Biomaterials Developed with an Oxyresveratrol-Rich Artocarpus lakoocha Extract Exhibiting High Phenolic and Flavonoid Contents, Strong Antioxidant Activity, and In Vitro Fibroblast Compatibility

Introduction

The skin, as the body’s primary protective shield, functions as a physiological barrier that safeguards the organism against environmental threats1. Skin barrier impairment can result from both intrinsic factors such as genetic mutations in filaggrin and extrinsic influences including excessive hygiene practices, low humidity, and exposure to surfactants. Repeated mechanical or chemical irritation can disrupt the lipid matrix and corneocyte cohesion, thereby weakening the barrier function and increasing susceptibility to dermatological disorders2. Maintaining the integrity of the skin barrier is essential, as it provides effective defense against microbial invasion, allergens, and chemical irritants3. Moreover, impaired barrier function has been associated with the pathogenesis of various dermatological conditions4. Globally, approximately 2% of hospitalized patients suffer from chronic skin wounds, including lacerations, burns, and pressure injuries underscoring the substantial need for effective wound management strategies5. Appropriate wound care accelerates healing and reduces recurrence and complications. Despite advances, conventional wound dressings often fail to adequately address complex healing scenarios due to limited adaptability, poor moisture regulation, and insufficient support for regenerative cell activity.

Due to their unique characteristics, including high flexibility, pronounced hydrophilicity, superior absorbency, and soft consistency, hydrogels have emerged as promising materials for advanced wound dressing applications6. These three-dimensional polymeric networks can be formulated from a wide range of natural and synthetic polymers, either individually or in combination. Synthetic polymers commonly used for hydrogel wound dressings include poly(ethylene glycol) (PEG), polyacrylamide (PAM), and polyvinyl alcohol (PVA), which provide mechanical stability and tunable swelling properties7. Natural polymers, such as chitosan, collagen, alginate, gelatin, hyaluronic acid, and cellulose derivatives, offer inherent biocompatibility and biodegradability, making them suitable for biomedical applications8,9. These hydrogels can be further functionalized with therapeutic agents, nanoparticles, or bioactive plant extracts to enhance their antimicrobial, anti-inflammatory, or pro-regenerative capabilities10,11. Despite these advantages, conventional hydrogels often exhibit limitations, such as relatively low mechanical strength, restricted pore size, and limited oxygen and nutrient diffusion, which can compromise their performance in deep or chronic wound environments12,13. To overcome these drawbacks, cryogels have emerged as promising alternatives. During cryogelation, polymerization occurs at subzero temperatures, resulting in the formation of interconnected macroporous networks characterized by rapid swelling kinetics, high permeability, and enhanced mechanical resilience. These characteristics make cryogels highly suitable for applications in wound healing, tissue engineering, and drug delivery14,15.

Artocarpus lakoocha Roxb. (A. lakoocha), a medicinal plant belonging to the Moraceae family, is predominantly distributed in tropical regions of Southeast Asia, India, and Thailand16,17. Commonly known as “monkey jack,” this species has been widely used in traditional medicine for the treatment of various ailments including inflammation, diarrhea, abscesses, and parasitic infections. Numerous studies have reported the pharmacological activities of extracts obtained from different parts of A. lakoocha, such as the leaves, bark, and heartwood, revealing antimicrobial, antioxidant, anti-inflammatory, hepatoprotective, and anticancer properties18,19,20. The major active compound of A. lakoocha is Oxyresveratrol, a polyphenolic stilbene with high antioxidant capacity, which has been shown to support wound healing by promoting fibroblast proliferation, collagen synthesis, and the upregulation of wound-related gene expressions such as VEGFR-2 and TGF-β121,22,23,24. Additionally, in vitro studies have demonstrated that ethanolic extracts of A. lakoocha significantly suppress LPS-induced inflammatory cytokines including IL-6, TNF-α, and MCP-1 in macrophage cell lines, suggesting its strong anti-inflammatory effects via modulation of PI3K/Akt and NF-κB pathways25,26. The antibacterial efficacy of A. lakoocha has been validated against several wound-associated pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, exhibiting average inhibition zones ranging from 10–20 mm27. In the present study, A. lakoocha extract was incorporated into PVA/collagen-based cryogels, and comprehensive physicochemical characterization and in vitro biological evaluations were conducted to assess their potential suitability as wound dressing materials. Specifically, this study aimed to determine the total phenolic content (TPC), total flavonoid content (TFC), phenolic profile (LC–MS Q-TOF), and antioxidant activity (DPPH) of the extract-enriched biomaterials, as well as to evaluate their effects on cytotoxicity, cell adhesion, and cytoskeletal organization using NIH-3T3 fibroblast cells in vitro.

Materials and Methods

Total Phenolic Content (TPC)

The total phenolic content of A. lakoocha extracts was determined using the Folin–Ciocalteu colorimetric method described by Singleton and Rossi (1965), with some modifications28. The A. lakoocha sample (100 µL) was mixed with 1 mL of deionized water and 250 µL of Folin-Ciocalteu’s reagent solution and 150 µL 7% (v/v) sodium carbonate (Na2CO3) solution. The mixture was incubated at room temperature for 45 min. The absorbance was measured at 750 nm using spectrophotometer (Shimadzu, Kyoto, Japan). The amount of TPC gallic acid equivalent (GAE) in the extract samples was calculated as “mg GAE/g dry extract”. The experiments were performed in triplicate.

Total Flavonoid Content (TFC)

The total flavonoid content of A. lakoocha extracts was determined using the Aluminum Chloride Colorimetric Method reported by Zhishen29. The A. lakoocha sample (100 µL) was mixed with 300 µL of 30% ethanol and 200 µL of 10% aluminium chloride (AlCl3) solution. The mixture was incubated for 10 minutes. After 10 min of incubation, 200 µL of 1 M sodium acetate (NaOAc) and 1 ml of H2O were added and incubated for 60 min. After that, the absorbance values of the samples were measured using a spectrophotometer at a wavelength of 415 nm. A calibration curve was created using a quercetin standard to calculate the total flavonoid content. A calibration curve was prepared with quercetin standard. The results were expressed as mg QE/g extract. The experiments were performed in triplicate.

DPPH Radical Scavenging Activity

The antioxidant capacity of the extracts was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method described by Blois, with some modifications30. For analysis, volumes of extract solutions prepared at different concentrations were taken and mixed with a 0.1 mM DPPH solution prepared in methanol. After the mixtures were homogenized, reactions were carried out in the dark due to the light sensitivity of DPPH, and the samples were incubated at room temperature for 30 minutes. The absorbance was measured at 517 nm by a spectrophotometer. IC₅₀ values were determined by nonlinear regression using GraphPad Prism 9.1.0 software. Butylated hydroxytoluene (BHT) served as a positive control. All experiments were performed in triplicate, and data were presented as mean ± standard deviation (SD). The radical scavenging activity was calculated using the following equation:

Inhibition (%) = [(Acontrol−Asample) / Acontrol] × 100

LC–MS Q-TOF Analysis for Chemical Profiling

The metabolite profile of A. lakoocha extracts was analyzed using LC–MS Q-TOF system (Agilent Technologies, USA). Chromatographic separation was performed on an Agilent Poroshell C18 column (3.0 × 100 mm, 2.7 μm). Two solvents, one containing 0.1% formic acid, and the other acetonitrile, were used as mobile phases during the analysis. The flow rate was set at 0.6 mL/min. Mass spectrometric data were acquired in both positive and negative electrospray ionization modes with these parameters: capillary voltage, 2000 V; drying gas (N₂), 11 L/min at 325 °C; nebulizer pressure, 35 psi; and collision energies of 10, 20, and 30 eV. Metabolite identification was performed using Agilent MassHunter software, supported by METLIN, PubChem, FooDB databases, and MS/MS fragmentation pattern analysis.

Synthesis of Artocarpus lakoocha Extract–Enriched Polymeric Cryogels

A 10% (w/v) poly(vinyl alcohol) (PVA) solution (Sigma-Aldrich, USA) was prepared by dissolving PVA in ultrapure water at 90 °C under constant stirring. Separately, a 1% (w/v) type I collagen solution (fish-derived, Sigma-Aldrich, USA) was prepared at 40 °C. The two solutions were then mixed at a 1:2 (v/v) ratio. Subsequently, A. lakoocha extract, characterized by an Oxyresveratrol-rich phenolic profile, was incorporated into the polymer mixture to obtain a final extract concentration of 1% (w/v). Glutaraldehyde (Merck, Germany) was added as a cross-linking agent, and the mixture was gently homogenized and immediately dispensed into 24-well plates. For comparison, control cryogels without A. lakoocha extract were prepared following the same protocol. The samples were frozen at −16 °C for 12 h to induce cryogelation.

Swelling Tests

Cryogels were dried with a lyophilizer (BK-FD10, Biobase) at −55 °C for 3 h. Dried cryogels were weighed (md) and immersed in phosphate-buffered saline (PBS) at 37 °C. At predetermined intervals, the swollen cryogels were weighed (msw). The swelling ratio (SR) was calculated using the following equation:

SR (%) = (msw- md) / md x 100 (Equation 1)

BET analysis

Specific surface area and porosity characteristics of the cryogels were evaluated using the Brunauer–Emmett–Teller (BET) method with nitrogen adsorption–desorption isotherms at 77 K31. Prior to analysis, samples were dried under vacuum at 40 °C for 24 h to remove residual moisture. The specific surface area of the samples was determined based on the volume of nitrogen adsorbed and expressed in units of m²/g.

Cell Culture Studies

NIH-3T3 human fibroblast cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 100 U/mL penicillin–streptomycin under standard conditions (37 °C, 5% CO₂). Prior to cell culture, A. lakoocha extract–enriched cryogels were sterilized by immersion in a graded ethanol series prepared at 60%, 70%, 80%, 90%, and 100% (v/v) for a total of 5 minutes at each stage32. For cell seeding, NIH-3T3 cells were placed on the biomaterials (13 mm diameter, 0.5 mm thickness) at a density of 1 × 10⁶ cells for each sample.

MTT Assay

The MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a colorimetric technique that determines cell viability based on the reduction of the MTT reagent by metabolically active mitochondria in viable cells33. MTT assay was performed at 24, 48 and 72 hours. The experimental setup was a positive control group (cells cultured without cryogel) and a negative control group (cryogel without cells). MTT solution (0.5 g/mL) was added to each well and the cells were incubated at 37 °C and 5% CO2 for 3 h. After incubation, the medium was removed and dimethyl sulfoxide (DMSO) was added and incubated for 30 min. The absorbance values were measured with a spectrophotometer (Shimadzu, Uvmini-1240) at a wavelength of 570 nm. Cell viability was calculated using the following formula:

Cell viability (%) = (Mean Abs570 of test group / Mean Abs570 of control group) × 100

In this formula, the "test group" refers to cells cultured on A. lakoocha extract–enriched cryogels, while the "control group" refers to cells cultured directly on the plate without any biomaterials32,34,35.

Immunofluorescence analyses

Immunofluorescence analysis was performed to evaluate the effects of A. lakoocha extract–enriched cryogels on the morphology of NIH-3T3 and on cell-cell and cell-matrix interactions. For this purpose, biomaterials samples were placed in 24-well culture plates and seeded with NIH-3T3 cells. The cells were cultured under incubation conditions for 48 hours. Following incubation, biomaterials were fixed in PBS containing 4% (v/v) paraformaldehyde for 30 minutes at room temperature. After fixation, samples were washed with PBS to remove residual fixatives. Cell were permeabilized using 0.1% (v/v) Triton X-100 in PBS for at 10 minutes. F-actin filaments were stained using Alexa Fluor 594-conjugated phalloidin, prepared in PBS containing 1% (w/v) bovine serum albumin (BSA) and Tween-20 (PBS-T), at a dilution of 1:1000. For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) was diluted 1:1000 in PBS and applied simultaneously. The staining solutions were incubated with the samples for 90 minutes in the dark at room temperature. After incubation, samples were washed with PBS and imaged using a fluorescence microscope (Dmil LED Fluo, Leica, Germany).

Phase Contrast Microscopy

To examine cell morphology on biomaterials, 13 mm diameter Thermanox™ coverslips (Thermo Fisher Scientific, USA) were placed on 24-well culture plates, and then cryogels samples were fixed on these coverslips. After 48 h of incubation, the samples were examined using a phase-contrast microscope (Leica DMiL LED Fluo, Germany).

Statistical Analysis

All statistical analyses were performed using SPSS software (version 20.0; IBM, Chicago, IL, USA). All findings are expressed as the mean values and standard deviation (SD) of three independent replicates. GraphPad Prism software (v8.4.3, GraphPad Software Inc., USA) was used for advanced comparisons and graphical presentations. Fluorescence intensity measurements were quantified using ImageJ software. For comparisons involving three or more groups, two-way ANOVA followed by Tukey’s post-hoc test was applied. A p-value of less than 0.05 was considered statistically significant.

Results

Determination of Total Phenolic and Flavonoid Contents and Assessment of Antioxidant Potential in Artocarpus lakoocha

The evaluation of A. lakoocha revealed a high content of phenolic and flavonoid compounds, along with substantial antioxidant capacity (Table 1). The total phenolic content (TPC), determined using the Folin–Ciocalteu method, was 541.3 ± 14.1 mg GAE/g extract, indicating a strong presence of phenolic constituents. The total flavonoid content (TFC), assessed by the aluminum chloride colorimetric assay, was 96.3 ± 5.1 mg QE/g extract, further supporting the phytochemical richness of the extract. Antioxidant activity was measured by DPPH radical scavenging assay and demonstrated an IC₅₀ value of 98.03 ± 0.57 µg/mL, reflecting significant free radical neutralizing ability. Compared to previous studies reporting TPC values ranging from 127.99 to 398.44 mg GAE/g and TFC values between 2.05 and 8.79 mg QE/g in ethanol-based extracts, the significantly higher levels observed in this study may be attributed to differences in extraction methodology, solvent polarity, and plant material19,36. The IC₅₀ value aligns with literature data reporting antioxidant activities within the range of ≤50–100 µg/mL for A. lakoocha extract assessed through DPPH and other radical scavenging assays37,38,39,40. These findings confirm the phytochemical richness and potent antioxidant potential of A. lakoocha extract, highlighting its promise as a functional ingredient in biomaterials aimed at enhancing wound healing.

Phytochemical Composition and Antioxidant Activity of Artocarpus lakoocha Extract

Main Phenolic Compounds Identified in Artocarpus lakoocha by LC–MS Q-TOF

Analysis revealed that Oxyresveratrol, Resveratrol, and Gallic acid were the primary bioactive phenolic compounds in the extract. Quantitative assessment revealed that Oxyresveratrol was present at the highest concentration, at 78.8 ± 9.1 µg/mg of extract. This was followed by gallic acid (9.2 ± 0.9 µg/mg) and resveratrol (4.5 ± 0.6 µg/mg) (Table 2). These values were calculated as approximately 78.8, 9.2, and 4.5 mg/g, respectively, on a dry weight basis, demonstrating that the extract is quite rich in phytochemicals.

Quantitative Analysis of Major Phenolic Compounds in Artocarpus lakoocha Extract

Morphological and Structural Characteristics of Artocarpus lakoocha Extract–Enriched Cryogels

Cryogels incorporating A. lakoocha extract were successfully synthesized using a PVA/collagen blend through the cryogelation process. The resulting biomaterials exhibited a macroporous structure with well-interconnected pores, as confirmed by visual inspection (Figure 1). Cryogels enriched with A. lakoocha extract, characterized by an Oxyresveratrol-rich phenolic profile, displayed a light yellow coloration. Importantly, the incorporation of the extract did not adversely affect the cryogel formation process. Both control and extract-enriched biomaterials maintained their three-dimensional structural integrity after repeated freeze–thaw cycles, indicating good structural stability.

Macroscopic view of cryogels prepared with A. lakoocha extract

Characterization Studies

Swelling ratio analysis demonstrated that cryogels incorporating A. lakoocha extract, characterized by an Oxyresveratrol-rich phenolic profile, exhibited a slightly reduced water uptake capacity compared to control cryogels. This minor reduction can be attributed to additional hydrogen-bonding interactions between phenolic functional groups present in the extract and the polymer matrix. Despite this decrease, the extract-enriched cryogels retained excellent hydration capacity. The swelling ratio of the control cryogel was 420 ± 3.2%, whereas cryogels enriched with A. lakoocha extract exhibited a swelling ratio of 415 ± 4.5%. Brunauer–Emmett–Teller (BET) analysis revealed distinct surface and porosity characteristics for the extract-enriched cryogels, indicating the formation of a highly porous network suitable for biomedical applications. The specific surface area of the control cryogel was determined to be 12.8 ± 0.9 m²/g, while cryogels incorporating A. lakoocha extract exhibited a higher specific surface area of 15.2 ± 2.1 m²/g. This increase suggests an expanded surface area that may facilitate improved cell adhesion. All BET measurements were performed in triplicate, and the results are presented as mean ± standard deviation. Overall, the incorporation of A. lakoocha extract with an Oxyresveratrol-rich phenolic profile led to a modest increase in specific surface area without compromising the hydration properties of the cryogels.

MTT Assay

The effects of control cryogels and cryogels incorporating A. lakoocha extract, characterized by an Oxyresveratrol-rich phenolic profile, on cell proliferation were evaluated using the MTT assay at 24, 48, and 72 hours. As shown in Table 3, compared with the control group (100%), cell proliferation in the control cryogel group was reduced by 3.8% ± 2.1%, 10.4% ± 0.8%, and 12.5% ± 1.4% at 24, 48, and 72 hours, respectively. These reductions were statistically significant at 48 and 72 hours, whereas no significant difference was observed at 24 hours. In contrast, cryogels incorporating A. lakoocha extract exhibited a significant proliferative effect. Cell proliferation increased by 8.7% ± 2.2%, 12.9% ± 2.2%, and 10.3% ± 2.4% at 24, 48, and 72 hours, respectively, compared to the control group (p < 0.0001). Overall, these findings indicate that while the control cryogel slightly suppressed cell proliferation over time, the incorporation of A. lakoocha extract with an Oxyresveratrol-rich phenolic profile significantly enhanced cell viability in a time-dependent manner, with the most pronounced effect observed at 48 hours (Figure 2, Table 3).

Cell viability of NIH-3T3 fibroblast cells cultured on control cryogels and A. lakoocha extract–enriched cryogels at 24, 48, and 72 hours. Cell proliferation was assessed using the MTT assay, and data are presented as mean ± standard deviation (n = 3).

Cell Viability of NIH-3T3 Cells on Cryogels at 24, 48, and 72 Hours

Immunofluorescence Analysis of NIH-3T3 Cells on Artocarpus lakoocha Extract–Enriched Cryogels

Immunofluorescence imaging demonstrated that NIH-3T3 fibroblast cells adhered to and spread effectively on both control cryogels and cryogels incorporating Artocarpus lakoocha extract after 48 hours of incubation (Figure 3). F-actin staining (red) revealed well-organized cytoskeletal structures, with cells exhibiting typical fibroblast morphology. Notably, cells cultured on extract-enriched cryogels, characterized by an Oxyresveratrol-rich phenolic profile, displayed more pronounced actin filament organization and extensive lamellipodia formation, indicating enhanced cell–substrate interactions and increased migratory potential compared to control cryogels. DAPI staining (blue) showed a uniform distribution of nuclei throughout the cryogel matrix, suggesting a high density of viable cells. Furthermore, cells cultured on A. lakoocha extract–enriched cryogels formed more interconnected cellular networks, reflecting enhanced cell–cell communication and a favorable microenvironment for tissue integration. Overall, these findings indicate that A. lakoocha extract–enriched cryogels effectively support fibroblast attachment, proliferation, and cytoskeletal organization, which are critical parameters for wound healing applications.

Immunofluorescence images of NIH-3T3 fibroblast cells cultured on cryogel matrices after 48 hours. (A) Control cryogel and (B) Artocarpus lakoocha extract–enriched cryogel. F-actin filaments were stained with phalloidin (red), and cell nuclei were counterstained with DAPI (blue), demonstrating cell adhesion, spreading, and cytoskeletal organization within the cryogel microarchitecture.

Discussion

The findings obtained in the study indicate that the extract of A. lakoocha plant has high phytochemical content and strong antioxidant potential. The total phenolic content was determined as 541.3 ± 14.1 mg GAE/g using the TPC assay, while the total flavonoid content was measured as 96.3 ± 5.1 mg QE/g extract using the TFC assay. These values determined by the used experimental methods were significantly higher than the literature studies, where values in ethanol-based extracts of A. lakoocha plant were found to be 127.99 – 398.44 mg GAE/g by the TPC method and 2.05 – 8.79 mg QE/g by the TFC method19,36. Such discrepancies between the results obtained from the current study and those from literature studies may be attributed to differences in experimental methods, such as differences in extraction methods and solvent polarity, as well as differences in the geographical origin of the plants used and harvesting conditions10,41. The findings of this study suggest that the use of more polar solvents and optimized extraction protocols, including longer extraction times than those used in previous studies, may have increased the yield of the bioactive compounds present42,43.

Consistent with its elevated phenolic and flavonoid content, the extract exhibited substantial antioxidant activity, as demonstrated by the DPPH radical scavenging assay with an IC₅₀ value of 98.03 ± 0.57 µg/mL. This result indicates a strong free radical neutralizing capacity and is consistent with previously reported IC₅₀ values for A. lakoocha extracts, which typically range from ≤50 to 100 µg/mL37,38,39,40. Antioxidant activity is generally correlated with phenolic content due to the electron-donating properties of phenolic hydroxyl groups, which can stabilize free radicals44,45. Therefore, the high antioxidant efficacy observed in this study is likely associated with the extract’s elevated phenolic and flavonoid composition46.

Furthermore, LC–MS Q-TOF analysis revealed that the most abundant phenolic compound in the extract was Oxyresveratrol (78.8 ± 9.1 µg/mg), followed by gallic acid (9.2 ± 0.9 µg/mg) and resveratrol (4.5 ± 0.6 µg/mg). These compounds are well known for their antioxidant and anti-inflammatory activities, which may collectively contribute to the overall bioactivity profile of the extract47,48. Notably, Oxyresveratrol, a Resveratrol analogue with additional hydroxyl groups, has been reported to exhibit strong antioxidant activity and potential relevance for skin-related application in various studies, making its high presence in A. lakoocha particularly relevant for biomedical applications, including wound healing49,50.

In summary, the high levels of phenolic and flavonoid compounds, coupled with potent antioxidant activity and the presence of bioactive metabolites such as Oxyresveratrol and Gallic acid, underscore the potential of A. lakoocha extract as a valuable natural source for therapeutic applications. These results support its further development as a functional ingredient in antioxidant-rich formulations, particularly for use in biomaterials aimed at promoting tissue repair and enhancing wound healing outcomes51,52,53.

A. lakoocha extract extract-enriched cryogels were successfully synthesized using a PVA/Collagen blend via a freeze–thaw cryogelation technique. The fabrication process resulted in macroporous cryogels with interconnected structures, visually confirming the integrity and uniformity of the formed network. Control and extract-enriched cryogels maintained their three-dimensional structure after repeated freeze–thaw cycles54,55.

Swelling analysis revealed that incorporation of A. lakoocha extract led to a slight reduction in water absorption capacity compared to control cryogels (415 ± 4.5% vs. 420 ± 3.2%). This decrease, although minimal, may be attributed to additional hydrogen-bonding interactions between phenolic hydroxyl groups present in the extract and functional groups within the polymer matrix such as –OH groups in PVA and amide (–NH–CO–) and carboxyl (–COOH) groups in type I collagen54,56,57. Such interactions likely reduced the number of available hydrophilic sites capable of binding water molecules, leading to a modest decrease in swelling behavior. Nevertheless, the extract–enriched polymeric cryogels preserved excellent hydration capacity, a desirable feature for wound dressing applications where moisture retention is essential for maintaining a moist healing environment and promoting tissue regeneration52,55.

BET analysis provided further insights into the structural properties of the cryogels. Interestingly, the specific surface area of cryogels increased upon A. lakoocha incorporation (from 12.8 ± 0.9 m²/g in the control to 15.2 ± 2.1 m²/g in the extract–enriched cryogels), suggesting that the addition of the extract may contribute to changes in porosity and surface roughness56,58. This increase in surface area is of particular relevance for biomedical applications, as it may facilitate improved cell adhesion, nutrient exchange, and bioactive compound diffusion across the cryogel network59,60. The presence of phenolic compounds could influence pore formation during cryogelation by altering ice crystal nucleation and growth dynamics, thus subtly modulating the internal microarchitecture of the resulting biomaterial31,58. Overall, the physicochemical characterization results support the suitability of A. lakoocha-enriched cryogels as promising candidates for biomedical applications, particularly in wound healing. Their favorable swelling behavior, enhanced surface area, and stable macroporous structure suggest that these cryogels can effectively support cell–material interactions while simultaneously delivering antioxidant and bioactive molecules derived from the extract.

The MTT assay results revealed a distinct difference in cellular responses between control cryogels and A. lakoocha-enriched cryogels over the 72-hour evaluation period. Control cryogels exhibited a slight reduction in cell proliferation compared to the baseline, which may be attributed to limited nutrient diffusion and initial cell adaptation to the three-dimensional macroporous structure13,15. This decrease is consistent with previous reports indicating that synthetic or semi-synthetic cryogels, despite their interconnected porosity, can transiently restrict metabolic activity during early incubation phases due to surface chemistry and hydrophilicity variations14. Conversely, the incorporation of A. lakoocha extract markedly enhanced cell viability at all time points, with the most pronounced effect observed at 48 hours. This finding is consistent with previous studies reporting the bioactive potential of phenolic and flavonoid compounds present in A. lakoocha, which exhibit antioxidant and anti-inflammatory activities conducive to cellular proliferation and wound healing31,38. Polyphenolic constituents such as Oxyresveratrol and stilbene derivatives, previously identified in A. lakoocha, are known to modulate oxidative stress pathways and promote cell survival through upregulation of pro-regenerative signaling cascades. These mechanisms may contribute to the observed enhancement in cell proliferation.

Immunofluorescence analysis provided valuable insights into the cytocompatibility and morphological behavior of NIH-3T3 cultured on cryogel matrices. Immunofluorescence imaging further corroborated the MTT results by demonstrating enhanced cell adhesion and spreading on A. lakoocha-enriched cryogels. Both control and extract–enriched cryogels supported fibroblasts attachment; however, cells on extract–enriched cryogels exhibited more pronounced F-actin organization and extensive lamellipodia formation, indicative of stronger substrate interactions and enhanced migratory potential14. Cytoskeletal integrity and lamellipodia extension are critical determinants of fibroblasts motility, a process essential for re-epithelialization during wound healing61. The improved cytoskeletal integrity on A. lakoocha-integrated cryogels may be attributed to the bioactive phytochemicals, such as Oxyresveratrol and flavonoids, known for their antioxidant and anti-inflammatory properties38. These compounds can modulate oxidative stress and inflammatory signaling, thereby promoting cell survival and enhancing actin dynamics31. Additionally, the formation of interconnected cellular networks observed in the extract-loaded group suggests improved cell–cell communication and tissue-like organization within the macroporous cryogel matrix11. These findings reflect the synergistic influence of cryogel microarchitecture, which supports oxygen and nutrient diffusion, and the bioactivity of incorporated phytochemicals that modulate cellular signaling12,14. Future studies should further investigate the molecular pathways regulating cytoskeletal rearrangement and intercellular communication in response to bioactive extract incorporation.

genAI

No artificial intelligence-based tools or generative AI technologies were used in this study. The entire content of the manuscript was originally prepared, reviewed, and approved by both authors.

Funding

The work was supported by Adana Alparslan Türkeş Science and Technology University Scientific Research Projects Coordination Unit (Grant Number: 24303002 | Recipient: Sibel ÖZDAŞ, PhD).

Conflict of interest statement

The authors declare that they have no conflict of interest.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Author contributions

Conceptualization: Sibel ÖZDAŞ (SÖ); Data curation: SÖ, Gözde Baydemir PEŞİNT (GBP)

Formal analysis: Ahmet Burak ALTUNSÖZ (ABA), İpek CANATAR (İC); Funding acquisition: SÖ; Investigation: ABA, SÖ, İC, GBP; Methodology: ABA, İC, SÖ; Project administration: ABA, SÖ; Resources: ABA, SÖ, İC, GBP; Software: ABA, İC; Validation: ABA, SÖ; Visualization: ABA, SÖ; Writing – original draft: ABA, SÖ, İC, GBP; Writing – review & editing: ABA, SÖ, İC, GBP