SSP–CG scaffolds: a synergistic approach to enhance wound healing and tissue repair

Abstract

The development of advanced biomaterials with multifunctional properties is essential to address the complex challenges of impaired wound healing and tissue regeneration. This study introduces a novel composite scaffold (SSP–CG), in which silk sericin (SS) and polyvinyl alcohol (PVA) form the SSP component, while copper nanoparticles (CuNPs) and gallic acid (GA) constitute the CG component. SS provides biocompatibility and biodegradability, while PVA enhances structural integrity. CuNPs and GA impart antimicrobial and antioxidant activity, respectively, making the scaffold highly suitable for biomedical applications. The scaffold features an optimal pore size (96 ± 19 μm) and pore volume, promoting cell infiltration and nutrient diffusion. In vitro degradation studies revealed a controlled, sustained profile over 6 weeks, ideal for long-term therapeutic use. A gradual and prolonged release of GA ensured continuous antioxidant activity, confirmed by a DPPH assay showing significant free radical scavenging activity (40.5 ± 2.1%). In vitro studies further confirmed excellent biocompatibility, with optimal cell adhesion, proliferation, and viability while maintaining the environment for tissue regeneration. In vivo studies demonstrated superior wound healing outcomes for the SSP–CG scaffold compared to both positive and negative controls, with histological analysis further confirming enhanced tissue regeneration and reduced inflammation. This first-of-its-kind integration of SS, PVA, CuNPs, and GA highlights the synergistic benefits of these components, offering a promising solution for advanced wound healing and tissue regeneration. These findings suggest that SSP–CG scaffolds could contribute to next-generation biomaterials tailored for chronic wound management and regenerative therapies.

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1. Introduction

The skin, the largest organ in the human body, serves as the first line of defence for the body against external threats and harmful substances. The skin and its appendages are crucial for maintaining physiological function and protecting internal organs from hazardous external conditions.^1,2 Skin, being the outermost layer, is the most susceptible organ and may easily be injured. This barrier is compromised when the dermis is injured. It is essential to encourage quick wound closure and skin regeneration to repair the barrier function and avoid potential contamination, dehydration, heat loss, and other harm.^3,4 Trauma-induced soft tissue injury has been one of the most important issues globally for the past several years. These injuries affect many tissue types, including muscle, skin, and nerve. Effective management of wounds is essential for the prevention of complications that may include infections, prolonged healing times, or the development of chronic wounds. Presently, biomaterials or scaffolds that mimic the three-dimensional (3D) porous natural extracellular matrix (ECM) to produce a microenvironment that facilitates cell migration and proliferation and exhibit intrinsic antimicrobial properties are of great interest in the field of regenerative medicine.^5–7 An ideal biomaterial should keep the wound interface moist, facilitate gas exchange, act as a barrier to bacteria, and eliminate excess exudates. It should be non-toxic and should not cause any harmful allergic reactions. It should also be non-sticky to prevent trauma during removal. Further, it should be produced from commonly available materials with minimum processing requirements and excellent antibacterial and wound healing potential. An appropriate scaffold with a porous structure can direct vascular infiltration and promote cell migration, making it a perfect dermal alternative for wound healing. Numerous in vitro studies have found that scaffolds with larger pores promote cell motility, adhesion, and proliferation.^8–11

SS is one of the best natural substances with exceptional characteristics, making it an ideal candidate for wound dressing. SS is a hydrophilic protein that makes up 25–30% of the cocoon and exhibits a variety of qualities, including antibacterial, antioxidant, and UV light protection. SS has a high affinity for biomolecules and is biodegradable and biocompatible. SS is widely used as one of the primary biologically active ingredients in wound dressings because of its immunomodulatory and reactive oxygen species (ROS) clearance properties.^12–14 SS possesses mitogenic and cytoprotective properties for keratinocytes and fibroblasts, attracting them to the skin for tissue repair and development. To aid in wound healing, topical administration of sericin-based wound dressings speeds up collagen deposition and skin tissue re-epithelialization.¹⁵ Another polymer, PVA, has garnered attention for being a biodegradable synthetic polymer known for its nontoxicity, water solubility, biocompatibility, and gel-forming ability.¹⁶ Besides, the hydroxyl groups present in PVA enable the formation of hydrogen bonds with biopolymers, thus promoting the production of environmentally friendly materials. Although PVA lacks natural bioactivity, which restricts its use as a bioactive dressing for sophisticated wound healing. It is typically combined with biomaterials or bioactive compounds to make it more effective, to speed the healing process, and to improve cellular interactions.^17,18

Nanomaterials like CuNPs have been receiving a lot of scientific attention. Copper has a complicated function in different cells, affecting the mechanisms of action of growth factors and cytokines, and is engaged in every step of the wound healing process.^19,20 CuNPs have been demonstrated to induce angiogenesis by altering hypoxia-inducible factor (HIF-1a) expression and regulating vascular endothelial growth factor (VEGF) secretion.²¹ Additionally, CuNPs have recently been identified as antibacterial agents. Cu has also been demonstrated to have a function in wound healing by influencing the expression of 84 genes involved in angiogenesis and wound repair.²² GA is a naturally occurring phenolic chemical found mostly in fruits, leaves, and wildflowers. GA has been proven to possess antioxidant, anti-inflammatory, analgesic, and anti-diabetic effects.²³ GA has been shown to enhance wound contraction and minimize re-epithelialization time in excision wounds. According to studies, gallic acid is an efficient antibacterial agent against bacteria isolated from wounds. GA has the potential to become a valuable agent for wound therapy due to its advantageous properties.^24,25

SS–PVA scaffolds crosslinked with natural agents such as genipin and glycerin have been shown to yield three-dimensional matrices with tunable porosity, enhanced moisture retention, and mechanical stability suitable for wound healing applications.²⁶ Prior work embedding metal nanoparticles into polymeric dressings has demonstrated that CuNPs impart potent antimicrobial activity and pro-angiogenic effects, as seen in nanoparticle hydrogels that achieve antibacterial activity and accelerated wound closure.²⁷ GA has been incorporated into PVA–alginate hydrogel membranes to enable controlled polyphenol release and strong antioxidant/anti-inflammatory responses, resulting in faster epithelialization and reduced irritation.²⁸ Metal-only composites, such as electrospun polycaprolactone dressings embedded with copper oxide nanoparticles or copper nanostructures, were leveraged for antibacterial and pro-angiogenic effects.²⁹ Moreover, SS composites combined with silver or chitosan have been explored for antimicrobial action.³⁰ However, to our knowledge, no prior study has simultaneously integrated both CuNPs and GA within an SS–PVA scaffold network.

This study introduces a novel SSP–CG scaffold developed from SS and PVA, incorporating GA and CuNPs. It serves as a biocompatible and mechanically robust scaffold supporting cell attachment, proliferation, and tissue regeneration, along with the controlled degradation of the scaffold material and seamless integration with host tissue. The SSP–CG scaffold would synergistically improve tissue regeneration while exhibiting potent antimicrobial activity and structural design to mimic native extracellular matrices, making it a potential candidate for future wound care and regenerative medicine applications.

2. Methodology

2.1. Materials

Silk cocoons were provided by the Temperate Sericulture Research Institute, SKUAST-Kashmir. PVA (Cat# 363138), gelatin (porcine type A, Cat# G1890), ascorbic acid (Cat# A5960), and glutaraldehyde solution (50 wt%, Cat# 380855) were purchased from Sigma Aldrich. Copper sulfate pentahydrate (CuSO₄·5H₂O, Cat# PCT0104), sodium hydroxide pellets (NaOH, Cat# MB095), and sodium borohydride (NaBH₄, Cat# GRM10345) were procured from HiMedia Laboratories. The cell culture chemicals, including Dulbecco's Modified Eagle Medium (DMEM, Cat# D0822), fetal bovine serum (FBS, Cat# F2442), and antibiotics (penicillin–streptomycin, Cat# A5955), were purchased from Sigma Aldrich. Fluorescein diacetate (FDA, Cat# F7378) and propidium iodide (PI, Cat# P4864) were purchased from Sigma Aldrich. Escherichia coli (MTCC 443) and Staphylococcus aureus (MTCC 737) were procured from the Microbial Type Culture Collection (MTCC), Chandigarh, India. Primary fibroblasts were obtained as a kind gift from the Reproductive Biotechnology Lab, SKUAST-Kashmir. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under standard conditions (37 °C, 5% CO₂).

2.2. Scaffold fabrication

SS was isolated by the HTHP degumming process. Briefly, 3 g of silk cocoons were cut into pieces, and 100 mL of deionized water was added to it. The solution was autoclaved for 60 min. Following autoclaving, the solution was filtered to obtain sericin and fibroin. The SS solution was lyophilized to obtain powder. CuNPs were synthesized using a chemical method. Briefly, 0.02 M ascorbic acid was dissolved in 50 mL deionized water, and to it, 40 mL of 0.01 M CuSO₄·5H₂O was slowly added under continuous stirring at room temperature. 30 mL of 1 M NaOH was added slowly for adjustment of pH adjustment and stirred at room temperature for 30 minutes. Lastly, 50 mL of 0.1 M NaBH₄ was added under continuous stirring, and the color turned into blue-red-brown, confirming the completion of the reaction.

For the development of scaffolds, 2% (w/v) SS and 5% (w/v) PVA were dissolved separately in autoclaved distilled water under continuous stirring at 60 °C to make homogenous solutions. The two solutions were mixed, and 0.1% (w/v) CuNPs in gelatin solution were added with continuous stirring so that CuNPs are dispersed uniformly throughout the mixture. The resultant mixture was crosslinked with 0.4% glutaraldehyde and poured into 25 mm² cell culture dishes and placed in −40 °C overnight. Following freezing, the scaffold was lyophilized. The lyophilized scaffold was placed in a solution of 2% (w/v) gallic acid to allow for gallic acid uptake. The scaffold was removed, blotted to remove excess moisture, and again lyophilized to develop into a gallic acid-enriched scaffold (SSP–CG scaffold).

2.3. Characterization of CuNPs

Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and energy-dispersive X-ray spectroscopy (EDX) were used to confirm the structural and functional characteristics of CuNPs. The functional groups of CuNPs were identified by FTIR. For FTIR, samples were dried, mixed with KBr, pressed into a pellet, and scanned from 4000 to 400 cm⁻¹. For SEM analysis, dried CuNPs were placed on a carbon-coated aluminium stub coated with gold. The images were taken to check the particle shape and integration. Further, EDX analysis was done to check the elemental composition of these CuNPs.

2.4. Characterization of scaffold

2.4.1. FTIR. The FTIR of the SSP–CG scaffold was carried out on the Shimadzu IRSpirit FTIR spectrometer. For FTIR, the sample was dried, ground thoroughly, mixed into a KBr pellet, and analyzed directly with an ATR accessory. The scans were done at 4 cm⁻¹ resolution over the range of 4000–400 cm⁻¹. The prepared scaffold sample was then analyzed with automatic background subtraction, yielding the final spectrum.

2.4.2. SEM. The surface morphology of the SSP–CG scaffold was investigated using SEM (Zeiss EVO 18). 2 mm thick scaffold samples were taken and slowly dehydrated by adding ethanol at increasing concentrations: 20%, 40%, 60%, 80%, and finally 100%. Each step was done for 20 minutes, then vacuum-dried overnight. The samples were coated using the sputter coater with a thin layer of gold for 90 seconds and scanned at an accelerating voltage of 15 kV to visualize surface morphology. Pore sizes across different scaffold regions were analyzed using ImageJ software.

2.4.3. Porosity and pore volume of scaffolds. The porosity of SSP–CG scaffold was measured by the cyclohexane absorption method. Discs of about 10 mm diameter and 2 mm thickness were cut from the scaffold. Dry weight of the scaffold discs was measured and then immersed in cyclohexane for 1 hour at room temperature. After 1 hour, the weight of the swollen disc was taken (W_s), and porosity was calculated as:
where W_s is the weight of the swollen scaffold, and W_d is the weight of the dry scaffold.

For calculating pore volume, the following formula was used,

ρ is the density of cyclohexane, and W_c is the weight of cyclohexane inside the cryogel, W_c = W_s − W_d.

2.4.4. Swelling kinetics. The swelling kinetics of the SSP–CG scaffold was determined to measure the ability of the scaffold for solvent uptake. The scaffolds were cut into discs of 10 mm diameter and 2 mm thickness. The dry weights of these discs were measured. The discs were then immersed in 0.1 M PBS for 30 seconds and measurements were taken of the wet weight at 30-second intervals until an equilibrium swelling state (We) was reached. The process was repeated twice to study the effect of multiple swelling and deswelling cycles on solvent uptake. Samples with equal dimensions and weights were repeated three times to minimize errors from experiments.

The solvent uptake percentage was calculated by the following formula:

where W_t is the weight of the scaffold at a regular interval of solvent uptake, W_d is the weight of the dry disc, and W_e is the weight of the disc at equilibrium.

The swelling ratio of the scaffold was obtained by the formula.

W_s is the weight of the swollen scaffold, and W_d is the weight of the dry scaffold.

2.4.5. Degradation analysis. For degradation analysis, the SSP–CG scaffold discs of 10 mm diameter and 2 mm thickness were taken and weighed to measure the initial dry weight (W_i). These discs were treated with an ethanol gradient (20–100%) followed by exposure to UV light to sterilize the scaffold discs. The discs were dried and incubated in 0.1 M PBS in autoclaved Eppendorf tubes at 37 °C. To determine the degree of degradation, samples were taken at different time points for 6 weeks. Each sample collected was vacuum dried and weighed again to measure the final dry weight (W_f). The degree of degradation was then calculated using the following formula:
W_i and W_f are the initial dry weight before incubation and final dry weight after incubation, respectively.

2.5. Gallic acid encapsulation efficiency and release

The encapsulation efficiency (EE) of the GA in the SSP–CG scaffold was determined using an extractant of ethanol–water–acetic acid solvent mixture set at 42 : 50 : 8, v/v/v.³¹ The SSP–CG scaffolds weighing 500 mg were washed with 5 mL of the solvent mixture to release GA present on the surface of the scaffold. The supernatant containing released GA was collected and analyzed to determine the surface GA content. Each supernatant (200 μL) containing GA was mixed with 1 mL distilled water and 100 μL Folin–Ciocalteu reagent diluted 1 : 1, v/v with distilled water. The incubation was done at room temperature for 5 min. After incubation, 2 mL of sodium carbonate solution (0.1 g mL⁻¹) was added, followed by incubation at room temperature for 60 minutes. This solution was later centrifuged at 2500 × g for 10 minutes. For release kinetics, the SSP–CG scaffold was mixed with 5 mL of extractant and put on a shaker for 30 minutes to release GA, filtered, and the supernatant was collected to determine the total content of GA present in the scaffold.

The absorption was taken at 725 nm using a spectrophotometer (Thermo Evolution 201) to determine the GA content. EE was calculated using the formula:

2.6. Hemolysis assay

Hemolysis is a process where the red blood cells (RBCs) rupture, releasing their contents into the surrounding plasma. In this study, the hemocompatibility of the SSP–CG scaffold was evaluated using a hemolytic assay. For this, 5 mL of human blood was collected from a healthy volunteer, followed by centrifugation at 3000 rpm for 5 minutes. The RBC pellet was then resuspended in phosphate-buffered saline (PBS). SSP–CG scaffolds were cut into 1 × 1 cm pieces and incubated in the RBC solution for 60 minutes at 37 °C. Water was used as the positive control, while PBS served as the negative control. The experiment was conducted in triplicate. After the incubation, the samples were centrifuged at 2500 rpm for 5 minutes, and the supernatant was collected for OD measurement at 540 nm. Hemolysis was calculated using the following equation:

2.7. DPPH assay

The free radical scavenging activity of the SSP–CG scaffold was determined using the DPPH assay. In brief, a 0.35 mM DPPH solution was made by dissolving DPPH in methanol. Then, 2 mg of the scaffold was mixed with 1 mL of the DPPH solution. The blend was vortexed well to achieve good interaction between the scaffold and the DPPH solution. The blend was incubated in the dark at room temperature for 20 minutes to facilitate the reaction. Post-incubation, the absorbance of the treated SSP–CG scaffold (Abs_sample) was recorded at 517 nm with a UV-visible spectrophotometer. The absorbance of the untreated DPPH solution (Abs_control) was recorded as a control. The antioxidant activity of the scaffold was measured by the following formula:

2.8. Cytotoxicity assay

SSP–CG scaffolds with 5 mm diameter and 2 mm thickness were prepared and sterilized in an ethanol gradient followed by UV light. These discs were immersed in DMEM at 37 °C for 24 hours to extract substances from the scaffold into the media. Fibroblast cells were seeded in a 24-well tissue culture plate at a density of 1 × 10⁴ cells per well. The cells were allowed to grow for 12 hours with 200 μL of conditioned media that was collected from the scaffolds. After 12 hours, the cells were washed with PBS, and the morphology of the cells was examined under a Leica microscope to check for any toxic effects.

2.9. Live–dead assay

SSP–CG scaffolds with 5 mm diameter and 2 mm thickness were prepared and sterilized in an ethanol gradient followed by UV light. These discs were immersed in DMEM at 37 °C for 24 hours to extract substances from the scaffold into the media. Fibroblasts were seeded at 1 × 10⁴ cells per well on 96-well plates and incubated overnight at 37 °C in 5% CO₂. After cell attachment, the medium was replaced with scaffold-conditioned media and cells were incubated for 24 hours. Following treatment, cells were gently washed with PBS and stained with fluorescein diacetate at 37 °C for 10 min. Without an intermediate wash, PI was added and incubated for an additional 5 min in the dark. Samples were then rinsed with PBS and immediately imaged using a fluorescence cell imager (Cytation 5). Live cells emitted green fluorescence (FDA⁺), whereas dead cells displayed red fluorescence (PI⁺), enabling a clear assessment of cell viability following exposure to the scaffold-conditioned media.

2.10. Antibacterial activity of SSP–CG scaffold

A zone of inhibition assay was performed to assess the antibacterial efficacy of the SSP–CG scaffold. In this assay, LB-agar plates were uniformly seeded with Staphylococcus aureus and Escherichia coli cultures at a concentration of 10⁸ CFU mL⁻¹. Circular discs equal in size to the control discs prepared from the SSP–CG scaffold and copper nanoparticles were then placed on the agar surface. The plates were incubated at 37 °C overnight. Following incubation, the clear zones formed around each disc were measured to determine the extent of bacterial growth inhibition, providing a quantitative evaluation of the scaffold's antibacterial properties.

2.11. In vivo study

All animal experiments were carried out as per the guidelines of the Institute Animal Ethical Committee (IAEC), SKUAST-Kashmir, under protocol number SKUAST/IAEC-17/2023/14. 8-Week-old male Wistar rats were kept in standard laboratory conditions and acclimatized for 10 days before the experiment. Rats were anesthetized with intramuscular injection of xylazine (10 mg per kg⁻¹ body weight) and ketamine (80 mg per kg⁻¹ body weight). After anesthesia, the dorsal surface of the rat was shaved and then disinfected with 70% ethanol. A 1 cm diameter full-thickness wound was made using a biopsy punch. Rats were randomly divided into three groups (n = 6 per group): a negative control group, a positive control group (Betadine ointment), and an SSP–CG scaffold group. After the surgery, tramadol (20 mg per kg⁻¹ body weight) was administered intramuscularly for analgesia. Wound healing was monitored daily, and images were taken on day 0, day 7, and day 14.

2.12. Histological analysis

Animals were euthanized at 14th day of the experiment by CO₂ asphyxiation followed by cervical dislocation. Tissue samples at the wound site were collected from each group and preserved in 10% formalin. Samples then underwent embedding into paraffin wax blocks after being fixed. Microsections were prepared at a thickness of 5 μm, following which sections were stained using hematoxylin and eosin, and histological images were taken on a Leica DM 2500 microscope.

2.13. Statistical analysis

Statistical analyses were performed in GraphPad Prism 7.0. All experiments were carried out in triplicate, and results are reported as mean ± standard deviation. Comparisons among two or more groups were made by two-way ANOVA followed by Tukey's post-hoc test. A p-value ≤0.05 was considered statistically significant.

3. Results and discussion

3.1. Characterisation of CuNPs

The morphology and particle size of the synthesized copper nanoparticles were analyzed by SEM. The SEM images showed that the copper nanoparticles had irregular and interconnected morphology displaying slight agglomeration (Fig. 1b). The average particle size of the nanoparticles was found to be 24.9 ± 4 nm, indicating that the nanoparticles were successfully synthesized at the nanoscale level. The degree of aggregation may be associated with high surface energy as well as inter-particle interaction, which is typical in nanoscale materials. Such a morphology is very suitable for applications requiring high surface area, such as catalysis and antimicrobial coatings. EDX spectroscopy was used to determine the elemental composition of the copper nanoparticles. The EDX spectrum indicates strong peaks for Cu at around 0.93 keV, 8 keV, and 9 keV, which suggests that Cu is the main constituent in the nanoparticles (Fig. 1c). In addition, a minor peak is seen at about 0.5 keV corresponding to oxygen (O), and this may be indicative of some surface oxidation or trace amounts of copper oxide on the surface of the nanoparticles. Strong peaks of copper and very low intensity for the oxygen peak suggest that the composition of the nanoparticles is largely pure metallic copper with little oxidation. This minimal surface oxidation can be due to exposure during the synthesis and handling to atmospheric conditions. In general, EDX analysis confirms that these copper nanoparticles are highly pure and can be used where minimal elemental contamination is present. FTIR was used to evaluate the surface functional groups as well as the chemical bonds in the copper nanoparticles. The FTIR spectrum showed several distinct peaks at 3311 cm⁻¹, 1393 cm⁻¹, 1032 cm⁻¹, and 610 cm⁻¹ (Fig. 1d). The peak at 3311 cm⁻¹ is typically attributed to O–H stretching vibrations, which may indicate the presence of hydroxyl groups on the nanoparticle surface, possibly due to atmospheric moisture or minor oxidation. The broad peak at 1393 cm⁻¹ could correspond to C–O stretching or C–H bending. This suggests the presence of some residual carbonate or organic material that had been left from the synthesis process. The broad band at 1032 cm⁻¹ is associated with C–O or C–O–C stretching vibrations, suggesting some form of surface-bound organic material may be present, but the intensity is minor. The peak at 610 cm⁻¹ is consistent with Cu–O stretching vibrations, which could also suggest the formation of copper oxides on the nanoparticle's surface, probably as a consequence of slight oxidation due to exposure to air.

Fig. 1 (a) Representative images of the reaction mixture after copper nanoparticle synthesis, showing the characteristic color change indicative of copper nanoparticle formation. (b) SEM micrograph demonstrating the morphology and aggregation state of the synthesized copper nanoparticles. (c) EDX spectrum confirming the elemental composition of the copper nanoparticles, including the presence of copper (Cu) peaks. (d) FTIR spectrum displaying characteristic functional groups associated with the copper nanoparticles.

3.2. Scaffold characterization

3.2.1. FTIR. The FTIR pattern of the SSP–CG scaffold exhibits a number of significant absorption peaks, verifying its structural content and the successful incorporation of its components (Fig. 2a). The broad absorption peak at 3257.7 cm⁻¹ reflects O–H and N–H stretching, emphasizing the strong hydrogen bonding between sericin, gelatin, and PVA. The presence of characteristic amide I (1628.8 cm⁻¹) and amide II (1543.1 cm⁻¹) peaks confirm the presence of proteinaceous components, whereas C–O stretching at 1248.7 cm⁻¹ and 1077.2 cm⁻¹ confirms the contribution of GA and PVA. The presence of the peak at 2117.4 cm⁻¹ is also indicative of possible interaction between CuNPs and other biomolecules, signifying the successful incorporation of nanoparticles. The peaks in the region of 965.2–809 cm⁻¹ also confirm the aromatic ring structure of GA, further confirming its incorporation. These spectral results confirm the synthesis of a biocompatible, antioxidant-dense, and structurally stable scaffold, which renders it a potential candidate for tissue engineering and wound healing.

Fig. 2 FTIR and SEM images of the SSP–CG scaffold with incorporated nanoparticles (a) FTIR spectrum of the SSP–CG scaffold confirming the characteristic functional groups corresponding to the different components of the scaffold. The broad peak at 3257.7 cm⁻¹ corresponds to O–H/N–H stretching, while 1628.8 cm⁻¹ and 1543.1 cm⁻¹ indicate amide bonds. Peaks at 1248.7 cm⁻¹ and 1077.2 cm⁻¹ confirm PVA and GA, while 2117.4 cm⁻¹ suggests CuNPs interactions (b) (scale bar = 100 μm): low-magnification view showing the highly porous, sponge-like architecture of the SSP–CG scaffold (c) (scale bar = 50 μm): Higher-magnification view illustrating the interconnected pores and revealing finer structural details, including nanoparticle aggregates embedded within the SSP–CG scaffold.

3.2.2. SEM. The SEM images reveal a porous, interconnected structure characteristic of scaffolds intended for tissue engineering applications. The surface of the SSP–CG scaffold shows a highly porous structure, with pore sizes varying between 96 ± 19 μm, facilitating an ideal environment for cell attachment and nutrient flow (Fig. 2b and c). The porous structure can be attributed to the freeze-drying process utilized during scaffold preparation, which induces the formation of interconnected pores. This structural feature is beneficial for tissue regeneration, as it allows for cell infiltration and vascularization, crucial for effective tissue integration. The presence of uniformly distributed pores throughout the scaffold suggests that the blend of sericin and PVA created a stable matrix, suitable for potential biomedical applications such as wound healing and tissue engineering.

3.2.3. Porosity measurement. The porosity and pore volume of the SSP–CG scaffold were assessed using the cyclohexane displacement method. This technique involved immersing the scaffold in cyclohexane for 1 hour to allow for complete penetration of the solvent into the porous structure. The porosity of the scaffold was determined to be 90.4 ± 1.67%, indicating a highly porous network. This high porosity enhances the ability of the SSP–CG scaffold to transport nutrients and oxygen, which is beneficial for cell proliferation and tissue growth.

The scaffold also exhibited a pore volume of 14.37 ± 0.48 mm³ mg⁻¹, which provides a substantial surface area for cellular attachment and nutrient exchange (Fig. 3a). The interconnected pores are crucial for facilitating the absorption and retention of exudates, ensuring a moist environment that supports the wound healing process. This combination of high porosity and significant pore volume underscores the suitability of the SSP–CG scaffold for wound management applications, as it promotes optimal conditions for tissue regeneration.

Fig. 3 (a) A bar graph showing the SSP–CG scaffold's mean percentage porosity and pore volume, highlighting a high porosity relative to the measured pore volume. (b) Water uptake capacity over multiple time points (0–150 seconds), demonstrating rapid fluid absorption and retention by the SSP–CG scaffold.

3.2.4. Swelling kinetics. The water uptake capacity of the SSP–CG scaffold was assessed over multiple cycles, and the results are presented in triplicate to ensure accuracy and reproducibility. The data reveal a rapid increase in water uptake capacity, achieving an average maximum of approximately 95.3 ± 0.7% within 150 seconds for each cycle (Fig. 3b). For the first cycle, the mean water uptake reached 83.6 ± 2.4% at 30 seconds, 89.6 ± 1.9% by 60 seconds, and stabilized at 93.3 ± 1.5% at 90 seconds, ultimately achieving 95.1 ± 0.7% by 120 seconds and 95.3 ± 0.7% at 150 seconds, indicating near-complete hydration. This pattern was consistently observed across subsequent cycles, with slight variations. The swelling ratio of the SSP–CG scaffold was found to be 16.6 ± 0.6. These findings indicate the water retention capacity of the SSP–CG scaffold and their ability to rehydrate rapidly and consistently across multiple cycles. This rehydration ability highlights the potential of the SSP–CG scaffold for applications requiring repeated hydration, such as wound healing, where sustained moisture maintenance is crucial for tissue repair and regeneration.

3.2.5. Degradation studies. The degradation kinetics of the SSP–CG scaffold were systematically evaluated over 14 days under physiological conditions (37 °C in 0.1 M phosphate-buffered saline). The degradation profile shows a steady decrease in mass with a consistent increase in degradation percentage over time (Fig. 4a). After 1 week, the SSP–CG scaffold exhibited an average weight loss of 9.2 ± 0.4%, suggesting minimal initial degradation, which may be beneficial for applications requiring early-stage material stability. The mean degradation reached 21.6 ± 0.7% by week 3, 26.2 ± 0.8% by week 4, and 28.9 ± 0.9% by week 5. By the end of the 6 weeks, the SSP–CG scaffold demonstrated a mean degradation of 31.1 ± 0.5%, reflecting a predictable and controlled degradation pattern. These findings suggest that the SSP–CG scaffold possesses a stable and controlled degradation profile, which is advantageous for biomedical applications that require gradual matrix breakdown and sustained release of bioactive compounds.

Fig. 4 (a) In vitro degradation profile of the SSP–CG scaffold over six weeks, illustrating a progressive increase in the percentage of degradation with time. (b) Cumulative release of gallic acid from the SSP–CG scaffold over 120 hours, demonstrating a gradual and sustained release profile. (c) Hemolysis assay comparing the SSP–CG scaffold with positive and negative controls, where the y-axis represents the percentage of hemolysis relative to the positive control (set at 100%).

3.3. Encapsulation and release kinetics of gallic acid

The EE of the SSP–CG scaffold was found to be extremely high, at 89.3 ± 1.51%, indicating strong encapsulation and retention of GA within the scaffold matrix. The high EE indicates the structural stability and encapsulation capability of the SSP–CG scaffold to accommodate stable loading of GA within the matrix rather than on the surface. High EE supports the prospect of the SSP–CG scaffold in controlled and sustained release therapeutics, whereby slow release of GA is expected to be credited with its action. The mean percentage release of the drug was measured over 5 days, with a 12-hour interval (Fig. 4b). The data reveal a smooth and progressive increase in the cumulative release of the GA with time, from 0% at 0 hours to progressively reaching 79.67 ± 2.05% at 120 hours. These findings suggest a sustained release profile, with an initial rapid release period followed by a slow rise, suggesting a controlled and sustained release mechanism of the GA over the period of 5 days. This pattern of release can suggest the potential for a sustained therapeutic effect of GA.

The sustained GA release profile is expected to correlate strongly with enhanced wound healing efficacy. The initial burst release within the first 24 hours aligns with the acute inflammatory phase of wound healing, where high GA concentrations can mitigate excessive ROS and inflammation, preventing tissue damage. The subsequent slow release over a period of 5 days helps in promoting angiogenesis, collagen deposition, and epithelialization, as the antioxidant effects of GA protect newly formed tissue. This temporal release pattern mirrors the sustained delivery systems reported in other wound healing studies, where controlled release of bioactive molecules enhances tissue regeneration by maintaining a consistent therapeutic effect.^28,32 The ability of the SSP–CG scaffold to mimic native ECM, combined with GA's bioactive properties, positions it as an ideal platform for supporting these cellular processes.

3.4. Hemolytic assay

The hemocompatibility of the SSP–CG scaffold was assessed through a hemolytic assay performed on human red blood cells (RBCs). When a foreign material interacts with the RBCs, this may cause damage to the cell membrane because of the release of hemoglobin. According to the American Society for Testing and Materials (ASTM), hemolysis under 2% qualifies a biomaterial as hemocompatible. Fig. 4c shows 0% hemolysis for the negative control and 100% hemolysis for the positive control. In comparison with these controls, the SSP–CG scaffold exhibited a hemolysis percentage of 0.80 ± 0.12%, confirming hemocompatibility and suitability as the matrix for tissue regeneration.

3.5. DPPH assay

The SSP–CG scaffold possessed strong free radical scavenging activity. Following the scavenging activity formula, the SSP–CG scaffold indicated a free radical scavenging activity of 40.5 ± 2.1%. This finding emphasizes the scaffold's free radical neutralizing capability, which is due to the synergistic action of gallic acid, a strong phenolic antioxidant, and copper nanoparticles, which would facilitate electron transfer reactions. The antioxidant activity of the scaffold makes it a strong candidate for wound healing and tissue regeneration applications where oxidative stress suppression is essential.

3.6. Cytotoxicity assay

Following an incubation period of fibroblast cells with conditioned media prepared from the SSP–CG scaffold, the cells were washed with PBS to remove any residual media. The morphology of the cells was normal and healthy, with the cells well spread and elongated, displaying characteristics of viable fibroblasts (Fig. 5). There were no clear signs of toxicity, such as shrinkage, detachment from the surface, or rounding—features typically observed in cells subjected to toxic materials. The cells maintained a normal morphology, suggesting that these materials exerted no adverse effects on cellular integrity or function. There were no distinct alterations in cell density or growth pattern, which indicates that the scaffold-derived conditioned media were not inhibitory to cell proliferation. This strongly indicates that the developed scaffold is non-toxic, qualifying as an excellent biocompatible material.

Fig. 5 Morphology of fibroblasts exposed to scaffold-conditioned medium: representative micrographs of fibroblasts cultured for 12 hours in (a) standard growth medium and (b) SSP–CG scaffold-conditioned medium. Images were acquired on an inverted phase-contrast microscope at 5× magnification (scale bar = 50 μm). Both panels show typical spindle-shaped morphology and comparable cell density, indicating that soluble factors released from the SSP–CG scaffold do not adversely affect cell attachment or proliferation.

3.7. Live dead assay

The cytocompatibility of the SSP–CG scaffold was evaluated using a live/dead assay with FDA and PI staining on fibroblasts cultured in scaffold-conditioned media. FDA, converted by intracellular esterases in live cells, produced green fluorescence, while PI selectively stained the nuclei of membrane-compromised (dead) cells red. Both control and SSP–CG groups exhibited a dense population of viable fibroblasts with strong green fluorescence, indicating that scaffold exposure did not adversely impact cell health (Fig. 6). Only a few red-stained dead cells were observed, confirming minimal cytotoxicity. Additionally, the fibroblasts retained their typical elongated and spindle-shaped morphology. Quantitative analysis revealed a slight but statistically insignificant reduction in cell viability in the SSP–CG group compared to the control, with both maintaining viability around 90%. These findings demonstrate that the SSP–CG scaffold is biocompatible and conducive to fibroblast growth, supporting its potential use in wound healing applications.

Fig. 6 Live/dead viability assay of fibroblasts cultured in control and SSP–CG scaffold-conditioned media. (a) Fluorescence images showing viable cells stained green with FDA and dead cells stained red with PI. Overlays (FDA/PI) indicate predominance of live cells with minimal dead cells in both groups. Scale bar: 200 μm. (b) Quantitative cell viability analysis indicating high viability (around 90%) in both control and SSP–CG groups.

3.8. Antibacterial assay

The antibacterial efficacy of the SSP–CG scaffold was assessed using a zone of inhibition assay against Staphylococcus aureus and Escherichia coli. Both the SSP–CG scaffold and copper nanoparticle discs exhibited distinct zones of inhibition, confirming their antimicrobial potential. CuNPs exhibited antibacterial activity against both Staphylococcus aureus and Escherichia coli, with a slightly larger zone of inhibition observed in the scaffold, likely due to the combinational effect of copper nanoparticles and gallic acid. The SSP–CG scaffold demonstrated a clear and measurable zone of inhibition for both bacterial strains, indicating the effective release and activity of copper nanoparticles embedded within the scaffold matrix. Similarly, copper nanoparticles alone showed strong antibacterial activity, further validating their contribution to the scaffold's antimicrobial performance (Fig. 7). These findings suggest that the SSP–CG scaffold, with its integrated copper nanoparticles, possesses broad-spectrum antibacterial properties, making it a promising material for use in infection-prone wound healing applications.

Fig. 7 Zone of inhibition assay demonstrating the antibacterial activity of CuNPs, SSP–CG scaffold, and standard antibiotics against Escherichia coli (left) and Staphylococcus aureus (right). Each agar plate was divided into four quadrants containing: copper nanoparticles (CuNPs), SSP–CG scaffold, and antibiotic controls—kanamycin (Kan) and erythromycin (Ery). Both CuNPs and the SSP–CG scaffold exhibited clear zones of inhibition against E. coli and S. aureus, indicating strong antibacterial activity.

3.9. Wound healing

Wounds were divided into three groups on the 0th day: negative control, positive control, and SSP–CG scaffold-treated. By 7 days, the negative control was still significantly inflamed, with significant redness and tissue necrosis, indicating poor healing. The positive control group showed improvement, but it still possessed some yellow necrotic tissues and inflammation. However, the SSP–CG scaffold group showed a less inflamed wound and evidence of the presence of red granulation tissues, indicating an active healing process. By 14 days, the Negative Control group was mostly unhealed, thickly scabbed, and still having damaged tissues. The Positive Control group showed considerable size reduction with scabs and early healing of the tissue. The SSP–CG scaffold group exhibited the most healing advancement, with small scabs and a significant reduction in the area of the wound, which highly suggests significantly improved tissue regeneration (Fig. 8). These findings indicated that scaffold treatment accelerates and enhances wound healing compared to both control groups.

Fig. 8 (a) Representative images showing the wound healing progression in a rat model with different conditions. The wounds were divided into three groups: negative control (untreated), positive control (commercial wound dressing), and SSP–CG scaffold. The healing of the wounds was assessed, and the SSP–CG scaffold showed better wound healing as compared to controls. (b) Quantitative analysis of wound area (%) over time. Data are presented as mean values for each group (negative control, positive control, and SSP–CG scaffold) at day 0, day 7, and day 14. The results demonstrate the comparative efficacy of the SSP-CC Scaffold in promoting wound healing relative to the control groups.

3.10. Histological analysis

Histological analysis reveals substantial variation among the three groups: negative control, positive control, and SSP–CG scaffold-treated. The negative control demonstrates severe tissue damage with disrupted epidermal layers, acute inflammation, and poor structural organization, suggesting that healing is delayed. The positive control exhibits moderate re-epithelialization, tissue remodelling, and collagen formation. In contrast, in the SSP–CG scaffold-treated group, the tissue regeneration was well organized, with new skin structures such as hair follicles and connective tissue appearing more intact, indicating enhanced healing and tissue repair (Fig. 9). These findings suggest that the scaffold is much better at promoting wound healing than the remaining groups in terms of faster tissue regeneration and structural restoration.

Fig. 9 Representative H&E-stained histological sections of wound sites in three groups: negative control, positive control, and biomaterial. (A) The negative control (scale bar = 100 μm) exhibits extensive tissue disruption with minimal organized repair. (B) The positive control (scale bar = 50 μm) section shows partial re-epithelialization and moderate tissue remodeling. (C) The SSP–CG scaffold (scale bar = 100 μm) demonstrates more advanced tissue organization and regeneration.

4. Conclusion

The development of SSP–CG scaffolds incorporated with CuNPs and GA represents a significant advancement in the field of regenerative medicine and wound healing. The integration of SS and PVA enhances the scaffold's biocompatibility and biodegradability, providing a more physiologically relevant extracellular matrix-like environment for cellular activities. The incorporation of CuNPs not only imparts strong antimicrobial properties but also interacts electrostatically with the hydroxyl and amine groups of SS and PVA, contributing to mechanical reinforcement and improved network cohesion. The addition of GA, a potent antioxidant and anti-inflammatory agent, synergistically complements the therapeutic properties of copper nanoparticles, ensuring enhanced cellular protection while promoting angiogenesis and tissue remodelling. The sustained and controlled release of GA from the scaffold ensures enhanced fibroblast proliferation, efficient collagen synthesis, and reduced inflammation without cytotoxic effects, addressing critical challenges in wound management. These synergistic interactions result in a scaffold that mimics the extracellular matrix both structurally and functionally. In vitro and in vivo studies demonstrated accelerated wound closure and improved tissue regeneration, underscoring the multifunctional benefits of SSP–CG scaffolds in wound environments. Importantly, no component incompatibility was observed during scaffold fabrication, indicating formulation stability. Moreover, previous studies have shown that CuNPs enhance wound repair through multifaceted mechanisms: they upregulate angiogenic factors like VEGF and HIF-1α, suppress pro-inflammatory cytokines (IL-1β, TNF-α, NF-κB), and reinforce extracellular matrix synthesis via collagen and integrin induction.^33,34 Similarly, GA is well-documented to promote fibroblast proliferation, support collagen formation, and exhibit potent antioxidant and anti-inflammatory effects, contributing to accelerated tissue regeneration.³² These mechanistic insights provide a strong rationale that the SSP–CG scaffold's therapeutic effects stem from established molecular pathways validated in similar biomaterial systems. This study lays the foundation for future clinical translation of SSP–CG scaffold as a promising platform for advanced tissue engineering and wound healing therapies. This integration of bioactive compounds and biopolymers enhances the potential of precision biomaterials for regenerative medicine.

Conflicts of interest

The authors declare that they have no competing interests.

Data availability

The data that support the findings of the study are included in the manuscript.

Acknowledgements

This work was supported by the NMHS grant (NMHS/2023-24/SC-XI/SG89/04/144/232), and we gratefully acknowledge NMHS for their support in enabling this research. We also acknowledge and extend our appreciation to the Researchers Supporting Project Number (RSPD2025R709), King Saud University, Riyadh, Saudi Arabia, for funding this study.

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Footnote

† Equal contribution.

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