Clinical ready-to-use autologous adipose tissue-derived matrix collagen membrane and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a clinical ready-to-use autologous adipose tissue-derived matrix collagen membrane and application thereof. The clinical ready-to-use autologous adipose tissue-derived matrix collagen membrane provided by the invention is mainly composed of autologous adipose tissue-derived collagen, and contains a plurality of key adhesion proteins, glycosaminoglycans, growth factors and other bioactive components. The preparation method comprises the following steps: by taking autologous adipose tissue as a raw material, breaking cells by a mechanical method, repeatedly cleaning to remove cell components such as cell debris and oil drops, and mechanically pressing through a mold to form a diaphragm with uniform thickness, regular shape and rough surface. The membrane effectively overcomes the limitation of the existing acellular matrix biological membrane, can load various bioactive substances to enhance the function, can be stored at low temperature for a long time without influencing the repair effect, and greatly enhances the convenience of subsequent use.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and more specifically, relates to a clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane and its application. Background Technology

[0002] The extracellular matrix (ECM) is a core component of the cellular microenvironment, providing structural support and biochemical signals for cells, and playing a crucial role in tissue repair and regeneration. Autologous ECM is considered an ideal material for regenerative medicine due to its complete lack of immune rejection and high bioactivity. Liposuction and abdominoplasty produce large amounts of adipose tissue, most of which is discarded as medical waste, except for a small portion used for fillers. Adipose tissue ECM is rich in type I / III collagen, fibronectin, elastin, and various growth factors, possessing excellent bioactivity and adipogenic induction capabilities, making it a highly promising source of autologous ECM for development.

[0003] Currently, ECM-based decellularized matrix biomaterials (such as porcine dermis, bovine pericardium, human cadaver skin, and human amniotic membrane) are widely used in wound dressings, tissue patches, and regenerative scaffolds. Although these materials possess good biocompatibility and biodegradability, they still have significant limitations: animal-derived materials pose immunogenicity risks, and their composition and mechanical properties differ from human-derived tissues; while human cadaver-derived materials face challenges such as scarcity, high cost, and ethical issues, making it difficult to meet the needs of large-scale and personalized clinical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a clinically ready-to-use autologous fat tissue-derived matrix collagen membrane and its application.

[0005] This invention provides a clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane. The preparation method of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane is as follows: fat fibers are separated from autologous adipose tissue and washed, fat cells are broken until the fat fibers turn white, purified fat fibers are obtained after washing, and the purified fat fibers are wrapped in a porous mesh and pressed to obtain a clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane.

[0006] The clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane provided by this invention is mainly composed of collagen derived from autologous adipose tissue and contains various key adhesion proteins, as well as bioactive components such as glycosaminoglycans and growth factors. Using autologous adipose tissue as raw material, cells are mechanically broken down, and cell debris and oil droplets are repeatedly washed away. Then, the membrane is mechanically pressed using a fixed mold to form a membrane with uniform thickness, regular shape, and a rough surface. This clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane is derived from the patient's own body, eliminating the risk of immune rejection and pathogen transmission. The preparation process is simple and rapid, and it can be cryopreserved, greatly improving its clinical applicability and convenience.

[0007] Furthermore, the porous mesh is a 100-mesh to 300-mesh mesh.

[0008] Furthermore, the pressurization condition is 0.5 × 10⁻⁶. 6 kp~1.5×10 6 kp.

[0009] Furthermore, the tensile strength of the clinically ready-to-use autologous fat tissue-derived matrix collagen membrane is 300 kPa to 400 kPa, and the elongation at break is 4% to 6%.

[0010] The present invention also provides the application of the aforementioned clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane in the preparation of drug carriers.

[0011] The present invention also provides the application of the aforementioned clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane in the preparation of wound dressings.

[0012] The present invention also provides the application of the aforementioned clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane in the preparation of biomaterial prostheses.

[0013] The present invention also provides the application of the aforementioned clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane in the preparation of soft tissue regeneration scaffolds.

[0014] The present invention has the following beneficial effects: This invention is the first to propose the concept of "clinically ready-to-use autologous fat tissue-derived matrix collagen membrane" and establishes a rapid preparation process based primarily on purely physical methods, enabling immediate preparation and application. This technology transforms surgical waste fat into standardized, immediately applicable regenerative medicine products, not only achieving high-value utilization of waste resources but also effectively solving the problems of source limitations, immune risks, and costs associated with traditional ECM materials, thus possessing significant clinical translational value.

[0015] Original innovation and cost advantage: Transforming clinical waste fat into high-value-added biomaterials, with wide availability and low cost, realizing "turning waste into treasure".

[0016] Excellent biocompatibility: derived from the patient's own body, with no risk of immune rejection or pathogen transmission, and extremely high safety.

[0017] True clinical readiness: The preparation process is simple and quick, supporting immediate intraoperative preparation and long-term cryopreservation, greatly improving clinical applicability and convenience.

[0018] Excellent structure and function: The membrane has suitable mechanical strength, bioactivity and degradation properties, and can effectively promote angiogenesis and adipose tissue regeneration.

[0019] Flexible and scalable applications: The product has diverse forms and functions, and can be used independently, as an active factor carrier, or combined with other materials to meet diverse clinical scenarios. Attached Figure Description

[0020] Figure 1 Typical appearance images of each key stage in the preparation of clinically ready-to-use adipose tissue-derived matrix (ADF) are shown. A is a typical appearance image of each key stage in the preparation of clinically ready-to-use adipose tissue-derived matrix (ADF) from human liposuction fat, and B is a typical appearance image of each key stage in the preparation of clinically ready-to-use adipose tissue-derived matrix (ADF) from abdominoplasty fat.

[0021] Figure 2 The images show the histological staining results at different stages of ADF preparation. A is an H&E staining image, B is a Masson staining image, and C is an Oil Red O staining image.

[0022] Figure 3 The images show the physicochemical properties of ADF at different stages of its preparation process. In the images, A is the infrared spectral analysis image, B is the mechanical property test image, C is the ADF image of different shapes, D is the folded ADF image, and E is the ADF image of the recovered shape.

[0023] Figure 4 The images show the morphology of ADF, where A is the surface microstructure of ADF under a scanning electron microscope, B is the cross-sectional microstructure of ADF under a scanning electron microscope, and C is a typical morphology image under a laser confocal microscope.

[0024] Figure 5 The graphs show the ADF swelling performance, where A is a macroscopic representation of ADF swelling performance and B is a quantitative statistical graph of ADF swelling performance.

[0025] Figure 6The images show staining patterns of live and dead cells. In this image, A shows the staining patterns of live and dead cells after co-culturing different cell types with ADF; B shows the survival rate statistics of HaCaT cells; C shows the survival rate statistics of NFb cells; D shows the survival rate statistics of ADSCs cells; and E shows the survival rate statistics of HUVEC cells.

[0026] Figure 7 The graphs show hemolysis test results, where A is the hemolysis test result for ADF and B is the hemolysis rate statistical graph. Figure 8 The images show H&E stained histological sections of the major organs (heart, liver, spleen, lung, and kidney) 12 weeks after ADF was subcutaneously implanted in nude mice.

[0027] Figure 9 The diagrams show the co-culture experiments of ADF and vascular endothelial cells. In the diagrams, A represents vascular endothelial cell proliferation, B represents vascular endothelial cell migration, and C represents vascular endothelial cell tube-forming ability.

[0028] Figure 10 The diagram shows the co-culture experiment of ADF and adipose-derived mesenchymal stem cells. In the diagram, A is the proliferation of adipose-derived mesenchymal stem cells, B is the migration of adipose-derived mesenchymal stem cells, and C is the adipogenic differentiation capacity of adipose-derived mesenchymal stem cells.

[0029] Figure 11 The figures show the experimental results of ADF implanted in nude mice at weeks 2, 4, 12, and 24. In the figures, A shows the morphological retention of ADF in nude mice at weeks 2, 4, 12, and 24 after implantation; B shows the volume retention rate; C shows the material plasticity results after 24 weeks of implantation; and D shows the mechanical property test results.

[0030] Figure 12 The images show histological analysis at weeks 2, 4, 12, and 24 after ADF implantation. In the images, A is an H&E staining image, B is a Masson staining image, C is a Perilipin immunofluorescence staining image, D is a quantitative statistical graph of the adipogenic differentiation area, and E is a quantitative statistical graph of the Perilipin immunofluorescence staining area.

[0031] Figure 13 The images show the effect of ADF implantation at 2 weeks and 12 weeks post-implantation. In the images, A is the immunofluorescence staining of CD31 (red) and α-SMA (green) at 2 weeks and 12 weeks post-implantation, B is the statistical graph of cell infiltration density, C is the quantitative statistical graph of total blood vessel density, and D is the quantitative statistical graph of mature blood vessel density.

[0032] Figure 14 The images show immunohistochemical images of collagen. In A, the results of the immunohistochemical detection of collagen were observed at weeks 2, 4, 12, and 24 after ADF implantation. In B, the quantitative statistical graph of collagen replacement rate was presented.

[0033] Figure 15 The following are statistical graphs showing the results of the wound healing experiment. In the graphs, A is a macroscopic photograph of the wound healing of a full-thickness skin defect model in nude mice, B is a curve of wound closure fitting, and C is a statistical graph of wound closure rate.

[0034] Figure 16 The images show histological analysis of the skin at the wound site. A is an H&E staining image, B is a statistical image of granulation tissue thickness, C is a Masson staining image, D is a quantitative statistical image of collagen deposition, E is a statistical image of the ratio of mature blood vessels, and F is an immunofluorescence staining image of CD31 (red fluorescence) and α-SMA (green fluorescence). Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0036] In the following examples, "clinically ready-to-use adipose tissue-derived extracellular matrix film" will be uniformly referred to as "Adipose tissue-derived extracellular matrix film" (ADF), and will be referred to as "ADF" in the following text. ADF frozen at -20°C (frozen adipose tissue-derived extracellular matrix film, F-ADF) will be referred to as "F-ADF" in the following text.

[0037] Example 1: Preparation of ADF.

[0038] I. Preparation of ADF using human liposuction fat as raw material.

[0039] 1. Obtaining ECM-rich adipose fibers: From the adipose tissue obtained through conventional liposuction, large adipose fibers rich in extracellular matrix (ECM) are physically sorted using a syringe needle and forceps, and labeled as ECM-rich fat. The results are as follows: Figure 1 As shown in A.

[0040] 2. Preliminary Cleaning and Breakdown: The aforementioned fat fibers were placed in physiological saline or deionized water and repeatedly inverted and shaken to remove residual blood and swelling fluid, causing the fibers to clump together. The fibers were then removed with tweezers and the moisture was removed using absorbent material; at this point, the tissue appeared yellowish-white. Subsequently, a high-speed cutting method was used for preliminary breakdown to destroy the fat cells, and the tissue was labeled ECM and oil. The results are as follows... Figure 1 As shown in A.

[0041] 3. Deep decellularization and purification: ECM fibers and lipids are separated using tweezers. Then, the tissue is pressed and abraded through a 1mm mesh to further break down residual fat cells until the tissue turns white and no yellow lipid droplets are visible to the naked eye. At this point, it is labeled as ECM with little oil. The results are as follows: Figure 1 As shown in A.

[0042] 4. Compression Molding: Wrap the ECM with little oil in 200-mesh medical polyethylene mesh and place it in a stainless steel mold (purchased from Tianjin Hengyueda Technology Co., Ltd.), with filter paper lining the top and bottom. In a 1×10... 6 Press under KP pressure for 5 minutes, then release the pressure and remove the wrapping mesh to obtain the final ADF product, which is white in appearance and has a rough surface. The result is as follows: Figure 1 As shown in A.

[0043] II. Preparation of ADF using fat from abdominoplasty.

[0044] 1. Acquisition and Preliminary Processing: The adipose tissue removed during abdominoplasty was cut into approximately 1cm × 1cm tissue blocks (Smallfat deposits), mechanically broken down into a paste, and coarse adipose fibers were separated to obtain the ECM-enriched portion, labeled as ECM and oil. Results are as follows: Figure 1 As shown in B 2. Decellularization and Purification: ECM fibers and lipids were separated using tweezers. The tissue was then pressed and abraded through a 1mm mesh to further break down residual fat cells until the tissue turned white and no visible yellow lipid droplets remained. At this point, it was labeled "ECM with little oil." The results are as follows: Figure 1 As shown in B.

[0045] 3. Compression Molding: The purified fatty fibers are wrapped in 200-mesh medical polyethylene mesh, placed in a specific mold, and lined with filter paper on the top and bottom. (The process is repeated in the original text.) 6 Press down under KP pressure for 5 minutes, then release the pressure and remove the wrapping mesh to obtain the final ADF product. The result is as follows. Figure 1 As shown in B.

[0046] Example 2: Histological verification of the ADF preparation process.

[0047] Histological examinations were performed on samples from each stage of ADF preparation using liposuction fat as raw material. For example... Figure 2 As shown, HE staining revealed the gradual removal of cell nuclei; Masson staining showed good preservation of collagen fibers; and Oil Red O staining confirmed the effective removal of lipid droplets. The results indicate that this preparation process can gradually and effectively remove cellular components and lipids.

[0048] Example 3: Characterization of the basic physicochemical properties of ADF.

[0049] The prepared ADF underwent a systematic test of multiple physicochemical properties, and was scanned using a Fourier transform infrared spectroscopy (FTIR) instrument. The characteristic absorption peaks (amide I band, amide II band, and 1250 cm⁻¹) were analyzed. -1 and 1030cm -1 The characteristic peaks of glycosaminoglycans were observed to verify the successful preservation and structural integrity of the main components of the extracellular matrix (collagen and glycosaminoglycans). Tensile tests were performed, applying unidirectional tension to the samples using a mechanical testing machine until fracture. The tensile strength (unit: kPa) and elongation at break (%) of the samples were measured to evaluate their mechanical strength and flexibility. Physical manipulations were performed on the samples, including cutting them into different shapes and subjecting them to deformation treatments such as folding and crumpling, followed by observation of their shape recovery. The processability, flexibility, and shape memory capacity of the material were visually assessed. Specific results are as follows:

[0050] FTIR (Full-Time Infrared Spectroscopy): such as Figure 3 As shown in A, the spectrum shows the amide I band (1600~1700 cm⁻¹). -1 ), Amide II band (1480~1580cm) -1 This indicates that the protein structure is intact; 1250cm -1 With 1030cm -1 The characteristic peaks indicate the retention of glycosaminoglycans (GAGs). These results validate the successful retention of major ECM components such as collagen in ADF.

[0051] Mechanical properties: Figure 3 As shown in Figure B, the tensile test shows that the ADF has a tensile strength of 350 kPa and an elongation at break of 5%, demonstrating good mechanical strength.

[0052] plasticity: Figure 3 As shown in C, D, and E, ADF can be cut into different shapes and can basically return to its original shape after folding and crumpling, demonstrating excellent plasticity and shape memory ability.

[0053] Example 4: Microstructure and swelling properties of ADF.

[0054] After freeze-drying and gold sputtering, the surface morphology of ADF samples was observed using scanning electron microscopy. The microscopic topology of the sample surface, such as the distribution of uneven regions and the density and looseness of collagen fibers, was characterized. Three-dimensional scanning imaging of the sample surface was performed directly using laser confocal microscopy to further confirm and visualize the irregular topology of the sample surface. The dried ADF samples were weighed (initial dry weight, W0) and then immersed in PBS. At specific time points, they were removed, excess liquid was absorbed, and they were weighed again (wet weight, W1) until the weight no longer increased significantly. The swelling rate is usually calculated using the formula: Swelling rate = [(W1-W0) / W0] × 100%. The sample's ability and rate of liquid absorption were measured, and its hydrophilicity and liquid retention capacity were evaluated by calculating the swelling rate. Specific results are as follows:

[0055] Microstructure: Scanning electron microscopy (SEM) revealed that the ADF surface exhibits a regular uneven structure, with dense collagen fibers in the concave areas and a relatively loose structure in the convex areas, such as... Figure 4 As shown in A and B. This rough structure facilitates integration with host tissue after implantation and prevents displacement. Laser confocal microscopy images further confirm its irregular surface topology, such as... Figure 4 As shown in C.

[0056] Swelling properties: Swelling experiments show that, Figure 5 As shown in A, ADF reaches swelling equilibrium within 24 hours. Figure 5 As shown in B, the weight increases by 150% after absorbing water, indicating moderate hydrophilicity.

[0057] Example 5: Biocompatibility evaluation of ADF.

[0058] The biocompatibility of ADF and its cryopreserved sample F-ADF is systematically evaluated through three dimensions: in vitro cell compatibility testing, in vitro blood compatibility testing, and in vivo systemic biocompatibility assessment, to ensure its safety as a biomaterial.

[0059] Cell compatibility evaluation experimental design: Cells were co-cultured with the material using a direct contact method. The effect of the material on cell viability was assessed using live and dead cell staining and quantitative analysis. ADF and F-ADF were cut to the appropriate size for the cell culture plate, sterilized with UV light or ethylene oxide, and placed at the bottom of the wells. For the experimental group, cells were seeded and cultured on the material surface. A mixed staining solution of Calcein-AM (labeling live cells, producing green fluorescence) and propidium iodide (PI, labeling dead cells, producing red fluorescence) was used. The culture medium was discarded, and staining working solution was added, followed by incubation in the dark for 30 minutes. Subsequently, observation and photography were performed using a fluorescence microscope or laser confocal microscope. Cell viability quantification method: A cell counting kit (CCK-8) was used. After specific time points of co-culture, CCK-8 solution was added, and after incubation, the absorbance value at 450 nm was measured using a microplate reader. Survival rate (%) = (Absorbance of experimental group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group) × 100%. The negative control group consists of cells cultured on ordinary culture plates.

[0060] Blood compatibility evaluation experimental design: An in vitro hemolysis experiment was conducted to assess whether contact between the material and blood would cause erythrocyte rupture and hemolysis. Fresh anticoagulated rabbit whole blood was taken and diluted with physiological saline. Experimental group: ADF and F-ADF samples were immersed in diluted blood. Negative control: An equal volume of physiological saline, theoretically not causing hemolysis. Positive control: An equal volume of deionized water or 1% Triton X-100 solution, theoretically causing 100% hemolysis. All test tubes were incubated in a 37℃ water bath for 2 hours. After incubation, the solutions in each tube were centrifuged, and the supernatant was collected. The absorbance (OD) of the supernatant at 545 nm (the characteristic absorption wavelength of hemoglobin) was measured using an ELISA reader. Hemolysis rate (%) = [(OD experimental group - OD negative control) / (OD positive control - OD negative control)] × 100%.

[0061] Systemic biocompatibility evaluation experimental design: An in vivo animal implantation experiment was conducted to observe the long-term systemic effects of the material on major organs. BALB / c mice were used. After anesthesia, skin preparation, and disinfection, sterilized ADF and F-ADF samples were implanted subcutaneously through small incisions. Patients were observed for 12 weeks post-implantation. After 12 weeks, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were removed and fixed in formalin. The fixed organs were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). The organ sections were observed under a light microscope, focusing on the presence of pathological damage such as inflammatory cell infiltration, tissue degeneration, necrosis, and fibrosis. Specific results are as follows:

[0062] Cell compatibility: Live and dead cell staining showed that various cell types co-cultured with ADF and its frozen sample F-ADF, such as Figure 6As shown in Figure A, no obviously red fluorescently labeled dead cells were observed, such as Figure 6 As shown in B, C, D, and E, cell viability statistics confirm its excellent cell compatibility.

[0063] Blood compatibility: such as Figure 7 As shown, the hemolysis experiment showed that the hemolysis rate of the ADF and F-ADF groups was comparable to that of the negative control (PBS) and much lower than that of the positive control (Triton X-100), proving that they were not hemolytic.

[0064] Systemic biosafety: such as Figure 8 As shown, after 12 weeks of implantation of ADF and F-ADF into nude mice, H&E staining sections of major organs showed no pathological damage, indicating that their in vivo application has good biocompatibility.

[0065] Example 6: In vitro biological functions of ADF.

[0066] This study evaluated the biological functions of adenosine dehydrogenase (ADF) and its cryopreserved sample F-ADF in promoting angiogenesis and adipogenesis using in vitro angiogenesis and adipogenesis models, respectively, to provide in vitro evidence for their application in tissue regeneration. Angiogenesis promotion capacity evaluation experimental design: Human umbilical vein endothelial cells (HUVECs) were used as a model. Through a co-culture system, the promoting effect of ADF on angiogenesis was assessed at three key stages: cell proliferation, migration, and tube formation. HUVECs were seeded in wells containing ADF / F-ADF samples (experimental group) and ordinary culture plates (control group). After 24 hours of culture, the effect of the materials on endothelial cell proliferation activity was quantitatively assessed using the EdU method. First, HUVECs were seeded into culture plates and cultured until a dense monolayer formed. Then, uniform scratches were made on the cell layer using a sterile pipette tip. After removing cell debris from the scratches, the culture medium was replaced with either the test material extract or the control group, and the cells were cultured in an incubator. Images of the scratches at fixed time points (0, 12, and 24 hours) were captured under a microscope. Finally, the closure rate of the scratch area in each group was measured and compared using image analysis software to quantitatively evaluate the material's promoting effect on cell migration. Pre-cooled Matrigel was spread into well plates and cured. HUVECs were then seeded onto the gel. ADF / F-ADF and culture medium were mixed at a weight-to-volume ratio of 0.2 g:1 mL, ensuring complete immersion of ADF / F-ADF in the medium. Extraction was carried out continuously at 37°C in a shaker for 24 hours. The extract was then filtered through a 0.22 μm microporous membrane for sterilization and mixed with an equal volume of ordinary culture medium to obtain a culture medium containing the ADF / F-ADF extract. The experimental group used culture medium containing ADF / F-ADF extract, while the control group used ordinary culture medium. After incubation for several hours, the cells were observed and photographed under a microscope. The number of closed tubular structures, nodes, and total tube length formed in each field of view were counted. The in vivo angiogenesis process was simulated to assess the material's ability to promote the formation of tubular structures by endothelial cells. For adipogenesis promotion, adipose-derived mesenchymal stem cells (ADSCs) were used as a model, and the effects of ADF on ADSC proliferation, migration, and differentiation into adipocytes were evaluated through a co-culture system. The cell proliferation assay was the same as the EdU method in the angiogenesis assay. ADSCs were co-cultured with ADF / F-ADF material, and cell proliferation activity was detected at specific time points. For cell migration assays, the migration of ADSCs was observed using ADF / F-ADF material or its extract as a chemotactic source. Serum-free cell suspensions were added to the upper chamber of the Transwell, and complete culture medium containing ADF / F-ADF extract or directly containing the material sample was added to the lower chamber. After incubation for a certain period, the cells were fixed, stained, and the number of cells that passed through the polycarbonate membrane and reached the lower chamber was counted. Adipogenic differentiation induction and identification: After co-culturing ADSCs with the material to a certain density, the medium was replaced with Gibco™ human mesenchymal stem cell adipogenic differentiation induction medium.The experimental and control groups used the same induction culture medium to eliminate the influence of the inducer itself, focusing on the effect of the microenvironment provided by the material on differentiation. The medium was changed every two days for 21 days of induction. After induction, the culture medium was discarded, and cells were fixed with 4% paraformaldehyde (v / v). Then, Oil Red O working solution was used to stain the cells to mark intracellular lipid droplets (which appeared red). After microscopic imaging, the Oil Red O dye in the cells was dissolved in isopropanol, and its absorbance at 510 nm was measured for semi-quantitative analysis. Specific results are as follows:

[0067] Angiogenesis-promoting ability: such as Figure 9 As shown in the co-culture experiment with vascular endothelial cells, both ADF and F-ADF can significantly promote the proliferation, migration, and in vitro tube formation of endothelial cells.

[0068] Lipogenesis-promoting ability: such as Figure 10 As shown in the experiment, co-culture with adipose-derived mesenchymal stem cells (ADF) and F-ADF can effectively promote the proliferation, migration and adipogenic differentiation of stem cells.

[0069] Example 7: In vivo degradation and tissue regeneration of ADF.

[0070] The degradation characteristics and tissue regeneration capacity of ADF and its cryopreserved sample F-ADF in vivo were evaluated through systematic animal experiments. Seven-week-old male BALB / c nude mice were used as animal models. Materials were cut to uniform specifications and implanted into the subcutaneous tissue of the mouse back under general anesthesia. Multiple observation time points (2, 4, 12, and 24 weeks) were established, with sufficient sample size at each time point to ensure statistical power. During implantation, the animals' health status and macroscopic changes at the implantation site were observed regularly. At the predetermined time points, the animals were euthanized, and the implant and surrounding tissue were completely removed. The obtained samples were processed in three parts: one part was immediately subjected to mechanical property testing to assess changes in tensile strength and elongation at break; another part was fixed with 4% paraformaldehyde for subsequent histological analysis; and the third part was cryopreserved at -80°C for molecular biological analysis. Histological analysis included paraffin section preparation and various staining methods. Hematoxylin-eosin staining was used to observe the basic structure and cell infiltration of the material; Masson staining was used to show the distribution and regeneration of collagen fibers; immunohistochemical staining employed species-specific collagen antibodies to distinguish between host-derived collagen and implant material-derived collagen, thereby accurately calculating the host integration rate. Furthermore, immunofluorescence staining was used to label mature adipocytes with Perilipin antibody and vascular structures with CD31 and α-SMA antibodies to assess the progress of adipogenesis and vascularization. For data analysis, image analysis software was used to quantitatively analyze tissue sections, including the percentage of newly formed adipose tissue area, vascular density, and host collagen replacement rate. Mechanical testing data were acquired using a material testing machine and analyzed using specialized software. All data are expressed as mean ± standard deviation, and appropriate statistical methods were used for inter-group comparisons, with p < 0.05 considered statistically significant. Specific results are as follows:

[0071] Morphological and mechanical evolution: such as Figure 11 As shown, the implant maintained good structural integrity over 24 weeks, with a slight initial increase in volume followed by stabilization, and a retention rate of approximately 140%. Even after 24 weeks, the material remained malleable. Mechanical testing revealed a decrease in tensile strength (from 383.35±16.41 kPa to 298.73±18.26 kPa), but an increase in elongation at break (from 5.36±0.52% to 7.15±1.54%), indicating benign remodeling and increased flexibility within the body.

[0072] Tissue regeneration and remodeling: such as Figure 12As shown, H&E and Masson staining revealed a significant amount of newly formed adipose tissue and collagen deposition within the implant over time. Perilipin immunofluorescence confirmed active adipogenesis, with the positive area increasing significantly from 17.91±7.87% at 12 weeks to 55.51±11.87% at 24 weeks.

[0073] Cell infiltration and vascularization: such as Figure 13 As shown, DAPI staining revealed that cells infiltrated from the periphery to the center, and by 12 weeks they were evenly distributed. Immunofluorescence showed that the density of both total vessels (CD31⁺) and mature vessels (α-SMA⁺CD31⁺) increased significantly over time.

[0074] Host remodeling: such as Figure 14 As shown, species-specific immunohistochemistry revealed that host-derived collagen gradually replaced the ADF scaffold, with a replacement rate as high as 93.10±3.21% by week 24, achieving good host integration and functional remodeling.

[0075] Example 8: ADF promotes the repair of full-thickness skin defects.

[0076] This study systematically evaluated the efficacy of ADF and its cryopreserved sample F-ADF in promoting the repair of full-thickness skin defects by establishing a nude mouse full-thickness skin defect model. The experiment used a nude mouse full-thickness skin defect model, and the experimental animals were randomly divided into four groups: the ADF experimental group, the F-ADF experimental group, the acellular dermal matrix positive control group (ADM), and the blank control group. A rectangular full-thickness skin defect with a diameter of 15 mm and a side length of 15 mm was established, and the corresponding materials were precisely implanted. The wound healing process was observed regularly. On postoperative days 0, 4, 7, 11, 14, and 18, the wound was photographed using a high-definition digital camera, and the wound area was calculated using ImageJ software to quantify the wound closure rate and assess the impact of different treatments on the healing speed. For histological analysis, wound tissue samples were obtained on postoperative days 7 and 14. The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then prepared into serial sections with a thickness of 5 μm. Core indicators of wound healing, including granulation tissue thickness, inflammatory cell infiltration, and epithelial regeneration, were observed using hematoxylin-eosin staining. Masson staining was used to assess collagen fiber deposition, distribution, and maturity, with a focus on the ratio of type I to type III collagen to determine tissue repair quality. To further investigate angiogenesis and maturation, immunofluorescence staining analysis was also performed. All vascular endothelial cells were labeled with anti-CD31 antibody, while perivascular cells were labeled with anti-α-smooth muscle actin (α-SMA) antibody. The ratio of mature vessels (expressing both CD31 and α-SMA) to total vessels (CD31 positive) was observed and calculated using laser confocal microscopy to objectively assess differences in vascular maturation among different treatment groups. Wound closure rate, granulation tissue thickness, epithelial regeneration length, collagen deposition area, and mature vessel density were expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons, with p < 0.05 considered statistically significant. This comprehensive experimental design allows for a complete and objective evaluation of the overall performance of ADF materials in promoting skin defect repair across multiple dimensions, from macroscopic healing to microscopic structure, and from tissue regeneration to vascular maturation. Specific results are as follows:

[0077] Wound closure rate: Compared with acellular dermal matrix (ADM) and the blank control group, such as Figure 15 As shown, the ADF and F-ADF groups exhibited faster wound closure speed and significantly higher closure rates. There was no significant difference between the ADF and F-ADF groups, indicating that cryopreservation did not affect their activity.

[0078] Histological analysis: such as Figure 16 As shown, H&E staining revealed that the ADF and F-ADF groups had thicker granulation tissue and better epithelial regeneration on days 7 and 14. Masson staining showed earlier and more mature type I collagen deposition.

[0079] Vascular maturity: such as Figure 16 As shown, immunofluorescence analysis revealed that the ratio of mature blood vessels in the wound was significantly higher in the ADF and F-ADF groups than in the ADM group.

[0080] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0081] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0082] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane, characterized in that, The preparation method of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane is as follows: fat fibers are separated from autologous adipose tissue and washed, fat cells are broken until the fat fibers turn white, purified fat fibers are obtained after washing, and the purified fat fibers are wrapped in a porous mesh and pressed to form a clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane.

2. The clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane according to claim 1, characterized in that, The porous mesh is a 100-300 mesh mesh.

3. The clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane according to claim 1, characterized in that, The pressurization condition is 0.5 × 10⁻⁶. 6 kp~1.5×10 6 kp.

4. The clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane according to claim 1, characterized in that, The tensile strength of the clinically ready-to-use autologous fat tissue-derived matrix collagen membrane is 300 kPa to 400 kPa, and the elongation at break is 4% to 6%.

5. The application of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane as described in claim 1 in the preparation of drug carriers.

6. The application of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane as described in claim 1 in the preparation of wound dressings.

7. The application of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane as described in claim 1 in the preparation of biomaterial prostheses.

8. The application of the clinically ready-to-use autologous adipose tissue-derived matrix collagen membrane as described in claim 1 in the preparation of soft tissue regeneration scaffolds.

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