“Revitalizing” Alginate Films: Control of Texture, Hemo- and Cellular Compatibility via Addition of Cellulose Nanocrystals

Abstract

The multifactorial modification of the structure and properties of alginate matrix was conducted using partially acetylated cellulose nanocrystals. Fourier-transform infrared spectroscopy and thermogravimetric analysis indicated the absence of chemical interactions between the polymer matrix and the filler. The surface texture was examined using optical microscopy and scanning electron microscopy, along with a reconstruction of its 3D model. With an increase in the content of nanoparticles in the composite, the following was revealed. Firstly, the roughness and density of the arrangement of surface elements increased, while their size decreased. Secondly, at pH values < 7, the puncture resistance increased, whereas the swelling coefficient of the films decreased. In Hanks solutions, the low solubility of the films was established, as well as a higher swelling coefficient at pH > 7. Thirdly, the contribution of donor centers to the free surface energy, cytocompatibility of composite films, and adhesion of fibroblasts to the surface increased. The hematological tests of the composites showed a procoagulant effect. Summarizing the data, we propose a model that explains the influence of nanocrystals and their concentration on the formation of the observed composites’ structure and their physicochemical and biological properties. The main driving forces of structurization are the factor of the excluded volume and interactions in a heterogeneous colloidal system.

1. Introduction

Crosslinked polysaccharide gels, in the form of films, are elastic, hydrophilic, and biocompatible materials that are widely utilized in the biomedical and pharmaceutical fields. The modification of these hydrogel films with various nanoparticles to impart new characteristics and properties is a notable recent trend, which expands their applications in medicine [1,2].

Currently, a new paradigm in tissue restoration is gaining popularity, utilizing biomaterials of both artificial and natural origin that are capable of adhesion and supporting cell growth. Fibroblasts play a crucial role in the restoration of the extracellular matrix in the skin and soft tissues. The fibroblast response is influenced by the design of the biopolymer material, including the size and number of pores [3], hydrophobicity, and the nature and density of spatial distribution of chemical functional groups [4], as well as the mechanical properties of the biomaterial [5]. In addition to their excellent biocompatibility, the ability of polysaccharide gels to induce fibroblast cellular responses is of significant interest in the fields of regenerative medicine, tissue engineering, and cell engineering.

An aspect inextricably linked to the regulation of cellular functions is the hemocompatibility of polysaccharide gels intended for biomedical applications—a crucial property for materials that come into contact with blood [6]. From this perspective, the negative implications for medical practice include the potential to activate blood clotting, induce thrombosis, and promote the hemolysis of erythrocytes. The evaluation of hemocompatibility involves studying the effects of the material on the clotting of whole blood or plasma through in vitro experiments utilizing various coagulation tests [7].

Alginate gels, both in their pure form and when filled with nanoparticles, have found various applications in biomedical research and cell technologies. These hydrogels exhibit chemical resistance, biocompatibility, biodegradability, and pH sensitivity. However, they are also characterized by a low mechanical strength, high fragility, and inertness towards cells, necessitating modifications to promote adhesion and support cell growth [8,9,10]. These limitations hinder the advancement of such materials.

Composites of nanoparticles with alginate are intriguing because varying the component ratios can influence the rheological properties of gels, as well as regulate their mechanical properties and biocompatibility. The impact of nanoparticles in composite films on the life cycle of fibroblasts and hemocompatibility is of significant scientific and practical interest for selecting options and developing novel types of polysaccharide gel films. Currently, this field has considerable gaps in knowledge, although individual components have been studied with the necessary depth.

The choice of nanoparticle fillers for alginate matrices is extensive; however, polysaccharide nanocrystals offer several advantages in this application:

• Biocompatibility and biodegradability enhance the potential of filled gels for integration with living systems;
• The chemical and functional similarities with the filled alginate gel, along with the presence of a large number of hydroxyl groups, enhance the embedding of particles into the matrix;
• The anisotropic morphology of the most common nanocrystals presents additional opportunities for regulating the rheology of gels, as well as the orientation of particles and agglomerates. This may contribute to the development of materials with anisotropic properties, such as enhanced mechanical characteristics;
• The high intrinsic mechanical strength of nanocrystals.

Cellulose nanocrystals (CNCs) and chitin nanocrystals meet the criteria for biocompatibility and biodegradability, and these can range from satisfactory to good depending on the functional structure of the surface and morphology in various systems and models. CNCs are generally significantly less toxic compared to other anisotropic particles [11,12]. A notable characteristic of CNC as a filler in alginate matrices is its negative surface charge, which facilitates mixing and promotes a more uniform distribution of particles throughout the volume of the alginate gels. Numerous practical applications and novel materials based on CNCs have been proposed, including hydrogels, films, composites, and biocompatible emulsions [13,14,15]. Gels containing particles have been successfully utilized for the immobilization of mammalian cells. The incorporation of cellulose nanoparticles represents a promising approach for the targeted modification of the topography, morphology, and physical properties of alginate materials, thereby regulating the functional activity and differentiation of dermal fibroblasts.

The overwhelming majority of existing studies on CNC composites incorporating alginate have utilized cellulose nanocrystals with a sulfated surface. Concurrently, the surface structure and morphology of nanoparticles significantly influence both their properties and the characteristics of the resulting composites. The authors of [2] concluded that at a particle concentration of ≤3 wt.%, CNCs with an average length-to-diameter ratio (L/D) of 37.58 ± 20.37 and the lowest surface charge density (a zeta potential of approximately −20 mV) among the tested types of CNCs exhibited the most substantial increase in the Young’s modulus and tensile strength of the dry alginate composite film. Conversely, the incorporation of CNCs with the highest L/D ratio of 55.27 ± 26.54 (a zeta potential of approximately −50 mV) resulted in a significantly greater enhancement in the water resistance, water contact angle, and glass transition temperature of the film. This improvement may be attributed to an increase in the number of new hydrogen bonds formed between the CNCs and the alginate matrix, which can reduce the interaction between alginate and water.

Gel composites made from various polymers, combined with cellulose nanocrystals, demonstrate significant potential for the immobilization of mammalian cells [16]. Gelatin composites reinforced with carbonyl-modified CNCs enhanced adhesion and proliferation in three cell lines, including fibroblasts [17]. A recent study [18] investigated a poly (N-isopropylacrylamide) composite containing partially carboxylated CNCs for its compatibility with fibroblasts. The results indicated that a hydrogel composed of 0.25 wt.% PNIPAAm and 0.5 wt.% CNCs exhibited superior compatibility with fibroblasts compared to CNCs alone. Furthermore, the incorporation of CNCs into alginate-based gels increased the resistance to rapid degradation in biological environments and enhanced fibroblast infiltration upon contact with the hydrogel [19].

A synergistic effect in the polymer–CNC system has been demonstrated across various systems. In this context, the polymer, such as alginate, not only serves a structural role but also mitigates some of the negative aspects associated with nanocrystals, particularly their poor film-forming properties. Conversely, cellulose nanocrystals significantly enhance the physical and physicochemical properties of the polymer matrix, while simultaneously promoting the fibroblast cell cycle.

To date, at least two significant issues in scientific research have been identified regarding nanoparticle-filled polysaccharide gels as matrices for the fibroblast life cycle. Both of these issues stem from the overarching thesis that the structure of the film serves as a signal for cells at various levels, including the chemical composition, functional characteristics, and nanoscale morphology of the gel components. For instance, alginates exhibit different mannuronic to guluronic (M/G) link ratios, while CNC particles differ in their functional group composition, morphology, and hydrophilic–lipophilic balance (HLB). The topography of the adhesive surface, the presence and size of pores, and the mechanical properties of the matrix are largely influenced by the nanoparticle fillers. These factors act as signals for fibroblasts, as well as for other cells and blood components, significantly influencing their responses and prompting changes in their functional activity. Optimal parameters facilitate normal adhesion and support the complete life cycle of cells, ensuring hemocompatibility and effective tissue regeneration. Conversely, unsuitable parameters can lead to inadequate adhesion and negatively impact the subsequent regeneration process following the implantation of the material, primarily due to alterations in local concentrations of cytokines and growth factors induced by the specific structure.

This study focuses on the multifactorial modification of the structure and properties of an alginate matrix through the incorporation of cellulose nanoparticles. The aim is to impart new physicochemical and biofunctional properties to the matrix. The primary focus of this work lies in developing film materials with wound-healing capabilities, a low hemotoxicity, and hemostatic properties, including medical biomimetic implants that regulate the functional activity and differentiation of fibroblasts.

The innovative aspect of this approach lies in the use of partially acetylated CNCs (paCNCs), which possess many characteristics similar to those of other nanosized cellulose materials, while also exhibiting a distinct HLB due to the presence of acetate groups on their surface.

The complexity of this approach involves evaluating the effects of paCNC additives on their surface microtopology and physicochemical properties, as well as assessing how modifications to the composite structure influence cell viability, fibroblast adhesion, and the hemocompatibility of composite film materials.

2. Materials and Methods

2.1. Materials

Alginic acid sodium salt and hydrogen peroxide (35 wt.%) were purchased from Alfa Aesar. The molecular weight (Mw) of the alginic acid was determined by gel permeation chromatography and was equal to 4.1 × 10⁵ g/mol. The ratio of 1,4-linked residues of β-D-mannuronic acid (M structural units) and α-L-guluronic acid (G structural units) was determined by 13C NMR spectroscopy. Signals related to the C4 atoms of M and G units were used for the calculation [20,21]. The obtained M/G ratio for the used sodium alginate was 1.67 ± 0.17. The detailed procedures of the NMR and gel permeation chromatography are given in the Supplementary Materials.

CaCl₂∙6H₂O was acquired from Reakor (Moscow, Russia). Phosphotungstic acid, acetic acid, octanol-1, ethanol, and trisodium citrate (Na₃C₆H₆O₇) were purchased from Vekton (Saint Petersburg, Russia). Deionized water was used in the experiment.

Biological assay. Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), and Hanks’ Balanced Salt Solution (HBSS) were obtained from Gibco (New York, USA). Trypsin-EDTA (tr-EDTA) and phosphate buffer solution (PBS) were acquired from StemCell (Massachusetts, USA). Sodium dodecyl sulfate (SDS), MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Thiazole Blue), 4′,6-Diamidino-2-phenylindole dihydrochloride, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), and rhodamine were obtained from Sigma. Potassium chloride, potassium hydroxide, and ethyl alcohol undenatured (95%) were obtained from Biolot (Saint Petersburg, Russia) and used in the study. Penicillin–streptomycin (PS) was purchased from PanEco (Moscow, Russia).

paCNC preparation. paCNCs were isolated from cotton cellulose by previously described methods, with simultaneous solvolysis and acetylation in situ [22]. Briefly, cotton cellulose was solvolyzed in the acetic acid/octanol-1/0.25 mol% phosphotungstic acid system in the presence of hydrogen peroxide. The solvent ratio of acetic acid/octanol-1 was (10:1 v/v) per part by mass of cellulose. Solvolysis was carried out for 60 min at the boiling temperature of the mixture (115 °C). The reaction mixture was separated by centrifugation, and the precipitate was washed with ethyl alcohol and finally purified by dialysis (CelluSep 7000 kDa membranes). The concentration of particles in the resulting sol was 2–3 wt.% (gravimetric determination).

2.2. Methods

Polysaccharide solution and film preparation. The Alg-Na solution was prepared by completely dissolving alginate in water heated to 70 °C with vigorous stirring for approximately 4 h. To obtain alginate films without nanoparticles (AlgF), a 2 wt.% aqueous solution of Alg-Na was used. For the preparation of composite films, Alg-Na was dissolved in a calculated volume of water, accounting for its additional amount contained in the paCNC hydrosol, ensuring that the final concentration of Alg-Na in all solutions remained at 2 wt.%. The calculated mass of paCNC particles (1, 5, 10, and 30 wt.% of the mass of Alg-Na) was added as a hydrosol after the complete dissolution of Alg-Na, also at 70 °C with rapid stirring. The resulting solutions were stirred for an additional 60 min to cool them to 40 °C, after which they were poured directly into polypropylene Petri dishes. The volume of the solution was carefully measured using electronic scales to ensure that the mass of dry Alg-Na was 52 mg for an area of 10 cm² in each individual dish. The resulting Alg/paCNC gels were dried at 40 ± 1 °C in a convection drying oven until reaching a humidity of 5 ± 2%. Subsequently, to crosslink and impart water insolubility to the gel, the films, which were easily separated from the Petri dishes, were immersed for 60 min in an 8 wt.% aqueous solution of CaCl₂ to ensure the complete progression of the ion-exchange reaction in composites with varying swelling capacities and to maintain the reproducibility of the films’ properties. Afterward, the films were washed in water to remove excess CaCl₂, with the wash water monitored for Ca²⁺ content through conductometric analysis. The resulting crosslinked gels were placed on a polypropylene surface and dried at 40 ± 1 °C in a convection drying oven until achieving a humidity of 4 ± 1%. The thickness of the films was measured using a micrometer, yielding an average thickness of 60 ± 15 μm. These values were also confirmed by cross-sectional SEM images (Figure S3). The resulting films were designated as AlgCNC-xx, where xx is the percentage content of paCNC relative to alginate—for example, AlgCNC-10.

An analysis of paCNC surface ester groups content was determined by conductometric titration (conductometer “Expert-002”, Econiks-Expert, Moscow, Russia). Hydrosol containing 100 mg of CNCs (5–7 mL) and 2 mL of a 0.2 M NaOH aqueous solution was stirred at 60 °C for 24 h to saponify the acetyl groups. The mixture was then diluted to 20 mL with deionized water and further acidified with 6 mL HCl (0.1 M). The acidified suspension was titrated with NaOH (0.01 M). The volume of titrant used to neutralize the free acetic acid corresponded to the linear region where the conductivity of the solution remained unchanged. The degree of substitution (acetoxy groups relative to the total mass of the cellulosic material) was calculated based on the acetic acid content:

$$\mathrm{DS}={\displaystyle \frac{162\mathsf{\omega }\left(\mathrm{A}\mathrm{c}\right)100}{24300-43\mathsf{\omega }\left(\mathrm{A}\mathrm{c}\right)}}$$

(1)

where DS—degree of acetylation and ω(Ac)—acetoxy groups in the sample, wt.%.

A quantitative analysis of the aldehyde groups in the CNCs was determined by the titrimetric method based on the condensation of hydroxylamine (NH₂OH‧HCl) in the presence of triethylamine (TEA) with carbonyl groups.

Detailed information about zeta potential measurements, XRD, atomic force microscopy (AFM), transmission electron microscopy (TEM), FTIR and UV–Vis spectra, scanning electron microscopy (SEM), synchronous thermal analysis, swelling, and solubility is given in the Supplementary Materials.

Optical microphotographs of the film surface were obtained using an Olympus BX53M microscope equipped with a 50× objective and a 20 MP camera. The 3D relief model was reconstructed using the original software based on a multifocal image stack, with a step size of 0.8 μm along the Z-axis and a nominal spatial resolution of 0.1 μm/pixel. The cloud of generated points was transformed into a 3D surface model using CloudCompare v.2.11.1 software, which involved creating a grid with a step size of 1 μm. Smoothing of the resulting surface was performed using MeshLab v.2023.12d software, employing the Laplace smoothing method. Subsequently, the original image was applied as a texture to the generated surface using Rhinoceros v.6.36 software. Xerogels for optical microscopy (OM) were prepared from standard Alg-Na solutions, with the addition of a specific amount of paCNC sols. A thin layer of the solution was applied to a glass substrate for micropreparations, dried in a convection oven at 40 °C, and then immersed in an 8 wt.% aqueous solution of CaCl₂ for 10 min. Afterward, the sample was washed in water to remove excess salts and dried for 2 h at 40 °C.

Mechanical properties and puncture strength measurements were conducted following the methodology outlined in study [23] using a TA.XTplusC Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK). Film samples were immersed in buffer solutions with pH levels of 5.5 and 8.0 at 25 °C for 4 h. The thickness of the films with dimensions of 20 × 20 mm was assessed using a micrometer with an accuracy of 0.01 mm. Subsequently, the films were clamped between two plates with a hole of 10 mm in diameter. Puncture testing was performed using a cylindrical probe with a diameter of 2 mm at a speed of 60 mm/min. All measurements were repeated at least five times to ensure accuracy.

The determination of the contact angle of wetting for test liquids was conducted using the Phoenix MT(M.A.T.) (Surface Electro Optics, Gyeonggi-do, Republic of Korea) instrument complex, employing the sessile drop method. More detailed information is given in the Supplementary Materials.

2.3. Biological Assay

In this work, we performed a comprehensive biological study of our materials. These tests included morphometric characteristics of the cells, cytotoxicity, cell adhesion to alginate-based films, hemocompatibility, a blood recalcification time (BRT) test, activated partial thromboplastin time (APTT), and the hemolysis of erythrocytes. A detailed description of these procedures is given in the Supplementary Materials.

3. Results

3.1. CNC Characterization

CNCs are typically produced by treating cellulose with sulfuric acid, resulting in partially sulfated nanocrystals. This treatment shifts their HLB towards hydrophilicity. Acetylated CNC possesses additional hydrophobic domains, which is confirmed by a more effective stabilization of the water–oil interface in emulsions [24]. The method we used for obtaining paCNCs involves a one-pot acetylation process combined with controlled solvolysis, eliminating the drying stage that can lead to irreversible particle aggregation. Partial acetylation does not significantly affect colloidal stability. The resulting particles exhibit a rod-shaped morphology and a high degree of ordering, with a crystallinity index of 0.90 ± 0.02 (Figure S1). A statistical analysis of AFM and TEM micrographs indicates that the starting cotton cellulose used for isolating paCNCs yields particles with a relatively small length (L)/thickness (W) ratio, where L = 170 ± 35 nm and W = 8.5 ± 2.0 nm. The degree of acetylation in the paCNC samples throughout the entire volume of the particles, including internal zones, is 13.2 per 100 anhydroglucose units. The content of acetate groups present solely on the particle surface can be estimated based on the data presented in work [25]. In that study, calculations were performed for cellulose nanocrystals with a similar morphology and geometric dimensions; the increasing coefficient for the modification of groups only on the surface of the particles was estimated to be 1.5. Thus, the DS (surface) can be estimated at 20 acetate groups per 100 anhydroglucose units. The amount of carbonyl groups, primarily terminal ones, localized on the reducing ends of cellulose chains is 1.7 ± 0.3 wt.%.

3.2. Structure and Physicochemical Properties of Composite Films

3.2.1. FTIR and UV–Vis Spectroscopy

The FTIR spectra of alginate films without nanoparticles (AlgF), lyophilized paCNCs, and composite AlgCNC films are presented in Figure 1a. The spectrum of AlgF exhibits several characteristic bands, including a maximum at 1612 cm⁻¹, which is associated with the antisymmetric vibrations of the carboxylate anion. Additionally, there is a band at 1421 cm⁻¹ corresponding to the symmetric vibrations of the carboxylate anion, along with bands with maxima at 1300 cm⁻¹, 1100 cm⁻¹, and 1037 cm⁻¹, which are attributed to the deformation vibrations of C–C–H, C–O–C, and the stretching vibrations of pyranose rings, respectively. The group of signals characteristic of polysaccharides in the region of 950–812 cm⁻¹, which includes oscillations of the C–O bond and deformation vibrations of the C1–H anomeric centers of the polysaccharides, indicates the presence of residues of mannuronic and α-L-guluronic acids [26,27,28]. Furthermore, the FTIR spectrum of the pure alginate film contains a broad absorption band with a maximum at 3362 cm⁻¹, corresponding to the stretching vibrations of the O−H bond, as well as bands at 2930 cm⁻¹, which are associated with the asymmetric and symmetric stretching vibrations of the C−H bond.

The absorption peaks observed in the paCNC spectrum (Table S2) are primarily characteristic of cellulose materials [29,30]. A notable feature of the FTIR spectra for this type of cellulose nanoparticle is the presence of absorption bands with maxima at 1730 cm⁻¹ and 1249 cm⁻¹. These signals can be attributed to the stretching vibrations of the C-O bond in the ester group [31,32], which result from the in situ esterification of cellulose with acetic acid residues during cellulose solvolysis.

The nature of the spectra of the AlgCNC composite films generally corresponds to that of the spectrum of the main component (alginic acid) detected in the AlgF sample (Figure 1a). Notably, no significant contributions from the absorption bands of cellulose were detected, even in highly filled samples. The absence of new absorption bands or shifts in characteristic regions in the FTIR spectrum was noted upon the introduction of paCNCs, which may indicate the absence of ionic interactions between the alginate matrix and the paCNCs. The primary distinction between the spectra of composite films containing nanoparticles and those of AlgF and paCNCs is the pronounced tendency for the maximum of the 3600–3200 cm⁻¹ band (λ_max) to shift to longer wavelengths as the paCNC content in the film increases: AlgF at 3358 cm⁻¹; AlgCNC-1 at 3354 cm⁻¹ (∆ = 4 cm⁻¹); AlgCNC-5 at 3342 cm⁻¹ (∆ = 16 cm⁻¹); AlgCNC-10 at 3340 cm⁻¹ (∆ = 18 cm⁻¹); and AlgCNC-30 at 3338 cm⁻¹ (∆ = 20 cm⁻¹). The observed “red shift” indicates an extension [33] and a “loosening” of the network of intermolecular hydrogen bonds within the alginate matrix upon the addition of paCNCs. This suggests a change in the number and arrangement of interactions among the hydroxyl groups of the alginic acid, with reduced involvement of carboxyl groups. The weakening of intra- and intermolecular hydrogen bonds due to the presence of paCNCs results in an increase in the bond length of the OH groups. According to the data presented, the most distinct shift of λ_max was observed in the sample containing 5 wt.% paCNCs, whereas a further increase in the particle content shifted the band further only slightly. Therefore, it can be concluded that at this concentration, the primary deformation of bonds within the AlgCNC matrix occurs, and further additions of nanoparticles lead to a less pronounced weakening of the hydrogen bond network.

The UV–Vis transmission spectra of the AlgF and AlgCNC alginate films are presented in Figure 1b. The spectra of all the samples exhibit a shoulder at 250 nm, likely due to the presence of carbonyl groups in the alginate. Films containing 1 and 5 wt.% of paCNCs demonstrated greater optical transparency compared to the unfilled alginate film. This enhancement can be attributed to the structuring of the film upon the introduction of a small quantity of nanoparticles, resulting in a more homogeneous structure with fewer optical defects that facilitate light scattering. However, increasing the paCNC content in the films to 10 wt.% and 30 wt.% resulted in a decrease in transparency. This reduction may be attributed to the formation of larger aggregated structures that increase surface roughness and introduce additional defects within the film structure. Furthermore, as the concentration of particles increases, light scattering on these particles also increases, further diminishing the films’ transparency.

3.2.2. Surface and Microstructure of Films

Surface relief is of primary importance during the cell attachment stage. Figure 2a–c illustrate the generated textures of swollen hydrogel films filled with paCNCs according to optical microscopy data. Due to the lack of significant height variations on the film surface and issues with image focusing, the texture image of AlgF and AlgCNC-1 could not be obtained.

The results indicate that AlgCNC-5 films displayed the largest height difference, measuring up to 3.6 μm. In contrast, AlgCNC-10 samples had a smoother surface with a height difference of up to 1 μm. AlgCNC-30 films exhibited a smaller height difference, ranging from 2 to 2.4 μm.

The most significant feature of films with the maximum content of nanoparticles was the increased heterogeneity and roughness of their surfaces. Specifically, these films exhibited a greater number of height variations per unit area, significantly smaller dimensions, and a higher frequency of element repetition. For instance, a standard relief element (either a rise or a depression) in AlgCNC-5 and AlgCNC-10 films measured approximately 12–20 μm, with a similar distance between individual elements. In contrast, the highly filled AlgCNC-30 film displayed relief elements measuring 4–10 μm, accompanied by a high density of their arrangement.

The analysis of scanning electron and optical micrographs (Figure 3) revealed an increase in morphological differences and the emergence of new structures as the nanoparticle content in the composites increased. Samples without nanoparticles exhibited the presence of globular structures approximately one micrometer or smaller in size on the surface of the films (Figure 3a). The introduction of 1 wt.% of paCNCs altered the nature of the surface inhomogeneities; their size decreased to around 0.5 μm, and their shape became oblong (Figure 3b). The surface of the films appeared more uniform, with a reduced roughness. Films containing 5 wt.% paCNCs displayed a dense structure with a different type of inhomogeneity, attributed to the incorporation of paCNCs into the polymer matrix (Figure 3c). As the nanoparticle content continued to increase, the films became rougher and more uneven (Figure 3d,e), corroborating the findings from the surface texture analysis of swollen films conducted using optical microscopy and software methods (Figure 2).

The comparison of SEM and OM data (Figure 3d,e) leads to the conclusion that there is an increase in the number of light-scattering centers with increasing concentration of paCNCs. Overall, the results from microscopy, surface texture analysis, and UV spectroscopy correlate well with one another. The homogeneous structure of the films at a low paCNC content (1 and 5 wt.%) enhances transparency, while the rough surface with a heterogeneous structure at higher paCNC concentrations (10 and 30 wt.%) results in increased opacity. Imaging of the films in transmitted light also reveals a gradual increase in heterogeneities in the samples as the paCNC content increases (see Figure S2 in the Supplementary Materials).

SEM imaging of the films in BSE mode showed the absence of impurity phases in the samples (Figure S2).

3.2.3. Synchronous Thermal Analysis of Films

The change in sample mass with increasing temperature (Figure 4a) for all the samples can be divided into three distinct sections: 25–60 °C (mass remains unchanged), 60–208 °C (uniform mass decrease, largely independent of the amount of paCNCs), and 208–300 °C (sharp mass decrease of samples, dependent on the degree of filling of paCNCs with alginate). Up to 208 °C, all mass losses are attributed to the removal of water molecules adsorbed in the polymer matrix. The gradual nature of the mass decrease, without abrupt changes, suggests a lack of free water molecules in the films. After 190 °C, the decomposition of glycosidic bonds in the cellulose component of the material, along with the decarboxylation, decarbonylation, and dehydration of alginate, occurred. The introduction of paCNCs resulted in a reduction in mass loss during thermal decomposition at 300 °C compared to the alginate films: 40 wt.% for AlgF and 34 wt.% for AlgCNC-30. Concurrently, the inflection points at which the intensive decomposition process commenced shifted to higher temperatures, from 208 °C for AlgF to 214 °C for both AlgCNC-10 and AlgCNC-30.

Figure 4b and Table S3 present the differential scanning calorimetry data. Within the temperature range of 25–60 °C, a mild exothermic effect was observed, which was associated with structural rearrangements in the alginate polymer matrix leading to the formation of more kinetically favorable supramolecular structures [34]. At temperatures of 130–131 °C, an endothermic effect characteristic of the alginate matrix was observed, which was defined as the glass transition temperature (T_g) [35]. Furthermore, the position of T_g was found to be independent of the paCNC content, indicating that the mobility of the polymer chain elements remained unchanged with the introduction of nanoparticles. This finding suggests the absence of chemical interactions between the acid-base centers of the polyelectrolyte molecules of the alginate and the cellulose nanocrystals. Additionally, the authors of [36] claim that the glass transition temperature is not detectable in samples containing free water, as the endothermic effect associated with the removal of free water overlaps any signal related to the glass transition. The results obtained corroborate the FTIR spectroscopy data, which indicate that in the studied films, water molecules interact with the electron donor centers (–COO⁻) in the alginate molecule and on the paCNC surface through electrostatic forces, such as hydrogen bonds.

After reaching 190 °C, a series of exothermic and endothermic processes were observed in AlgCNC films, including water evaporation, intermolecular interactions, melting (Table S3), and the decomposition of the CNC and alginate matrix. Consequently, it is challenging to identify the key factors influencing the thermal processes in the films within the temperature range of 190–250 °C. Generally, it can be noted that the introduction of paCNCs lacked an effect on the onset temperatures of the thermal decomposition and melting of the alginate matrix. The decrease in ΔH_m (Table S3) with an increase in the content of nanocrystals may be attributed to a reduction in the proportion of alginate in the sample. The DSC curve for AlgF at 206 °C exhibits an exothermic peak (Figure 2b), which aligns with previously published data for alginate [35]. This phenomenon can be associated with the fact that in alginate films, Ca²⁺ ions bind approximately 40% of the COONa groups [37], resulting in non-crosslinked sections of polymer chains that are more susceptible to thermal decomposition. The incorporation of cellulose nanocrystals organizes the polyelectrolyte molecules through hydrogen bonding, leading to the formation of more homogeneous structures.

Thus, the data from the synchronous thermal analysis indicate that the studied films lack water molecules that are unbound to the electron donor centers of alginate and paCNCs. The introduction of cellulose nanocrystals into the alginate matrix left the mobility of the polymer chains unaffected, due to the absence of a chemical interaction between them. Cellulose nanocrystals assemble polyelectrolyte molecules through hydrogen bonds, resulting in the formation of more homogeneous supramolecular structures.

3.2.4. Swelling, Solubility, and Mechanical Properties of Films

Swelling and Solubility of Films at Different pH Values

Graphs illustrating the dependence of the swelling coefficient and solubility of the studied systems on the paCNC content are shown in Figure 5.

Based on the data presented in Figure 5, groups can be identified that either exhibit statistically significant differences or lack such differences. In this study, a significance level of p < 0.050 was adopted to define statistical significance. Calculations were performed by comparing solubility and swelling coefficient values for the same composition in different media (aqueous vs. Hanks solutions) or between compositions with varying paCNC content in the same medium. A detailed description of the results of the statistical data processing is provided in the Supplementary Materials. Based on this statistical analysis of changes in the solubility and swelling ratio of alginate films under various conditions, it can be concluded that the swelling coefficient of pure alginate films was directly influenced by the pH of the system. Specifically, a higher pH in the Hanks solution correlated with an increased swelling coefficient, reaching a maximum of approximately 500% at pH 8.0. In contrast, the swelling coefficient of AlgF in pure water was significantly lower, at around 90%. The swelling behavior of AlgCNC films was predominantly pH-independent but was influenced by the nanocrystal concentration within the polymer matrix, with swelling generally decreasing as the paCNC content increased. An exception to this trend was observed in the AlgCNC-1 films, which exhibited a sharp increase in their swelling coefficient in both water and a solution with a pH of 5.5. For this particular composition, a 100% increase in the swelling coefficient was noted compared to the original alginate matrix. This anomaly contrasts with the overall trend of decreasing swelling coefficients. We attribute this phenomenon to the partial loosening of the alginate matrix structure following the incorporation of 1 wt.% of nanoparticles, which is also confirmed by cross-sectional SEM micrographs of the films (Figure S3). In solutions with pH > 7, no anomalies were observed; the swelling coefficient reached its maximum, likely due to the basic nature of these Hanks compositions, which enhanced the swelling of alginic acid.

In general, the data obtained correlate well with the results presented in [38,39], where the swelling coefficient of hydrophilic films increased in dilute solutions containing electrolytes. The presence of both singly and doubly charged cations and anions in Hanks solutions contributes to the so-called “salt effect”, causing more pronounced swelling compared to that observed in pure water. This phenomenon also accounts for the lower swelling coefficients observed in pure water across all compositions, as these effects are absent in that medium.

The low solubility of all types of the studied films in water was observed. AlgF films exhibited the highest solubility across all media, and in Hanks solutions, the solubility of alginate films was directly influenced by the pH of the system. The general trend indicated that the solubility of the films increased with rising pH levels. In AlgCNC composite films, even with a 1 wt.% of paCNCs, the solubility in Hanks solutions decreased, although it still depended on the pH of the system. However, further increases in paCNC content resulted in the solubility of the films becoming independent of the pH in the Hanks solutions.

In reference [40], it was demonstrated that the swelling coefficient and solubility of alginate films crosslinked with Ca²⁺ ions increased with rising pH levels in simulated physiological environments. The authors attribute this phenomenon to the enhanced ionization of carboxyl groups on the alginate surface at elevated pH values, which facilitates improved swelling and the dissolution of the alginate polymer matrix. In the case of pure alginate films, our data align completely with the conclusions of that study.

In the case of composite films, the overall decrease in solubility and swelling, as well as the gradual reduction in their dependence on pH, are directly related to the increased content of cellulose nanocrystals (CNCs) in the film, consistent with the findings in [41]. Simultaneously, cellulose nanocrystals remain stable under the specified conditions. The literature indicates [42] that for many types of CNCs, significant solubility and swelling occur only at extreme pH values (below 1 or above 13). In contrast, at pH values typical of physiological environments (pH 2–8), the solubility and swelling of CNC particles are negligibly low and do not significantly impact the water absorption of the polymer matrix.

Mechanical Properties of Films

Alginate and CNC are pH-sensitive polymers, as the degree of dissociation of their acid-base centers is dependent on the pH of the aqueous medium. For these polymeric materials, the degree of interparticle interaction, which affects their strength characteristics, can be regulated by altering the pH [43]. The literature indicates that for alginate, the pK_a (COOH) ranges from 3.5 to 4.1 [44,45], while for CNC, the pK_a (COOH) ranges from 3.9 to 4.1 [24]. A decrease in the pH of alginate solution below these pK_a values results in the formation of highly viscous systems due to the development of denser supramolecular structures, which occurs as the charge of the polyelectrolyte molecule diminishes [46]. Consequently, a puncture test of the films was conducted after isothermal holding at 25 °C in buffer solutions with pH values of 5.5 (where the active centers of alginate and CNC are protonated) and 8.0.

An increase in pH from 5.5 to 8.0 (Figure 6) for AlgF resulted in a partial disruption of the crosslinking arrangement of alginate units involving Ca²⁺ ions, as well as an increase in the number of dissociating groups within the polyelectrolyte molecule. This change also enhanced the repulsion between the polymer chains. Collectively, these effects contributed to the formation of more elastic supramolecular structures with improved resistance to deformation. Consequently, the puncture resistance of the AlgF increased from 0.50 to 0.74 MPa (Figure 6). The puncture resistance values of the AlgF are consistent with the literature data [23].

When negatively charged paCNCs are introduced into a solution of negatively charged polyelectrolyte molecules of Na alginate, new supramolecular structures are formed. In these structures, rod-shaped crystals serve as reinforcing agents without establishing interparticle interactions. The variation in strength characteristics relative to particle concentration indicates that for like-charged structures, there is a minimum value (1 wt.%) associated with a deficiency of reinforcing elements and an optimum value (10 wt.%) at which a balance is achieved between the electrostatic repulsion of the composite’s structural elements and the reinforcing effect of the rod-shaped crystals at pH 5.5. An increase in pH to 8.0 results in a higher degree of dissociation of the like-charged structures, disrupting this balance and causing the optimum to shift toward higher concentrations of paCNCs (AlgCNC-30). The increased mutual repulsion between paCNC and alginate polyelectrolyte molecules at elevated pH levels apparently leads to the formation of more elastic structures.

Thus, it has been established that in the case of the alginate/CNC system, the microstructure and associated strength characteristics can be regulated by both the ratio of the initial components and the pH of the medium.

3.2.5. Contact Angle

The values of the contact angles of wetting of droplets of diiodomethane, water, glycerol, ethylene glycol, and formamide on the surface of alginate and CNC/alginate composite films with different paCNC content are shown in

Table 1. Contact angle values of test liquids and surface free energy components (mJ/m²) for AlgF and AlgCNC composites.

Sample	Diiodo-Methane, °	Water, °	Glycerol, °	Ethylene Glycol, °	Formamide, °	γsLW	γs+	γs−
AlgF	33.5 ± 0.2	51.9 ± 0.4	49.6 ± 0.3	36.1 ± 0.4	36.8 ± 0.5	42.94	0.45	24.64
AlgCNC-1	34.2 ± 0.3	32.2 ± 0.4	56.9 ± 0.4	42.1 ± 0.5	42.1 ± 0.4	42.76	0.15	31.42
AlgCNC-5	33.0 ± 0.3	38.5 ± 0.4	59.9 ± 0.4	39.6 ± 0.4	35.7 ± 0.5	43.85	0.07	41.66
AlgCNC-10	32.2 ± 0.3	29.2 ± 0.4	67.2 ± 0.4	36.3 ± 0.4	28.4 ± 0.5	42.17	0.05	44.13
AlgCNC-30	30.1 ± 0.3	36.4 ± 0.4	50.4 ± 0.4	39.7 ± 0.4	38.2 ± 0.5	41.98	0.03	47.53

. The obtained values of γ_s^LW, γ_s⁺, and γ_s⁻ depending on the content of paCNC sin the films are also presented in Table 1. Figure 7 shows the graphs of the dependence of γ_s⁺ and γ_s⁻ on the content of paCNCs in the films.

The contact angle values for the wetting of droplets of diiodomethane, water, glycerol, ethylene glycol, and formamide on the surfaces of alginate and mixed films with varying concentrations of paCNCs are presented in Table 1. The corresponding values of γ_s^LW, γ_s⁺, and γ_s⁻, as a function of paCNC content in the films, are also included in Table 1. Figure 7 illustrates the relationship between γ_s⁺ and γ_s⁻ and the paCNC content in the films.

As illustrated in Table 1 and Figure 7, the non-polar component γ_s^LW values of the surfaces of pure alginate films (AlgF) and their composites with CNC (AlgCNC) are virtually unchanged with an increasing paCNC content. The observed pattern indicates the similar nature of the Lifshitz–van der Waals electrostatic interactions for both AlgF and AlgCNC composites. However, the donor and acceptor components of the free surface energy vary with increasing paCNC content in the material. As shown in the dependencies presented in Figure 7, the donor component increases, while the acceptor component decreases with a higher nanoparticle content. Chibowski and Perea-Carpio concluded that one of the key factors influencing the increase in the donor component is the presence of trace amounts of sorbed water on the film surface, as well as the presence of electron–donor functional groups on the surface, such as amino and hydroxyl groups [47]. The data obtained from solubility, film swelling, and thermal analysis indicate that the amount of residual moisture in the films decreases with increasing paCNC content but remains within the range of 8.5% to 12.6% for all the compositions. Consequently, the factor of moisture sorbed by the polymer composite plays a role in altering the donor and acceptor components of the free energy of the film surface. Additionally, it is also demonstrated that the presence of electron–acceptor functional groups on the surface, such as carboxyl and sulfate, increases the acceptor component of the surface free energy [47]. Thus, Ca²⁺–alginate contain electron–acceptor functional groups (carboxyl groups) on their surface, contributing to the acceptor component of the free surface free energy. Conversely, the presence of electron–donor functional groups on the paCNC surface (hydroxyl groups) enhances the donor component of the surface free energy.

As known from the literature, the donor component of the free surface energy of CNCs exhibits high values, while the acceptor component is low [48]. In contrast, alginate has a higher acceptor component of its free surface energy compared to cellulose-based materials [49]. These properties are attributed to the influence of functional groups (hydroxyl/carboxyl) on the polar components of the materials’ free surface energy. The decrease in the acceptor component with increasing paCNC content in mixed films can be explained by the fact that a higher paCNC content increases the contact area of nanoparticles with the external environment (Figure 3). This occurs not only due to the proportional increase in nanoparticle content within the matrix but also because of electrostatic repulsion between similarly charged alginate and paCNCs. This repulsion leads to greater enrichment of the film surface with CNCs and their aggregates.

Thus, the observed dependencies illustrate the influence of paCNCs and alginate on the donor and acceptor properties of the film surface. An increase in paCNC content results in an increase in the donor component and a decrease in the acceptor component. When the paCNC content exceeds 30%, the acceptor component approaches near-zero values, while the donor component reaches a plateau. This phenomenon minimizes the effect of alginate on the acceptor properties of the film. Based on the presented data, it can be concluded that further increasing the paCNC content in the film beyond 30% will lack any effect on the nature of surface donor–acceptor interactions.

3.2.6. Adhesion of Fibroblasts to Films and Its Components

Morphometric Characteristics and Growth Activity of Fibroblasts

In the control group, after 24 h of fibroblast culturing, more than 90% of the cells exhibited a spindle shape, with an average cell length, including filopodia, of 94 ± 18 μm (Figure 8a). These data provide evidence of normal cellular growth activity.

It was observed that after 24 h of culturing fibroblasts with AlgF, the cells exhibited spindle-shaped forms with a length of 71 ± 13 μm (Figure 8b). In the population of fibroblasts incubated for 24 h with AlgCNC-10, the length of the cells along the longest axis was 72 ± 11 μm (Figure 8c). In the population of fibroblasts incubated with AlgCNC-30, the cell length measured 81 ± 15 μm (Figure 8d). Thus, the morphometric characteristics of the cells indicate that AlgF, AlgCNC-10, and AlgCNC-30 did not affect cell growth activity after 24 h of co-incubation.

At the next stage of this study, we assessed the effects of the biomaterial specimens on the metabolic activity of fibroblasts. Cytotoxicity was evaluated based on cell viability in comparison to control samples. An activity level relative to the controls of less than 30% indicates severe cytotoxicity, between 30 and 60% indicates moderate cytotoxicity, between 60 and 90% indicates slight cytotoxicity, and greater than 90% indicates non-cytotoxicity [50,51].

Figure 9 presents the results of fibroblast MTT assays conducted after 24 and 48 h of incubation with AlgF, AlgCNC-10, and AlgCNC-30 samples. A reduction in cell viability of 30–40% compared to the control was observed for AlgF and AlgCNC-10 after 24 h of incubation. However, cell viability recovered after 48 h of incubation with AlgCNC-10, while it did not recover for AlgF. Notably, the AlgCNC-30 samples exhibited no cytotoxicity after both 24 and 48 h of incubation with fibroblasts. These results evaluated the metabolic activity of the cells and found that they had slight to no cytotoxicity.

Cell Adhesion to Hydrogel Biomaterials’ Surface

In the control group, after 6 h of incubation, fibroblasts efficiently adhered to the plastic surfaces. The number of cells that adhered to the plastic surface was 60 ± 9 cells per 0.2 mm² (Figure S3a). After 6 h of incubation, 7 ± 2 (Figure S3b), 13 ± 3 (Figure S3c), and 23 ± 4 fibroblasts (Figure S3d) were observed adhering to the same surface area of AlgF, AlgCNC-10, and AlgCNC-30 samples, respectively. Thus, it was determined that the surface properties of the hydrogels inhibited fibroblast adhesion. However, increasing the concentration of cellulose nanocrystals in the hydrogels up to 30 wt.% improved fibroblast adhesion.

It is known that alginate is composed of inert monomers that inherently lack the bioactive ligands necessary for cell anchoring [52,53]. Our results align with the existing literature, which indicates that cell adhesion on hydrogel surfaces is low. Various methods have been employed to enhance adhesion to alginate, including the integration of graphene oxide [54], modification with natural and synthetic peptides [55], regulation of zeta potentials and surface charges [56], and formation of composite hydrogels from two or more different polymers [57]. We found that the addition of cellulose nanocrystals to alginate hydrogels significantly increased cell adhesion to the hydrogel surfaces, which can be utilized to regulate the functional properties of hydrogel biomaterials.

Thus, a comparison of the biocompatibility of AlgF, AlgCNC-10, and AlgCNC-30 (Figure S3b–d) demonstrated that all the studied biomaterials did not affect cell growth activity after 24 h of co-incubation. The metabolic activity of the cells after 48 h of co-incubation showed only slight changes, indicating that these alginate hydrogels are considered to have slight to no cytotoxicity.

3.2.7. Hemocompatibility of Composite Films

In this study, two coagulation tests—BRT and APTT—along with an assessment of the effect on erythrocyte hemolysis, were employed to investigate the in vitro compatibility of films with specific blood components.

Blood coagulation in the presence of carbohydrate-containing materials can be evaluated using the BRT test [58]. In the present study, the incubation of blood with AlgF and AlgCNC films (S₁ = 0.5 cm × 0.5 cm = 0.25 cm² and S₂ = 0.75 cm × 0.75 cm = 0.56 cm²) for both 10 min and 20 min resulted in a significant reduction in coagulation time (p < 0.05) in the BRT test compared to the incubation of blood without samples (Figure 10). Doubling the area of the films led to a notable decrease in coagulation time during blood incubation for both 10 min and 20 min with the initial alginate AlgF and the AlgCNC-1 and AlgCNC-30 composites. For all the samples, including AlgCNC-5 and AlgCNC-10, a significant decrease in coagulation time—ranging from 3.1 to 7.2 times—was observed when the area was increased to S₂ and the incubation time extended to 20 min, compared to S₁ and a 10 min incubation. Blood incubation with AlgCNC-5 (S₁ or S₂) for 10 min resulted in a significant reduction in coagulation time, nearly two-fold, compared to the initial alginate. A similar effect was noted upon incubating blood for 20 min with films of a 0.25 cm² area; with an area of 0.56 cm², the effect was pronounced but not statistically significant.

The studied samples demonstrated an accelerated blood coagulation effect (procoagulant effect). This effect of the AlgCNC-5 film on S₁ (10 min and 20 min) and S₂ (10 min) was slightly, but significantly, more pronounced than that observed for the same parameters with AlgF.

Having documented the procoagulant effect of the films during incubation with blood containing all the cellular and plasma components, we decided to investigate the outcome of incubation with plasma devoid of platelets. Would this effect be replicated? Several authors have demonstrated that chitosan derivatives promote the aggregation of erythrocytes and platelets [59,60]. Therefore, it is possible that alginate films may not influence plasma coagulation.

Based on the fact that the films were incubated in 0.5 mL of blood for the BRT test, and considering the ratio of the volume of constituent elements to plasma (hematocrit), the optimal volume required for incubating films in plasma was calculated to be 0.28 mL.

The APTT test indicated that after 1 min of incubating films (S₁ and S₂) with plasma, no significant differences were observed compared to the control without film nor during the incubation with films based on the initial alginate (Figure 11). However, a significant reduction in coagulation time was noted when analyzing plasma samples after 5 min of incubation with films (S₁ and S₂). Incubation for 10 min with S₁ did not yield the same effect as the 5 min incubation with S₂. It was observed that after 5 min of plasma incubation with the films, the modified AlgCNC-1 ÷ 30 films demonstrated a greater reduction in clotting time (for S₁: 15.69 ± 4.74 s, 15.09 ± 3.14 s, 13.79 ± 1.96 s, 20.8 ± 4.34 s; for S₂: 4 ± 0 s, 5.4 ± 1.4 s, 4 ± 0 s, 4.85 ± 0.85 s) compared to the films based on the initial alginate (for S₁: 23.58 ± 4.41 s; for S₂: 18.69 ± 4.94 s).

Thus, the incubation of plasma with S₂ samples AlgCNC-1–AlgCNC-30, which contain cellulose nanocrystals, resulted in a procoagulant effect that was independent of the paCNC content. This effect was more pronounced than that observed after plasma incubation with the initial alginate.

Regardless of the intended effect on blood or plasma coagulation, materials should not provoke the destruction of erythrocytes [61]. This consideration applies to three categories of materials: (1) those designed to stop bleeding or exhibit procoagulant activity on their surface [62]; (2) those with anticoagulant activity on their surface [63]; and (3) those that do not affect blood or plasma coagulation [58].

Figure S5 illustrates the impact of the studied films on the OD of the supernatant (SN) obtained from the centrifugation of erythrocyte suspensions. The OD of the supernatant after incubating S₁ films with the erythrocyte suspension (OD-SN-S₁) reached values of 0.132 ± 0.03 relative units to 0.202 ± 0.053 relative units, which did not significantly differ from the OD of the control supernatant with PBS without film (0.202 ± 0.038 relative units). In contrast, the OD of the supernatant after incubation with the erythrocyte suspension of films S₂ (OD-SN-S2) AlgCNC-1, AlgCNC-10, and AlgCNC-30 was significantly higher than that of the same samples with a two-fold smaller area of S₁, measuring 0.207 ± 0.015 relative units, 0.237 ± 0.027 relative units, and 0.251 ± 0.016 relative units for S₂, compared to 0.132 ± 0.03 relative units, 0.140 ± 0.024 relative units, and 0.148 ± 0.024 relative units for S₁, respectively.

A slight yet significant increase in the optical density of the supernatant was observed with a two-fold increase in the size of films containing 10 and 30 wt.% paCNCs. The degree of hemolysis during the incubation of the erythrocyte suspension with S₁ and S₂ films was measured at 0.01 ± 0.01% to 1.12 ± 0.45% and 0.38 ± 0.38% to 1.55 ± 0.43%, respectively.

Polysaccharides are advantageous compounds for the development of biomaterials due to their availability, low immunogenicity, and biodegradability. Common chemical modifications of cellulose, chitosan, dextran, hyaluronan, and alginate, along with their related changes in biological activity, potential methods of functionalization, and possible biomedical applications, such as drug delivery systems and tissue engineering, have been previously described in [64,65,66].

Polymer biomaterials with universal properties are extensively utilized in medical practice and biological research. When the development of polymer biomaterials involves contact with blood, it is essential to first evaluate their in vitro hemocompatibility, which refers to their effects on the cellular and plasma components of blood [67]. This evaluation is crucial, because the interaction of any foreign material with blood can activate platelets, initiate a cascade of serine proteinases within the blood coagulation system, and lead to the formation of fibrin clots (the primary constituents of blood clots), as well as the destruction of erythrocytes (hemolysis) [61,68]. To assess the impact of these materials on blood and plasma coagulation, several tests are employed, including blood coagulation time, plasma coagulation time, prothrombin time, thrombin time, and activated partial thromboplastin time [60,69,70].

Alginate-based films are utilized in the development of non-toxic biomaterials [71,72]. The literature review in [73] provides a comprehensive examination of advancements in the creation of hydrogels derived from natural polysaccharides, including chitosan, alginate, hyaluronic acid, cellulose, and dextran, in conjunction with catechol extracted from mussels. The authors highlight several derivatives of chitosan and alginate that enhance blood coagulation in vitro.

In this study, we demonstrate that the incubation of blood (for 10 or 20 min) or plasma (for 1, 5, or 10 min) from individuals with the tested films results in a reduction in coagulation time in both the BRT and APTT tests, indicating a procoagulant effect. Its extent is influenced by the surface area of the films and the duration of their incubation in blood or plasma. Notably, the procoagulant effect observed during the incubation of plasma with AlgCNC-1–AlgCNC-30 films was more pronounced than that seen with films based on the initial alginate.

Li Z. et al. [74] have developed gels derived from chitosan that rapidly halt bleeding and demonstrate exceptional wound-healing properties. Salmasi, S. S. et al. [75] demonstrated the procoagulant activity of a membrane composed of kappa-carrageenan and carboxymethyl chitosan; in vitro incubation of citrate blood with this membrane resulted in coagulation.

Hemolysis, the destruction of the erythrocyte membrane, can occur when blood comes into contact with a foreign surface. Intravascular hemolysis may contribute to the formation of ADP-mediated blood clots [76].

In this study, it was demonstrated that the degree of hemolysis in erythrocyte suspensions, following incubation with the examined films, was independent of their surface area and remained below 2%. Warale D. et al. [77] developed nanocomposite films derived from alginate, which exhibited a degree of erythrocyte hemolysis ranging from 0.40 ± 0.02% to 4.20 ± 0.03%. Saleem A. et al. [71] observed a degree of hemolysis of less than 10% when films composed of alginate and okra hydrogel interacted with erythrocytes. Salmasi S. S. et al. [75] created a membrane utilizing kappa-carrageenan and carboxymethyl chitosan, which demonstrated a degree of erythrocyte hemolysis of less than 2%.

4. Discussion

A comparative study of the alginate composite film materials reveals significant changes in both their physicochemical properties and the nature of their interactions with fibroblasts and blood components, depending on the content of cellulose nanocrystals. In this section, we will present a model of the composite structure that aligns with the experimental observations.

The available data indicate the following general trends regarding the changes in the physicochemical, mechanical, and biological characteristics of swollen composite films as the content of paCNC increases:

(1)

A decrease in the swelling coefficient and solubility of films at pH levels below 7;

(2)

An increase in the mechanical strength of the swollen composite hydrogel matrix.

A notable characteristic of these two trends is the presence of extremes in films composed of AlgCNC-1, which contradicts the overall trend. Clearly, these extremes signify a pronounced difference in the materials’ structure, stemming from both the original alginate and composites with a higher content of paCNC. These differences are characterized by a loosening of the structure, resulting in a loss of mechanical strength and an increase in the swelling index;

(3)

A decrease in the acceptor component of free surface energy, accompanied by an increase in the donor component, is observed in composites;

(4)

An increase in fibroblast adhesion to the hydrogel surface, along with a general tendency to enhance the procoagulant effect of the films, is observed.

Changes in the composite structure, influenced by the concentration of paCNCs, are directly reflected in alterations to the surface texture. Furthermore, the texture of film materials is fundamentally important for their interactions with living cells and blood. Depending on the mass ratio of alginate to paCNC, three distinct types of surface textures are formed:

(1)

With a 0 and 1 wt.% paCNC content, the texture of the film in its swollen state is smooth and dense, exhibiting minimal optical inhomogeneities primarily determined by the alginate components;

(2)

At a 5 and 10 wt.% paCNC content, the surface texture of the films in the swollen state displays a limited number of relatively large, sparse elements, along with the emergence of relatively rare groups of optical inhomogeneities;

(3)

At a 30 wt.% paCNC content, a significant change in surface texture is observed. The surface texture of these films in the swollen state exhibits a higher density of smaller relief elements and a greater number of optical inhomogeneities. The SEM micrographs reveal spindle-shaped structures on the micrometer scale.

Comparing these findings allows us to confidently assert a correlation between optical inhomogeneities and relief elements. The three texture types also correlate well with changes in the physicochemical and biological characteristics of the composite films. Furthermore, based on the results obtained from Fourier-Transform Infrared Spectroscopy (FTIR) and thermogravimetric analysis (TGA), we draw a clear conclusion regarding the absence of significant chemical interactions between alginate macromolecules and paCNCs, as well as a weakening of hydrogen bonds. Both alginate and paCNCs possess a negative charge, which inhibits their proximity to one another. Their functional composition and analytical data indicate the likelihood of only weak non-covalent interactions, such as hydrogen bonds.

Other authors have noted a strong influence of CNC additives on the physicochemical and mechanical properties of alginate materials, as detailed in the introduction, which includes a review of the relevant literature in the field. Significant observations pertinent to the topic under discussion were made in study [78]. The authors suggest that phase formation and/or an increase in pore size occur with a higher content of nanocelluloses in the alginate matrix. Consequently, highly filled composite cryogels containing partially oxidized CNCs exhibit increased moisture absorption. Furthermore, the interaction among cellulose nanocrystals intensifies as their concentration in the gel rises, leading to poor particle dispersion and phase formation. This phenomenon results in a decrease in the values of the dynamic modulus (storage modulus). In direct contact tests, this type of cryogel demonstrates a moderate cytotoxic effect on fibroblasts; however, cell growth remains stable, and the cell population is higher than that observed in pure alginate gel. It has been shown that the addition of cellulose nanocrystals or nanofibers can enhance the attachment, spreading, and growth of fibroblasts on alginate gels. Despite the differences in the methods of preparation and forms of alginate composites, these findings exhibit significant similarities to the results of our work.

As a result of the conclusions drawn from this study, we propose a model for the structure of the composites developed herein, as well as the mechanisms of their formation based on the alginate/CNC ratio.

In the proposed scheme (Figure 12), the surface and interior of the composite are represented as two conventional planes. The upper plane corresponds to the texture plane, while the lower plane generalizes the interior. The initial alginate matrix, AlgF, possesses a unique structure formed by entanglements within a concentrated polymer solution. Certain sections of the polymer chains are coordinated by Ca²⁺ ions according to the “egg box” type. Such a conventional unit cell of the matrix is illustrated in the schemes below the planes. The introduction of 1 wt.% of negatively charged rod-shaped nanoparticles (AlgCNC-1) into the alginate matrix results in their dispersion, followed by the incorporation of paCNCs into the cell, which partially weakens the hydrogen bonds and disrupts the entanglement network of alginate macromolecules. The disintegration of the model unit cell is attributed to spatial factors. Firstly, there is the exclusion of polymer segments from particulate volumes, a phenomenon known as depletion interaction [79]. Secondly, the electrostatic repulsion forces between like-charged polymers and nanoparticles dominate over the adhesion forces, primarily hydrogen bonds. Under these conditions, the emergence of structural defects and partial destruction of the existing framework of the alginate matrix is not compensated for by the formation of a new, more stable framework due to the low concentration and isolation of paCNCs within the volume. The weakening of the structure, in turn, leads to a decline in the mechanical properties of the composite, an increase in swelling capacity due to reduced connectivity, and the enhanced mobility of the matrix elements.

An increase in the content of paCNCs (AlgCNC–5–10) leads to a critical concentration, which, in the presence of a polyelectrolyte, results in nanoparticle aggregation, flocculation, and the formation of “bulges”. The flocculated “bulge” contains both paCNC aggregates and, apparently, captured alginate macromolecules. The sorption of polymers onto the particles is excluded [80]. According to previous calculations using the DLVO method [81], cellulose nanocrystals within these formations exhibit a range of mutual orientations, from cross to parallel. It is the formation of “bulges” with dimensions of about 15 μm, containing thousands of particles, that accounts for the observed heterogeneities in the surface relief of films with this component ratio, as shown in the micrographs and calculated 3D relief models (Figure 2a,b and Figure 3c,d). Concurrently with the formation of “bulges”, thinner “bridges” of aggregated particles are created, extending throughout the composite structure and forming a new framework. The second process involves the compaction of alginate chains, as the excluded volume occupied by particles reduces the available volume for the non-sorbed polymer. The development of a new framework and the compaction of alginate macromolecules align well with the observed improvements in the mechanical properties of the modified films, as well as a decrease in their solubility and swelling coefficient. Furthermore, the positioning of “bulges” of flocculated paCNCs on the surface correlates with a decrease in the acceptor component and an increase in the donor component of the surface energy. The formation of phases within the “bulges” accounts for the increase in the number of optical inhomogeneities observed in the presented micrographs (Figure 3).

Finally, in highly filled AlgCNC-30 films, the formation of structure is primarily guided by paCNCs. The increase in the number of particles, the convergence of like-charged “bulges”, and the reduction in free space lead to the compression of the electric double layer of particles. The convergence of individual paCNCs within the “bulges” induces the compression of these structures, resulting in an increase in their density and a reduction in their average size to approximately 5 μm. Consequently, the observed film texture exhibits a frequent arrangement of smaller elements (Figure 2c). In the SEM micrographs (Figure 3e), “bulges” with a spindle-shaped morphology are clearly visible, while optical microscope images reveal an abundance of phase inhomogeneities. The latter is attributed not only to the large number of “bulges” but also to the higher density of paCNC swithin them. The closer arrangement and formation of a network of bridges between the “bulges” create a rigid framework, which accounts for the low mobility of the AlgCNC-30 elements, resulting in the increased fragility of the matrix and reduced swelling coefficients. The arrangement of the collapsed “bulges” on the film surface enhances the surface’s affinity for fibroblasts, thereby explaining their good adhesion. It can be inferred that this affinity is influenced by the rough surface, with densely located centers of a predominantly donor nature. Cell adhesion typically increases with a higher surface roughness [82]. An increase in the donor component (γ_s⁻) enhances fibroblast and other cell adhesion through the formation of hydrogen bond networks or increased protein adsorption, while the concurrent decrease in the acceptor component (γ_s⁺) reduces nonspecific interactions. These results align with findings from studies [83,84], which show that the most effective adhesion of living cells, particularly fibroblasts, occurs on test surfaces enriched with -NH₂ or -OH groups. These groups exhibit high values of the surface free energy donor parameter (γ⁻) in the acid-base component (γᴬᴮ).

The formation of such “bulges” from paCNCs on the surface correlates well with the increased ability of filled films to coagulate blood. Thus, in reference [11] it has been demonstrated that paCNCs possess procoagulant activity, which we attribute to the presence of aldehyde groups in their composition [22], as well as to the formation of additional hydrophobic domains on their surface. paCNCs, like all cellulose nanocrystals, have hydrophobic planes [85], but acetylation additionally increases their ability for hydrophobic interactions, which has previously been proven in the stabilization of emulsions [24]. It is known that these directions of the chemical modification of polysaccharide materials enhance procoagulant activity [86].

Thus, a model is proposed that effectively explains the structural and physicochemical changes in alginate matrices, as presented in this study and previous research, upon the addition of cellulose nanoparticles. This model also elucidates the effects observed upon contact with fibroblasts and blood cells. In the proposed model, paCNCs interact weakly with the alginate matrix through chemical means, while the primary forces influencing structural formation are those characteristic of like-charged mixed colloidal systems in the presence of electrolytes. These forces include the excluded volume effect, flocculation, and electrostatic interactions.

5. Conclusions

The incorporation of cellulose nanocrystals into alginate composites is an effective method for modulating the properties of the materials. Of particular interest is the ability to regulate biocompatibility with cells and living tissues.

The properties, structure, and texture of the formed films are highly dependent on the alginate/CNC ratio. It has been established that the introduction of 1 wt.% CNCs leads to a restructuring of the alginate matrix, a decrease in puncture strength, and an increase in the swelling coefficient; however, it has little effect on the texture parameters and interactions with fibroblasts and blood cells. In contrast, highly filled composites (30 wt.% CNCs) exhibit a rigid framework and a high affinity for fibroblasts. The cellulose nanocrystals are responsible for imparting new properties to the material. Within the composite, they form structures that influence its physicochemical and mechanical properties, while on the surface, they determine its biological properties, including procoagulant characteristics.

This set of methods demonstrates the absence of chemical interactions between the polymer and cellulose filler. The developed model of composite formation accounts for the differences in mechanisms caused by the concentration of CNC in the volume, as well as the electrostatic interactions between alginate and CNCs, and between CNC particles themselves. The flocculation of particles in the materials, beginning at 5 wt.%, along with a reduction in the volume available for occupation by alginate macromolecules, leads to the formation of various structures both within the material and on the surface of the films. The identified patterns hold practical significance for the development of new biomedical materials based on cellulose nanocrystals. The studied films may be promising for the creation of wound dressing materials that do not damage the erythrocyte membrane, exhibit procoagulant activity, and possess the ability to support the cell cycle of fibroblasts. The most predictable area of application is fibroblast-colonized wound film hydrogels. The resulting composites are of particular interest for creating coatings for purulent infected wounds due to their increased mechanical strength at pH 5.5.

It is possible that the increased adhesion of fibroblasts on the surface of alginate films filled with paCNCs is not the only functional reaction of cells. Additional research, for example in the area of assessing the differentiation of fibroblasts into a smooth muscle phenotype (myofibroblasts), will allow the creation of biomimetic material for regulating the functional activity of fibroblasts. These studies are particularly important for the treatment and prevention of fibrotic diseases or, conversely, for stimulating the formation of extracellular matrix proteins and the stimulation or regeneration of damaged tissues.

Overall, the films remain optically transparent in the visible spectrum, making them suitable for applications that require the visual monitoring of the processes occurring beneath them, such as wound healing, tissue repair, and food safety assessment.