Metformin-induced stem cell-derived extracellular matrix material, and preparation method and application thereof

Abstract

The application discloses a metformin-induced stem cell-derived extracellular matrix material and a preparation method and application thereof, and relates to the technical field of biological materials. The preparation method comprises the following steps: inoculating mesenchymal stem cells on a pretreated culture carrier, and using a culture system containing metformin to induce and treat the mesenchymal stem cells; adding ascorbic acid to induce and culture extracellular matrix secretion; carrying out decellularization treatment and nuclease treatment on the metformin-induced extracellular matrix; collecting the metformin-induced stem cell-derived extracellular matrix, mixing the metformin-induced stem cell-derived extracellular matrix with an aqueous medium, and enriching and forming an extracellular matrix precipitate through centrifugal treatment; and sequentially carrying out freezing treatment and thawing treatment on the extracellular matrix precipitate to obtain the metformin-induced stem cell-derived extracellular matrix material. The metformin is used to induce the mesenchymal stem cells to secrete an engineered extracellular matrix, and the engineered extracellular matrix is used to promote the repair of bone defects, skeletal muscle injuries and musculoskeletal joint defects in a diabetic environment.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to extracellular matrix materials derived from metformin-induced stem cells, their preparation methods, and applications. Background Technology

[0002] Diabetes mellitus is a common chronic metabolic disease. A prolonged high-sugar environment not only disrupts glucose and lipid metabolism but also affects the normal repair processes of bone and skeletal muscle tissue. In a diabetic environment, bone tissue is prone to bone loss, osteoporosis, and increased bone fragility. Skeletal muscle injuries are also prone to delayed repair, increased fibrosis, and insufficient muscle fiber maturation. Because bone and skeletal muscle are structurally and functionally interdependent, the repair of diabetes-related bone injuries, muscle injuries, and musculoskeletal defects is significantly more difficult. How to promote musculoskeletal tissue regeneration and restore motor function within the pathological microenvironment of diabetes remains a pressing issue in the field of tissue repair.

[0003] The extracellular matrix (ECM) is a crucial component of the cellular microenvironment, providing biochemical signals and physical support for cell adhesion, migration, proliferation, differentiation, and tissue remodeling. Mesenchymal stem cell-derived ECM contains various matrix components such as collagen, fibronectin, glycosaminoglycans, and elastin, and can mimic the natural tissue microenvironment to some extent. However, under pathological conditions such as diabetes, the local tissue microenvironment is often accompanied by dysregulation of cellular energy metabolism, decreased mitochondrial function, and enhanced abnormal glycolysis. Relying solely on ordinary ECM materials to provide passive support or adhesion sites is insufficient to fully meet the comprehensive needs for cellular metabolic regulation, osteogenic differentiation, and myogenic differentiation in the repair of diabetic musculoskeletal defects.

[0004] Currently, the preparation of extracellular matrix (ECM) materials derived from mesenchymal stem cells (MSCs) typically focuses on collecting naturally secreted ECM or combining ECM with hydrogel matrices to improve plasticity and filling properties. However, these methods often lack active regulation of the metabolic state of the source cells and the matrix secretion phenotype, resulting in ECMs that are deficient in functional guidance, component enrichment, and molding stability. Furthermore, direct drug administration is easily limited by factors such as rapid local diffusion and short retention time, while live cell transplantation is easily affected by cell viability, colonization efficiency, and the pathological microenvironment. Therefore, there is an urgent need to construct an engineered ECM material that does not rely on continuous survival of live cells and direct drug release, while retaining induced repair signals, enriching ECM components, and forming a stable gel structure for the repair of bone defects, fractures, skeletal muscle injuries, and musculoskeletal fusion defects in diabetic settings. Summary of the Invention

[0005] One objective of the first aspect of this invention is to provide a method for preparing extracellular matrix material derived from metformin-induced stem cells, thereby solving the technical problems in the prior art where ordinary extracellular matrix materials have insufficient functional guidance, limited ECM enrichment and gelation capabilities, and are difficult to effectively regulate mitochondrial metabolism in damaged cells under diabetic conditions and promote musculoskeletal tissue repair.

[0006] Another objective of the first aspect of this invention is to further meet the differentiated needs of different stages of musculoskeletal injury repair in diabetes for matrix remodeling, cell adhesion, ECM deposition and metabolic microenvironment regulation.

[0007] The second aspect of the present invention aims to provide an extracellular matrix material derived from metformin-induced stem cells prepared according to the above-described preparation method.

[0008] The third aspect of this invention aims to provide the use of the above-mentioned metformin-induced stem cell-derived extracellular matrix material in the preparation of materials or formulations for the repair of diabetes-related musculoskeletal defects.

[0009] According to the first aspect of the present invention, the present invention provides a method for preparing extracellular matrix material derived from metformin-induced stem cells, comprising the following steps: The culture medium is pretreated to obtain a pretreated culture medium; Mesenchymal stem cells were seeded onto the pretreated culture medium and induced using a culture system containing metformin. After the mesenchymal stem cells reach a preset degree of fusion, ascorbic acid is added to induce extracellular matrix secretion culture, so that the mesenchymal stem cells secrete to form metformin-induced extracellular matrix. The metformin-induced extracellular matrix was subjected to decellularization and nuclease treatment to obtain a metformin-induced stem cell-derived extracellular matrix. The metformin-induced stem cell-derived extracellular matrix was collected and mixed with an aqueous medium, then enriched by centrifugation to form an extracellular matrix precipitate. The extracellular matrix precipitate was subjected to freezing and thawing treatments in sequence, so that the enriched extracellular matrix precipitate was physically cross-linked to form extracellular matrix material derived from metformin-induced stem cells.

[0010] Optionally, the concentration of metformin in the culture system is any value between 400 μM and 600 μM.

[0011] Optionally, the induction time of the mesenchymal stem cells by metformin is any value between 60h and 84h.

[0012] Optionally, metformin may be added during the proliferation culture period from the time the mesenchymal stem cells are seeded until the preset degree of fusion is reached, during the extracellular matrix induction culture period after the addition of ascorbic acid, or during the entire culture period from the time the mesenchymal stem cells are seeded until the end of the induction culture on the pretreated culture medium.

[0013] Optionally, the mesenchymal stem cells are bone marrow mesenchymal stem cells, and the seeding density of the mesenchymal stem cells is 1000 cells / cm². 2 -10000 pieces / cm 2 Any value among them.

[0014] Optionally, the preset fusion degree is any value between 80% and 95%, and the concentration of ascorbic acid is any value between 50 μM and 200 μM.

[0015] Optionally, the preprocessing includes: The culture carrier was coated with a gelatin solution, followed by cross-linking with glutaraldehyde and blocking with ethanolamine; among these, The mass concentration of the gelatin solution is any value between 0.1% and 0.5%, the mass concentration of the glutaraldehyde is any value between 0.5% and 2%, and the concentration of the ethanolamine is any value between 0.5M and 2M.

[0016] Optionally, the freezing temperature is -60℃ to -100℃, the freezing time is 6h to 24h, and the thawing temperature is 25℃ to 40℃.

[0017] According to a second aspect of the present invention, the present invention also provides an extracellular matrix material derived from metformin-induced stem cells prepared according to the preparation method described above.

[0018] According to a third aspect of the present invention, the present invention also provides the use of the above-mentioned metformin-induced stem cell-derived extracellular matrix material in the preparation of materials or formulations for the repair of diabetes-related musculoskeletal defects.

[0019] This invention utilizes a continuous process involving metformin induction, extracellular matrix secretion, decellularization, centrifugation enrichment, and freeze-thaw gelation to transform the regulatory effect of metformin on the metabolic state and secretory function of mesenchymal stem cells into the compositional and structural advantages of cell-free extracellular matrix materials. Compared with extracellular matrix derived from ordinary mesenchymal stem cells, the cryogel obtained in this embodiment not only retains natural matrix components such as collagen, fibronectin, and elastin in the extracellular matrix, but also, through metformin induction, endows it with engineered matrix characteristics more suitable for cell adhesion, tissue repair, and regulation of the microenvironment in diabetic injuries. Furthermore, centrifugation enriches the dispersed extracellular matrix components derived from metformin-induced stem cells into a high-density precipitate. Then, through freezing and thawing, the enriched extracellular matrix undergoes physical cross-linking under the action of ice crystal displacement, local concentration, and fiber network rearrangement, forming an extracellular matrix cryogel with a three-dimensional network structure. This cryogel combines ECM component enrichment, structural shaping, and local tissue filling adaptability, making it better suited for microenvironment reconstruction at defect sites.

[0020] Furthermore, by regulating the timing of metformin addition, this invention can achieve phased regulation of the composition, structure, and bioactivity of Met-ECM, thereby improving the adaptability of the cryogel obtained from subsequent decellularization, centrifugation enrichment, and freeze-thaw gelation to the microenvironment of diabetic musculoskeletal injury.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is a schematic flowchart of a method for preparing extracellular matrix material derived from metformin-induced stem cells according to an embodiment of the present invention; Figure 2 This is a schematic flowchart of a method for preparing extracellular matrix material derived from metformin-induced stem cells according to an embodiment of the present invention. Figure 3 This is an evaluation result of the intervention effect of metformin concentration on mitochondrial-related expressed genes according to an embodiment of the present invention; Figure 4 This is the evaluation result of the intervention effect of metformin at different induction times according to Example 1 and Comparative Example 1 of the present invention; Figure 5 This is an evaluation of the effect of metformin on iterative bone marrow mesenchymal stem cell intervention in Example 1 and Comparative Example 1 of the present invention; Figure 6 This is an evaluation of the intervention effect of metformin on mitochondrial and extracellular matrix-related functional genes in Example 1 and Comparative Example 1 of the present invention; Figure 7 This is an evaluation of the intervention effect of metformin on matrix remodeling-related enzyme molecular markers in Example 1 and Comparative Example 1 of the present invention; Figure 8 This is the result of the Western blot experiment on the protein immunoblotting of metformin on mitochondrial function in Example 1 and Comparative Example 1 of the present invention; Figure 9 This is an evaluation of the intervention effect of metformin at different addition times on genes related to extracellular matrix synthesis and degradation according to Examples 1-3 and Comparative Example 2 of the present invention; Figure 10 The results of light microscopy, scanning electron microscopy, transmission electron microscopy and immunofluorescence observations of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention; Figure 11 The results are the DNA content determination results before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention; Figure 12 The results are the determination results of glycosaminoglycan content before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention; Figure 13 The results are the collagen content determination results before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention; Figure 14 This is a light micrograph of the extracellular matrix material after decellularization according to Embodiment 1 of the present invention; Figure 15 This is a light micrograph of the extracellular matrix cryogel prepared according to Example 1 of the present invention; Figure 16 This is a biocompatibility test of the extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 of the present invention; Figure 17 The results of alizarin detection are from the extracellular matrix materials prepared according to Example 1 and Comparative Examples 1-2 of this invention. Figure 18 This is an evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Examples 1-2 of the present invention on genes related to promoting osteodifferentiation; Figure 19 This is an immunofluorescence staining image showing that the extracellular matrix material prepared according to Example 1 and Comparative Examples 1-2 of the present invention promotes myogenic differentiation of mouse C2C12 cells. Figure 20This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Examples 1-2 of the present invention on promoting myoblastic differentiation of mouse C2C12 cells; Figure 21 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on restoring the osteogenic differentiation ability of bone marrow mesenchymal stem cells damaged by high sugar and high lipid. Figure 22 This is an evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on the recovery of myogenic differentiation ability of C2C12 cells in mice with high glucose and high lipid injury; Figure 23 This describes the effect of extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on the mitochondrial oxygen consumption rate of cells damaged by high sugar and high lipid. Figure 24 Evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on enhancing the mitochondrial oxidative phosphorylation capacity of cells damaged by high sugar and high lipid; Figure 25 This describes the effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on the extracellular acidification rate of cells damaged by high sugar and high lipids. Figure 26 This is an evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on reducing the level of glycolysis in cells damaged by high sugar and high lipids; Figure 27 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on promoting tissue repair and recovery of motor function after diabetic muscle loss; Figure 28 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on improving grip and gait function in diabetic mice after muscle injury. Figure 29 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on promoting bone tissue repair after bone injury in diabetic mice. Detailed Implementation

[0023] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, it should be noted that, for ease of description, only the parts relevant to this application are shown in the accompanying drawings, not the entire structure. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0025] The terms “comprising” and “having”, and any variations thereof, used in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0026] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0027] Figure 1 This is a schematic flowchart illustrating a method for preparing extracellular matrix material derived from metformin-induced stem cells according to an embodiment of the present invention. Figure 2 This is a schematic flowchart illustrating a method for preparing extracellular matrix material derived from metformin-induced stem cells according to an embodiment of the present invention.

[0028] like Figure 1 and Figure 2 As shown, the present invention provides a method for preparing extracellular matrix material derived from metformin-induced stem cells, comprising the following steps: Step S100: Pre-treat the culture medium to obtain a pre-treated culture medium; Step S200: Mesenchymal stem cells are seeded onto a pretreated culture medium and induced to develop in a culture system containing metformin. Step S300: After the mesenchymal stem cells reach the preset degree of fusion, ascorbic acid is added to induce extracellular matrix secretion culture, so that the mesenchymal stem cells secrete to form metformin-induced extracellular matrix. Step S400: Decellularize and nuclease the metformin-induced extracellular matrix to obtain metformin-induced stem cell-derived extracellular matrix; Step S500: Collect metformin-induced stem cell-derived extracellular matrix and mix it with an aqueous medium, then centrifuge to enrich and form an extracellular matrix precipitate; Step S600: The extracellular matrix precipitate is subjected to freezing and thawing treatments in sequence, so that the enriched extracellular matrix precipitate is physically cross-linked to form metformin-induced stem cell-derived extracellular matrix material, namely Met-ECM material (Metformin-induced extracellular matrix).

[0029] In this embodiment, the method for preparing metformin-induced stem cell-derived extracellular matrix material involves first pretreating the culture carrier with gelatin coating, glutaraldehyde crosslinking, and ethanolamine blocking to construct a suitable culture interface for mesenchymal stem cell adhesion and extracellular matrix secretion. Subsequently, mesenchymal stem cells are seeded onto the pretreated culture carrier, and a metformin-containing culture system is used to induce their formation. Metformin regulates the metabolic state and matrix secretion behavior of mesenchymal stem cells during cell proliferation and / or extracellular matrix formation. Once the mesenchymal stem cells reach a predetermined degree of fusion, ascorbic acid is added to promote the secretion and deposition of collagen and related extracellular matrix components. The extracellular matrix (ECM) is formed by the accumulation of cells, resulting in metformin-induced extracellular matrix (ECM). Cellular components and residual nucleic acids are then removed through decellularization and nuclease treatment to obtain cell-free metformin-induced stem cell-derived ECM. This ECM is then collected and mixed with an aqueous medium, and centrifuged to enrich the dispersed ECM components, forming an ECM precipitate. Finally, the ECM precipitate undergoes freezing and thawing, allowing the enriched ECM precipitate to physically cross-link during the freeze-thaw process, forming a metformin-induced stem cell-derived ECM cryogel, i.e., metformin-induced stem cell-derived ECM material, or Met-ECM material. Here, the ECM material is in a gel state.

[0030] In this embodiment, a continuous process involving metformin induction, extracellular matrix secretion, decellularization, centrifugation enrichment, and freeze-thaw gel formation transforms the regulatory effect of metformin on the metabolic state and secretory function of mesenchymal stem cells into the compositional and structural advantages of cell-free extracellular matrix materials. Compared with extracellular matrix derived from ordinary mesenchymal stem cells, the cryogel obtained in this embodiment not only retains natural matrix components such as collagen, fibronectin, and elastin in the extracellular matrix, but also, through metformin induction, endows it with engineered matrix characteristics more suitable for cell adhesion, tissue repair, and regulation of the microenvironment in diabetic injuries. Furthermore, centrifugation enriches the dispersed extracellular matrix components derived from metformin-induced stem cells into a high-density precipitate. Then, through freezing and thawing, the enriched extracellular matrix undergoes physical cross-linking under the action of ice crystal displacement, local concentration, and fiber network rearrangement, forming an extracellular matrix cryogel with a three-dimensional network structure. This cryogel combines ECM component enrichment, structural shaping, and local tissue filling adaptability, making it better suited for microenvironment reconstruction at defect sites.

[0031] In this embodiment, the metformin-induced stem cell-derived extracellular matrix cryogel does not merely function as a passive scaffold material, but rather regulates the energy metabolism of cells under high glucose and high lipid injury conditions. It enhances the oxidative phosphorylation capacity of damaged cells and reduces their glycolysis levels, promoting a shift from a stress-induced, inefficient glycolytic metabolic state to a more stable and efficient mitochondrial oxidative phosphorylation metabolic state, thereby achieving mitochondrial metabolic reprogramming. Since bone formation, fracture healing, muscle fiber maturation, and musculoskeletal regeneration all depend on sufficient mitochondrial energy supply and metabolic homeostasis, this cryogel can provide a favorable metabolic microenvironment for osteogenic differentiation of bone marrow mesenchymal stem cells, myogenic differentiation of skeletal muscle cells, and regeneration of damaged tissue under diabetic conditions, thereby promoting the repair of diabetes-related bone defects, fractures, skeletal muscle injuries, and musculoskeletal defects.

[0032] Furthermore, by transforming the metformin-induced cellular secretory microenvironment into a cell-free, engineered extracellular matrix material that can be preserved, enriched, and gelled, this material avoids the dependence of live cell transplantation on cell survival, colonization, and phenotypic stability. It also overcomes the limitations of conventional extracellular matrix materials, such as insufficient functional targeting, difficulty in enriching ECM content, and limited gelation properties, making it more suitable for repairing bone defects, fractures, skeletal muscle injuries, and musculoskeletal fusion defects in diabetic settings.

[0033] In step S100, pretreatment of the culture medium creates a functional interface on its surface that facilitates the adhesion, spreading, and long-term secretion of extracellular matrix by mesenchymal stem cells (MSCs). Gelatin coating provides adhesion sites similar to the natural extracellular matrix, improving the initial adhesion efficiency of MSCs on the culture medium surface. Glutaraldehyde cross-linking enhances the stability of the gelatin layer during culture, reducing coating shedding during subsequent medium changes, induction, and decellularization. Ethanolamine blocking neutralizes or blocks residual aldehyde groups, reducing the toxicity of residual glutaraldehyde to cells, thus providing a stable and low-toxicity culture interface for subsequent metformin induction and ECM deposition.

[0034] In this embodiment, the mass concentration of the gelatin solution is any value between 0.1% and 0.5%, the mass concentration of glutaraldehyde is any value between 0.5% and 2%, and the concentration of ethanolamine is any value between 0.5M and 2M. That is, the mass concentration of the gelatin solution can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, or any other value between 0.1% and 0.5%. The mass concentration of glutaraldehyde can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, or 2.0%, or any other value between 0.5% and 2%. The concentration of ethanolamine can be 0.5M, 0.8M, 1.0M, 1.2M, 1.5M, 1.8M, or 2.0M, or any other value between 0.5M and 2M. By controlling the gelatin solution, glutaraldehyde, and ethanolamine within the aforementioned ranges, a continuous pretreatment system capable of forming an adhesion layer, achieving cross-linking stability, and blocking residues can be constructed on the culture carrier surface. Gelatin provides a biomimetic matrix interface suitable for mesenchymal stem cell adhesion and spreading; glutaraldehyde enhances the structural stability of the gelatin coating layer during long-term culture and decellularization; and ethanolamine further blocks residual aldehyde groups to reduce cytotoxicity. The synergistic effect of these three components endows the culture carrier with cell adhesion, coating stability, and biocompatibility, thereby promoting the formation of a uniformly distributed, fully deposited, and easily decellularized, scraped, collected, and centrifuged engineered extracellular matrix by mesenchymal stem cells under metformin induction. This provides a stable foundation for the preparation of high ECM-content extracellular matrix cryogels.

[0035] In step S200, mesenchymal stem cells are induced using a metformin-containing culture system. This allows for the regulation of cellular energy metabolism, mitochondrial function, and secretory phenotype before or during ECM secretion. The resulting ECM is no longer the matrix deposit found under ordinary culture conditions, but rather an engineered extracellular matrix regulated by metformin metabolism. The mechanism lies in metformin's ability to regulate AMPK-related energy metabolism pathways, improve cellular metabolic homeostasis, and further influence the expression and secretion of ECM-related components such as collagen, fibronectin, and matrix remodeling enzymes. This results in a more targeted repair mechanism and enhanced microenvironment regulation capabilities in the obtained ECM.

[0036] In step S300, ascorbic acid is added after the mesenchymal stem cells reach a preset fusion level, which further promotes the secretion, deposition, and maturation of the extracellular matrix. Ascorbic acid is an important cofactor in the hydroxylation of proline and lysine during collagen synthesis, which is beneficial for the stable formation and fibrotic deposition of collagen molecules. Therefore, this step can improve the amount and structural integrity of ECM formation, allowing metformin-induced mesenchymal stem cells to fully deposit matrix components such as collagen and fibronectin, providing a foundation for subsequent decellularization to obtain Met-ECM with a continuous fibrous network structure.

[0037] In step S400, decellularization removes cellular components such as the cell membrane, cytoplasm, and nucleus while preserving the main structural and functional components of the extracellular matrix as much as possible. Nuclease treatment further degrades residual DNA, reducing cellular residues and potential immune stimulation risks in the material. This transforms the metformin-induced cellular secretory environment into a cell-free ECM material, allowing the resulting material to primarily rely on extracellular matrix components for tissue repair and microenvironment regulation, rather than depending on live cell transplantation or direct metformin administration. This avoids the problems of low cell viability and susceptibility to pathological microenvironment in live cell transplantation, and also avoids the issues of insufficient local retention, short duration of action, and difficulty in maintaining effective concentrations associated with direct metformin administration. Here, decellularization is performed using a decellularization solution containing Triton and NH4OH, and nuclease treatment is performed using deoxyribonuclease.

[0038] In this embodiment, the decellularization solution includes PBS, Triton (0.1%-1% by volume), and NH4OH (5mM-50mM), and the concentration of deoxyribonuclease is 50U / mL-200U / mL. The volume fraction of Triton can be 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.8%, or 1.0%, or any other value between 0.1% and 1%; the concentration of NH4OH can be 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, or 50mM, or any other value between 5mM and 50mM; and the concentration of deoxyribonuclease can be 50U / mL, 80U / mL, 100U / mL, 120U / mL, 150U / mL, 180U / mL, or 200U / mL, or any other value between 50U / mL and 200U / mL.

[0039] In this embodiment, by setting the components of the decellularization solution within the aforementioned ranges, PBS provides a buffer system close to the physiological environment for the decellularization reaction. Triton primarily disrupts the cell membrane and removes membranous and cytoplasmic components, NH4OH further assists in cell lysis and the release of nuclear components, and deoxyribonuclease degrades residual nucleic acids. Through the combination of the above components and concentration ranges, it is possible to effectively remove cell and nucleic acid residues while preserving as much as possible the extracellular matrix fiber network and functional matrix components induced by metformin. This results in Met-ECM with both low cell residue and good biosafety, as well as structural integrity suitable for subsequent scraping, centrifugation, enrichment, and freeze-thaw physical cross-linking into a gel.

[0040] In step S500, decellularized Met-ECM is collected by cell scraping, mixed with an aqueous medium, and then centrifuged. This process concentrates and precipitates the fibrous, membrane-like, or sheet-like ECM components that were originally attached to the surface of the culture carrier, forming an extracellular matrix precipitate with a higher ECM content. This step is a key enrichment step in the preparation of the cryogel in this application. Its function is not merely to collect materials, but to compress and enrich the dispersed Met-ECM components through centrifugation, increasing the ECM content per unit volume and providing a foundation for the subsequent formation of a cryogel with sufficient structural strength and biological activity. Here, the collection method is to collect metformin-induced stem cell-derived extracellular matrix by cell scraping.

[0041] In this embodiment, the aqueous medium is at least one of deionized water, purified water, physiological saline, or phosphate buffer. The centrifugation speed is 2000 rpm / min-5000 rpm / min, and the centrifugation time is 5 min-15 min. The aqueous medium can be deionized water, purified water, physiological saline, or phosphate buffer, or any combination of two or more of these. The centrifugation speed can be 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, or 5000 rpm, or any other value within the range of 2000 rpm / min-5000 rpm. The centrifugation time can be 5 min, 6 min, 8 min, 10 min, 12 min, 14 min, or 15 min, or any other value within the range of 5 min-15 min. Synergistic control of aqueous media, centrifugation speed, and centrifugation time enables the effective enrichment of metformin-induced ECM, which was originally attached to the surface of the culture carrier or dispersed in the aqueous phase, into an extracellular matrix precipitate with relatively intact structure, suitable water content, and high ECM content. This precipitate is more likely to form a stable extracellular matrix cryogel during subsequent freezing and thawing through ice crystal displacement, local concentration, and fiber entanglement, thereby improving the enrichment degree, structural continuity, and local tissue filling adaptability of the cryogel.

[0042] In step S600, the enriched ECM precipitate undergoes freezing and thawing treatment, which utilizes the freeze-thaw process to promote physical cross-linking and network reconstruction of the ECM components. During freezing, ice crystals form in the aqueous medium. The growth of these ice crystals displaces and concentrates the ECM fibers and protein components in the interstitial regions of the ice crystals, further enriching the ECM components locally. During thawing, after the ice crystals disappear, the previously compressed and concentrated ECM fibers undergo entanglement, rearrangement, and non-covalent interactions such as hydrogen bonding / electrostatic interactions, thereby forming a cryogel with a three-dimensional network structure. This process does not require the introduction of additional chemical cross-linking agents, which helps maintain the natural biological activity of ECM and reduces the risk of residual chemical cross-linking.

[0043] Figure 3 This is an evaluation result of the intervention effect of metformin concentration on mitochondrial-related expressed genes according to an embodiment of the present invention.

[0044] In a further embodiment, the concentration of metformin in the culture system is any value within the range of 400 μM to 600 μM. That is, the concentration of metformin can be 400 μM, 450 μM, 500 μM, 550 μM, or 600 μM, or any other value within the range of 400 μM to 600 μM. In this embodiment, by controlling the concentration of metformin within the range of 400 μM to 600 μM, the inducing effect of metformin on the metabolic state and extracellular matrix secretion phenotype of mesenchymal stem cells can be fully utilized without significantly inhibiting the growth state of mesenchymal stem cells. This allows the cells to secrete engineered extracellular matrix with stronger repair orientation, such as OPA1, SIRT3, and ATP5A.

[0045] like Figure 3 As shown, the expression levels of mitochondrial energy metabolism-related genes such as OPA1, SIRT3, and ATP5A were dose-dependently upregulated with increasing metformin concentration. The 500 μM treatment group showed significantly higher levels of all indicators compared to the low concentration group, indicating that the above concentration range can more effectively enhance the induction effect of metformin on mesenchymal stem cells, thus providing suitable induction conditions for the subsequent preparation of Met-ECM materials with good cell adhesion, metabolic regulation, and musculoskeletal repair potential. Specifically, OPA1 protein is mainly involved in mitochondrial inner membrane fusion, mitochondrial cristae structure maintenance, and mitochondrial network stability; its increased expression usually indicates enhanced mitochondrial morphological integrity and mitochondrial homeostasis. SIRT3 protein is a deacetylase mainly located in mitochondria, capable of regulating mitochondrial energy metabolism, antioxidant defense, and oxidative phosphorylation; its increased expression usually indicates improved mitochondrial metabolic regulation and antioxidant capacity. ATP5A protein is an α-subunit-related protein of mitochondrial ATP synthase complex V, involved in ATP generation; its increased expression usually reflects enhanced mitochondrial oxidative phosphorylation and energy supply capacity.

[0046] Figure 4 The results are evaluations of the intervention effects of metformin at different induction times according to Example 1 and Comparative Example 1 of the present invention.

[0047] In a further embodiment, the induction time of metformin on mesenchymal stem cells is any value between 60h and 84h. That is, the induction time of metformin on mesenchymal stem cells during proliferation can be 60h, 72h, or 84h, or any other value within the range of 60h-84h. In this embodiment, by controlling the induction time of metformin on mesenchymal stem cells during proliferation within the range of 60h-84h, metformin can continuously act on mesenchymal stem cells before the cells reach a preset degree of fusion, fully regulating their mitochondrial function, energy metabolism state, and subsequent extracellular matrix secretion phenotype, enabling the cells to form a stable engineered secretory state before entering the ascorbic acid-induced ECM deposition stage.

[0048] In other embodiments, if the induction time is too short, metformin will not adequately regulate the cellular metabolic state and ECM-related expression, resulting in limited functionalization of the subsequently formed Met-ECM; if the induction time is too long, it may increase cellular metabolic stress or affect the cell proliferation and spreading state, which is not conducive to the formation of a uniform, continuous and fully deposited extracellular matrix.

[0049] like Figure 4 As shown, compared with the corresponding C group, the expression levels of P16 and P53 in the M group decreased at different induction times, indicating that metformin treatment can reduce the senescence and stress damage of mesenchymal stem cells during induction culture. P21 expression increased in the shortest treatment time, but decreased with prolonged induction time, suggesting that the effect of metformin is time-dependent. Appropriately extending the induction time helps cells transition from an early stress-regulated state to a relatively stable low-senescence state. In summary, controlling the metformin induction time within a reasonable range can reduce P16 / P21 / P53-related cellular senescence and stress signals, maintain good metabolic activity and secretory state of mesenchymal stem cells, and thus facilitate the subsequent formation of stable and highly functionalized Met-ECM materials. Here, 24h-C, 48h-C, and 72h-C indicate no metformin was added during the corresponding induction period, while 24h-M, 48h-M, and 72h-M indicate metformin was added during the corresponding induction period.

[0050] In a further embodiment, metformin is added during the proliferation culture period after mesenchymal stem cell seeding until a predetermined degree of confluence is reached, during the extracellular matrix induction culture period after the addition of ascorbic acid, or throughout the entire culture period from the seeding of mesenchymal stem cells to the end of the induction culture on the pretreated culture medium. In this embodiment, by controlling the timing of metformin addition, the composition, structure, and bioactivity of Met-ECM can be controlled in stages, improving the adaptability of the cryogel obtained after subsequent decellularization, centrifugation enrichment, and freeze-thaw gelation to the microenvironment of diabetic musculoskeletal injury.

[0051] Adding metformin during the proliferation culture period after mesenchymal stem cell seeding until the predetermined fusion level is reached can regulate cell metabolic activity and the expression of matrix remodeling-related molecules during cell expansion and spreading, enabling cells to form a functional state more suitable for ECM deposition before subsequent induction culture. Adding metformin during extracellular matrix induction culture after the addition of ascorbic acid can directly act on the massive secretion and deposition phase of ECM, promoting the expression and enrichment of adhesive and repair matrix components such as fibronectin and type III collagen, and regulating the expression of matrix degradation / remodeling-related enzymes, making the resulting ECM more conducive to cell adhesion, matrix deposition, and the construction of a tissue repair microenvironment. Adding metformin throughout the entire culture period from mesenchymal stem cell seeding to the end of extracellular matrix induction culture can continuously cover the processes of cell proliferation, secretory phenotype establishment, and ECM deposition and maturation, resulting in extracellular matrix with good matrix remodeling capacity, structural component deposition capacity, and adhesion signal enrichment characteristics.

[0052] In a further embodiment, the mesenchymal stem cells are bone marrow mesenchymal stem cells, and the seeding density of the mesenchymal stem cells is 1000 cells / cm². 2 -10000 pieces / cm 2 Any value in the range, i.e., the seeding density of mesenchymal stem cells, can be 1000 cells / cm². 2 2000 pieces / cm 2 3000 pieces / cm 2 4000 pieces / cm 2 5000 pieces / cm 2 6000 pieces / cm 2 8000 pieces / cm 2 Or 10,000 / cm 2 It can also be 1000 pieces / cm 2 -10000 pieces / cm 2 Any other value. In this embodiment, the above seeding density range can achieve a balance between cell viability, culture period, ECM deposition amount and ECM structural uniformity, so that bone marrow mesenchymal stem cells can form a uniformly distributed, fully deposited, and easily processed, scraped, collected, centrifuged, and freeze-thawed engineered extracellular matrix under metformin induction, thereby improving the ECM enrichment, structural continuity and musculoskeletal repair adaptability of the obtained extracellular matrix cryogel.

[0053] In this embodiment, by selecting bone marrow mesenchymal stem cells (ECMs) as the source cells for the extracellular matrix, their strong osteogenic differentiation potential, paracrine regulatory capacity, and extracellular matrix secretion capacity can be utilized to make the extracellular matrix formed after metformin induction more suitable for the repair of bone defects, fractures, skeletal muscle injuries, and musculoskeletal fusion defects in the context of diabetes. The ECM derived from bone marrow mesenchymal stem cells contains collagen, fibronectin, elastin, and various matrix regulatory signals, which can provide biomimetic support for subsequent cell adhesion, migration, osteogenic differentiation, and tissue microenvironment reconstruction.

[0054] In other embodiments, if the seeding density is too low, the cell coverage will be insufficient, the intercellular signal communication will be weak, and the subsequent ECM deposition will be low and discontinuous, which is not conducive to the formation of a complete Met-ECM network after decellularization. If the seeding density is too high, the cells will reach an over-fusion state too quickly, which will easily cause local nutrient consumption, accumulation of metabolic waste and uneven cell state, thereby affecting the metformin induction effect and the stability of ECM component composition.

[0055] In a further embodiment, the preset fusion degree is any value between 80% and 95%, and the concentration of ascorbic acid is any value between 50 μM and 200 μM. That is, the preset fusion degree can be 80%, 82%, 85%, 88%, 90%, 92%, or 95%, or any other value between 80% and 95%, and the concentration of ascorbic acid can be 50 μM, 80 μM, 100 μM, 120 μM, 150 μM, 180 μM, or 200 μM, or any other value between 50 μM and 200 μM. In this embodiment, when the preset fusion degree and the ascorbic acid concentration work synergistically, the former ensures that mesenchymal stem cells enter the ECM secretion stage under suitable cell density and intercellular interaction conditions, while the latter further promotes the synthesis, secretion, and deposition of collagen and related matrix components. The combination of the two can improve the deposition, continuity and structural integrity of metformin-induced extracellular matrix, making the resulting Met-ECM more suitable for subsequent decellularization, scraping and collection, centrifugation enrichment and freeze-thaw physical cross-linking into gel, thereby improving the enrichment degree and structural stability of ECM components in extracellular matrix cryogel.

[0056] In a further embodiment, the freezing temperature is -60°C to -100°C, the freezing time is 6h to 24h, and the thawing temperature is 25°C to 40°C. The freezing temperature can be -60°C, -70°C, -80°C, -90°C, or -100°C, or any other value from -60°C to -100°C; the freezing time can be 6h, 8h, 10h, 12h, 16h, 20h, or 24h, or any other value from 6h to 24h; and the thawing temperature can be 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, or 40°C, or any other value from 25°C to 40°C. In this embodiment, by synergistically combining freezing temperature, freezing time, and thawing temperature, a cryogel with a three-dimensional network structure can be formed by the precipitation of metformin-induced extracellular matrix after centrifugation through ice crystal formation, ECM extrusion and concentration, fiber network rearrangement, and physical cross-linking. This improves the degree of ECM enrichment and gel formation without the need for additional chemical cross-linking agents, while preserving as much of the metformin-induced repair matrix components and functional matrix signals as possible. This enhances the microenvironment regulation ability and local filling adaptability of the resulting cryogel in the repair of diabetic musculoskeletal tissue.

[0057] The present invention also provides a metformin-induced stem cell-derived extracellular matrix material prepared according to the preparation method.

[0058] This invention also provides the application of the above-mentioned metformin-induced stem cell-derived extracellular matrix material in the preparation of materials or formulations for the repair of diabetes-related musculoskeletal defects. In this embodiment, the application of the metformin-induced stem cell-derived extracellular matrix material in the repair of diabetes-related musculoskeletal defects means that the material is no longer merely used as a passive scaffold for defect filling, but rather regulates the metabolic state and differentiation function of diabetic damaged cells through metformin-induced engineered ECM signals, thereby simultaneously achieving matrix support, microenvironment reconstruction, and mitochondrial metabolic reprogramming at the defect site, thereby promoting the repair of diabetes-related musculoskeletal tissue.

[0059] The technical solution of this application will be further described below with reference to specific embodiments.

[0060] Example 1 The preparation method of metformin-induced stem cell-derived extracellular matrix material includes the following steps: Step S100: Prepare a regular cell culture dish, add 0.2% gelatin, incubate at 37°C for 1 hour, discard the gelatin, wash once with PBS, incubate at room temperature with 1% glutaraldehyde for 30 minutes, discard the glutaraldehyde, wash once with PBS, incubate at room temperature with 1M ethanolamine for 30 minutes, discard the ethanolamine, wash three times with PBS. If not used immediately, add an appropriate amount of PBS to cover the culture dish and temporarily store at 4°C.

[0061] Step S200: Take the pretreated culture dish, aspirate the PBS, and add adult BMSCs at 3000 / cm³. 2 Cells were seeded at a density of 100 μM on pretreated culture dishes and treated with 500 μM metformin for 3 days.

[0062] Step S300: When the cell density reaches about 90%, add 100 μM ascorbic acid and 500 μM metformin as an induction solution to further induce the cells to secrete ECM. The induction period is 6 days, and the induction solution is changed every 3 days.

[0063] Step S400: When induction is complete, discard the old culture medium, add decellularization solution, incubate at 37°C for 5 min, confirm complete cell removal under a microscope, remove the decellularization solution, add 100 U / mL deoxyribonuclease, incubate at 37°C for 1 h, discard the deoxyribonuclease, wash three times with PBS, and finally store at 4°C for later use. Here, the decellularization solution consists of 99.5% PBS, 0.5% Triton X-ray, and 20 mM NH4OH. The decellularization solution should be prepared fresh for each use.

[0064] Step S500: Collect the decellularized Met-ECM with a cell scraper and transfer it to a 1.5 mL centrifuge tube. Add deionized water and centrifuge the tube at 3500 rpm / min for 8 min.

[0065] Step S600: The resulting Met-ECM precipitate in the tube is then frozen overnight at -80°C and thawed at 37°C. At this point, the precipitate is transformed into Met-ECM gel through physical cross-linking. The Met-ECM gel is then carefully removed with tweezers and stored at -20°C for later use.

[0066] Example 2 The only difference between Example 2 and Example 1 is that metformin is added during the proliferation culture in step S200, while no metformin is added during the induction in step S300.

[0067] Example 3 The only difference between Example 3 and Example 1 is that metformin is not added in step S200, while metformin is added at the same time as VC in step S300.

[0068] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that metformin was not added in steps S200 and S300, and ascorbic acid was not added in step S300.

[0069] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that metformin was not added in steps S200 and S300, while ascorbic acid was added only in step S300.

[0070] Figure 5 This is an evaluation of the effect of metformin on iterative bone marrow mesenchymal stem cell intervention in Example 1 and Comparative Example 1 of the present invention. Figure 6 This invention evaluates the intervention effect of metformin on functional genes related to mitochondrial and extracellular matrix synthesis, as described in Example 1 and Comparative Example 1 of the present invention. Figure 7 This is an evaluation of the intervention effect of metformin on matrix remodeling-related enzyme molecular markers in Example 1 and Comparative Example 1 of the present invention. Figure 8 The results are from Western blot experiments on the effects of metformin on mitochondrial function according to Example 1 and Comparative Example 1 of the present invention.

[0071] First, the Met-ECM materials prepared in Example 1 and Comparative Example 1 were evaluated for their effect of metformin intervention on bone marrow mesenchymal stem cells, and the results were as follows: Figures 5 to 8 The test results are shown below. The proteomics analysis method is as follows: 25 mM dithiothreitol, 100 mM iodoacetamide, phenol extraction buffer, 50 mM ammonium bicarbonate, and a series of reagents required for in-gel enzymatic hydrolysis (decolorizing solution, reducing agent, alkylating reagent, dehydrating solution, enzymatic hydrolysis covering solution, peptide extraction solution) were prepared. Proteins were extracted using the phenol extraction buffer. After dissolving the sample in mass spectrometry phase A, the peptide concentration was determined using a micro-UV-Vis spectrophotometer and stored at -80℃ for later use. If it was a peptide solution, it was thawed, mixed, quantified, and also stored at -80℃. Before mass spectrometry injection, iRT internal standard was added to each sample at a volume ratio of 1:20 for chromatographic system calibration and quantitative quality control. A C18 analytical column (15 cm × 100 μm, 1.9 μm) was used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% FA acetonitrile solution. A gradient elution program was set for high-resolution mass spectrometry detection. It should be noted that the detection method for protein detection involved in this application is the same as above.

[0072] like Figure 5 As shown in the figure, P16, P21, and P53 are all biomarkers related to cell senescence, cell cycle arrest, and stress damage. Higher expression levels generally indicate more severe cell senescence or stress. As can be seen from the figure, in different passage stages of P9, P10, and P11, Example 1 showed a decrease in the relative expression levels of P16, P21, and P53 compared to Comparative Example 1. The decreasing trend was more pronounced for P21 and P53, indicating that metformin intervention can alleviate the senescence and stress damage of bone marrow mesenchymal stem cells during passage, maintain good cell functional activity, and provide a cellular basis for the subsequent formation of stable and functional Met-ECM.

[0073] like Figure 6As shown, Example 1 upregulated the expression levels of mitochondrial antioxidant / anti-stress-related factors such as SIRT3, NRF2, CAT, and SOD2 compared to Comparative Example 1, indicating that metformin can enhance cellular antioxidant defense and mitochondrial protection capabilities. Simultaneously, it upregulated mitochondrial respiratory chain and oxidative phosphorylation-related factors such as ND4, SDHA, ATP5A, and COX4, suggesting enhanced cellular energy metabolism and ATP production. Furthermore, the increased levels of extracellular matrix structure / adhesion-related factors such as FN1, COL3A1, and ELN indicate that metformin can promote the deposition of repair-type matrix components. Increased levels of PGC-1α, TFAM, MFN1, and OPA1 suggest that it can also enhance mitochondrial biogenesis, mitochondrial fusion, and mitochondrial homeostasis. Overall, Example 1 demonstrates that it can enhance the musculoskeletal repair-related functions of Met-ECM from multiple levels, including antioxidant activity, mitochondrial energy metabolism, ECM deposition, and mitochondrial homeostasis.

[0074] like Figure 7 As shown in the figure, ADAMTS1, MMP2, and MMP13 are all proteases related to extracellular matrix degradation / remodeling. Excessive expression of these proteases usually indicates enhanced ECM degradation or matrix disruption. As can be seen from the figure, compared to Comparative Example 1, the relative mRNA expression levels of ADAMTS1, MMP2, and MMP13 in Example 1 were significantly reduced. This indicates that metformin intervention can inhibit excessive matrix degradation and abnormal matrix remodeling, which is beneficial for maintaining the structural stability and deposition integrity of the metformin-induced extracellular matrix, thereby improving matrix retention in subsequent centrifugation enrichment, freeze-thaw gelation, and tissue repair applications.

[0075] like Figure 8 As shown, based on the results of Western blot analysis, the overall protein expression of catalase, mitochondrial transcription factor A, superoxide dismutase 2, and glutathione peroxidase 4 was enhanced in Example 1, while β-actin remained basically stable. This indicates that metformin treatment can enhance the antioxidant defense, mitochondrial homeostasis maintenance, and mitochondrial function improvement capabilities of bone marrow mesenchymal stem cells, supporting the induction of metabolically regulatory Met-ECM at the protein level.

[0076] Figure 9 This study evaluates the intervention effects of metformin at different addition times on genes related to extracellular matrix synthesis and degradation, as described in Examples 1-3 and Comparative Example 2 of the present invention. Figure 10 These are the light microscopy, scanning electron microscopy, transmission electron microscopy, and immunofluorescence observation results of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention. Figure 11 These are the DNA content determination results before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention. Figure 12 These are the results of glycosaminoglycan content determination before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention. Figure 13 These are the collagen content determination results before and after decellularization treatment according to Example 1 and Comparative Example 2 of the present invention. Figure 14 This is a light micrograph of the extracellular matrix material after decellularization according to Embodiment 1 of the present invention. Figure 15 This is a light micrograph of the extracellular matrix cryogel prepared according to Example 1 of the present invention.

[0077] Next, the extracellular matrix materials prepared in Examples 1-3 and Comparative Example 2 were subjected to relevant tests and characterizations to obtain the following results: Figures 9 to 15 The test results are shown.

[0078] like Figure 9 As shown, ADAMTS7, MMP2, and MMP14 are matrix degradation / remodeling-related enzymes. In Example 3, the expression of ADAMTS7, MMP2, and MMP14 was reduced overall, suggesting that adding metformin during induction is beneficial to reduce excessive ECM degradation. COL1 did not change significantly among the groups, indicating that metformin does not simply increase the expression of all collagen. COL3A1 was significantly increased in Examples 2, 3, and 1, and FN1 was significantly increased in Examples 3 and 1, indicating that adding metformin during or throughout the induction process is more beneficial to enriching repair-type and adhesion-type ECM components such as type III collagen and fibronectin.

[0079] like Figure 10 As shown, both Comparative Example 2 and Example 1 formed extracellular matrix network structures after decellularization. However, Example 1 exhibited a more pronounced and continuous fibrous ECM structure under light, scanning electron, and transmission electron microscopy, with a stronger immunofluorescence signal. This indicates that the Met-ECM obtained after metformin induction has a richer fibrous network and matrix component deposition, which is beneficial for subsequent cell adhesion, matrix support, and cryogel formation. Here, Comparative Example 2-before and Example 1-before represent the test results of Comparative Example 2 and Example 1 before decellularization, respectively; Comparative Example 2-after and Example 1-after represent the test results of Comparative Example 2 and Example 1 after decellularization, respectively.

[0080] like Figure 11 As shown, the DNA content of Comparative Example 2 and Example 1 decreased significantly from a high level to an extremely low level after decellularization treatment, indicating that the decellularization treatment can effectively remove residual cellular nucleic acids. This result demonstrates that the preparation process of this application can transform the metformin-induced cellular secretion environment into a cell-free ECM material with low DNA residue, thereby improving the biosafety of the material. Here, Comparative Example 2-before and Example 1-before represent the test results of Comparative Example 2 and Example 1 before decellularization treatment, respectively; Comparative Example 2-after and Example 1-after represent the test results of Comparative Example 2 and Example 1 after decellularization treatment, respectively. Figure 12 and Figure 13 Similarly.

[0081] like Figure 12 As shown, the overall glycosaminoglycan content decreased after decellularization, but the glycosaminoglycan content before and after decellularization in Example 1 was higher than that in the corresponding ratio, and Example 1 maintained a high glycosaminoglycan level after decellularization. The results indicate that metformin induction is beneficial for increasing the content of matrix components such as glycosaminoglycans in ECM and ensuring their good retention after decellularization, thereby enhancing the material's water retention, matrix microenvironment regulation ability, and tissue repair adaptability.

[0082] like Figure 13 As shown, the collagen content decreased after decellularization compared to before decellularization. However, the collagen content in Example 1 was higher than that in the comparative example both before and after decellularization, indicating that metformin induction can promote the deposition of collagen-like ECM components and retain a high collagen level after decellularization. This result suggests that the Met-ECM obtained in Example 1 has a higher content of structural matrix components, which is beneficial for the formation of a stable ECM fiber network and subsequent freeze-thaw physical cross-linking.

[0083] like Figure 14 As shown, the extracellular matrix material obtained after decellularization in Example 1 exhibits a translucent, sheet-like, or membrane-like structure and can be grasped with forceps, indicating that the decellularized Met-ECM was not completely fragmented or dissolved, but retained a certain degree of continuity and integrity. This result demonstrates that the decellularization process of this invention can effectively preserve the extracellular matrix network structure induced by metformin while removing cellular components, providing a structural basis for subsequent scraping, collection, centrifugation enrichment, and freeze-thaw gelation.

[0084] like Figure 15 As shown, the extracellular matrix cryogel prepared in Example 1 formed white gel-like clumps at the bottom of the centrifuge tube, indicating that after centrifugation enrichment, freezing, and thawing, the decellularized Met-ECM can be transformed from a dispersed or sheet-like extracellular matrix into a gel material with a certain volume and morphology retention capacity. This result demonstrates that centrifugation can enrich Met-ECM components, and the freeze-thaw process can promote physical cross-linking and structural rearrangement of the enriched extracellular matrix, thereby forming a collectable, storable Met-ECM cryogel with local filling potential.

[0085] Figure 16 This is a biocompatibility test of the extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 of the present invention.

[0086] The extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 were subjected to biocompatibility tests, and the results were as follows: Figure 16 The test results.

[0087] like Figure 16As shown in the figure, the OD values ​​indicate that after bone marrow mesenchymal stem cells (BMSCs) were seeded into the materials prepared in Comparative Example 1, Comparative Example 2, and Example 1, the OD values ​​of each group continued to increase as the culture time increased from day 1 to day 7. This indicates that none of the three materials exhibited significant cytotoxicity and all were able to support BMSC adhesion and proliferation, demonstrating basic biocompatibility. Further comparison of the different material groups shows that the OD values ​​of the Example 1 group at each time point were generally higher than or no lower than those of Comparative Example 1 and Comparative Example 2. On day 1, the OD values ​​of all groups were low, indicating that the cells were in the initial adhesion stage. On day 3, the OD values ​​of the Example 1 group were close to those of the Comparative Example 2 group and higher than those of the Comparative Example 1 group, indicating that the material of Example 1 could support early BMSC proliferation. On days 5 and 7, the OD values ​​of the Example 1 group further increased and were significantly higher than those of the Comparative Example 1 group. On day 7, the OD values ​​were also higher than those of the Comparative Example 2 group, indicating that the material of Example 1 had a stronger promoting effect on the continuous proliferation of BMSCs.

[0088] The above results demonstrate that the metformin-induced stem cell-derived extracellular matrix material prepared in Example 1 exhibits good biocompatibility and is more beneficial for maintaining BMSC activity and promoting BMSC proliferation compared to the comparative material. This may be because the metformin induction process enriches the resulting Met-ECM with functional matrix components and matrix signals that are more conducive to cell adhesion, spread, and proliferation, thereby providing BMSCs with a biomimetic support that more closely resembles the tissue repair microenvironment, which is beneficial for subsequent osteogenic differentiation, matrix deposition, and repair of diabetic musculoskeletal defects.

[0089] Figure 17 The results of alizarin detection are from the extracellular matrix materials prepared according to Example 1 and Comparative Examples 1-2 of this invention. Figure 18 This is an evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Examples 1-2 of the present invention on genes related to promoting osteodifferentiation.

[0090] The effects of the extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 on bone marrow mesenchymal stem cell intervention were evaluated, and the results were as follows: Figure 17 and Figure 18 The test results are shown.

[0091] like Figure 17 As shown in the staining results, Comparative Example 1 showed less red mineralization deposition, Comparative Example 2 showed enhanced red staining, and Example 1 showed the most obvious and densely distributed red mineralization area. Quantitative results showed that the matrix mineralization level of Example 1 was significantly higher than that of Comparative Example 1 and Comparative Example 2, indicating that the metformin-induced extracellular matrix material prepared in Example 1 can more effectively promote calcium salt deposition and osteoid matrix formation in the later stages of osteogenic differentiation of bone marrow mesenchymal stem cells. This result indicates that Met-ECM has a stronger bone mineralization-promoting effect than ordinary materials, which is beneficial for the repair of diabetes-related bone defects or fractures.

[0092] like Figure 18 As shown, ALP is an early marker of osteogenic differentiation, RUNX2 is a key transcription factor for osteogenic differentiation, and COL1A1 is the type I collagen α1 chain, an important structural component of bone matrix formation. As can be seen from the figure, compared with Comparative Example 1, Comparative Example 2 showed a certain increase in the expression of ALP, RUNX2, and COL1A1, with a more significant increase in RUNX2. In Example 1, ALP, RUNX2, and COL1A1 were all further increased, especially RUNX2, indicating that the material in Example 1 can simultaneously promote early osteogenic differentiation, osteogenic transcriptional regulation, and bone matrix deposition. Figure 17 The mineralization results indicate that the Met-ECM prepared in Example 1 can enhance the osteogenic differentiation capacity of bone marrow mesenchymal stem cells from two aspects: osteogenic gene expression and matrix mineralization.

[0093] Figure 19 These are immunofluorescence staining images showing the promotion of myogenic differentiation of mouse C2C12 cells by the extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 according to the present invention. Figure 20 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Examples 1-2 of the present invention on promoting myogenic differentiation of mouse C2C12 cells.

[0094] The effects of the extracellular matrix materials prepared in Example 1 and Comparative Examples 1-2 on mouse C2C12 cells were evaluated, and the results were as follows: Figure 19 and Figure 20 The test results are shown.

[0095] The immunofluorescence staining method is as follows: After decellularization, the PBS was aspirated, and the sample was fixed with 4% paraformaldehyde at room temperature for 10 min. The fixative was removed, and the sample was washed three times with PBS. The sample was blocked with 1% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibody against elastin (diluted 1:200) was added, and the sample was incubated overnight at 4°C. The sample was washed twice with PBS, and secondary antibody Alexa Fluor 488 (diluted 1:500) was added. The sample was incubated at room temperature for 1 h, washed three times with PBS, and the expression of elastin was observed under a fluorescence microscope, and images were acquired. It should be noted that the immunofluorescence staining method used in this application is the same as above.

[0096] like Figure 19 As shown, Figure 19The results of immunofluorescence staining for C2C12 cell myogenic differentiation are shown. Myosin heavy chain is used to reflect myotube formation and myofibril maturation, fibrous actin is used to display the cytoskeleton and cell morphology, and the cell nucleus is used to display cell number and distribution. As can be seen from the figure, in Comparative Example 1, the myosin heavy chain and fibrous actin signals are weak, and there are fewer cord-like myotube structures; in Comparative Example 2, cell arrangement and myotube-like structures are enhanced; in Example 1, the positive signal of myosin heavy chain and fibrous actin structure are more obvious, and more continuous and extended myotube-like structures can be seen in the combined figure, indicating that the Met-ECM material prepared in Example 1 can more effectively promote C2C12 cell myogenic differentiation and myotube-like structure formation.

[0097] like Figure 20 As shown, MyoD is an early regulator of myoblastic differentiation, MyoG is myopoietin, which participates in the advancement of myoblastic differentiation and myotube formation, and MHC2 is a myosin heavy chain-related marker for mature myotubes. As can be seen from the figure, compared with Comparative Example 1, the expression of MyoD, MyoG, and MHC2 in Example 1 group was all increased, indicating that the material in Example 1 can simultaneously promote the initiation, advancement, and myotube maturation of C2C12 cells. In Comparative Example 2 group, MHC2 expression was also significantly increased, but MyoG expression was lower than in Example 1, indicating that the material in Example 1 provides a more balanced regulation of the myoblastic differentiation process, which is beneficial for promoting the development of C2C12 cells from myoblastic initiation to myotube formation and maturation.

[0098] comprehensive Figure 19 and Figure 20 It can be seen that the Met-ECM material prepared in Example 1 can enhance the myogenic differentiation ability of C2C12 cells, which is beneficial to the repair of diabetes-related skeletal muscle damage.

[0099] Figure 21 This study evaluates the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on restoring the osteogenic differentiation capacity of bone marrow mesenchymal stem cells damaged by high glucose and high lipid. Figure 22 This study evaluates the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on restoring the myogenic differentiation ability of C2C12 cells in mice with high glucose and high lipid injury.

[0100] The intervention effects of the Met-ECM materials in Example 1 and Comparative Example 2 on cells damaged by high glucose and high lipid levels were evaluated in an in vitro diabetes model, and the results were as follows: Figure 21 and Figure 22The test results are shown. The BSA group is the control group, whose cells were not damaged by high glucose and high lipids and were treated only with bovine serum albumin. HGPA is the treatment of high glucose and high lipid-damaged cells with a combination of 25 mM glucose and 250 μM palmitic acid (solvent is BSA). HGPA-Comparative Example 2 is the seeding of high glucose and high lipid-damaged cells onto the extracellular matrix prepared in Comparative Example 2. HGPA-Example 1 is the seeding of high glucose and high lipid-damaged cells onto the extracellular matrix prepared in Example 1.

[0101] like Figure 21 As shown in the results of Alizarin Red staining, the BSA group showed more significant mineralization, while the HGPA group showed a significant reduction in red mineralization, indicating that high glucose and high lipid injury inhibited the osteogenic mineralization capacity of bone marrow mesenchymal stem cells. Mineralization in the HGPA-Comparative Group 2 showed some recovery, but the recovery was limited. Red mineralization in the HGPA-Example 1 group was further enhanced. Quantitative results showed that the matrix mineralization level in the HGPA group was significantly lower than that in the BSA group, while the HGPA-Example 1 group was significantly higher than that in the HGPA group. This indicates that the Met-ECM prepared in Example 1 can improve the late-stage osteogenic differentiation mineralization capacity of bone marrow mesenchymal stem cells under high glucose and high lipid injury conditions, which is beneficial for osteoid matrix formation. In other words, Met-ECM can significantly restore the decreased osteogenic mineralization capacity caused by high glucose and high lipid injury, and shows a better recovery trend than ordinary ECM.

[0102] like Figure 22 As shown in the immunofluorescence results, myosin heavy chain was used to reflect myotube formation and myofibril maturation, fibrous actin was used to display the cytoskeleton, and the cell nucleus was used to display cell distribution. The figure shows that the BSA group formed relatively obvious myosin heavy chain positive cord-like structures; the HGPA group showed a significant decrease in myosin heavy chain signal and a reduction in myotube-like structures, indicating that high glucose and high lipid injury inhibits myoblastic differentiation of C2C12 cells; the HGPA-comparative Example 2 group showed some recovery in myosin heavy chain signal and myotube length; the HGPA-Example 1 group showed more obvious myosin heavy chain positive cord-like structures and a further increase in myotube length. Quantitative results showed that the myotube length in the HGPA group was lower than that in the BSA group, and the HGPA-Example 1 group was significantly higher than that in the HGPA group, indicating that the Met-ECM prepared in Example 1 can promote myoblastic differentiation and myotube-like structure formation in C2C12 cells damaged by high glucose and high lipid injury, which is beneficial for the repair of diabetes-related skeletal muscle injury.

[0103] therefore, Figure 21 and Figure 22 Together, they demonstrate that the Met-ECM obtained in Example 1 can simultaneously improve osteogenic mineralization and myogenic differentiation in an in vitro diabetic injury model, reflecting its application basis for repairing musculoskeletal defects in diabetes.

[0104] Figure 23This describes the effect of extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on the mitochondrial oxygen consumption rate of cells damaged by high glucose and high lipids. Figure 24 The intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on enhancing the mitochondrial oxidative phosphorylation capacity of cells damaged by high glucose and high lipids was evaluated. Figure 25 This describes the effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on the extracellular acidification rate of cells damaged by high glucose and high lipids. Figure 26 This is an evaluation of the intervention effect of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on reducing the level of glycolysis in cells damaged by high sugar and high lipid.

[0105] To verify whether Met-ECM can improve energy metabolism disorders in cells damaged by high glucose and high lipids and promote the recovery of cells from an abnormal glycolysis-dependent state to a mitochondrial oxidative phosphorylation metabolic state, the Seahorse cellular energy metabolism analysis system was used to detect oxygen consumption rate and extracellular acidification rate, as shown in the following figures. Figures 23 to 26 The test results are shown.

[0106] like Figure 23 As shown, the overall OCR of the HGPA group was lower than that of the BSA group, indicating that high glucose and high lipid damage inhibited mitochondrial oxygen consumption and oxidative phosphorylation. The HGPA-comparative group 2 showed some recovery compared to the HGPA group, but the improvement was limited. The HGPA-Example 1 group showed the highest OCR peak after the addition of the mitochondrial uncoupling agent FCCP, indicating that the Met-ECM prepared in Example 1 can significantly enhance the maximum mitochondrial respiration capacity of cells damaged by high glucose and high lipid. After the addition of antimycin A / rotenone, the OCR of all groups decreased to a lower level, indicating that the detected changes in oxygen consumption mainly originated from mitochondrial respiration.

[0107] like Figure 24 As shown, regarding basal mitochondrial respiration levels, the HGPA group showed a significant decrease compared to the BSA group, indicating that high glucose and high lipid damage reduced the cell's basal oxidative phosphorylation capacity. The HGPA-Comparative Example 2 group showed no significant difference from the HGPA group, while the HGPA-Example 1 group showed a significant increase, approaching or recovering to the BSA group level. Regarding mitochondrial reserve respiration capacity, the HGPA-Example 1 group was significantly higher than the BSA group, the HGPA group, and the HGPA-Comparative Example 2 group, indicating that the Met-ECM prepared in Example 1 can not only restore basal mitochondrial respiration but also improve the cell's metabolic reserve capacity under increased energy demand.

[0108] like Figure 25As shown, the ECAR level in the HGPA group significantly increased after the addition of antimycin A / rotenone, indicating that high glucose and high lipid damage caused cellular metabolism to favor glycolysis. The ECAR levels in both the HGPA-Comparative Example 2 group and the HGPA-Example 1 group were lower than those in the HGPA group, with the HGPA-Example 1 group showing the lowest overall ECAR level, approaching that of the BSA group. After the addition of 2-deoxyglucose, the ECAR levels in all groups decreased significantly, indicating that this acidification signal mainly originates from glycolysis. These results demonstrate that Met-ECM can reduce abnormal glycolytic activity induced by high glucose and high lipid damage.

[0109] like Figure 26 As shown, the basal and compensatory glycolysis levels in the HGPA group were significantly higher than those in the BSA group, indicating that high glucose and high lipid damage led to increased cellular dependence on glycolysis for energy. The HGPA-Comparative Group 2 partially reduced glycolysis levels, while the HGPA-Example 1 group showed a more significant decrease, with both basal and compensatory glycolysis levels approaching or falling below those in the BSA group. These results demonstrate that the Met-ECM prepared in Example 1 can inhibit the abnormally enhanced glycolytic metabolism in cells damaged by high glucose and high lipid damage and promote the recovery of cellular energy metabolism towards mitochondrial oxidative phosphorylation.

[0110] In summary, the Met-ECM prepared in Example 1 can enhance the mitochondrial oxidative phosphorylation capacity of cells damaged by high sugar and high lipids, reduce the level of abnormal glycolysis, thereby improving cellular energy metabolism disorders and promoting mitochondrial metabolic reprogramming.

[0111] Figure 27 This study evaluates the interventional effects of the extracellular matrix materials prepared according to Example 1 and Comparative Example 2 of the present invention on promoting tissue repair and recovery of motor function after diabetic muscle loss. Figure 28 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on improving grip and gait function in diabetic mice after muscle injury.

[0112] Muscle tissue samples were harvested and observed in diabetic mice with muscle defects in the blank group, gel group, gel-combined extracellular matrix material group (Extracellular matrix material group of Example 2), and gel-combined extracellular matrix material group (Extracellular matrix material group of Example 1) for four weeks. Muscle electrical stimulation response tests, grip strength tests, and gait function tests were also performed. The results are as follows: Figure 27 and Figure 28 The test results are shown below. Here, muscle defects and fractures were first modeled in diabetic mice. The control group received no treatment or intervention. The gel group consisted of mice with musculoskeletal injuries injected with 5% methacrylamide gelatin. The gel-comparative example 2 group received 100 μL of the extracellular matrix material from Comparative Example 1 and 5% methacrylamide gelatin. The gel-Example 1 group received 100 μL of the extracellular matrix material from Example 1 and 5% methacrylamide gelatin.

[0113] Specifically, 8-week-old male C57BL / 6 mice were ordered from the Animal Center of Soochow University. After one week of acclimatization to the environment with a normal diet, they were fed a high-fat diet for six weeks. Fasting (but not water) began around 8 PM the night before STZ administration. A diabetic mouse model was induced by two intraperitoneal injections of streptozotocin (STZ: 100 mg / kg) (one day apart). After the injections, the mice were promptly given water and feed to establish a mouse model of high glucose and high fat damage. The method for establishing muscle defects and fractures in diabetic mice is as follows: Mice are placed prone with their limbs fixed to a sterile operating table. The skin on the left femur is prepared using a small animal razor. Then, cotton swabs are used to disinfect the area three times with 1% povidone-iodine and once with alcohol. After the alcohol dries, a 0.8–1 cm incision is made along the transverse axis of the femur near the hip joint using a No. 11 scalpel. A pre-fabricated sterile intramedullary nail is used, with the tail end held parallel to the nail using a needle holder. A hole is drilled near the hip joint to access the medullary cavity, and the intramedullary nail is shortened. The knee joint is passively moved to check for any joint restriction. The incision is then repositioned using toothed forceps and the wound is sutured. Mice were placed in a supine position with their limbs fixed to a sterile operating table. The skin on the medial side of the right femur was prepared using a small animal razor. Subsequently, a cotton swab soaked in 1% povidone-iodine was used for disinfection three times, followed by one wipe with alcohol. After the alcohol dried, a 0.8–1 cm incision was made along the longitudinal axis of the femur using a No. 11 scalpel. The skin was bluntly dissected with toothed forceps to expose the quadriceps femoris muscle. A 3 mm muscle punch was inserted close to the upper surface of the quadriceps femoris muscle and rotated to remove the muscle. After muscle removal, the midshaft of the femur was exposed. A transverse fracture of the midshaft of the femur was induced by vertically clamping the midshaft with microscissors and applying force, and the presence of bone fragments was examined. After modeling, the operating area was rinsed with 1% ceftriaxone PBS buffer, and the fascia and skin were sutured sequentially. After suturing, the mice were placed on a warming blanket to restore body temperature.

[0114] like Figure 27 As shown in the gross images of the surrounding muscle tissue samples, the tissue integrity and repair morphology of the defect area in the gel composite Example 1 group were superior to those in the blank group and the gel-only group. Quantitative results showed that the muscle electrical stimulation response intensity in the gel composite Example 1 group was significantly higher than that in the blank group, the gel group, and the two comparative gel composite groups, indicating that the material of Example 1 can more effectively restore the electrophysiological response capacity of damaged muscles. Simultaneously, the grip strength level in the gel composite Example 1 group was the highest, suggesting that it can improve limb strength recovery after diabetic muscle injury. These results indicate that the Met-ECM material obtained in Example 1 can promote tissue repair after diabetic muscle defects and improve muscle function recovery.

[0115] like Figure 28As shown in the gait footprint diagram, the footprint distribution in the blank group and the gel group was relatively irregular, indicating poor gait stability after injury. The gait in the gel-comparative example 2 group showed improvement. The footprint distribution in the gel-comparative example 1 group was more continuous and regular, indicating a better recovery effect on limb motor coordination. The quantitative results on the right side show that although the stride length and swing length of the injured hind limb in the gel group showed an increasing trend compared to the blank group, the improvement was limited. The gel-comparative example 2 group showed further improvement in these two indicators, indicating that the material in comparative example 2 can improve the range of motion and gait control of the injured hind limb to a certain extent. In contrast, the stride length and swing length of the injured hind limb in the gel-example 1 group reached a higher level, and the difference was significant compared to the blank group, indicating that the material in example 1 can more effectively promote the recovery of gait function in the injured hind limb of mice after diabetic muscle injury, and improve limb motor coordination and functional reconstruction.

[0116] Therefore, the Met-ECM material prepared in Example 1 can not only promote local muscle tissue repair, but also improve functional recovery after diabetic muscle loss from multiple levels such as muscle electrophysiological response, grip strength and gait function.

[0117] Figure 29 This is an evaluation of the intervention effect of the extracellular matrix material prepared according to Example 1 and Comparative Example 2 of the present invention on promoting bone tissue repair after bone injury in diabetic mice.

[0118] To evaluate the promoting effect of the extracellular matrix material prepared in Example 1 of this invention on bone tissue regeneration and trabecular bone structure reconstruction after diabetic bone injury, Micro-CT three-dimensional reconstruction and bone morphological parameter analysis were performed on diabetic bone injury mice in the blank group, gel group, gel-combined control group (Extracellular matrix material of Example 2), and gel-combined control group (Extracellular matrix material of Example 1). The results are as follows: Figure 29 The test results are shown.

[0119] like Figure 29As shown in the Micro-CT 3D reconstruction images, the blank group and the gel group showed less new bone formation in the bone defect area, indicating insufficient defect repair. The gel composite control group showed improved bone tissue repair in group 2. The gel composite control group (Example 1) showed more significant bone tissue filling in the defect area and more complete bone repair morphology. Quantitative results showed that the bone volume fraction (BV / TV) of the gel composite control group (Example 1) was significantly higher than that of the blank group, the gel group, and the gel composite control group (Example 2), indicating that the material in Example 1 can promote new bone formation and increase bone mass. Simultaneously, the structural model index (SMI) of the gel composite control group (Example 1) decreased, suggesting that the trabecular structure of new bone changed from a loose rod-like structure to a denser plate-like structure, improving the quality of bone tissue structure. These results indicate that the Met-ECM material prepared in Example 1 can promote bone tissue regeneration and trabecular structure reconstruction after diabetic bone injury, thereby improving the repair effect of bone defects in a diabetic environment.

[0120] In summary, the results of the examples and comparative studies demonstrate that this invention, through metformin-induced secretion of engineered extracellular matrix by bone marrow mesenchymal stem cells, followed by decellularization, centrifugation enrichment, and freeze-thaw gelation, yields a Met-ECM material with low DNA residue, ECM component enrichment, three-dimensional gel formation, and good biocompatibility. This material can upregulate repair / adhesive matrix components and mitochondrial function-related factors, inhibit excessive matrix degradation and cellular senescence / stress-related signals, and enhance oxidative phosphorylation capacity and reduce abnormal glycolysis levels under high glucose and high lipid injury conditions, thereby promoting mitochondrial metabolic reprogramming. Further in vitro osteogenic and myogenic experiments, as well as validation using diabetic mouse muscle and bone injury models, show that the Met-ECM obtained in Example 1 can simultaneously improve osteogenic differentiation, myogenic differentiation, muscle function recovery, and bone tissue regeneration, indicating its suitability for repairing diabetes-related musculoskeletal defects.

[0121] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0122] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing an extracellular matrix material derived from metformin-induced stem cells, characterized in that, Includes the following steps: The culture medium is pretreated to obtain a pretreated culture medium; Mesenchymal stem cells were seeded onto the pretreated culture medium and induced using a culture system containing metformin. After the mesenchymal stem cells reach a preset degree of fusion, ascorbic acid is added to induce extracellular matrix secretion culture, so that the mesenchymal stem cells secrete to form metformin-induced extracellular matrix. The metformin-induced extracellular matrix was subjected to decellularization and nuclease treatment to obtain a metformin-induced stem cell-derived extracellular matrix. The metformin-induced stem cell-derived extracellular matrix was collected and mixed with an aqueous medium, then enriched by centrifugation to form an extracellular matrix precipitate. The extracellular matrix precipitate was subjected to freezing and thawing treatments in sequence, so that the enriched extracellular matrix precipitate was physically cross-linked to form extracellular matrix material derived from metformin-induced stem cells.

2. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 1, characterized in that, The concentration of metformin in the culture system is any value between 400 μM and 600 μM.

3. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 2, characterized in that, The induction time of the mesenchymal stem cells by metformin is any value between 60h and 84h.

4. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 3, characterized in that, The metformin is added during the proliferation culture of the mesenchymal stem cells from seeding to the point of reaching the preset fusion degree, during the extracellular matrix induction culture after the addition of ascorbic acid, or during the entire culture process from seeding of the mesenchymal stem cells to the end of the induction culture on the pretreated culture medium.

5. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 4, characterized in that, The mesenchymal stem cells are bone marrow mesenchymal stem cells, and the seeding density of the mesenchymal stem cells is 1000 cells / cm². 2 -10000 pieces / cm 2 Any value among them.

6. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 5, characterized in that, The preset fusion degree is any value between 80% and 95%, and the concentration of ascorbic acid is any value between 50 μM and 200 μM.

7. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to any one of claims 1-6, characterized in that, The preprocessing includes: The culture carrier was coated with a gelatin solution, followed by cross-linking with glutaraldehyde and blocking with ethanolamine; among these, The mass concentration of the gelatin solution is any value between 0.1% and 0.5%, the mass concentration of the glutaraldehyde is any value between 0.5% and 2%, and the concentration of the ethanolamine is any value between 0.5M and 2M.

8. The method for preparing extracellular matrix material derived from metformin-induced stem cells according to claim 5, characterized in that, The freezing temperature is -60℃ to -100℃, the freezing time is 6h to 24h, and the thawing temperature is 25℃ to 40℃.

9. The extracellular matrix material derived from metformin-induced stem cells prepared by the preparation method according to any one of claims 1-8.

10. The use of the metformin-induced stem cell-derived extracellular matrix material of claim 9 in the preparation of materials or formulations for the repair of diabetes-related musculoskeletal defects.

Images