Application of Sr-Se@SiO2-loaded acellular matrix and adipose-derived mesenchymal stem cells in sequential treatment of volumetric muscle loss

Abstract

The application belongs to the field of medical material preparation, and particularly relates to application of a decellularized matrix loaded with Sr-Se@SiO2 and adipose-derived mesenchymal stem cells in sequential treatment of volumetric muscle loss. On the basis of Sr-Se@SiO2 nanocomposites, a composite filling scaffold is developed in combination with a tissue engineering scaffold, and the role of the composite filling scaffold in VML treatment is studied. The filling material is composed of three parts, namely, the nanomaterial Sr-Se@SiO2 with multiple functions such as anti-inflammatory response and promotion of vascularization, ADSCs with myogenic differentiation potential, and a dECM composite scaffold for loading contents. The composite filling scaffold provided in the application will improve the problems in traditional VML treatment, and realize sequential treatment of personalized guided VML.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical material preparation, specifically relating to the application of decellularized matrix-loaded Sr-Se@SiO2 and adipose-derived mesenchymal stem cells in the sequential treatment of volumetric muscle defects. Background Technology

[0002] Skeletal muscle typically possesses a high regenerative capacity after minor injuries. However, volumetric muscle loss (VML) is a type of skeletal muscle damage caused by primary or secondary injury that exceeds the body's natural repair capabilities, ultimately leading to long-term disability of the limb. VML resulting from traffic accidents, natural disasters, and gunfights can affect hundreds of millions of people, causing chronic pain and organ dysfunction. The muscle repair process following VML injury is characterized by excessive inflammation and excessive collagen deposition. In such cases, the rate of fibrous tissue formation exceeds the rate of myoblast differentiation and maturation, resulting in thickened, non-functional scars that severely hinder myotube fusion, ultimately leading to permanent muscle loss and lifelong disability. Disability caused by VML prevents patients from returning to work, placing a heavy burden on patients and society.

[0003] VML (Vascular Muscle Mutilation) results in severe loss of satellite cells and their niche elements, including the basal layer, mesenchymal cells, and vascular network, leading to impaired muscle tissue regeneration. Simultaneously, after VML occurs, skeletal muscle activates its repair system; this pathophysiological process occurs in three phases: inflammation, new tissue formation, and tissue remodeling. Current treatments for VML include physical therapy, scar tissue debridement, and muscle transposition. However, because these methods cannot reverse the pathological changes following VML injury, their success rates are low. Successful treatment requires substantial muscle formation to replace lost tissue, adequate vascularization to supply blood flow, nerve innervation to generate action potentials, and effective immune regulation to reduce fibrosis and support tissue regeneration.

[0004] Therefore, exploring the repair mechanisms of VML and seeking new treatments for VML are of great significance to human health and social development. Summary of the Invention

[0005] To address the aforementioned issues, this invention develops a composite scaffold based on Sr-Se@SiO2 nanocomposite materials and combines it with a tissue engineering scaffold, and investigates its role in the treatment of vulvovaginal lymphoma (VML). This scaffold consists of three parts: Sr-Se@SiO2 nanomaterials with multiple functions such as anti-inflammatory response and pro-angiogenesis; ADSCs with myogenic differentiation potential; and a dECM composite scaffold for loading contents. The composite scaffold provided by this invention will improve the difficulties in traditional VML treatment and enable personalized, guided sequential treatment of VML.

[0006] In a first aspect, the present invention provides a method for preparing a Sr-Se@SiO2-dECM tissue engineering scaffold, the method comprising the following steps: Step 1: Prepare Se@SiO2 nanoparticles using the reverse microemulsion method; Step 2: Dissolve the Se@SiO2 nanoparticles obtained in Step 1 with SrCl2·6H2O and PVP in deionized water and stir until homogeneous. Add ammonia dropwise, stir, and place in a polytetrafluoroethylene reactor. React at 130-150℃ for 4-6 h. Wash with deionized water to obtain Sr-Se@SiO2 nanoparticles. Step 3: Place the Gelma sample in a pretreated tissue culture polystyrene plate, and simultaneously place ADSCs into the tissue culture polystyrene plate. Add growth medium and culture until the ADSCs fuse with the Gelma sample. Then add ascorbic acid for 6-8 days to obtain ADSCs loaded on gelatin sheets. Step 4: Place the ADSCs obtained in Step 3 in the extraction buffer and incubate for 3-8 minutes, then fix with glutaraldehyde to obtain dECM derived from ADSCs. Step 5: Uniformly spray the Sr-Se@SiO2 nanoparticles obtained in Step 3 onto the ADSCs-derived dECM obtained in Step 4 to obtain the Sr-Se@SiO2-dECM tissue engineering scaffold with nanostructure.

[0007] In some embodiments, step 1 includes the following steps: adding hexanol, Triton X-100, and hexane to deionized water and stirring until homogeneous; adding copper selenide solution and stirring for 3-5 minutes; adding tetraethyl orthosilicate and stirring for 3-5 minutes; adding ammonia water to make the solution dark blue; stirring for 20-28 hours; and then washing with anhydrous ethanol by centrifugation to obtain the final product.

[0008] In some embodiments, the volume ratio of n-hexanol, Triton X-100, and n-hexane is 1:1:10, the volume ratio of copper selenide solution to n-hexanol is 3:2, and the volume ratio of copper selenide solution to tetraethyl orthosilicate is 10-12:1.

[0009] In some embodiments, the preparation method of the copper selenide solution includes placing Se and NaOH in deionized water, heating to 45-55°C, stirring until the selenium is completely dissolved, then sequentially adding N2H4·H2O, CuCl2·2H2O, and 2 mL of oleylamine (OA) to the mixed solution, maintaining at 80-120°C for 2-5 h, washing with ethanol 2-4 times to obtain Cu2-xSe nanoparticles, and then dispersing Cu2-xSe in n-hexane.

[0010] In some embodiments, the mass ratio of Se@SiO2 nanoparticles to SrCl2·6H2O is 1:3-5, the mass ratio of Se@SiO2 nanoparticles to PVP is 95-105:1, the mass-to-volume ratio of Se@SiO2 nanoparticles to ammonia (25%-30%) is 20-80 mg:1 mL, the reaction temperature in the polytetrafluoroethylene reactor is 120-160℃, and the reaction time is 2-8 h.

[0011] In some embodiments, the method for preparing the Gelma sample includes pouring a 5-15 wt% Gelma solution into a mold, exposing it to ultraviolet light for crosslinking to obtain a sample, and lyophilizing the sample overnight to obtain the Gelma sample.

[0012] In a second aspect, the present invention also provides a Sr-Se@SiO2-dECM tissue engineering scaffold prepared by the preparation method described in the first aspect.

[0013] Thirdly, the present invention also provides the application of the Sr-Se@SiO2-dECM tissue engineering scaffold described in the second aspect in the repair or treatment of volumetric muscle defects.

[0014] In some embodiments, the Sr-Se@SiO2-dECM tissue engineering scaffold promotes angiogenesis in the repair of volumetric muscle defects.

[0015] Compared to existing technologies, this invention is the first to combine Sr-Se@SiO2, a nanomaterial with multiple functions such as anti-inflammatory and pro-angiogenic properties, ADSCs with myogenic differentiation potential, and a dECM composite scaffold for loading contents, to obtain a composite filling scaffold. This allows for the slow release of Sr and Se while overcoming the difficulties of angiogenesis in the repair of volumetric muscle defects. This invention will improve the difficulties of traditional VML treatment, enabling personalized sequential treatment of VML, and providing a new tissue engineering approach to solving the clinical challenges of VML treatment. Attached Figure Description

[0016] Figure 1 The images show the morphology of Sr-Se@SiO2 nanoparticles; A and B are TEM images of Sr-Se@SiO2 nanoparticles, and C is the elemental distribution diagram of Sr-Se@SiO2 nanoparticles.

[0017] Figure 2The percentages of Se and Sr released from Sr-Se@SiO2 nanoparticles at different times are: A. the percentage of Se released from Sr-Se@SiO2 nanoparticles at different times, and B. the percentage of Sr released from Sr-Se@SiO2 nanoparticles at different times.

[0018] Figure 3 The image shows the preparation and characterization of ADSCs-Sr-Se@SiO2-dECM; A. the preparation process of micropatterned GelMA gelatin scaffold, B. surface electron microscopy image of GelMA gelatin scaffold loaded with dECM, C. CCK8 determination of the effect of different concentrations of Sr-Se@SiO2 on ADSCs cell viability, D. swelling ratio of different scaffold materials, E. mechanical properties of GelMA gelatin scaffold: (1) oscillation frequency (2) oscillation amplitude (3) stress-strain curve.

[0019] Figure 4 To assess the toxicity of co-cultured dECM / Sr-Se@SiO2 with ADSCs, the study included: A. Staining images of live and dead cells after 3 days of co-culture; B. Cell proliferation assays using CCK8 after 1, 3, 5, and 7 days of co-culture. C: Control; d: dECM; S: Sr-Se@SiO2; d+S: dECM+Sr-Se@SiO2.

[0020] Figure 5 The effect of dECM / Sr-Se@SiO2 on the myogenic differentiation level of ADSCs was investigated. A and B involved immunofluorescence detection and quantitative analysis of the expression levels of MYOD, Desmin, and MyHC; C and D involved Western blotting detection and quantitative analysis of the expression levels of MYOD, MYOG, and Desmin. C: Control; d: dECM; S: Sr-Se@SiO2; d+S: dECM+Sr-Se@SiO2.

[0021] Figure 6 This study investigated the effects of dECM / Sr-Se@SiO2 on intracellular oxidative stress levels and mitochondrial activity in ADSCs. A and B involved mitochondrial fluorescent probe staining to detect and quantify mitochondrial numbers. C and D involved detecting and quantifying ROS levels in ADSCs one day after H2O2 induction. C: Control; d: dECM; S: Sr-Se@SiO2; d+S: dECM+Sr-Se@SiO2; H: H2O2; H+d: H2O2+dECM; H+S: H2O2+Sr-Se@SiO2; H+d+S: H2O2+dECM+Sr-Se@SiO2.

[0022] Figure 7To assess the angiogenesis-promoting effect of dECM / Sr-Se@SiO2; A, B. Transwell assay to detect and quantify the migration ability of HUVECs after 1 day of co-culture; C, D. Immunofluorescence assay to detect and quantify the expression levels of CD31 and VEGF. Wherein, C: Control, d: dECM, S: Sr-Se@SiO2, d+S: dECM+Sr-Se@SiO2.

[0023] Figure 8 For animal experiments; including A. Establishment of animal models: VML; filling with materials of different components, B. Gross specimens of the tibialis anterior muscle of 6 groups of rats after 2 weeks, 4 weeks and 8 weeks.

[0024] Figure 9 For animal experiments; H&E staining and Masson staining of the tibialis anterior muscle at 2 and 4 weeks.

[0025] Figure 10 The expression levels of MyHC and CD31 in tissues were detected by immunofluorescence and Western blotting. Specifically, A. Immunofluorescence was used to detect and quantify the expression levels of MyHC, α-SMA, and CD31 in the tibialis anterior muscle at 2 weeks, and B. Western blotting was used to detect and quantify the expression levels of MYOD and CD31 in the tibialis anterior muscle at 2 weeks. Detailed Implementation

[0026] The specific embodiments of the present invention are described below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0027] Example 1: Preparation and Characterization of Sr-Se@SiO2-dECM 1. Preparation of Se@SiO2 First, 0.3 g Se and 15 g NaOH were placed in 40 mL of deionized water and heated to 50 °C, stirring until the selenium was completely dissolved. Then, 2 mL N2H4·H2O, 0.568 g CuCl2·2H2O, and 2 mL oleylamine (OA) were added to the mixture sequentially, and the mixture was maintained at 100 °C for 2 h. After washing three times with ethanol, Cu2-xSe nanoparticles were obtained, and then the Cu2-xSe was dispersed in 20 mL of n-hexane.

[0028] Subsequently, Se@SiO2 nanospheres were prepared using a reverse microemulsion method. 0.9 mL of deionized water was added to a 100 mL single-necked flask, followed by 3 mL of n-hexanol and then 3 mL of Triton X-100. 30 mL of n-hexane was added to the flask, and the mixture was stirred with a magnetic stirrer. Then, 2 mL of the synthesized copper selenide solution was added and stirred. After 5 min, 0.18 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise to the flask, ensuring thorough mixing. After another 5 min, 0.18 mL of ammonia was slowly added dropwise to the flask, immediately turning the solution a dark blue color. The mixture was stirred for 24 h. After the reaction was complete, the nanospheres were washed three times by centrifugation with anhydrous ethanol (11000 rpm, 15 min) to obtain the final Se@SiO2 nanospheres, which were then dispersed in anhydrous ethanol.

[0029] 2. Preparation of Sr-Se@SiO2 10 mg SrCl2·6H2O, 40 mg Se@SiO2, and 0.1 g PVP were dissolved in 30 mL of deionized water and stirred for 1 h. Then, 1 mL of ammonia (30%) was added dropwise and stirred for 10 min. The mixture was then placed in a 50 mL polytetrafluoroethylene reactor and reacted at 140 °C for 5 h in a heating oven. After washing three times with deionized water, Sr-Se@SiO2 nanoparticles were obtained.

[0030] Morphological analysis of Sr-Se@SiO2 was performed using transmission electron microscopy, including its size, morphology, particle size, distribution, and particle size range. The elemental composition, chemical valence state, and relative abundance on the surface of the solid material were investigated using photoelectron spectroscopy.

[0031] Figure 1 In the images A and B, we see transmission electron microscopy (TEM) images of Sr-Se@SiO2. The particle size of Sr-Se@SiO2 is approximately 250 nm. After the addition of Sr, the silica shell transforms into a rough, urchin-like morphology. Figure 1 C is the elemental distribution diagram of Sr-Se@SiO2. In the diagram, Si, O and Sr elements are uniformly distributed throughout the nanoparticles, with small Se particles scattered within them, indicating that Sr-Se@SiO2 nanoparticles were successfully synthesized.

[0032] 3. Preparation of dECM derived from ADSCs Mold with aligned microgroove pattern: The mold with a width of 15 mm, a length of 15 mm, and a height of 20 mm was prepared by 3D printing technology. The linear pattern has a width of 100 μm, a gap of 200 μm, and a depth of 100 μm.

[0033] A 10 wt% Gelma solution was poured into a mold and exposed to UV light (405 nm, 30 mW / cm²) for 5 minutes to crosslink. The samples were pre-frozen at -20°C for 30 minutes, then lyophilized overnight. The lyophilized samples were then swollen with sterile water, stored in PBS containing 1% penicillin and streptomycin, and irradiated with UV light for 30 minutes before use. Tissue culture polystyrene (TCPS) plates were pretreated with 0.25% agarose at 37°C for 1 hour, and then patterned Gelma samples were placed on them. ADSCs were placed on the pretreated plates and cultured in growth medium until they reached 90% confluence, followed by treatment with 100 μM L-ascorbic acid for 7 days. Cell-free ECMs were obtained by culturing cells in extraction buffer (PBS solution of 0.5% Triton X-100 and 20 mM NH4OH, pH 7.4) at 37°C for 5 minutes. The ECMs were washed three times with PBS to remove residual extraction buffer solution and then stored under sterile conditions at 4°C.

[0034] The dECM scaffold samples were fixed with 2.5% glutaraldehyde and then dehydrated under conditions of increasing ethanol concentration (50%, 75%, 80%, 95%, 100%). Scanning electron microscopy was used for analysis.

[0035] 4. Preparation of Sr-Se@SiO2-dECM Sr-Se@SiO2 nanocomposite was uniformly sprayed onto the surface of ADSCs-derived dECM scaffolds to form tissue engineering scaffolds with a nanostructure and specific striped grooves. The microstructure of Sr-Se@SiO2 on the ECM surface was observed using scanning electron microscopy, and the release rates of Sr and Se, as well as the degradation rate of the scaffold material, were detected. The results are as follows: Figure 2 As shown.

[0036] 5. Preparation of ADSCs-containing Sr-Se@SiO2-dECM ADSCs were uniformly filled and laid on the surface of the prefabricated Sr-Se@SiO2-dECM scaffold, and the ADSCs and Sr-Se@SiO2-dECM scaffolds were stacked in multiple layers to complete the formulation of the tissue engineering block graft.

[0037] Scanning electron microscopy was used to detect dECM-GelMA, Sr-Se@SiO2-dECM-GelMA, and ADSCs-Sr-Se@SiO2-dECM-GelMA, respectively.

[0038] The results are as follows Figure 3As shown in the figure, scanning electron microscopy revealed that the dECM-loaded GelMA gelatin scaffold exhibited a porous structure, which is beneficial for the adhesion, proliferation, and differentiation of ADCSs. Experiments determined that the optimal concentration of Sr-Se@SiO2 for ADSC cell viability and proliferation was 20 μg / ml, and this concentration was used in subsequent cell experiments. Furthermore, the dECM-loaded GelMA gelatin scaffold exhibited good swelling ratio and mechanical properties.

[0039] Example 2: Evaluation of the effect of Sr-Se@SiO2-dECM on cell viability Grouping: Negative control group, dECM group, Sr-Se@SiO2 group, dECM+Sr-Se@SiO2 group Experimental methods: Cell viability was detected by CCK8 and live / dead cell staining on days 1, 3, 5, and 7.

[0040] The results are as follows Figure 4 As shown, cell live / dead staining and CCK-8 assays indicated that dECM and Sr-Se@SiO2 had no significant toxicity to ADSCs and could significantly promote the proliferation of ADSCs. Among them, dECM was more effective than Sr-Se@SiO2 in promoting the proliferation of ADSCs.

[0041] Example 3: Assessment of the effect on the myogenic differentiation level of ADSCs

[0042] Grouping: Negative control group, dECM group, Sr-Se@SiO2 group, dECM+Sr-Se@SiO2 group Experimental methods: Immunofluorescence and Western blotting were used to detect the expression levels of MYOD, MYOG, Desmin, and MyHC.

[0043] The results are as follows Figure 5 As shown, immunofluorescence and Western blotting were used to detect the expression levels of myogenic markers (MYOD, MYOG, Desmin, MyHC), indicating that dECM and Sr-Se@SiO2 significantly promoted myogenic differentiation of ADSCs.

[0044] Example 4: Assessment of the effect on intracellular oxidative stress levels and mitochondrial activity in ADSCs

[0045] Grouping: Negative control group, dECM group, Sr-Se@SiO2 group, dECM+Sr-Se@SiO2 group

[0046] Experimental methods: Fluorescent probe staining was used to detect the number and activity of mitochondria in cells, the level of ROS in cells and mitochondria, and changes in mitochondrial membrane potential; Western blotting was used to detect antioxidant levels (SOD-1); mitochondrial respiratory chain activity was detected; the state of mitochondria in cells was observed by transmission electron microscopy; ADSCs treated with Sr-Se@SiO2 were collected and sequenced to further clarify the mechanism by which nanocomplexes regulate mitochondrial function.

[0047] The results are as follows Figure 6 As shown, the red fluorescent probe of mitochondria indicates that both dECM and Sr-Se@SiO2 can promote the number and activity of mitochondria in ADSCs, with Sr-Se@SiO2 having a more significant effect. After inducing ROS production in ADSCs with hydrogen peroxide for one day, ADSCs were co-cultured with dECM and Sr-Se@SiO2 for three days, and the intracellular ROS levels were then measured. It was found that both dECM and Sr-Se@SiO2 could reduce ROS production in ADSCs, with Sr-Se@SiO2 having a more significant effect.

[0048] Example 5: Evaluation of the pro-angiogenic effect of the material

[0049] Grouping: Negative control group, dECM group, Sr-Se@SiO2 group, dECM+Sr-Se@SiO2 group

[0050] Experimental methods: scratch assay, migration assay, and tube formation analysis; immunofluorescence and Western blotting were used to detect the expression levels of CD31 and VEGF in human umbilical vein endothelial cells (HUVECs); sequencing was used to further clarify the mechanism by which the nanocomplex promotes angiogenesis.

[0051] The results are as follows Figure 7 As shown, the Transwell assay demonstrated that dECM and Sr-Se@SiO2 significantly promoted the migration of human umbilical vein endothelial cells (HUVECs). Immunofluorescence detection of the expression levels of angiogenesis markers (CD31, VEGF) revealed that Sr-Se@SiO2 significantly promoted the angiogenesis of HUVECs, while the angiogenesis ability of dECM was not significant.

[0052] Example 6 Animal Experiment

[0053] Grouping: 45 animals were divided into four groups: sham surgery group, VML group, VML+dECM group, VML+dECM+ADSCs group, VML+dECM+Sr-Se@SiO2 group, and VML+dECM+ADSCs+Sr-Se@SiO2 group, with 5 animals in each group. Samples were collected after 2, 4, and 8 weeks of rearing. Biological function tests included muscle function testing, gait analysis, and color Doppler ultrasound observation of local blood perfusion in the lower limbs.

[0054] like Figure 8 As shown in Figure A, the VML animal model was established. A muscle defect area of ​​approximately 10mm × 7mm × 3mm (length × width × depth) was surgically removed from the anterior tibial muscle (TA) of the rat. A scaffold material containing ADSCs and Sr-Se@SiO2 was implanted into the treatment group. Figure B shows the gross appearance of rat TA specimens obtained at 2, 4, and 8 weeks after modeling. It can be seen that the muscle tissue at the defect site was basically restored after 8 weeks of modeling.

[0055] Example 7: Staining of tissue sections

[0056] HE staining was used to observe the cross-sectional area of ​​muscle fibers and angiogenesis; Masson staining was used to observe the distribution of fibrous connective tissue and newly formed muscle fibers.

[0057] The results are as follows Figure 9 As shown, HE staining indicated that after 2 weeks and 4 weeks of modeling, the cross-sectional area and number of regenerated myofibrils were significantly improved when dECM scaffolds, ADSCs, and Sr-Se@SiO2 were present simultaneously. Masson staining showed that the area of ​​fibrous connective tissue was significantly reduced when dECM scaffolds, ADSCs, and Sr-Se@SiO2 were present simultaneously, and more neocapillary formation was observed when Sr-Se@SiO2 was loaded (black arrows).

[0058] Example 8: Analysis of tissue myogenesis and angiogenesis levels

[0059] Immunofluorescence and Western blotting were used to detect the expression levels of MyHC and CD31 in the tissues.

[0060] Analysis results as follows Figure 10 As shown, immunofluorescence and Western blotting were used to detect the expression levels of myogenic markers (MYOD, MyHC) and angiogenic markers (CD31, α-SMA). It was found that the simultaneous presence of dECM scaffolds, ADSCs, and Sr-Se@SiO2 promoted muscle and blood vessel regeneration after muscle tissue loss.

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a Sr-Se@SiO2-dECM tissue engineering scaffold, characterized in that, The preparation method includes the following steps: Step 1: Prepare Se@SiO2 nanoparticles using the reverse microemulsion method; Step 2: Dissolve the Se@SiO2 nanoparticles obtained in Step 1 with SrCl2·6H2O and PVP in deionized water and stir until homogeneous. Add ammonia dropwise, stir, and place in a polytetrafluoroethylene reactor. React at 130-150℃ for 4-6 h. Wash with deionized water to obtain Sr-Se@SiO2 nanoparticles. The mass ratio of Se@SiO2 nanoparticles to SrCl2·6H2O is 1:3-5, the mass ratio of Se@SiO2 nanoparticles to PVP is 95-105:1, the mass-volume ratio of Se@SiO2 nanoparticles to ammonia is 20-80 mg:1 mL, and the mass percentage of ammonia is 25%-30%. Step 3: Place the Gelma sample in a pretreated tissue culture polystyrene plate, and simultaneously place ADSCs into the tissue culture polystyrene plate. Add growth medium and culture until the ADSCs fuse with the Gelma sample. Then add ascorbic acid for 6-8 days to obtain ADSCs loaded on the Gelma sample. Step 4: Place the ADSCs loaded on the Gelma sample obtained in Step 3 in the extraction buffer and incubate for 3-8 minutes to obtain the dECM of ADSCs loaded on the Gelma sample; the extraction buffer is a PBS solution containing 0.5% Triton X-100 and 20mM NH4OH. Step 5: Uniformly spray the Sr-Se@SiO2 nanoparticles obtained in Step 2 onto the dECM derived from ADSCs and loaded on the Gelma sample obtained in Step 4 to obtain the Sr-Se@SiO2-dECM tissue engineering scaffold with nanostructure.

2. The preparation method according to claim 1, characterized in that, Step 1 includes the following steps: adding hexanol, Triton X-100, and hexane to deionized water and stirring until homogeneous; adding copper selenide solution and stirring for 3-5 minutes; adding tetraethyl orthosilicate and stirring for 3-5 minutes; adding ammonia water to make the solution dark blue; stirring for 20-28 hours; and then washing with anhydrous ethanol by centrifugation to obtain the final product.

3. The preparation method according to claim 1, characterized in that, The method for preparing the Gelma sample includes pouring a 5-15 wt% Gelma solution into a mold, exposing it to ultraviolet light for cross-linking to obtain a sample, and then freeze-drying the sample overnight to obtain the Gelma sample.

4. The Sr-Se@SiO2-dECM tissue engineering scaffold prepared by any of the preparation methods described in claims 1-3.

5. The use of the Sr-Se@SiO2-dECM tissue engineering scaffold according to claim 4 in the preparation of medical composite materials for repairing or treating volumetric muscle defects.

6. The application according to claim 5, characterized in that, The Sr-Se@SiO2-dECM tissue engineering scaffold promotes angiogenesis in the repair of volumetric muscle defects.

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