Skeletal muscle structure and microenvironment bionic sponge and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to skeletal muscle structure and microenvironment bionic sponge and a preparation method and application thereof. The skeletal muscle microenvironment-like decellularized ECM and a directional freezing technology are utilized to successfully construct the skeletal muscle structure and microenvironment double bionic decellularized ECM / sodium alginate sponge, the physiological skeletal muscle microenvironment can be fully simulated, and the migration, proliferation and differentiation of muscle satellite cells are effectively promoted, so that the regeneration, maturation and differentiation of muscle fibers in the sponge are promoted. Therefore, the invention can effectively solve the dilemma and limitation of fibrosis of the current decellularized ECM in VML treatment application, promote regeneration and repair of damaged tissues of the VML, provide a new treatment approach for regeneration and functional recovery of muscular tissues of the VML, lay a theoretical and technical foundation for research and development of the decellularized ECM with clinical potential application value and a scaffold thereof, and have a wide application prospect. Therefore, the method has important clinical potential significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterials, and particularly relates to a skeletal muscle structure and microenvironment biomimetic sponge as well as a preparation method and application thereof. BACKGROUND

[0002] As a frontier field of biomedical research, tissue engineering, combined with biomaterials, stem cells, active factors, etc., can effectively promote the regeneration and repair of tissues and organs, and even achieve functional remodeling. Based on this, the regeneration and repair treatment of tissue engineering in volumetric muscle loss (VML) has also been studied, and ideal results have been achieved, opening up new ideas and laying a foundation for theoretical knowledge for the treatment of VML. However, in the process of actual clinical application, tissue engineering in the treatment of VML still needs to solve many difficulties and technical bottlenecks. For example, the current decellularized extracellular matrix (dECM) tissue engineering technology can effectively promote the regeneration and repair of VML muscle tissue, and even promote the recovery of local function. However, in-depth research has found that the repair of VML tissue and function is not achieved by promoting muscle fiber regeneration and repair, but by causing an inflammatory response through the dECM scaffold, and then promoting fibrous tissue deposition and scar formation to achieve partial tissue and functional repair. The fundamental reason for this result is that the structure and composition of the current dECM scaffold are quite different from the anatomical structure and composition of the original skeletal muscle. After being implanted into the body, it cannot promote the migration of muscle satellite cells in the original muscle tissue into the scaffold and proliferate and differentiate into muscle fibers, but instead causes an inflammatory response, leading to fibrous tissue deposition in the dECM scaffold to form scar tissue, thereby inhibiting the regeneration and repair of functional muscle tissue. SUMMARY

[0003] Therefore, based on the defects of the above-mentioned dECM scaffold, the present application intends to extract a cell-derived decellularized ECM with a basal membrane-like property according to the physiological muscle satellite cell distribution microenvironment (distributed between the sarcolemma and the basement membrane), combine the skeletal muscle fiber anatomical structure, and construct a biomimetic decellularized ECM / sodium alginate sponge, so as to promote the migration of muscle satellite cells into the decellularized ECM / sodium alginate sponge and proliferate and differentiate into muscle fibers, thereby promoting the regeneration and repair of VML damaged tissue.

[0004] The first aspect of the present application aims to provide a preparation method of a skeletal muscle structure and microenvironment biomimetic sponge.

[0005] The second aspect of the present application aims to provide a skeletal muscle structure and microenvironment biomimetic sponge.

[0006] The third aspect of the present application aims to provide the preparation method of the first and second aspects of the present application, and the application of the biomimetic sponge.

[0007] The fourth aspect of the present application aims to provide a product.

[0008] To achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a preparation method of a skeletal muscle structure and microenvironment biomimetic sponge, comprising the following steps: 1) Decellularized ECM is dissolved by pepsin to prepare a decellularized ECM solution; 2) Sodium alginate powder is weighed, stirred and dissolved in double-distilled water to prepare a sodium alginate solution; 3) The decellularized ECM solution prepared in step 1) is mixed with the sodium alginate solution prepared in step 2), and stirred to prepare a decellularized ECM / sodium alginate solution; 4) The decellularized ECM / sodium alginate solution prepared in step 3) is prepared into a decellularized ECM / sodium alginate frozen solid matrix with a directional structure by directional freezing technology; 5) The decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) is placed into a pre-cooled CaCl2 solution to cross-link and prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) is frozen at-80℃, and freeze-dried to prepare a decellularized ECM / sodium alginate sponge.

[0009] In some embodiments of the present application, the decellularized ECM includes cell-derived decellularized ECM or tissue-derived decellularized ECM. It has a basement membrane-like composition and structural characteristics, and is used for biomimicry of the skeletal muscle microenvironment of the sponge.

[0010] In some embodiments of the present application, the cell-derived decellularized ECM is derived from any one of stem cells, fibroblasts and tumor cells.

[0011] In some embodiments of the present application, the preparation of the decellularized ECM in step 1) comprises the following steps: The tissue or cultured cells are subjected to decellularization treatment by a decellularization reagent to obtain the decellularized ECM.

[0012] In some embodiments of the present application, the decellularization reagent includes at least one of ammonia, deoxycholic acid sodium, DNase -I and Triton X-100.

[0013] In some embodiments of the present application, the preparation of the decellularized ECM in step 1) comprises the following steps: After cell digestion, resuspension, and adherent expansion culture, differentiation culture is performed to form primitive endoderm-like cells; the primitive endoderm-like cells are subjected to decellularization treatment using a decellularization reagent, thereby obtaining the decellularized ECM.

[0014] In some embodiments of the present application, the preparation method of the decellularized ECM has been patented (202211710997.0).

[0015] In some embodiments of the present application, the pepsin dissolution solution is a 0.05-0.2 M HCl solution, and the pepsin concentration is 1-2 mg / mL. The decellularized ECM is dissolved and stirred at 37°C for 24-48 h.

[0016] In some embodiments of the present application, in step 3), the concentration of the sodium alginate solution is 1%-4%, and the concentration of the decellularized ECM is 0 mg / mL-2.0 mg / mL (excluding 0).

[0017] In some embodiments of the present application, in step 4), the cooling rate of the directional freezing is 10-15°C / min.

[0018] In some embodiments of the present application, the device for directional freezing is as follows: (1) The bottom layer is a cold source device for containing a freezing source, which includes but is not limited to liquid nitrogen or dry ice, etc. (2) The middle layer is a heat conducting layer, such as a metal heat conductor such as iron, copper, and aluminum, etc. (3) The upper layer is a mold layer for containing the freezing substance. The bottom surface of the mold in this layer is in contact with the upper surface of the heat conducting layer, and the outer periphery is a heat insulation layer.

[0019] In some embodiments of the present application, the freezing device contains a liquid nitrogen freezing source, which is cooled to the mold layer through a copper plate. The decellularized ECM / sodium alginate solution contained in the mold is gradually frozen from the bottom layer of the mold upwards until all the contained solution is frozen into a solid matrix.

[0020] In some embodiments of the present application, the cooling rate of the directional freezing is 10-15°C / min.

[0021] In some embodiments of the present application, in step 5), the concentration of the CaCl2 solution is 0.05 mol-0.2 mol; preferably, 0.1 mol-0.15 mol.

[0022] In some embodiments of the present application, in step 5), the CaCl2 solution is in a 0°C ice-water mixed state.

[0023] In some embodiments of the present invention, in step 5), the crosslinking time of the CaCl2 solution is 12-24 h; preferably 6-18 h; more preferably 9-15 h. The ambient temperature is 4 °C.

[0024] In a second aspect, the present invention provides a skeletal muscle structure and microenvironment biomimetic sponge, prepared by the preparation method of the first aspect of the present invention.

[0025] A third aspect of the present invention provides a method for preparing the first aspect of the present invention and / or the application of the skeletal muscle structure and microenvironment biomimetic sponge of the second aspect of the present invention in any one of (1) to (9): (1) Preparation of VML regeneration and repair transplantation scaffold; (2) In vitro study on skeletal muscle cell proliferation and differentiation; (3) Construct an in vitro 3D muscle tissue model; (4) Cell culture; (5) Prepare products from cultured cells; (6) Promotes the expression of cellular functions; (7) Prepare products that promote cell function expression; (8) Muscle tissue repair; (9) Prepare products for muscle tissue repair.

[0026] A fourth aspect of the present invention is to provide a decellularized ECM / sodium alginate sponge product comprising the first aspect of the present invention.

[0027] In some embodiments of the present invention, the product applications include, but are not limited to, muscle tissue organoids, muscle tissue 3D models, and muscle tissue substitutes.

[0028] In some embodiments of the present invention, the product has the function of culturing muscle cells and promoting the functional expression of muscle cells.

[0029] The beneficial effects of this invention are: This invention utilizes decellularized ECM with a skeletal muscle-like microenvironment and targeted cryotherapy to successfully construct a decellularized ECM / sodium alginate sponge that mimics both skeletal muscle structure and microenvironment. This sponge effectively simulates the physiological skeletal muscle microenvironment, promoting the migration, proliferation, and differentiation of muscle satellite cells, thereby facilitating the regeneration and maturation of muscle fibers within the sponge. Therefore, the skeletal muscle-like structure and microenvironment-mimicking decellularized ECM / sodium alginate sponge constructed in this invention can effectively address the current limitations and challenges of fibrosis in the treatment of venous fibrosis (VML) with decellularized ECM, promoting the regeneration and repair of damaged VML tissue, and providing a new therapeutic approach for the regeneration and functional recovery of VML muscle tissue. This lays the theoretical and technical foundation for the development of decellularized ECM and its scaffolds with potential clinical applications, thus possessing significant clinical potential. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 SEM analysis of directional (A) and non-directional (B) decellularized ECM / sodium alginate sponge and pore size analysis of directional decellularized ECM / sodium alginate sponge (C).

[0031] Figure 2 FTIR detection of directional and non-directional decellularized ECM / sodium alginate sponge.

[0032] Figure 3 Targeted and non-targeted decellularized ECM / sodium alginate sponge C2C12 culture CCK-8 assay. Detailed Implementation

[0033] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0034] The specific process for decellularized ECM extraction is as follows: (1) Resuscitate and expand mouse teratoma cells (F9 cells): Add F9 cells to complete culture medium (using high glucose DMEM as the basic culture medium, with double antibiotics accounting for 1% of the culture medium volume and FBS accounting for 10% of the culture medium volume) and culture at 37℃ for 30h; (2) Digest the cells from step (1) (add 1 mL of digestion solution (0.25% Trypsin-0.53 mM EDTA) and digest at room temperature for 1 min), centrifuge at 800 rpm for 5 min, discard the supernatant, add 5 mL of complete culture medium containing 10% serum to resuspend the cells and count them; (3) The cells counted in step (2) were divided into groups of 4.5 × 10⁻⁶. 6 Re-inoculate 10cm culture dishes, add complete culture medium, and incubate at 37℃ for 24 hours in an incubator. (4) Replace the culture medium in step (3) with differentiation medium (based on DMEM (high glucose, 4 g / L) culture medium, wherein the double antibiotic accounts for 1% of the culture medium volume, FBS accounts for 10% of the culture medium volume, the concentration of all-trans retinoic acid is 0.1 μM and the concentration of bibutyryl-cyclic adenosine monophosphate is 250 μM), and culture F9 cells for differentiation for 3 days to form primitive endoderm-like cells; (5) Wash the primitive endoderm-like cells obtained in step (4) twice with deionized water, add ammonia water with a concentration of 25mM, and place the cells on a decolorizing shaker at 4℃ for 1 hour to remove cells. (6) Place the cell sample after decellularization in step (5) into a dialysis bag (1000D) and stir in deionized water at room temperature (about 25°C) for 24 hours, changing the water 5 to 10 times during the process; (7) The cell sample washed in step (6) was freeze-dried at -80°C to obtain decellularized extracellular matrix containing laminin and collagen IV, i.e., decellularized ECM.

[0035] Example 1 A method for preparing a biomimetic sponge with skeletal muscle structure and microenvironment includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (0.4 mg / mL: 2%). 4) The decellularized ECM / sodium alginate solution prepared in step 3) is used to prepare a decellularized ECM / sodium alginate cryo-solid matrix with a directional structure by directional freezing technology; 5) Place the decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a pre-cooled CaCl2 solution (0℃ ice-water mixture, 0.12mol) and crosslink (4℃, 12h) to prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare decellularized ECM / sodium alginate sponge.

[0036] Example 2 A method for preparing a biomimetic sponge with skeletal muscle structure and microenvironment includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (0.8 mg / mL: 2%). 4) The decellularized ECM / sodium alginate solution prepared in step 3) is used to prepare a decellularized ECM / sodium alginate cryo-solid matrix with a directional structure by directional freezing technology; 5) Place the decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a pre-cooled CaCl2 solution (0℃ ice-water mixture, 0.12mol) and crosslink (4℃, 12h) to prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare decellularized ECM / sodium alginate sponge.

[0037] Example 3 A method for preparing a biomimetic sponge with skeletal muscle structure and microenvironment includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (1.0 mg / mL: 2%). 4) The decellularized ECM / sodium alginate solution prepared in step 3) is used to prepare a decellularized ECM / sodium alginate cryo-solid matrix with a directional structure by directional freezing technology; 5) Place the decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a pre-cooled CaCl2 solution (0℃ ice-water mixture, 0.12mol) and crosslink (4℃, 12h) to prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare decellularized ECM / sodium alginate sponge.

[0038] Example 4 A method for preparing a biomimetic sponge with skeletal muscle structure and microenvironment includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (2.0 mg / mL: 2%). 4) The decellularized ECM / sodium alginate solution prepared in step 3) is used to prepare a decellularized ECM / sodium alginate cryo-solid matrix with a directional structure by directional freezing technology; 5) Place the decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a pre-cooled CaCl2 solution (0℃ ice-water mixture, 0.12mol) and crosslink (4℃, 12h) to prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare decellularized ECM / sodium alginate sponge.

[0039] Comparative Example 1 A method for preparing a skeletal muscle-inspired sponge (without ECM) includes the following steps: 1) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare a 2% sodium alginate solution; 2) The sodium alginate solution prepared in step 1) is used to prepare a sodium alginate frozen solid matrix with an oriented structure by directional freezing technology; 3) The sodium alginate frozen solid matrix prepared in step 2) was placed in a pre-cooled CaCl2 solution (0℃ ice-water mixture, 0.12 mol) and crosslinked (4℃, 12 h) to prepare sodium alginate hydrogel; 4) The sodium alginate hydrogel prepared in step 3) was frozen at -80℃ and freeze-dried for 3 days to prepare sodium alginate sponge.

[0040] Comparative Example 2 A method for preparing non-directional sodium alginate sponge includes the following steps: 1) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare a 2% sodium alginate solution; 2) Place the sodium alginate solution prepared in step 1) into a mold and place it in a -80℃ freezer to prepare a non-directional sodium alginate frozen solid matrix; 3) Place the sodium alginate frozen solid matrix prepared in step 2) into a room temperature CaCl2 solution (0.12 mol) and crosslink it (12 h) to prepare sodium alginate hydrogel; 4) The decellularized ECM / sodium alginate hydrogel prepared in step 3) was frozen at -80℃ and freeze-dried for 3 days to prepare sodium alginate sponge.

[0041] Comparative Example 3 A method for preparing non-directional decellularized ECM / sodium alginate sponge includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (0.4 mg / mL: 2%). 4) Place the decellularized ECM / sodium alginate solution prepared in step 3) into a mold and place it in a -80℃ freezer to prepare a non-directional decellularized ECM / sodium alginate frozen solid matrix. 5) The non-oriented decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) was placed in a room temperature CaCl2 solution (0.12 mol) and cross-linked (12 h) to prepare a non-oriented decellularized ECM / sodium alginate hydrogel; 6) The non-oriented decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare a non-oriented decellularized ECM / sodium alginate sponge.

[0042] Comparative Example 4 A method for preparing non-directional decellularized ECM / sodium alginate sponge includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (0.8 mg / mL: 2%). 4) Place the decellularized ECM / sodium alginate solution prepared in step 3) into a mold and place it in a -80℃ freezer to prepare a non-directional decellularized ECM / sodium alginate frozen solid matrix. 5) Place the non-oriented decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a room temperature CaCl2 solution (0.12 mol) and crosslink (12 h) to prepare a non-oriented decellularized ECM / sodium alginate hydrogel; 6) The non-oriented decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare a non-oriented decellularized ECM / sodium alginate sponge.

[0043] Comparative Example 5 A method for preparing non-directional decellularized ECM / sodium alginate sponge includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (1.0 mg / mL: 2%). 4) Place the decellularized ECM / sodium alginate solution prepared in step 3) into a mold and place it in a -80℃ freezer to prepare a non-directional decellularized ECM / sodium alginate frozen solid matrix. 5) Place the non-oriented decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a room temperature CaCl2 solution (0.12 mol) and crosslink (12 h) to prepare a non-oriented decellularized ECM / sodium alginate hydrogel; 6) The non-oriented decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare a non-oriented decellularized ECM / sodium alginate sponge.

[0044] Comparative Example 6 A method for preparing non-directional decellularized ECM / sodium alginate sponge includes the following steps: 1) Decellularized ECM was dissolved in pepsin (0.1M HCl, 1mg / mL) to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare a decellularized ECM / sodium alginate solution (2.0 mg / mL: 2%). 4) Place the decellularized ECM / sodium alginate solution prepared in step 3) into a mold and place it in a -80℃ freezer to prepare a non-directional decellularized ECM / sodium alginate frozen solid matrix. 5) Place the non-oriented decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a room temperature CaCl2 solution (0.12 mol) and crosslink (12 h) to prepare a non-oriented decellularized ECM / sodium alginate hydrogel; 6) The non-oriented decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried for 3 days to prepare a non-oriented decellularized ECM / sodium alginate sponge.

[0045] Example 1: Non-directional and directional decellularized ECM / sodium alginate sponge and its SEM detection and pore size analysis By comparing Examples 1-4 to Comparative Examples 1-6, Figure 1 It is evident that freezing and directional freezing techniques can prepare non-directional and directional decellularized ECM / sodium alginate sponges with different concentration gradients. Scanning electron microscopy results show that the cross-section of the directional decellularized ECM / sodium alginate sponge exhibits a smooth and uniform honeycomb network structure, while the longitudinal section shows parallel tubular structures. Figure 1 (A), and the statistical results of the pore diameter show that the honeycomb pore diameter is uniformly distributed at around 90-100 μm ( Figure 1 (C). In contrast to directional decellularized ECM / sodium alginate sponges, non-directional decellularized ECM / sodium alginate sponges exhibit an irregular, porous structure of varying sizes on their surface. Figure 1 (B)

[0046] Example 2: FTIR detection of decellularized ECM / sodium alginate sponge By comparing Examples 1-4 to Comparative Examples 1-6, Figure 2 It is evident that the decellularized ECM used possesses distinct extracellular matrix characteristic functional groups, such as 1640 cm⁻¹. -1 The amide I functional group at the location, 1539cm -1 The amide II functional group at the position, 1090 to 1375 cm -1 The amide III functional groups between regions and 3435cm -1 There are obvious NH functional groups. With the preparation of decellularized ECM / sodium alginate composite sponges, the composite material's spectral results show: 1640 cm⁻¹ -1 The amide I functional group at the location, 1539cm -1 The amide II functional group at the position, 1090 to 1375 cm -1 Amide III functional groups were present in both regions of the decellularized ECM / sodium alginate sponge. Simultaneously, at 1595 cm⁻¹... -1 A distinct high absorption peak appears at 1632 cm⁻¹, and at 1632 cm⁻¹... -1 and 1537cm -1 Two distinct shoulder peaks appeared at 3600 cm⁻¹, both characteristic absorption peaks of the amide carbonyl group in decellularized ECM molecules, indicating a significant complexation between sodium alginate and decellularized ECM. Furthermore, the results also showed that at 3600 cm⁻¹... -1 up to 3200 cm -1The presence of a high absorption peak further indicates that sodium alginate interacts with decellularized ECM to form hydrogen bonds. Therefore, the above results suggest that decellularized ECM exists within the decellularized ECM / sodium alginate sponge and interacts with sodium alginate to form a composite scaffold material.

[0047] Example 3: Decellularized ECM / Sodium Alginate Sponge Cell Culture and CCK-8 Detection Examples 1-4 to Comparative Examples 1-6 were used to culture C2C12 cells, and cell proliferation was detected at corresponding time points. The results showed (e.g.) Figure 3 Over time, both directed and non-directed decellularized ECM / sodium alginate sponge cells gradually proliferated, but the proliferation of cells in the directed decellularized ECM / sodium alginate sponge was significantly greater than that in the non-directed decellularized ECM / sodium alginate sponge, indicating that the directed structure effectively promoted cell proliferation. Furthermore, in both directed and non-directed decellularized ECM / sodium alginate sponges, the addition of decellularized ECM effectively promoted cell proliferation with continued culture time. In the directed decellularized ECM / sodium alginate sponge, the addition of decellularized ECM showed concentration specificity for cell proliferation, with the best effect observed at 0.8 mg / ml. Further increases in concentration inhibited cell proliferation. However, in the non-directed decellularized ECM / sodium alginate sponge, the effect of decellularized ECM was inconsistent with that of the directed decellularized ECM / sodium alginate sponge, showing a cell-promoting effect, but only at high concentrations was the promotion effective. Therefore, the above results indicate that decellularized ECM / sodium alginate sponge can promote cell proliferation, and its effect is correlated with the sponge's structure.

[0048] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a biomimetic sponge with skeletal muscle structure and microenvironment, comprising the following steps: 1) Decellularized ECM was dissolved with pepsin to prepare a decellularized ECM solution; 2) Weigh out sodium alginate powder, stir and dissolve it in double-distilled water to prepare sodium alginate solution; 3) Mix the decellularized ECM solution prepared in step 1) with the sodium alginate solution prepared in step 2), and stir until homogeneous to prepare the decellularized ECM / sodium alginate solution; 4) The decellularized ECM / sodium alginate solution prepared in step 3) is used to prepare a decellularized ECM / sodium alginate cryo-solid matrix with a directional structure by directional freezing technology; 5) Place the decellularized ECM / sodium alginate frozen solid matrix prepared in step 4) into a pre-cooled CaCl2 solution to crosslink and prepare a decellularized ECM / sodium alginate hydrogel; 6) The decellularized ECM / sodium alginate hydrogel prepared in step 5) was frozen at -80℃ and freeze-dried to prepare decellularized ECM / sodium alginate sponge.

2. The preparation method according to claim 1, characterized in that: The preparation of decellularized ECM in step 1) includes the following steps: Decellularized ECM is obtained by treating tissues or cultured cells with a decellularization reagent.

3. The preparation method according to claim 2, characterized in that: The decellularization reagent includes ammonia, sodium deoxycholate, and DNase. -I and at least one of Triton X-100.

4. The preparation method according to claim 1, characterized in that: In step 3), the concentration of the sodium alginate solution is 1%-4%; The concentration of the decellularized ECM is 0 mg / mL to 2.0 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step 4), the cooling rate of the directional freezing is 10-15℃ / min.

6. The preparation method according to claim 1, characterized in that: In step 5), the concentration of the CaCl2 solution is 0.05 mol to 0.2 mol.

7. The preparation method according to claim 5, characterized in that: The cross-linking time of the CaCl2 solution is 12-24 h, and the ambient temperature is 4℃.

8. A skeletal muscle structure and microenvironment biomimetic sponge, prepared by the method of any one of claims 1 to 7.

9. The application of the preparation method according to any one of claims 1 to 7 or the skeletal muscle structure and microenvironment biomimetic sponge according to claim 8 in any one of (1) to (9): (1) Preparation of VML regeneration and repair transplantation scaffold; (2) In vitro study on skeletal muscle cell proliferation and differentiation; (3) Construct an in vitro 3D muscle tissue model; (4) Cell culture; (5) Prepare products from cultured cells; (6) Promotes the expression of cellular functions; (7) Prepare products that promote cell function expression; (8) Muscle tissue repair; (9) Prepare products for muscle tissue repair.

10. A product comprising the skeletal muscle structure and microenvironment biomimetic sponge of claim 8.

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