Polyvinyl alcohol/alginate/gelatin hydrogel-based CaSiO₃ designed for accelerating wound healing

2.1 Materials

Sodium alginate (SA, Mw 120,000 g·mol⁻¹), gelatin (Gel, 300 g Bloom), PVA (Mw 84,000), CS (CaSiO₃, particle size 1–5 µm), calcium chloride (CaCl₂), and all other reagents utilized in this study were sourced from Aladdin (Shanghai, China). GTA was acquired from Sigma Aldrich, while the cell counting kit-8 (CCK-8) was procured from Yasen Biotechnology (Shanghai, China).

2.2 Scaffold preparation

PVA weighing 1.2 g was dissolved in 30 mL of ultrapure water. The mixture was heated to 95℃ and stirred continuously using magnetic stirrers for 2 h until the PVA completely dissolved. Once dissolved, the solution was cooled to 37℃ before adding 0.6 g of SA. Stirring was continued for another 2 h. Following this, 2.4 g of gelatin was incorporated into the mixture. CS (CaSiO₃) powder was then mixed into the composite solution at different weight concentrations: 0.1, 0.3, and 0.5 wt%. This resulted in a hydrogel solution containing CaSiO₃, which was poured into molds and soaked in a 0.6% calcium chloride solution for 2 h. After soaking, the scaffolds were rinsed three times with ultrapure water to remove any residual solution on the surface. Subsequently, the scaffolds were soaked in a 0.4% GTA solution for 3 h to further cross-link the materials. Finally, they were rinsed three times with deionized water and frozen for 4 h at −20°C to completely crystallize the internal water of the molds. They were dried in a freeze dryer at −80°C for 24 h prior to use.

The resulting PVA/SA/gelatin hydrogel scaffolds containing CaSiO₃ were designated as PSG, PSG-0.1%, PSG-0.3%, and PSG-0.5%, respectively, based on their CaSiO₃ concentrations.

2.3 Characterization of PSG/CS composite scaffolds

The lyophilized hydrogels underwent fracture to obtain sections, which were then coated with a thin gold layer using vacuum sputtering. This preparation enabled the observation of their morphology and microstructure after freeze-drying through a scanning electron microscope (SEM, Hitachi, S-4800, Japan). Additionally, Fourier transform infrared (FTIR) spectroscopy was performed to investigate the potential organic–inorganic interactions within the PSG-CS scaffolds. This analysis covered a wavelength range of 500–4,000 cm⁻¹ (FTIR; Nicolet iS50, Thermos Scientific, USA). Furthermore, X-ray diffraction (XRD) analysis was performed on the powdered PSG-CS scaffolds using an X-ray diffraction analyzer (DX-2700B, Hao Yuan Instrument Co., Ltd, China). The powdered samples were exposed to CuKα-radiation at a voltage of 40 kV and a current of 30 mA, with a scanning angle ranging from 10° to 70°.

2.4 In vitro mechanical properties and the water contact angle of the hydrogel

The mechanical properties, specifically compression strength, Young’s modulus, and stress–strain, of the PSG/CS hydrogel were assessed using a Universal Material Tester (EZ-Test-5N, Shimadzu, Kyoto, Japan). The hydrogel membranes were precisely cut into a dumbbell shape, measuring 6 cm in length, 2 cm wide at the ends, narrowing to 1 cm in the middle, and maintaining a uniform thickness of 2 mm [27]. Cylindrical specimens of the gels with varying CS contents were prepared using molds of 10 mm height and 15 mm diameter [28]. During testing, a constant tensile pull rate of 50 mm·min⁻¹ was applied until complete rupture of the hydrogel occurred, while the compressive speed was set at 10 mm·min⁻¹. Each mechanical test was conducted on at least three specimens to ensure reliability. Additionally, the water contact angle of the hydrogel was measured using a contact angle measuring device (Portsmouth device, USA), employing the Laplace–Young method for accurate results.

2.5 In vitro swelling, degradation, and ions release of the hydrogel

The swelling behaviors of hydrogels were evaluated in the following steps [29]. PSG/CS scaffolds were weighed (100 mg) and immersed in 5 mL of phosphate buffer saline (PBS, pH 7.4, 37℃). At predetermined time points, the scaffolds were removed, tap-dried over filter paper to remove water from the surface of the scaffold, and weighed. The experiments were performed in triplicate, and the swelling ratio was calculated using the following formula:

The weight was W t when absorption equilibrium was reached, and the initial weight was W 0 .

For the in vitro degradation assay [30], scaffolds were immersed in simulated body fluid (SBF) solution (pH = 7.4) with a weight-to-volume ratio of 1 g/20 mL and accurately weighed ( W 1 ) at 37℃ at different times (0, 1, 3, 7, 14 and 21 days). The scaffolds were freeze-dried, and the residual weight ( W r ) was measured. Meanwhile, the SBF solution was collected to determine their released silicate ions using an inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7850, USA). The degradation degree of hydrogels was calculated based on the following formula:

2.6 Cell culture (L929)

L929 cells were acquired from the Chinese National Immortalized Cell Bank and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% fetal bovine serum and 1% penicillin/streptomycin solution (P/S). The cells were incubated in a controlled environment of 5% CO₂ at 37℃. The medium was refreshed every 48 h to ensure optimal growth conditions.

2.7 CCK-8 assay

The CCK-8 assay kit was employed to assess cell proliferation at 1, 3, and 5 days, following the manufacturer’s recommended protocol. In summary, mouse fibroblast L929 cells were seeded at a density of 3 × 10⁵ cells/well in 24-well plates and incubated with hydrogel extracts. Subsequently, the cell culture media were substituted with CCK-8 solution and incubated for an hour. The absorbance of the resulting supernatant was then measured at 450 nm using a microplate reader (Epoch2, Bio-Tec Instruments, USA) to determine the level of cell proliferation.

2.8 In vitro cytocompatibility assay (AM/PI staining)

In vitro cell assays were conducted by co-culturing the hydrogel extract with L929 cells. Initially, 1 g of the hydrogel was incubated in 5 mL of DMEM cell culture medium (pH 7.4) for 24 h. Prior to use, the solution was filtered through sterile filters. The cells were seeded in 24-well plates at a density of 3 × 10⁵ cells per well and incubated for 24 h in a cell incubator maintained at 37℃ with 5% CO₂. Subsequently, the hydrogel extracts were introduced to the 24-well culture plate, with 100 μm added to each well. Cell viability was assessed using AM/PI staining for 15 min at specific time points of 1, 3, and 5 days. Each group was replicated three times, and the cells were washed three times with PBS before being visualized under an inverted optical microscope (Leica DMI 4000, Germany) to observe live cells.

2.9 Cell morphology

L929 cells were seeded onto PSG/CS composite hydrogel scaffolds in 24-well plates at a density of 5 × 10⁵ cells per well and incubated under controlled conditions of 37°C and 5% CO₂. Following 3 days of incubation, the culture medium was aspirated, and the cells were gently washed twice with sterile PBS. Subsequently, 200 μm of 2.5% GTA was added to each well for fixation, and the plates were stored at 4°C. After 12 h, GTA was removed, and the scaffolds were subjected to freeze-drying. They were then sputter-coated with gold and examined using SEM(Hitachi S-4800, Japan) to observe the morphology of the L929 cells.

2.10 Cell scratch experiment

In summary, L929 cells were plated in a 6-well plate at a density of 5 × 10⁵ cells per well. Once the cells had reached a minimum of 90% confluence, three scratches were created per well using a 200 μL pipette tip. The wells were then gently washed with PBS to eliminate any detached cells. Subsequently, 2 mL of the hydrogel extracts were carefully added for co-incubation. At designated time points (0, 12, 24, and 48 h), an inverted optical microscope was utilized to capture images of the scratched regions, with three replicate samples per group. The wound area was precisely measured using ImageJ software, and the percentage of wound closure was determined using the following formula:

2.11 In vivo wound healing experiments

To assess the wound-healing potential of the hydrogels in vivo, male Sprague–Dawley rats weighing between 200 and 220 g were employed. The animal experiments were conducted in accordance with the approved guidelines provided by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu. Throughout the study, the rats were provided with a standard pellet diet and water ad libitum. The animals were randomly divided into five groups, with six rats in each group. Anesthesia was induced via an intraperitoneal injection of a ketamine (100 mg·kg⁻¹) and xylazine (10 mg·kg⁻¹) mixture. The dorsal fur of the rats was shaved using an electric razor, and the exposed skin was sterilized with 70% ethanol. Subsequently, two square wounds (10 mm × 10 mm) were surgically created on the dorsum of each rat using a skin needle biopsy punch. Each wound was dressed with either sterile gauze (serving as the control) [31] or one of the hydrogel formulations: PSG, PSG-0.1%, PSG-0.3%, or PSG-0.5%. The dressings were secured in place with an elastic adhesive bandage and changed daily. Wound progression was documented by taking photographs on days 3, 6, 9, and 12. ImageJ software was utilized to measure the wound size accurately.

2.12 Histological analysis

Skin tissues surrounding the wounded areas were harvested from the rats on days 6 and 12. These tissues were then promptly fixed in a 10% neutral buffered formalin solution and subsequently embedded in paraffin using standard laboratory procedures. The wound tissues were carefully sectioned into 5 μm-thick slides for detailed analysis. To evaluate the wound-healing process, hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were employed. Additionally, Ki67 and CD31 immunohistochemical staining were utilized to identify newly proliferated cells and regenerated blood vessels at the wound site, respectively.

2.13 Statistical analysis

All values were expressed as mean ± standard deviation. Student’s t-test, followed by regression analysis, was used for the calculation of p-values to examine the significant differences between experimental data, and p < 0.05 was considered significant. All statistical analysis was performed using the GraphPad Prism version5.1.

3.1 Scaffold preparation and characterization

In the present study, the scaffold was meticulously prepared using a blend of PVA, alginate, and gelatin in a ratio of 2:1:4 dissolved in deionized water, employing the freezing-drying method. To achieve gelation, SA was initially cross-linked with 0.6% calcium chloride; the gel capacity of different groups of hydrogels at different times is shown in Figure S1. Subsequently, GTA was used to cross-link gelatin, leveraging the reaction between the aldehyde group of GTA and the amino groups present in various proteins. PVA plays a pivotal role in regulating scaffold degradation. Consequently, the combination of PVA and the double cross-linked alginate/gelatin matrix ensures optimal mechanical strength while mimicking the ECM microenvironment. To optimize the CS content for tissue regeneration, composite scaffolds incorporating varying amounts of CS (0, 0.1, 0.3, and 0.5 wt%) were prepared and characterized, and the states of hydrogel in various stages are shown in Figure S2.

3.2 SEM

SEM analysis revealed that as the CS content increased, the surface morphology of the hydrogel became increasingly rough (Figure 2a1–a4). The cross-sectional view exhibited a network structure conducive to oxygen exchange and the transport of medicinal nutrients. This structure also possesses the ability to absorb wound exudate while maintaining an appropriate moisture level (Figure 2b1–b4). Notably, when the CS content reached 0.5%, the three-dimensional cavity structure of the hydrogel underwent significant shrinkage, with CS evenly distributed throughout the scaffold. Conversely, at CS content below 0.1%, the degradation rate of the scaffold may be accelerated.

Figure 2

SEM images of PSG/CS composite scaffolds. Surface (a₁–a₄) and cross-sectional (b₁–b₄) FTIR spectra of PVA, SA, Gel, and CaSiO3 (c), and composite PSG with different CS ratios (d); and (e) XRD patterns of CaSiO3, PSG, PSG-0.1%, PSG-0.3%, and PSG-0.5%.

3.3 FT-IR spectroscopy

To investigate the intermolecular interactions within the blended compounds, FT-IR spectroscopy was employed to characterize the chemical groups of the hydrogel. The results are presented in Figure 2c and d. Pure PVA exhibited transmittance bands at 3,434 cm⁻¹ for the O–H group, 2,913 cm⁻¹ for C–O stretching, and 1,097 cm⁻¹ for C–H group vibrations. SA peaks were observed at 1,623 cm⁻¹ for the carboxyl bond C–O, and 2,038 and 2,360 cm⁻¹ for antisymmetric and symmetric vibrations, respectively. Characteristic peaks of gelatin were identified at 3,427 cm⁻¹ for the N–H stretching of the amide bond, 1,616 cm⁻¹ for the C–O group of amide I, 1,418 cm⁻¹ for the N–H group of amide II, and 1,031 cm⁻¹ for the NH group of amide III. CS absorption peaks at 706 and 610 cm⁻¹ corresponded to the peak vibrations of Ca–O and Si–O–Si, respectively. The peaks at 1,420, 1,300, 1,047, and 950 cm⁻¹ were attributed to symmetric stretching in CS molecules.

The FT-IR spectra of the PSG hydrogel scaffold revealed several notable changes. The O–H peak shifted to 3,271 cm⁻¹, the amide I peak at 1,616 cm⁻¹ broadened significantly, the amide II peak moved from 1,418 to 1,444 cm⁻¹, and the weak peak associated with SA disappeared. These observations confirmed the formation of hydrogen bonds between PVA, gelatin, and SA [32,33]. Additionally, the PSG/CS spectra exhibited stronger peaks at 3,260, 1,628, 1,029, and 818 cm⁻¹, representing Si–OH, C–H, Si–O–Si, and Si–O, respectively. These results clearly indicate the successful incorporation of CS into the PSG scaffold without compromising its structural integrity. Furthermore, the difference in intensity between PSG-0.5% and PSG-0.1% suggests that varying concentrations of CS can be loaded into PSG. The presence of Si–OH is reported to enhance the mechanical properties of hydrogels by providing additional covalent bonds between gelatin [34].

3.4 XRD

The composites exhibited an amorphous state without being incorporated into CS. However, XRD analysis revealed characteristic diffraction peaks at 2θ values of 23.1°, 25.2°, 26.7°, 28.8°, 29.9°, and 39° (Figure 2e) when added to CS. These peaks closely matched the standard diffraction pattern of CS (CCDC:75-1396), indicating that the double cross-linking method did not disrupt the crystalline structure of CS.

3.5 Mechanical properties of the hydrogel in vitro

Mechanical properties, such as the compression strength and tensile strength of the hydrogels, were assessed. As illustrated in Figure 3a and b, the composite scaffolds demonstrated significantly higher compressive strengths compared to the pure PSG scaffolds. Notably, when the CS content exceeded 0.3%, the compressive strength plateaued and remained relatively stable, comparable to that of the 0.5% CS composite. Similarly, the tensile strength of the composite scaffolds surpassed that of the pure PSG scaffolds, with the 0.3% CS composite scaffolds exhibiting the highest tensile strength. Stress–strain curves also proved this (Figure S5), showing better elasticity. This enhancement in mechanical properties is primarily attributed to the uniform distribution of CS within the hydrogel network, which reinforces the scaffold’s resistance to deformation and improves the mechanical properties.

Figure 3

Physiochemical properties of the composite scaffolds in vitro. (a) Compressive strength of scaffolds (n = 4). (b) Tensile strength of scaffolds (n = 3). (c) Water contact angle of scaffolds (n = 3). (d) Swelling of scaffolds (n = 3). (e) Degradation of scaffolds (n = 3). (f) Accumulated concentration of released SiO 3 2 − ions. **P < 0.01 and ***P < 0.001.

3.6 In vitro water contact angle of the hydrogel

The water contact angles formed between a droplet of DMEM and the surfaces of PSG, PSG-0.1%, PSG-0.3%, and PSG-0.5% were found to be 58.5 ± 1.6°, 61.8 ± 3.6°, 67.6 ± 4.1°, and 77.2 ± 0.8°, respectively (Figure 3c and Figure S3). Although the addition of CS led to a gradual decrease in hydrophilicity, the contact angles remained below 90°, indicating that the composite hydrogels retained excellent hydrophilic properties even after the incorporation of CS.

3.7 In vitro swelling, degradation, and ion release of the hydrogel

During the initial soaking stage (<1 h), the hydrogel scaffold exhibited a rapid swelling rate. Within 5 h, the scaffold achieved considerable absorption capacity, reaching up to 855% (Figure 3d and Figure S4). As the CS content increased in the PSG-0.1%, PSG-0.3%, and PSG-0.5% composites, the equilibrium swelling rates of the hydrogels decreased to 806, 588, and 506%, respectively. This trend suggests that the swelling behavior of the hydrogels can be effectively modulated by varying the CS content. Composite hydrogels with higher swelling rates demonstrated enhanced fluid exchange capabilities [35], facilitating transport between cells and matter.

The in vitro degradation assay revealed a decreasing trend with lower CS content (Figure 3e). After soaking in PBS solution for 21 days, the degradation rates of the scaffolds were 78.90 ± 1.82% (PSG), 68.32 ± 5.61% (PSG-0.1%), 45 ± 5.82% (PSG-0.3%), and 23 ± 3.02% (PSG-0.5%), respectively. Despite the hydration of CS and the continuous release of calcium ions, the hydrogels maintained structural stability throughout the degradation process.

Furthermore, the ion release from different composite scaffolds was evaluated. It was observed that both SiO 3 2 − and Ca²⁺ ions were consistently released from all composite scaffolds. As the CS content increased, the ion concentration gradually increased, providing crucial support for collagen fiber formation and tissue regeneration (Figure 3f and Figure S6).

3.8 Biocompatibility of the hydrogels

3.8.1 Cytocompatibility and cell morphology of hydrogels

To assess the cytocompatibility of hydrogels, a hydrogel leaching test was conducted [36]. The results, presented in Figure 4a, demonstrate that the viability of L929 cells gradually increased in all four groups after incubation with hydrogel leach liquor for 1, 2, 3, and 5 days. This suggests good biocompatibility of the hydrogels. Notably, after 5 days of coincubation, the PSG/CS group exhibited a higher number of cells compared to the control and PSG groups. Furthermore, live/dead staining images in Figure 4b revealed enhanced cell growth and proliferation in hydrogel extract over 5 days. Specifically, the PSG-0.5 group displayed the highest fluorescence intensity among all groups, indicating that CS may enhance cell proliferation. Additionally, Si ions released from silicate-based bioceramics not only attract fibroblasts but also augment the secretion of ECM-related proteins from these cells [37]. After co-culturing the scaffold with cells for 3 days and dehydrating with alcohol for 2 h, the SEM images in Figure 4c revealed the cell morphology of the scaffold [38]. Compared to PSG, scaffolds doped with CS exhibited a rough and porous surface, facilitating better cell pseudopod adhesion to the scaffold’s micropores. This was more conducive to cell adhesion and growth into the scaffolds. CS inorganic nanoparticles have been reported to interact with fibroblasts, potentially contributing to the increased rate of cell adhesion in the PSG/CS scaffold group [39].

Figure 4

The effect of composite scaffolds on L929 cells. (a) Proliferation of L929 cells after being treated with the leaching solution of different hydrogels for 1, 3, and 5 days (n = 3). (b) AM/PI staining of L929 cells after being treated with the leaching solution of different hydrogels for 1, 3, and 5 days (n = 3). (c) Morphology of dead cells of L929 cells cultured on different scaffolds after co-culture for 5 days (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

3.8.2 Cell migration (in vitro scratch wound assay)

L929 cells were used in the scratch assay to evaluate the effect of the hydrogel formulation on cell migration [40]. As shown in Figure 5a, compared with the control and PSG groups, the L929 cells grew and migrated significantly faster in the PSG/CS groups. This is attributed to the CS loaded into the hydrogels, which can promote the proliferation and migration of fibroblast cells. Furthermore, the quantitative evaluation of the scratch area healing rate showed that the healing percentage of cell scratch in the PSG-0.3 group was 66.35% at 48 h (Figure 5b). In comparison, the healing percentages in the control and PSG groups were only 51.69 and 34.59%, respectively. The rate of cell proliferation continuously increased with the extension of time and the increase of CS concentration. Silicate-based composition has been reported to promote cell proliferation through stimulation of growth factors and also activation of certain cell signaling pathways like extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3-kinase) [22].

Figure 5

(a) Pictures of cell wound scratch assay with varying hydrogel treatments at different times (n = 3); scale bar = 200 μm. (b) Quantitative data of the cell wound scratch area.

3.9 In vivo animal wound model

3.9.1 In vivo wound repair efficacy

A full-thickness excision wound model on SD rats was established to assess the wound healing potential of hydrogels in vivo. Representative images captured on post-treatment days 3, 6, 9, and 12 are presented in Figure 6a. As shown in Figure 6b, the wound area gradually decreased in all five groups over time. Notably, after 6 days, the PSG-0.3% group exhibited a smaller wound area (61.42%) compared to the control (46.59%), PSG (51.44%), PSG-0.1% (52.56%), and PSG-0.5% (60.58%) groups. This trend persisted on the day 9, with the PSG-0.3% group demonstrating a significantly faster healing rate. By day 12, wounds treated with PSG-0.3% had completely closed, forming smooth epidermal tissue. In contrast, 82.34 and 85.16% of the wounds in the control and PSG groups, respectively, remained open with uneven scarring. These findings suggest that CS inherently contributes to wound healing. Previous reports have highlighted the hemostatic effects of the polymer matrix and its ability to provide a warm, moist wound environment, making hydrogels an effective wound dressing material [41]. In the present study, CS demonstrated a significant wound-healing promotion effect, likely due to its combined actions in cell proliferation and ECM formation. The hydrogel-based dressings maintained an optimal moisture level, facilitating wound exudate management.

Figure 6

(a) Photographs of the wounds and traces of wound closure on days 0, 3, 6, 9, and 12 (n = 6, *P < 0.05, **P < 0.01). (b) In vivo wound closure rates during day 12. (c) H&E staining and Masson staining of the wound sites on days 6 and 12. Scale bars = 400 μm.

To corroborate these findings, H&E staining and Masson’s trichrome staining were performed after 6 and 12 days of treatment (Figure 6c). H&E staining revealed notable tissue destruction in the control group compared to the PSG-0.3% group on both days 6 and 12. Masson’s trichrome staining was used to assess collagen deposition and distribution, showing intact epidermis and well-ordered collagen fibers in the PSG-0.3% group. Conversely, in the control and PSG groups, few collagen fibers and abundant granulation tissue were observed. These staining results suggest that the PSG/CS hydrogel enhances wound healing by promoting re-epithelialization and collagen deposition.

3.9.2 Ki67 and CD31 expression during the healing process

The underlying mechanisms of hydrogel-mediated wound healing were further explored through Ki67 and CD31 immunohistochemical staining (Figure 7a and b). The semi-quantitative analysis is presented in Figure 7c and d. The Ki67 antibody was used to label cell nuclei associated with mitosis, identifying actively proliferating cells such as keratinocytes, fibroblasts, and vascular endothelial cells, which are crucial for wound healing. As shown in Figure 7a and c, wounds treated with PSG/CS hydrogel exhibited higher Ki67 expression than other groups after 6 and 12 days of treatment. The addition of CS effectively enhanced cell proliferation and differentiation, demonstrating good affinity [42] and promoting tissue regeneration.

Figure 7

(a) Immunohistochemical images of Ki67 expression on days 6 and 12; scale bars = 400 μ m . (b) Immunohistochemical images of CD31 expression on days 6 and 12; scale bars = 400 μ m . (c) Quantitative analysis of Ki67 positive cells. (d) Quantitative analysis of CD31 positive cells.

Angiogenesis, evaluated by CD31 immunohistochemical staining at 6 and 12 days post-surgery (Figure 7b and d), plays a vital role in wound healing. CD31 is a key marker of vascular endothelial cells and is essential for vascular regeneration [43]. Weak positive staining for CD31 was observed in the control and PSG groups, whereas wounds treated with PSG/CS showed the strongest immunohistochemical intensity for CD31 on days 7 and 14. This indicates the superiority of PSG/CS in accelerating wound healing and enhancing angiogenesis.

In summary, the PSG/CS hydrogel, with its bioactive, self-healing, and biocompatible properties, effectively promotes infected wound healing by simultaneously upregulating Ki67 and CD31 production.