Dressings that promote wound healing in diabetic patients

Abstract

This invention relates to a dressing that promotes wound healing in diabetic patients. The method involves dissolving chitosan in hydrochloric acid at room temperature to obtain a chitosan solution; dissolving loxoprofen in ethanol activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide and adding it dropwise to the chitosan solution; then adding ethanol while stirring at high speed with a magnetic rod to form a transparent solution, followed by low-speed stirring in the dark; drying the solution in an oven to form a solid film, and thoroughly washing it in ethanol to obtain a chitosan-loxoprofen conjugate; adding chitosan-loxoprofen to phosphate buffer to form a chitosan-loxoprofen solution; dissolving genipin in phosphate buffer to obtain a genipin solution; mixing the chitosan-loxoprofen solution and the genipin solution at high speed, cross-linking, and then freezing to obtain a chitosan-loxoprofen / genipin sponge scaffold. The dressing of this invention can effectively relieve chronic inflammation, significantly improve skin tissue regeneration, and stimulate angiogenesis.

Description

Technical Field

[0001] This invention relates to a disease treatment drug, specifically a drug for promoting wound healing in diabetic patients. Background Technology

[0002] Diabetic wounds are a common and serious complication of diabetes and remain a leading cause of amputation and death worldwide, primarily caused by peripheral nerve damage, vascular dysfunction, and wound infection. The wound healing process is complex and dynamic, involving the coordinated action of various cells and signaling molecules to repair damaged tissue, and can generally be divided into three interrelated phases: inflammation, proliferation, and remodeling. For diabetic wounds, several distinct characteristics emerge, including severe and persistent inflammation, impaired angiogenesis, and impaired reepithelialization. These factors collectively contribute to the challenge of achieving effective wound closure. Persistent inflammation delays the progression from inflammation to proliferation, a key factor affecting the normal healing of diabetic wounds. Furthermore, the proliferation and migration of cells are suppressed due to the large release of inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6. Macrophages are one of the key immune cells regulating the inflammatory process and can be activated and polarized into different phenotypes, mainly divided into the classical activated M1 phenotype with pro-inflammatory effects and the alternative activated M2 phenotype with anti-inflammatory capabilities. During the proliferative phase, M2 macrophages significantly influence vascular bed regeneration, cell proliferation, and suppress inflammatory responses, thereby promoting wound healing. Due to the complex inflammatory immune microenvironment resulting from intense inflammatory stimulation in diabetic wounds, M1 macrophages become overactivated and dominant. This imbalance and glucose-induced immunosuppression further contribute to chronic inflammation, impaired angiogenesis, and delayed healing in diabetic wounds. To address this issue, strategies to reverse this immunosuppression and promote M2 polarization have emerged as effective methods to facilitate the transition from the inflammatory phase to the proliferative phase and further improve diabetic wound healing.

[0003] Loxoprofen (LOX), 2-(4-((2-oxocyclopentyl)methyl)phenyl)propionic acid, was marketed in Japan in 1986 as an anti-inflammatory drug by Daiichi Sankyo Co., Ltd. LOX is a prodrug of the non-selective NSAID benzoxoprofen; it is one of the most widely used nonsteroidal anti-inflammatory drugs (NSAIDs) for reducing inflammation, pain, and postoperative tissue adhesions. Macrophages are the primary target for LOX's anti-inflammatory effects. However, LOX is typically delivered orally via tablets; repeated oral administration of LOX can lead to gastric mucosal damage and upper gastrointestinal bleeding, thus limiting its oral use. Furthermore, oral administration makes it difficult to control the sustained release of the drug at the target site in the body. Therefore, developing a drug suitable for diabetic wound healing, minimizing adverse reactions, and ensuring therapeutic efficacy is a topic that needs to be discussed here. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a dressing that promotes wound healing in diabetic patients.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A dressing for promoting wound healing in diabetic patients, characterized in that the dressing is prepared by the following steps:

[0007] Step 1: Dissolve 1.61 g of chitosan in 100 mL of hydrochloric acid solution at room temperature by continuous stirring to prepare a chitosan solution;

[0008] Step 2: Dissolve 0.246 g, 1 mM loxoprofen in 20 mL of ethanol, then add 0.96 g, 6.18 mmol of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.28 g, 5.95 mmol of N-hydroxysuccinimide to obtain a loxoprofen solution with the carboxyl group activated. Add the loxoprofen solution dropwise to the chitosan solution in Step 1, and then add 80 mL of ethanol while stirring at 800 rpm with a magnetic rod to ensure uniform dispersion and the formation of a clear solution.

[0009] Step 3: In the dark, keep the transparent solution from Step 2 at 25 °C and stir at 200 rpm for 6 hours.

[0010] The solution was then air-dried in a 37 °C oven until it became a solid film, and then thoroughly washed several times in ethanol to obtain a solid film chitosan-loxoprofen conjugate.

[0011] Step 4: Add the chitosan-loxoprofen conjugate obtained in Step 3 to 80 mL of phosphate buffer. The amount of chitosan-loxoprofen added is 1.6 g. Stir magnetically until the chitosan-loxoprofen solution is dissolved at 2% w / v.

[0012] Step 5: Dissolve 0.2 g of genipin in 40 mL of phosphate buffer to prepare a 0.5% w / v genipin solution;

[0013] Step six: The chitosan-loxoprofen solution and genipin solution prepared in step four are mixed at a volume ratio of 4:1 by high-speed stirring, poured into a mold for cross-linking for 24 h, and then placed in a -80℃ freezer for freezing. Finally, the chitosan-loxoprofen / genipin sponge scaffold is prepared by freeze drying.

[0014] A novel freeze-dried scaffold was developed using the aforementioned technical approach, through amino cross-linking of gardenia extract genipin (GP) with chitosan-loxoprofen conjugate (CS-LOX). This design integrates covalently grafted LOX into a mechanically stable network formed by precise genipin-mediated cross-linking to achieve sustained anti-inflammatory drug release. Transcriptomic analysis (RNA-seq) showed that the CS-LOX / GP scaffold significantly downregulated the TNF, IL-17, and NF-κB signaling pathways in macrophages, promoting a therapeutic M1 to M2 phenotypic transition. In a rat diabetic wound model, CS-LOX / GP treatment effectively alleviated chronic inflammation, significantly improved skin tissue regeneration, and stimulated angiogenesis.

[0015] The present invention will now be further described with reference to the accompanying drawings. Attached Figure Description

[0016] Figure 1 shows the physicochemical properties characterization of the scaffold;

[0017] Figure 2 shows the results of the antibacterial properties and hemostasis tests;

[0018] Figure 3 shows the results of immunofluorescence staining analysis of wound healing quality. Figure 1 ;

[0019] Figure 4 shows the results of immunofluorescence staining analysis of wound healing quality. Figure 2 ;

[0020] Figure 5 shows a gross photograph of the CS / GP and CS-LOX / GP stents 7 days after subcutaneous implantation;

[0021] Figure 6 shows the histological images of H&E and Masson trichrome staining on day 7;

[0022] Figure 7 shows high-magnification images of cells in the fibrous capsule and scaffold; Figure 8 shows the number of cells in the fibrous capsule.

[0023] Figure 9 shows the number of cells in the scaffold;

[0024] Figure 10 is a quantitative diagram of the thickness of the fibrous capsule. Detailed Implementation

[0025] Example 1: Preparation of CS-LOX / GP foam scaffold.

[0026] The CS-LOX / GP scaffold was synthesized via a two-step method. First, loxoprofen (LOX) was coupled to chitosan (CS) via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) / N-hydroxysuccinimide (NHS) carbodiimide chemistry in an ethanol / water solution. Briefly, 1.61 g of CS (-10 mM repeating unit) was dissolved in 100 mL of 0.1 M hydrochloric acid (HCl) solution at room temperature with continuous stirring. Simultaneously, 0.246 g of LOX (1 mM) was dissolved in 20 mL of ethanol activated by EDC (0.96 g) / NHS (0.28 g) and added dropwise to the chitosan (CS) solution. Then, 80 mL of ethanol was added under high-speed stirring with a magnetic rod to allow for uniform dispersion and the formation of a clear solution. The reaction was carried out in the dark at 25 °C with low-speed stirring for 6 hours. The solution was air-dried in a 37 °C oven until it became a solid film, and then thoroughly washed several times in ethanol to obtain the CS-LOX conjugate.

[0027] CS-LOX conjugates were crosslinked with genipin to prepare CS-LOX / GP sponge scaffolds. In short, 1.6 g of CS-LOX was added to 80 mL of phosphate-buffered saline (PBS) (1X) and magnetically stirred until dissolved to form a CS-LOX solution (2% w / v). Subsequently, 0.2 g of GP was dissolved in 40 mL of phosphate-buffered saline (PBS) (1X) to prepare a GP solution (0.5% w / v). The CS-LOX solution (2% w / v) and the genipin (GP) solution (0.5% w / v) were mixed at a volume ratio of 4:1 with high-speed stirring, poured into a mold, and crosslinked for 24 h. The mixture was then frozen overnight at -80 °C and finally lyophilized to prepare the CS-LOX / GP sponge scaffold.

[0028] Example 2: Fabrication of the CS / GP stent.

[0029] 1.6 g of CS was dissolved in 80 mL of hydrochloric acid (0.1 M) and stirred at high speed at room temperature until completely dissolved to prepare a chitosan (CS) solution (2% w / v). The CS solution and GP solution were mixed at a volume ratio of 4:1 by high-speed stirring, poured into a mold for crosslinking for 24 h, and then frozen overnight at -80°C. Finally, the mixture was lyophilized to prepare a CS / GP scaffold as a control group.

[0030] Example 3: Physicochemical analysis of CS-LOX / GP sponge scaffold.

[0031] To elucidate the successful grafting of LOX and to identify the physical and chemical properties of the scaffold, FTIR and XRD tests were performed. Furthermore, the morphology, pore size, porosity, density, degradation, drug release, and water absorption of the scaffold were investigated. Detailed experimental procedures are provided in the supplementary information. The Fourier Transform Infrared (FTIR) spectroscopy method for the CS-LOX / GP sponge scaffold is detailed in the literature (https: / / doi.org / 10.1021 / acsbiomaterials.4c00882). CS, LOX, CS-LOX, GP, CS-GP, and CS-LOX / GP were scanned using a Nicolet IS50 infrared spectrometer (Thermo Fisher Scientific). The FTIR spectra had a resolution of 0.5 cm⁻¹ and a scanning range of 4000 to 600 cm⁻¹. X-ray diffraction (XRD) spectra of CS, LOX, and CS-LOX were determined using an X-ray diffractometer (D8 Advance, Bruker, Germany), and the crystal phase of the samples was detected in the 2θ range of 5° to 60°.

[0032] The microstructure of the scaffolds was observed using scanning electron microscopy (SEM). CS-GP and CS-LOX / GP scaffolds were coated with gold, placed on conductive tape, and imaged using a scanning electron microscope (XL desktop, Phenom, Netherlands) at an accelerating voltage of 5 kV. Twenty wells were randomly selected from individual scaffold samples, and their pore sizes were measured using ImageJ and statistically analyzed.

[0033] The porosity and density of the scaffold were determined using the liquid displacement method described above (https: / / doi.org / 10.1016 / j.actbio.2025.02.043). In short, the length (d), width (l), and height (h) of the scaffold were measured, and a dry sample (m0) was weighed and then immersed in ethanol for 1 hour. The scaffold was removed from the ethanol and weighed (m1). The porosity of the scaffold was calculated using the following formula: Porosity (%) = ((m - m)) / (ρ × d × l × h) × 100%; the density of the scaffold was calculated using the following formula: ρ = (m) / (d × l × h) × 100%; three parallel samples were tested for each group of samples.

[0034] The volume expansion ratio and water absorption rate of the scaffold were determined by measuring the length (d0), width (l0), height (h), and mass (md) of the dried sample. The sample was incubated in deionized water for different time periods. After gently wiping off the surface moisture with absorbent paper, the wet weight (mw) was measured. When the sample reached its maximum water absorption state, its diameter (d) and height (h) were measured. The volume expansion ratio was calculated using the following formula: Volume expansion ratio (%) = ((d × l × h - d × l × h )) / (d × l × h ) × 100%; the water absorption rate was calculated using the following formula: Water absorption capacity (%) = ((mw - md)) / (md) × 100%; three parallel samples were tested for each group of samples.

[0035] The degradability of the scaffold was assessed by cutting the dried scaffold into approximately 10 mg pieces, weighing them (m1, n = 3), and incubating them in 2 mL of phosphate-buffered saline (PBS) at 37 °C and 100 rpm. Samples were collected on days 7, 14, 21, and 28, lyophilized, and weighed (m0). The remaining mass was calculated using the following formula: Mass remaining = m1 / m0 × 100%.

[0036] Drug release behavior of the stent: 10 mg LOX was weighed and LOX standard solutions of different concentration gradients were prepared. The absorbance of the solutions was measured at 220 nm using a UV spectrophotometer (TU-1810), and a standard curve was plotted. The absorbance of the supernatant of the degradation samples was measured on days 1, 4, 7, 14, 21, and 28 to evaluate the release behavior of LOX (n=3).

[0037] Fourier transform infrared (FTIR) spectroscopy analysis revealed characteristic absorption peaks for the typical functional groups in the untreated chitosan (CS) sample: amide I (C=O stretching vibration) at 1652 cm⁻¹, amide II (N–H bending vibration) at 1590 cm⁻¹, and amide III (C–N stretching vibration) at 1374 cm⁻¹. In the loxoprofen (LOX) sample, the stretching vibration peak at 2967 cm⁻¹ indicates the presence of a –OH group; a characteristic stretching vibration peak for the carboxyl group (–C=O) appears at 1540 cm⁻¹, while the peak at 1698 cm⁻¹ corresponds to the carbonyl group (–C=O) stretching vibration in the cyclopentanone structure. In the spectrum of the CS-LOX covalent conjugate, the increased absorption peak intensities at 1622 cm⁻¹ and 1521 cm⁻¹ indicate the formation of amide I and amide II bonds between the carboxyl group of LOX and the amino group on the CS molecular chain. These results indicate that a grafting reaction occurred between LOX and CS via amide bonds. The characteristic transmission peaks of genipin appeared at 1679 cm⁻¹ and 1618 cm⁻¹, corresponding to the C=C stretching vibrations of the carboxymethyl group and the aromatic ring, respectively (DOI:10.1039 / d2ma00536k). With the formation of crosslinks between CS and GP, the intensities of the amide I and amide II absorption peaks of CS-GP and CS-LOX / GP were enhanced, indicating that the amino groups in the chitosan molecule reacted with the carboxymethyl groups in the genipin molecule to form a secondary amide structure. Furthermore, the C=O stretching vibration peak of the secondary amide in the hydrogel overlapped with the C=C stretching vibration peak of the olefin ring in the genipin molecule in the infrared region, resulting in a slight broadening of the amide I absorption band profile.

[0038] X-ray diffraction (XRD) patterns showed that LOX exhibited multiple narrow and sharp diffraction peaks, indicating good crystallinity. In the XRD patterns of chitosan (CS) and its derivatives, a distinct diffraction peak was observed at approximately 2θ = 20°, representing its partially crystalline structure. However, after grafting with the drug (LOX), this peak gradually broadened, indicating a decrease in the crystallinity of chitosan. This result suggests that the successful modification of chitosan with LOX disrupted its original highly crystalline structure, leading to a disturbance of the ordered arrangement of the CS macromolecular chains and thus reducing overall crystallinity.

[0039] The microstructure of the scaffolds was examined using SEM, and the results showed that all scaffolds exhibited typical porous morphology. Compared with the CS / GP scaffold, the density, porosity, pore size, and volume expansion rate of the drug-grafted CS-LOX / GP scaffold did not change significantly (Figure 1 shows the density, porosity, and pore size of CS / GP and CS-LOX / GP; Figure 10, p > 0.05). We further characterized the water absorption rate of the scaffolds, which affects the absorption of tissue exudates and the change in scaffold volume. The water absorption rate of the hydrogel was evaluated over 120 minutes, as shown in the water absorption curves of CS / GP and CS-LOX / GP in Figure 1. Both CS / GP and CS-LOX / GP rapidly absorbed water within 20 minutes and gradually reached water absorption equilibrium; the water absorption curves of the two scaffolds were similar.

[0040] The degradation performance of the scaffolds is shown in Figure 1, which illustrates the in vitro degradation curves of CS / GP and CS-LOX / GP. On day 28, the CS / GP hydrogel degraded to 55.4 ± 9.1% of its initial mass, while the CS-LOX / GP hydrogel degraded to 60.4 ± 4.3% of its initial mass. We also assessed the cumulative release of LOX from the CS-LOX / GP hydrogel over a period of up to 28 days (Figure 1 shows the in vitro drug release curves of CS-LOX / GP over 28 days). In the first 7 days, the CS-LOX / GP hydrogel released approximately 50% of the LOX. Over the next 28 days, LOX release was relatively slow, but by day 28, it was almost completely released. Overall, the in vitro release of LOX exhibited a slow and sustained drug delivery pattern.

[0041] Example 4: In vitro biocompatibility and biological function of the scaffold.

[0042] The biocompatibility and biological function of the scaffold were evaluated through a series of in vitro assays, including antibacterial tests, blood compatibility assessment, live / dead staining, cell proliferation assays, macrophage phenotype analysis, macrophage ROS detection, macrophage inflammatory cytokine expression, scratch healing assays, migration assays, and in vitro angiogenesis-related gene expression tests. In addition, transcriptomic analysis was performed on untreated and CS-LOX / GP-treated RAW264.7 cells. Detailed experimental procedures are provided in the supplementary information.

[0043] 1. Antibacterial performance test of the stent.

[0044] The in vitro antimicrobial activity of the scaffold was evaluated against *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 25923). Bacteria were cultured overnight in LB broth at 37 °C in a shaker. The scaffold was then mixed with 200 μL of bacterial inoculum (10⁸ cfu / mL) and incubated at 37 °C for 12 h. Subsequently, 100 μL of each diluted bacterial culture was inoculated onto LB agar and incubated at 37 °C for 12 h. Colonies were imaged using an automated colony counter (Shineso Science & Technology Co., Ltd., Hangzhou). Imagej counted the colonies to calculate normalized survival rate (n=3).

[0045] 2. Blood compatibility test of stent.

[0046] In vitro hemolysis experiments were performed using 2% (v / v) suspension of red blood cells (RBCs). Red blood cells were obtained from fresh anticoagulated whole blood of healthy rats, diluted with physiological saline, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the cells were washed three times with physiological saline to achieve a final suspension of 2% (v / v). During the experiment, 200 μL of pre-warmed (37 ℃, 30 min) scaffold sample (10 mm long, 5 mm wide, 3 mm high) was mixed with 500 μL of the red blood cell suspension and incubated at 37 ℃ for 1 h. After incubation, the sample was centrifuged at 3000 rpm, the supernatant was collected, and its absorbance at 540 nm was measured to assess the amount of hemoglobin released. Physiological saline and deionized water served as negative and positive controls, respectively. The hemolysis rate was calculated using the following formula: Hemolysisrate = (As-An) / (Ap-An); where A is the absorbance of the experimental group supernatant, A is the absorbance of the negative control, and A is the absorbance of the positive control; three parallel samples were tested for each group of samples.

[0047] The hemostatic ability of the stent was assessed using a dynamic whole blood coagulation test. The stent was incubated at 37 °C for 5 minutes. 100 μL of fresh anticoagulated rabbit whole blood (supplemented with 10% sodium citrate) was added to the stent (10 mm long, 5 mm wide, 3 mm high) and gauze, respectively, followed by the addition of 10 μL of calcium chloride (CaCl2, 0.2 M) solution. The sample was then incubated at 37 °C for 10 minutes to allow blood coagulation. Afterward, 10 mL of deionized (DI) water was added to the sample, and the sample was shaken in a track shaker at 37 °C for 5 minutes to remove any unbound blood.

[0048] The absorbance (Dt) of the supernatant was measured at 540 nm. Deionized water and physiological saline were used as positive (Dm) and negative controls (Dn), respectively. The coagulation index (BCI) was calculated by the equation: BCI=(Dt-Dn) / (Dm-Dn)×100% (n = 3), and three parallel samples were tested for each group of samples.

[0049] The whole blood clotting time was tested by adding 2 mL of whole blood (containing 10% sodium citrate) to a centrifuge tube containing a scaffold (10 mm long, 5 mm wide, 3 mm high), followed by 60 μL of CaCl2 solution (0.25 mol L⁻¹). A gauze control group was cut into rectangular slices 10 mm long and 5 mm wide. The centrifuge tubes were inverted, and clot formation was recorded each time (n = 3).

[0050] Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were used to assess the coagulation activation pathway and clotting time of the stent. Whole blood from healthy New Zealand rabbits (containing 3.8% sodium citrate, 9:1 ratio) was centrifuged at 3000 rpm for 15 minutes to obtain platelet-free plasma. The stent was then incubated with 100 μL of platelet-free plasma at 37°C for 2 minutes. For the control group, gauze was cut into rectangles 10 mm long and 5 mm wide. Next, 100 μL of PT reagent was added to the incubated hydrogel, and the time required for clot formation in the platelet-free plasma was recorded. To determine APTT, 100 μL of APTT reagent was added to the hydrogel, and then incubated at 37°C for 3 minutes. Subsequently, 100 μL of 0.025 M CaCl2 solution was added, and APTT was recorded. For TT measurement, 200 μL of platelet-free plasma was added to a hydrogel and incubated at 37°C for 2 minutes. Then, 200 μL of TT reagent was added, and TT was recorded (n=3).

[0051] 3. Cell compatibility test of scaffold.

[0052] The scaffold was incubated for 24 hours in a complete medium consisting of high glucose DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin to prepare the scaffold conditioned medium.

[0053] Mouse fibroblasts (NIH3T3, ATCC), human umbilical vein endothelial cells (HUVECs, ATCC), and mouse macrophages (RAW 264.7, ATCC) were provided by the Cell Bank of the Chinese Academy of Sciences for cell compatibility assessment. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37 °C in a humidified incubator containing 5% CO2. NIH3T3 and RAW264.7 cells were seeded at a density of 0.8 × 10⁶ cells / well in 48-well plates and cultured in a CO2 incubator at 37 °C. After the cells were firmly attached, they were replaced with conditioned medium containing various scaffolds. Cell proliferation was assessed using the Cell Count Kit-8 (CCK-8) assay on days 1 and 4. In short, aspirate culture medium and add 200 μL of CCK-8 working solution (serum-free medium: CCK-8 solution = 9:1, v / v) to each well, then incubate for 1 hour and 30 minutes. Collect approximately 100 μL of solution from each well and measure absorbance at 450 nm using a microplate reader (Thermo Fisher Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). Perform live / dead staining with calcein AM and propidium iodide (PI), image cells using a fluorescence microscope (DMi, Leica, Germany), and count cell viability (n = 3) using ImageJ.

[0054] Simultaneously, the scaffolds were sliced ​​into thin slices (1 mm thick) and sterilized by irradiation of both sides with ultraviolet (UV) light for 12 hours. The sterilized scaffolds were placed in 48-well cell culture plates, fixed with sterile stainless steel rings, and 600 μL of the corresponding complete culture medium was added to each well. The scaffolds were incubated at 37°C and 5% CO2. NIH3T3 and HUVECs were seeded on the scaffolds (cell density = 2 × 10⁴ cells / well) and cultured in an incubator for 1 and 4 days. On day 4, cells were stained with DAPI / F-actin for cytoskeleton staining and imaged using a fluorescence microscope. Cell proliferation was assessed using a CCK-8 assay on days 1 and 4 (n = 3).

[0055] 4. Anti-inflammatory and antioxidant performance test of the stent.

[0056] The anti-inflammatory and antioxidant capabilities of the scaffold were assessed using lipopolysaccharide (LPS)-stimulated macrophages. Briefly, RAW 264.7 cells (3 × 10⁶ cells / well) were seeded in six-well plates and cultured for 12 hours. The cells were then incubated for 8 hours in serum-free medium containing 100 ng / mL LPS. The LPS-containing medium was then removed, and the cells were incubated in scaffold-conditioned medium for 24 hours.

[0057] The cell pellet was resuspended in 1 mL of phosphate-buffered saline (PBS 1X) containing 1 μg PE anti-mouse CD86 antibody and 1 μg FITC anti-mouse CD2066 antibody (BioLegend) for immunophenotyping analysis. To maintain cell viability and prevent non-specific antibody binding, the resuspended cells were incubated on ice for 30 min, followed by two washes to remove unbound antibodies. CD86 and CD206 expression was analyzed using a CytoFLEX flow cytometer (Becton Dickinson, USA) and analyzed using FlowJo (FlowJo V 10) (n=3).

[0058] Antioxidant performance was assessed using a 2′,7′-dichlorodihydrofluorescein (DCFH-DA) assay kit (Beyotime, China) to determine intracellular reactive oxygen species (ROS) levels. ROS levels were detected using an inverted fluorescence microscope and quantified by flow cytometry (n=3).

[0059] According to the manufacturer's instructions (n=3), the NO level in the centrifuged supernatant was determined using a nitric oxide (NO) detection kit (Beyotime, China).

[0060] RAW 264.7 cells were cultured to extract RNA. Total RNA was isolated from the samples using the EZ-press RNA purification kit (EZBioscience, Roseville, MN, USA). RNA was then reverse transcribed using the Color Reverse Transcription Kit (EZBioscience) to generate complementary DNA (cDNA). Quantitative qRT-PCR analysis was then performed using 2×ColorSYBR Green qPCR Master Mix (EZBioscience). The relative expression levels of the target genes (n=3) were assessed using the 2–ΔΔCt method. Primer sequences are shown in Appendix S1, with GAPDH used as an internal control gene.

[0061] Table S1. Different types of primers used for RAW 264.7 quantitative RT-PCR of cells.

[0062] Gene names Sequences Forward 5'- AGGTCGGTGTGAACGGATTTG -3’Reverse 5'- TGTAGACCATGTAGTTGAGGTCA -3’ Forward 5’- CTGCAAGAGACTTCCATCCAG -3’Reverse 5’- AGTGGTATAGACAGGTCTGTTGG -3’ Forward 5’- CAGGCGGTGCCTATGTCTC -3’Reverse 5’- CGATCACCCCGAAGTTCAGTAG -3’ Forward 5’- TGGAGAGTGTGGATCCCAAG -3’Reverse 5’- GGTGCTGATGTACCAGTTGG -3’ Forward 5’-CAGGTGTGCTTCTGCCAAGAT -3’Reverse 5’- GGTAGGTATCCGTCATGGTCT-3’ Forward 5’- CTTACTGACTGGCATGAGGATCA -3’Reverse 5’- GCAGCTCTAGGAGCATGTGG -3’ Forward 5’- TCTCACCTCCCAACTGCTTC -3’Reverse 5’- ACTTTGCCGAGCTGTCCTTGGAGA -3’ Forward 5’- CTCCAAGCCAAAGTCCTTAGAG -3’Reverse 5’- GGAGCTGTCATTAGGGACATCA -3’ .

[0063] 5. RNA sequencing of the scaffold (RNA-seq).

[0064] In RNA sequencing (RNA-seq) analysis, RAW264.7 cells activated with LPS and treated with scaffold-conditioned medium were used, and RNA was extracted by lysis using TRIZOL reagent. The RNA-seq analysis was then performed by Shanghai Xihua Yanchuang Biotechnology Co., Ltd.

[0065] 6. Preparation of conditioned medium for macrophages.

[0066] RAW264.7 cells were seeded at a density of 3 × 10⁶ cells / well in 6-well plates. After adhesion, fresh medium containing lipopolysaccharide (LPS, 100 ng / mL) was added for 12 h of stimulation. Subsequently, the cells were gently washed with PBS 1X, and 1.5 mL of scaffold conditioned medium was added to each well. The cells were cultured for another 24 hours, after which the medium was collected. The treated medium from each group was filtered through a 0.22 μm filter and then mixed with other cell culture media at a 1:1 volume ratio to prepare macrophage conditioned medium for subsequent experiments.

[0067] 7. In vitro Transwell migration of stents.

[0068] The migration ability of HUVECs was assessed using a Transwell migration assay. Transwell chambers were placed in 24-well cell culture plates. 200 μL of cell suspension (n = 3, 2 × 10⁶ cells per well) was added to each chamber, and 800 μL of different macrophage conditioned medium was added to the lower wells. The culture plates were incubated at 37 °C for 12 h. Cells were fixed with 4% paraformaldehyde (PFA) for 30 min and stained with 100 μL of 1% crystal violet solution for 5 min. Cells on the upper side of the membrane were then carefully removed with a moistened cotton swab, and cells on the lower side were observed using an optical microscope (EclipseNikon, Japan). Finally, 10% acetic acid solution was added to dissolve the crystal violet dye on the lower surface of the chamber, and the OD value was measured at 590 nm.

[0069] 8. Healing of external scratches on the stent.

[0070] HUVECs were cultured in 24-well cell culture plates (n=3, 3 × 10⁴ cells per well) and grown to 90% confluence. A uniform scratch was made on the cell monolayer using a sterile 200 μL pipette. Cells were washed with phosphate-buffered saline (PBS) to remove debris, and then 1 mL of the appropriate macrophage conditioned medium was added to each well. Cell migration was observed under an optical microscope at t=0 and t=24 hours. The scratch area at t=0 (P0) and t=24 hours (Pt) was counted using ImageJ, and cell migration rate was calculated using a formula (n=3). 9. Expression of genes related to in vitro angiogenesis in stents.

[0071] Expression of angiogenesis-related genes in HUVECs was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The genes detected included vascular endothelial growth factor (VEGF), kinase insertion domain receptor (KDR), endothelial nitric oxide synthase (eNOS), and hypoxia-inducible factor (HIF). Briefly, HUVECs were seeded in 6-well plates and cultured to confluence (n = 3, 3 × 10⁶ cells per well). The original medium was then replaced with macrophage conditioned medium, and culture continued until day 5. Total RNA was extracted from the cells using TRIzol reagent, and the extracted RNA was reverse transcribed into cDNA using the RevertAidFirst Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR reactions were performed using a NovoStart® SYBR qPCR SuperMix Plus (Novozymes Biotechnology, Shanghai) on an AppliedBiosystems™ 7500 real-time quantitative PCR system. Gene expression levels were normalized using the internal reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used in this study are shown in Table S2.

[0072] Table S2 Summary of different types of primers used for quantitative RT-PCR of HUVEC

[0073] Gene names Sequences Forward 5'- CAAGCTCATTTCCTGGTATGACAAT -3’Reverse 5'- GTTGGGATAGGGCCTCTCTTG -3’ Forward 5’- TATGCGGATCAAACCTCACCA -3’Reverse 5’- TCGTTCTGTTCTTTTTAGGGACAC - 3’ Forward 5’ - ATCCAGTGGGGGAAGCTGCA -3′Reverse 5’- GGGAACACTGTGATGGCCGAG -3′ Forward 5’- AACAGCACATTTGTCAGGGTCCA -3’Reverse 5’- CTGGGGGTGGGTAACCAAGGTA -3’ Forward 5′- AGTTCCGCAAGCCCTGAAAGC -3′Reverse 5′- GCAGTGGTAGTGGTGGCATTAGC-3′ .

[0074] 10. Conclusion.

[0075] 1) Regarding the in vitro biocompatibility and biological function of the stent.

[0076] The stents were tested for antibacterial activity and their hemostatic capacity was assessed. Compared with the control group, the CS / GP and CS-LOX / GP stent groups showed significantly fewer bacterial colonies. The normalized survival rates of *E. coli* co-cultured with the CS / GP and CS-LOX / GP stents were 11.04 ± 0.94% and 10.37 ± 1.53%, respectively, and those of *Staphylococcus aureus* were 11.59 ± 1.34% and 10.62 ± 3.32%, respectively. The CS-LOX / GP stent exhibited significant antibacterial activity.

[0077] The in vitro coagulation function of the stents was assessed using the blood coagulation index (BCI). Compared to the gauze in the control group, both stent groups promoted clot formation. The BCI values ​​for the control group, CS / GP, and CS-LOX / GP stents were 106.4 ± 3.8%, 52.2 ± 1.3%, and 49.3 ± 7.2%, respectively. The in vitro hemostatic performance of the hydrogels was evaluated by measuring clotting time. All samples formed clots after a period of blood incubation, and quantitative results indicated that the CS / GP and CS-LOX / GP stents had accelerated clotting times. Furthermore, blood compatibility was also an important performance indicator. The hemolysis test showed that the solution in the positive control group turned dark red due to red blood cell rupture, while no significant color change was observed in the negative control group or any stent group. Quantitative analysis showed that the hemolysis rates of both the CS / GP and CS-LOX / GP stents were within the international standard range of ≤5%, indicating that all material groups had good blood compatibility (hemolysis rate shown in Figure 2). To assess the impact of the materials on extrinsic and intrinsic coagulation pathways, prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured. Compared with the control group, the APTT values ​​of CS / GP and CS-LOX / GP stents were significantly reduced, while the PT and TT values ​​showed no significant differences (see Figure 2 for stent activated partial thromboplastin time / prothrombin time / thrombin time).

[0078] The APPT value of the CS-LOX / GP group (40.3 ± 1.5 s) was significantly smaller than that of the Control group (45.3 ± 0.6 s), but comparable to that of the CS / GP group (40.7 ± 1.2 s). There were no significant differences in PT values ​​among the CS-LOX / GP, CS / GP, and Control groups (22.7 ± 1.2 s, 21.3 ± 1.2 s, and 21.3 ± 3.1 s, respectively). Furthermore, there were no significant differences in TT values ​​among the CS-LOX / GP, CS / GP, and Control groups (15.3 ± 1.2 s, 14.7 ± 1.2 s, and 14.3 ± 2.1 s, respectively). These results indicate that the CS-LOX / GP stent can activate the intrinsic coagulation pathway.

[0079] Cell compatibility of the scaffolds was assessed using NIH3T3 cells, RAW 264.7 cells, and HUVECs. Both NIH3T3 fibroblasts and RAW 264.7 macrophages maintained good cell viability in the scaffold-conditioned medium. From day 1 to day 4, both cell types showed a majority of viable cells (green staining) and a small number of dead cells (red staining), with an increase in cell number. CCK-8 assays also showed a sustained increase in cellular metabolic activity, with no significant differences between groups. On day 4, cell viability was above 90% in all groups. Simultaneously, NIH3T3 cells and HUVECs were seeded onto the scaffolds. On day 4, DAPI / F-actin staining showed that some cells also adhered to the scaffold pore walls and exhibited a spindle-shaped morphology, indicating cell migration within the scaffold pores. CCK-8 assays also showed that cells in the scaffolds proliferated over time from day 1 to day 4.

[0080] 2) Targeting in vitro immune regulation.

[0081] The conversion of macrophages from the M1 to the M2 phenotype is considered a potential anti-inflammatory treatment. Therefore, we investigated the effect of the CS-LOX / GP scaffold on macrophage polarization under the LPS challenge. Flow cytometry analysis showed that after CS-LOX / GP treatment, the proportion of M1 macrophages (CD86+) changed (4.87%), while the proportion of M2 macrophages (CD206+) increased (3.8%). Furthermore, quantitative data from the flow cytometry results showed a statistically significant increase in the M2 / M1 ratio (0.739±0.077) in the CS-LOX / GP group compared to the Control (0.052±0.004) and CS / GP (0.276±0.081) groups. Flow cytometry analysis showed that the percentages of M2 macrophages (CD 206+) and M1 macrophages (CD 86+) were significantly increased after treatment with CS-LOX / GP.

[0082] To further elucidate the role of CS-LOX / GP in macrophage polarization and the regulation of inflammatory mediators, RT-PCR was used. The results showed that in the CS-LOX / GP group, the expression of pro-inflammatory genes IL-6, TNF-α, IL-1β and CCR7 was significantly downregulated, while the expression of anti-inflammatory genes IL-4, IL-10 and ARG1 was significantly upregulated, highlighting the strong immunomodulatory and pro-repair capabilities of the CS-LOX / GP scaffold.

[0083] Previous studies have shown that NO production plays an important physiological role in inflammation. High concentrations of NO are cytotoxic to cells and are associated with various inflammatory diseases. Our study demonstrates that the application of the CS-LOX / GP scaffold significantly reduces NO levels, thereby alleviating cellular oxidative stress damage. Excessive ROS accumulation in diabetic wounds not only oxidizes surrounding lipids and proteins, causing cell damage, but also promotes the chemotaxis of inflammatory factors to the wound site, thus hindering skin regeneration (DOI: 10.1038 / s41467-020-16544-7). Therefore, RAW 264.7 cells were cultured in a pathological oxidative microenvironment induced by 100 ng / mL LPS, and intracellular ROS levels were assessed using DCFH-DA. Results showed that flow cytometry quantification revealed a significantly lower percentage of FITC-positive channels in the CS-LOX / GP scaffold group compared to other groups. Furthermore, DCFH-DA staining revealed that the CS-LOX / GP scaffold group exhibited significantly lower green fluorescence compared to the control and CS / GP groups. This finding highlights the strong ROS scavenging ability of the CS-LOX / GP scaffold and underscores its potential as an effective antioxidant in wound dressing applications.

[0084] To further investigate whether cytokines and growth factors produced by macrophages activated by the CS-LOX / GP scaffold contribute to tissue repair, we cultured tissue-repair-related HUVECs in conditioned medium (CM) with pretreated macrophages. The effects of different CM treatments on HUVECs were investigated using in vitro scratch assays and Transwell migration assays. After co-culturing HUVECs with CM treated with the CS-LOX / GP scaffold for 24 h, cell migration was significantly higher than in other groups. Transwell migration assays also showed similar results, with the CS-LOX / GP group significantly enhancing cell migration. We further performed qRT-PCR to assess the expression of angiogenesis-related genes in HUVECs; we found that the CS-LOX / GP group significantly upregulated the mRNA expression of angiogenesis-related genes, including VEGF, eNOS, KDR, and HIF. These data suggest that the CS-LOX / GP scaffold may enhance the angiogenesis capacity of endothelial cells through paracrine factors released by macrophages, showing great promise in promoting wound healing.

[0085] To elucidate the molecular mechanism by which the CS-LOX / GP scaffold mediates macrophage anti-inflammatory activity, transcriptomic analysis was performed. The analysis revealed distinct transcriptomic profiles between the LPS and LPS+CS-LOX / GP groups. Principal component analysis (PCA) further highlighted the transcriptional alterations induced by CS-LOX / GP treatment. Volcano plot analysis revealed that 1436 genes were upregulated and 709 genes were downregulated in the CS-LOX / GP group compared to the LPS group. To further explore the biological functions and key enrichment pathways of these differentially expressed genes (DEGs), KEGG and GO pathway enrichment analyses were performed. KEGG enrichment analysis showed that genes with significantly downregulated expression between the CS-LOX / GP group and the control group included IL-17, TNF, NF-κB, and various signaling pathways. On the other hand, the differentially upregulated gene expression between the CS-LOX / GP group and the control group in KEGG enrichment analysis was mainly related to cellular signaling and metabolism, such as ECM-receptor interaction, lysine degradation, alpha-Linolenicacid metabolism, and the oxytocin signaling pathway. In GO enrichment analysis, the downregulated genes between the CS-LOX / GP group and the control group were mainly enriched in biological processes related to inflammation and immune responses, such as inflammatory response, immune response, and response to lipopolysaccharide. The upregulated genes between the CS-LOX / GP group and the control group were mainly enriched in those related to tissue remodeling and cellular regulation of inflammation, such as extracellular matrix organization and cellular component morphogenesis. Furthermore, gene set enrichment analysis (GSEA) showed downregulation of the TNF, IL-17, and NF-κB signaling pathways, consistent with the results of KEGG and GO enrichment analyses. In summary, these results indicate that CS-LOX / GP materials play a positive regulatory role in inflammation relief, and the mechanism may be related to the regulation of key gene transcription.

[0086] Example 5; Animal Experiment; This animal research protocol has been approved by the Ethics Committee of Wenzhou Medical University and conducted in accordance with the "Guideline for the Care and Use of Laboratory Animals" (National Institutes of Health, 8th Edition, 2011 Revision).

[0087] 1. Subcutaneous implantation of scaffolds in rats.

[0088] Prior to implantation, scaffolds with a diameter of 10 mm and a height of 2 mm were prepared and sterilized by irradiation of both sides with ultraviolet (UV) light for 12 hours. Six 6–8 week old male SD rats were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. and housed in a standard environment with a temperature of 22–25 ℃, humidity of 40–70%, and a 12-hour light / dark cycle. Before surgery, the fur on the rats' backs was shaved and disinfected. After anesthesia with isoflurane, a small incision of approximately 1 cm was made along the midline of the back. Subsequently, two symmetrical subcutaneous sacs were formed on both sides of the incision using blunt dissection, and scaffolds were implanted in each sac. After implantation, the incision was sutured with 7–0 sutures. On the 7th day post-surgery, the animals were sacrificed, and the scaffolds along with surrounding tissues were removed, fixed in 4% paraformaldehyde, and then embedded in paraffin for subsequent histological analysis. Histological staining assessment of tissue reaction: Hematoxylin-eosin (H&E) staining and Masson's trichrome staining were used to analyze the tissue reaction induced by the scaffolds. Paraffin-embedded samples were sectioned to a thickness of 5 μm and subjected to H&E staining according to standard procedures. The number of cells in the scaffold and the density of cells in the surrounding fibrous capsules were further analyzed based on the H&E images. In addition, Masson's trichrome staining was performed to assess collagen deposition and fibrous capsule formation around the hydrogel.

[0089] Masson staining images were used to measure the thickness of the fibrous capsule surrounding the scaffold, and related image analysis was also performed using ImageJ (n = 3). Finally, the major organs, including the heart, liver, spleen, lungs, and kidneys, were retrieved and subjected to H&E staining.

[0090] 2. Streptozotocin (STZ)-induced diabetic rats.

[0091] A diabetic model was established using a cohort of 36 male SD rats aged 6 to 8 weeks. The rats were housed under controlled environmental conditions, maintained at 22 ± 2°C, with three animals per cage to ensure sufficient space and social interaction. The diabetic rat model was established by intraperitoneal injection of streptozotocin. Successful establishment of the diabetic model was indicated by three consecutive measurements of fasting blood glucose levels greater than 16.7 mmol / L.

[0092] 3. Wound healing assessment.

[0093] After isoflurane induction anesthesia, the dorsal fur of the rats was carefully removed using an electric animal razor, and the surgical area was disinfected with 75% alcohol. The rats were then divided into three groups: control, CS-GP, and CS-LOX / GP, with 12 animals in each group. In each rat, a full-thickness skin wound with a diameter of 20 mm was created on the back using a sterile biopsy punch. The wound was then covered with a suitable scaffold, bandaged, and secured with sutures. Wound healing progress was recorded at 0, 3, 7, 10, and 14 days post-healing by taking digital photographs of the wound, and the relative wound area was calculated using ImageJ. The wound size percentage was calculated using the formula: Wound closure rate = At / A0 × 100%, where A(0) and A(3,7,14,21) represent the wound area on day 0 and days 3, 7, 10, and 14, respectively.

[0094] 4. Histological and immunofluorescence analysis.

[0095] Animal healing tissues were harvested on postoperative days 7 and 14 and subjected to a series of histological preparations. First, samples were stained using hematoxylin-eosin (H&E) and Masson's trichrome staining. Subsequently, immunofluorescence (IF) staining was performed to assess the phenotype of macrophages and the extent of tissue repair in the damaged skin tissue. Paraffin sections were dewaxed, clarified three times in xylene for 15 minutes each, then rehydrated twice in absolute ethanol for 5 minutes each, rehydrated once each in 85% ethanol and 75% ethanol for 5 minutes, and finally rehydrated once in distilled water. Different slides were then incubated overnight at 4 °C with primary antibodies against α-smooth muscle actin (α-SMA) and CD31; CD86 and CD206; and VEGF and F4 / 80, respectively. After rinsing three times in PBS (pH 7.4) for 5 minutes each time, the slides were incubated with specific secondary antibodies at room temperature for 50 minutes. Subsequently, after further washing in PBS, the slides were incubated with DAPI in the dark for 10 minutes. Morphological and quantitative analysis was performed on the stained sections under a Ci-S microscope (Nikon). (DOI: 10.1186 / s12951-025-03274-5).

[0096] 5. In vivo biocompatibility of the stent.

[0097] To determine the in vivo biocompatibility and foreign body response of the scaffolds, we subcutaneously implanted CS / GP and CS-LOX / GP scaffolds into SD rats for 7 days. The results are shown in Figures 5–10. In the CS / GP group, inflammatory features such as redness and swelling were observed around the subcutaneous site. However, this did not occur in the CS-LOX / GP scaffold. Both scaffolds still showed relatively complete bulging during the first 7 days; this indicates that the scaffolds did not undergo extensive degradation during the first 7 days (gross images of CS / GP and CS-LOX / GP scaffolds on day 7 after subcutaneous implantation in Figure 5). Figure 6 shows low-power images of H&E staining and Masson trichrome staining of the two scaffolds on day 7 after implantation. Compared with the CS / GP group, the CS-LOX / GP group showed a milder inflammatory response, which can be observed in H&E staining. Masson trichrome staining also showed that the CS-LOX / GP group induced less inflammation than the CS / GP group. On day 7 post-implantation, there were significant differences in inflammatory cell density within the fibrous capsule: CS / GP group = 8.3 ± 0.4 (×10³ cells / mm²), CS-LOX / GP group = 4.6 ± 1.1 (×10³ cells / mm²). Intrastrap inflammatory cell densities were: CS / GP group = 2.70 ± 0.6 (×10³ cells / mm²), CS-LOX / GP group = 1.3 ± 0.4 (×10³ cells / mm²); the inflammatory cell density in the CS-LOX / GP group was significantly lower than that in the CS / GP group. Interface analysis of the fibrous capsule thickness also showed a significant reduction in thickness in the CS-LOX / GP group (333 ± 28 μm) compared to the CS / GP group (473 ± 52 μm). Furthermore, no significant lesions, such as tissue necrosis and edema, were observed in major organs including the heart, liver, lungs, spleen, and kidneys.

[0098] 6. Evaluation of the regeneration effect of diabetic wounds in rats.

[0099] To evaluate the ability of the CS-LOX / GP scaffold to promote full-thickness skin wound healing in diabetic rats, we conducted in vivo experiments in an STZ-induced diabetic SD rat model. Wound healing was observed on days 0, 3, 7, 10, and 14. By day 3, the wound closure rate in the CS-LOX / GP scaffold group was significantly higher than that in the CS / GP group (29 ± 6%) and the Control group (11 ± 4%), reaching 49 ± 6%. By day 14, the wounds in the CS-LOX / GP group were almost completely healed, while wounds in other groups still showed delayed healing. To further elucidate the role of the scaffold in promoting chronic wound healing, we performed histological analysis. H&E staining results showed that, compared with the Control and CS / GP groups, the wounds in the CS-LOX / GP scaffold-treated group exhibited more neovascularization and hair follicle formation, and less inflammatory cell infiltration. By day 14, the wounds in the CS-LOX / GP scaffold-treated group had achieved complete re-epithelialization, exhibiting typical epidermal structure and morphological characteristics. Furthermore, H&E staining on day 14 revealed that the inflammatory cell density at the wound site was 0.63 ± 0.17 (×10³ cells / mm²), significantly lower than that in the Control group (2.08 ± 0.14 (×10³ cells / mm²) and the CS / GP group (1.72 ± 0.1 (×10³ cells / mm²)). Masson's trichrome staining was used to assess granulation tissue thickness to determine collagen deposition and scar length, important indicators of wound healing. Compared to the Control and CS / GP groups, the CS-LOX / GP scaffold treatment group exhibited thicker granulation tissue and significantly narrower wound edges. Dense and orderly collagen deposition was observed beneath the epidermis in this group, indicating more mature skin regeneration. In contrast, other treatment groups showed poorer skin regeneration, characterized by sparse collagen deposition and prominent wound edges. Furthermore, quantitative analysis showed that on day 14, the length of the unhealed wound in the CS-LOX / GP treatment group was only (1.64 ± 0.39 mm), which was significantly lower than that in the Control group (5.67 ± 0.61 mm) and the CS / GP group (4.36 ± 0.54 mm).

[0100] Based on observed H&E and Masson staining results, the inflammatory response in diabetic wounds treated with the CS-LOX / GP scaffold was significantly reduced. To further assess the inflammatory status during the healing process, immunofluorescence staining was performed on days 7 and 14 using CD206 (green) and CD68 (red). Compared with other groups, the CS-LOX / GP group showed a significantly higher percentage of CD206 (M2) expression in CD68-labeled macrophages (Figure 3 shows IF staining on days 7 and 14, revealing double staining of CD86+ and CD206+, and Figure 4 shows the ratio of CD86+ positive macrophages to CD206+ positive macrophages), indicating that CS-LOX / GP treatment effectively alleviated the inflammatory response during the healing process in diabetic mice. In vitro studies have shown that the CS-LOX / GP scaffold enhances the expression of angiogenesis-related genes in HUVECs when cultured in LPS-induced macrophage conditioned medium, prompting us to explore angiogenesis and the formation of neovascular networks in vivo. CD31 and α-SMA were stained on days 7 and 14 to assess the angiogenic capacity of the CS-LOX / GP scaffold. Figure 3The images show CD31 and α-SMA double staining on days 7 and 14, and Figure 4 shows the quantitative data of newly formed blood vessels corresponding to α-SMA staining and the quantitative data of mature blood vessels corresponding to CD31 staining. Compared with the Control group and the CS / GP group, the CS-LOX / GP scaffold treatment group showed significantly enhanced angiogenesis, with a wider range of CD31 and α-SMA positive areas, strongly indicating its superior pro-angiogenic activity and further accelerating the wound closure process. To investigate whether the enhanced angiogenesis in the CS-LOX / GP group in diabetic wounds is related to macrophage behavior, tissue samples were collected on days 7 and 14 and subjected to double immunofluorescence staining for VEGF (vascular endothelial growth factor, red) and F4 / 80 (macrophage marker, green). Compared with the Control group and the CS / GP group, the CS-LOX / GP group had higher VEGF fluorescence intensity, indicating higher VEGF expression levels in these groups. Furthermore, the proportion of VEGF-expressing macrophages in the CS-LOX / GP group was significantly higher than in the other four groups. This finding suggests that the CS-LOX / GP group can effectively promote VEGF secretion by macrophages during wound healing in diabetic mice (Figure 3 shows VEGF and double F4 / 80 staining on days 7 and 14; Figure 4 shows the ratio of VEGF-positive macrophages to F4 / 80-positive macrophages). Therefore, the above results validate that the CS-LOX / GP scaffold can significantly promote angiogenesis and functional vascular network construction in diabetic wounds. This effect is mainly attributed to the enhanced VEGF secretion induced by the CS-LOX / GP scaffold in M2 macrophages.

[0101] Persistent chronic inflammation and excessive ROS production in diabetic wounds impair the activity of growth factors, inhibit angiogenesis, and significantly increase the risk of infection. This cascade of events ultimately leads to prolonged healing time for diabetic wounds. To address these challenges, a CS-LOX / GP scaffold with inflammation-modulating properties was developed by covalently coupling LOX to CS and then cross-linking the coupling with GP. It exhibits hemostatic and antibacterial capabilities, sustains LOX release, and displays anti-inflammatory and antioxidant properties, collectively promoting the healing of diabetic wounds.

[0102] Chemical modification of CS primarily targets its -NH2 groups, as these groups mainly contribute to its crystallinity. Carboxymethylation is typically the most common method to improve the water solubility of CS. Furthermore, the CS-drug conjugate approach has gained interest in CS modification because it not only increases its water solubility but also introduces bioactive agents or reagents to enhance the biological properties of CS. Our results demonstrate that CS-LOX conjugates are a successful method for CS modification and further GP crosslinking of CS. FTIR spectroscopy confirmed the successful grafting of LOX in the CS-LOX conjugates, which disrupts the strong intermolecular and intramolecular hydrogen bonds of the CS chain. This alteration significantly reduces the crystallinity of CS. Meanwhile, CS-based scaffolds are renowned for their excellent antibacterial activity, primarily due to the interaction between the polycationic amino groups on CS and negatively charged macromolecular residues on the bacterial cell surface, altering cell membrane permeability, leading to bacterial metabolic disturbances, and ultimately bacterial death. However, our recent study indicates that hydrogels prepared by consuming free amino groups in IBU-CS during MA modification result in a significant reduction in antibacterial activity. However, the antibacterial performance of the CS-LOX / GP scaffold obtained by lyophilizing GP-crosslinked CS-LOX can reach about 90% (as shown in Figure 2, survival rates of Escherichia coli and Staphylococcus aureus). This is likely due to the large pore size of the scaffold (as shown in Figure 1), allowing bacteria to exert their antibacterial function not only through contact with the scaffold surface but also through contact with the free amino groups inside the scaffold. The successful synthesis and characterization of the CS-LOX / GP scaffold confirms its structural stability, biocompatibility, and suitability for wound healing applications. Degradation characteristics demonstrate its ability to remain stable during wound healing (Figure 1 shows the in vitro degradation curves of CS / GP and CS-LOX / GP), while the controlled release of LOX within 28 days minimizes the need for frequent dressing changes. Figure 1 (In vitro drug release curves of CS-LOX / GP during 28 days). Optimal water absorption and pore size support a clean wound environment and promote cell migration, both of which are essential for wound healing and tissue regeneration.

[0103] In diabetic patients, uncontrolled bleeding and infection can significantly delay wound healing and increase the risk of trauma-related death. High blood glucose levels in diabetic patients lead to excessively thick blood, metabolic disturbances, reduced clotting factors, and slowed blood flow, all of which impair coagulation function. Although the body initiates a coagulation cascade after bleeding, severe or uncontrollable bleeding often requires external hemostatic materials. The CS-LOX / GP scaffold exhibits remarkable extracorporeal hemostatic properties, promoting rapid hemostasis at diabetic wound sites (Figure 2: Coagulation Index and Clotting Time values). Positively charged CS molecular chains attract negatively charged cell walls, promoting cell adhesion. The interaction between CS and red blood cells leads to cell aggregation and subsequent blood coagulation. In addition to the hemostatic function of the organic components, the CS-LOX / GP scaffold provides a larger surface area for platelet adhesion and aggregation. Furthermore, the scaffold's ability to absorb more water supports its ability to absorb blood like a sponge, forming a physical barrier that impedes blood flow and promotes local hemostasis.

[0104] Prolonged and excessive inflammation is detrimental to successful wound healing in diabetic patients. Maintaining the balance between M1 and M2 macrophage populations is crucial for controlling inflammation and promoting tissue repair. However, this balance is disrupted in diabetic wounds, leading to a decrease in the immunomodulatory activity of macrophages. Previous studies have found that LOX can regulate the inflammatory response by inducing the transformation of M1 macrophages into M2 macrophages. Therefore, in this study, we found that under in vitro LPS stimulation, the CS-LOX / GP scaffold effectively induced macrophage polarization towards the M2 phenotype and inhibited ROS production by modulating the TNF, IL-17, and NF-κB signaling pathways. In vivo results showed that CS-LOX / GP scaffold treatment successfully activated macrophage polarization towards the M2 phenotype and partially inhibited M1 macrophage activation, reducing the inflammatory response and thus modulating immune dysregulation in wound repair.

[0105] In natural skin tissue, a complex capillary network is crucial for maintaining normal metabolism, facilitating the efficient exchange of nutrients, gases, and metabolic waste. Therefore, vascularization of regenerating skin tissue is a key component of the wound regeneration process after injury. Previous studies have shown that pro-regenerative macrophages can secrete pro-angiogenic and neurotrophic factors, thereby promoting new blood vessel formation after injury. However, diabetic wounds often exhibit impaired angiogenesis due to factors such as hydrolyzed growth factors, leading to delayed healing and increased risk of infection. Here, we investigated the effects of CS-LOX / GP scaffolds on angiogenesis. First, all scaffolds exhibited excellent biocompatibility when co-cultured with HUVECs. Furthermore, in the inflammatory microenvironment, CS-LOX / GP scaffolds induced the conversion of M1 macrophages to M2 macrophages, thereby significantly enhancing the angiogenic behavior of HUVECs through paracrine mechanisms, including migration, upregulation of angiogenesis-related factors, and scratch healing capacity. In vivo results support the role of M2 macrophages in enhancing angiogenesis. In vivo, CS-LOX / GP scaffold-induced M2 macrophages secrete abundant VEGF at the wound site, promoting angiogenesis and elongation from existing vessels into the wound. The increased number of α-SMA-positive vessels confirms this enhanced angiogenesis. Therefore, CS-LOX / GP-induced M2 polarization enhances the angiogenic capacity of HUVECs and further accelerates the healing process of diabetic wounds by promoting angiogenesis through VEGF secretion.

[0106] In summary, the CS-LOX / GP scaffold demonstrates significant potential for accelerating diabetic wound healing. By modulating the NF-κB signaling pathway, it effectively polarizes macrophages towards the M2 phenotype, reduces inflammation, and increases intralesional angiogenesis and collagen deposition, ultimately leading to satisfactory wound healing outcomes in diabetic patients. With the sustained release of LOX and the significant antibacterial effect of CS, this multifunctional scaffold achieves promising healing outcomes by modulating macrophages.

Claims

1. A dressing to promote healing of a diabetic wound, characterized in that: The dressing is prepared by the following steps: Step one, at room temperature, 1.61 g of chitosan is dissolved in 100 mL of hydrochloric acid solution by continuous stirring to prepare a chitosan solution; Step two, 0.246 g of 1 mM loxoprofen is dissolved in 20 mL of ethanol, then 0.96 g of 6.18 mmol (1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 0.28 g of 5.95 mmol N-hydroxysuccinimide are added to obtain a loxoprofen solution with activated carboxyl group, the loxoprofen solution is added dropwise to the chitosan solution in step one, and 80 mL of ethanol is added with a magnetic bar at a high speed of 800 rpm to ensure uniform dispersion and form a clear solution; Step three, the transparent solution in step two is kept in the dark at 25 ℃ for 6 hours with stirring at 200 rpm; Then the solution is air-dried in a 37 ℃ oven until it becomes a solid film, and then washed thoroughly in ethanol several times to obtain a solid film of chitosan-loxoprofen conjugate; Step four, the chitosan-loxoprofen conjugate prepared in step three is added to 80 mL of phosphate buffer, and the chitosan-loxoprofen added is 1.6 g, and the magnetic stirring is until the chitosan-loxoprofen solution is 2% w / v; Step five, 0.2 g of genipin is dissolved in 40 mL of phosphate buffer to prepare a 0.5% w / v genipin solution; Step six, after mixing the chitosan-loxoprofen solution prepared in step four with the genipin solution at a volume ratio of 4:1 with high speed stirring, pour into the mold, crosslink for 24 h, then put into the-80 ℃ refrigerator to freeze, and finally freeze-dried to prepare a chitosan-loxoprofen / genipin sponge scaffold.

Images