Materials. Poly (deca-4,6-diynedioic acid) (PDDA) was synthesized following previously reported protocols35. Chitosan (average MW of 100,000, 85% deacetylation degree) was purchased from Golden-shell pharmaceutical (Zhejiang, China). 3,3’,5,5’-Tetramethyl benzidine (TMB) Substrate was purchased from BOSTER Biological Technology (Wuhan, China). Superoxide anion scavenging capacity assay kit, crystal violet was purchased from Solarbio Science & Technology (Beijing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Energy Chemical (Shanghai, China). Matrix gel was purchased from Corning Inc. (NY, USA) and 24-well transwell chambers were purchased from Labselect (Beijing, China). The glucometer and glucose test strips were purchased from Sinocare Co., Ltd. (Changsha, China).
Cell lines. The murine fibroblast NIH3T3 cells were purchased from the American Type Culture Collection, the HUVECs were kindly provided by Dr. Jianglin Wang. Cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin at 37°C in 5% CO2 atmosphere.
Animals. BALB/c mice (female, 18–20 g) were purchased from the Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). Mice were housed on a 12/12 light/ dark cycle with constant temperature (22°C) and humidity (50–60%), allowed to feed food and water ad libitum. Two Bama pigs (male, 18–20 kg) were obtained from Hubei Yizhicheng Biotechnology Co., Ltd (Hubei, China). The pigs were fed with food and water ad libitum and housed at a temperature of 22° ± 2°C and a relative humidity of 45 to 55% with a 12-hour light/12-hour dark cycle. The animal experiments were approved by the Institutional Animal Care and Ethic Committee of Huazhong University of Science and Technology ([2022] IACUC Number: 3568) and Institutional Animal Care and Ethic Committee of Hubei Yizhicheng Biotechnology Co., LTD (IACUC Issue No. WDRM 202312002).
Radicals scavenging activity. Superoxide radical -scavenging assay: The elimination capability of PDDA against ·O2− was assessed by measuring the content of generated red azo compounds using a superoxide anion scavenging capacity assay kit. After mixing the reagents and PDDA (0, 1, 2, 5 mg mL− 1) for 30 min, the absorbance value at 530 nm of the mixed solutions was recorded and the scavenging efficiency of ·O2− was calculated as follows:
·O2− scavenging rate (%) = [(AbsBlank – AbsPDDA)/AbsBlank] × 100%.
Hydroxyl radical-scavenging assay
The elimination capability of PDDA against ·OH was assessed by TMB chromogenic assay. PDDA (0, 20, 50, 100 µg mL− 1) were mixed with 100 µM H2O2, 100 nM Fe2+ and 500 µg mL− 1 TMB in 0.2 M sodium acetate-acetic acid buffer (pH 3.6), and the absorbance at 652 nm was recorded every 10 min for a total of 60 min on the microplate reader (Varioskan LUX, Thermo Scientific). The scavenging efficiency of ·OH was calculated as follows
·OH scavenging rate (%) = [(AbsBlank – AbsPDDA)/AbsBlank] × 100%.
Hypochlorous acid-scavenging assay
Hypochlorous acid (HOCl) was prepared just before the experiment by adjusting the pH of a 10% (v/v) solution of NaOCl to 6.2 with 0.2 M sodium acetate-acetic acid buffer (pH 3.6) and the concentration of HOCl was determined by TMB using the absorbance at 652 nm. The scavenging efficiency of ClO− was calculated as follows
ClO− scavenging rate (%) = [(ConBlank - ConPDDA)/ConBlank] × 100%.
Con: concentration change of HOCl compared to initial concentration.
Characterization of PDDA degradation products of Fenton reaction. 40 µM of FeSO4·7H2O was added to an aqueous PDDA dispersion (10 mL, 1 mg mL− 1), and diluted hydrochloric acid was added to adjust the pH value to 4. After stirring and dispersing, 200 µL of 30% (w/w) H2O2 was added every 12 h and stirred at room temperature for 48 h. The reaction was terminated after another 72 hours. The reaction mixture was added with NaOH until the pH value reached 12, then filtered to remove FeSO4·7H2O through a 0.22 µm nylon filter film and lyophilized into degradation products. Lyophilized degradation product was dissolved in 450 µL of deuterium water for 1H and 13C NMR (400 MHz) measurement (Agilent 400-MR 400 MHz). High resolution mass spectroscopy (HR-MS) was conducted on an Orbitrap LC/MS (Q Exactive) (Thermo Fisher Scientific, USA) with an electrospray interface, a Q Exactive mass analyser and an ion detector working in negative ion mode (M−) and an Ultimate 3000 UPLC system with a mass detector.
Quantitative analysis of PDDA degradation products over time. Diluted hydrochloric acid was added to water to adjust the pH to 4 and prepared to use. A PDDA film (10 mg) was placed in a covered vial containing 10 mL of deionized water (pH 4), followed by the addition of 10 µM of FeSO4·7H2O and 100 µL of 30% (w/w) H2O2. The vial was kept at room temperature and steady, and the supernatant was collected every 24 h. Simultaneously, the remaining PDDA film was reintroduced into the 10 µM of FeSO4·7H2O and 100 µL of 30% (w/w) H2O2 for further degradation, repeating this cycle for 168 hours. The reaction mixture collected was filtered with a 0.22 µm membrane and then lyophilized to obtain the degradation products. Lyophilized degradation products were dissolved in 450 µL of deuterium water and 0.25 mg of DMSO was added as an internal standard for 1H NMR (400 MHz) measurement (Agilent 400-MR 400 MHz).
Scratch wound healing assay. For the scratch wound healing assay, HUVECs were seeded at a density of 1 × 105 cells per well in 24 well plates and scratched using a sterile 10 µL pipette tip, then washed with PBS to remove unattached cells. Subsequently, the attached cells were treated with or without PDDA degradants. Photographs of the HUVECs were taken 24 hours after the initial wounding. The migration rate was determined by calculating the ratio of the closed area to the initial wound area.
Real-time quantitative PCR (RT-qPCR) analysis. Total RNA was extracted from the cells or tissues using RNA isolater Total RNA Extraction Reagent (Vazyme) following the manufacturer's instructions. 1 µg mRNA for each sample was reverse-transcribed into cDNA using the Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen), and then utilized for quantitative PCR using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen). All primers were synthesized by Huayu Gene Biological Technology (Wuhan, China). The sequences of the primers for cell experiments were as follows:
VEGFA-forward primer | 5’-TATGCGGATCAAACCTCACCA-3’ |
VEGFA-reverse primer | 5’-CACAGGGATTTTTCTTGTCTTGCT-3’ |
Angpt2-forward primer | 5’-TTATCACAGCACCAGCAAGC-3’ |
Angpt2-reverse primer | 5’-TTCGCGAGAACAAATGTGAG-3’ |
GAPDH-forward primer | 5’-GAGTCAACGGATTTGGTCGT-3’ |
GAPDH-reverse primer | 5’-TTGATTTTGGAGGGATCTCG-3’ |
The sequences of the primers for diabetic mouse were as follows:
IL-1β-forward primer | 5’-TGGACCTTCCAGGATGAGGACA-3’ |
IL-1β-reverse primer | 5’-GTTCATCTCGGAGCCTGTAGTG-3’ |
IL-12A-forward primer | 5’-GCCAGTCCCGAAACCTGCTG-3’ |
IL-12A-reverse primer | 5’-GCTGGTTTGGTCCCGTGTGA-3’ |
IL-6-forward primer | 5’-AGGAGTGGCTAAGGACCAAGA-3’ |
IL-6-reverse primer | 5’-GACCACAGTGAGGAATGTCCA-3’ |
VEGFA-forward primer | 5’-CAGGCTGCTCTAACGATGAA-3’ |
VEGFA-reverse primer | 5’-CAGGAATCCCAGAAACAACC-3’ |
Col-1-forward primer | 5’-CCCAGAGTGGAACAGCGATTAC-3’ |
Col-1-reverse primer | 5’-TGTCTTGCCCCATTCATTTGTC-3’ |
GAPDH-forward primer | 5’-TCAACGGCACAGTCAAGG-3’ |
GAPDH-reverse primer | 5’-ACTCCACGACATACTCAGC-3’ |
The sequences of the primers for diabetic pig were as follows:
TLR-4-forward primer | 5’-TGGTGTCCCAGCACTTCATA-3’ |
TLR-4-reverse primer | 5’-CGGCATGACTCCTCAGAAAC-3’ |
IL-1β-forward primer | 5’-TGGCCCACACATGCTGAA-3’ |
IL-1β-reverse primer | 5’-CCTTGCACAAAGCTCATGCA-3’ |
VEGFA-forward primer | 5’-TATGCGGATCAAACCTCACCA-3’ |
VEGFA-reverse primer | 5’-CACAGGGATTTTTCTTGTCTTGCT-3’ |
Col-1-forward primer | 5’-AGTGTGAGGCCACGCATGAGC-3’ |
Col-1-reverse primer | 5’-GGTTTCCTGGTCGGTGGGTGA-3’ |
TGFβ1-forward primer | 5’-GTGGCTGTCCTTTGATGT-3’ |
TGFβ1-reverse primer | 5’-CGTGGAGTGTGTTATCTTTG-3’ |
IGF2-forward primer | 5’-GTGCTGCTATGCTGCTTACCG-3’ |
IGF2-reverse primer | 5’-CCGCAGACAAACTGGAGGG-3’ |
GAPDH-forward primer | 5’-GGTCACCAGGGCTGCTTTTA-3’ |
GAPDH-reverse primer | 5’-CCTTGACTGTGCCGTGGAAC-3’ |
Each qPCR reaction contained 30–45 ng of cDNA. Quantification of gene expression was carried out using the 2–ΔΔCt method, with gene expression levels normalized to the housekeeping gene GAPDH.
Transwell migration assay. For cell migration assay, HUVECs were seeded into the upper chamber with a density of 1 × 104 cells per well with serum-free culture medium; the lower chamber was added with or without PDDA degradants. After 24 hours, the migrated cells kept on the upper surface filter were removed by cotton swab, while the migrated cells on the bottom side of the filter were stained with a 1% crystal violet solution for 15 mins. The number of migrated cells was photographed and counted under an optical microscope (Mingmei, Guangdong, China).
Tube formation assay. For tube formation assay, 50 µL per well of thawed Matrigel was added into a pre-cooled 96-well plate and incubated for 1 hour at 37°C. Then, HUVECs were seeded with 1 × 104 cells per well into the Matrigel-coated chamber incubated with or without PDDA degradants. To assess the tube formation, HUVECs were imaged under microscope and quantified using ImageJ software.
Synthesis of CS films, CN films, and PCD. Thoroughly cleaning the glass plate, after the glass plate is completely dry, add 1.0% CS water solution to it. Shake the solution to eliminate any bubbles and then evaporate the solvent at 60°C using a casting method to obtain CS films. After obtaining the CS film, add a 0.1 M NaOH solution and soak it for 15 minutes to ensure thorough wetting of the entire film. Rinse the film with ultrapure water and allow it to dry to obtain CN films. For the PCD, add 1.67 mg/mL PDDA water solution in a 1:10 mass ratio (PDDA:CS), wait for 15 minutes to allow sufficient interaction between PDDA and the CS film, and then dry to obtain PCD. Peel the films from the glass plate, rinse them three times with ethanol to eliminate excess salt, and then subject them to vacuum drying to obtain the final CS films, CN films, and PCD.
Mechanical strength measurement. CS films, CN films and PCD were prepared in rectangular shape with 2 cm × 1 cm. Mechanical properties of the films were evaluated using Universal testing machine under ambient temperature and normal humidity conditions with a stretching speed of 20 mm/min.
Scanning electron microscopy (SEM). The morphology of the surface and cross-section were characterized by SEM (FEI Quanta 200). All the samples were sputter-coated with platinum, the surface imaging was examined at 10 kV, the cross-section imaging was examined at 15 kV.
Water contact angle assay. The contact angles were determined using sessile-drop method under room temperature. Prior to measurement, the untreated CS films, CN films, and PCD were prepared on glass slides. During the measurement, 1 µL of ultrapure water was dropped onto the film surface, and the contact angle was immediately recorded using a camera.
Swelling rate. The mass of each lyophilized CS films, CN films, and PCD was weighed and denoted as m0. Subsequently, the samples were then put into 0.9% NaCl solution immediately under room temperature and took out after being soaked in 0.9% NaCl solution for 30 minutes. The weights of swollen CS films, CN films, and PCD were measured and denoted as mt. The swelling rate was calculated as follows:
Swelling rate (%) = (mt – m0)/m0 ×100%.
Porosity measurement. The porosity of the films was determined using a solvent exchange method. Three sets of CS films, CN films, and PCD were prepared, the mass of each film was measured and recorded as W1. The films were placed into 5 mL of ethanol, the total mass of the films and ethanol was recorded as W2. The films were then immersed in ethanol for 48 hours to ensure thorough solvent exchange. After solvent exchange, the films were removed, and the remaining ethanol mass was measured and recorded as W3. The calculation formula for film porosity is as follows:
Porosity (%) = [(W2 - W1 - W3)/(W2 - W3)] × 100%.
In vivo hemostatic assay. In vivo mouse tail hemostatic ability: A mouse tail model (female, BALB/c mice, 16 to 20 g) was used to investigate the in vivo hemostatic ability of CS films, CN films, and PCD. Pre-weighed filter paper was placed under the tails of the mice, the tails were cut approximately 2 cm from the tip after by surgical scissors. The tails were left undisturbed for 15 seconds to ensure normal bleeding. After 15 seconds, CS films, CN films, and PCD applied to the bleeding wound, the nontreatment wound served as the negative control. The bleeding time for each group of mice was recorded, once the tail bleeding was completely stopped, the total mass of the blood-soaked filter paper was measured again. The bleeding volume from the mouse tails was determined by subtracting the initial filter paper mass from the final mass. Photographs of the filter paper were taken for each group after hemostasis.
In vivo mouse liver hemostatic ability
After extracting the liver from mice (female, BALB/c mice, 16 to 20 g), pre-weighed filter paper was placed under the mouse liver. Subsequently, a wound was created at the site of the mouse liver using a needle from a syringe. The liver was left undisturbed for 15 seconds to ensure normal bleeding. After 15 seconds, the nontreatment wound served as the negative control, while each experimental group applied the corresponding film to the bleeding wound. The bleeding time for each group of mice liver was recorded. After liver bleeding was completely stopped, the total mass of the blood-soaked filter paper was measured again. The bleeding volume from the mouse liver was determined by subtracting the initial filter paper mass from the final mass. Photographs of the filter paper were taken for each group after hemostasis.
Antimicrobial activity assay. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were chosen to verify the antibacterial activity of wound dressings. Two kinds of bacterial were cultured until the turbidity of the bacterial suspension to OD600 = 0.1. Subsequently, the bacterial was collected and resuspended using PBS buffer. The resuspended bacteria were incubated with CS film, CN film, and PCD respectively in 12-well plates. After 2 h, the Colony-forming unit (CFU) of each group were measured through drop plate method. The inhibition rate (%) was calculated as below:
Inhibition rate (%) = (CFUBlank – CFUSample)/CFUBlank × 100
In vitro biocompatibility. CS films, CN films, and PCD were fully immersed in DMEM culture medium at room temperature for 7 days to obtain the extraction solution. NIH/3T3 cells were then diluted with either DMEM culture medium or the extraction solution and seeded into 96-well plate at a density of 3,000 cells per well. The cells were then incubated at 37°C in a 5% CO2 atmosphere for 24 and 48 hours. After the respective time points, 0.5 mg mL− 1 MTT was added to each well and cultured for 4 h. Following incubation, the MTT was removed and replaced with 150 µL of DMSO per well to dissolve the formazan salt. The absorbance at 570 nm was measured using a microplate reader.
Hemolysis assay. The red blood cells (RBCs) were obtained by centrifuging blood at 1500 rpm for 10 min, they were PBS-rinsed thrice and diluted to the concentration of 5% v/v. The films were incubated in 0.9% NaCl solution at 37°C for 30 min, 150 µL of the extract solution and 150 µL of the RBC suspension were mixed and incubated at 37 ℃ for 3 h, then it was centrifuged at 1500 rpm for 10 min. The obtained supernatants were reading absorption at 540 nm with a microplate reader, 0.1% Triton X-100 and 0.9% NaCl solution were used as positive and negative controls, respectively. The hemolysis rate (%) was calculated as below:
Hemolysis rate (%) = (ODsample – ODnegative)/(ODpositive – ODnegative) × 100
In vivo diabetic mouse skin wound healing study. The animals were administered STZ (50 mg/kg; Yeasen, Shannghai) mixed in sodium citrate buffer daily for five consecutive days. All of the treated mice with plasma glucose levels ≥ 16.7 mM for 2 consecutive days under normal condition were considered diabetic 2 weeks after the first STZ injection. Animals were maintained in a diabetic state for the wound healing experiment. The STZ-induced diabetic mice were anesthetized under sterile conditions, and then a full-thickness wound was made on the center of dorsum to remove the epidermis and superficial parts of the dermis by using a 10-mm biopsy punch. The animals were randomly divided into four groups: control group, CS films, CN films, and PCD. Digital images of the wound area were captured on the day of surgery and every other day after wounding. Harvested wound tissues were either fixed in 4% PFA in PBS, or flash-frozen in liquid nitrogen different evaluations, including H&E, DHE, Masson's trichrome, immunohistological staining and mRNA expression analysis.
In vivo diabetic porcine skin wound healing study. Male bama miniature pigs were subjected to a single intravenous administration of STZ (150 mg/kg) mixed in a sodium citrate buffer to induce diabetes, which was sustained for more than 2 weeks before the initiation of the wounds. Under sterile conditions, diabetic pigs were anesthetized, and six 1.5 cm² full-thickness wounds were created on the dorsal region on each side. To prevent collateral effects, the wounds were spaced more than 2 cm apart. Each individual square was marked with a sterile surgical marker. Using a No. 11 blade scalpel, a dermal full-thickness wound (approximately 0.5 cm deep) was meticulously created, removing the skin through careful dissection with skin forceps and double-blade cutting scissors, control, CS films, CN films, and PCD were applied to the wounds. A cotton dressing was used to cover and completely shield the wounded area, securing it with an elastic net for additional protection of the dermal wounds. To address potential bacterial infections, pigs received antibiotic injections during the first three days. Digital images of the wound area were captured on the day of surgery and every 2 days thereafter. Wounds were harvested 15 days post-surgery. The harvested wound tissues were either fixed in 4% PFA in PBS or flash-frozen in liquid nitrogen for further H&E, DHE, Masson's trichrome staining and mRNA expression analysis. Blinded evaluations were conducted for scoring wound histology according to standard (Table S1)18.
In vivo biosecurity. The healthy mice were anesthetized under sterile conditions, and then a full-thickness wound was made on the center of dorsum, the animals were randomly treated with CS films, CN films, and PCD. After 7 days, the mice were sacrificed, the major organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining.