3D printed antibacterial and anti-inflammatory scaffold containing vanillin-loaded Soluplus nanomicelles for healing of infected wounds

The skin serves as a protective barrier, blocking exogenous and pathogenic microorganisms from entering the body. When severe damage occurs, these microorganisms can infiltrate the tissue, leading to infection. Infected wounds deviate from normal healing processes due to intense inflammation, causing delayed tissue repair. In critical cases, severe infection can result in tissue loss or even fatal outcomes¹. The most common approach to combating infections is the administration of antibiotics. However, oral administration or injection of antibiotics reduces bioavailability in the wound site, and their use is always limited due to the probability of bacterial resistance^2,3.

Beyond infection control, supporting the regeneration of damaged skin tissue is a vital clinical approach. In recent decades, advancements in skin tissue engineering have emerged to overcome the limitations of conventional wound treatment methods⁴. 3D printing is a pivotal technique in tissue engineering, enabling the rapid and precise fabrication of customized scaffolds with exceptional structural integrity and reproducibility⁵. Natural biomaterials, such as biopolymers, are favored in biomedical applications like wound healing due to their renewability, biodegradability, biocompatibility, and ability to be metabolized in the human body, while also minimizing immune response compared to synthetic alternatives⁶. Alginate (Alg) is a linear anionic polysaccharide derived from brown algae, consisting of β-1,4-linked D-mannuronic acid and β-1,4-linked L-guluronic acid units. The deprotonated carboxyl groups in α-L-guluronic acid provide a negative charge, facilitating cross-linking with divalent cations such as Ca²⁺. This unique chemical composition, characterized by excellent gelation properties and high viscosity, has positioned Alg as a promising material for biomedical applications, especially in the fabrication of 3D-printed tissue engineering constructs⁷. Additionally, Alg possesses hemostatic properties and enhances wound healing by modulating chemokine expression, promoting anti-inflammatory markers such as CXCL4, CCL3, CCL12, and CXCL12, while suppressing pro-inflammatory ones like CXCL5, CXCL8, CCL1, CCL2, CCL5, and CCL11^8,9. However, one significant drawback of Alg-based biomaterials in treating infected wounds is their lack of antibacterial properties¹⁰. Fucoidan (F), a sulfated biopolymer derived from brown algae, is both biodegradable and biocompatible. It demonstrates strong antibacterial properties and excellent cytocompatibility, contributing to wound healing by regulating inflammation through the modulation of pro-inflammatory factors and cytokine secretion. Moreover, F can play a crucial role in the early stages of wound repair by acting as an effective hemostatic agent^5,11.

As previously noted, conventional antibiotics frequently encounter the significant hurdle of bacterial resistance, while systemic administration compromises their bioavailability. To overcome the bioavailability challenges, antibacterial delivery systems have been seamlessly integrated with tissue-engineered constructs to enhance the effectiveness against infected wounds. Notably, natural products represent a rich source of novel compounds with potent antibacterial properties, which minimize the possibility of bacterial resistance¹². Vanillin (Vn) (4-hydroxy-3-methoxybenzaldehyde), a phenolic aldehyde extracted from vanilla orchid pods and commonly used as a flavoring agent in the food industry, exhibits favorable biocompatibility compared to other phenolic compounds¹³. Due to its phenolic groups and lipophilic nature, it is also recognized as a potent antibacterial agent against both Gram-negative and Gram-positive bacteria^14,15. However, the lipophilic nature of Vn poses challenges for its incorporation into hydrophilic polymer solutions. To facilitate the loading of lipophilic compounds in aqueous systems, amphiphilic nanocarriers are commonly utilized. Amphiphilic polymers, such as Soluplus® (Sol), can undergo self-assembly to form nanomicelles with a hydrophilic shell and a lipophilic core. Sol is a biocompatible copolymer composed of polyvinylcaprolactam, polyvinylacetate, and polyethylene glycol, where polyethylene glycol serves as the hydrophilic portion, while the polyvinylcaprolactam-polyvinylacetate side chains form the hydrophobic segments^16,17.

Building on the aforementioned points, this study focuses on the fabrication of a 3D-printed Alg-F scaffold incorporating Vn-loaded nanomicelles and evaluating its effectiveness in treating infected wounds. To the best of our knowledge, no prior research has examined the application of 3D-printed Alg-F scaffolds for wound healing, particularly the encapsulation of Vn within Sol nanomicelles and its integration into skin tissue engineering scaffolds. This innovative strategy presents a distinct approach to enhancing infected wound healing by integrating a 3D-printed alginate–fucoidan scaffold with vanillin-loaded nanomicelles as a targeted drug delivery system, thereby bridging a specific gap not addressed in the current literature.

Materials

Sodium alginate (Alg) was purchased from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China (Mw = 153,300 g/mol, the ratio of mannuronic acid (M blocks) to guluronic acid (G blocks), i.e., M/G ratio = 0.8). Soluplus® (Sol) was purchased from BASF Company (Ludwigshafen, Germany) and Vanillin (Vn) from Merck (Germany). Fucoidan (F) (≥ 85% purity, ∼72 kDa) and dimethyl thiazole diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich, USA. For animal studies, ketamine (Rotexmedica, Germany), and xylazine (Rompun®, Germany) were also obtained.

Synthesis and characterization of Vn-loaded nanomicelles

For the synthesis of nanomicelles, the thin film method similar to previous studies was used^16,18. Briefly, 17.25 g of Sol was dissolved in 100 mL of acetone in a round-bottomed flask at room temperature for 2 h. Then, under mechanical stirring, Vn was added to the Sol solution at a concentration of 10% w/w, and the resulting solution was stirred for another hour. The resulting solution was then placed in an oven at 40 °C overnight, allowing the solvent to evaporate off, forming a thin film at the bottom of the flask. The obtained film was subsequently hydrated with 10 mL of water, subjected to sonication for 10 min, and then stirred using a mechanical stirrer at 400 rpm for 20 min.

Field Emission Scanning Electron Microscopy (FE-SEM, FEI Quanta 450, USA) was used for evaluation of nanomicelles morphology. To prepare the sample, 20 µL of the nanomicellar suspension was deposited onto a silicon wafer chip and left to air dry at ambient temperature. Subsequently, the sample was affixed to SEM stubs using double-sided adhesive tape. Before imaging, the samples were coated with a thin layer of gold via sputtering under vacuum conditions. Finally, Image J software was used to measure the size of nanomicelles.

Fabrication of the scaffolds

For the fabrication of scaffolds via 3D printing, a 10% w/v solution of Alg in distilled water was first prepared at 40 °C for 4 h. Additionally, 10% wt of F and 5% wt of Vn-loaded nanomicelles (VnNMs) were mixed with the Alg solution for 2 h to obtain a homogeneous ink suitable for the 3D printing process. The polymer concentrations were selected based on printability in pilot experiments as well as previous studies.

The scaffolds were fabricated using an extrusion-based 3D bioprinter (Abtin III- Abtin Teb Fanavar, Iran) and designed from an STL file created via computer-aided design (CAD). Initially, the as-prepared bioinks (Alg, Alg-F, and Alg-F-VnNMs) were loaded into the printer’s reservoir. The scaffolds, measuring 20 × 20 × 3 mm, were printed with alternating layers oriented at 0° and 90°, utilizing a 300 µm inner diameter nozzle at a speed of 6 mm/s under ambient conditions. Upon completion of printing, the scaffolds were promptly immersed in a 0.25 molar calcium chloride solution for 24 h, followed by rinsing with distilled water and undergoing freeze-drying¹⁹. Consequently, the Alg, Alg-F, and Alg-F-VnNMs scaffolds were successfully fabricated.

Characterization of the scaffolds

The 3D-printed scaffolds were coated with a thin layer of gold using a sputter coater for imaging via scanning electron microscopy (SEM) and morphological analysis. Imaging was conducted at an accelerating voltage of 20 kV. The diameter of strands and channels was measured using ImageJ software.

To evaluate the scaffolds’ swelling behavior, 10 × 10 mm samples were first weighed to determine their initial dry weight (W₀) and then submerged in 50 mL of distilled water at 37 °C. At intervals of 12, 24, 36, 48, 60, and 72 h, the scaffolds were removed, excess surface water was wiped out, and they were reweighed to obtain their swollen weight (W_S). Equation (1) was employed to calculate the swelling²⁰, and three independently fabricated scaffolds were considered as replicates:

Biodegradability

To evaluate the biodegradation process, 10 × 10 mm scaffolds were fabricated for testing. Initially, the samples were weighed to determine their baseline dry weight (W₁) before being immersed in 10 mL of phosphate-buffered saline (PBS) and maintained at 37 °C for 1, 3, 5, and 7 days. At predetermined time intervals, the scaffolds were removed and subjected to freeze-drying. Their post-degradation dry weight (W₂) was precisely measured using a high-precision 5-decimal laboratory balance. The degradation (weigh loss) of the scaffolds at each time point was calculated utilizing Eq. (2)¹⁹, and three scaffold samples were used as replicates:

Vn encapsulation efficiency and release

To determine the encapsulation efficiency, 10 mg of VnNMs was dissolved in 20 mL of acetone at room temperature for 48 h. Subsequently, a sample of the medium containing Vn was collected, and the Vn concentration in the medium was measured using UV–Vis spectrophotometry at a wavelength of 231 nm²¹. The encapsulation efficiency of Vn in nanomicelles was then calculated using Eq. (3):

To evaluate the release of Vn from the Alg-F-VnNMs scaffold, 10 × 10 mm samples were incubated in 20 mL of PBS solution for 1, 3, 5, and 7 days. At each specified time point, 3 mL of the medium was withdrawn and replaced with fresh PBS. The Vn concentration in the collected medium was measured using a UV–Vis spectrophotometer at a wavelength of 231 nm²¹, and a cumulative release profile of Vn was plotted. Each sample group was evaluated in triplicate.

Assessment of antibacterial efficacy

The antibacterial activity of the engineered scaffolds was examined using the inhibition zone method against Staphylococcus aureus MTCC 1688 (S. aureus, Gram-positive) and Escherichia coli DH5 alpha (E. coli, Gram-negative). For this assessment, the scaffolds (10 × 10 mm) were placed on agar inside a Petri dish containing 15 μL of bacterial suspension at a concentration of 1 × 10⁵ CFU/mL. After incubating the samples at 37 °C for 24 h, the inhibition zones formed around the scaffolds were visually inspected and recorded through photography.

To ensure accurate assessment of antibacterial effectiveness, the antibacterial properties of the scaffolds were also evaluated against S. aureus and E. coli following the protocol outlined by Rafieerad et al.²². Each scaffold, with a size of 1 cm², was placed in the wells of a 24-well culture plate, and 1 mL of bacterial suspension at a concentration of 10⁸ CFU/mL was introduced. The samples were then incubated for 24 h. After incubation and subjecting the samples to ultrasonic vibration for 10 min, the remaining viable bacteria were quantified using the serial dilution method. The antibacterial activity was subsequently determined using Eq. (4):

In this equation, “B” represents the count of viable bacteria in the control well, which lacks scaffolds, while “A” denotes the number of viable bacteria present in the well containing scaffold within the culture medium. Antibacterial assessments were performed with three replicates per group.

In vitro cytocompatibility evaluation

For the in vitro cell experiments, Human keratinocyte (HaCaT) cells (sourced from the Pasteur Institute, Iran) were expanded in T75 culture flasks using DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, maintaining incubation conditions until the cell layer reached approximately 70% confluency. To prepare the scaffolds for cell-based studies, samples measuring 10 × 10 mm were fabricated and subsequently sterilized via UV irradiation, exposing each side for 20 min before conducting biological assessments. All sample groups were tested using three independent replicates.

Cytotoxicity analysis was carried out using the MTT assay. In this procedure, approximately 2 × 10⁵ cells were seeded directly onto the scaffolds, which had been positioned within a 24-well culture plate. At days 1 and 7 post-seeding, MTT reagent (5 mg/mL) was introduced into each well, followed by a 4-h incubation period to allow the formation of formazan crystals. The resulting crystals were solubilized using DMSO, and the optical absorbance was recorded at 570 nm using a microplate reader, providing quantitative insights into cell viability.

Cell adhesion assay was performed to evaluate the interaction between HaCaT cells and the scaffold surfaces. First, 2 × 10⁵ cells were seeded onto the scaffolds and maintained in standard culture conditions for 24 h, allowing sufficient time for cell adhesion and initial interactions with the material. Following the incubation period, the cell-seeded scaffolds underwent fixation using a 3% glutaraldehyde solution at a controlled temperature of 4 °C for 2 h^23,24,25. This fixation process preserved cellular morphology and attachment integrity, ensuring that cell-scaffold interactions remained stable for further evaluation. Next, the samples were subjected to a graded ethanol dehydration process, where increasing concentrations of ethanol were sequentially applied to gently remove cellular moisture, preventing structural alterations. Once dehydration was complete, the samples were left to dry under ambient conditions at room temperature, maintaining their stability for imaging. Finally, the dried scaffolds were examined using SEM to capture images of cell attachment, morphology, and interaction with the scaffold surfaces.

Hemolysis and blood clotting time assays

To assess the blood compatibility of the developed scaffolds, a hemolysis test was conducted in accordance with ASTM F756 guidelines. Freshly collected blood samples were transferred into EDTA-coated tubes and centrifuged at 2000 rpm for 8 min to separate plasma, which was subsequently discarded. The remaining red blood cell (RBC) pellet was then diluted in phosphate-buffered saline (PBS) at a 1:9 ratio. In parallel, scaffold specimens measuring 10 × 10 × 3 mm were immersed in normal saline and incubated at 37 °C for 30 min to equilibrate. Each scaffold was then placed into individual wells of a 24-well plate, followed by the addition of 1 mL of the prepared RBC suspension. The samples were incubated at 37 °C for 1 h to allow interaction. After incubation, the supernatant was centrifuged under the same conditions, and the absorbance of the supernatant was measured at 540 nm to quantify hemoglobin release. For reference, 1% Triton X-100 (100 μL) served as the positive control (representing complete hemolysis), while PBS (100 μL) was used as the negative control (indicating no hemolysis). The percentage of hemolysis was calculated using Eq. (5)²⁶:

where As, An, and Ap represent the absorbance values of the test sample, negative control, and positive control, respectively.

Blood clotting time was determined by monitoring the cessation of blood flow following recalcification²⁷. In brief, 2 mL of whole blood anticoagulated with 10% (v/v) sodium citrate was added to glass tubes containing 10 mg of each test sample. Medical gauze was used as the control. To initiate coagulation, 60 μL of calcium chloride solution (0.25 M) was introduced into each tube. The tubes were gently tilted every 15 s, and the clotting time was recorded as the point at which blood flow visibly stopped. All experiments were performed in triplicate, and average values were reported.

In vivo animal studies

Four months old Wistar rats, with an average weight of 300 ± 50 g, were obtained from the animal nest of the Faculty of Biological Sciences, Mustansiriyah University, Baghdad, Iraq, and housed in individual cages under standard room temperature conditions (~ 25 °C), maintaining a 12-h light/dark cycle. They had unrestricted access to standard rodent chow and water. All animal experiment procedures were in accordance with EU Directive 2010/63/EU and ARRIVE guidelines, and the experimental methods were approved by the Animal Care Committee of the Iraqi National Center for Drugs Control and Research. A total of 24 rats were randomly assigned to four groups (six rats in each group): control (untreated), Alg, Alg-F, and Alg-F-VnNMs. A random number generator was used to allocate animals to each group, ensuring equal distribution and minimizing selection bias. To induce anesthesia, ketamine (80 mg/kg) and xylazine (10 mg/kg) were administered via intraperitoneal injection. Prior to surgery, the fur was removed to ensure a clear working area. Once full anesthesia was achieved, a full-thickness wound was created using a biopsy punch with a diameter of 10 mm on the dorsal skin of each rat. The fabricated scaffolds were then applied to the wounds, while an untreated wound was maintained as a control. The applied scaffolds remained in place throughout the 14-day study period and were not replaced. They were secured over the wound sites using sterile gauze dressings, which were changed every 3 days under aseptic conditions, while the scaffolds themselves remained undisturbed. In the control group, the untreated wounds were covered with a simple sterile gauze dressing. The dressing was changed every 3 days under aseptic conditions. Prior to applying the new gauze, the wound area was gently rinsed with sterile normal saline to maintain cleanliness and reduce the risk of infection. After 14 days, the wound surfaces were imaged for calculation of wound closure rate, and the animals were euthanized with 100% carbon dioxide inhalation using a CO₂ chamber and the wound tissue was harvested and fixed in 10% formalin, followed by paraffin embedding and sectioning at a thickness of 5 μm. Subsequent histopathological examinations were carried out using hematoxylin and eosin (H&E) staining as well as Masson’s trichrome staining. The stained tissue sections were then imaged under a light microscope and the histopathological evaluation was performed by an independent pathologist who was blinded to the treatment groups to minimize assessment bias. Quantitative analysis of wound healing parameters was performed using ImageJ software. The percentage of re-epithelialization was calculated from histological sections stained with H&E by measuring the length of newly formed epithelium relative to the total wound width. Collagen deposition was quantified from Masson’s Trichrome-stained sections by calculating the collagen-positive area as a percentage of the total tissue area.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

The quantitative real-time PCR (qRT-PCR) method was employed to assess the expression levels of transforming growth factor-β (TGF-β), a gene involved in cellular proliferation and tissue regeneration, alongside tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which function as inflammatory cytokines. To extract total RNA, tissue samples were homogenized and processed using TRIzol reagent, following the manufacturer’s protocol. The quantity and purity of the isolated RNA were evaluated via spectrophotometric analysis (Nanodrop 2000, Thermo Scientific, USA). For cDNA synthesis, the iScript™ cDNA Synthesis Kit (Bio-Rad) was utilized. Gene expression levels were subsequently quantified using qRT-PCR, performed on a real-time PCR system (Bio-Rad) with three replicates for each sample. The housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene, ensuring normalization of gene expression measurements. The primer sequences applied in this study are listed in Table 1²⁸.

Statistical analysis

In this study, all numerical values are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 8.0). For comparisons between two groups, an unpaired Student’s t-test was used to assess statistical differences. For comparisons involving more than two groups, one-way ANOVA was applied to detect significant variations, followed by Tukey’s post-hoc test to correct for multiple comparisons. Statistical significance was set at p ≤ 0.05. Accordingly, *, **, ***, and **** denote p-values of ≤ 0.05, ≤ 0.01, ≤ 0.001, and ≤ 0.0001, respectively.

Characterization

In this research, Sol nanomicelles incorporating Vn were synthesized via the thin film method for integration into 3D-printed scaffolds. Figure 1 illustrates the FESEM image of these nanomicelles. As observed, the Sol nanomicelles containing Vn exhibited a perfectly spherical morphology, with no indications of agglomeration or aggregation. Based on image, their average diameter was determined to be 80.2 ± 11.9 nm. A similar structural characteristic was reported in a related study by Piazzini et al.¹⁶ for Sol-based nanomicelles.

FE-SEM image of Vn-loaded Sol-based nanomicelles.

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SEM images of the 3D-printed scaffolds are presented in Fig. 2a. These mesh-like structures are fabricated through a repetitive arrangement of perpendicular strands across multiple layers. All scaffolds possess open and interconnected channels and demonstrate a relatively uniform morphology. The strand diameters measured for the Alg, Alg-F, and Alg-F-VnNMs scaffolds were 413.7 ± 26.4 µm, 289.2 ± 64.2 µm, and 275.2 ± 82.1 µm, respectively. Also, the channel diameters for these scaffolds were determined to be 369.8 ± 37.0 µm, 684.5 ± 28.8 µm, and 656.2 ± 44.1 µm, respectively. Consequently, scaffolds incorporating F exhibited significantly thinner strands and wider channels compared to the Alg scaffold (p < 0.001). This effect is likely due to an increase in the polymer solution concentration and the viscosity of the printing ink following the addition of F. As the viscosity of the 3D printing ink rises, the nozzle output diminishes under constant pressure, leading to the formation of thinner strands and larger channels²⁹. It is worth noting that the addition of nanomicelles did not cause a significant change in the structure of the Alg-F-VnNMs scaffold compared to the Alg-F scaffold.

(a) SEM images showcasing the microstructure of the fabricated scaffolds, (b) swelling behavior of the scaffolds in distilled water, (c) weight loss of the scaffolds due to degradation in PBS, and (d) release profile of Vn from the Alg-F-VnNMs scaffold in PBS.

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Wound exudates, the secretion of tissue fluid following an injury, facilitate the transfer of biochemical substances and foster a suitable environment for wound recovery. However, an overabundance of wound exudates can extend the inflammatory phase and impede recovery, especially in cases of chronic wounds³⁰. The swelling behavior of the fabricated scaffolds was evaluated based on their water uptake ability over 72 h (Fig. 2b). All scaffolds exhibited a significantly increasing swelling trend in a similar manner, with the distinction that equilibrium swelling was achieved for the Alg scaffold after 60 h and for the Alg-F and Alg-F-VnNMs scaffolds after 48 h. Accordingly, the highest swelling rate was measured at 349.7 ± 21.6% for the Alg scaffold, while for the Alg-F and Alg-F-VnNMs scaffolds, the swelling rates were 277.0 ± 18.1% and 294.3 ± 24.1%, respectively. The significantly higher swelling capacity of the Alg scaffold compared to the others may be attributed to its thicker strands and greater water absorption ability. After reaching equilibrium swelling, the swelling of the Alg scaffold remained nearly constant (with a slight decrease) until 72 h, whereas the swelling of the Alg-F and Alg-F-VnNMs scaffolds noticeably declined until 72 h. This reduction may be due to the degradability of these scaffolds affecting the swelling rate.

Biodegradability

A skin tissue scaffold must maintain sufficient stability to support the adhesion and colonization of native cells across its surface and within its structure, while degrading at an appropriate rate. This controlled degradation facilitates the release of loaded molecules and enables the regeneration of native tissue in a sequential manner³¹. According to Fig. 2c, all fabricated scaffolds experienced a progressive weight reduction over 7 days of immersion in PBS. The degradation of the Alg, Alg-F, and Alg-F-VnNMs scaffolds after 7 days was measured to be 21.4 ± 2.1%, 33.5 ± 4.4%, and 38.0 ± 2.25%, respectively. Based on the results, the incorporation of F and Sol-based nanomicelles into the Alg scaffold led to an increase in biodegradability. Following the cross-linking of the scaffolds, covalent cross-links between calcium ions and guluronic acid blocks in alginate enhanced the stability of the scaffolds³², however, F and nanomicelles, as additives, did not participate in such linkages; therefore, the degradation rate of the Alg-F and Alg-F-VnNMs scaffolds was higher than that of the scaffold made from pure Alg.

Vn release profile

In this study, the encapsulation of Vn in Sol nanomicelles was carried out to achieve stability and optimal dispersion of Vn in the hydrophilic Alg-based solution. The encapsulation efficiency of Vn in Sol nanomicelles was measured to be 64.8 ± 3.9%. Figure 2d represents the release profile of Vn from the Alg-F-VnNMs scaffold. The release of Vn on the first day was recorded as 17.5 ± 4.1%. Following a sustained manner, the release of Vn reached 80.6 ± 5.3% by day 7. The two primary factors contributing to the release of Vn from the Alg-F-VnNMs scaffold were degradation and swelling, accompanied by the elution of nanomicelles. A controlled and sustained release of the loaded antibacterial agent within the skin tissue scaffold at the wound site is considered a clinical advantage, as it enhances bioavailability and effectiveness while reducing side effects³³.

Assessment of antibacterial efficacy

The antibacterial properties of the fabricated scaffolds were examined through the inhibition zone method and bacterial viability assessment. Figure 3a displays images of the inhibition zones formed by different scaffolds against S. aureus and E. coli. As observed, the Alg scaffold, due to its lack of antibacterial properties, did not produce any inhibition zone against the bacteria. In contrast, the Alg-F and Alg-F-VnNMs scaffolds led to inhibition zones of 17.8 ± 2.1 mm and 21.4 ± 1.15 mm against S. aureus, and 17.9 ± 2.0 mm and 23.2 ± 0.9 mm against E. coli, respectively (Fig. 3b). Figure 3.c depicts the antibacterial activity of different scaffolds in contact with S. aureus and E. coli. Consistent with the results of the inhibition zone test, the Alg scaffold exhibited no antibacterial properties against the bacteria, whereas the Alg-F and Alg-F-VnNMs scaffolds demonstrated considerable antibacterial efficacy. Notably, the scaffold containing VnNMs showed stronger antibacterial activity compared to the Alg-F scaffold, both against S. aureus (p < 0.05) and E. coli (p < 0.001). Stroescu et al.²¹ prepared chitosan-Vn films and demonstrated that the resulting films produced an inhibition zone of 11 ± 1.3 mm against E. coli after 24 h of incubation. Also, Ibrahim et al.³⁴ developed a chitosan/polyvinyl alcohol/vanillin hydrogel loaded with L-arginine and reported that the fabricated hydrogel exhibited an inhibition zone of 31.32 ± 3.87 mm against S. aureus and 21.92 ± 5.21 mm against E. coli after 24 h of incubation. Therefore, these literature-reported inhibition zone values show a comparable trend to those observed in the current study.

(a) Images of the inhibition zones generated by different scaffolds against S. aureus and E. coli, (b) inhibition zone diagram, and (c) antibacterial activity of the scaffolds against S. aureus and E. coli.

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The antibacterial effect of the Alg-F scaffold was solely attributed to the presence of F. The antibacterial properties of F are closely linked to glycoprotein receptors found on the surface of polysaccharides. These receptors exhibit a remarkable ability to bind with key bacterial structures—including the cell wall, cytoplasmic membrane, and DNA—ultimately disrupting essential bacterial functions and inhibiting growth³⁵. It was revealed that F possess the ability to hinder the growth of both Gram-positive and Gram-negative bacteria, showcasing their potent and wide-ranging antibacterial efficacy³⁶. On the other hand, the release of Vn was responsible for the stronger antibacterial activity of Alg-F-VnNMs scaffold compared to Alg-F scaffold. This plant-derived phenolic formaldehyde compound causes antibacterial activity by pore-forming and disrupting the integrity of the bacterial membrane, as well as inhibiting energy metabolism^13,15. It has been demonstrated that Vn can act synergistically with other antibacterial agents, enhancing their effectiveness and leading to bacterial eradication^12,14. Therefore, the enhanced antibacterial activity of the Alg-F-VnNMs scaffold compared to other scaffolds is entirely justified. In this study, antibacterial activity was assessed against two bacterial strains, S. aureus and E. coli, which are commonly involved in wound infections. However, to better understand the broader antibacterial potential in wound healing applications, further evaluation against a more diverse set of pathogenic strains is needed.

In vitro cytocompatibility evaluation

The HaCaT cells viability and their adhesion to the scaffold surfaces were evaluated as indicators of cytocompatibility. The MTT assay results, shown in Fig. 4a, demonstrate that all scaffolds exhibited appropriate cellular compatibility, with no scaffold-induced cytotoxicity observed. Overall, the Alg-F and Alg-F-VnNMs scaffolds showed higher levels of cell viability and proliferation compared to the Alg scaffold (p < 0.05). On day 1, there was no significant difference in cell viability between the Alg-F and Alg-F-VnNMs scaffolds (p > 0.05); however, after 7 days, cell viability on the Alg-F-VnNMs scaffold increased significantly compared to the Alg-F scaffold (p < 0.05).

(a) Viability assessment of HaCaT cells cultured on different scaffolds for 1 and 7 days, and (b) SEM images illustrating HaCaT cell adhesion on scaffolds after 24 h of culture.

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Morphological analysis of cultured cells after 24 h on the scaffolds was performed using SEM imaging, with the results presented in Fig. 4b. Based on the findings, cells cultured on the scaffolds are observable at varying densities with almost spherical morphology. Weak cellular adhesion occurred on the Alg scaffold, which is consistent with other studies that have also reported poor cell adhesion on pure Alg surfaces^37,38. SEM images indicate that cells adhered to the surfaces of Alg-F and Alg-F-VnNMs scaffolds with higher density and broader coverage. Interestingly, cellular expansion appeared to be more prominent on the Alg-F-VnNMs scaffold compared to the Alg-F scaffold.

The increase in surface hydrophilicity with the incorporation of F³⁹ and the role of F as a preventive agent against cell apoptosis induced by oxidative stress⁴⁰ were the two key factors contributing to the enhanced cellular behavior in the Alg-F scaffold compared to the Alg scaffold. According to Ryo et al.⁴¹, F mitigates oxidative stress in HaCaT cells by modulating the expression of heme oxygenase-1 (HO-1) and superoxide dismutase-1 (SOD-1). This regulation is mediated through the Nrf2/ERK signaling pathway, which plays a crucial role in cellular defense mechanisms against oxidative damage. Furthermore, it has been reported that Vn (in non-toxic concentrations) not only promotes stemness and self-renewal in HaCaT keratinocytes by regulating the expression of Oct-4, Nanog, and phosphorylated Oct-4 (p-Oct-4), thereby facilitating their regenerative capacity and long-term maintenance, but also enhances the expression of E-cadherin in HaCaT cells, allowing them to tightly integrate with surrounding cells and more effectively receive signals from their niches⁴². Thus, the improved HaCaT cell behavior observed on the Alg-F-VnNMs scaffold compared to other scaffolds was a predictable outcome, attributed to the controlled release of Vn from this scaffold.

Hemolysis and blood clotting time assays

The hemolytic potential of the 3D-printed scaffolds was assessed to evaluate their compatibility with blood, and the corresponding data are presented in Fig. 5a. Hemolysis was quantified by measuring the amount of hemoglobin released, which reflects the extent of red blood cell membrane damage. According to established criteria, materials are generally considered blood-compatible if their hemolysis rate remains below 5%⁴³, with some reports suggesting a stricter threshold of 2%²⁶. In the present study, all scaffold samples exhibited hemolysis rates below 2%, indicating excellent hemocompatibility and classifying them as non-hemolytic.

Bar graph representation of blood–scaffold interaction assessments: (a) Hemolysis assay results reflecting the hemocompatibility of the fabricated scaffolds, and (b) Blood clotting time assay results.

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As illustrated in Fig. 5b, the 3D-printed scaffolds demonstrated a notable enhancement in blood coagulation efficiency compared to the control group (sterile gauze). The clotting times recorded for the Alg, Alg-F, and Alg-F-VnNM scaffolds were 238.3 ± 14.7, 252.6 ± 9.0, and 256.6 ± 15 s, respectively, substantially shorter than the clotting time observed for control (291.3 ± 13.2 s). The reduced clotting time observed for the scaffolds relative to the control can be attributed to multiple factors, including the release of Ca²⁺ ions from calcium chloride used as a crosslinking agent during scaffold fabrication, which may promote coagulation upon blood contact, and the enhanced platelet adhesion supported by the scaffold’s high biocompatibility. Importantly, the reduction in clotting time was significantly greater for the Alg scaffold compared to the Alg-F and Alg-F-VnNMs scaffolds (p < 0.05), likely due to the presence of fucoidan in these scaffolds. Fucoidan contains sulfated moieties known to impart anticoagulant properties, thereby attenuating coagulation⁴⁴.

In vivo animal studies

In vivo animal studies were conducted to evaluate full-thickness wound healing following the application of the fabricated scaffolds in a rat model over a 14-day period. On day 14 post-injury, the wound sites in different rats were photographed to assess the macroscopic appearance and quantify wound closure following the creation of circular excisional wounds with a diameter of 10 mm (Fig. 6a,b). All wounds demonstrated typical healing characteristics, with no visible abnormalities in color, texture, or surrounding tissue. Quantitative analysis of wound closure revealed a statistically significant enhancement in healing in the Alg and Alg-F groups compared to the control group (p < 0.05 and p < 0.01, respectively). Remarkably, the Alg-F-VnNMs group exhibited a pronounced pro-healing effect, outperforming all other groups. By day 14, approximately 95.8 ± 2.8% of the wound area had closed in this group, indicating a highly accelerated regenerative response.

(a) Representative macroscopic images of wound sites in rats on day 14 post-injury, illustrating the visual appearance of healing across experimental groups, and (b) quantitative analysis of wound closure percentage at day 14.

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The histopathological results of the animal study are presented in Fig. 7. In Fig. 7a related to H&E-stained tissue sections, the essential histological attributes, including the keratin (Kr) layer, epidermis (Ep.D), dermis (D), hypodermis (H.D), regenerated area (R.A), neovascularization area (N.V), hair follicle (H.F), sebaceous gland (Sb.G) and sweat gland (Sw.G) are clearly marked in the images for each group. Also, inflammatory cells (If.C) were indicated by arrows. In the control group, Ep.D and Kr layers did not form after 14 days and are not visible in the images, and only a wound crust and large areas of N.V along with a lot of If.Cs are recognizable. In the group treated with the Alg scaffold, the Ep.D layer developed but lacked structural integrity, while the N.V regions were smaller and If.Cs were fewer compared to those in the control group. All three layers of H.D, D, and Ep.D were well formed in the groups treated with the Alg-F and Alg-F-VnNMs scaffolds, the N.V. areas and If.Cs were greatly reduced, and even H.Fs can be detected in the tissue slice images of these groups. However, the formation of an integrated and developed Kr layer, negligible number of If.Cs, and the presence of other skin appendages, including Sb.G and Sw.G, confirms the more complete wound healing of the Alg-F-VnNMs group compared to the Alg-F group after 14 days. A noteworthy point is that balancing angiogenesis and regression is essential for proper tissue repair. In the final stages of healing process, apoptosis in capillaries drives vascular regression, restoring density to normal levels. While early angiogenesis supports healing, delayed regression increases the risk of hypertrophic scars⁴⁵. Therefore, the presence of extensive N.V areas in tissue sections of the control and Alg groups after 14 days indicates that the natural healing process has not been completed or that the healing has been delayed. In this study, the first H.F reaching the skin surface was considered as the R.A boundary, and the distance between the R.As on both sides of the wound actually indicates the unrepaired area. In the control group, there was no R.A with the mentioned features. The diameter of unrepaired area for the Alg, Alg-F, and Alg-F-VnNMs groups was measured to be 2674.87, 1628.32, and 258.89 µm, respectively, showing almost complete regeneration in the Alg-F-VnNMs group. Also, quantitative analysis of re-epithelialization based on H&E-stained tissue sections in Fig. 7b demonstrated a markedly improved epithelial coverage in all treatment groups compared to the control. The highest level of re-epithelialization was observed in the group treated with the Alg-F-VnNMs scaffold, showing a statistically significant enhancement relative to the control (p < 0.0001).

Histological evaluation and quantitative analysis of wound healing following a 14-day in vivo examination: (a) H&E-stained tissue slices (some inflammatory cells are marked with arrows), (b) quantitative analysis of re-epithelialization, (c) Masson’s trichrome-stained tissue slices, and (d) quantification of collagen content per area.

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As depicted in Fig. 7c, the tissue sections of various groups stained with Masson’s trichrome reveal collagen deposition and maturation, distinctly visualized in blue. Collagen deposition in the control group lacks organization, whereas in the R.As of the Alg group, collagen bundles exhibit increased thickness compared to the control. Quantitative assessment of collagen deposition per area, based on Masson’s trichrome-stained sections, revealed significantly higher collagen accumulation in all treatment groups compared to the control (Fig. 7d). Collagen content in the Alg-F group was significantly greater than that in the Alg group (p < 0.05), while the Alg-F-VnNMs group exhibited even higher levels than Alg-F (p < 0.05). Accordingly, the Alg-F-VnNMs group demonstrated the most pronounced collagen synthesis among all experimental groups. As can be seen in the tissue sections stained with Masson’s trichrome, the N.V. regions in both the control and Alg treated groups appear distinctly visible. Although N.V regions remain visible in the Alg-F treated group, their presence is far less distinguishable in the Alg-F-VnNMs group. Also, the Alg-F and Alg-F-VnNMs groups exhibit a markedly greater density and more structured arrangement of collagen bundles compared to both the control and Alg groups. These findings indicate that the Alg-F-VnNMs group successfully achieved almost full wound healing within 14 days.

The expression of TGF-β, TNF-ɑ, and IL-1β genes in wound tissues were assessed via qRT-PCR and the results are presented in Fig. 8. The role of TGF-β in wound healing is essential, as it governs keratinocyte cell cycle regulation, facilitates re-epithelialization, and orchestrates key processes such as angiogenesis, inflammation, and granulation tissue development⁴⁶. All treated groups had higher levels of TGF-β expression than the control group. The level of TGF-β expression in the Alg-F and Alg-F-VnNMs groups was higher than that in the Alg group, and the highest level of TGF-β expression was associated with the Alg-F-VnNMs scaffold-treated group. The expression of pro-inflammatory cytokines TNF-ɑ, and IL-1β was also significantly lower in the treated groups than in the control group. The Alg-F scaffold significantly reduced the expression of pro-inflammatory genes compared to the Alg scaffold, and the lowest expression of these pro-inflammatory genes was related to the group treated with the Alg-F-VnNMs scaffold.

Quantitative analysis of TGF-β, TNF-ɑ, and IL-1β genes’ expression at the wound tissues in different groups.

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It is reported that F exhibits promising therapeutic potential for treating wounds and burns, as its heparin-like properties stimulate TGF-β. Additionally, it plays a crucial role in facilitating fibroblast migration and activation within injured tissue⁴⁷. It has also been established that the anti-inflammatory mechanism of F is based on the downregulation of inflammatory mediators such as IL-1β, IL-6, IL-8, TNF-α, nitric oxide (NO), prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), while simultaneously upregulating interferon gamma (IFN-γ) and IL-10, thereby playing a crucial role in modulating inflammatory pathways⁴⁸. On the other hand, studies have confirmed that Vn effectively suppresses pro-inflammatory cytokines such as IL-1β and TNF-α, while simultaneously enhancing the secretion of IL-4, IL-10, and TGF-β⁴⁹. This dual action reinforces its anti-inflammatory properties and contributes to wound healing acceleration. de Aragão Tavares et al.⁵⁰ demonstrated that the presence of Vn in a chitosan-based membrane played a pivotal role in modulating gene expression in diabetic wound tissues by suppressing TNF-α and IL-1β, while concurrently upregulating TGF-β, thereby fostering a more conducive environment for tissue regeneration.

These results highlight the effectiveness of the Alg-F-VnNMs scaffold in wound healing and skin tissue regeneration, demonstrating its superior performance compared to other scaffolds assessed within a 14-day evaluation period. This enhanced performance is attributed to the synergistic effect of the presence of F and Vn, which significantly contributed to the accelerated repair process.

In this study, a 3D-printed Alg-F-VnNMs scaffold was developed for wound management and skin repair. The scaffold exhibited favorable swelling and degradation behavior, enabling controlled Vn release over seven days. The synergistic effects of F and Vn contributed to potent antibacterial activity without inducing cytotoxicity, while also promoting cellular adhesion and proliferation. Histopathological and molecular analyses confirmed accelerated wound healing, reduced inflammation, and improved organized tissue regeneration, including collagen deposition and skin appendage formation. These results underscore the Alg-F-VnNMs scaffold’s potential in skin tissue engineering and regenerative medicine.

Despite the promising outcomes, this study has certain limitations that warrant consideration. The antibacterial assessment was restricted to two bacterial strains, and the in vivo experiments were conducted using a non-infected wound model. Moreover, several evaluations including drug release and biodegradability were performed under in vitro conditions, which may not fully replicate the complexity of in vivo environments. Looking ahead, addressing these limitations through broader pathogen profiling, infected wound models, and clinical validation will be essential to fully assess the translational impact of this platform. The customizable nature of the scaffold offers promising avenues for personalized treatment strategies in complex wound healing scenarios.

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Authors and Affiliations

1. Department of Clinical Laboratory Sciences, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq

   Zainab Farooq Shafeeq & Fitua Al-Saedi

2. Department of Microbiology, College of Science, Al Karkh University for Science, Baghdad, Iraq

   Eslah Shakir Rajab

3. Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq

   Mastafa H. Al-Musawi

4. Human Anatomy Department, College of Medicine, Mustansiriyah University, Baghdad, Iraq

   Farah E. Ismaeel

5. Department of Textile Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

   Shima Najafi

6. Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran

   Maryam Vesal

7. Department of Pathology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

   Salar Nasr Esfahani

8. Center for Advanced Materials and Structures, School of Science and Technology, The University of Georgia, 0171, Tbilisi, Georgia

   Fariborz Sharifianjazi

9. Department of Civil Engineering, School of Science and Technology, The University of Georgia, 0171, Tbilisi, Georgia

   Fariborz Sharifianjazi

10. School of Medicine, Georgian American University, 10 Merab Aleksidze Str., 0160, Tbilisi, Georgia

    Ketevan Tavamaishvili

11. Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran

    Marjan Mirhaj & Mohamadreza Tavakoli

Contributions

Conceptualization: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., M.V., S.N.E., F.Sh., K.T., M.M., M.T. Funding acquisition: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., F.Sh., K.T. Data curation: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., M.V., S.N.E., F.Sh., K.T., M.M., M.T. Visualization: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., M.V., S.N.E., F.Sh., K.T., M.M., M.T. Investigation: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., M.V., S.N.E., F.Sh., K.T., M.M., M.T. Validation: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., M.M., M.T. Formal analysis: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., M.M., M.T. Methodology: Z.F.Sh., F.Al-S., E.Sh.R., M.H.Al-M., F.E.I., Sh.N., M.V., S.N.E., F.Sh., K.T., M.M., M.T. Supervision: M.T., M.H.Al-M., M.M. Resources: M.T., M.H.Al-M., M.M., F.Sh., K.T. Project administration: M.T., M.M. Software: Sh.N., M.V., S.N.E., M.M. Writing—original draft: Z.F.Sh., F.Al-S. Writing—review and editing: M.T., M.H.Al-M., M.M.

Corresponding authors

Correspondence to Mastafa H. Al-Musawi, Marjan Mirhaj or Mohamadreza Tavakoli.

Competing interests

The authors declare no competing interests.

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