" polymers, including cellulose and its derivatives [1]. Of particular interest are materials based on bacterial cellulose (BC) [2], which is a product of the vital activity of Acetobacteraceae bacteria (Gram-negative anaerobic bacteria) in carbon-containing nutrient media. Typically,

Thermal Analysis
Thermogravimetric (TGA) and differential thermal (DTA) analyses were performed to determine the concentration of remaining water in the films and the exact contents of ceria in the nanocomposite materials. The effect of CeO 2 on the kinetics of thermal decomposition of the samples was assessed. A DTG-60 thermal analyser (Shimadzu, Kyoto, Japan) was used, the samples (~5 mg) being heated in air up to 600 • C at a rate of 5 • C/min.

Swelling Properties
To determine equilibrium water absorption, the samples, having first been dried at 150 • C, were weighed and then immersed in the distilled water. The swollen samples were periodically removed from the water, wiped with a tissue, and weighed. The experiment continued until a constant weight had been attained.
Polymers 2021, 13, 1999 4 of 18 Water absorption W m was calculated using the following equation: where m s and m d are the weights of the swollen and dry samples, respectively.

Mechanical Properties
An AG-100kNX Plus setup (Shimadzu, Kyoto, Japan) operating in a uniaxial extension mode was used to study the mechanical characteristics of the films. Strip-like samples (2 × 30 mm 2 ) were stretched at room temperature at a rate of 10 mm/min, according to ASTM D638 requirements. The Young's modulus E, the break stress σ b , and the ultimate deformation ε b were determined.

Cell Proliferation Control Techniques Cell Culture
The experiments were conducted using mouse mesenchymal stem cells (MSC) at the 3rd passage, harvested from the embryonic skeleton of 13 day-old embryos of B10GFP/ Balb/c hybrid mice carrying a green fluorescent protein (GFP). The cells were received from the vivarium of ICB RAS (Pushchino, Moscow Region, Russia). The study was conducted according to the guidelines of EU directive 2010/63 of European Parliament and council of Europe Union 22 September 2010 for the protection of animals used for scientific purposes, and approved by the Institutional Ethics and biosafety committee of Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences. The animals were fed twice a day. Animal maintenance was in accordance with the rules of Good Laboratory Practice (GLP) and the Order of the Ministry of Health of the Russian Federation No. 199n "Rules of Good Laboratory Practice".
Using this type of cell was regarded as being expedient due to the need to avoid applying fluorescent dyes in order to prevent the coloration of the samples under study. Mice were euthanized by cervical dislocation on the 13th day of gestation. Their uteri with embryos were isolated. After tissue dissociation in 0.25% trypsin-0.02% EDTA solution (NPO "PanEco", Moscow, Russia) for 30 min at 37 • C, the cells were collected by centrifugation at 1500 rpm for 2 min. Then, the cells were re-suspended to obtain a single-cell state in a DMEM/F12 medium (NPO "PanEco", Russia) supplemented with 10% of fetal bovine serum (FBS) (in 1:1 ratio). The resulting suspensions were transferred into vials and cultivated in a 5% CO2 atmosphere at 37 • C in DMEM containing 10% FBS (Hy-Clone, Logan, MI, USA) and 100 units/mL of penicillin/streptomycin, with the addition of 2 mM L-glutamine. When a subconfluent state was reached, the cells were treated with 0.25% trypsin-EDTA solution and seeded into vials in a 1:3 ratio. The cultivation was carried out in a DMEM/F12 medium containing 10% FBS (HyClone, Logan, MI, USA) and 100 units/mL of penicillin/streptomycin, with the addition of 2 mM L-glutamine. Cultures of 2-5 passages were used in the research. The analysis of the viability of cell cultures was carried out using the resazurin assay. After 24-96 h of cultivation, the cell culture medium was replaced with one containing 0.02% solution resazurin and the MSC were further cultured.

Fluorescent Microscopy
The films were cut into 0.8 × 0.8 cm 2 fragments and placed in the wells of a 24-well plate, under sterile conditions. One sample was assigned to each test point. A sterile cover glass was used as a reference sample. Cells were seeded on the substrates' surface. The initial cell seeding density was 30 × 10 4 cells/cm 2 . The morphological assessment of the cells cultivated on the surface of the test materials was implemented on the 1st, 2nd, 3rd, and 4th day of cultivation in fluorescent light, using an inverted microscope Zeiss Axiovert 200 (Carl Zeiss, Oberkochen, Germany).

Scanning Electron Microscopy
The cells were plated on the substrates and cultivated for 24 to 96 h. The samples were then washed twice for 30 min in phosphate-buffered saline (PBS) at pH 7.4 and dehydrated by being passed through alcohol solution of increasing concentration: 30%, 50%, 70%, and 80% for 30 min, and 90% and 100% for 30 min (twice). At the final stage, the samples were placed in a hexamethyldisilazane solution for 24 h. In order to avoid charge accumulation during scanning, the samples were coated with a 2 nm gold layer by ion sputtering in an argon atmosphere (0.1 mm Hg). A SUPRA-55VP (Carl Zeiss, Oberkochen, Germany) microscope was used in the experiments.

Reverse Transcription Polymerase Chain Reaction (PCR-RT)
Reverse transcription was performed with a Sileks kit (Sileks, Moscow, Russia), using an oligo(dT) primer, according to the manufacturer's protocol. The cDNAs produced served as a real-time PCR matrix. For PCR reaction, a mixture was used with SYBR Green dye (Syntol, Moscow, Russia). To analyze the expression activity of 93 key genes under cell cultivation on the CeO 2 -containing scaffolds, a CFX-96 amplifier ((BioRad, Irvin, CA, USA) or an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) was used. The expression of 96 genes responsible for key cell processes (Supplementary Table S1) was thus determined. The genes that were analyzed were selected from the database https: //dataanalysis2.qiagen.com/pcr (accessed on 16 June 2021) for PCR profiling of different biological processes. The level of gene transcription was normalized according to the levels of expression of housekeeping genes β-actin, rplp0 (ribosomal protein, large, P0), and gapdh (glyceraldehyde-3-phosphate dehydrogenase). Gene-specific primers were selected using the Primer Express program (Applied Biosystems, Waltham, MA, USA). Each measurement was made twice (internal repetition) and averaged for two independent samples. A sample without a reverse transcription stage was used as the control. The expression data obtained were analyzed using online services https://dataanalysis2.qiagen.com/pcr, mayday-2.14 software (Center for Bioinformatics, Tübingen, Germany) and Genesis software.

SEM
SEM images of the films fabricated ( Figure 1) show clearly the structural elements of the films, namely tightly-packed fibrils. Comparing the morphologies of the composite and pristine BC films, there is no noticeable effect of CeO 2 nanoparticles on the packing type, except that the BC fibrils in the composite films seem to be aggregated more closely. No evident aggregates of CeO 2 nanoparticles are seen on the composite films' surfaces.

XRD Analysis
Obviously, the SEM technique does not enable individual filler nanoparticles to be distinguished. Their presence in the films is only evident from the comparative analysis of the XRD patterns of the films and pristine CeO 2 powder ( Figure 2). Well-resolved reflections at 2θ near 14.4 • , 16.7 • , and 22.4 • are revealed in all XRD patterns of BC-based films. According to the data of X-ray scattering, the structure of these specimens is attributed to highly crystalline cellulose I, most probably Iα allomorph [37].
The XRD pattern for CeO 2 powder shows sharp reflections at 2θ = 28.7, 32.97, and 47.5 deg. attributed to (111), (200), and (220) planes, respectively, corresponding to the cubic fluorite crystal structure (ICDD PDF card #34-394, data from NIST (National Institute of Standards and Technology, Gaithersburg, Maryland, Boulder, CO, USA)). The full width at half-maximum (FWHM) of the (111) peak was used in the calculation of the apparent transverse D111 sizes of CeO 2 crystals, using Scherrer's equation [38]. The D 111 size was found to be~5.4 nm. The same reflections, along with those of the BC, can be seen in the patterns of the nanocomposite films (Figure 2b-d). Their intensities increased linearly with the concentration of ceria nanoparticles in the BC matrix.

SEM
SEM images of the films fabricated ( Figure 1) show clearly the structural elements of the films, namely tightly-packed fibrils. Comparing the morphologies of the composite and pristine BC films, there is no noticeable effect of CeO2 nanoparticles on the packing type, except that the BC fibrils in the composite films seem to be aggregated more closely. No evident aggregates of CeO2 nanoparticles are seen on the composite films' surfaces.

XRD Analysis
Obviously, the SEM technique does not enable individual filler nanoparticles to be distinguished. Their presence in the films is only evident from the comparative analysis of the XRD patterns of the films and pristine CeO2 powder ( Figure 2). Well-resolved reflections at 2θ near 14.4°, 16.7°, and 22.4° are revealed in all XRD patterns of BC-based films. According to the data of X-ray scattering, the structure of these specimens is attributed to highly crystalline cellulose I, most probably Iα allomorph [37].

XRD Analysis
Obviously, the SEM technique does not enable individual filler nanoparticle distinguished. Their presence in the films is only evident from the comparative a of the XRD patterns of the films and pristine CeO2 powder ( Figure 2). Well-resol flections at 2θ near 14.4°, 16.7°, and 22.4° are revealed in all XRD patterns of BC films. According to the data of X-ray scattering, the structure of these specimen tributed to highly crystalline cellulose I, most probably Iα allomorph [37].  The XRD pattern for CeO2 powder shows sharp reflections at 2θ = 28.7, 32.97, and 47.5 deg. attributed to (111), (200), and (220) planes, respectively, corresponding to the cubic fluorite crystal structure (ICDD PDF card #34-394, data from NIST (National Institute of Standards and Technology, Gaithersburg, Maryland, Boulder, CO, USA)). The full width at half-maximum (FWHM) of the (111) peak was used in the calculation of the apparent transverse D111 sizes of CeO2 crystals, using Scherrer's equation [38]. The D111 size was found to be ~5.4 nm. The same reflections, along with those of the BC, can be seen in the patterns of the nanocomposite films (Figure 2b-d). Their intensities increased linearly with the concentration of ceria nanoparticles in the BC matrix. Figure 3 shows the results of energy dispersive X-ray analysis (EDX). This method enables the distribution of elements on the surface of a sample to be mapped. The results confirm the presence and increasing concentration of cerium (the areas appearing as speckled yellow in Figure 3c,e) in the samples, images of the latter being shown in Figure  3b,d. The information on the total percentage of Ce can also be obtained by EDX analysis. It was derived that the weight amounts of Ce are the following: 0.81 wt.% for the composite film containing 1% CeO2, and 4.02 wt.% for the 5% CeO2 film.  Figure 3 shows the results of energy dispersive X-ray analysis (EDX). This method enables the distribution of elements on the surface of a sample to be mapped. The results confirm the presence and increasing concentration of cerium (the areas appearing as speckled yellow in Figure 3c,e) in the samples, images of the latter being shown in Figure 3b,d. The information on the total percentage of Ce can also be obtained by EDX analysis. It was derived that the weight amounts of Ce are the following: 0.81 wt.% for the composite film containing 1% CeO 2 , and 4.02 wt.% for the 5% CeO 2 film.

Thermal Analysis
TGA was used to precisely determine the nanofiller concentration in the nanocomposite films prepared ( Figure 4). The shape of the BC weight loss curve in air ( Figure 4a) is attributable to the complete decomposition of the polymer upon heating to 500 • C, accompanied by the removal of the gaseous products. A different outcome is observed from the TGA of the composite films (Figure 4a), viz., after total volatilization of the organic part of the material, stable inorganic residues were obtained, whose concentrations in the films (1.0 wt.%, 3.05 wt.%, and 4.90 wt.%) corresponded to those used in the fabrication of the composite samples.
The TGA curves also enable an evaluation of the weight of water remaining in the as-prepared films. The curves show an initial weight loss step below 120 • C, followed by a plateau at higher temperatures, where the weight did not change until the start of thermal decomposition. The weight loss in all of the samples in this low-temperature region was 2.0-2.5%. The TGA curves corroborate the literature data [18,39] on the catalytic activity of ceria in decomposition reactions of a number of organic compounds. Comparing the TGA results for the pristine BC film and the composites with various CeO 2 contents, it is noticeable that intense decomposition processes shifted to lower temperatures as ceria concentration increased. The deterioration in the thermal stability of the samples was accompanied by a decrease in the indices τ 5 and τ 10 (the temperature values at which a polymer or a composite loses 5% and 10% of its initial weight, respectively, due to thermal destruction processes) ( Table 1).

Thermal Analysis
TGA was used to precisely determine the nanofiller concentration in the na posite films prepared ( Figure 4). The shape of the BC weight loss curve in air (Fi is attributable to the complete decomposition of the polymer upon heating to 50 companied by the removal of the gaseous products. A different outcome is observ the TGA of the composite films (Figure 4a), viz., after total volatilization of the part of the material, stable inorganic residues were obtained, whose concentration films (1.0 wt.%, 3.05 wt.%, and 4.90 wt.%) corresponded to those used in the fab of the composite samples.
The TGA curves also enable an evaluation of the weight of water remainin as-prepared films. The curves show an initial weight loss step below 120 °C, follo a plateau at higher temperatures, where the weight did not change until the start mal decomposition. The weight loss in all of the samples in this low-temperatur was ~2.0-2.5%. The TGA curves corroborate the literature data [18,39] on the cata tivity of ceria in decomposition reactions of a number of organic compounds. Com the TGA results for the pristine BC film and the composites with various CeO2 c it is noticeable that intense decomposition processes shifted to lower temperatures as ceria concentration increased. The deterioration in the thermal stability of the samples was accompanied by a decrease in the indices τ5 and τ10 (the temperature values at which a polymer or a composite loses 5% and 10% of its initial weight, respectively, due to thermal destruction processes) ( Table 1).    A detrimental effect of CeO 2 on the thermal stability of BC is evident from the DTA curves of the nanocomposite films ( Figure 4b). Indeed, an increase in ceria concentration from 0 to 5 wt.% led to a gradual shift of exothermic peaks toward the low-temperature region, the peaks being ascribed to the heat effect of thermal destruction. In accordance with previously reported data [40,41], the thermo-oxidative destruction of BC and BC-based nanocomposites occurred in two steps (two peaks on the DTA curves corresponding to these steps are in the 300-330 • C and 420-480 • C temperature regions). The increase in ceria content is seen to result in both a shift of the exothermic peaks to lower temperatures and a redistribution of the peak heights: a low-temperature peak became higher, whilst a high-temperature peak tended to become lower and smoother. In accordance with the well-known scheme of cellulose destruction-the Broido-Shafizadeh model [42]-the main reaction taking place in the low-temperature region (up to 350 • C) is the thermally stimulated decomposition of cellulose macrochains, resulting in the formation of monomeric and oligomeric fragments (the so-called "sirup" fraction). At the next (high-temperature) stage, these fragments decompose further, producing a variety of gaseous products. Based on the results obtained in the experiments on which this paper reports, it can be inferred that CeO 2 causes deep depolymerization of BC, and full decomposition of the oligomers into monomers during the first stage of the destruction, thus facilitating the second stage.
It is worth mentioning that all of the aforementioned processes start at temperatures higher than 180-200 • C, and this should be taken into account when thermally sterilizing nanocomposite BC-ceria materials for biomedical applications.

Mechanical Properties
The successful practical use of the composite materials devised requires certain mechanical characteristics of the films they form. The mechanical properties of the BC-ceria composite materials with different CeO 2 content are summed up in Table 2. The data given in Table 2 reveal both the high stiffness (Young's moduli of all the materials are higher than 5 GPa) and good strength characteristics (mechanical strength reaches 100-110 MPa) of the films. The incorporation of ceria in the BC matrix is seen to lead to a gradual (as CeO 2 concentration grows) increase in the elastic modulus (5 wt.% of CeO 2 augment the modulus by 27%). Such a pronounced rise in stiffness cannot be attributed to the reinforcing action of the nanofiller on BC matrix, as CeO 2 nanoparticles, which have quasi-spherical shapes (aspect ratio is~1) and are taken in rather small amounts, do not provide a reinforcing effect. A sensible explanation is that additional links between the BC macrochains form via the active surface of the nanoparticles when the nanocomposite is fabricated. It should also be pointed out that ceria does not cause a substantial decrease in the ultimate deformation of the films. This confirms the good compatibility between both components of the nanocomposites.

Ability to Swell in Water
The discussion above concerns the mechanical properties of dry films (according to TGA, water content is~2.0-2.5%). However, the capability of a material to absorb water is of crucial importance in a number of practical applications, including biomedicine. Therefore, the current study considered the impact of ceria upon this property of the BC films.
It is evident from the swelling curves ( Figure 5) that the films demonstrated a limited ability to swell in aqueous media, viz. the ultimate amount of water in the matrix BC in a swollen state was only 3.25 times higher than the weight of the dry polymer. Apparently, such a limited ability to swell is determined by the characteristics of the intermolecular bonds formed during fabrication of the films. A fairly interesting phenomenon observed in the experiments was the fall in the ultimate amount of water in a swollen material as CeO2 content increased ( Figure 5). This observation corroborates the hypothesis about additional intermolecular interactions, the CeO2 nanoparticles acting as links. A similar reduction in the degree of swelling in water was observed in nanocomposite films consisting of a mixture of chitosan with cellulose acetate filled with CeO2 nanoparticles [43].
An assessment of the mechanical properties of the swollen films is important in the context of the practical use of the materials. A technique was developed for performing mechanical tests on the swollen samples. Their mechanical characteristics are shown in Table 2. The results show explicitly that, even in an extremely swollen state, the films possessed a certain mechanical rigidity. The elastic moduli of the samples fell to 0.03-0.04 of that of a dry film but were of the same order of magnitude as the modulus of the LDPE film (170-300 MPa). It should be pointed out that a less pronounced decrease in the elastic modulus caused by swelling was registered in the nanocomposite films in comparison with the pristine BC sample. Such a trend was observed as ceria concentration in the material was augmented: the ratio of the elastic modulus of a film in a fully swollen state, to that of a dry film increased from 0.032 to 0.044 when the CeO2 content changed from 0 to 5 wt.% (Table 2). This clearly reflects a gradual decrease in the degree of swelling of the films with a rise in the concentration of the nanoparticles in the BC matrix. Despite a fairly significant drop in stiffness, the swollen films were characterized by a mechanical strength of 4-6 MPa, the strength being high enough for the material to be used in some biomedical applications. A fairly interesting phenomenon observed in the experiments was the fall in the ultimate amount of water in a swollen material as CeO 2 content increased ( Figure 5). This observation corroborates the hypothesis about additional intermolecular interactions, the CeO 2 nanoparticles acting as links. A similar reduction in the degree of swelling in water was observed in nanocomposite films consisting of a mixture of chitosan with cellulose acetate filled with CeO 2 nanoparticles [43].
An assessment of the mechanical properties of the swollen films is important in the context of the practical use of the materials. A technique was developed for performing mechanical tests on the swollen samples. Their mechanical characteristics are shown in Table 2. The results show explicitly that, even in an extremely swollen state, the films possessed a certain mechanical rigidity. The elastic moduli of the samples fell to 0.03-0.04 of that of a dry film but were of the same order of magnitude as the modulus of the LDPE film (170-300 MPa). It should be pointed out that a less pronounced decrease in the elastic modulus caused by swelling was registered in the nanocomposite films in comparison with the pristine BC sample. Such a trend was observed as ceria concentration in the material was augmented: the ratio of the elastic modulus of a film in a fully swollen state, to that of a dry film increased from 0.032 to 0.044 when the CeO 2 content changed from 0 to 5 wt.% ( Table 2). This clearly reflects a gradual decrease in the degree of swelling of the films with a rise in the concentration of the nanoparticles in the BC matrix. Despite a fairly significant drop in stiffness, the swollen films were characterized by a mechanical strength of 4-6 MPa, the strength being high enough for the material to be used in some biomedical applications.

MSC Proliferation
Among the possible applications of BC-ceria nanocomposite materials, their use in bioengineering as scaffolds for the proliferation of stem cells seems to be very promising. One of the main, and most auspicious, prospects for steady progress in modern medicine is associated with the development, and improvement, of the technology for growing cells of this kind. BC is known to be an interesting and promising material in the manufacture of such substrates [31][32][33]44]. This is due to the peculiarities of the structure of the material, its excellent biocompatibility combined with its high bioresorption ability, its non-cytotoxicity, and the possibility of its preparation in an extra-pure state. There were good reasons to combine such merits of BC matrices with those inherent in ceria in biological systems.
Experiments were carried out on MSC proliferation on substrates made of bare BC and nanocomposites containing up to 5 wt.% of CeO 2 . Figure 6 shows the data on mouse MSC proliferation activity on the surface of bacterial cellulose, the latter containing various concentrations of CeO 2 nanoparticles. The results were recorded on the 1st, 2nd, 3rd, and 4th day of cultivation under fluorescent light.  The CeO2-containing scaffolds demonstrated good adhesion with the mouse MSC. It is well known that the adhesion of cells to the scaffold surface is strongly affected by the micro-relief and texture of the latter [45]. The addition of CeO2 nanoparticles to the BC matrix is supposed to create a surface structure that is most suitable for the formation of focal contacts of the cell filopodia, followed by the protrusion of the cell front. A certain electric charge on the scaffold surface also ensures optimal conditions for proliferation [43]. In the case of BC-CeO2 nanocomposite materials, the electric charge might be provided by the nanoparticles [46].
The adhesive characteristics of the composite scaffolds differ considerably, depending on ceria content. The minimum concentration of the nanoparticles (1 wt.%) provided adhesion for a substantial part of the cell culture; however, after 24 h, morphological features of the cells differed from those on the reference substrate. In particular, the cells did not exhibit the characteristic protrusions and formed multicellular aggregates, whose size decreased with an increase in ceria concentration in the scaffold. This might be explained by weak surface micro-relief, as well as by insufficient adhesion in initial sites for the formation of a fully-fledged extracellular matrix. Further cultivation on the scaffolds demonstrated that the cells spread effectively on the substrate even at small CeO2 concentrations. This process, however, took longer than that on the scaffolds with 3 and 5 wt.% of ceria. The CeO 2 -containing scaffolds demonstrated good adhesion with the mouse MSC. It is well known that the adhesion of cells to the scaffold surface is strongly affected by the micro-relief and texture of the latter [45]. The addition of CeO 2 nanoparticles to the BC matrix is supposed to create a surface structure that is most suitable for the formation of focal contacts of the cell filopodia, followed by the protrusion of the cell front. A certain electric charge on the scaffold surface also ensures optimal conditions for proliferation [43]. In the case of BC-CeO 2 nanocomposite materials, the electric charge might be provided by the nanoparticles [46].
The adhesive characteristics of the composite scaffolds differ considerably, depending on ceria content. The minimum concentration of the nanoparticles (1 wt.%) provided adhesion for a substantial part of the cell culture; however, after 24 h, morphological features of the cells differed from those on the reference substrate. In particular, the cells did not exhibit the characteristic protrusions and formed multicellular aggregates, whose size decreased with an increase in ceria concentration in the scaffold. This might be explained by weak surface micro-relief, as well as by insufficient adhesion in initial sites for the formation of a fully-fledged extracellular matrix. Further cultivation on the scaffolds demonstrated that the cells spread effectively on the substrate even at small CeO 2 concentrations. This process, however, took longer than that on the scaffolds with 3 and 5 wt.% of ceria.
It should be pointed out that the scaffolds with high concentrations of CeO 2 nanoparticles (3 and 5 wt.%) exhibited notably higher cell viability after 96 h of cultivation than the scaffolds with 0 and 1 wt.% of the nanoparticles. The quantity of cells on these scaffolds was also substantially higher than that of the reference sample (cover glass). Thus, it can be inferred that ceria nanoparticles embedded in a scaffold ensure an increase in the rate of mouse MSC proliferation. This confirms the stimulating action of the hybrid scaffolds in comparison with those with no ceria. No difference was observed between the substrates with high CeO 2 concentrations and the reference sample, at the early stages of cultivation (12 and 24 h), but longer cultivation periods demonstrated the well-defined stimulating effect of the former.
SEM images of the mouse MSC on the surface of BC film, with maximal content of CeO 2 nanoparticles on the 4th day of cultivation, are shown in Figure 7, and can be compared to those cultivated on bare BC and glass substrate. SEM images of the mouse MSC on the surface of BC film, with maximal content of CeO2 nanoparticles on the 4th day of cultivation, are shown in Figure 7, and can be compared to those cultivated on bare BC and glass substrate. Analysis of the SEM images reveals the developed surface microstructure of the scaffolds. Long-term cultivation (96 h) resulted in migration and proliferation activity of the mouse MSC on the hybrid CeO2-containing substrates. This is evident from the formation of more focal contacts, as well as from an increase in the protrusion area. The cell mono- Analysis of the SEM images reveals the developed surface microstructure of the scaffolds. Long-term cultivation (96 h) resulted in migration and proliferation activity of the mouse MSC on the hybrid CeO 2 -containing substrates. This is evident from the formation of more focal contacts, as well as from an increase in the protrusion area. The cell monolayer formed on the BC substrates with high CeO 2 concentrations (3-5 wt.%) had morphological features typical of actively dividing cells, these features being less pronounced for scaffolds containing 0 and 1 wt.% of ceria.
Quantitative analysis of the number of cells adhered to CeO 2 -containing scaffolds showed that all the scaffolds provided effective cell adhesion. The analysis of proliferative activity was carried out during four days of cultivation by resazurin test (Figure 8). After 48 h of cultivation, the CeO 2 -containing scaffolds provided a significant increase in the number of cells, which was confirmed by increased values of resazurin fluorescence. The results obtained show a dose-dependent effect of ceria-containing substrates on cell culture proliferation.  The molecular mechanisms of stimulation of MSCs proliferation in the presence of cerium dioxide nanoparticles are associated with the regulation of MSCs redox status in particular; nanoceria reduces the level of intracellular reactive oxygen species (ROS), and a high ROS concentration inhibits the rate of proliferation. Most likely, biological activity of nanoceria with regard to MSCs is connected to mixed cerium valence state in cerium dioxide nanoparticles [29]. For example, nanoceria has been shown to possess anti-inflammatory properties [47]; the treatment with nanoceria reduced both the level of IL-6 and inflammatory markers in the blood of mice in both in vitro and in vivo experiments [27]. Enhanced bone regeneration due to the activation the ERK signaling pathway by nanoceria has been also reported [28]. In our manuscript, using the PCR-RT, we confirm the contribution of cerium oxide to the acceleration of the MSCs proliferation through the regulation of genes associated with antioxidant enzymes (Figure 9a).
The polymerase chain reaction method (PCR-RT) was used to study the expression of gene clusters (panel of 93 genes) encoding antioxidant enzymes (the family of glutathione peroxidases and peroxiredoxins) and genes involved in the regulation of ROS methabolism (SOD-1, SOD-2, catalase, and thioredoxins, etc.). We also analyzed the genes The molecular mechanisms of stimulation of MSCs proliferation in the presence of cerium dioxide nanoparticles are associated with the regulation of MSCs redox status in particular; nanoceria reduces the level of intracellular reactive oxygen species (ROS), and a high ROS concentration inhibits the rate of proliferation. Most likely, biological activity of nanoceria with regard to MSCs is connected to mixed cerium valence state in cerium dioxide nanoparticles [29]. For example, nanoceria has been shown to possess anti-inflammatory properties [47]; the treatment with nanoceria reduced both the level of IL-6 and inflammatory markers in the blood of mice in both in vitro and in vivo experiments [27]. Enhanced bone regeneration due to the activation the ERK signaling pathway by nanoceria has been also reported [28]. In our manuscript, using the PCR-RT, we confirm the contribution of cerium oxide to the acceleration of the MSCs proliferation through the regulation of genes associated with antioxidant enzymes (Figure 9a).
The polymerase chain reaction method (PCR-RT) was used to study the expression of gene clusters (panel of 93 genes) encoding antioxidant enzymes (the family of glutathione peroxidases and peroxiredoxins) and genes involved in the regulation of ROS methabolism (SOD-1, SOD-2, catalase, and thioredoxins, etc.). We also analyzed the genes responsible for mitochondrial dysfunction, as this is the main cellular organoid involved in ROS formation. These genes are responsible for the activation of a number of cascade pathways, including apoptosis, necrosis, antioxidant system, differentiation, proliferative and migration activities, the repair system, and pro-inflammatory response. PCR-RT was used to study the cells cultured on substrates containing 0 and 5% CeO 2 (Figure 9). After 24 h of cultivation, activation was observed for peroxiredoxin groups, and genes PRDX1, NCF1, Nos2, Tro, Noxo1, Noxa1, AOX1, FTH1, Ngb, and Nqo1. After 96 h of cultivation, NCF1, Tro, Noxa1, FTH1, and Nqo1 also retained a high level of expression. According to PCA analysis (Figure 9b), ceria-containing substrates demonstrated a significant difference in gene expression pattern in comparison with ceria-free substrates. The CeO 2 -containing scaffolds significantly increased the integral index of the upregulation in the expression pattern of the selected genes, which indicates a positive effect of ceria-modified scaffolds for the metabolic microenvironment and MSCs redox status. To summarize, it can be inferred that MSC proliferation proceeds more effectively on the scaffold with the highest CeO2 concentration used in the study, i.e., 5 wt.%. Such a scaffold is characterized by a water absorption value that is high enough to ensure nutrient exchange capacity, and that is sufficient to enable intense proliferation. It may be assumed that a further increase in ceria content would facilitate a stimulating activity of the To summarize, it can be inferred that MSC proliferation proceeds more effectively on the scaffold with the highest CeO 2 concentration used in the study, i.e., 5 wt.%. Such a scaffold is characterized by a water absorption value that is high enough to ensure nutrient exchange capacity, and that is sufficient to enable intense proliferation. It may be assumed that a further increase in ceria content would facilitate a stimulating activity of the scaffold, its high biodegradability, and the maintenance of cytoneutrality.

Conclusions
A method of fabrication of BC-CeO 2 composite films has been proposed and implemented to prepare biologically active materials capable of enduring mechanical stress. A combination of XRD and SEM analyses corroborated the uniform distribution of the nanoparticles in the BC matrix. Simultaneous TGA and DTA revealed the pronounced catalytic activity of ceria regarding the thermo-oxidative destruction processes in BCbased nanocomposites.
The materials obtained exhibited a limited ability to swell in an aqueous medium, the addition of ceria to the BC matrix causing a decrease in the equilibrium concentration of water in the swollen films. The water content changed from 325 to 210% of a dry sample's mass with a change from the bare BC to the film with 5 wt.% of ceria.
CeO 2 nanoparticles imparted BC films with enhanced stiffness, the maximum value of the latter being observed in the nanocomposite with 5 wt.% of the filler (27% higher than that of the unfilled BC film). Such an increase in stiffness of the material, along with a decrease in its ability to swell, points to the additional intermolecular interactions via the active surfaces of CeO 2 nanoparticles. The swollen films have a stiffness that is equal to 0.03-0.04 of that of the dry films, the elastic moduli and strength values being 170-300 MPa and 4-6 MPa, respectively. Such characteristics are similar to those of a number of human tissues, such as muscles, cartilages, and ligaments [48].
It was concluded, after examination of the nanocomposite materials regarding their use as bioresorbable scaffolds for the mouse MSC proliferation, that ceria positively affected the cells' attachment to a substrate, followed by their growth. The ceria-containing scaffolds possess a high degree of adhesion with regard to the stem cells via the formation of the focal contact of cell filopodia on their surface. The scaffold with the highest CeO 2 concentration (5 wt.%) demonstrated the best cell viability after 96 h of cultivation. A cell monolayer formed on this substrate had morphological features typical of an actively dividing culture. The number of cells on the composite substrates was considerably higher than that on a reference sample (glass substrate). Thus, it can be inferred that the stem cells proliferate faster in the presence of CeO 2 nanoparticles. This confirms the stimulating effect of the hybrid substrates compared to the bare BC. The nanocomposite sample with 5 wt.% of ceria was characterized by a water absorption value that is considered to be high enough to provide the good nutrient exchange capacity needed for intensive cell proliferation. BC-based composites containing 5% CeO 2 provided upregulation of the main clusters of genes responsible for proliferation, migration, metabolism of reactive oxygen species, and downregulation of proapoptotic, proinflammatory and mitochondrial dysfunction genes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/polym13121999/s1, Table S1: Selected gene groups for PCR-RT analysis.  Data Availability Statement: Data sharing not applicable. No previously reported data were analyzed in this study. Data sharing is not applicable to this article.