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Preparation and in vitro validation of ascorbyl palmitate-enriched marine collagen/alginate aldehyde hydrogels/cryogels for wound healing dressings
* Corresponding author: E-mail address: gmalsnany@pnu.edu.sa (G. Al-Senani)
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Received: ,
Accepted: ,
Abstract
The present study involves the preparation of oxidized alginate/collagen (ADA/COL) hydrogels, their conversion into cryogels via a freeze-drying process, and the enrichment of these hydrogels with ascorbyl palmitate (AAP) to create a multifunctional material with enhanced properties for promoting wound healing. Both of ADA/COL and AAP-loaded cryogels were characterized in detail for their gel content, zeta potential, and morphology using scanning electron microscopy (SEM), while the chemical integrity was assessed by FTIR spectroscopy. Additionally, the in vitro study was performed in terms of water uptake, biodegradability, cytocompatibility on human skin fibroblasts (HSF), and the capability to inhibit microbial growth. The AAP-loaded samples showed a higher gel fraction, with the 20AAP-loaded cryogel formulation achieving the maximum value of 91.2 ± 7.1% and –7.36 ± 1.24 mV surface charge. The average pore diameter of the ADA/COL cryogels was 254 ± 65 µm, while the AAP loaded-cryogels showed irregular and small pores in the range of 25±7 μm. Besides their antimicrobial activities, the AAP-loaded cryogels demonstrated an impressive wound closure rate of 97.8 ± 2.8% over 48 h in a HSF migration assay. Overall, the study results highlight the significant advantages of using cryogels, especially those loaded with AAP, to address several critical requirements for effective wound healing, including superior water uptake, biocompatibility with human cells, and infection control.
Keywords
Antimicrobial activity
Bioactive wound dressing
Cryogels
Marine collagen
Oxidized alginate
1. Introduction
Bioactive wound dressing materials are indeed highly sought after because they play a dual function in wound healing and the inhibition of microbial growth [1,2]. Reactive oxygen species (ROS) have played a dual role in the wound healing process, acting as both essential signaling molecules and potential sources of damage, depending on their levels [3]. Indeed, excessive ROS release leads to oxidative stress, characterized by prolonged inflammation and cellular dysfunction [4]. Therefore, the free-radical scavenging activity (antioxidant) offers an effective treatment strategy to promote the performance of wound healing materials via minimizing cellular oxidative stress in the wound bed [5-8]. As one of the natural biomolecules widely present in living organisms, vitamin C (also known as ascorbic acid (AA)) has obvious application in the control of the wound healing process due to its antioxidant, antibacterial, and anti-inflammation activities [5,9]. In addition, AA acts as a cofactor for collagen synthesis; thereby, it is rapidly consumed during the healing process. Actually, AA is highly sensitive to environmental factors like heat, light, oxygen, and metal ions, making it unstable in many formulations. However, stabilization techniques such as using derivatives, encapsulation, and proper packaging can enhance its shelf life and efficacy [10-12]. Understanding and controlling these factors is essential for maintaining the potency of vitamin C in medical products [13]. Ascorbic acid 6-palmitate (also known as ascorbyl palmitate, AAP) is a fat-soluble (lipophilic) derivative of AA (vitamin C), formed by the esterification reaction between AA and palmitic acid. It is widely applied in numerous food, cosmetics, and pharmaceutical formulations due to its antioxidant properties [14,15].
Due to their biocompatibility and porosity, cryogels are used as scaffolds for cell growth and tissue regeneration applications [16,17]. A cryogel is a type of hydrogel formed at sub-zero temperatures through a cryogelation process. Cryogels are unique soft materials, characterized by their highly interconnected porous structure and mechanical resilience. These properties arise during the process of cryogelation, where a solution containing a polymer or other material is frozen, and the gel network forms within the frozen matrix. Upon thawing, the frozen solvent (typically water) leaves behind a porous structure [18]. The crosslinking that occurs between polymer chains helps them to keep water molecules within their internal structure. In addition, cryogels possess features of fluid transport and responsive features in response to different stimuli, such as pH, temperature, redox, light, and electric/magnetic fields. Indeed, the polymer cryogel materials possess excellent biocompatibility, physicochemical features, and high strength for a variety of technical and medical applications [19-22], including wastewater treatment, sensors, drug delivery vehicles, bioscaffolds, wound care materials, and medical textiles.
Marine collagen is a promising bioactive ingredient with significant benefits for skin, joint, and overall health due to its high bioavailability and rich content of type I collagen [23,24]. As a sustainable and effective alternative to other collagen sources, it is widely used in cosmetics, supplements, and therapeutic applications [25]. Marine collagen is derived from bones, skin, scales, and other parts of marine organisms, primarily fish. It has gained attention over the past years due to its potential health outcomes, particularly in skin tissue healing, joint function, and overall wellness. Among different collagen types, type I collagen is the most abundant form of collagen found in marine collagen [20,26]. Compared to collagen derived from other sources (such as bovine or porcine), marine collagen is considered to have better bioavailability. Its smaller peptide size allows for more efficient absorption into the bloodstream after oral consumption [26,27]. Sourced from scales, skin, and bones of sea fish, marine collagen is often seen as a sustainable alternative because it makes use of biomass from the seafood industrial field, reducing waste [28,29]. However, its fast dissolution and degradation rates, as well as inherently low mechanical strength, limit the use of marine collagen in living tissue engineering and other related biomedical applications [30]. To overcome these shortcomings, collagen-based materials have been combined with other biopolymers, such as polysaccharides [31]. The combination of marine collagen with polysaccharides not only resembles the glycoproteins portion in the native extracellular matrix (ECM) but also brings novel synergistic features that would otherwise be impossible to deliver using one of them [32].
Over the past decades, different polysaccharide polymers have been used in combination with synthetic polymers, natural proteins, or inorganic nanoparticles through chemical or physical crosslinking to create hybrid/composite cryogels to fabricate novel materials with unique properties and desirable functions [33-35]. Alginate, a naturally occurring water-soluble polysaccharide produced by algae, is a linear biopolymer of (1,4)-β-d-mannuronic acid (M) and α-l-guluronic acid (G) residues in the chemical form of consecutive homogeneous units (MM- or GG) and alternative blocks (MG- or GM) [36]. Chemical modification of alginate is usually performed to improve its biodegradation and biological activity, thereby broadening its application range. Fortunately, the secondary hydroxyl (−OH) substituents at C−2 and C−3 in the alginate molecule can be modified through periodate oxidation, which leads to the introduction of two aldehyde residues on the uronic units [37-39]. The fresh oxidized aldehyde groups are highly reactive and can link intramolecular bonding to other moieties or neighboring polymer containing hydroxyl or amine groups by the Schiff base reaction [40]. In this study, oxidized alginate/collagen (ADA/COL) hydrogels at different mass ratios of ADA/COL were successfully prepared using Schiff’s base reaction and zinc ions, followed by the freeze-drying process to obtain ADA/COL cryogels. The effect of ascorbyl palmitate (AAP) on the surface morphologies, biodegradation rate, water absorption, and antibacterial potential of ADA/COL cryogels was systematically studied. The microbial activity results indicated that AAP-enriched cryogels exhibited a marked effect against Aspergillus fumigatus, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. With human skin fibroblasts (HSF), the MTT assay results indicate a stimulatory effect of AAP functionalization.
2. Materials and Methods
2.1. Material
Alginate aldehyde (ADA, 20% aldehyde content and medium viscosity), zinc chloride, and 6-O-Palmitoyl-L-ascorbic acid (AAP) were obtained from Sigma-Aldrich (USA). Cod Collagen Peptide (COL) with a Mw of 3000 Da was purchased from Beijing SEMNL Biotechnology Co (China). Ethyl alcohol solution (96%) was provided by Carlo Erba Reagents. The research experiments were accomplished at the Natural and Health Sciences Research Center.
2.2. Fabrication of ascorbyl palmitate-enriched COL/ADA cryogels
The ADA was crosslinked with marine collagen (COL), leading to the formation of 3D crosslinked hydrogels that are subsequently freeze-dried to obtain highly porous cryogels. Two types of cryogel were made by mixing the ADA solution with the COL solution. The weight ratio of ADA/COL was adjusted at 50/50 and 75/25 wt%. In a typical procedure, COL and ADA powder were weighed and dissolved separately in deionized water to obtain 10% (w/v) polymer solutions. Once the polymer solutions were prepared, the COL solution was slowly added to the ADA solution and vortexed to ensure homogeneity in all the solutions to form a homogenous mixture. The obtained ADA/COL combination was vigorously agitated for 20 min at 40°C. Then, cylindrical molds of 12 mm diameter were loaded with the ADA/COL polymer blends and frozen at -20°C for 24 h. The frozen cryogels were kept overnight at −20°C in an ethanol solution containing 0.1 M ZnCl2 and then washed three times with a 70% ethanol solution at room temperature. Finally, the cryogel scaffolds were freeze-dried at -52°C for 48 hrs.
For preparing the AAP-loaded cryogels, the AAP-ADA/COL weight ratios were fixed at 40:40:20 and 45:45:10 wt%. The predetermined amount of AAP powder was dissolved in an ethanol solution at 40°C and then added to the ADA solution. The obtained blend solution was stirred for 30 mins at 40°C to form a homogenous mixture. After that, 10 mL of COL solution (10%) was gradually mixed into the AAP/ADA solution under constant stirring. The obtained blends were also mixed at 40°C for 20 mins and then frozen at −20°C before crosslinking and lyophilization steps. The cryogels were named as 75ADA/25COL, 50ADA/50COL, 10AAP-50ADA/50COL, and 20AAP-50ADA/50COL.
2.3. Characterization
2.3.1. Microstructural characterization
ATR-FTIR/Varian IR spectrometers (Agilent, Santa Clara, CA) spectroscopy was used to characterize the obtained ADA/COL cryogel samples. The morphology and pore diameters were explored by a field emission scanning electron microscopy (SEM: Jeol JXA 840, Japan). The zeta potential of the ADA/COL cryogels was recorded via dynamic light scattering (DLS) (Malvern Zetasizer NanoZS, Worcestershire, UK). The dried cryogels were suspended in 2 mL of potassium chloride solution (KCl: 1 mM) and analyzed by a zeta dip cell.
2.3.2. Determination of gelation (GEL %)
The freeze-dried ADA/COL cryogel samples were dried at 60°C for 24 hrs and weighed. After that, 1 g of each sample was submerged in 100 mL deionized water on a shake plate set at 50 rpm for 24 hrs to remove uncrosslinked components of the cryogels. The gelled parts, crosslinked gel, were collected using Whatman No. 4 filter paper and then dried at 60°C and weighed. The gel percent (GEL %) in the cryogel was obtained using Eq. (1).
Where Wg and Wd represent the gelled component dry weight and the cryogel sample original dry weight, respectively.
2.3.3. Cryogel scaffolds swelling ratio (SR)
The prepared cryogels scaffold with different ADA/COL contents were dried at 60°C and immersed in water until equilibrium at 37 oC. After 24 hrs, all the cryogel samples were removed from the water and were slowly blotted with a wet filter paper to absorb excess water on the surfaces of the samples. The data were determined as mean ± standard deviation (SD) based on three independent samples. The swelling ratio was obtained as the amount of water per unit mass of the dry cryogel using the following expression (Eq. 2):
Where Wd is the original dry weight and Ww is the weight of the sample after soaking for 24 h in the water.
2.3.4. Hydrolytic degradation of cryogel scaffolds
The degradation test was performed under hydrolytic environments in a buffered solution of PBS. The dry samples were added to plastic vials containing 5 mL of PBS medium. The degradation vials were then sealed and incubated for different time intervals, including 1, 3, 6, 12, and 24 days. The PBS medium was refreshed every 3 days. At each time period, the wet cryogel samples were gently removed and rinsed with distilled water to eliminate the degradation products. After that, the wet cryogels were dried at 40°C for 36 hrs and then weighed. The degradation rate was obtained from the weight loss percentage (WL%) using Eq. (3).
Where Wd is the original cryogel weight and Wdd is the dry weight of the sample after each incubation interval period in PBS medium.
2.3.5. Antimicrobial test
The ability of the as-prepared AAP-loaded ADA/COL cryogels to inhibit the microbial proliferation was performed for different pathogen strains, including Gram-negative bacterium (Pseudomonas aeruginosa), Gram-positive bacterium (Staphylococcus aureus), unicellular fungi (Candida albicans), and multicellular fungus (Aspergillus fumagitus) [41]. The activation of each pathogen was done by inoculating bacterial strains in the nutrient broth medium for 24 hrs at 37°C. On the other hand, the fungal strains were inoculated in a Potato Dextrose Broth (PDB) medium for 48 hrs at 28°C under a moderate shaking process. Screening of the APP-tested samples was preliminarily done at different concentrations by using Mueller Hinton Broth (MHB) via a turbidimetric method. In addition, the cryogels were tested against pathogens by using Mueller Hinton Agar (MHA) based on the diameter of the inhibition zone [42].
2.3.6. Cytotoxicity study
HSF cells were seeded in 96-well cultures containing Dulbecco’s Modified Eagle Medium (DMEM) at a density of 3 × 103 cells per well. The DMEM medium was supplemented with glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), fetal calf serum (10%), and fungizone (1.25 µg/mL) in a humidified, 5% (v/v) CO2 atmosphere. After 24 hrs, the confluent HSF cells were washed three times with DMEM and then cultured for 72 hrs in a culture medium containing the cryogel extracts that were collected by incubating the ADA-COL and AP-loaded cryogels in culture DMEM medium for 72 hrs at 37°C. The cell viability was obtained after 72 hrs using MTT kits in which the DMEM medium was removed and replaced by 100 μL fresh medium loaded with MTT solution (20 μL). The cell palates were kept at 37°C for another 4 hrs in the dark. Then, the added medium was removed again and replaced by 100 μL/well of DMSO solution to dissolve the newly formed formazan crystal. After slow shaking for 10 mins, the absorbance was recorded at 570 nm with a Thermo Fisher microplate reader (USA). The cytotoxicity was introduced as the percentage of HSF cell viability in comparison to the blank control (cell incubated without cryogel extracts). Three parallel experiments were done for cryogels.
2.3.7. Wound closure assay (Migration assay)
For the in vitro wound healing assay, also known as the cell migration assay, HSF cells were seeded in a 12-well plate at a density of 7x105 cells/well and incubated overnight in DMEM medium at 37°C and 5% CO2 atmosphere. After that, the confluent HSF cells were scratched and washed with PBS medium thrice. The control wells were replenished with DMEM, whereas other wells were treated with DMEM media containing the cryogel extracts of the as-prepared ADA/COL and AAP-loaded cryogels. Cell images were captured through an inverted optical microscope at different time periods. The obtained images were characterized via the Image Analyzer Program (MII Image View software, version 3.7). The wound closure rate is introduced as the percentage of area reduction of scratched area, as shown in Eq. (4).
Where A0 is the scratched area immediately after the scratching process (time zero), and At is the scratched area at different time intervals.
2.3.8. Statistical analysis
All experiments were carried out in triplicates (n=3), and data are reported as mean ± standard deviation (SD). Statistical analysis was reported by a Prism GraphPad software. Differences between groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test to compare multiple groups. A p-value of <0.05 was determined statistically significant.
3. Results and Discussion
3.1. Characterization of the obtained ADA/COL and AAP-loaded cryogels
Natural hydrogels are usually used for drug delivery, tissue engineering, and wound healing applications due to their physicochemical properties, smart features, controlled degradation, and excellent biocompatibility. Indeed, Schiff base reaction is of particular importance for the development of natural hydrogels from protein and polysaccharide. The present study involves the preparation of ADA/COL hydrogels based on the condensation reactions between the amine residues (-NH2) present in lysine amino acids of collagen biomacromolecules and the aldehyde residues (-C=O) found on polysaccharide chains as a consequence of the oxidation process. This process results in Schiff base formation. Although cryogels are a type of hydrogel formed under freezing temperatures. The process involves freezing a polymer solution (or wet hydrogel), which leads to phase separation into a polymer-rich phase and a polymer-poor phase (frozen solvent). In the case of water-soluble polymers, the ice crystals of water act as pores, forming templates, creating a homogenous and porous 3D structure as they undergo sublimation during the drying process [18].
In this study, the effect of ADA and AAP contents on the gel percent in the obtained APP-loaded and unloaded ADA/COL cryogels are shown in Table 1. However, there were no obvious changes in the Gel % with increasing ADA content. The AAP-loaded samples exhibited higher Gel % with maximum values of 91.2±7.1 % for 20AAP-50ADA/50COL, which could be due to the hydrophobic nature of AAP. As shown in Table 1, the results of zeta potential demonstrated that ADA/COL and AAP-loaded cryogels exhibit negative surface charges with a polydispersity index (PDI) value lower than 1. The zeta potential of 75ADA/25Col and 50ADA/50Col were –3.71 ± 1.22 mV and –1.28 ±0.72 mV, respectively. The AAP-loaded cryogels were changed from –4.25 ± 0.93 mV for 10AAP-loaded cryogels to –7.36 ± 1.24 mV for 20AAP-loaded cryogels. In agreement with previous studies, the decrease of surface charge among the ADA/COL and AAP-loaded cryogel samples could be due to the ability of Zn2⁺ to neutralize the negative charges on carboxylate groups of alginate and AAP, reducing the magnitude of the zeta potential [43,44]. It is also important to mention that the introduction of aldehyde groups (-CHO) does not directly contribute to the zeta potential because aldehydes are neutral. However, they can react with amines to form Schiff’s bases, which may alter the overall charge distribution [45,46].
| Sample name | Gel % | Zeta – potential [mV] | PDI |
|---|---|---|---|
| 75ADA/25COL | 74.5±7.5 | –3.71 ± 1.22 | 0.98 |
| 50ADA/50COL | 72.5±2.1 | –1.28 ±0.72 | 0.61 |
| 10AAP-50ADA/50COL | 86.4±3.9 | –4.25 ± 0.93 | 0.87 |
| 20AAP-50ADA/50COL | 91.2±7.1 | –7.36 ± 1.24 | 0.78 |
The morphology of cryogel scaffolds based on ADA/COL and AAP-loaded ADA/COL samples was investigated using SEM, whereby cross-sections of the samples were thoroughly examined and presented in Figure 1. However, all the samples display porous structures with heterogeneous and interconnected pore microstructures. It was observed that the COL content results in some morphological changes. As shown in Figure 1, the morphology of the ADA/COL cryogels revealed an interconnected porous structure with an even distribution of pores with an average pore size of 254±65 µm for the 75ADA/25COL sample and 114±35 for the 50ADA/50COL samples. Compared to ADA/COL samples, the AAP-loaded cryogel samples exhibited irregular and small pores, and the range of pore size was 45±11 μm for 10AAP-50ADA/50COL cryogels. Additionally, further increase of AAP contents resulted in an obvious decrease of pore diameter to 25±7 μm, and submicrosize pores were observed on the pore walls of APP-containing samples. However, SEM micrographs revealed the formation of interconnected porous structures in all cryogels. The cryogel with higher ADA content showed a homogenous porous structure with thin walls in comparison to the other samples. The incorporation of AAP into the ADA/COL cryogels results in smaller pore sizes with thick walls, as shown in Figure 1. Furthermore, the SEM images of the AP-ADA/COL samples showed obvious aggregates of fine particles embedded in the pores and the walls of the sample, indicating a homogenous distribution of AAP within the scaffolds. The obtained ADA/COL and AAP-loaded cryogels have a porous structure with interconnected pores, allowing for efficient nutrient and oxygen diffusion and waste removal, which are critical for tissue regeneration application [47]. The porous structure also facilitates cell migration, infiltration, and angiogenesis (formation of new blood vessels).
- SEM micrographs of the obtained cryogel scaffolds at different magnifications (a, b) 75ADA/25COL, (c, d) 50ADA/50COL, (e, f) 10AAP-50ADA/50COL, and (g, h) 20AAP-50ADA/50COL.
Figure 2 shows FTIR spectra of the obtained ADA/COL and AAP-loaded ADA/COL cryogel samples. The spectrum of COL showed amide III band (3284 cm−1), -CH2 stretching (2940 cm−1), amide I band (1630 cm−1), amide II band (1532 cm−1), -CH2 bending (1437 cm−1), C-N stretching peak and (1248 cm−1). The ADA/COL FTIR spectra possess the amide characteristic bands with some modification according to COL concentration. There was a clear change in the intensity of the peaks around 1600 and 1400 cm−1, which could be due to the interaction between gelatin amine groups and aldehyde groups of alginates and can also be related to the presence of ionic crosslinking of alginate carboxylic groups and zinc ions. In addition, the band at 1024 cm−1 related to C–O–C (cyclic ether) stretching vibrations of alginate was observed for ADA/COL samples.
- FTIR spectra of (A) COL, (B) 50ADA/50COL, (C) 75ADA/25COL, (D) 10AAP-50ADA/50COL, and (E) 20AAP-50ADA/50COL.
3.2. Water uptake and degradation
Maintaining a moist wound surround provides an optimal environment for all phases of the healing process, from debridement and granulation to re-epithelialization and remodeling [48,49]. A moist wound environment maintains optimal hydration levels, softening necrotic tissue and eschar. This allows endogenous enzymes such as proteases (e.g., matrix metalloproteinases) and phagocytic cells to break down and remove dead tissue more efficiently. Promotes cleaner wound beds without the need for mechanical or surgical debridement, reducing patient discomfort [50]. In this study, water uptake of the as-prepared ADA/COL and AAP-loaded cryogels was carried out in acetate (pH 3.4) and tris HCl-buffer (pH 7.4) and was determined by analyzing the changes in sample weight. The water uptake capacity of ADA/COL and AAP-loaded cryogels was found to be pH-sensitive. Figure 3 represents the water uptake of the as-prepared cryogel samples after incubation in the buffer systems for 24 hrs. The ADA/COL cryogel sample gradually swelled after 24 hrs to a level of 734.6±90% and 523.2±76.5% in acetate and tris HCl-buffer, respectively. Both alginate and collagen are highly hydrophilic, enabling the cryogels to imbibe large amounts of wound exudate while maintaining structural integrity. The water uptake capacity of AAP-loaded cryogels decreased to 670.3±79.3 and 609.4 ±50.1 for 10AAP- and 20AP-loaded cryogels in acetate buffer at pH 3.4, whereas the water uptake values of AAP-loaded tris HCl-buffer was reduced to 495.8±39 and 470.9±53% for 10AP- and 20AP-loaded cryogels. In agreement with previous studies, the water uptake values were lower at the incubation of cryogel samples in tris buffer (pH 7.4) than in acetate buffer at pH 3.4 [51,52]. This could be attributed to the fact that the crosslinking between ADA and COL via Schiff base linkage is favorable in basic conditions. Therefore, the cryogel loses its stability in acidic environments due to the limited Schiff base linkage.
- Water uptake and weight loss of the ADA/COL and AAP-loaded cryogels.
Biodegradation plays a critical role in wound healing, particularly when considering the use of biodegradable materials in advanced wound care products such as dressing materials [53]. Weight loss is a well-known in vitro approach to probe the biodegradation behavior of a specific biomaterial under biological conditions. As shown in Figure 3, the results revealed changes in the weight loss (%) among the cryogel samples over 3 days of incubation in tris-buffer and acetate-buffer. In general, The ADA/COL cryogels showed a higher degradation rate in acidic conditions. For instance, the weight loss rate for ADA/COL samples was 43.7% and 20.8% at pH 3.4 and 7.4, respectively. In addition, the weight loss of the 20AAP-loaded cryogel was around 50 % in acetate buffer, which was slightly higher than 10AAP-loaded cryogel of 45.4%. Indeed, the biodegrading potential and high-water uptake capacity of ADA/COL cryogels make them highly promising for wound healing. The aldehyde modification allows ADA to form dynamic pH-sensitive imine bonds (Schiff bases) with collagen at the wound bed. These bonds can gradually degrade, releasing alginate and collagen fragments. In addition, the reversible Schiff base crosslinks between ADA and collagen allow the cryogel to swell without breaking down prematurely, increasing its ability to retain water.
Overall, the cryogels display pH-sensitive features, which are beneficial for wound healing. Wound environments often have varying pH levels, and the ability of the cryogels to respond to these changes ensures optimal performance in different wound conditions. This high water uptake aligns with the requirements for wound dressings to manage exudates and maintain a hydrated environment, which is crucial for promoting healing.
3.3. Antibacterial activity
The microbial activities of ADA/COL cryogel against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumagitus were determined by using the Agar disc diffusion technique. The potential activity of the obtained cryogels was determined by calculating the diameter of the clear zone, the inhibition zone, and around the tested samples, as given in Table 2. However, the ADA/COL cryogel showed antibacterial activity against S. aureus only. The APP-loaded ADA/COL cryogels showed high activities against Staphylococcus aureus, Candida albicans, and Aspergillus fumagitus and limited activity against Pseudomonas aeruginosa, as shown in Table 2. It can be clearly seen that the inhibition zone increases with the increase of APP loading in the ADA/COL cryogels. Therefore, 20AAP-loaded cryogels exhibited acceptable antimicrobial activities, which makes them of great potential to fight against infection in wound healing applications.
| Sample code | Inhibition zone (mm) | |||
|---|---|---|---|---|
| S. aureus | P. aeruginosa | C. albicans | A. fumigatus | |
| AAP | 4±1.7 | -- | 2±0.4 | 5±0.7 |
| 50ADA/50COL | 1±0.5 | -- | -- | -- |
| 75ADA/25COL | 1.3±0.6 | -- | -- | -- |
| 10AAP-50ADA/50COL | 2.8±0.9 | -- | 0.8±0.1 | 4.7±0.9 |
| 20AAP-50ADA/50COL | 3.2±0.4 | 0.8±0.3 | 1.2±0.3 | 4.9±1.4 |
3.4. Cell viability
Improving skin cell growth is crucial in the wound healing process, as fibroblasts are key players in producing the ECM, promoting angiogenesis, and synthesizing collagen to strengthen the healing tissue [54]. Strategies to enhance fibroblast activity and proliferation include optimizing the wound environment, utilizing growth factors, and incorporating bioactive molecules. Figure 4 shows the HSF cell viability after 72 hrs of incubation with the cryogel-extracted medium. The cell viability assay demonstrated that the incorporation of AAP accelerates the proliferation of HSF cells.
- HSF cell viability after 3 days of incubation with extracted medium from various ADA/COL and AAP-ADA/COL cryogel samples. (a) The HSF cell viability using the MTT assay after 3 days. Three independent experiments were carried out for each sample. When compared to the corresponding controls, **indicates non-significant (P > 0.05) and * indicates significance (P < 0.05). The extent of cell staining with calcein: (b) control, (c) ADA/COL, and (d) 10AAP- ADA/COL and (e) 20AAP-ADA/COL scaffolds.
3.5. Cell migration assay
A wound closure assay, also known as a migration assay or scratch assay, is a simple and broadly used in vitro method to examine cell migration, a critical process in wound healing, cancer metastasis, and other physiological and pathological conditions [55]. It provides insights into how cells migrate to close a gap, mimicking the natural wound-healing process. In addition, this assay remains a robust and flexible in vitro method to investigate cellular motility and dynamics critical to wound healing and other physiological or pathological processes [56,57]. Thus, the current study was directed to examine the effect of ADA/COL and AP-ADA/COL cryogel samples on HSF cell migration. As shown in Figure 5, the control group was associated with wound closure rates of 47.1±2.8 and 81.8±6% after 24 and 48 hrs, respectively. While ADA/COL cryogels showed lower wound closure rates after 24 and 48 hrs of 29.4±0.23 and 71.3±5.45%, respectively. On the other side, the AAP-loaded cryogel showed a better wound closure rate of 69.1±1.24 and 97.8±2.8% after 24 and 48 hrs, respectively. The loading of AAP into ADA/COL cryogels significantly promotes fibroblast cell invasion and migration, creating a favorable environment for tissue repair. This is attributed to the antioxidant properties of AAP, the biocompatibility of the ADA/COL matrix, and the porous structure of the cryogels.
- Wound closure rate over 2 days after exposure to HSF cells to extracted medium ADA/COL and 20AAP-loaded cryogel samples. When compared to the control samples, **denotes non-significant (P > 0.05) and * denotes significance (P < 0.05).
4. Conclusions
In summary, the study successfully developed highly stable cryogels based on marine ADA/COL gel using a freeze-drying technique. The incorporation of AAP had significant synergistic effects on both the gel content and the porous structure of the cryogels. The obtained cryogels displayed pH sensitive features with excellent water absorption capability of 734.6±90% (in pH 3.4) and 523.2± 76.5% (in pH 7.4). The AAP-loaded cryogels demonstrate antimicrobial effects against a range of pathogens, including S. aureus, C. albicans, and A fumigatus. Moreover, the AAP-loaded cryogels reveal the dual role of promoting HSF growth and migration. These findings underscore the potential of AAP-enriched cryogels as advanced wound dressings that promote healing and prevent infections, making them a valuable tool for managing complex wounds. Thus, further in vivo studies and clinical trials will be crucial for translating this innovative material into clinical practice, offering a new and effective option for wound healing.
Acknowledgment
This research was funded by the Deanship of Scientific Research and Libraries at Princess Nourah bint Abdulrahman University, through the “Nafea” Program, Grant No. (NP-45-025).
CRediT authorship contribution statement
Ghadah M. Al Senani: Conceptualization, Methodology, Visualization, Investigation, Supervision, Data curation, Software, Validation, Writing-Original draft preparation, Writing-Reviewing and Editing. Salhah D. Al-Qahtani: Conceptualization, Methodology, Data curation, Writing-Reviewing and Editing. Hesah M. AlMohisen: Conceptualization, Methodology, Data curation, Writing-Original draft preparation.
Declaration of competing interest
The authors declare that they have no competing interests.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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