Thermosensitive decellularized matrix hydrogels, their preparation methods and applications

Abstract

This invention discloses a thermosensitive decellularized matrix hydrogel, its preparation method, and its application, relating to the field of material preparation technology. The method includes the following steps: human bone marrow mesenchymal stem cells are cultured in a serum-free culture medium containing ascorbic acid to obtain a biomass matrix material; the biomass matrix material is added to a decellularization solution for decellularization treatment to obtain a decellularized matrix membrane; the decellularized matrix membrane is freeze-dried and ground to obtain a decellularized matrix powder; the decellularized matrix powder is added to a pepsin hydrochloric acid solution and mixed evenly to obtain a decellularized matrix solution; the pH and osmotic pressure of the decellularized matrix solution are adjusted, gelled at 37°C, and stored at 4°C to obtain a thermosensitive decellularized matrix hydrogel. This application prepares a hydrogel with natural extracellular matrix characteristics, capable of rapid liquid-solid phase transition at body temperature, and possesses advantages such as high biocompatibility, excellent temperature response performance, and effective promotion of tissue repair.

Description

Technical Field

[0001] This invention relates to the field of materials preparation technology, and in particular to thermosensitive decellularized matrix hydrogels, their preparation methods, and applications. Background Technology

[0002] Hydrogels are polymeric materials with a three-dimensional network structure formed by cross-linking of each other through covalent bonds, hydrogen bonds, or van der Waals forces, using water as the dispersion medium. Due to their unique properties such as high viscoelasticity, high water content, and environmental responsiveness, they have broad application prospects in biomedical fields such as tissue engineering, drug sustained release, and biosensors.

[0003] Traditional hydrogels can be divided into synthetic polymer hydrogels and natural polymer hydrogels. Synthetic hydrogels are mostly homopolymers and copolymers of acrylamide and its derivatives, and also include synthetic materials such as polyvinyl alcohol and polyphosphazene. Natural polymer materials such as chitosan, dextran, guar gum, collagen, and proteins are also frequently used to prepare hydrogels. However, these traditional hydrogels have significant shortcomings in terms of biocompatibility, response speed, and mechanical strength, making it difficult to meet clinical needs. Although researchers have modified traditional hydrogels in various ways to approximate the properties of the extracellular matrix, these modified hydrogels still show significant differences from the natural extracellular matrix in terms of ultrastructure and biological function, particularly in terms of poor biocompatibility and tissue repair effects. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a thermosensitive decellularized matrix hydrogel, its preparation method, and its application, aiming to improve the biocompatibility and wound repair effect of decellularized matrix hydrogel.

[0005] The first aspect of this invention provides a thermosensitive decellularized matrix hydrogel, comprising the following steps: S1. Human bone marrow mesenchymal stem cells were cultured in a serum-free culture medium containing ascorbic acid to obtain biomass matrix material; S2. The biomass matrix material is added to a decellularization solution for decellularization treatment to obtain a decellularized matrix membrane; S3. The decellularized matrix membrane is freeze-dried and ground to obtain decellularized matrix powder; S4. Add the decellularized matrix powder to the pepsin hydrochloric acid solution and mix well to obtain a decellularized matrix solution; S5. Add NaOH to adjust the pH of the decellularized matrix solution to 7.2-7.4, then add 10×PBS solution, place at 37℃ to form a gel, and store at 4℃ to obtain a thermosensitive decellularized matrix hydrogel.

[0006] Using the above technical solution, human bone marrow mesenchymal stem cells are cultured to construct a biomass matrix. Ascorbic acid is used to promote collagen synthesis and form a dense matrix layer. A decellularized solution selectively dissolves cell membrane components while preserving the complete collagen fiber network. The freeze-dried matrix powder is partially enzymatically hydrolyzed by pepsin to form soluble collagen fragments. After pH adjustment to neutral, a buffer salt solution is added, and a three-dimensional network structure is formed through temperature-sensitive self-assembly. This process mimics the formation mechanism of the natural extracellular matrix, achieving a phase transition from solution to gel at a body temperature of 37°C. The hydrogel constructed in this application has a three-dimensional structure highly similar to the natural extracellular matrix, providing a biomimetic microenvironment for cell growth. The temperature-responsive characteristics allow the material to gel in situ after injection, effectively filling irregular wounds. Decellularization eliminates immunogenicity, and the matrix components retain bioactive factors, promoting the regeneration and repair of damaged tissue. This preparation method avoids the use of synthetic cross-linking agents, ensuring the safety and biodegradability of the material.

[0007] Optionally, the concentration of ascorbic acid in the serum-free culture medium is 100 μg / mL.

[0008] Through the above technical solution, ascorbic acid, a water-soluble vitamin, serves as a key cofactor in extracellular matrix synthesis, promoting the hydroxylation reaction of collagen. Serum-free culture medium refers to cell culture medium that does not contain animal serum, avoiding interference from serum components in subsequent decellularization processes. When culturing human bone marrow mesenchymal stem cells, ascorbic acid is added to the serum-free culture medium to achieve a final concentration of 100 μg / mL. This concentration range effectively maintains cell viability and promotes extracellular matrix secretion, optimizing extracellular matrix synthesis efficiency and avoiding cell damage or matrix structure loosening caused by inappropriate ascorbic acid concentration. If the stimulation concentration is below 100 μg / mL, the resulting decellularized matrix membrane will be correspondingly thinner, while avoiding cytotoxicity caused by excessively high concentrations. During culture, ascorbic acid activates key enzymes such as prolyl hydroxylase, promoting the cross-linking and deposition of collagen fibers, providing a structural basis for the subsequent formation of decellularized matrix materials.

[0009] Optionally, the decellularization solution contains 3% TritonX-100 + 2% SDS.

[0010] In the above technical solution, TritonX-100 refers to a nonionic surfactant, specifically polyethylene glycol octylphenyl ether, which achieves cell lysis by disrupting the lipid bilayer structure of the cell membrane. SDS refers to sodium dodecyl sulfate, specifically an anionic surfactant, which promotes the removal of cell debris by dissolving membrane proteins and nucleoproteins. The synergistic effect of the two can reduce damage to the extracellular matrix protein structure while ensuring decellularization efficiency.

[0011] Specifically, the biomass matrix material was immersed in a mixed solution containing 3% Triton X-100 and 2% SDS. The surfactants selectively dissolved cell membrane lipids and proteins, enabling rapid removal of cellular components. Triton X-100 preferentially acted on the cell membrane lipid layer, while SDS further decomposed residual protein components. The proportional combination of the two surfactants avoided matrix fiber breakage caused by excessively high concentrations of a single reagent. Through the synergistic ratio of the two surfactants, a more complete decellularization effect was achieved while reducing the total reagent concentration. The decellularization process effectively balanced the contradiction between cell component removal efficiency and matrix structural integrity, resulting in tightly packed and unbroken collagen fibers. This provides a high-quality raw material basis for the subsequent preparation of hydrogels with biomimetic structures.

[0012] Optionally, the culture of the human bone marrow mesenchymal stem cells includes the following steps: adding serum-free culture medium containing ascorbic acid to the human bone marrow mesenchymal stem cells and culturing for 10 days, changing the culture medium every other day; aspirating the culture medium containing ascorbic acid, rinsing with PBS solution, and then immersing in ultrapure water at 37°C for 10 minutes to obtain biomass matrix material.

[0013] Using the above technical solution, serum-free culture medium containing ascorbic acid is continuously applied to the cell culture system for a 10-day cycle. During this period, a stable microenvironment is maintained by regularly changing the culture medium. Before the addition of ascorbic acid (pre-VC), bone marrow mesenchymal stem cells are spindle-shaped and relatively tightly packed, but intercellular spaces still exist. After 5 days of ascorbic acid stimulation, the outline of bone marrow mesenchymal stem cells becomes blurred, and the extracellular matrix fills the intercellular spaces. After 10 days of ascorbic acid stimulation, bone marrow mesenchymal stem cells are tightly packed together and secrete a large amount of extracellular matrix. If the stimulation exceeds 10 days, the extracellular matrix membrane at the base of the cells gradually rolls inward from the periphery, leading to the destruction of the overall structure of the decellularized matrix membrane. After the matrix secretion stage is completed, culture residues are removed through a stepwise washing process: first, the surface is rinsed with a buffer solution, followed by immersion in ultrapure water under constant temperature conditions. This phased processing method effectively removes impurities while avoiding damage to the extracellular matrix from strong chemical reagents, ultimately obtaining structurally intact biomass matrix materials. This achieves efficient preparation of biomass matrix materials, and the obtained materials have lower immunogenicity and higher structural integrity. It avoids the use of organic solvents or strong detergents, making the final product more suitable for constructing biomimetic hydrogel systems and providing a high-quality raw material basis for subsequent decellularization processing.

[0014] Optionally, the decellularization process includes the following steps: after removing ultrapure water from the bio-based matrix material, adding a decellularization solution, extracting at 37°C for 5 minutes, and then washing three times with PBS solution to obtain a decellularized matrix membrane.

[0015] The above technical solution first reduces moisture interference in the matrix material by physically removing ultrapure water during the decellularization process. Then, a decellularization solution is added to rupture cell membranes and dissolve cell contents. A short extraction at a constant temperature of 37°C for 5 minutes ensures efficient decellularization while preventing degradation of matrix components due to prolonged high-temperature exposure. After extraction, three PBS rinse steps gradually remove residual chemical reagents and cell debris, ultimately yielding a structurally intact decellularized matrix membrane. This achieves a highly efficient yet gentle decellularization process, significantly reducing the risk of damage to matrix proteins from chemical reagents, while the step-by-step rinsing effectively controls the residue content.

[0016] Optionally, the decellularized matrix membrane can be stored at 4°C for 2-3 weeks.

[0017] Using the above technical solution, decellularized matrix membranes, after freeze-drying and grinding, form a porous fibrous structure. The low-temperature environment reduces the hydrolysis rate of collagen fibers and inhibits enzyme activity. When the membrane is placed in a 4°C environment, the internal water content remains in a non-frozen state, which avoids mechanical damage to the fiber network caused by ice crystal formation and maintains the natural conformation of extracellular matrix proteins. The storage period is set at 2-3 weeks to ensure that the material retains complete bioactive sites and mechanical support functions during subsequent hydrogel preparation. This effectively solves the problem of short-term deterioration of decellularized matrix materials, providing a stable raw material guarantee for subsequent hydrogel preparation.

[0018] Optionally, the concentration of the pepsin hydrochloric acid solution is 1 mg / mL; The concentration of the decellularized matrix solution is 10 mg / mL.

[0019] Through the above technical solution, when decellularized matrix powder is mixed with pepsin hydrochloric acid solution, the concentration of pepsin, the mass-to-volume ratio, and the solution concentration are controlled to ensure that the decellularized matrix is ​​moderately enzymatically hydrolyzed under acidic conditions, forming a homogeneous solution with controllable viscosity. In this process, too low a pepsin concentration leads to insufficient enzymatic hydrolysis efficiency, while too high a concentration may destroy the active ingredients in the matrix. The selection of the mass-to-volume ratio range directly affects the homogeneity of the solution and subsequent gelation properties. If the concentration of the decellularized matrix solution is too high, such as 20 mg / mL, the structure is too dense, affecting cell migration and nutrient diffusion, potentially causing premature gelation. If the concentration is too low, such as 5 mg / mL, the formed hydrogel is too soft and cannot provide sufficient mechanical support for the cells, making it difficult to form a stable three-dimensional network structure. Through the synergistic control of the above parameters, a homogeneous solution suitable for temperature-sensitive gelation is finally obtained. This solves the problems of poor solution homogeneity and unstable gelation properties caused by unclear enzymatic hydrolysis conditions in traditional hydrogel preparation processes, achieving efficient dissolution and homogenization of the decellularized matrix, and providing reliable technical support for the preparation of temperature-sensitive hydrogels with stable three-dimensional network structures.

[0020] Optionally, in step S5, the molar concentration of the NaOH solution is 0.1 mol / L, and the volume ratio of the NaOH solution to the decellularized matrix solution is 10:1. The volume ratio of the 10×PBS solution to the decellularized matrix solution is 9:1.

[0021] Through the above technical solution, during the pH adjustment process of the decellularized matrix solution, a 0.1 mol / L NaOH solution is added at a volume ratio of 10:1, which can precisely control the neutralization reaction process and avoid local over-alkaliness leading to protein denaturation. Subsequently, a 10×PBS solution is added at a volume ratio of 9:1. Through the osmotic pressure balance effect of the buffer system, the solution forms a stable three-dimensional network structure at 37°C. This mixing ratio ensures a balanced distribution of intermolecular forces during gelation and provides suitable physical stability for storage at 4°C. This application effectively solves the problem of uneven gelation caused by insufficient pH adjustment precision in the preparation of decellularized matrix hydrogels. At the same time, by optimizing the buffer system ratio, the thermal response characteristics and low-temperature storage stability of the hydrogel are significantly improved, providing reliable physical performance assurance for subsequent biomedical applications.

[0022] A second aspect of the present invention provides a thermosensitive decellularized matrix hydrogel, prepared according to any of the preparation methods described above, wherein the hydrogel undergoes a liquid-solid phase transition at body temperature.

[0023] Through the above technical solution, the preparation process of decellularized matrix hydrogels preserves the natural three-dimensional structure and bioactive components of the extracellular matrix through decellularization. Under body temperature conditions, collagen fibers in the matrix solution spontaneously cross-link through hydrogen bonds and hydrophobic interactions to form a network structure, thereby achieving a liquid-to-solid transition. This phase transition characteristic allows the material to be delivered to the damaged site via injection and form a stable three-dimensional scaffold structure in vivo.

[0024] A third aspect of the present invention provides the application of a thermosensitive decellularized matrix hydrogel prepared according to any of the above-described preparation methods in materials for repairing uterine damage.

[0025] Through the aforementioned technical solution, the thermosensitive decellularized matrix hydrogel, upon contact with the uterine injury site, undergoes a liquid-to-solid transformation triggered by body temperature, forming a three-dimensional scaffold structure that matches the morphology of the injury. The collagen, glycoproteins, and other components retained in the decellularized matrix mimic the physicochemical properties of the natural extracellular matrix, guiding the directional migration of endometrial epithelial cells and the secretion of repair-related factors. The hydrogel's degradation rate is synchronized with the tissue regeneration process, avoiding secondary surgical intervention. This achieves dynamic biomimetic repair of the uterine injury microenvironment, accelerating endometrial functional reconstruction through the synergistic effect of physical barrier construction and bioactivity, while avoiding the mechanical damage or repair delays caused by excessive rigidity or degradation rate mismatches resulting from traditional materials.

[0026] The beneficial effects of this invention are as follows: by culturing human bone marrow mesenchymal stem cells, decellularizing them, and using a temperature-sensitive gelling process, a hydrogel with the characteristics of a natural extracellular matrix is ​​prepared. This hydrogel can achieve rapid liquid-solid phase transition at body temperature and has the advantages of high biocompatibility, excellent temperature response performance, and effective promotion of tissue repair. Attached Figure Description

[0027] Figure 1 This is an immunofluorescence identification image of hBMSC-dECMM in Example 1 of the present invention; Figure 2 This is a macroscopic schematic diagram of the decellularized morphological characterization and component detection of hBMSC-dECMM in Example 1 of the present invention; Figure 3 This is a scanning electron microscope (SEM) image of the decellularized morphology and component detection of hBMSC-dECMM in Example 1 of the present invention; Figure 4 This is a transmission electron microscope (TEM) image of the decellularized morphology and component detection of hBMSC-dECMM in Example 1 of the present invention; Figure 5 This is a schematic diagram of the state of the hydrogel prepared in Example 1 of the present invention.

[0028] Figure 6This is a scanning electron microscope image of the hydrogel prepared in Example 1 of the present invention.

[0029] Figure 7 This is a schematic diagram showing the detection results of the erosion rate and degradation rate of the hydrogel prepared in Example 1 of the present invention.

[0030] Figure 8 This is a schematic diagram of the dECMH biocompatibility test results of the hydrogel prepared in Example 1 of the present invention.

[0031] Figure 9 This is a schematic diagram of the DNA residue detection results of hBMSC-dECMM in Example 1 of the present invention; Figure 10 This is a schematic diagram of the quantitative detection results of total protein, collagen and GAGs in hBMSC-dECMM in Example 1 of the present invention; Figure 11 This is a schematic diagram of the quantitative detection results of EGF, HGF and TGF-β factors in hBMSC-dECMM in Embodiment 1 of the present invention; Figure 12 This is a schematic diagram of HE staining (longitudinal section) comparing the thickness of rat endometrial tissue before and after surgery in Example 1 of the present invention; Figure 13 This is a schematic diagram of HE staining of the mid-segment of the uterus in mice 3 days after surgery, according to Example 1 of the present invention; Figure 14 This is a schematic diagram showing the observation of endometrial thickness and the statistical results of endometrial gland count in each model group of mice 3 days after surgery in Example 1 of this invention.

[0032] Figure 15 These are light micrographs of human bone marrow mesenchymal stem cells from Example 1 of the present invention cultured in serum-free medium containing ascorbic acid for 0, 5, and 10 days.

[0033] Figure 16 Light micrographs of human bone marrow mesenchymal stem cells from Examples 1 and 1-2 of this invention cultured for 10 days in serum-free medium containing 30 μg / mL, 60 μg / mL, and 100 μg / mL ascorbic acid. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The method for preparing a thermosensitive decellularized matrix hydrogel provided in this invention includes the following steps: S1, P3 generation human bone marrow mesenchymal stem cells were used at 10 5 The cells were seeded in 100 mm dishes at a concentration of 100 μg / mL. After the cell confluence was greater than 90%, the cells were cultured in serum-free medium containing 100 μg / mL ascorbic acid for 10 days, with the medium being changed every other day. The ascorbic acid-containing culture medium was then aspirated, and the cells were rinsed with PBS solution and then immersed in ultrapure water at 37°C for 10 min to obtain the biomass matrix material. S2. Add the biomass matrix material to the decellularization solution, extract at 37°C for 5 min, and then wash 3 times with PBS solution to obtain cell-derived decellularized matrix membrane. The content of the decellularization solution is 3% Triton X-100 + 2% SDS. S3. The decellularized matrix membrane is freeze-dried in a freeze dryer and then ground in a grinder at 60 Hz for 120 s to obtain decellularized matrix powder. The particle size of the decellularized matrix powder is: S4. Add pepsin to HCl solution to obtain pepsin hydrochloric acid solution with a concentration of 1 mg / mL. Add the decellularized matrix powder to pepsin hydrochloric acid solution and mix evenly to obtain decellularized matrix solution with a concentration of 10 mg / mL. Transfer to a container and store at 4°C. S5. Add 0.1 mol / L NaOH solution to adjust the pH of the decellularized matrix solution to 7.2-7.4, with a volume ratio of NaOH solution to decellularized matrix solution of 10:1; then add 10×PBS solution, with a volume ratio of 10×PBS solution to decellularized matrix solution of 9:1, place at 37℃ to form a gel, and store at 4℃ to obtain a thermosensitive decellularized matrix hydrogel (i.e., hBMSC-dECMM).

[0036] Information on the raw materials involved in this invention is shown in Table 1.

[0037] Table 1 Raw Material Information The preparation method of the thermosensitive decellularized matrix hydrocoagulation involved in this invention will be specifically described below with reference to specific embodiments and comparative examples: Example 1

[0038] The preparation method of thermosensitive decellularized matrix hydrogel includes the following steps: S1, P3 generation human bone marrow mesenchymal stem cells were used at 10 5The cells were seeded in 100 mm dishes at a concentration of 100 μg / mL. After the cell confluence was greater than 90%, the cells were cultured in serum-free medium containing ascorbic acid at a concentration of 100 μg / mL for 10 days, with the medium being changed every other day. The ascorbic acid-containing culture medium was then aspirated, and the cells were rinsed with PBS solution and then immersed in ultrapure water at 37°C for 10 min to obtain the biomass matrix material. S2. Add the biomass matrix material to the decellularization solution, extract at 37°C for 5 min, and then wash 3 times with PBS solution to obtain cell-derived decellularized matrix membrane. The content of the decellularization solution is 3% Triton X-100 + 2% SDS. S3. The decellularized matrix membrane is freeze-dried in a freeze dryer and then ground in a grinder at 60 Hz for 120 s to obtain decellularized matrix powder with a particle size of 300 μm. S4. Add pepsin to 0.05M HCl solution to obtain a pepsin hydrochloric acid solution with a concentration of 1 mg / mL. Add the decellularized matrix powder to the pepsin hydrochloric acid solution and mix evenly to obtain a decellularized matrix solution with a concentration of 10 mg / mL. Transfer the solution to a container and store it at 4°C. S5. Add 0.1 mol / L NaOH solution to adjust the pH of the decellularized matrix solution to 7.2-7.4, with a volume ratio of NaOH solution to decellularized matrix solution of 10:1; then add 10×PBS solution, with a volume ratio of 10×PBS solution to decellularized matrix solution of 9:1, place at 37℃ to form a gel, and store at 4℃ to obtain a thermosensitive decellularized matrix hydrogel.

[0039] Comparative Example 1 The method for preparing the thermosensitive decellularized matrix hydrogel differs from that in Example 1 in that the concentration of ascorbic acid is 30 μg / mL.

[0040] Comparative Example 2 The method for preparing the thermosensitive decellularized matrix hydrogel differs from that in Example 1 in that the concentration of ascorbic acid is 60 μg / mL.

[0041] The preparation methods of the thermosensitive decellularized matrix hydrogels in Comparative Examples 1-2 are the same as those in Example 1.

[0042] Performance testing

[0043] The following performance tests were conducted on the thermosensitive decellularized matrix hydrogels provided in Example 1 and Comparative Examples 1-2 of this invention.

[0044] 1. Evaluation of decellularization effect The expression of Collagen I, Collagen IV, Laminin, and Fibronectin in the decellularized matrix membranes of Example 1 was detected by immunocytochemical staining. Primary antibodies (Anti-fibronectin 1:100), (Anti-collagen I 1:250), (Anti-laminin 1:50), and (Anti-collagen IV 1:100) were added to the samples, respectively, and incubated at 4°C for 24 h. Then, the corresponding secondary antibodies were added and incubated at 4°C in the dark for 12 h. A suitable amount of DAPI was added for mounting, and the samples were observed and photographed under a fluorescence microscope. Figure 1 As shown. (Through) Figure 1 It can be seen that the decellularized matrix membrane contains abundant fibronectin, laminin, collagen I, and collagen IV. The fibronectin is regularly arranged in a filamentous pattern with a certain directionality, while laminin, collagen I, and collagen IV appear as cloud-like or clump-like structures. No DAPI positive marker was observed, indicating that a decellularized matrix membrane with its original structure and biological activity was successfully obtained.

[0045] The morphological characterization of decellularized matrix membranes was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0046] Samples were fixed with 4% glutaraldehyde at 4°C for 2–4 h, followed by fixation with 1% osmium tetroxide at room temperature in the dark for 2 h. Finally, a gradient ethanol dehydration process was performed (30%, 50%, 70%, 80%, 95% sequentially), 10 min each time, followed by overnight immersion in anhydrous ethanol. The ethanol was replaced with fresh anhydrous ethanol the next day, and the samples were air-dried at room temperature. After crystal plating and mounting, the samples were observed under a scanning electron microscope (SEM). Samples prepared for TEM were dehydrated with anhydrous ethanol in a gradient manner, followed by resin infiltration and embedding polymerization. Embedded tissue blocks were trimmed and routinely ultrathin sections (70 nm thick) were prepared. These sections were stained with 2% uranium acetate saturated alcohol solution in the dark and observed and photographed under a TEM microscope. The results are as follows: Figure 2-4 As shown, through Figure 2 As can be seen, after decellularization, a smooth and viscous decellularized matrix membrane is visible to the naked eye at the bottom of the culture dish; through Figure 3 It can be seen that the decellularized matrix membrane has a dense fibrous network structure, with spherical substances of varying sizes distributed between the network fibers. Figure 4 It can be seen that the nanofibers of the decellularized matrix membrane have alternating light and dark horizontal stripes.

[0047] DNA content determination.

[0048] DNA was extracted from decellularized matrix membrane flaps, and the DNA content was measured using a NanoDrop fluorescence spectrophotometer. The results are as follows: Figure 9 As shown, through Figure 9 It can be seen that, compared with the hBMSC group, there was virtually no DNA residue on the decellularized matrix membrane.

[0049] Content detection of relevant components.

[0050] Approximately 10 mg of sample powder was added to 150 μL of radioimmunoprecipitation assay buffer (P003B, Beyotime Biotechnology) and lysed at 4°C for 24 h, followed by centrifugation at 12,000 rpm for 10 min. The supernatant was collected for protein content determination using the BCA Protein Assay Kit (P0012, Beyotime Biotechnology). The precipitate was resuspended at 37°C with stirring in 100 μL of a solution containing 8 M urea, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol (pH 8.0) for 2 h. The supernatant was collected for collagen content determination using the hydroxyproline assay kit (K555-100). The concentration of sulfated GAG was determined using the dimethyl methylene blue assay kit (JM9048, Hepeng Biological). The sample powder was dissolved in 0.5% bovine serum albumin / PBS solution, incubated at 37°C for 24 h, and centrifuged at 12,000 rpm for 10 min. Collect the supernatant and determine the concentrations of EGF, TGF-β, and HGF using an EGF enzyme-linked immunosorbent assay (ELISA) kit (ELHVEGF-1, RayBiotech), a TGF-β ELISA kit (ELH-TGFb1-1, RayBiotech), and an HGF ELISA kit (ELH-HGF-1, RayBiotech), respectively. The results are as follows: Figure 10 and Figure 11 As shown, through Figure 10 and Figure 11 It can be seen that the total protein content of hBMSC-dECMM is 362±68 mg / g, the collagen content is 98±8.2 mg / g, and the GAGs content is 1.25±0.33 mg / g. The EGF content is (8.02±1)¹⁰. -2 ng / mg, HGF content was (82.6±14)10 -2 The TGF-β content was (28.6±5.8) ng / mg and 10 ng / mg. -2The results indicate that the levels of matrix proteins and cytokines are high, and they possess high biological activity.

[0051] 2. Physicochemical properties characterization of dECMH.

[0052] Morphological observations of dECMH.

[0053] The macroscopic and microscopic morphology of the hydrogel was recorded using a camera and an optical microscope, respectively. The surface morphology and cross-sectional characterization of the material were observed using a scanning electron microscope (the hydrogel samples were soaked in deionized water for 24 hours after preparation and then observed directly in a swollen state). For SEM observation, the gel precursor solution was poured into 48-well plates, and the plates were placed in a 37°C incubator for 1 hour. After complete gelation, the plates were removed, and electron microscopy specimens were prepared according to the following steps: The samples were fixed with 0.25% glutaraldehyde overnight, then dehydrated in a gradient of ethanol solutions of different concentrations, and finally dehydrated once in anhydrous ethanol solution. All samples were then freeze-dried overnight. The freeze-dried samples were cut open with a sharp knife to expose the gel interior, cut into 5×5 mm pieces, sputter-coated with gold, and observed under a scanning electron microscope.

[0054] pass Figure 5 It can be seen that the prepared hydrogel exhibits good fluidity at 4℃ and can pass smoothly through an 18G syringe needle. Under in vitro conditions at 37℃, it can autonomously assemble into a gel with a specific shape; the formed gel no longer has fluidity and can be held with tweezers without damaging its shape. Figure 6 As shown, using scanning electron microscopy to observe its surface ultrastructure, it can be seen that the internal fibers are intertwined with each other, forming porosity of varying sizes, which has more advantages in promoting cell growth and nutrient transport.

[0055] Detection of erosion rate and degradation rate of hydrogels.

[0056] The prepared hydrogel sample was immersed in deionized water to remove excess monomer. The residual liquid on the surface was blotted off with filter paper, and the mass taken at this point was recorded as the initial weight W0 of the hydrogel. 1 mL of a 1×1×1 cm³ hydrogel sample (n=3) was placed in 10 mL of PBS solution (pH 7.4 / 5.5) and collagenase solution (10 U / mL, 5 U / mL), respectively, and continuously shaken at low speed at 37℃. Samples were removed at different time points (1, 3, 5, 7, 10, 15, 20 d) and (6, 12, 24, 36, 48, 60, 72 h), dried with filter paper, and weighed using an electronic balance. This weight was recorded as Wnd. The results are shown in Table 2 below. Figure 7As shown. Calculate the erosion rate and degradation rate using the formula: (W0-Wnd) / W0 × 100%.

[0057] Table 2 Statistical results of erosion rate and degradation rate at different time points like Figure 7 As shown in Table 2, the degradability of hydrogels is crucial because they can be replaced by regenerating tissue. Extracellular matrix hydrogels degrade rapidly in the presence of natural collagenases in vivo. Approximately 90% of dECMH was degraded after 72 hours in the presence of collagenase (10 U / mL). However, the erosion rate of dECMH was slower. After 20 days in PBS, the erosion rate was close to 90% at pH 5.5 and 60% at pH 7.4, indicating the stability of the hydrogels in the absence of enzymes.

[0058] 3. dECMH biocompatibility testing Remove the HaCaT cell culture dish from the cell culture incubator, discard the culture medium, and digest the cells with 0.25% trypsin. Stop the digestion with complete culture medium. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium and mix well. Immerse the hydrogel material in 75% ethanol for 20 min, irradiate with UV light in a clean bench for 15-20 min, then wash three times with PBS. Then, aspirate 10 µL of EMCs cell suspension, inject it into a cell counting chamber, and dilute to a density of 5 × 10⁻⁶. 3 The culture medium was added at a rate of 1 / mL to each well of a 24-well plate and co-cultured with the hydrogel material at 37°C for 1, 3, and 5 days. The culture medium was then discarded, and 500 µL of the prepared CCK-8 solution was added to each well. The plates were then incubated for 4 h. Finally, 200 µL was taken from each well and added to a 96-well plate, and the OD was measured at 450 nm.

[0059] like Figure 8 As shown, cell compatibility tests indicated that dECM hydrogel (10 mg / mL) did not exhibit any significant toxicity to HaCaT cells.

[0060] 4. Uterine function recovery assessment Animal models and grouping Twenty female SD rats (200-220 g, 8 weeks) with normal estrous cycles (40 uteruses) were divided into four groups: Group A (normal control group); Group B (dECMH); Group C (dECMH & dECMM); and Group D (natural repair group). Tissue samples were collected 3 days post-surgery. After anesthetizing the rats in each group with isoflurane (2-3%, inhalation), the uterus was exposed through a midline incision in the lower abdomen of Groups B, C, and D. The right uterus was cut along its long axis, and the endometrium was scraped away with a T10 scalpel blade until the uterine surface was rough and bleeding. Hemostasis was achieved with saline-soaked gauze, and the sutures were closed with 6-0 absorbable sutures. The left uterus was treated in the same manner. In group A rats under anesthesia, only the lower abdominal wall was incised and sutured, without treating the uterus. In group B, after closing the uterine cavity, dECMH was injected into the cavity until saturation; after 2 minutes, the hydrogel could not be aspirated, indicating gelation, and the abdominal wall was then sutured layer by layer. In group C, after closing the uterine cavity, dECMH hydrogel was injected into the uterine cavity, and then dECMM (3 cm in diameter) was placed around the uterine incision. In group D, the uterine incision was sutured, the uterine cavity was closed, and the abdominal wall was sutured layer by layer, allowing the damaged endometrium to repair itself naturally. Animals receiving the same treatment were placed in the same cages. To ensure the survival of the rats after surgery, several measures were taken, including maintaining body temperature with postoperative infrared irradiation, feeding glucose saline to replenish fluids and energy, and timely changing of the lining to prevent wound infection.

[0061] Evaluation of the in vivo uterine regeneration effect.

[0062] Histological analysis was used to further evaluate the repair effect of the dECM material. Hematoxylin and eosin (H&E) staining was performed on the longitudinal and cross-sectional sections of the mid-uterine segment to observe endometrial regeneration and remodeling 3 days post-surgery. H&E was used to assess endometrial thickness and glandular count. Figure 12 As shown, this invention constructed a severe uterine injury model in rats. Preoperative (left image) HE staining histological observation showed that the endometrial structure was intact, the endometrial surface was wavy, and the glandular epithelium and luminal epithelial cells were intact; postoperative (right image) the entire uterine wall was significantly thinned, the endometrial continuity was damaged, wrinkles were reduced, and the glandular epithelium and endometrial epithelial cells showed a low columnar change. Figure 13 and Figure 14 As shown, 3 days post-surgery, there was no significant difference in endometrial thickness between the dECMH group and the dECMH&dECMM group and the normal control group (left side). Using a 20× objective lens, the number of endometrial glands per field of view was observed; there was no significant difference between the dECMH group and the normal control group in the number of endometrial glands. The endometrial thickness in the natural repair group (right side) was significantly thinner than the other three groups, and the number of glands (18.2±2.9) was also significantly less than the other three groups.

[0063] Figure 15 The image shows a scanning electron microscope (SEM) image of human bone marrow mesenchymal stem cells (BMSCs) cultured for 10 days in serum-free medium containing ascorbic acid (100 μg / mL) as in Example 1. Before the addition of ascorbic acid (pre-VC), the cell confluence was approximately 90%, and the BMSCs were spindle-shaped and relatively tightly packed, but intercellular spaces still existed. After 5 days of ascorbic acid stimulation (VC-5d), the outline of the BMSCs became blurred, and the extracellular matrix filled the intercellular spaces. After 10 days of ascorbic acid stimulation (VC-10d), the BMSCs were tightly packed together and secreted a large amount of extracellular matrix. If the stimulation exceeded 10 days, the basal matrix membrane gradually rolled inward from the periphery towards the center, leading to the overall structural destruction of the decellularized matrix membrane.

[0064] Figure 16 The images show scanning electron microscope (SEM) images of human bone marrow mesenchymal stem cells from Examples 1 and 2, cultured in serum-free medium containing ascorbic acid for 10 days. From left to right, the concentrations of ascorbic acid are 30 μg / mL, 60 μg / mL, and 100 μg / mL, respectively. It can be seen that when the concentration of ascorbic acid is too low, the decellularized matrix membrane formed becomes correspondingly thinner.

[0065] Finally, it should be noted that the above descriptions are merely optional examples of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive decellularized matrix hydrogel, characterized in that, Includes the following steps: S1. Human bone marrow mesenchymal stem cells were cultured in a serum-free culture medium containing ascorbic acid to obtain biomass matrix material; The culture of the human bone marrow mesenchymal stem cells includes the following steps: The human bone marrow mesenchymal stem cells were cultured in serum-free medium containing ascorbic acid for 10 days, with the medium being changed every other day. The culture medium containing ascorbic acid was aspirated, and the material was rinsed with PBS solution and then immersed in ultrapure water at 37°C for 10 min to obtain the biomass matrix material. The concentration of ascorbic acid in the serum-free culture medium is 100 μg / mL; S2. The biomass matrix material is added to a decellularization solution for decellularization treatment to obtain a decellularized matrix membrane; The decellularization solution contained 3% Triton X-100 + 2% SDS; S3. The decellularized matrix membrane is freeze-dried and ground to obtain decellularized matrix powder; S4. Add the decellularized matrix powder to the pepsin hydrochloric acid solution and mix well to obtain a decellularized matrix solution; S5. Add NaOH to adjust the pH of the decellularized matrix solution to 7.2-7.4, then add 10×PBS solution, place at 37℃ to form a gel, and store at 4℃ to obtain a thermosensitive decellularized matrix hydrogel.

2. The method for preparing the thermosensitive decellularized matrix hydrogel according to claim 1, characterized in that, The decellularization process includes the following steps: After removing the ultrapure water from the bio-based matrix material, a decellularization solution was added, and the mixture was extracted at 37°C for 5 minutes. Then, it was rinsed three times with PBS solution to obtain a decellularized matrix membrane.

3. The method for preparing the thermosensitive decellularized matrix hydrogel according to claim 1, characterized in that, The decellularized matrix membrane can be stored at 4°C for 2-3 weeks.

4. The method for preparing the thermosensitive decellularized matrix hydrogel according to claim 1, characterized in that, The concentration of the pepsin hydrochloric acid solution is 1 mg / mL; The concentration of the decellularized matrix solution is 10 mg / mL.

5. The method for preparing the thermosensitive decellularized matrix hydrogel according to claim 1, characterized in that, In step S5, the molar concentration of the NaOH solution is 0.1 mol / L, and the volume ratio of the NaOH solution to the decellularized matrix solution is 10:

1. The volume ratio of the 10×PBS solution to the decellularized matrix solution is 9:

1.

6. A thermosensitive decellularized matrix hydrogel, characterized in that, The hydrogel is prepared according to any one of claims 1-5, and undergoes a liquid-solid phase transition at body temperature.

7. The application of a thermosensitive decellularized matrix hydrogel prepared by the method according to any one of claims 1-5 in the preparation of materials for repairing uterine damage.

Images