Compound polysaccharide-fish skin source collagen scaffold as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a compound polysaccharide-fish skin source collagen scaffold as well as a preparation method and application thereof. According to the preparation method, tilapia skin is taken as a raw material, collagen is extracted and prepared under the condition that the temperature is lower than the denaturation temperature of the collagen, then astragalus polysaccharide and angelica polysaccharide are selected as drugs carried by the collagen scaffold, and the fish-derived collagen scaffold with biological activity is constructed and used for loading bone marrow mesenchymal stem cells (BMSCs). The oxidative stress injury of the BMSCs can be effectively reduced, the proliferation of the BMSCs can be promoted, the diabetic ulcer wound surface can be repaired, and the wound surface healing can be promoted. The stem cell scaffold with biological activity is constructed by utilizing the traditional Chinese medicines astragalus polysaccharide and angelica polysaccharide for the first time, and the advantages of the traditional Chinese medicines are exerted; meanwhile, the collagen adopted by the invention is derived from fish skin instead of mammals, so that religious barriers, mammalian infectious diseases and the like are avoided.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composite polysaccharide-fish skin-derived collagen scaffold, its preparation method, and its application. Background Technology

[0002] Diabetic foot is an infection, ulcer, and / or deep tissue destruction of the foot associated with localized neuropathy and peripheral vascular disease in the distal lower extremities. The International Diabetic Foot Working Group defines diabetic foot ulcer (DFU) as a wound involving the full thickness of the skin below the ankle in diabetic patients. DFU is a serious complication of diabetes and a major cause of disability and death in diabetic patients, with an estimated incidence of 15%. It is characterized by its difficulty in treatment, high recurrence rate, and high amputation rate. DFU can lead to amputation, loss of walking and working ability, and shortened life expectancy. Coupled with the enormous medical expenses, it places a huge burden on patients both physically and mentally, and economically, severely impacting their quality of life. With the accelerating aging of the population, the incidence rate is rising year by year. Currently, DFU has become a major cause of chronic, refractory wounds in my country and a global problem that urgently needs to be solved.

[0003] The pathogenesis of diabetic foot ulcers is not yet fully understood. The generally accepted view is that the formation of diabetic wounds is mainly due to ischemia, hypoxia, a hyperglycemic environment, and a persistently expanding inflammatory response. These mechanisms lead to inflammatory cell infiltration, degradation of the cellular matrix and growth factors that promote wound repair, impaired angiogenesis, reduced collagen formation, poor granulation tissue growth, and wound healing arrest at one or more stages of the healing process. The pathogenesis of diabetic foot ulcers (DFU) is complex, the condition is recurrent, and its clinical treatment is very challenging.

[0004] Currently, there are many methods for treating diabetic ulceration (DFU), such as local debridement, topical application of growth factors, closed negative pressure drainage, vascular intervention, and flap repair. However, these methods are time-consuming, prone to recurrent ulcers, and expensive. The pathogenesis of DFU is not yet fully understood, and the disease course is long and recurrent, making it difficult to achieve good results with a single treatment method. Clinically, multiple treatment methods are often combined. With the development of tissue engineering technology, skin tissue engineering cell scaffolds and seed cells for treating diabetic ulcers have become a focus of attention. There are three main factors in tissue engineering: seed cells, cell scaffolds, and growth factors. Seed cells are the foundation of tissue engineering technology. Ideal seed cells should have certain characteristics: ① easy to obtain, minimal damage to the donor, abundant supply, and low cost; ② strong cell proliferation capacity and good biological activity; ③ able to maintain the original cell function and repair cartilage defects with high quality after re-implantation. Currently, the most commonly used seed cells for skin tissue engineering technology are bone marrow-derived mesenchymal stem cells (BMSCs) and fibroblasts. Fibroblasts, as the main repair cells in wound healing, participate in the entire healing process. However, their excessive proliferation can lead to hypertrophic scars and scar contractures. Furthermore, excessive deposition of advanced glycation end products (AGEs) in the local skin of diabetic patients can cause morphological changes in fibroblasts and inhibit their ability to synthesize collagen, thus affecting wound healing. Therefore, the application of fibroblasts in DFU (Disease-Induced Fusion). Biogenic mesenchymal stem cells (BMSCs), on the other hand, are abundant, easy to obtain, low-cost, and possess multi-directional differentiation potential. Even in the ulcer microenvironment, BMSCs can not only transform into fibroblasts but also promote fibroblast growth. BMSCs themselves are immunomodulatory cells with immunosuppressive effects that do not disappear with differentiation, thus allowing for both autologous and allogeneic reimplantation. It is precisely because of these advantages that BMSCs have become one of the most commonly used seed cells in skin tissue engineering.

[0005] However, stem cells (BMSCs) suffer repeated oxidative stress damage during in vitro isolation and culture. Furthermore, transplanted BMSCs exhibit poor survival rates and limited ability to differentiate into desired cell types, often hindering their application. To address these issues, some researchers have attempted to enhance the repair capacity of BMSCs through pre-stimulation with monomeric components of traditional Chinese medicine (TCM). TCM monomers and their active ingredients can play an important role in promoting the healing of diabetic foot ulcers. However, problems such as low solubility, poor stability, slow onset of action, and low bioavailability greatly limit the application of TCM active ingredients. Therefore, finding a safe and effective method for treating diabetic foot ulcers is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a composite polysaccharide-fish skin collagen scaffold, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a method for preparing a composite polysaccharide-fish skin-derived collagen scaffold, comprising the following steps:

[0009] Tilapia skin collagen solution and polysaccharide solution containing astragalus polysaccharide and angelica polysaccharide were mixed, and then subjected to static standing, centrifugation, drying and cross-linking in sequence to obtain the composite polysaccharide-fish skin collagen scaffold.

[0010] Preferably, the concentration of fish skin collagen in the fish skin collagen solution is 2 wt.%-10 wt.%; and the mass ratio of Astragalus polysaccharide to Angelica polysaccharide in the polysaccharide solution is 1.25:1.

[0011] More preferably, the concentration of fish skin collagen in the fish skin collagen solution is 6 wt.%.

[0012] Preferably, the crosslinking agent used is genipin, the time is 30 minutes, and the temperature is 37°C.

[0013] Preferably, the centrifugation conditions are: 4℃, 4000×g centrifugation for 20min.

[0014] Preferably, the method for preparing the fish skin collagen solution includes the following steps: removing cells from fresh fish skin using a decellularization method, followed by washing, degreasing, soaking, crushing, filtering, adding acetic acid solution, first extraction, first centrifugation, second extraction, second centrifugation, enzymatic hydrolysis, adding NaCl, third centrifugation, dialysis, concentration, filtration, drying, and dissolving to obtain the fish skin collagen solution.

[0015] Preferably, the degreasing process uses 10% n-butanol as the solvent and takes 24 hours.

[0016] The solvent used for soaking was a 0.1 mol / L NaOH aqueous solution, and the soaking time was 24 hours.

[0017] The first extraction was performed at a temperature of 4°C for 48 hours.

[0018] The centrifugation includes a first centrifugation and a second centrifugation;

[0019] The conditions for the first centrifugation, the second centrifugation, and the third centrifugation are all: centrifugation at 10000 rpm for 30 min;

[0020] The second extraction was performed at a temperature of 4°C for 24 hours.

[0021] The enzyme used in the enzymatic hydrolysis is pepsin; the final concentration of the pepsin is 1‰; the pH of the enzymatic hydrolysis is 2.0, and the time is 2 days.

[0022] This invention provides a composite polysaccharide-fish skin collagen scaffold prepared using the above-described preparation method.

[0023] This invention provides the application of the above-mentioned composite polysaccharide-fish skin collagen scaffold in any of the following:

[0024] (1) Promotes the proliferation of bone marrow mesenchymal stem cells;

[0025] (2) Application in promoting wound healing;

[0026] (3) Repairing diabetic ulcers;

[0027] (4) Prepare products that promote the proliferation of bone marrow mesenchymal stem cells;

[0028] (5) Prepare products that promote wound healing;

[0029] (6) Prepare products for repairing diabetic ulcer wounds.

[0030] More preferably, the diabetic ulcer is a diabetic foot ulcer.

[0031] This invention provides a composite polysaccharide scaffold loaded with bone marrow mesenchymal stem cells. The preparation method of the composite polysaccharide scaffold includes the steps of seeding the bone marrow mesenchymal stem cells into the above-mentioned composite polysaccharide-fish skin collagen scaffold and culturing them to obtain the composite polysaccharide scaffold.

[0032] The present invention provides the application of the above-described composite polysaccharide scaffold in any of the following:

[0033] (1) Application in promoting wound healing;

[0034] (2) Repairing diabetic ulcer wounds;

[0035] (3) Prepare products that promote wound healing;

[0036] (4) Prepare products for repairing diabetic ulcer wounds.

[0037] More preferably, the diabetic ulcer is a diabetic foot ulcer.

[0038] The present invention discloses the following technical effects:

[0039] This invention uses tilapia skin as raw material to extract and prepare collagen under conditions below the collagen denaturation temperature. Then, astragalus polysaccharides and angelica polysaccharides are selected as drugs to modify the collagen scaffold, constructing a bioactive fish-derived collagen scaffold for loading bone marrow mesenchymal stem cells (BMSCs). This effectively reduces oxidative stress damage to BMSCs, promotes their proliferation, and repairs diabetic ulcers, promoting wound healing. This invention is the first to utilize the traditional Chinese medicines astragalus polysaccharides and angelica polysaccharides to construct a bioactive stem cell scaffold, leveraging the advantages of traditional Chinese medicine. Furthermore, the collagen used in this invention is derived from fish skin, not mammals, avoiding religious barriers and mammalian-derived infectious diseases. A sufficient number of rat bone marrow mesenchymal stem cells were successfully isolated and cultured. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is an image showing the appearance of a collagen scaffold (6% scaffold).

[0042] Figure 2 Electron micrograph of a 2% scaffold;

[0043] Figure 3 Electron micrograph of 4% of the scaffold

[0044] Figure 4 Electron micrograph of a 6% scaffold;

[0045] Figure 5 Electron micrograph of 8% of the scaffold;

[0046] Figure 6 Electron micrograph of 10% of the scaffold;

[0047] Figure 7 Electron micrograph of the Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold;

[0048] Figure 8 Observational images of BMSCs;

[0049] Figure 9 This is a flow cytometry analysis result;

[0050] Figure 10 A statistical graph of positive somatic cells;

[0051] Figure 11 The results of cell proliferation assay using the CCK-8 assay are shown. Detailed Implementation

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0055] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0056] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0057] Unless otherwise specified, all components used in this invention are commonly purchased by those skilled in the art; all methods used in this invention are methods well known to those skilled in the art.

[0058] Example 1

[0059] 1. Extraction of fish skin collagen and preparation of fish skin collagen scaffolds

[0060] 1.1 Extraction of Collagen from Fish Skin

[0061] Fresh fish skin was washed with cooled pure water to remove dirt, mud, and foreign matter, and the scales were scraped off. It was then cut into small pieces. Cells were removed from the tissue using a decellularization method. The decellularized tissue was washed with cooled pure water and then defatted for 24 hours with 10% (v / v) n-butanol (1g:20mL, w / v, i.e., the mass-to-volume ratio of the washed tissue to 10% (v / v) n-butanol is 1g:20mL) (changing the solution every 8 hours). The tissue was repeatedly rinsed with deionized water until odorless. After rinsing, the tissue was soaked in a 0.1mol / L NaOH aqueous solution for 24 hours (1g:20mL, w / v, i.e., the mass-to-volume ratio of the rinsed tissue to the 0.1mol / L NaOH aqueous solution is 1g:20mL) to remove impurities (changing the solution every 8 hours). Finally, the tissue was washed with water. The solution was brought to pH 7-8, water was drained, and the mixture was then homogenized in a tissue homogenizer. After washing and filtering, the tissue fragments were mixed with a 0.5 mol / L acetic acid solution (1 g: 50 mL, w / v) to form a homogenate. The mixture was then extracted with magnetic stirring at 4°C for 48 h, followed by centrifugation at 10,000 rpm for 30 min to obtain a precipitate and centrifugal supernatant 1. The precipitate was then mixed with a 0.5 mol / L acetic acid solution (1 g: 50 mL, w / v, i.e., the mass-to-volume ratio of the precipitate to the 0.5 mol / L acetic acid solution was 1 g: 50 mL) to form a homogenate. The mixture was then extracted with magnetic stirring at 4°C for 24 h, followed by centrifugation at 10,000 rpm for 30 min to obtain centrifugal supernatant 2. The centrifugal supernatant 1 and centrifugal supernatant 2 were then combined to obtain the crude collagen extract from fish skin, which is acid-soluble collagen.

[0062] Pepsin was added to the crude collagen extract from fish skin to achieve a final concentration of 1 wt‰, pH of 2.0, and an extraction time of 2 days, resulting in an enzymatically hydrolyzed collagen solution. Then, NaCl was added to the enzymatically hydrolyzed collagen solution to a final concentration of 0.9 mol / L, centrifuged at 10,000 rpm for 30 min, the precipitate was collected and dissolved in 0.5 mol / L acetic acid solution, dialyzed with 0.1 mol / L acetic acid solution for 1 day, and then dialyzed with distilled water for 2 days (the semipermeable membrane has a molecular weight cutoff of 100 kDa) to obtain purified collagen solution; the purified collagen solution was concentrated at 4℃ using a 50 kDa membrane until the protein mass concentration in the solution reached 2%; the concentrated solution was filtered through a 0.22 μm plate membrane for sterilization, 1% (w / v) coconut shell (pharmaceutical grade) activated carbon was added, stirred and filtered to remove the pyrogen; finally, it was freeze-dried in a vacuum freeze dryer for 72 h and sterilized by γ-rays generated by Co60 at a dose of 25 kGy / h, and then packaged to obtain a sponge-like freeze-dried scaffold derived from tilapia skin collagen.

[0063] Fish skin collagen sponge-like freeze-dried scaffolds were cut into four equal parts, cross-linked with genipin solution at 37℃ for 30 min, with a final genipin concentration of 1 wt.% during the cross-linking process, and rotated once every 10 min. The scaffolds were then soaked in anhydrous ethanol for 5 min, washed three times with distilled water (10 min each time), incubated overnight at -20℃, and finally freeze-dried in a vacuum freeze dryer for 24 h. 60 Sterilized by Co-γ-ray sterilization, sealed and stored at room temperature for later use, and subjected to electron microscopy and porosity testing, and subsequent experiments were conducted.

[0064] 1.2 Screening for the optimal concentration of collagen

[0065] A sponge-like freeze-dried scaffold derived from tilapia skin was weighed and dissolved thoroughly in 0.05 mol / L acetic acid at 4°C to prepare collagen solutions with concentrations of 2 wt.%, 4 wt.%, 6 wt.%, 8 wt.%, and 10 wt.%, respectively. Five types of collagen sponge scaffolds without polysaccharide drugs were obtained and designated as 2% scaffold, 4% scaffold, 6% scaffold, 8% scaffold, and 10% scaffold. The appearance of the scaffolds was observed, and scanning electron microscopy and scaffold porosity were measured to preliminarily determine the required scaffold concentration for subsequent experiments.

[0066] 1.3 Preparation of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold

[0067] Weigh out the spongy freeze-dried collagen sponge from tilapia skin, dissolve it thoroughly in 0.05 mol / L acetic acid at 4℃ to prepare a collagen scaffold solution of the appropriate concentration selected in step "1.2 Screening of Optimal Collagen Concentration". Weigh out 6 mg of two polysaccharides in different ratios (Astragalus:Angelica = 5:1, 2.5:1, 1.25:1, m / m) and dissolve them separately in 10 mL of distilled water to prepare solutions. Then mix the two solutions at a volume ratio of 1:1, stir well, and let stand. After centrifugation at 4000×g for 20 min at 4℃, add 100 μL of the mixed solution to a 24-well plate, cover, and place in a -20℃ freezer overnight. Freeze-dry in a vacuum freeze dryer for 72 h to prepare the polysaccharide scaffold material. Cut each scaffold material into 4 pieces, cross-link them with genipin solution, using the same method as in step "1.1 Extraction of Fish Skin Collagen", and then... 60 Sterilized by Co-γ-ray sterilization, sealed and stored at room temperature for later use, three different Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds were obtained, and then subjected to electron microscopy and porosity analysis.

[0068] 1.4 Scanning electron microscopy observation of different Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds

[0069] Different Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold samples were rehydrated in PBS and pre-fixed at 4°C for 24 h in 4% glutaraldehyde-sucrose-added dimethylarsine buffer, followed by fixation in osmium tetroxide for 2 h. The samples were then dehydrated by immersion in 30%, 50%, 70%, 85%, 95%, and 100% (v / v) ethanol solutions for 10 min each. After soaking in a mixture of isoamyl acetate and ethanol (isoamyl acetate:ethanol = 1:1) for 10 min, the scaffolds were embedded in isoamyl acetate, allowed to stand for 10 min, and then dried at the CO2 critical point. Gold was deposited using an ion sputtering apparatus. The surface morphology of the scaffolds was observed under a scanning electron microscope (SEM). Five types of collagen sponge scaffolds without drug loading and polysaccharide drugs (2% scaffold, 4% scaffold, 6% scaffold, 8% scaffold, and 10% scaffold) were used as a control group, and the pore size of the scaffolds was measured.

[0070] 1.5 Determination of porosity of different protein scaffolds

[0071] The porosity of the Astragalus polysaccharide-Angelica polysaccharide-fish collagen scaffold was determined using the ethanol permeation method. A 10 mL centrifuge tube was filled with ethanol and weighed (denoted as W1). The Astragalus polysaccharide-Angelica polysaccharide-fish collagen scaffold was weighed (denoted as Ws) and then immersed in the ethanol-filled centrifuge tube. The tube was then sonicated for 30 min, and the tube was filled with ethanol again and weighed (denoted as W2). After removing the ethanol-filled sample, the remaining ethanol and the centrifuge tube were weighed (denoted as W3). Meanwhile, unloaded collagen sponge scaffolds (2%, 4%, 6%, 8%, and 10% scaffolds) were used as a control group. The experiment was repeated three times, and the average value was used to calculate the porosity. The formula for calculating the porosity is:

[0072] .

[0073] 2. Results

[0074] 2.1 General appearance and electron microscopic structure of drug-free collagen sponge scaffolds at different concentrations

[0075] Drug-free collagen sponge scaffolds (2% scaffold, 4% scaffold, 6% scaffold, 8% scaffold, and 10% scaffold) are white in color and soft and porous. Figure 1 When the collagen concentration was 2 wt.% and 4 wt.%, the scaffold was relatively loose, and electron microscopy revealed that the internal pores were extremely irregular, with stacked and interwoven sheet-like structures. The size and shape of the pores varied significantly, and the continuity was poor. Figure 2 and Figure 3 At a concentration of 6 wt.%, the scaffold fibers are more densely packed, and the pore structure becomes smaller and more uniformly distributed. Figure 4At a concentration of 8 wt.%, the scaffold pores exhibited an irregular shape and a relatively dense porous state, with weaker connectivity between pores compared to the 6 wt.% scaffold. Figure 5 At a concentration of 10 wt.%, the porosity is relatively high, but a dense porous state is observed, and the pore size is relatively uneven. Figure 6 Therefore, a 6 wt.% stent was selected for subsequent experiments.

[0076] 2.2 Electron Microscopic Appearance and Results of the Astragalus Polysaccharide-Angelica Polysaccharide-Fish Skin Collagen Composite Scaffold

[0077] The three types of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds obtained in step "1.3 Preparation" were all light yellow in color, tough in texture, and under electron microscopy, the fibers of the scaffolds were densely arranged, and the pore structure decreased in size and was evenly distributed. Figure 7 The pore size is 50μm-150μm.

[0078] 2.3 Average porosity of the Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold

[0079] The average porosity of the three Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds obtained in step “1.3 Preparation of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds” was (78.520±3.210)%, and the average porosity of the unloaded collagen sponge scaffolds (2% scaffold, 4% scaffold, 6% scaffold, 8% scaffold and 10% scaffold) was (76.890±2.950)%, respectively. There was no statistically significant difference between the two (t=1.674, P=0.123).

[0080] Example 2

[0081] 1. Isolation, culture and identification of BMSCs

[0082] 1.1 Isolation and Culture

[0083] Five SD rats weighing 50-100g were anesthetized and euthanized by cervical dislocation. Under aseptic conditions, the bilateral femurs and tibias were removed and rinsed twice with sterile phosphate buffer. The ends were cut open, and the bone marrow was repeatedly flushed out with 10mL of DMEM / F12 culture medium containing 10% fetal bovine serum.

[0084] BMSCs were isolated and cultured using a whole bone marrow adherent separation and selection method: Bone marrow fluid was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and an appropriate amount of DMEM / F12 was added. The cells were then centrifuged again at 1200 rpm for 5 min, the supernatant was discarded, and the cells were counted and adjusted to a density of 5 × 10⁶ cells / mL. 5Cells were cultured at a density of 10 cells / mL, and a complete medium containing 15% FBS and DMEM / F12 was added to prepare a cell suspension. After thorough mixing, the cells were seeded into culture flasks and immediately placed in a 37°C, 5% CO2 incubator for routine culture. The medium was completely replaced for the first time after 48 hours, and then every 48-72 hours thereafter. When the cells reached 85% confluence in 7-8 days, they were routinely digested using 0.25% trypsin-EDTA, with digestion time controlled within 30 seconds to 1 minute. The passage ratio was 1:2 or 1:3. The fetal bovine serum albumin (FBS) concentration was adjusted to 10% during passage culture. Cell growth was observed daily under an inverted microscope and recorded by photography. Third-generation BMSCs with relatively uniform morphology, strong refractive index, and no granules in the cytoplasm, indicating no signs of aging, were retained for later use.

[0085] 1.2 Identification of BMSCs

[0086] Morphological observation: The morphology, proliferation rate, and population growth pattern of adherent cells were observed daily under an inverted microscope and photographed for record-keeping.

[0087] Cellular markers such as CD105, CD73, CD90, CD45, CD34 and CD14 were detected by flow cytometry.

[0088] 2. Results

[0089] 2.1 General Observation

[0090] Inverted microscopy revealed that BMSCs were spindle-shaped or fusiform, adhered well to the culture wall, and formed colonies. With prolonged culture time, cell density increased and confluence improved, indicating that BMSCs possess strong proliferative capacity. Third-generation BMSCs exhibited uniform morphology and high refractive index, making them suitable for subsequent experiments. Figure 8 ).

[0091] 2.2 Flow cytometry results

[0092] Flow cytometry results showed that the positive rates of characteristic BMSC markers CD105, CD73, and CD90 were all ≥90%, while the positive rates of hematopoietic stem cell and immune cell-related markers CD45, CD34, and CD14 were all ≤5%. This indicates that the isolated and cultured cell population consisted of high-purity BMSCs, containing little or no non-target cell types.

[0093] Flow cytometry results showed that the positive rates of characteristic BMSC markers CD105, CD73, and CD90 were all ≥90%, while the positive rates of hematopoietic stem cell and immune cell-related markers CD45, CD34, and CD14 were all ≤5%, indicating that the isolated and cultured cell population was high-purity BMSCs, containing little or no non-target cell types. Figure 9 and Figure 10 ).

[0094] Example 3: Cell adhesion on protein scaffold materials

[0095] The support material has undergone 60 After sterilization by Coγ-ray sterilization, under aseptic conditions, one piece each of the drug-free collagen sponge scaffold (6% scaffold) and the three types of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds obtained in step "1.3 Preparation of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold" in Example 1 were placed in a culture dish, soaked in 4 mL of 0.01 mol / L PBS, and incubated overnight at 37°C in a CO2 incubator. Third-generation BMSCs were collected, and after reaching 85% confluence, the original solution was discarded. The cells were washed twice with 0.01 mol / L PBS, digested with 0.25% trypsin for 30 seconds to terminate digestion, and the cells were gently pipetted to mix thoroughly. The cell density was adjusted to 2 × 10⁻⁶ cells / year. 9 Cells / L, 2 mL of culture medium was inoculated onto each material, and cultured routinely for 8 hours. Complete culture medium was added to a final volume of 4 mL, and cultured further, changing the medium every other day. After 3 days of co-culturing with the material, the material was removed, fixed with 2.5% glutaraldehyde, and observed under a scanning electron microscope. Electron microscopy results showed that after 3 days of co-culturing with cells on drug-free collagen sponge scaffolds (6% scaffold) and the three types of Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds, stem cells adhered relatively uniformly to the surface of both materials. The cell nuclei were of uniform size. Cells adhered and grew on both materials, indicating good biocompatibility between the materials and cells.

[0096] Example 4: CCK-8 assay for BMSC proliferation on scaffold material

[0097] Weigh out the tilapia-derived sponge-like lyophilized scaffold derived from tilapia skin collagen in step "1.1 Extraction of Fish Skin Collagen" of Example 1, and dissolve it thoroughly in 0.05 mol / L acetic acid at 4°C to prepare a 6 wt.% collagen scaffold solution. Separately weigh out 6 mg of each of two polysaccharides in different ratios (Astragalus: Angelica = 5:1, 2.5:1, 1.25:1, m / m), 6 mg of Astragalus polysaccharide, and 6 mg of Angelica polysaccharide, and dissolve them separately in 10 mL of distilled water to prepare solutions. Then, mix the two solutions at a volume ratio of 1:1, stir well, and let stand. After centrifugation at 4°C for 20 min at 4000×g, add 100 μL of the mixed solution to a 24-well plate, cover, and place in a -20°C freezer overnight. Then, freeze-dry in a vacuum freeze dryer for 72 h to prepare the polysaccharide scaffold material. Each scaffold material was cut into four pieces and cross-linked with genipin solution, following the same method as in step "1.1 Extraction of fish skin collagen". The scaffolds were then sterilized using 60Co γ-ray sterilization, sealed, and stored at room temperature for later use, resulting in five different Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffolds, designated as Group 1 (Astragalus polysaccharide:Angelica polysaccharide = 5:1), Group 2 (Astragalus polysaccharide:Angelica polysaccharide = 2.5:1), Group 3 (Astragalus polysaccharide:Angelica polysaccharide = 1.25:1), Astragalus polysaccharide-scaffold group, and Angelica polysaccharide-scaffold group.

[0098] Under aseptic conditions, different sterilized scaffold materials were cut into 3mm×3mm×1mm rectangular blocks and placed in 96-well plates, divided into 14 groups. Group 1 consisted of scaffolds (6% scaffold, prepared as in Example 1) + complete culture medium; Group 2 consisted of complete culture medium; Group 3 consisted of Angelica sinensis polysaccharide-scaffolds; Group 4 consisted of Astragalus membranaceus polysaccharide-scaffolds; Group 5 was compatibility group 1 (Astragalus membranaceus polysaccharide: Angelica sinensis polysaccharide = 5:1); Group 6 was compatibility group 2 (Astragalus membranaceus polysaccharide: Angelica sinensis polysaccharide = 2.5:1); Group 7 was compatibility group 3 (Astragalus membranaceus polysaccharide: Angelica sinensis polysaccharide = 1.25:1); and Groups 8-14 were the corresponding groups of Groups 1-7 with added stem cells. Each group had 15 wells, and each well in the cell-added groups was seeded with approximately 3 × 10⁶ cells (3rd generation BMSCs). 3Cells were cultured at 37°C in a CO2 incubator, with the medium changed every other day. After 24 hours of co-culturing, five wells from each group were sampled. The material from the test wells was transferred to new wells, and 100 μL of complete culture medium was added. For the cell-free group, the original solution was aspirated, and 100 μL of complete culture medium was added. 10 μL of CCK-8 was added to each test well, and the cells were incubated at 37°C in a 5% CO2 incubator for 3 hours. The liquid from each well was then transferred to another 96-well plate, and the absorbance at 450 nm was measured using a microplate reader. Cells and material were co-cultured for 48 hours and 72 hours, and the same method was used to measure the absorbance at 450 nm from five wells in each group, and cell viability (proliferative activity) was calculated. Each experiment was repeated three times. Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 21.0 software. All tests were performed using one-way ANOVA, and P < 0.05 was considered statistically significant.

[0099] The formula for calculating cell viability is as follows:

[0100] ;

[0101] Wherein, A (scaffold): absorbance of wells containing scaffold, culture medium, cells and CCK-8; A (blank): absorbance of wells containing culture medium, cells and CCK-8; A (no scaffold): absorbance of wells containing cells and CCK-8.

[0102] The results of cell proliferation assay using the CCK-8 assay are shown in Table 1 and... Figure 11 As described above, cells were seeded onto the scaffold material. Over time, cell proliferation in combination group 3 and the scaffold + complete culture medium group was significantly higher than that in the control group. Specifically, cell proliferation in combination group 3 was significant (P<0.05), while cell proliferation in combination groups 1, 2, the Astragalus polysaccharide scaffold group, and the Angelica polysaccharide scaffold group decreased. At the same time point, cell proliferation in combination group 3 seeded onto the material was significantly higher than that in other groups (P<0.05). In combination group 3, cell proliferation was most significant when the mass ratio of Astragalus polysaccharide to Angelica polysaccharide was 1.25:1, superior to other combination groups, with a statistically significant difference (P<0.05). Cell proliferation in the Astragalus polysaccharide scaffold group, the Angelica polysaccharide scaffold group, and at ratios of 5:1 and 2.5:1 was weaker than that in the scaffold group and the stem cell group alone, indicating that higher concentrations of Astragalus polysaccharide and Angelica polysaccharide may inhibit cell activity.

[0103] Table 1. Cell proliferation activity on different groups of materials ( ±s)

[0104] 24h 48h 72h Group 1 0.080±0.010 0.075±0.008 0.007±0.006 Group 2 0.075±0.009 0.070±0.007 0.065±0.005 Group 3 0.085±0.011 0.080±0.009 0.075±0.007 Group 4 0.060±0.008 0.055±0.006 0.050±0.004 Group 5 0.007±0.009 0.065±0.007 0.060±0.005 Group 6 0.080±0.010 0.075±0.008 0.060±0.006 Group 7 0.065±0.009 0.060±0.007 0.070±0.005 Group 8 0.321±0.008 0.418±0.018 0.682±0.021 Group 9 0.310±0.013 0.403±0.015 0.554±0.028 Group 10 0.320±0.015 0.330±0.020 0.480±0.030 Group 11 0.305±0.017 0.310±0.023 0.410±0.035 Group 12 0.301±0.016 0.390±0.022 0.490±0.032 Group 13 0.315±0.012 0.415±0.018 0.510±0.025 Group 14 0.450±0.006 0.700±0.015 1.150±0.018

[0105] Example 5 Animal Model Experiment

[0106] To comprehensively evaluate the effect of the Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold on the in vivo healing efficiency of diabetic wounds, this embodiment used 6-8 week old SPF-grade BALB / c male mice. Diabetes was induced using STZ combined with a high-sugar, high-fat diet. Based on this, a full-thickness diabetic wound model of MRSA infection was constructed. The specific steps are as follows:

[0107] 1. Constructing a diabetic mouse model: Prepare a citric acid solution with a concentration of 21 g / L and a sodium citrate solution with a concentration of 29.4 g / L. Mix the citric acid solution and sodium citrate solution at a volume ratio of 1:1.32, and adjust the pH to 4.2-4.5 to obtain a sodium citrate buffer solution for later use. Dissolve 100 mg of streptozotocin powder in 10 mL of freshly prepared sodium citrate buffer solution, filter it through a 0.22 μm filter to prepare a sterile STZ solution, and store it at 4 °C protected from light.

[0108] Seven-week-old male BALB / c mice were purchased and acclimatized for one week before the experiment began. After a 12-hour fast, mice were intraperitoneally injected with a prepared streptozotocin solution at a dose of 50 mg / kg. Injections continued for five days, while a high-sugar, high-fat diet was provided concurrently. After the injections, 5 mm of the mouse tail was removed and 2 μL of blood was collected. Blood glucose concentration was measured using a glucometer. Mice with a blood glucose concentration of 16.7 mmol / L or higher were considered to have met the target blood glucose level and were placed in the diabetes group. Mice that did not meet the target level continued to receive streptozotocin injections until their blood glucose levels met the standard. Blood glucose concentration was measured every three days. Mice whose blood glucose concentration remained above 16.7 mmol / L for two consecutive weeks were confirmed to have successfully established a diabetes model and maintained a stable hyperglycemic state.

[0109] 2. Construction of a diabetic full-thickness infected wound model: A 30 mg / mL sodium pentobarbital solution was prepared using physiological saline. After thorough mixing, the mice were intraperitoneally injected with 1 mL / kg of sodium pentobarbital solution for general anesthesia. Anesthetized mice were fixed on an operating table, and a 6 mm diameter full-thickness skin wound was constructed on the paw using sterile ophthalmic scissors. 1×10 6 An MRSA solution of CFU / mL was inoculated into the wound to construct an MRSA-infected diabetic wound.

[0110] 3. Grouping and Treatment: Mice with successfully established diabetic wounds were randomly divided into six groups, including a control group (saline group), a simple scaffold group (6% scaffold, prepared as in Example 1), and five treatment groups (Angelica polysaccharide-scaffold group, Astragalus polysaccharide-scaffold group, combination group 1 (Astragalus polysaccharide:Angelica polysaccharide = 5:1), combination group 2 (Astragalus polysaccharide:Angelica polysaccharide = 2.5:1), and combination group 3 (Astragalus polysaccharide:Angelica polysaccharide = 1.25:1, the scaffold preparation method was the same as in Example 4), with six mice in each group. Treatment: First, the scaffolds of different groups were precisely applied to the wound surface and changed every 24 hours. The wound area was quantitatively analyzed using ImageJ software, and the wound healing rate was calculated on days 7 and 14 after treatment. The control group was given an equal volume of PBS.

[0111] The wound healing rate is calculated using the following formula:

[0112] .

[0113] 4. Results

[0114] The wound healing rates on days 7 and 14 after medication are shown in Table 2.

[0115] Table 2. Average wound healing rate under different treatments (n=6)

[0116] deal with 7-day wound healing rate (%) 14-day wound healing rate (%) control group 39.8 53.8 Angelica polysaccharide-scaffold group 49.3 60.4 Astragalus polysaccharide-scaffold group 51.4 59.9 Formula 1 (Astragalus polysaccharide: Angelica polysaccharide = 5:1) 48.7 70.7 Formula 2 (Astragalus polysaccharide: Angelica polysaccharide = 2.5:1) 49.8 73.3 Formula group 3 (Astragalus polysaccharide: Angelica polysaccharide = 1.25:1) 59.2 80.6 Simple stent group 53.1 75.4

[0117] As shown in Table 2, the healing rate of combination group 3 (Astragalus polysaccharide: Angelica polysaccharide = 1.25:1) was the highest, reaching 80.6%, significantly higher than the other groups. Therefore, the Astragalus polysaccharide-Angelica polysaccharide-fish skin collagen composite scaffold provided by this invention exhibits excellent effects in promoting wound healing.

[0118] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a composite polysaccharide-fish skin-derived collagen scaffold, characterized in that, Includes the following steps: Tilapia skin collagen solution and polysaccharide solution containing astragalus polysaccharide and angelica polysaccharide were mixed, and then subjected to static standing, centrifugation, drying and cross-linking in sequence to obtain the composite polysaccharide-fish skin collagen scaffold.

2. The preparation method according to claim 1, characterized in that, The concentration of fish skin collagen in the fish skin collagen solution is 2wt.%-10wt.%; the mass ratio of Astragalus polysaccharide to Angelica polysaccharide in the polysaccharide solution is 1.25:

1.

3. The preparation method according to claim 1, characterized in that, The crosslinking agent used was genipin, the time was 30 minutes, and the temperature was 37°C.

4. The preparation method according to claim 1, characterized in that, The centrifugation conditions were: 4℃, 4000×g for 20 min.

5. The preparation method according to claim 1, characterized in that, The method for preparing the fish skin collagen solution includes the following steps: removing cells from fresh fish skin using a decellularization method, followed by washing, degreasing, soaking, crushing, filtering, adding acetic acid solution, first extraction, first centrifugation, second extraction, second centrifugation, enzymatic hydrolysis, adding NaCl, third centrifugation, dialysis, concentration, filtration, drying, and dissolving to obtain the fish skin collagen solution.

6. The preparation method according to claim 5, characterized in that, The degreasing process uses 10% n-butanol as the solvent and takes 24 hours. The solvent used for soaking was a 0.1 mol / L NaOH aqueous solution, and the soaking time was 24 hours. The first extraction was performed at a temperature of 4°C for 48 hours. The conditions for the first centrifugation, the second centrifugation, and the third centrifugation are all: centrifugation at 10000 rpm for 30 min; The second extraction was performed at a temperature of 4°C for 24 hours. The enzyme used in the enzymatic hydrolysis is pepsin; the final concentration of the pepsin is 1‰; the pH of the enzymatic hydrolysis is 2.0, and the time is 2 days.

7. A composite polysaccharide-fish skin collagen scaffold prepared using the preparation method according to any one of claims 1-6.

8. The application of the composite polysaccharide-fish skin collagen scaffold according to claim 7 in any of the following applications; (1) Promotes the proliferation of bone marrow mesenchymal stem cells; (2) Promotes wound healing; (3) Repairing diabetic ulcers; (4) Prepare products that promote the proliferation of bone marrow mesenchymal stem cells; (5) Prepare products that promote wound healing; (6) Prepare products for repairing diabetic ulcer wounds.

9. A composite polysaccharide scaffold loaded with bone marrow mesenchymal stem cells, characterized in that, The method for preparing the composite polysaccharide scaffold includes the steps of seeding the bone marrow mesenchymal stem cells into the composite polysaccharide-fish skin collagen scaffold of claim 7 and culturing them to obtain the composite polysaccharide scaffold.

10. The application of the composite polysaccharide scaffold of claim 9 in any of the following applications; (1) Promotes wound healing; (2) Repairing diabetic ulcer wounds; (3) Prepare products that promote wound healing; (4) Prepare products for repairing diabetic ulcer wounds.

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