Synthesis of acrylate β-cyclodextrin (Ac-β-CD)
100 mL of DMF was used to dissolve 7 g of β-CD, followed by the addition of 5 mL of TEA to the solution. After stirring and cooling the resulting blend to a temperature of 0 °C, an additional 5 mL of acrylic acid was introduced. After being stirred for 10 h, the solution was filtered to eliminate trimethylamine hydrochloride. The clear solution obtained was subsequently concentrated to a volume of around 20 mL using a vacuum rotary evaporator. Then, the solution was gradually added to 600 mL of acetone, resulting in the precipitation of the altered β-CD. Subsequently, the solid was washed multiple times with acetone and subjected to vacuum drying for a duration of 72 h.
Preparation of hydrogel
Phosphate Buffered Saline (PBS) was used to dissolve Gelatin and Ac-β-CD, resulting in solutions containing 8% (w/v) gelatin and 10% (w/v) Ac-β-CD at a temperature of 37 °C. Afterwards, the compound I2959 was introduced at a 0.05% (w/v) concentration. Then, the resultant blend was transferred into Polyvinyl Chloride (PVC) molds at a temperature of 37 °C, and subsequently cooled down to 25 °C.To promote the development of supramolecular hydrogels, the mixture was subjected to ultraviolet (UV) light with a wavelength of 390 nm at an intensity of 5 mW/cm2 for a duration of 3 min at a temperature of 25 °C.
Rheological characterization
Rheological measurements were conducted using an Anton Paar MCR301 rheometer equipped with 25 mm diameter plates. The hydrogels were evenly spread between the plates with a 0.2 mm gap size. Gel behavior was observed over time through time sweeps performed at a strain level of 0.1% and a frequency of 10 Hz. The sample underwent sequential shear with a strain of 0.1% for 120 seconds, followed by 1000% strain for 60 seconds, for a total of 4 cycles, to conduct shear thinning tests. By performing time sweeps at a fixed frequency of 10 Hz, recovery of storage (G’) and loss modulus (G”) were monitored.
Tensile and compression mechanical analysis
The MACH-1 Micromechanical System was used to perform tensile tests on samples measuring 5 mm in width, 2 mm in thickness, and 10 mm in length. The specimens were firmly fastened, and the measure of tensile force was recorded while applying an extension rate of 1 mm/s. The samples were subjected to tensile fatigue tests, where they were exposed to a tensile strain of 60% at 25 °C or 100% at 37 °C for 10 cycles. Each cycle lasted for 30 s, and the tests were conducted at the same load speed. The MACH-1 Micromechanical System was used to conduct compression tests on samples measuring 3 cm in diameter and 3 mm in thickness. To measure the compressive characteristics, the specimens were compressed at a rate of 1 mm/s.
Hydrogel swelling/degradation test
The prepared hydrogels (200 µl) were immersed in 1 mL PBS at 37 °C. Subsequently, at specific time intervals of 1/2/6/12/24 hours, the surface water was removed, and the swelling samples were weighed. The swelling ratio was computed using the formula: swelling ratio = (Wt − Wd) / Wd × 100%, where Wd signified the dry weight of the hydrogel, and Wt represented the swollen weight. In addition, the degradation of the hydrogel was evaluated at specific time points of day 2, 5, 7, 19, and 14. After eliminating the surface supernatant, the weight of the hydrogel was measured, and the degradation ratio was calculated by the formula: degradation ratio = W1/W0 × 100%, where W0 indicated the initial wet weight of the hydrogel, and W1 represented the wet weight at a given time point.
LipoSDF&RG1some synthesis and drug loading
To form anionic lipoSDF&RG1somes, chloroform (Sigma, 10 mg/mL) was used to dissolve cholesterol, which was then combined with DSPC and DSPG at a molar ratio of 2:7:1, with both compounds at a 10 mg/mL concentration. The solution was left on a rotary evaporator overnight at 60 °C while slowly decreasing the pressure to 70 mbar using a nitrogen stream in order to eliminate the chloroform. Afterwards, the resulting slender lipid layer was rehydrated using a 1 mL solution of SDF-1α(12.5 µg/mL, Peprotech) and RG1(1 mg/mL, MCE) in PBS. To create lipoSDF&RG1somes of consistent size, the solution was vigorously mixed to create an emulsion and then passed through 200 nm polycarbonate membranes (Avanti, Canada) a total of 11 times.
LipoSDF&RG1 characterization
The LipoSDF&RG1 and hydrogel were analyzed under a Tecnai G2 20 transmission electron microscope (FEI, USA) after being treated with 1% uranyl acetate for staining to observe their morphology. The LipoSDF&RG1’s size distribution and zeta potential were measured using a Malvern Zetasizer (Nano ZS, Malvern, U.K.). Weighed a certain mass of LipoSDF&RG1 and hydrogel accurately and placed them inside a dialysis bag (5000Da). Every group was submerged in a flask filled with one liter of PBS mixture and vigorously agitated at a temperature of 37 degrees Celsius (100 revolutions per minute). This dialysis bag was taken out of the beaker at specific time intervals (specifically 6, 12, 24, 48, 96 h, 7, 14, and 21 days). Then, ten millilitres of the release medium solution was accurately drawn. The above-release medium solution was evaporated to dryness, then 0.5mL absolute ethanol was added and fully dissolved, and the supernatant was taken after rapid centrifugation (10000 rpm, 30 min). Following the filtration process, high performance liquid chromatography was utilized to detect SDF-1α/RG1, and subsequently, the drug release rate was determined for each group. The calculation of EE% is done indirectly using the formula : EE % = (m1 − m2) / m1 × 100.
The initial mass of SDF-1α/RG1 used for membrane rehydration is represented by m1, while the mass of unencapsulated SDF-1α/RG1 identified through liquid chromatography is denoted as m2.
Cell culture and in vitro culture of hydrogels loaded with liposomes and ADSCs
The macrophage RAW264.7 cell line, obtained from mouse macrophages, and the HUVEC cell line, obtained from human umbilical vein endothelial cells, were acquired from the Cell Bank of the Chinese Academy of Science in Shanghai, China. The two cell lines were grown in a full medium that included 10% fetal bovine serum (FBS, Gibco, United States). Cells were subjected to D-Glucose (MCE, China) at 35 mmol/l concentration to simulate hyperglycemic conditions, while the control group was treated with glucose at a level of 5.6 mmol/l. Stem cells derived from mouse adipose tissue (ADSCs) were obtained from fat tissues located near the skin surface. The belly fat from 8-week-old C57BL/6 mice was gathered and placed in clean Petri dishes containing phosphate-buffered saline (PBS, Gibco, USA). After being minced and washed in Hank’s solution containing collagenase type II (Sigma-Aldrich, USA), the tissues underwent digestion at 37 °C for 40–90 min until they reached a uniform texture. The process of cell isolation involved the use of a 70 μm nylon mesh for centrifugation and filtration. Afterwards, the cells were treated with a solution containing erythrocyte lysis buffer. Then, they were filtered again through a 40 μm cell strainer and suspended in a complete medium made up of DMEM enriched with 10% fetal bovine serum (FBS, Gibco, United States), 100 µg/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated in a humid environment at a temperature of 37 °C and 5% CO2, with regular medium changes occurring every 2–3 days. Passaging of cells occurred once they achieved a confluence level of 70–80%.
The lipoSDF&RG1 were incorporated into the hydrogel solution to achieve appropriate drug concentrations of SDF-1α (approximately 5 µg/mL) and RG1 (approximately 400 µg/mL) in the hydrogel, and this drug-loaded liposome hydrogel was designated as the Gel@lipoSDF&RG1 group. For the Gel@lipoSDF&RG1/ADSCs group, adherent ADSCs were first digested and centrifuged. ADSCs at a concentration of 10,000 per microliter were uniformly mixed into the gelatin hydrogel, irradiated with 390 nm ultraviolet light (5 mW/cm², 3 min), followed by the addition of cell culture medium and incubation in a cell incubator for culture and subsequent experiments.
Assessment of the cell viability
After isolation and culture of ADSCs cells to a certain number and state. The prepared gelatin hydrogel was co-cultured with ADSCs cells using a transwell cell culture plate. ADSCs cells without the addition of hydrogel for co-culture were set as the control group (PBS was added). On the 1st and 3rd days after the end of co-culture, Calcein/PI staining kits (Beyotime, China) were used to stain the two groups of cells respectively according to the instructions of the reagent manufacturer. Finally, the staining results were observed and recorded under a fluorescence microscope, and the survival and death of cells were analyzed.
Furthermore, to investigate whether the hydrogel loaded with ADSCs can maintain the viability of the encapsulated cells during the injection process, after the synthesis of the hydrogel, we injected it through a G20 needle into a cylindrical mold to reshape the hydrogel. The reshaped hydrogel was cultured in vitro for 1 day and compared with the non-injected hydrogel cultured in vitro for 1 day through Calcein/PI staining and CCK8 assay. The Calcein/PI staining method was the same as mentioned above. When using the CCK-8 kit (Beyotime, China) to evaluate cell viability, the samples were cultured in a medium containing 10% CCK-8 solution at 37 °C for 2 h. The absorbance of the samples at 450 nm was measured using a microplate reader from BioTek, USA.
Flow cytometry
LPS stimulation induced RAW 264.7 cells to assume the M1 phenotype for a duration of 24 h, followed by a subsequent treatment of PBS, Gel, Gel@LipoSDF&RG1, or Gel@LipoSDF&RG1/ADSCs for an additional 24-hour period. After undergoing treatment and culturing, RAW 264.7 cells were collected, rinsed, and suspended in flow tubes. Cells were blocked by incubating with blocking buffer (Beyotime, China) for 20 min. Then, they were incubated with anti-F4/80 antibody (1:150, BioLegend, USA) conjugated with PE, anti-CD86 antibody (1:150, BioLegend, USA) conjugated with allophycocyanin (APC), and anti-CD206 antibody (1:150, BioLegend, USA) conjugated with FITC for 30 min. The analysis was performed using a BD flow cytometer and evaluated using FlowJo software.
Reverse transcription-PCR
According to the protocol provided by the reagent supplier, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to measure the mRNA expression levels. The primer sequences for each gene were as follows: Nestin (forward primer: 5′GCAGAGAAGACAGTGAGGCAGATG-3′; reverse primer: 5′-GGAGGCAGGAGACTTCAGGTAGAG-3′), Vimentin (forward primer: 5′-CTGCTGGAAGGCGAGGAGAG-3′; reverse primer: 5′-TCAACCGTCTTAATCAGGAGTGTTC-3′), and TUBB3 (forward primer: 5′-CAGCGATGAGCACGGCATAGAC-3′; reverse primer: 5′-CCAGGTTCCAAGTCCACCAGAATG-3′).
Cell immunofluorescent staining
Mesenchymal stem cells were placed onto confocal culture dishes with a concentration of either 4 × 104 or 2 × 104 cells per dish. The cells underwent treatment with PBS, Gel, or Gel@LipoSDF&RG1 for a period of 7 days. Afterward, the cells were rinsed with PBS in a gentle manner, then fixed in 4% paraformaldehyde for a duration of 15 min. Subsequently, they were subjected to 15 min of permeabilization with 0.1% Triton X-100, followed by a 30-minute blocking step using 10% goat serum (Boster, China). Overnight, the samples were then incubated overnight at 4 °C with primary antibodies targeting Nestin (Abcam, England) and β3-tubulin (Cell Signaling, China). The primary antibodies were detected by incubating them with FITC-conjugated goat anti-mouse IgG H&L (Beyotime, China) and CY3-conjugated goat anti-rabbit IgG H&L (Boster, China) at 25 °C for 2 h. In the end, the cells were stained again with DAPI and captured using a confocal fluorescence microscope from Olympus in Japan. Similarly, raw 264.7 cells were treated by different groups and then subjected to immunofluorescent staining using iNOS and Arg1 antibodies (Abcam, England).
Wound healing assay
HUVECs were cultured and seeded in six-well plates until they reached 90% confluence. Next, a sterile micropipette tip with a volume of 200 µL was utilized to generate an accurate scratch that was perpendicular to the surface of the well plate. Afterwards, the cell culture medium was discarded, and the plates were washed with PBS (three times). A medium without serum was included, and photographs were regularly captured at specific time intervals for documentation purposes. The extent of wound closure was quantified using ImageJ software for accurate analysis and assessment.
Tube formation assay
To assess the formation of the functional capillary network, HUVECs were treated differently and subsequently seeded in Matrigel-coated 96-well plates. Following a 6-hour incubation at 37 °C, images of formation of capillary-like structures were captured using an inverted microscope. The number of capillaries formed was quantified using ImageJ software, providing quantitative data for analysis.
EdU incorporation assay
Cell proliferation was assessed using EdU, a thymidine nucleotide analog, which was introduced into the cells. Following a 2-hour incubation period with EdU, HUVECs were fixed with 4% paraformaldehyde. Visualization of EdU incorporation was accomplished using an incorporation assay from manufacturer.
Transwell assay
The Transwell assay was conducted using 24-well Transwell chambers. HUVECs, suspended in a serum-free medium, were introduced into the upper compartment, while the lower compartment was filled with a complete medium. Different groups received equal volumes of PBS, hydrogel, Gel@LipoSDF&RG1, or Gel@LipoSDF&RG1/ADSCs added to the lower chamber. After incubating for 24 h, cells on the upper surface of the filter were carefully wiped off with a cotton swab. The cells that migrated to the lower surface were then stained with a 0.5% crystal violet solution. These migrated cells were subsequently observed and analyzed using an optical microscope.
Establishment of diabetic mice wound model
Approval for animal experiments on diabetic mice wound model was granted by the IACUC of Tongji Medical College, Huazhong University of Science and Technology (NO. 4083). 6-week-old male C57BL/6J mice were made diabetic by feeding them a high-fat diet for 6 weeks and then injecting them with streptozotocin (STZ; 40 mg kg− 1 day− 1) for 7 days intraperitoneally. The diagnosis of diabetes was confirmed based on consistently high fasting blood glucose levels above 15.9 mmol/L. The mice were sedated with sodium pentobarbital (50 mg/kg; Sigma Aldrich), and circular skin wounds with a diameter of around 1 cm were surgically made on their dorsal region. Following the surgery, multiple subcutaneous injections totaling 100µL of drug or PBS were administered around the wound sites. Photographs of the wound were captured on days 0, 3, 7, and 14,, and the wound closure progress was evaluated using ImageJ software from Media Cybernetics, USA.
Histological analysis
We collected wound tissue samples from mice on days 3, 7, and 14 and promptly fixed them in 4% paraformaldehyde. After dehydration, these samples were embedded in paraffin sections for subsequent Hematoxylin and Eosin (HE) staining and Masson’s trichrome staining. Additionally, for partial wound tissue paraffin sections obtained on day 14 postoperatively, antigen retrieval was performed by incubating in citrate buffer for 15 min, followed by blocking with goat serum for 30 min. Subsequently, the sections were incubated overnight at 4 °C with anti-CD31 antibody (1:100, Abcam), followed by DAB and hematoxylin staining. Finally, CD31-positive cells were counted under the microscope to evaluate the angiogenesis of the wound. Additionally, immunofluorescence staining using antibodies against INOS, Arg1, Nestin, and β-3 tublin (1:200, Abcam) were performed to assess their expression levels.
Statistical analysis
All the quantitative data were presented as mean ± standard deviation (SD). The significance was determined using the two-tailed student T-test or one-way ANOVA, with P < 0.05. Each experiment was repeated at least three times.
Western blot analysis
Wound tissue samples were collected from each mouse group on the 10th day. The tissues were lysed with a buffer containing 1% protease inhibitors (Servicebio, China). Proteins were separated using SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with specific antibodies against ACTB, IL-6, and TNF-α (1:1000, Wanleibio, China). Following this, the membranes were washed and incubated for one hour at 23 °C with a goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:1000, Cell Signaling, USA). Chemiluminescence was detected using the Western Blotting Detection kit for ECL (Byotime, China) as per the manufacturer’s instructions to visualize the proteins.
