Carboxymethyl chitosan (Macklin, China), Sodium alginate (Macklin, China), D-glucono-δ-lactone (GDL, Sigma Aldrich, United States), RIPA buffer (Beyotime, China). Cell Counting Kit-8 (Solarbio, China), Masson’s Trichrome Stain Kit (Solarbio, China). BCA Protein Assay Kit (Beyotime, China), Minimum essential medium (MEM, Gibco, United States), Fetal bovine serum (FBS, Gibco, United States). Clarity Western ECL Substrate (Shanghai, Bole Life Medical Products Co. Ltd., China), anti-CD31 (Abcam, UK) and α-SMA (Cell Signaling Technology, United States), VEGF (Proteintech, United States), PGP9.5 (Abcam, UK), Catalase (Cell Signaling Technology, United States), GAPDH (Proteintech, United States), anti-rabbit and anti-mouse secondary antibodies (Cell Signaling Technology, United States). All other chemicals were of analytical grade.

50 g of A. membranaceus and 50 g of P. notoginseng were extracted with 600 mL of 75% ethanol three times. The filtrates of traditional Chinese medicine were concentrated using a rotary evaporator. The concentrated extract was evaporated by a nitrogen blower until complete drying.

2 mL of 2% SA and 2 mL of 2% CS were mixed until obtaining a homogeneous suspension. 1 mL of GDL solution (20 mg/mL) was added to the CMCS/SA solution with constant stirring in the ice water bath for 1 min to form CS hydrogels.

2% SA and 2% CS were mixed separately by a filtered solution of AP (125 mg/mL). 2 mL of 2% SA-125 mg/mL AP and 2 mL of 2% CS-125 mg/mL AP were mixed until obtaining a homogeneous suspension. 1 mL GDL solution (20 mg/mL) was added to the CMCS/SA solution and maintained under stirring in the ice water bath for 1 min to form APCS hydrogels with AP concentration of 100 mg/mL.

Both the CS hydrogel and the APCS hydrogel were cut into cross-sectional slices of 2 mm thickness and 25 mm diameter. The storage modulus (G′) and loss modulus (G″) of hydrogels with a rheometer (AR 2000, TA Instruments) under the frequency range of 0.1–10 Hz were measured at 37°C and a constant strain, and a strain sweep on hydrogels were carried out from 0.1% to 10% at a frequency of 1 Hz.

The samples were tested for their compressive properties using a universal tensile testing machine (Instron3345). Hydrogel samples of diameter 5 mm and height 8 mm were placed between compression plates with 100 N load cells. The compressive stress-strain behavior of the samples was plotted and used to calculate compressive strength and modulus.

All samples were covered with a gold layer, and the structural morphology of CS hydrogels and APCS hydrogels were observed by scanning electron microscopy (SEM, inspect F50, FEI).

Flux dialysis was used to conduct the drug release experiment. The dialysis bag with 2 mL of APCS was put into 50 mL of PBS at 37°C and stirred continuously with a magnetic stirrer. 1 mL was collected and immediately replaced with an equal amount of fresh PBS at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h, respectively. The oleanolic acid release was obtained by detecting absorbance at 523 nm using an ultraviolet spectrophotometer (Agilent Technologies). All the experiments were repeated three times, and the cumulative drug release amount was determined with the standard calibration curve.

L929 cells were purchased from Stem Cell Bank, Chinese Academy of Sciences, and cultured in MEM with 10% FBS, 1% penicillin, and 1% streptomycin. Cells were grown at 37°C with 5% CO2. L929 cells (1 × 10⁵ cells/well) were seeded on the 96-well plate, and cultured for 24 h. The supernatant was replaced by the various concentrations (0.0125, 0.025, 0.05, 0.075, 0.15, and 0.3% v/v) of the CS/APCS hydrogel leaching solution, or the various concentrations (12.5, 25, 50, 75, 150, and 300 mg/L) of AP solution. After treatment for 48 h, 100 μL of MEM containing 10% CCK-8 reagent was added to each well. The absorbance of optical density (OD) was recorded at 450 nm.

The L929 cells were planted on the 96-well plate with a number of 1 × 10³ cells/well, and cultured for 24 h. The supernatant was replaced by the CS/APCS hydrogel leaching solution (0.075% v/v) or the AP solution (75 mg/L), and the cells were cultured for 24 h, 48 h, and 72 h 100 μL of MEM with 10% CCK-8 reagent was added to each well. The absorbance was recorded at 450 nm.

L929 cells (5 × l0⁵ cells/well) were inoculated in the six-well plate. When L929 cells reached 100% confluence, the wound was created with a sterile 200 μL pipette tip. After the cells were washed with PBS and serum-starved for 5 h. The supernatant was replaced by the CS/APCS hydrogel leaching solution (0.075%v/v), or the AP solution (75 mg/L). After 0, 12, and 24 h, the scratched areas were photographed using an inverted microscope (Osaka, Japan) at ×40 magnification.

32 male Sprague-Dawley rats (180–220 g) were obtained from Beijing HFK Bio-Technology Co., Ltd. Rats were housed under standard laboratory conditions (22°C ± 2°C, 55%–60% relative humidity, and 12 h light/12 h dark cycle) and the rats received food and water ad libitum. All the animal experiments were performed following the Ethics Committee of the Experimental Animal Research Centre, Shenyang Medical College (protocol No.SYYXY2023030501). After 1 week of adaptive feeding, rats were fasted for 12 h and then anesthetized by intra-peritoneal injection of 10% chloral hydrate to induce anesthesia. The dorsal surface hair of the animals was shaved, and a full-thickness skin defect with a diameter of 20 mm was created using scissors, then the rats were returned to their cages individually. Animals were randomly divided into four groups (n = 8 per group): the NC group received no treatment, 0.1 mL of CS for the group CS, 0.1 mL of AP for the group AP, and 0.1 mL of APCS for the group APCS, drugs were administered once during the entire treatment period. Photographs were taken with a digital camera on days 0, 4, 7, 10, and 14. In each group, four rats were randomly euthanized on days 7 and 14 after starting the treatment. The tissues collected from the healing area adjacent to the wound site and normal skin. The collected samples were cut into two parts, half of the samples at −80°C for further molecular analyses, and the others fixed in a 10% neutral formalin solution for histological analysis.

After paraffin embedding, the samples were cut into a 5 μm thick section and stained with hematoxylin and eosin for the study of tissue appearance. Masson’s Trichrome Staining Kit was used to detect the content of collagen in wound healing. Angiogenesis in wound healing was confirmed by immunohistochemistry with antibodies CD31 (1:2000) and α-SMA (1:5000). Briefly, each section was incubated in 3% H2O2 for 10 min and in anti-CD31 or anti-VEGF at 4°C overnight. And then the slice was incubated with Poly-HRP Anti Mouse/Rabbit IgG for 30 min at 37°C.

Cells were seeded into 6-well plates at a density of 5 × 10⁵/well. After 24 h, the supernatant was replaced by the CS/APCS hydrogel leaching solution (0.075%v/v), or the AP solution (75 mg/L). Tissues or cells were homogenized in RIPA buffer, and the protein concentration of the supernatants was determined by BCA Kit. The samples were then moved to sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V and they were subsequently transferred to PVDF membranes at 80 V. Afterward, the membranes were blocked with skimmed milk for 1 h at room temperature. The membranes were with α-SMA (1:1000), VEGF (1:1000), PGP9.5 (1:5000), Catalase (1:1000), and GAPDH (1:50000) polyclonal primary antibodies at 4°C overnight. Corresponding anti-rabbit or anti-mouse secondary antibodies (1:3000) were incubated for 1 h at room temperature. The immunoreactive bands were developed with Clarity Western ECL Substrate and analyzed using ImageJ software.

All data were analyzed using GraphPad Prism 9 software (GraphPad, United States) and SPSS 23.0 software (IBM, United States). Results were expressed as mean ± SEM. Comparison between groups was conducted by one-way ANOVA, and P < 0.05 was considered significant.

In order to investigate the behavior of the hydrogel, we examined the gelation kinetics of CS hydrogels and APCS hydrogels by analysis of the storage modulus (G′) and loss modulus (G″). The frequency sweep profiles demonstrated that the hydrogels exhibited a value of G′ of 600 Pa (CS) and 1200 Pa (APCS) at 0.1 Hz. And the values of G′ and G″ for APCS hydrogels were always higher than that for CS hydrogels at the same frequency. Moreover, as G′ exceeded G″ over the entire frequency range, CS hydrogels, and APCS hydrogels showed a gel state. These data suggest that the CS hydrogels loaded with AP could increase the elastic free energy (Figure 1A). Meanwhile, a strain sweep on CS hydrogel or APCS hydrogel was carried out from 0.1% to 10% at a frequency of 1 Hz to determine the linear viscoelastic range (LVER). The amplitude sweep test showed that APCS hydrogels exhibited a similar LVER, compared to pure CS hydrogels (Figure 1B). The hydrogels showed fluid characteristics (G″ >G′) with a strain of more than 5%. In the compression test of hydrogels, the anti-pressure ability of the CS hydrogels was significantly reduced in about 30% strain, while the APCS hydrogels could over 60%, the findings revealed that the compressive strength of the hydrogel was significantly enhanced following the composite AP to the CS hydrogels (Figure 1C). We detected the amount of drug released as a function of time by drug release experiment. After 12 h of sustained release, the cumulative drug release rate was over 90%, which showed that the APCS hydrogel could quickly release the medicines in a simulated wound condition. The drug release rate reached equilibrium after 24h, so the hydrogel had a good controlled release behavior for the drug (Figure 1D). The pore size of hydrogels is a critical parameter. A large porosity structure was observed in the CS hydrogel, while the APCS hydrogel showed a narrower range of porosity structure because of the crossing-linked of AP and the CS (Figure 1E). These results showed that the APCS hydrogel displays higher compressive strength than CS hydrogel and could improve the structural connections. Hydrogels’ structure with interconnected pore structures facilitates water absorption, oxygen and nutrient exchange, and cell migration and growth, providing conditions for the rapid healing of wound tissues.

To investigate the biocompatibility of APCS hydrogels, we detected the cell viability by CCK-8 staining. The results displayed that the cell viability was more than 95% in all concentrations, and there was no observed cytotoxicity in the APCS group at the maximum concentration. Then, the in vitro proliferation was evaluated on L929 cells. Compared with the NC group, the CS and AP groups could have different degrees of promoting fibroblast proliferation after treatment for 24 h, the proliferation rate was 120% ± 1.2% and 134.9% ± 2.4%, respectively. The proliferation rate of the APCS group (143.5% ± 2.1%) was significantly higher than the CS and AP groups. At 48 h post-treatment, the cell proliferation rate of the CS, AP, and APCS groups (137.5% ± 3.0%, 147.6% ± 1.1%, and 169.8% ± 0.4%) increased gradually. For cultivating 72 h, the fibroblast proliferation rate of the group treated with the APCS hydrogel was significantly higher than the NC and CS groups, and the data reached 185.3% ± 2.2% (Figure 2A). These results suggested that APCS hydrogels implied favorable biocompatibility, CS hydrogels and AP solution could have different degrees of promoting the proliferation of fibroblasts, and the APCS group displayed especially outstanding effects on increasing the proliferation of L929 cells.

To assess the migration effects of APCS hydrogels on L929 cells, we examined the wound healing rate by scratch assay. After 12 h of CS hydrogels and AP solution treatment, different degrees of cell migration were observed at the edge of the scratch, the wound healing rate was 32.2% ± 1.9% and 43.8% ± 0.4%, respectively. The narrowest gap was observed in the APCS group (52.5% ± 0.8%). At 24 h post-treatment, the wound healing rates of the CS and AP groups were 57.5% ± 4.1% and 69.2% ± 3.3%, and the scratches were completely closed in the APCS group (100%). This indicated that CS hydrogels and AP solution could partly accelerate cell migration, and the APCS hydrogel was detected to significantly promote the migration of fibroblasts in vitro (Figures 2B, C). These data suggested that the application of hydrogel carriers helped AP play a better role in promoting the migration of fibroblasts.

The differentiation of fibroblasts into myofibroblasts plays an important role during the remodeling phase of wound repair, α-SMA is a crucial biomarker for fibroblast differentiation. In order to determine whether the APCS hydrogels induce the differentiation of L929 cells, we detected the production of α-SMA using Western blot analysis. The expression of α-SMA was rarely tested in the NC group, the level of α-SMA protein was elevated after treatment with the CS hydrogel, but it was significantly upregulated in the AP and APCS groups (Figures 2D, E). The results showed that treatment of AP solution and APCS hydrogels could effectively promote fibroblast differentiation in vitro.

To investigate the therapeutic effects of the APCS hydrogel in vivo experiments, the full-thickness wounds were created on the backs of rats and treated with the CS hydrogel, the AP solution, and the APCS hydrogel, respectively. Photographs were taken on the 0, 4, 7, 10, and 14 days. During the entire treatment period, no significant differences in wound size were detected between the CS and NC groups. After the treatment of CS hydrogels for 10 days, the wounds of rats showed significant healing compared with the NC group. Notably, the APCS group cloud effectively accelerates wound closure during the entire healing process (Figures 3A–C). These results suggested that the application of the CS hydrogel carrier helped AP achieve better efficacy in promoting wound repair, the APCS hydrogel showed significantly promoting effects on wound healing.

Re-epithelialization and collagen synthesis have been considered important processes of wound healing. In the hematoxylin-eosin (HE) staining of wound tissue on the seventh day, different degrees of inflammatory reaction were observed in each group. Compared with the NC and CS groups, neovascularization was discovered in the AP group. In contrast to the AP solution treatment, more angiogenesis appeared in the APCS group. On the 14th day, the neoepithelium length in the CS and AP groups was significantly longer in comparison with that in the NC group, and the APCS group had a longer neoepithelium than the CS and AP groups. Furthermore, thicker granulation tissue was observed in the AP and APCS groups than in the NC and CS groups. As the wounds did not completely heal, many inflammatory cells and inflammatory exudate were found in the NC and CS groups. However, fewer inflammatory cells were discovered in the AP and APCS groups (Figures 4A, C). These results showed that AP solution and APCS hydrogels were able to facilitate angiogenesis, and the treatment of the APCS hydrogel shortened the transition time from the inflammatory phase to the proliferative phase and accelerated wound healing.

In the Masson staining of wound tissue on the seventh day, slight collagen deposition was observed in each group. After the treatment of CS hydrogels and AP solution, new blood vessels were formed compared with the NC group. Compared with the CS and AP groups, the extent of neovascularization was enhanced greatly in the APCS group. Trends in angiogenesis of each group were consistent with the changes observed in H&E staining on the seventh day. Compared with the NC group, more little and loose collagen bundles appeared in the CS and AP groups on the 14th day, and numbers of thicker and denser collagen fibers were observed in the APCS group (Figures 4B, D). These results displayed that the treatment of APCS hydrogels promotes collagen synthesis in the wound healing process.

CD31 is a marker of neovascularization in wound healing, and it is mainly expressed by vascular endothelial cells in early angiogenesis. In the immunohistochemical detection of wound tissue on the 14th day, a few CD31 were expressed in the NC group. The CS, AP, and APCS groups showed dense positive immunoreactivity, and the expression of CD31 in the AP and APCS groups was distinctly increased (Figure 5A). These results proved that the treatment of AP solution and APCS hydrogels promoted the regeneration of capillaries. α-SMA is recognized as an important biomarker of mature blood vessels in the proliferative phase of wound healing, and it is mainly expressed in the vascular smooth muscle cells during later angiogenesis. In the immunohistochemical detection of wound tissue on the 14th day, the expression of α-SMA was detected in each group, and a number of blood vessels were formed in the APCS group’s wound tissue compared with the CS and AP groups (Figure 5A). These results indicated that the APCS hydrogel could promote the regeneration of new blood vessels and mature blood vessels in the wound repair process.

High levels of ROS cause severe tissue damage in the wound healing process, and catalase eliminates excessive ROS in skin wound repair. We assessed the efficacy of the APCS hydrogel under oxidative stress and found no significant differences in catalase expression between the CS group and the NC group. Compared with the NC group, the expression of catalase was upregulated in the AP group, and the APCS group showed the highest level of catalase (Figures 5B, C). These results suggested that the treatment of APCS hydrogel could promote the elimination of ROS by upregulating the expressions of catalase. Moreover, angiogenesis plays an important role during the proliferative stage of wound healing, the increased expression of VEGF is necessary for the formation of new blood vessels during the tissue repair process. We found the VEGF level of the CS group was similar to the NC group by Western blot analysis. The expression of VEGF in the AP and APCS groups was significantly upregulated (Figures 5B, D). These results showed that the AP solution and the APCS hydrogel could accelerate the healing in rats by promoting angiogenesis. There is abundant nerve regeneration during the proliferative stage of wound healing. PGP9.5 is an important marker of epidermal nerves. After 14 days of treatment, no significant differences in the expression of PGP9.5 were discovered between the CS group and the NC group, and the expression level of PGP9.5 in the AP, and APCS groups was significantly increased (Figures 5B, E). These results displayed that the treatment of AP solution and APCS hydrogel could accelerate nerve regeneration by upregulating the expressions of PGP9.5.