Kelp decellularized scaffold for promoting diabetic wound repair as well as preparation method and application of kelp decellularized scaffold

Abstract

The invention discloses a kelp decellularized scaffold for promoting diabetic wound repair as well as a preparation method and application of the kelp decellularized scaffold. The method comprises the following steps: preparing a decellularization reagent from a hydrogen bond donor and a hydrogen bond receptor; and carrying out decellularization treatment on the dewaxed kelp by using a decellularization reagent to finally obtain the kelp decellularization scaffold. The kelp decellularized scaffold has a stable three-dimensional structure and proper mechanical properties, can provide a support space for cell growth under the condition of not adding any cross-linking agent and can ensure the integrity in in-vitro application, and the retained bioactive components enable the kelp decellularized scaffold to have endogenous antioxidant, anti-inflammatory and immunoregulatory activities; the traditional Chinese medicine composition can relieve inflammatory response of wound parts by removing active oxygen, change paracrine of macrophages through immunoregulation to promote revascularization, promote wound contraction, granulation tissue regeneration, collagen deposition and reepithelization, and improve the healing speed and quality of diabetic wounds.

Description

Technical Field

[0001] The invention belongs to the field of medical devices, and in particular relates to a kelp decellularized scaffold for promoting diabetic wound repair, and a preparation method and application thereof. Background Art

[0002] Poor wound healing in diabetes is the most serious complication of diabetes, which imposes a heavy burden on patients and healthcare systems. Wound healing involves hemostasis, inflammation, cell proliferation and tissue remodeling. It is a highly organized biological process synergistically acted by growth factors, extracellular matrix (ECM), different tissues and cell lineages. When a wound is formed, immune cells are recruited to the site to fight against the stimulus, especially macrophages, which are a prerequisite for wound healing. Macrophages can be activated and switch to different phenotypes to adapt to the microenvironment of wound healing. M1 macrophages (pro-inflammatory phenotype) are activated by pro-inflammatory factors such as lipopolysaccharide (LPS) in the early stage of inflammation, and then fight infection and inflammation at the wound site by secreting pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β); as wound healing transitions from the inflammatory stage to the proliferative stage, macrophages polarize to the M2 (pro-healing) phenotype, which can secrete anti-inflammatory factors (interleukin-4, IL-4) and growth factors such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) to stimulate fibroblast growth and migration and angiogenesis. However, in diabetic wounds, the hyperglycemic microenvironment produces ROS and pro-inflammatory factors, which disrupt the M2 macrophage phenotype conversion, leading to cellular oxidative stress damage and long-term chronic inflammation. In addition, the failure to switch to the M2 phenotype leads to the inability to communicate with other cells (such as fibroblasts and endothelial cells), hinders cellular activities such as cell proliferation and migration, impairs angiogenesis, re-epithelialization, and ultimately prolongs the healing process. Therefore, strategies to reduce ROS production, induce macrophage polarization, activate HUVEC responses, and promote angiogenesis are promising approaches to accelerate the healing of chronic wounds in diabetes.

[0003] In recent years, bioactive dressings including hydrogels, scaffolds and films have mainly relied on the delivery of cytokines and bioactive substances to accelerate the healing of diabetic wounds. Despite many successes, these strategies have problems such as high cost, cumbersome preparation process and limited bioactivity. Decellularized extracellular matrix (dECM) with natural ECM structure has been shown to be a promising bioactive wound dressing for diabetic wound healing. Compared with synthetic materials, natural dECM not only provides a microenvironment similar to the natural target tissue, but also regulates various processes involved in wound healing, such as cell growth, migration, and differentiation. dECM is mainly extracted from animal and terrestrial plant tissues using conventional physical, chemical and biological methods. Animal-derived dECM is a unique combination of growth factors, but galactosidase (α-gal) and major histocompatibility complex (MHC) can cause immunogenic responses. Plant-derived dECMs have low immunogenicity, while traditional decellularization processes (reagents) significantly reduce their structural stability and mechanical properties and usually require coupling with other polymers, hindering their applicability in wound healing. Therefore, it is imperative to find a new decellularization method and decellularized material to prepare natural bioactive dECM dressings with appropriate mechanical properties and stable structure for the treatment of chronic wounds in diabetes. Summary of the invention

[0004] The purpose of the present invention is to provide a kelp decellularized scaffold for promoting diabetic wound repair, and its preparation method and application. The kelp decellularized scaffold has endogenous antioxidant, anti-inflammatory and immunomodulatory activities, and the preparation method is simple, mild and effective, and can accelerate diabetic wound healing.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a method for preparing a kelp acellular scaffold for promoting diabetic wound repair, comprising the following steps: (1) The hydrogen bond donor and the hydrogen bond acceptor are mixed uniformly at high temperature until a clear and transparent liquid state is present, and no crystals are precipitated after cooling, thereby obtaining a decellularization reagent; (2) After processing, wash the kelp and soak it in n-hexane to remove the wax on the surface; (3) Decellularizing the dewaxed kelp using the decellularizing reagent of step (1), and then washing and freeze-drying to obtain a kelp decellularized scaffold.

[0006] Furthermore, the hydrogen bond donor is at least one of glycerol and ethylene glycol, and the hydrogen bond acceptor is at least one of imidazole and choline chloride.

[0007] Furthermore, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1-7:2-7.

[0008] Preferably, the hydrogen bond donor is glycerol, and the hydrogen bond acceptor is imidazole; the molar ratio of imidazole to glycerol is 7:7.

[0009] Furthermore, in step (2), the immersion time in n-hexane is 15-20 minutes, and the immersion times are 2-3 times.

[0010] Furthermore, in step (3), the decellularization treatment time is 5-7 days, the temperature is 37-40°C, and the rotation speed is 180-200 r / min.

[0011] Furthermore, in the step (1), the high temperature is 80°C.

[0012] The present invention also provides a kelp decellularized scaffold prepared by the preparation method, wherein the fucoidan content is 1.5-9 μg / g, the polyphenol content is 200-740 μg / g and the flavonoid content is 10-40 μg / g.

[0013] The present invention also provides application of the acellular kelp scaffold in preparing a dressing for promoting wound healing and repair.

[0014] Furthermore, the wound is caused by diabetes.

[0015] Furthermore, the dressing is used by covering the wound for more than 10 days.

[0016] Furthermore, the kelp decellularized scaffold can promote the healing and repair of diabetic wounds by reducing oxidative stress at the diabetic wound site, avoiding the formation of chronic inflammation, regulating the polarization of macrophages to the M2 phenotype, and promoting angiogenesis.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The decellularization reagent of the present invention is simple to prepare, safe, degradable, and has low toxicity; (2) The decellularization reagent of the present invention can not only effectively decellularize, but also has a mild treatment process and will not destroy the bioactive components, structure and mechanical properties of dECM; (3) The dECM prepared by the present invention has a stable three-dimensional structure and suitable mechanical properties, can provide a support space for cell growth without adding any cross-linking agent, and can ensure integrity in in vitro applications; (4) The bioactive components retained in the dECM prepared by the present invention enable it to have endogenous antioxidant, anti-inflammatory and immunomodulatory activities; (5) The dECM prepared by the present invention can improve the speed and quality of diabetic wound healing by removing reactive oxygen species, alleviating inflammatory response at the wound site, and promoting angiogenesis, wound contraction, granulation tissue regeneration, collagen deposition and re-epithelialization through immune regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results are for DNA content in kelp decellularized scaffolds.

[0019] Figure 2 The results are for the content of bioactive substances in kelp decellularized scaffolds.

[0020] Figure 3a The SEM images of the kelp decellularized scaffolds of Example 2 and Comparative Example 2; Figure 3b This is the SEM image of the kelp decellularized scaffold of Comparative Example 1.

[0021] Figure 4 Effects of kelp decellularized scaffolds on the proliferation and migration of fibroblasts.

[0022] Figure 5 The growth of L929 cells in the Laminaria japonica decellularized scaffold.

[0023] Figure 6a The total antioxidant activity of Im-Gly2 in Laminaria japonica decellularized scaffolds evaluated by ABST method; Figure 6b The total antioxidant activity of Im-Gly2 in Laminaria japonica decellularized scaffolds evaluated by FRAP method; Figure 6c The effect of Im-Gly2 in kelp decellularized scaffold on cells under oxidative stress.

[0024] Figure 7 This is the effect of kelp decellularized scaffold Im-Gly2 on intracellular ROS.

[0025] Figure 8 The effect of Im-Gly2 on the kelp decellularized scaffold on the M2 phenotype of macrophages.

[0026] Fig. 9 The effect of Im-Gly2 in Laminaria japonica decellularized scaffold on the migration and tube formation of HUVECs in vitro.

[0027] Fig.10 The effect of kelp decellularized scaffold on wound healing in diabetic mice.

[0028] Fig.11This is the effect of kelp decellularized scaffold on oxidative stress and chronic inflammation in diabetic wound sites. The ROS expression area was 56.45% on the 7th day, the TNF-α positive area was 12.28% on the 7th day, and the TNF-α positive area was 7.16% on the 14th day.

[0029] Fig.12 This is the effect of kelp decellularized scaffold on the M2 phenotype and cytokine secretion of macrophages in diabetic wound sites, among which the CD86 positive area was 2.95%, the CD206 positive area was 8.95%, and the CD31 positive area was 29.23%; TNF-α was 272.37pg / mL, IL-1β was 59.8pg / mL, IL-4 was 63.14pg / mL, TGF-β was 1126.59pg / mL and VEGF was 294.27pg / mL. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0031] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0032] Since the electrolyte composition of seawater is similar to that of extracellular fluid, seaweed-derived materials show similarity with the human microenvironment and have no immunogenicity issues, and have gained more attention in wound repair. Among them, Laminaria japonica, as a common brown algae, is rich in alginate, fucoidan, polyphenols, and flavonoids. In previous studies, alginate plays a good role in the structural support of biomaterials. Bioactive substances such as fucoidan can exert immunomodulatory activity by regulating macrophage polarization and affecting macrophage paracrine secretion. Polyphenols and flavonoid compounds show intrinsic antioxidant activity by interrupting free radical chain reactions. Therefore, designing a new decellularized scaffold with intrinsic bioactivity derived from Laminaria japonica can be used to promote diabetic wound healing. However, due to the unstable 3D structure, the existing Laminaria japonica-derived dECM obtained by traditional chemical reagents is only used as a cross-linking agent. DES is a low-melting point mixture formed by hydrogen bond donors (such as glycerol, ethylene glycol) and hydrogen bond acceptors (such as imidazole, choline chloride), which has been used to extract algal DNA and proteins. The synthesis of DES is simple, biodegradable, economically feasible, and safe. Therefore, the present invention developed a Laminaria japonica decellularized scaffold (Im-Gly2) with intrinsic bioactivity using a natural mild deep eutectic solvent (DES) to accelerate the healing of diabetic wounds.

[0033] Example 1: Preparation and screening of DES decellularization reagents Studies have shown that DES prepared by imidazole and glycerol as well as choline chloride and ethylene glycol in a certain molar ratio can be used to extract algae DNA and protein. The standard for successful preparation of DES is that it is a transparent liquid at room temperature and no crystals are precipitated. Based on this condition, the present invention screened out 8 DES with different molar ratio components, as shown in Table 1 and Table 2.

[0034] Table 1 DES (imidazole: glycerol) molar ratio screening

[0035] Table 2 DES (choline chloride: ethylene glycol) molar ratio screening .

[0036] Example 2 A method for preparing a DES that can be used as a decellularizing agent comprises the following steps: (1) Mix imidazole and glycerol in a molar ratio of 7:7 in a beaker, and place the beaker in a water bath at 80°C until it becomes a clear liquid; (2) Take the beaker out of the water bath and place it at room temperature. If no crystals are seen after cooling to room temperature, it indicates that the DES has been successfully prepared.

[0037] Example 3 A method for preparing a kelp decellularized scaffold having intrinsic antioxidant, anti-inflammatory and immunomodulatory activities for promoting diabetic wound healing, the method comprising the following steps: (1) Use a punch to prepare the kelp into a circle with a diameter of 10 mm and wash it three times with deionized water; soak the cleaned kelp in n-hexane for 15 minutes and repeat three times, mainly to remove the wax on the surface of the kelp, which is beneficial for the subsequent decellularization treatment of DES; (2) Decellularization of Laminaria japonica was performed using the DES prepared in Example 2 at 37° C. and 180 r / min (constant temperature shaker) for 5 days; (3) The kelp decellularized scaffold was washed with deionized water for three times; then, the kelp decellularized scaffold Im-Gly2 was obtained by freeze-drying for subsequent research.

[0038] Example 4 In this example, the 8 kinds of DES in Example 1 were used as decellularizing agents to decellularize Laminaria japonica, and the steps were the same as those in Example 3 to obtain 8 kinds of Laminaria japonica decellularized scaffolds. It was evaluated whether the Laminaria japonica decellularized scaffolds met the decellularization standard (DNA content ≤ 50ng / mg tissue) to further screen the DES that can effectively decellularize and the Laminaria japonica decellularized scaffolds that meet the decellularization standard. The results are shown in FIG. Figure 1As shown, four DES decellularization reagents were successfully screened, namely imidazole: glycerol (7:4.5), imidazole: glycerol (7:7), choline chloride: ethylene glycol (1:2.5) and choline chloride: ethylene glycol (1:4); and then four kelp decellularized scaffolds were prepared, namely Im-Gly1, Im-Gly2, Ch-EG1 and Ch-EG2.

[0039] Comparative Example 1 The kelp decellularized scaffold was prepared by the traditional chemical decellularization method: first, a mixture of NaClO solution (20%, v / v) and NaOH (6%, v / v) was prepared, and the pre-treated kelp (the steps were the same as those in Example 3) was placed in the mixture, and the decellularization treatment was performed at 60-70°C for 3 hours. When the color of the kelp turned transparent, the kelp was taken out and repeatedly washed with deionized water, and freeze-dried for later use.

[0040] Comparative Example 2 The preparation steps of calcium alginate gel are as follows: (1) Preparation of 1% sodium alginate solution: Accurately weigh 2.16 g of sodium alginate and dissolve it in 100 mL of double distilled water; (2) Preparation of 5% calcium chloride solution: Accurately weigh 5.05 g of calcium chloride and dissolve it in 100 mL of double distilled water; (3) Preparation of calcium alginate gel: Pour 1% sodium alginate solution into a culture dish and freeze-dry it, and pour 5% calcium chloride solution into the resulting freeze-dried sample.

[0041] Example 5 1. Detection of bioactive substance content Determination of fucoidan content: The kelp decellularized scaffold was ground into powder and passed through a 60-mesh sieve. The kelp decellularized scaffold powder was mixed with DW (1:40 g / mL), extracted at 90 °C for 2 h, and centrifuged to obtain the supernatant for subsequent detection. The phenol-sulfuric acid method was used to detect the content of fucoidan in Im-Gly1, Im-Gly2, Ch-EG1, and Ch-EG2. The glucose solution (0.04 mg / mL) was diluted into different gradients, and then phenol (6% w / v, 1.0 mL) and concentrated sulfuric acid (5 mL) were added and reacted for 30 min. The absorbance was measured at 490 nm. A standard curve was drawn with the polysaccharide content as the abscissa and the optical density as the ordinate. DW (1 mL) was added to the sample solution (1 mL), and then phenol (6% w / v, 1 mL) and concentrated sulfuric acid (5 mL) were added and mixed thoroughly. After 30 minutes of reaction, the absorbance was measured at 490 nm.

[0042] Determination of polyphenol content: The kelp decellularized scaffold was ground into powder, passed through a 60-mesh sieve, and 60% ethanol was added at a solid-liquid ratio of 1:50 (g:mL). The mixture was extracted at 75°C for 70 min, and the supernatant was centrifuged for subsequent detection. The total phenols in Im-Gly1, Im-Gly2, Ch-EG1, and Ch-EG2 were determined by the Folin-Ciocalteu method using gallic acid as the standard. The sample (50 μL) or gallic acid solution (50 μL), 70% ethanol (200 μL), and Folin-Ciocalteu reagent (10% v / v, 1.0 mL) were mixed. Then, Na2CO3 (20% w / v, 750 μL) was added for 5 min. The absorbance was measured at 765 nm. The sample solution was replaced by 70% ethanol as the control group, and the Na2CO3 solution was replaced by DW as the blank group. A standard curve was drawn with gallic acid as the standard, and the results were expressed as total phenol content μg / g per gram of dry matter.

[0043] Flavonoid content determination: The kelp decellularized scaffold was ground into powder, passed through a 60-mesh sieve, and 60% ethanol was added at a solid-liquid ratio of 1:50 (g:mL). The mixture was extracted at 75°C for 70 min, and the supernatant was centrifuged for subsequent detection. 100 μL of sample was reacted with 100 μL of AlCl3·6H2O solution (2% w / v) in a 96-well plate for 15 min. The absorbance was measured at 430 nm. The reaction system of AlCl3·6H2O was replaced by 70% ethanol solution as a control group. The standard curve was drawn with quercetin as the standard, and the results were expressed as total flavonoid content (μg / g) per gram of dry matter.

[0044] The results of the test on the content of bioactive substances in the acellular kelp scaffolds are as follows Figure 2 As shown, fucoidan, polyphenols and flavonoids were all retained in the kelp decellularized scaffolds, but the contents of fucoidan, polyphenols and flavonoids in the four kelp decellularized scaffolds were different, which would affect the realization of their biological functions. Among them, the content of bioactive substances retained in Im-Gly2 was higher, with a fucoidan content of 8.7μg / g, a polyphenol content of 735.3μg / g and a flavonoid content of 40μg / g.

[0045] 2. SEM experiment SEM was performed on the kelp decellularized scaffolds, and the results were as follows Figure 3a As shown, the kelp decellularized scaffold Im-Gly2 has a stable porous three-dimensional structure with an average pore size of 80.7 μm, which is a pore size suitable for the growth of fibroblasts. This porous structure allows the kelp decellularized scaffold to absorb wound exudate and avoid accumulation of exudate to cause wound infection.

[0046] like Figure 3bAs shown, compared with the kelp decellularized scaffold prepared by DES decellularization, the structure of the kelp decellularized scaffold (Comparative Example 1) obtained by traditional chemical decellularization was obviously destroyed, and the residues of the decellularization agent could be observed in the scaffold.

[0047] 3. Mechanical properties test The kelp decellularized scaffold was made into a long strip with a length of 15 mm, a width of 10 mm, and a height of 2 mm. The sample was stretched at a speed of 5 mm / min using a universal testing machine (Instron-5944).

[0048] Results As shown in Table 3, the tensile strain range of the four kelp decellularized scaffolds obtained by DES was 30.0-36.4%, and Ch-EG2 reached a maximum value of 36.4%, which was higher than the composite decellularized grapefruit peel (DPP) / polymer (<25%), and comparable to the physical / chemically cross-linked chitin-based hydrogel dressing (fracture strain: 27-50%). The tensile stress range of the four kelp decellularized scaffolds obtained by DES was 133.8-167.2 kPa, of which the maximum value of Im-Gly2 was 167.2 kPa. The kelp decellularized scaffold Im-Gly2 had suitable mechanical properties, with a tensile stress of 167.2 ± 2.6 kPa, a tensile strain of 31.7 ± 3.2%, and a Young's modulus of 530.7 ± 62.7 kPa, which protected the wound from external stimuli during wound healing. The kelp decellularized scaffold obtained by conventional methods (Comparative Example 1) had a tensile strain of only 12.4%. At the same time, the Young's modulus of the kelp decellularized scaffold is 368.2 −530.7 kPa, which is 5 times higher than that of the kelp decellularized scaffold obtained by the traditional method (Comparative Example 1, 67.7 ± 1.6 kPa). Compared with the kelp decellularized scaffold prepared by DES decellularization, the mechanical properties of the kelp decellularized scaffold (Comparative Example 1) obtained by traditional chemical decellularization were obviously damaged, while the structure of the kelp decellularized scaffold prepared by DES decellularization remained intact.

[0049] Table 3: Mechanical properties test results

[0050] 4. Cell proliferation and migration CCK-8 and cell scratch assays were used to evaluate the effects of Laminaria japonica decellularized scaffolds on the proliferation and migration of fibroblast L929 cells. Figure 4 As shown in Figure 2, the kelp decellularized scaffold has good biocompatibility and can promote the proliferation and migration of fibroblasts. Figure 5As shown, the green column fluorescence represents the cells in the kelp decellularized scaffold. The more green fluorescence, the more living cells there are in the kelp decellularized scaffold, indicating that L929 cells can grow in the kelp decellularized scaffold. This proves that the kelp decellularized scaffold Im-Gly2 can support the growth of fibroblasts in the scaffold and promote the proliferation and migration of fibroblasts.

[0051] 5. Total antioxidant activity detection The present invention further evaluates the total antioxidant activity of the kelp decellularized scaffold by the ABST method and the FRAP method, and finally screens out the kelp decellularized scaffold Im-Gly2 with the strongest total antioxidant activity for subsequent research.

[0052] The results are as follows Figure 6a and 6b As shown, the total antioxidant activity of kelp decellularized scaffolds was detected by ABST and FRAP methods, and it was found that Im-Gly2 had the strongest total antioxidant activity, which was consistent with its higher content of bioactive substances.

[0053] like Figure 6c As shown, 500 μ m H2O2 induced cell oxidative stress damage, green fluorescence indicated living cells, and red fluorescence indicated dead cells, indicating that Im-Gly2 can reduce cell damage under oxidative stress.

[0054] 6. ROS detection The ROS scavenging ability of Im-Gly2 at the extracellular level was evaluated by DCFH-DA staining. 4 RAW264.7 cells were seeded at a density of 10 cells / well and cultured in DMEM complete medium containing 10% fetal bovine serum. After RAW264.7 cells attached, the cells were treated with Im-Gly2 extract with or without 200mM H2O2 for 24h. Serum-free DMEM complete medium was added to the control group. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10mM. The cell culture medium was removed and 200mL of diluted DCFH-DA was added. Incubate in a cell culture incubator at 37℃ for 20min. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Fluorescent images of the cells were captured using an inverted microscope.

[0055] The results are as follows Figure 7 As shown, 200 μ m H2O2 induced the production of intracellular ROS (reactive oxygen species). The stronger the green fluorescence, the higher the level of intracellular ROS, indicating that Im-Gly2 can effectively eliminate intracellular ROS.

[0056] 7. Macrophage polarization toward M2 phenotype (anti-inflammatory phenotype) experiment RAW264.7 is 1 × 10 5 Cells / well were seeded in 24-well plates and cultured for 24 h. Im-Gly2 was then added to cells with or without LPS (1 μg / mL) stimulation and the cells were incubated for another 24 h. The control group was supplemented with serum-free DMEM complete medium. Subsequently, the cells were washed twice with pre-cooled PBS. The cells were fixed with 4% formaldehyde solution at room temperature for about 20 min. Subsequently, the cells were washed twice with pre-cooled PBS. The membrane was permeabilized by adding 0.1% TritonX-100 solution (500 μL) for 15 min. Subsequently, the cells were washed twice with pre-cooled PBS. The cytoskeleton was stained with phalloidin-Alexa Fluor 555 for 30 min at room temperature in the dark. The cells were washed twice with pre-cooled PBS. The nuclei were stained with DAPI stain for 15 min. The cells were washed twice with pre-cooled PBS. Finally, images can be captured by fluorescence inverted microscopy. The results are as follows Figure 8 As shown, Im-Gly2 can promote the polarization of macrophages toward the M2 phenotype (anti-inflammatory phenotype).

[0057] 8. HUVECs migration and tube formation in vitro Human umbilical vein endothelial cells (HUVECs) were cultured at 1 × 10 5 Cells were seeded into 24-well plates at a density of 100 μL / well and cultured in a 37°C incubator. When the cell confluence reached 80%, the DMEM medium containing 10% FBS was replaced with DMEM medium containing 0.2% FBS, and the cells were cultured under this condition for 24 h. Matrix-Gel™ Matrigel was added to 96-well plates at 50 μL / well and then placed in a constant temperature incubator at 37°C for 45 minutes to solidify. The cells were trypsinized and diluted to 2×10 using CM with or without LPS. 5 cells / mL. The control group was supplemented with serum-free DMEM medium. The cell suspension (50 μL / well) was added to a 96-well plate. The 96-well plate was placed in a constant temperature incubator and incubated, and the tube formation was observed every hour. A fluorescent inverted microscope was used to record the optimal time for tube formation, and the tube formation was quantitatively analyzed using ImageJ software.

[0058] The in vitro migration and tube formation of HUVECs are closely related to the cytokines TNF-α, IL-1β, IL-4, TGF-β and VEGF secreted by macrophages. Fig. 9 As shown, Im-Gly2 can regulate macrophage polarization, change macrophage paracrine secretion and promote HUVECs migration and tube formation in vitro.

[0059] 9. Diabetic mouse model Diabetic mice were induced by intraperitoneal injection of STZ (50 mg / kg) for 4 consecutive days. Starting from the 7th day after the first STZ injection, the blood glucose level of mice was tested every other day. If the blood glucose level was higher than 16.8mM for more than two consecutive measurements, the mice were considered to have diabetes. All mice were anesthetized with sodium pentobarbital (50mg / kg) before surgery. The mice were then shaved, depilated and disinfected with 75% ethanol. A full-thickness circular wound with a diameter of 8mm was made and covered with Alginate gel and Im-Gly2. The wounds of the control group were not treated. The skin tissue of the wound of the mouse on the 7th day after surgery was taken, and the prepared tissue sections were placed in an incubation dish in a dim place containing 10μM DHE working solution and incubated at 37°C for 30 minutes. After incubation, the unadsorbed DHE was washed off with PBS. The sample was placed on a glass slide, and an appropriate fluorescence microscopy buffer (such as PBS) was added to observe the DHE staining under a fluorescence microscope. Skin tissues from the wounds of mice on the 7th and 14th days were taken for immunofluorescence (IF) staining to evaluate the inflammatory regulation and angiogenesis of the regenerated tissues, and paraffin-embedded wound tissues were stained with anti-TNF-α. Mouse skin tissues frozen at -80°C were taken, homogenized and the supernatant was taken. TNF-α, IL-1β, IL-4, TGF-β, and VEGF in the homogenate supernatant of mouse skin tissue were determined according to the instructions of the ELISA kit. At the same time, skin tissues from the wounds of mice on the 7th day were taken for IF staining to analyze the mechanism of Im-Gly2 in promoting diabetic wound healing. CD86 (M1) and CD206 (M2) were selected to evaluate the effect of Im-Gly2 on macrophage polarization, and CD31 was selected to evaluate the effect of Im-Gly2 on angiogenesis at the wound.

[0060] The wound healing of diabetic mice was observed for 14 consecutive days. The control group and the alginate gel group were used. Fig.10 It was shown that the wounds of diabetic mice treated with Im-Gly2 healed faster, with the healing rate reaching 94% on the 14th day.

[0061] like Fig.11 As shown, DHE staining was used to evaluate the ROS (reactive oxygen species) level in diabetic wound sites under different treatments. The stronger the red fluorescence, the higher the level of reactive oxygen species. The expression level of tumor necrosis factor-α (TNF-α) was closely related to chronic inflammation in the wound site. Red fluorescence indicated TNF-α. The higher the expression level, the more severe the inflammatory response.

[0062] like Fig.12As shown, CD86 is a red fluorescent marker for M1 macrophages; CD206 is a green marker for M2 macrophages. The stronger the green fluorescence, the more effective Im-Gly2 can be in regulating the polarization of macrophages in the wound site to the M2 phenotype. Im-Gly2 also affects the secretion of cytokines in the diabetic wound site. The expression of two types of pro-inflammatory factors, TNF-α and IL-1β, decreases, and the expression of three anti-inflammatory and healing cytokines, IL-4, TGF-β and VEGF, increases; the change in the expression level of cytokines in the wound site of Im-Gly2 can effectively alleviate the inflammatory response in the wound site and promote angiogenesis in the wound site. CD31 is a red fluorescent marker for new blood vessels. The stronger the red fluorescence, the more angiogenesis in the diabetic wound site.

[0063] In summary, the kelp decellularized scaffold Im-Gly2 prepared by the present invention retains rich bioactive substances, such as fucoidan (8.7 μg / g), polyphenols (735.3 μg / g) and flavonoids (40 μg / g); and has a stable porous three-dimensional structure with an average pore size of 80.7 μm, which is a pore size suitable for the growth of fibroblasts. This porous structure enables the kelp decellularized scaffold to absorb wound exudate and avoid the accumulation of exudate to cause wound infection. The kelp decellularized scaffold Im-Gly2 has suitable mechanical properties, with a tensile stress of 167.2 ± 2.6 kPa, a tensile strain of 31.7 ± 3.2%, and a Young's modulus of 530.7 ± 62.7 kPa, which protects the wound from external stimulation during the wound healing process; its degradation rate is low, and the degradation rate is only 30% on the 16th day, and the main structure is retained, which can avoid secondary damage to the wound caused by frequent dressing changes during the wound healing process. The kelp decellularized scaffold Im-Gly2 has excellent biocompatibility and blood compatibility, which also proves that DES is a safe decellularized agent; it also has good free radical scavenging ability, can reduce cell death under oxidative stress conditions, remove intracellular ROS and increase intracellular SOD enzyme activity. The kelp decellularized scaffold Im-Gly2 can promote macrophage polarization to M2 phenotype, increase the expression of IL-4, TGF-β and VEGF, reduce the expression of TNF-α and IL-1β, and show anti-inflammatory activity; it can promote HUVECs migration and tube formation in vitro by changing macrophage paracrine through immune regulation; it can also promote diabetic wound contraction, granulation tissue regeneration, collagen deposition and re-epithelialization. Therefore, Im-Gly2 can accelerate the healing of diabetic wounds by reducing oxidative stress in diabetic wounds, avoiding the formation of chronic inflammation, regulating macrophage polarization to M2 phenotype and promoting angiogenesis.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing a kelp decellularized scaffold for promoting diabetic wound repair, characterized in that: The following steps are involved: (1) The hydrogen bond donor and the hydrogen bond acceptor are mixed uniformly at high temperature until a clear and transparent liquid state is present, and no crystals are precipitated after cooling, thereby obtaining a decellularization reagent; (2) After processing, wash the kelp and soak it in n-hexane to remove the wax on the surface; (3) Using the decellularization reagent of step (1) to decellularize the dewaxed kelp, and then washing and drying to obtain a kelp decellularized scaffold.

2. The preparation method according to claim 1, characterized in that: The hydrogen bond donor is at least one of glycerol and ethylene glycol, and the hydrogen bond acceptor is at least one of imidazole and choline chloride.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1-7:2-7.

4. The preparation method according to claim 1, characterized in that: In the step (2), the immersion time in n-hexane is 15 minutes to 20 minutes, and the immersion times are 2 to 3 times.

5. The preparation method according to claim 1, characterized in that: In the step (3), the decellularization treatment time is 5-7 days, and the temperature is 37°C-40°C.

6. The kelp decellularized scaffold prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The acellular kelp scaffold has a fucoidan content of 1.5 μg / g to 9 μg / g, a polyphenol content of 200 μg / g to 740 μg / g, and a flavonoid content of 10 μg / g to 40 μg / g.

7. Use of the acellular kelp scaffold according to claim 6 in preparing a dressing for promoting wound healing and repair.

8. The use according to claim 7, characterized in that: The wounds were caused by diabetes.

9. The use according to claim 7, characterized in that: The dressing is used by covering the wound with the dressing for more than 10 days.

10. The use according to claim 8, characterized in that: The kelp decellularized scaffold can promote the healing and repair of diabetic wounds by reducing oxidative stress at diabetic wound sites, avoiding the formation of chronic inflammation, regulating the polarization of macrophages to M2 phenotypes, and promoting angiogenesis.

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