Dual Modulation of Chitosan/Keratin Hydrogels by Pluronic F-68 and Hexamethylene Diisocyanate for Tunable Wound Healing Scaffolds

Abstract

This study investigates the complementary modulation of chitosan/keratin hydrogels through the combined use of hexamethylene diisocyanate (HDI) and Pluronic F-68, aiming to tailor mechanical stability, porosity, and biological performance for wound healing applications. Covalent crosslinking by HDI enhanced network integrity and mechanical resistance, while Pluronic F-68 promoted micelle-templated porosity, increased hydration, and improved permeability. The hydrogels exhibited compressive strength values ranging from 33.5 to 65.9 MPa, swelling capacities of up to approximately 160%, and porosity values approaching 60%, depending on composition. Increasing HDI content reduced solubility to approximately 15%, indicating improved structural stability. All formulations were cytocompatible, maintaining cell viability above 70%, with Pluronic-containing systems reaching values close to 100%. These results demonstrate that the independent control of crosslinking density and porosity enables the identification of compositional balance zones that reconcile mechanical integrity and biological response, supporting the rational design of adaptable hydrogel platforms for wound healing.

Keywords:
Hydrogels; Pluronic F-68; Hexamethylene diisocyanate; Wound healing

1. Introduction

The skin is the largest organ of the human body and plays a pivotal role in protection, immune defense, and the regulation of fluid and thermal homeostasis. When this barrier is compromised—particularly in cases of extensive or chronic wounds such as severe burns, diabetic ulcers, and pressure injuries—the healing process becomes highly complex, often prolonged, and susceptible to infection and functional impairment¹-³. These challenges underscore the need for wound dressings and scaffolds capable not only of covering the lesion, but also of actively supporting tissue regeneration while maintaining structural integrity throughout the healing process.

Polymeric hydrogels have emerged as promising materials for wound management due to their three-dimensional architecture, high water content, and ability to mimic key features of the extracellular matrix. Their capacity to maintain a moist environment, facilitate gas exchange, and support cell adhesion, migration, and proliferation makes them particularly attractive for skin regeneration applications. However, despite these advantages, the performance of hydrogel-based systems is strongly dependent on their chemical composition and internal structure, which must be carefully tailored to address the competing biological and mechanical demands imposed by different wound environments⁴-⁷.

Among natural polymers, chitosan has been widely explored in wound healing applications due to its biocompatibility, biodegradability, antimicrobial activity, and intrinsic hemostatic properties, which are particularly advantageous for preventing infection and promoting early-stage wound stabilization. In addition, chitosan exhibits structural similarity to glycosaminoglycans present in the extracellular matrix, supporting cell adhesion and proliferation while offering high versatility for chemical modification and crosslinking⁸-¹⁰.

Keratin, in turn, provides functional attributes that complement those of chitosan and justify its incorporation into hydrogel-based scaffolds. As a structural protein derived from natural sources, keratin contains a high density of amino acid residues and bioactive motifs, including arginine–glycine–aspartic acid (RGD) sequences, which promote integrin-mediated cell adhesion and regulate cell–matrix interactions. Previous studies have demonstrated that keratin-based biomaterials support fibroblast attachment, migration, and proliferation, while also contributing to mechanical reinforcement and improved biocompatibility of composite polymeric matrices. Unlike other commonly used structural proteins, keratin exhibits enhanced structural stability and a distinct amino acid composition, making it particularly suitable for reinforcing hydrogel networks without compromising bioactivity¹¹,¹².

The combination of chitosan and keratin enables the integration of complementary biological and structural functions within a single hydrogel platform. While chitosan contributes antimicrobial activity and processability, keratin enhances cell-interactive properties and network cohesion through its proteinaceous nature and reactive functional groups. Beyond chemical composition, porosity and crosslinking density are widely recognized as two of the most critical parameters governing hydrogel performance. Porosity directly influences fluid absorption, nutrient diffusion, oxygen transport, and cell infiltration, whereas crosslinking density dictates mechanical strength, dimensional stability, swelling behavior, and degradation kinetics¹³-¹⁵. Importantly, these parameters are often antagonistic: increasing crosslinking density enhances mechanical robustness but restricts pore interconnectivity and cellular infiltration, whereas highly porous networks favor biological performance at the expense of mechanical strength. This inherent trade-off remains a major challenge in the design of hydrogel-based wound dressings and scaffolds.

In this context, strategies capable of independently tuning porosity and network stability within a single system are particularly relevant. Despite growing interest in chitosan- and keratin-based hydrogels, few studies have systematically explored approaches that allow simultaneous yet controllable modulation of these antagonistic parameters using complementary physicochemical routes. The present study proposes a rational design strategy based on the combined use of a chemical crosslinker and a porogenic agent to modulate hydrogel properties through distinct yet synergistic mechanisms. Hexamethylene diisocyanate (HDI) was selected to promote covalent crosslinking between chitosan and keratin chains, thereby controlling network density and mechanical stability. In contrast, Pluronic F-68 was incorporated as a non-covalent structural modifier, whose micellar organization acts as a temporary template for pore formation, enhancing hydration and mass transport without altering the underlying crosslinking chemistry. This complementary strategy, referred to here as “dual modulation”, does not denote multiple therapeutic pathways, but rather the integration of chemical and physical modulation routes to systematically tailor structure–property–biological relationships within the same hydrogel platform.

Accordingly, this study aims to systematically investigate the influence of HDI and Pluronic F-68 concentrations on the structural, physicochemical, mechanical, and biological performance of chitosan/keratin hydrogels. While previous chitosan- or keratin-based systems have demonstrated promising biological behavior and moisture management, they often rely on either chemical crosslinking or physical porosity control, resulting in an inherent trade-off between mechanical stability and biological performance. In this context, the novelty of the present work lies in the systematic evaluation of a dual physicochemical modulation strategy, in which covalent crosslinking and micelle-templated porosity are independently controlled within the same hydrogel platform, enabling the identification of compositional balance zones suitable for wound healing applications.

2. Materials and Methods

2.1. Materials

Chitosan (CAS N^o. 9012-76-4, Mn = 150,000 g/mol, degree of deacetylation 75–85%; Med Chem Express), keratin (CAS N^o. 69430-36-0, 99% purity; Yuantai Biological Technology Co., Ltd.), Pluronic F-68 (CAS N^o. 9003-11-6; Sigma-Aldrich), hexamethylene diisocyanate (CAS N^o. 822-06-0; Sigma-Aldrich), and distilled water were used as received.

2.2. Methods

2.2.1. Hydrogel preparation

The hydrogel preparation was performed in two experimental stages. The first stage aimed to evaluate the effect of different concentrations of hexamethylene diisocyanate (HDI) on the crosslinking of chitosan/keratin matrices. The second stage investigated the influence of Pluronic F-68 on the porosity and functional performance of the formulations.

2.2.1.1. Stage 1 – Variation of HDI concentration

Chitosan and keratin were mixed in a 1:2 (w/w) ratio, with a total polymer concentration of 5% (w/w) relative to the aqueous solution. Initially, keratin was dispersed in distilled water under magnetic stirring (2000 rpm, 15 min, room temperature), followed by the addition of chitosan and further stirring for 1 h. The mixtures were homogenized using an Ultra-Turrax at 10,000 rpm, and HDI was then added at different concentrations (1, 2.5, and 5% w/w, calculated relative to the total polymer mass). The solutions were cast into glass molds and dried in an oven at 50 °C until complete solvent removal, yielding solid polymeric scaffolds.

During hydrogel preparation, the aqueous medium exhibited a mildly acidic pH of approximately 5.0. At this pH, chitosan remained adequately solubilized due to protonation of its amino groups, considering that the pKa of chitosan is typically reported in the range of 6.3–6.5. The molecular weight and degree of deacetylation of the chitosan used in this study further favored chain hydration and solubility under near-neutral acidic conditions, enabling homogeneous mixing with keratin without phase separation or precipitation. Moreover, the subsequent covalent crosslinking induced by hexamethylene diisocyanate stabilized the polymeric network, ensuring the formation of a mechanically stable and insoluble hydrogel despite the moderate acidity of the processing medium.

2.2.1.2. Stage 2 – Incorporation of Pluronic F-68

Based on the results of the first stage, the formulation containing 1% HDI was selected and modified with different concentrations of Pluronic F-68 (5, 10, and 15% w/w relative to the polymer mass). The preparation followed the same procedure described above, with the addition of Pluronic F-68 during the initial mixing step. The resulting mixtures were homogenized with an Ultra-Turrax (10,000 rpm), cast into glass molds, and dried at 50 °C to obtain the final hydrogel samples.

It is important to note that all Pluronic-containing hydrogels were prepared using the previously optimized HDI concentration (1%), as the formation of a stable hydrogel network requires covalent crosslinking. Therefore, the effect of Pluronic was systematically evaluated on HDI-crosslinked matrices, enabling assessment of their combined structural and functional impact.

2.2.2 Characterization

The hydrogels were characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), swelling and solubility assays, porosity measurements, compressive strength testing, and cytocompatibility assays with L929 fibroblasts, following ISO 10993¹⁶ guidelines. Detailed procedures for each analysis are described in the following sections.

2.2.2.1 Fourier-transform infrared spectroscopy (FTIR)

Fourier-transform infrared spectroscopy was performed using a Perkin Elmer Spectrum system (v.10.4.2) equipped with an ATR accessory. Spectra were acquired in the range of 600–4000 cm⁻¹ at controlled room temperature (25 °C).

2.2.2.2 Scanning electron microscopy (SEM)

The surface morphology of lyophilized hydrogels was analyzed by SEM after cryofracture. Fragments were mounted on metallic stubs using conductive carbon tape, sputter-coated with palladium for 180 s (Leica EM ACE200), and imaged with an FEI Quanta 400 MLA microscope.

2.2.2.3 Swelling and solubility

To determine the swelling ratio, cylindrical samples (2 cm × 3 cm) were dried in a vacuum oven at 37 °C until constant weight (stable mass over three measurements). The dried samples were immersed in distilled water for 24 h at 37 °C, gently blotted with filter paper, and weighed. The mass difference was used to calculate water uptake.

The same samples were then kept immersed for an additional 48 h, followed by drying under the same conditions to calculate solubility, which was expressed as the percentage of mass loss relative to the initial dry weight. Complementary solubility assays were performed at 50 °C to evaluate the thermal influence on hydrogel stability.

2.2.2.4 Porosity

Porosity was determined by the liquid displacement method using ethanol as a non-solvent. Cylindrical samples (2 cm diameter × 3 cm height) were immersed for 30 min in absolute ethanol. The porosity was calculated according to Equation (1):

$$\begin{array}{c}Porosity\left(\%\right)=\\ \left(W2-W3-Ws\right)/\left(W1-W3\right)\times 100\%\end{array}$$(Eq 1)

In which W1 is the mass of the beaker with 20 mL of ethanol, W2 is the mass after immersion of the hydrogel, W3 is the mass after removing the hydrogel, and Ws is the mass of the dry sample.

2.2.2.5 Cytocompatibility (L929 fibroblasts)

Cytocompatibility was assessed using the MTT assay with fibroblasts of the L929 lineage. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic, under 5% CO₂ at 37 °C. Hydrogels were placed in 24-well plates seeded with 1 × 10⁴ cells/well. After 24 h incubation, the MTT solution (5 mg/mL) was added, and the plates were incubated for 4 h. The medium was removed, formazan crystals solubilized with DMSO, and absorbance measured at 570 nm. Cell viability was expressed as a percentage relative to the control (cells that had no contact with the material).

2.2.2.6 Compressive strength

Cylindrical hydrogel samples (10 mm radius, 5 mm thickness) were conditioned for 12 h in a chamber at 37 °C under controlled relative humidity (50 ± 5%). After conditioning, the samples were allowed to equilibrate at room temperature (23 ± 2 °C) prior to testing to ensure reproducibility and minimize variations related to moisture content. Axial compression tests were carried out at a crosshead speed of 0.1 mm/s with a maximum load of 10 N. The compressive modulus was determined from the linear region of the stress–strain curve, based on the mean of three replicates per group.

2.2.2.7 Statistical analysis

Data were expressed as mean ± standard deviation. One-way ANOVA followed by Tukey’s test was applied for comparisons, with significance set at 5%. Statistical power was estimated using G*Power (v.3.1.9.2), and all tests showed power above 0.8.

2.2.2.8 Multivariate analysis and visual representations

Experimental data were normalized using Z-score standardization to integrate and compare the overall performance of the developed systems. The analyzed variables included swelling, solubility, porosity, compressive strength, and cell viability. Three-dimensional surface plots were generated to visualize interactions between Pluronic F-68 and HDI concentrations and the resulting properties. Complementary visualizations included: (i) radar charts, providing a comparative overview of each formulation across variables, and (ii) heatmaps, highlighting performance patterns. All visualizations were created in Python 3.10.13 using the matplotlib (v.3.7.2) and seaborn (v.0.12.2) libraries in a Jupyter Notebook environment.

3. Results and Discussion

3.1. Fourier-transform infrared spectroscopy (FTIR)

FTIR spectroscopy was employed to investigate the molecular interactions within chitosan (CS), keratin (KE), and HDI-crosslinked hydrogels (Figure 1a). The spectra of the isolated biopolymers exhibited typical features of materials rich in hydroxyl and amine groups. In both cases, a broad band around 3400 cm⁻¹ was assigned to O–H and N–H stretching vibrations, together with absorptions at 2920–2850 cm⁻¹ corresponding to aliphatic C–H stretching. Bands at approximately 1650 and 1550 cm⁻¹ were attributed to Amide I (C=O stretching) and Amide II (N–H bending), respectively, which appeared more intense in keratin due to its proteinaceous nature¹⁷,¹⁸. Chitosan also exhibited characteristic absorptions in the region of 1070–1030 cm⁻¹, assigned to C–O–C stretching of the glycosidic ring¹⁹,²⁰.

Figure 1
Fourier-transform infrared (FTIR) spectra of chitosan/keratin-based hydrogels containing (a) different concentrations of hexamethylene diisocyanate (HDI) and (b) different levels of Pluronic and 1% HD.

In binary CS/KE hydrogels prepared without HDI, relevant spectral modifications were already observed. A new absorption band around 1106 cm⁻¹, absent in the isolated polymers, was attributed to overlapping C–O and C–N stretching modes, suggesting specific protein–polysaccharide interactions stabilized by hydrogen bonding²¹. Additionally, the band near 1340 cm⁻¹ became more pronounced, which can be ascribed to the superposition of C–N stretching vibrations from chitosan with the Amide III band of keratin, further supporting the formation of intermolecular hydrogen-bonded structures²¹. These changes indicate conformational rearrangements resulting from CS–KE interactions prior to chemical crosslinking.

The incorporation of hexamethylene diisocyanate induced marked changes in the carbonyl region of the spectra. A broad absorption between 1680 and 1730 cm⁻¹ was observed and assigned to overlapping contributions of urethane carbonyl groups (~1705–1720 cm⁻¹) and urea-derived structures (~1660–1680 cm⁻¹), formed by reactions between isocyanate groups and hydroxyl or amine functionalities of chitosan and keratin²²,²³. Importantly, the absence of the characteristic –NCO stretching band at approximately 2270 cm⁻¹ confirms effective consumption of HDI and successful covalent crosslinking within the hydrogel network²².

The asymmetric shape and broadening of the carbonyl absorption suggest the possible formation of secondary structures such as biuret and allophanate groups, which are known to arise in isocyanate-based systems, particularly under conditions of isocyanate excess or in the presence of moisture²³. In addition, a discrete band near 1625 cm⁻¹, absent in non-crosslinked systems, was attributed to substituted urea or biuret structures, further confirming the involvement of amino groups in the crosslinking reaction²²,²³. These spectral features are consistent with previously reported FTIR analyses of polyurethane and poly(urea)urethane systems derived from aliphatic diisocyanates²²,²³.

FTIR spectra of hydrogels containing Pluronic F-68 are shown in Figure 1b. The characteristic absorption bands associated with HDI crosslinking were preserved in all Pluronic-containing formulations, including the broad carbonyl region (1680–1730 cm⁻¹) and the absence of the –NCO band, indicating that the presence of the copolymer did not interfere with the covalent crosslinking mechanism²²,²³.

Compared with HDI-crosslinked hydrogels without Pluronic, increased intensities were observed in the regions around 2920–2850 cm⁻¹ and approximately 1100 cm⁻¹, which were attributed to aliphatic C–H stretching and C–O–C stretching vibrations from the poly(ethylene oxide) and poly(propylene oxide) segments of Pluronic F-68²⁴-²⁶. No significant changes were detected in the carbonyl region, confirming that Pluronic F-68 is physically incorporated into the hydrogel network through hydrogen bonding and chain entanglement, rather than covalent bonding²⁴,²⁵.

Slight narrowing and intensity reduction of the broad O–H/N–H band around 3400 cm⁻¹ were also observed, suggesting reorganization of hydrogen bonding interactions between the hydrophilic Pluronic domains and the biopolymer chains²⁶. Taken together, these results demonstrate that HDI governs the chemical crosslinking of the system, while Pluronic F-68 acts as a physical structural modifier, influencing microstructure and hydration without altering the underlying network chemistry.

3.2. Scanning Electron Microscopy (SEM)

Figure 2 shows the scanning electron micrographs of hydrogels formulated with different concentrations of Pluronic F-68 (5%, 10%, and 15%) and hexamethylene diisocyanate (HDI: 1%, 2.5%, and 5%). SEM micrographs highlighted distinct morphological trends associated with the balance between crosslinking and porogenic agents. Samples containing higher HDI concentrations displayed denser and smoother surfaces, consistent with a compact and highly reticulated network. In contrast, hydrogels rich in Pluronic exhibited a well-defined porous architecture likely arises from the micellar organization of Pluronic domains that destabilize during drying, leaving voids within the polymeric matrix. Similar observations were reported by Siboro et al., who demonstrated reduced porosity in alginate hydrogels with higher crosslinker content²⁷.

Figure 2
Scanning electron microscopies of hydrogels containing different HDI and Pluronic contents.

Such contrasting morphologies reveal the antagonistic yet complementary roles of both additives. While HDI increases rigidity and mechanical cohesion, Pluronic introduces microdomains that enhance porosity and water diffusivity. The inteplay between these two components suggests that the degree of pore interconnectivity can be tuned to suit the wound environment – high porosity for exudative wounds and denser structures for long-term barrier applications.

Likewise, Pinthong et al. found that the incorporation of Pluronic F127 promoted the formation of more stable and homogeneous pores during the synthesis of macroporous hydrogels²⁸, supporting the results observed in this study. Additionally, Abdollahi et al. highlighted that the micellar arrangement of Pluronic directly influences the microstructure of hydrogels, impacting not only pore formation but also functional aspects such as drug diffusion and cell–matrix interactions²⁹.

Overall, these findings indicate that Pluronic acts as an effective structural modulator, favoring the formation of a more open network—an essential feature for tissue engineering applications, where high porosity is critical for cell migration, nutrient exchange, and tissue regeneration support.

3.3. Swelling and solubility

The swelling behavior reflected the combined effects of crosslinking density and polymer-solvent interactions. Figures 3a e 3b present the swelling and solubility values of hydrogels formulated with different concentrations of Pluronic F-68 and hexamethylene diisocyanate (HDI). As HDI concentration increased from 0 to 5%, the degree of swelling dropped from ~150% to ~65%, demonstrating the formation of a dense covalent network that restricts water diffusion. Conversely, Pluronic addition increased swelling up to 160% at 15% concentration due to its hydrophilic ethylene oxide segments. Mechanistically, this dual effect can be attributed to two competing factors: (i) covalent crosslinking by HDI limits the availability of hydrophilic sites, and (ii) micellar domains from Pluronic create channels that facilitate water penetration.

Figure 3
Results of (a) swelling degrees and (b) solubility of different hydrogel formulations.

Solubility tests revealed similar antagonistic trends. The highest HDI concentration yielded minimal solubility (~15%), confirming greater resistance to unbound copolymer. These observations suggest that while Pluronic enhances hydration and short-term absorption capacity, HDI ensures long-term matrix stability. This trade-off is particularly relevant for wound dressings, where early hydration supports exudate absorption and late-stage prevents premature degradation.

The increase in swelling with higher Pluronic content agrees with findings by Filip et al., who reported that hydrogels containing hydroxypropylcellulose and Pluronic exhibited enhanced water absorption capacity as copolymer concentration increased³⁰. This behavior is associated with the micellar structure and hydrophilic nature of Pluronic, which favors fluid uptake and retention within the three-dimensional hydrogel network. However, such behavior must be interpreted cautiously, since, as reported by Abdollahi et al., in certain systems, the higher Pluronic content may induce network compaction depending on the interaction with other formulation components²⁹. In the present work, the presence of keratin, rich in amino groups, together with chitosan, appears to have promoted a more open network arrangement, even in the presence of HDI, particularly at moderate concentrations.

Regarding solubility, HDI demonstrated a predominant structuring role, reducing both swelling and solubility in a dose-dependent manner. This effect is well documented, as highlighted by Wei et al., who reported that isocyanates increase crosslinking density, rendering the polymer matrix more resistant to hydrolytic degradation and less prone to expansion³¹. The formulation with 5% HDI showed the lowest solubility, reinforcing the stabilizing effect of the crosslinker.

In contrast, the data indicated that Pluronic, although amplifying swelling capacity, also contributes to the release of soluble fractions, which is consistent with reports by Sharun et al.³² and Camana et al.³³, who emphasized that Pluronic, not being directly involved in covalent crosslinking, tends to gradually dissolve in aqueous media. This phenomenon may be advantageous in applications where gradual removal or natural degradation of the system is desirable, minimizing potential tissue toxicity.

Taken together, these findings indicate that the balance between HDI-mediated crosslinking and structural modification via Pluronic is essential to achieve hydrogels with properties tailored to specific wound conditions. For highly exudative wounds, high swelling capacity may be prioritized, even at the expense of stability. Conversely, in contexts requiring longer dressing permanence, such as high-friction or difficult-to-access regions, a more stable formulation is preferable. Therefore, the optimal choice depends on the clinical scenario and the characteristics of the wound or tissue targeted for regeneration.

3.4. Porosity of hydrogels

Figure 4 shows the porosity values of the hydrogels. Porosity measurements further clarified the structural competition between the two modifiers. Increasing HDI reduced porosity from ~58% to ~28%, likely because crosslinked network compresses the internal mesh, limiting void formation. Conversely, higher Pluronic content produced larger and more connected pores, reaching porosity values comparable to those of gelatin-methacrylate systems used tissue scaffolds (~60%). These findings indicate that the internal free volume can be finely adjusted by varying the Pluronic/HDI ratio. The microstructural evidence aligns with the functional need for porous yet mechanically stable scaffolds, where mass transport and mechanical endurance must coexist.

Figure 4
Degree of total porosity of chitosan and keratin-based hydrogel.

From a functional standpoint, pore architecture governs fluid transport, nutrient diffusion, and ultimately tissue ingrowth. Highly porous hydrogels (>50%) enable rapid oxygen and metabolite diffusion, creating a microenvironment favorable to cell migration and extracellular matrix deposition. However, excessive porosity may compromise mechanical stability and cause premature material deformation under physiological stress. On the other hand, low-porosity systems (≤30%) improve durability but limit cell penetration depth, leading to surface-dominated colonization. Thus, the ability to tune porosity through the Pluronic/HDI ratio provides a powerful handle for tailoring hydrogel performance according to wound type – more open networks for highly exudative or infected wounds, and denser configurations for regions requiring prolonged structural support.

This trend is consistent with the findings of Filip et al., who reported increased porosity in hydrogels containing Pluronic³⁰, and with Bova et al.³⁴, who highlighted the role of this copolymer in generating more porous structures that enhance fluid uptake and exchange with the external medium. On the other hand, Wei et al. demonstrated that isocyanates as crosslinkers promote denser and less expandable polymer networks, reducing pore volume³¹. Similarly, Bourguignon et al.³⁵ emphasized that the crosslinking degree directly affects both surface and internal porosity of hydrogels, impacting their overall functionality.

Therefore, the results demonstrate that porosity can be finely tuned through the balance between Pluronic and HDI. Formulations with higher porosity may be more suitable for exudative wounds, while more densely crosslinked compositions may provide greater mechanical integrity in areas subject to friction or clinical handling.

3.5. Cellular viability – L929

Figure 5 presents the cell viability results obtained by the MTT assay after 24 h of exposure of L929 fibroblasts to chitosan/keratin-based hydrogels containing different concentrations of hexamethylene diisocyanate (HDI) and Pluronic F-68. All formulations maintained cell viability above 70%, fulfilling the criteria for cytocompatibility according to ISO 10993¹⁶ guidelines, which recommend metabolic activity assays as an initial screening tool for evaluating the biological safety of newly developed biomaterials.

Figure 5
Viability of L929 cell lineage cells after 24 h of contact with the different hydrogels obtained.

Although a gradual reduction in metabolic activity was observed with increasing HDI concentration, this behavior is more plausibly associated with physical modifications of the hydrogel microenvironment rather than chemical cytotoxicity. Higher HDI contents promote increased crosslinking density, leading to stiffer and less porous networks, as demonstrated by the mechanical and porosity analyses. Such structural features can restrict nutrient and oxygen diffusion and limit cellular spreading, resulting in lower mitochondrial activity measured by the MTT assay. Similar correlations between increased matrix stiffness, reduced pore interconnectivity, and attenuated fibroblast response have been reported for highly crosslinked hydrogel systems¹⁵,¹⁶.

Figure 5 presents the cell viability results obtained by the MTT assay with L929 fibroblasts after 24 h of exposure to hydrogels containing different concentrations of Pluronic F-68 and hexamethylene diisocyanate (HDI).

In contrast, formulations containing Pluronic F-68 exhibited higher cell viability, with values approaching or exceeding 90%. This behavior can be attributed to the enhanced hydration, swelling capacity, and pore connectivity introduced by the micellar organization of the copolymer. More permeable and hydrated networks favor mass transport and create a microenvironment that supports fibroblast metabolic activity and viability. In addition, Pluronic-based systems are widely recognized for their biocompatibility and low protein adsorption, which contribute to improved cell–material interactions²⁹,³⁶,³⁷.

Notably, hydrogels combining moderate crosslinking (1% HDI) with Pluronic F-68 incorporation showed the most balanced biological response, indicating the existence of an optimal compositional window in which structural stability and cytocompatibility coexist. This observation reinforces that cellular response in hydrogel systems is governed not only by chemical composition, but also by the interplay between network architecture, stiffness, porosity, and hydration¹⁴-¹⁶,³⁸,³⁹. Within this framework, the MTT assay provides a meaningful first-level evaluation of cytocompatibility, while more advanced biological investigations may be pursued in future studies focused on specific cell–matrix interaction mechanisms.

3.6. Mechanical properties

Mechanical compression tests revealed compressive strength values ranging from 33.5 to 65.9 MPa (Figure 6). The positive correlation between HDI concentration and compressive strength values obtained from compression tests demonstrates the reinforcing role of covalent bonds that restrict polymer chain mobility. In contrast, Pluronic reduced mechanical resistance, as the pores introduced by its micellar domains act as stress concentrators that diminish load-bearing capacity. The intermediate formulation (1% HDI, 10% Pluronic) achieved an optimal balance, combining mechanical resilience (≈45 MPa) with structural flexibility and high porosity.

Figure 6
Results of compression tests (compressive strength) of the different hydrogel formulations.

At the molecular level, the mechanical response reflects competition between permanent covalent junctions introduced by HDI and transient physical interactions driven by the amphiphilic Pluronic domains. Under compressive load, HDI-rich network deform elastically until chains scission occurs, whereas Pluronic-rich systems can dissipate stress via reversible micelle rearrangement and hydrogen bond disruption. This dual mechanism explain failure as Pluronic content increases. Additionally, the combination of these two networks reduces stress concentration, allowing partial energy relaxation and recovery after deformation – an essential feature for dressings subject yo mechanical movement.

A mechanistic interpretation of this behavior suggests that the system follows a dual-network model: HDI contributes to the chemical crosslinking network, while Pluronic generates a physical network that dissipates stress through hydrophilic domains. The interplay between both networks defines the macroscopic mechanical performance. These findings are consistent with previous reports showing that higher crosslinking density improves the mechanical integrity of hydrogel systems as evaluated by compression tests³⁹,⁴⁰. In addition, similar reductions in compressive properties derived from compression tests associated with increased porosity have been reported in other polymeric and hydrogel systems¹²,⁴⁰.

When compared with previously reported chitosan- and protein-based hydrogels for wound healing, the compressive properties obtained in this study fall within or above the typical range described in the literature, while offering the additional advantage of tunable porosity and cytocompatibility, supporting their suitability for wound dressing applications.

3.7. Multivariate analysis of hydrogel properties

3.7.1. 3D response surface representations

To evaluate the influence of Pluronic F-68 and hexamethylene diisocyanate (HDI) concentrations on the overall performance of the developed hydrogels, three-dimensional response surface plots were constructed (Figure 7) for swelling, solubility, porosity, and cell viability. The X- and Y-axes represent the concentrations of Pluronic (5%, 10%, and 15%) and HDI (1%, 2.5%, and 5%), respectively, while the Z-axis corresponds to the experimentally obtained values for each property.

Figure 7
3D graphs containing the correlation of different properties with the porosities of the hydrogels obtained.

The multivariate analysis provided a holistic view of property interdependence. The 3D response surfaces demonstrated clear antagonistic effects: HDI concentration negatively correlated with swelling, porosity, and cell viability²¹,²⁵, while Pluronic exhibited positive correlations with these parameters but an inverse relationship with mechanical strength¹⁸,¹⁹,²⁶. The radar plot highlighted that no single formulation maximized all properties simultaneously, reinforcing the need for a tailored approach.

Together, these three-dimensional representations provide a clear visualization of the antagonistic effects between Pluronic and HDI, identifying balance zones where hydration, stability, and biocompatibility can be simultaneously optimized. This is noteworthy as these are key attributes for designing bioactive wound dressings and scaffolds for tissue engineering applications¹⁸,¹⁹,²¹,²⁶,³³.

3.7.1.1. Heatmap and Chart Graph Applied to the Analysis of Hydrogels

Figure 8 presents two complementary approaches of multivariate analysis: a correlation heatmap between the physicochemical and biological properties evaluated, and a radar plot summarizing the performance of the formulations across these variables. The color scale in the Z-score heatmap represents normalized values of each evaluated parameter relative to the dataset mean. Positive Z-score values (displayed in warmer colors) indicate that a given formulation exhibits values above the average for that property, whereas negative Z-score values (cooler colors) indicate values below the average. Values close to zero correspond to properties near the mean. This normalization allows direct visual comparison of trends among properties with different absolute units and magnitude, emphasizing the relative influence of HDI and Pluronic contents on the overall hydrogel performance rather than absolute values.

Figure 8
Heatmap and Chart Graph Applied to the Analysis of Hydrogels.

A critical insight derived from the heatmap is that mechanical integrity and biological compatibility are inversely linked in this system. Denser matrices limit hydration and cell infiltration, while highly porous structures reduce strength. This opposition suggests that optimal composition depends on the clinical goal. For chronic exudative wounds, formulations with high Pluronic and low HDI content would be preferred due to superior hydration and permeability. For mechanically stressed or long-term applications, slightly higher HDI levels ensure durability without significant cytotoxicity.

Together, these findings demonstrate that the synergistic modulation by HDI and Pluronic allows fine-tuning of hydrogel properties across a multidimensional design space. By correlating chemical structure, morphology, and biological response, this study provides a mechanistic framework for rationally designing adaptable wound-healing scaffolds.

4. Conclusion

This study demonstrated that the combined use of hexamethylene diisocyanate (HDI) and Pluronic F-68 provides an effective strategy for tailoring the structural, physicochemical, mechanical, and biological properties of chitosan/keratin-based hydrogels. Spectroscopic analyses confirmed the formation of covalent urethane and urea linkages induced by HDI, while morphological and physicochemical evaluations showed that Pluronic F-68 acts as a physical structural modifier, promoting micelle-templated porosity and enhanced hydration without interfering with the chemical crosslinking mechanism.

Mechanical compression tests revealed compressive strength values ranging from 33.5 to 65.9 MPa, depending on HDI and Pluronic concentrations. Increasing HDI content improved mechanical stability and reduced solubility to values as low as approximately 15%, indicating enhanced network integrity. In contrast, Pluronic incorporation increased water uptake and pore connectivity, leading to swelling values of up to ~160% and porosity values approaching 60%, favoring mass transport and permeability.

Cytocompatibility assays using L929 fibroblasts demonstrated that all hydrogel formulations were biologically safe, maintaining cell viability above 70% in accordance with ISO 10993¹⁶ guidelines. Pluronic-containing hydrogels exhibited higher metabolic activity, with cell viability values reaching close to 100%, whereas formulations with higher HDI content showed slightly reduced viability, which was associated with increased matrix stiffness rather than chemical cytotoxicity.

Overall, the results highlight the antagonistic yet complementary roles of HDI and Pluronic F-68 and demonstrate that compositional balance zones can be identified to reconcile mechanical stability, permeability, and cytocompatibility. This rational dual-modulation approach provides a quantitative framework for the design of adaptable hydrogel systems tailored to different wound healing requirements.

5. Acknowledgments

We thank Centro de Tecnologia Mineral (CETEM) for performing the microscopy analyses presented in this article, as well as Centro de Ciências da Saúde/Universidade Federal do Rio de Janeiro (CCS-UFRJ) and Universidade Estadual do Rio de Janeiro (UERJ) for their support in conducting the biological assays.

• Data Availability
  All data supporting the findings of this study have been published within the article itself.

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Edited by

• Associate Editor:
  Rodrigo Orefice.

• Editor-in-Chief:
  Luiz Antonio Pessan.

Data availability

All data supporting the findings of this study have been published within the article itself.

Publication Dates

• Publication in this collection
  01 May 2026
• Date of issue
  2026

History

• Received
  15 Nov 2025
• Reviewed
  28 Jan 2026
• Accepted
  25 Feb 2026