The synthesis of poly (glycerol-sebacate) (PGS) was according to Wang’s research (Wang Y D et al., 2002), then synthesized poly (glyceryl-sebacate-acrylate) (PGSA) by modifying PGS with acryloyl chloride (Nijst et al., 2007). The structure of PGS and PGSA was verified by ¹H nuclear magnetic resonance (¹H-NMR) and Fourier Transform Infrared (FTIR) spectrometry. The acrylation degree of PGSA was computed from the ¹H-NMR according to previous study (Chen et al., 2018).

The preparation method of nanospheres was as follows: At first, 30 mg PGSA was mixed in a 3 mL of ethanol and distilled water solution (V_E: V_W = 1:2) to dissolve into a homogeneous solution. Photoinitiator solution was prepared by dissolving I2959 powder with deionized water, then 40 μL I2959 (2 mg/mL) was mixed with the above PGSA solution. Magnetic stirring was performed while irradiating the above solution with a UV lamp, and PGSA nanospheres (PNs) were obtained after 30 s.

Hyaluronic acid (HA) was oxidized to oxidized hyaluronic acid (OHA) by sodium periodate (NaIO₄) (Hongyi et al., 2022). The structure of OHA was tested by ¹H-NMR and FTIR spectrometry. The oxidation degree of OHA was obtained through the hydroxylamine hydrochloride method (Hongyi et al., 2022). The proportion of aldehyde groups in the OHA molecular chain, is calculated as following: H A − ( C H O ) 2 + 2 N H 2 − O H · H C l → H A − ( C H = N − O H ) 2 + 2 H C l H C l + N a O H = N a C l + H 2 O n = ∆ V × C N a O H / 2 W = n × M H A − C H O + m × M H A o x i d a t i o n   d e g r e e 100 % = n / n + m = n / W − n × M H A − C H O / M H A + n

ΔV (mL) is the volume of sodium hydroxide solution consumed when adjusting the pH of the reaction solution; n (mol) is the number of repeating units of aldehyde groups on the OHA molecular chain; W (g) is the mass of OHA; M_HA-CHO = 375 g/mol is the molecular weight of the OHA repeating unit; M_HA = 379 g/mol is the molecular weight of the hyaluronic acid repeat unit.

The preparation of hydrogels was referred to Wang’s research (Wang et al., 2019), briefly summarized as follows: OHA was freeze-dried and dissolved into an 80 mg/mL solution by using distilled water. ε-EPL was dissolved into a solution by using the same method with a concentration of 50 mg/mL and 100 mg/mL. Then F127 was dissolved into a 400 mg/mL solution under 4°C. According to the volume ratio of F127: ε-EPL: OHA as 3:0:0, 3:0:1, 3:1:1, the F127 solution and the ε-EPL solution were sequentially mixed at 4°C, and the OHA solution was added after mixing evenly, then the solution was put in a thermostatic shaker for gelation, hydrogels were named as F127, FH, FHE5, FHE10, respectively.

PNs combined with FHE10 hydrogel were prepared by the above procedure. After mixing the F127 solution and the ε-EPL solution at 4°C, PNs were dispersed in the mixing solution. Then the OHA solution was mixed with above solution at 37°C to obtain the hybrid hydrogel.

The microstructure of PNs was observed by SEM and TEM, then the diameter of PNs was evaluated by ImageJ. The study of particle size and z-potential was using a DLS. The injectability of PNs was tested by needle injection to observe whether it can be extruded smoothly.

Using SEM to observe the microstructure of hydrogels, and the pore size was measured via ImageJ. Testing whether the FHE10 can be successfully extruded from a needle proves its injectability. Phosphate buffer solution (PBS) was dropped into the test tube with hydrogels several times until the volume no longer changes and then weighed the mass. The hydrogel was lyophilized and weighed, and its water absorption ratio was computed through formula (1): W a t e r   a b s o r p t i o n   r a t i o % = W − W 0 W 0 × 100 %   (1) When hydrogel was at swelling equilibrium, the weight was W, and when it was lyophilized, the weight was W₀.

The prepared hydrogel was weighed and placed in an environment of 37°C with a relative humidity of 70% and then the weight of it was recorded every 1 h. The water retention ratio was computed through formula (2): W a t e r   r e t e n t i o n   r a t i o % = W X − W W 0 − W × 100 %   (2)

In the start, the weight of the hydrogel was W₀, W_X was the weight of the hydrogel at the X hour, and after lyophilized the weight was W.

The free radical scavenging rate of hydrogel was measured by DPPH assay to evaluate its antioxidant capacity. The fresh 0.1 mM 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH)/ethanol solution was prepared in the darkness. 30 mg of hydrogel was mixed with 3 mL of DPPH solution and cultured for 3 h and 24 h in the dark. Then a UV-Vis spectrophotometer was applied to measure the absorbance of the solution at 517 nm. The scavenging rate was computed by formula (3): S c a v e n g i n g   r a t e % = A − A 0 A 0 × 100 %   (3)

The absorbance of the control group was A₀, and A was the absorbance of the samples incubated with DPPH solution after 3 and 24 h.

Blood compatibility test was carried out according to the method of GB/T 16886.4-2003 of China. The procedure is described below. 4 mL of carotid artery blood was collected from healthy SD rats, and anticoagulated whole blood was prepared using aqueous trisodium citrate solution, then freshly diluted anticoagulated whole blood was prepared using PBS buffered solution. The materials F127, FH, FHE5 and FHE10 were placed in centrifuge tubes, 3 mL of PBS solution was added to each of them, and the shaker’s temperature was set at 37°C. Then put the centrifuge tubes in the shaker, and incubated for 30 min. After 60 μL of freshly diluted anticoagulated whole blood was added to each of them, the incubation was continued for 1 h. The tubes were centrifuged at 2000 rpm/min for 5 min to get the supernatant, and then test the supernatant at 545 nm to get the absorbance value L. The same volume of distilled water and diluted anticoagulated whole blood was added to the tube as positive control group and the absorbance was tested as M. The same volume of PBS buffer solution and diluted anticoagulated whole blood was added to the tube as negative control group and the absorbance was tested as N. Three parallel samples were tested in each group. The hemolysis ratio was calculated by formula (4): H e m o l y s i s   r a t i o % = H X − H N H P × 100 %   (4)

H_X was the absorbance of the experimental group, and H_N, H_P was the absorbance of the negative and positive group respectively.

Escherichia coli and Staphylococcus aureus were selected to evaluate the antibacterial performance of FHE series hydrogels. The procedure was as follows: 0.5 g of each of F127, FH, FHE5, and FHE10 hydrogels were accurately weighed in a tube and sterilized under UV for 30 min 10 μL of bacterial suspension was placed on top of the hydrogels, and after 12 h of contact, 10 mL of PBS solution was added to the tube, vortexed for 2 min, and then 10 μL of the above vortexed solution was added to the culture medium, and the bacterial solution was evenly coated on the sheep blood agar (SBA) plates using the spread-plate method and placed in a constant temperature incubator for 24 h. After the culture was completed, the antibacterial effect of the hydrogel was evaluated by observing the growth of the photographed bacteria.

To assess the cytocompatibility of the materials, we selected human umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFF-1). HUVECs and HFF-1 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). HUVECs were taken as an example to illustrate the culture of cells. HUVECs were grown in the culture flasks in endothelial cell medium (ECM) with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) and 1% endothelial cell growth supplement (ECGS). The flask was placed in a CO₂ cell incubator.

The proliferation of cells co-cultured with materials was evaluated by a Cell Counting Kit-8 (CCK-8) assay. The appropriate amount of lyophilized samples F127, FH, FHE5, and FHE10 were weighed, and a solution with a concentration of 10 mg/mL by soaking in serum-free medium for 24 h were prepared. HUVECs were seeded on a 24-well plate at 1×10⁴ per well and placed in a CO₂ cell incubator for 24 h, then the ECM medium was replaced by the ECM medium containing materials to culture cells for 1, 3, and 5 days. At a set point in time, 450 μL of ECM medium and 50 μL of the pre-warmed CCK-8 solution was placed to each well, then placed in a cell incubator for 1 h. After that, the solution was added to a 96-well plate, and then a microplate reader was used to survey the absorbance value at 450 nm wavelength.

The cytoskeletal proteins and nuclei were labeled by FITC-labeled phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) respectively. The same samples were used as the CCK-8 assay. The nucleus staining was visualized under the DAPI channel (excitation = 405 nm; emission = 437-552 nm), and cytoskeletal proteins were visualized under the GFP channel (excitation = 488 nm, emission = 505-545 nm). HUVECs were planted in a 24-well plate at a density of 2×10⁴ per well, and placed in a CO₂ cell incubator for 3 days. The cells were first washed three times with ice PBS for 15 min, and then covered with 4% cold paraformaldehyde for 15 min. For permeabilization, cells were treated with 0.1% Triton X-100 for 15 min and blocked with 1% BSA for 1 h. Finally, cells were incubated with primary antibodies at 4°C overnight. The cells were washed three times with PBS for 15 min among each step. After primary antibody incubations, cells were washed for 3 × 5 min in PBS-T (0.1% Tween 20%). Subsequently, fluorescent-labeled secondary antibody was used to incubated with cells for 2 h in dark and then DAPI was used on the cells for 15 min. All steps were washed with PBS-T. The results were observed via a confocal microscope.

Cell migration was tested by the transwell assay to measure the influences of materials, taking HUVECs as an example. HUVECs were planted in the upper chamber of a 24-well transwell, and 5×10⁴ cells were plated in each well. 600 μL of ECM medium containing materials were placed to the lower chamber and the ECM medium to the control group. The same samples were used as the CCK-8 assay. The culture plate was taken out after 8 h of culture and the migrated cells in upper chamber were washed with PBS for 3 times and fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet staining solution for 5 min and dried overnight. The cells were observed and taken pictures under a microscope. Counting the number of migrated cells using ImageJ.

The materials’ ability to promote HUVECs angiogenesis was tested by using Matrigel in vitro. 100 μL of well-mixed Matrigel was placed in the 48-well plate, then the plate was put in a cell incubator for 30 min. Each well of the plate was seeded with 3×10⁴ cells and ECM with materials was added to it, the control group was contained with ECM medium. Then the well plate was put in a cell incubator for 6 h. After this, the vascularized network structure on the surface of Matrigel was observed and photographed using a light microscope, and vessels were counted by ImageJ.

To explore the effects of materials on the gene expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), HUVECs and HFF-1 were placed on 6-well plates at a density of 4 × 10 per well. After 3 days, RNA was collected from different cell samples with TRIzol reagent. qRT-PCR was then achieved with ChamQ SYBR qPCR Master Mix. The gene expression levels were normalized to GAPDH, and the expression of related genes was analyzed by the 2^−ΔΔt method. The sequences of target genes and internal reference genes are shown in Table 1.

All animal experiments were authorized by the Animal Research Ethics Committee of the First Affiliated Hospital of Zhejiang University and were performed by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Forty healthy rats were randomly selected as experimental rats and divided into four groups, which were marked as the control group, PNs group, FHE10 group, and FHE10@PNs group. A rat skin defect model was established based on previous research methods (Wang et al., 2013). Each rat was weighed and 4%Pentobarbital (40 mg/kg) was used to anesthetize by intraperitoneal administration. After the injection, the vital signs of the rat were noted. Two symmetrical circular marks with a diameter of 2 cm were made on the depilation area on the back of the rat, and the full-thickness skin was cut off. The bleeding was compressed by the gauze until there was no obvious bleeding. In the next step, the wound was covered with materials and wrapped with gauze. After the operation, penicillin was injected to prevent infection, and the health of the rats was closely observed. Rats in each group were sacrificed 7 and 14 days after surgery, and the skin tissues at the edge of the wound were collected for immunohistochemical analysis.

The healing of skin wounds on days 0, 3, 7, and 14 after the operation was recorded by a camera, and the area of the wound was calculated by ImageJ. The wound healing ratio was computed by formula (5): W o u n d   h e a l i n g   r a t i o % = S 0 − S S 0 × 100 %   (5)

Wound area on day 0 was represented by S₀, and the wound area on days 3, 7, and 14 was represented by S.

The skin tissues were completely immersed in 10% formalin solution overnight, then in graded alcohol (75%, 85%, 95%, 100%) for dehydrating. Paraffin was used to embedded the cleared tissues, and the tissue block was cooled overnight then cut into 5 μm of sections.

The regeneration of wound epidermis was assessed by staining the tissue sections with hematoxylin and eosin (H&E). H&E staining was carried out according to previous study (Yoon et al., 2016). For H&E staining, paraffin-embedded sections were first deparaffinized and hydrate to water. Then, nucleus was stained with Hematoxylin Solution (Solarbio, China) for 15 min followed by rinsing in running tap water. Next, the tissue section was differentiated by Differentiation Solution (Solarbio, China) for 3 min and re-dyed with Eosin Y Aqueous Solution (Solarbio, China) for 1 min. After the sections were dehydrated and sealed, photos were taken and analyzed. The wound reepithelialization ratio was calculated using formula (6): R e e p i t h e l i a l i z a t i o n   r a t i o % = L 0 − L L 0 × 100 %   (6)

L₀ represents the original wound length, and L represents the incompletely healed wound epidermis length.

Masson’s trichrome staining was accomplished on tissue sections to evaluate the collagen deposition. Masson’s trichome staining were carried out according to previous study (Zhao et al., 2017). For Masson’s trichome stain, a Masson trichrome staining kit (Sigma-Aldrich, America) was used to stain the tissue section according to the manufacturer’s protocol. To observe and photograph the stained sections with an optical microscope. The area of the blue part was counted and quantitatively analyzed using ImageJ to evaluate the collagen deposition rate in the new tissue.

CD31 immunohistochemical staining and CD31/α-SMA immunofluorescence staining were performed on tissue sections to evaluate angiogenesis in wound tissue. CD31 immunohistochemical staining were carried out according to previous study (Kong et al., 2018). As for CD31immunohistochemical staining: A two-step immunohistochemistry kit (Zhongshan Golden Bridge Biotechnology, China) was used for this study according to the manufacturer’s protocol. To observe and photograph the stained sections with a light microscope, and two pathologists were asked to count the number of blood vessels in the stained sections under double-blind conditions.

Three parallel samples were prepared for the experiments, and the mean ± standard deviation was employed as the analysis data. One-way ANOVA was applied to process statistical differences between groups. When p < 0.05 (*) p < 0.01 (**) the differences had statistical significance.