A vascularization-inflammation balance type back subcutaneous deep fat acellular matrix material and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a pig back subcutaneous deep layer adipose tissue derived vascularization-inflammation balanced type acellular matrix material and a preparation method and application thereof. The method takes pig back subcutaneous deep layer adipose tissue, and the adipose tissue is prepared into an acellular matrix through freezing and thawing, homogenization, degreasing, nuclease treatment and freeze drying. The obtained material has less residual DNA, low residual glycerin triester, retained collagen fibers and porous structure, and a swelling rate of about 303.64%. The results show that one week after injection, the CD86 and CD206 levels of the method are significantly lower than those of the pig greater omentum adipose acellular matrix group; three months after injection, the CD31, CD31 and alpha-SMA levels of the method are significantly higher than those of the control group. In summary, the material is relatively balanced between the 1-week CD86 / CD206 immune-related marker signal and the 3-month blood vessel formation and maturation related indicators, and can be used for preparing a soft tissue repair or tissue integration promoting material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a vascularized-inflammatory balanced deep subcutaneous fat decellularized matrix material of the back, its preparation method, and its application. Background Technology

[0002] Adipose tissue decellularized matrix is ​​an extracellular matrix material obtained by removing cells, lipids, and nucleic acids from adipose tissue through processes such as decellularization, delipidation, and nucleic acid degradation. It typically retains key extracellular matrix components such as collagen, elastin, and glycosaminoglycans, exhibiting good biocompatibility and significant application potential in soft tissue repair and adipose tissue engineering. Existing technologies typically design adipose tissue decellularized matrix based on factors such as decellularization efficiency, delipidation efficiency, injectability, degradation rate, mechanical support, and adipogenic potential. Publicly available solutions involve porcine adipose tissue cell-free matrix, adipose tissue extracellular matrix preparation, adipose tissue extracellular matrix scaffolds, omentum decellularized matrix soft tissue filling materials, and injectable decellularized matrix microparticles.

[0003] However, in soft tissue repair or subcutaneous tissue integration, implanted materials need to avoid inducing excessively strong early immune-related tissue responses while simultaneously supporting angiogenesis and tissue integration over a longer observation period. Adipose tissue from different anatomical sources may differ in tissue composition and extracellular matrix characteristics, but existing research on decellularized adipose matrix typically focuses on preparation processes, injectability, degradation, or mechanical properties, with less emphasis on parallel comparisons and source screening based on the specific anatomical origin of the adipose tissue, comparing early immune-related responses and long-term angiogenesis-related responses. Therefore, how to select the appropriate anatomical source of the raw material to achieve both early host response control and long-term angiogenesis-related tissue integration in decellularized adipose matrix remains a technical challenge. Summary of the Invention

[0004] To address the lack of existing decellularized adipose matrix materials that balance early immune-related tissue responses and long-term angiogenesis-related responses based on anatomical source selection, this invention provides a vascularization-inflammation balanced decellularized matrix material derived from deep subcutaneous adipose tissue of the pig's back, along with its preparation method and applications.

[0005] Specifically, this invention prepares decellularized matrices from porcine superficial subcutaneous fat, porcine deep subcutaneous fat, and porcine greater omentum fat under the same processing conditions, and compares their effects by subcutaneous injection in mice. One week after injection, dual immunofluorescence quantitative results for CD86 and CD206 showed that the CD86-positive and CD206-positive areas in the porcine deep subcutaneous fat decellularized matrices group were significantly lower than those in the porcine greater omentum fat decellularized matrices group. Three months after injection, dual immunofluorescence quantitative results for CD31 and α-SMA showed that the CD31-positive area and the CD31 / α-SMA double-positive area in the porcine deep subcutaneous fat decellularized matrices group were significantly higher than those in the porcine superficial subcutaneous fat decellularized matrices group and the porcine greater omentum fat decellularized matrices group. These results indicate that porcine deep subcutaneous fat decellularized matrices possess both relatively low 1-week CD86 / CD206 immune-related marker signals and relatively strong 3-month angiogenesis and vascular maturation-related indicators, thus identifying it as a preferred source of decellularized matrices among the three porcine adipose tissues.

[0006] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a method for preparing a decellularized matrix derived from deep subcutaneous adipose tissue of the pig's back, comprising the following steps: 1) Obtain the deep subcutaneous fat tissue from the back of the pig and cut it into pieces; 2) Repeated freeze-thaw treatment of the deep subcutaneous fat tissue on the back of the cut pig; 3) Add buffer solution, homogenize and break up the frozen and thawed deep subcutaneous adipose tissue of the pig back, and collect the precipitate by centrifugation; 4) Mix the precipitate with the degreasing agent and shake to perform degreasing treatment; 5) Remove the defatting reagent and add nuclease for nucleic acid degradation treatment; 6) After removing nucleases, washing, and freeze-drying, a decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back was obtained.

[0007] In some embodiments of the present invention, the pigs include, but are not limited to, domestic pigs, wild boars, and related breeds. Related breeds include 83 local pig breeds, 38 cultivated breeds, and 6 introduced breeds and their breeding lines recorded in the "National List of Livestock and Poultry Genetic Resources (2020)". The specific type of pig does not affect the implementation of the present invention.

[0008] In some embodiments of the present invention, in step 1), the deep subcutaneous adipose tissue of the pig's back is the inner back fat tissue closest to the back skeletal muscles in the subcutaneous adipose tissue located between the skin and the skeletal muscles of the pig's back.

[0009] In some embodiments of the present invention, the deep subcutaneous adipose tissue on the back of the pig is cleaned of blood, tissue debris and surface impurities before being cut into pieces.

[0010] In some embodiments of the present invention, the size of the cut pieces is 0.5–2 cm. 3 Preferably, it is a small piece of 1 cm × 1 cm × 1 cm.

[0011] In some embodiments of the present invention, in step 2), the repeated freeze-thaw cycles are performed using liquid nitrogen for rapid freezing, and are repeated 2 to 5 times, preferably 3 times.

[0012] In some embodiments of the present invention, in step 3), the buffer solution includes phosphate buffer, Tris buffer, HEPES buffer, or physiological saline.

[0013] In some embodiments of the present invention, the ratio of the buffer solution to the deep subcutaneous adipose tissue of the pig's back is (1-5) mL: 1g.

[0014] In some embodiments of the present invention, the homogenization speed is 10,000 to 15,000 rpm and the homogenization time is 10 to 20 min.

[0015] In some embodiments of the present invention, the centrifugation speed is 2000-4000 rpm, and the centrifugation time is 5-10 min.

[0016] In some embodiments of the present invention, in step 4), the degreasing agent includes isopropanol, ethanol, acetone, or a combination thereof.

[0017] In some embodiments of the present invention, the ratio of the degreasing agent to the precipitate is (1-5) mL: 1 g.

[0018] In some embodiments of the present invention, the temperature of the degreasing treatment is 25–42°C.

[0019] In some embodiments of the present invention, the degreasing treatment time is 24 to 72 hours.

[0020] In some embodiments of the present invention, in step 5), the nuclease includes deoxyribonuclease and ribonuclease.

[0021] In some embodiments of the present invention, the steps of removing the defatting reagent and nuclease described above are performed by washing with buffer solution.

[0022] In some embodiments of the present invention, the washing is an agitation wash for 2 to 10 minutes, repeated 6 to 8 times.

[0023] In a second aspect, the present invention provides a decellularized matrix derived from deep subcutaneous adipose tissue of the back of a pig, prepared by the preparation method described in the first aspect of the present invention.

[0024] In some embodiments of the present invention, the decellularized matrix derived from deep subcutaneous adipose tissue of the pig's back has one or more of the following quality characteristics: residual DNA content less than 50 ng / mg dry weight; residual triglycerides less than 5 mg / g dry weight; no obvious intact cell nuclei observed by HE staining and DAPI staining; no obvious lipid droplet residue observed by Oil Red O staining; Sirius red staining showing preserved collagen fiber structure; porous extracellular matrix structure under scanning electron microscopy; and high swelling capacity and porous structure suitable for tissue ingrowth. In a preferred embodiment, the residual DNA content of the decellularized matrix from deep subcutaneous adipose tissue of the pig's back is approximately 10–30 ng / mg dry weight, the residual triglycerides are approximately 2–5 mg / g dry weight, and the swelling rate is approximately 250–350%.

[0025] A third aspect of the present invention provides the use of decellularized matrix derived from deep subcutaneous adipose tissue of the pig's back in the preparation of soft tissue repair materials, tissue integration promoting materials, or vascularization-inflammation balanced soft tissue integration materials.

[0026] In some embodiments of the present invention, the decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back includes the decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back prepared by the preparation method described in the first aspect of the present invention.

[0027] A fourth aspect of the present invention provides a soft tissue repair or tissue integration material, said material comprising a decellularized matrix derived from deep subcutaneous adipose tissue of the pig back as described in the second aspect of the present invention, and pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0029] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China* (2020 edition, Volume IV, List of Pharmaceutical Excipients).

[0030] In some embodiments of the present invention, the soft tissue repair or tissue integration material may be in the form of porous sponge, granules, powder, paste, or injectable form.

[0031] The beneficial effects of this invention are: 1. This invention obtained objective indicators to support the screening of raw material sources through parallel in vivo comparisons of three porcine adipose-derived fat matrix materials. One week after injection, the CD86-positive and CD206-positive areas of the porcine dorsal deep subcutaneous adipose-derived fat matrix group were significantly lower than those of the porcine greater omentum adipose-derived fat matrix group, indicating that its CD86 and CD206 immune-related marker signals were lower than those of the greater omentum-derived material. Three months after injection, the CD31-positive area and the CD31 and α-SMA double-positive area of ​​this group were significantly higher than those of the porcine dorsal superficial subcutaneous adipose-derived fat matrix group and the porcine greater omentum adipose-derived fat matrix group, indicating that it had a stronger angiogenesis and vascular maturation-related response at this time point. Therefore, this invention provides a preferred porcine adipose-derived fat matrix material that is more suitable for soft tissue integration needs than simply high-vascularization materials.

[0032] 2. This invention starts from the anatomical source of the raw material and screens deep subcutaneous fat from the back of pigs under the same preparation process and in vivo evaluation conditions. The material from this source shows a relative balance between relatively low 1-week CD86 / CD206 immune-related marker signals and relatively strong 3-month angiogenesis and vascular maturation-related indicators, making it suitable as a soft tissue integrative material with a balanced vascularization-inflammation profile.

[0033] 3. The decellularized subcutaneous fat matrix from the pig's back prepared by this invention exhibits excellent decellularization and defatting effects. No obvious intact cell nuclei were observed after hematoxylin-eosin staining and DAPI staining, and no obvious lipid droplets were observed after Oil Red O staining. The residual DNA content was less than 50 ng / mg dry weight, and the residual triglyceride content was low, indicating that the method of this invention can effectively remove cells, nucleic acids, and lipid components, thereby reducing the risk of adverse host reactions caused by residual cells or lipids after implantation.

[0034] 4. This invention, while removing cells, nucleic acids, and lipids, effectively preserves the extracellular matrix structure derived from adipose tissue. Decellularized subcutaneous adipose matrix from the pig's back, after collagen staining, shows preserved collagen fiber structures, and a porous extracellular matrix network structure is visible under scanning electron microscopy, exhibiting certain swelling properties and compressive mechanical response. These structural and physical properties facilitate liquid absorption, adhesion to host tissue, support for host cell entry, and subsequent tissue integration.

[0035] 5. This invention records the temporal morphological changes of the material after implantation through gross observation at 1 week, 2 weeks, 3 weeks, 4 weeks and 3 months after injection, as well as HE staining and Masson staining at 1 week, 2 weeks, 3 weeks and 3 months. Furthermore, it uses CD86 / CD206 dual immunofluorescence quantitative quantification at 1 week to characterize early immune-related marker signals, and uses CD31 / α-SMA dual immunofluorescence quantitative quantification at 3 months to characterize angiogenesis and vascular maturation-related responses at this time point, providing in vivo evidence for its use as a soft tissue integration material or subcutaneous repair adjuvant material.

[0036] 6. The preparation method of this invention is relatively mild, has a clear process, and good reproducibility. The method includes steps such as adipose tissue acquisition, washing, cutting, freeze-thaw, buffer homogenization, centrifugation, isopropanol defatting, DNA / RNA enzyme treatment, washing, and freeze-drying. The obtained material can be further prepared into porous sponge-like, granular, powdered, microparticle-like, paste-like, or injectable forms, which facilitates subsequent development according to the needs of subcutaneous soft tissue integration, soft tissue repair assistance, or integration after tissue filling. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the preparation process of the porcine adipose tissue decellularized matrix material group of the present invention.

[0038] Figure 2 This diagram illustrates the sampling of superficial subcutaneous fat, deep subcutaneous fat, and omentum fat from the back of a pig.

[0039] Figure 3The image shows the hematoxylin-eosin staining results of three types of primitive adipose tissue and their corresponding decellularized adipose matrix.

[0040] Figure 4 Bright-field and DAPI staining results of three types of primitive adipose tissue and their corresponding decellularized adipose matrix.

[0041] Figure 5 The image shows the Oil Red O staining results of three types of primitive adipose tissue and their corresponding decellularized adipose matrix.

[0042] Figure 6 The image shows the Sirius red staining results of three types of adipose-derived matrix.

[0043] Figure 7 The image shows the results of residual DNA content detection in three types of adipose-derived matrix.

[0044] Figure 8 The image shows the DNA gel electrophoresis results of three types of adipose-derived matrices.

[0045] Figure 9 The figure shows the results of triglyceride residue detection in three types of adipose-derived matrix.

[0046] Figure 10 The images show the scanning electron microscope (SEM) results of three types of decellularized fat matrix. The dashed boxes indicate magnified results.

[0047] Figure 11 The figure shows the swelling properties of three types of adipose-derived matrices.

[0048] Figure 12 The figure shows the results of the compressive mechanical properties test of three types of decellularized fat matrices.

[0049] Figure 13 This is a gross observation of three porcine adipose-derived adipose matrix products in mice at 1, 2, 3, and 4 weeks after subcutaneous injection.

[0050] Figure 14 Gross observation, HE staining, and Masson staining results of subcutaneous injection of decellularized matrix from superficial subcutaneous fat, deep subcutaneous fat, and greater omentum fat in pigs one week after injection.

[0051] Figure 15 Gross observation, HE staining, and Masson staining results of subcutaneous injection of decellularized matrix of superficial subcutaneous fat, deep subcutaneous fat, and omentum fat of pigs two weeks after injection.

[0052] Figure 16Gross observation, HE staining, and Masson staining results of subcutaneous injection of decellularized matrix from superficial subcutaneous fat, deep subcutaneous fat, and omentum fat in pigs three weeks after injection.

[0053] Figure 17 Gross observation, HE staining, and Masson staining results of subcutaneous injection of decellularized matrix from superficial subcutaneous fat, deep subcutaneous fat, and omentum fat in pigs three months after injection.

[0054] Figure 18 The results of CD86 / CD206 double immunofluorescence staining and quantification of positive area were obtained one week after subcutaneous injection of decellularized matrix of superficial subcutaneous fat, deep subcutaneous fat, and omentum fat in pigs.

[0055] Figure 19 The results of CD31 / α-SMA double immunofluorescence staining and quantification of positive area were obtained 3 months after subcutaneous injection of decellularized matrix of superficial subcutaneous fat, deep subcutaneous fat, and omentum fat in pigs. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] Example 1: Preparation of decellularized fat matrix 1. Acquisition and pretreatment of different adipose tissues Fresh adipose tissue from healthy pigs at slaughterhouses was selected as raw material. Deep subcutaneous adipose tissue from the pig's back was repeatedly washed in PBS solution to remove blood, tissue debris, and surface impurities. After washing, the adipose tissue was cut into small pieces of approximately 1 cm × 1 cm × 1 cm for later use. For source screening, superficial subcutaneous adipose tissue from the pig's back and omentum were simultaneously obtained as comparative materials. Visible blood vessels in the omentum were removed before washing and cutting to reduce interference from vascular tissue in subsequent material structural characterization and in vivo reaction evaluation. A schematic diagram of the preparation process of the porcine-derived decellularized adipose matrix material of this invention is shown below. Figure 1 As shown in the diagram. The sampling diagrams for superficial subcutaneous fat, deep subcutaneous fat, and omentum fat from the back of a pig are as follows. Figure 2 As shown.

[0058] 2. Preparation of decellularized fat matrix Pretreated fat fragments from different sources were subjected to repeated freeze-thaw cycles, a total of 3 cycles. After each freeze-thaw cycle, PBS solution was added at a ratio of 1 g adipose tissue to 3 mL PBS solution, and homogenized using a JJ-2B homogenizer at 12000 rpm for 15 min to obtain adipose tissue homogenate. The homogenate was centrifuged at 25°C and 3000 rpm for 5 min, and the upper lipid layer and liquid fraction were discarded, collecting the white precipitate at the bottom. The white precipitate was then added to 99% isopropanol for degreasing by shaking at a mass-to-volume ratio of 1 g adipose tissue to 3 mL at 37°C for 48 h. After degreasing, the tissue was washed 6 to 8 times with PBS solution, each time with shaking for 5 min.

[0059] The washed precipitate was placed in a DNA / RNase solution for nucleic acid degradation. The enzyme solution contained 55 mM disodium hydrogen phosphate, 17 mM potassium dihydrogen phosphate, 4.9 mM magnesium sulfate heptahydrate, 15000 U DNase I, and 12.5 mg RNase A. DNase I was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China, catalog number D7076), and RNase A was derived from bovine pancreas and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, catalog number R104906). The samples were treated at 37°C for 16 h, and then washed 6 to 8 times with PBS solution, each time with shaking for 5 min. Finally, the samples were lyophilized at -70°C, under a vacuum of 1 Pa, for 12 h to obtain porous, sponge-like decellularized subcutaneous fat matrix from the deep subcutaneous fat layer of the pig's back, decellularized subcutaneous fat matrix from the superficial subcutaneous fat layer of the pig's back, and decellularized omentum fat matrix from the pig.

[0060] Example 2: Decellularization, defatting, and collagen retention effect detection 1. Experimental Methods Decellularized subcutaneous deep fat matrix, decellularized subcutaneous superficial fat matrix, and decellularized omental fat matrix samples prepared in Example 1 were collected, and corresponding original adipose tissue samples were used as controls. After fixation, dehydration, embedding, and sectioning, the samples were stained with HE, DAPI, and Sirius red, respectively; after cryopreservation and embedding, the samples were stained with Oil Red O.

[0061] 2. Experimental Results Staining results as follows Figures 3-6As shown, the original adipose tissue clearly shows cellular and nuclear structures, while after decellularization, no intact cell nuclei were observed in the decellularized matrix of the deep subcutaneous adipose tissue on the pig's back, indicating a significant reduction or near-complete removal of cellular components. DAPI staining results showed no obvious nuclear fluorescence signal in the decellularized matrix. Sirius red staining results showed that the decellularized matrix retained collagen fiber structure. Oil Red O staining results showed no obvious lipid droplet residue in the decellularized matrix.

[0062] Example 3: Detection of Residual DNA Content and Triglyceride Residue The residual DNA content in the samples was detected using a DNA content detection kit, and the results were normalized to the sample dry weight. The detection results are as follows: Figure 7 As shown, the residual DNA content in the decellularized matrix of deep subcutaneous fat in the back of the pig was approximately 24.17 ng / mg dry weight, which is lower than 50 ng / mg dry weight. DNA gel electrophoresis results are as follows... Figure 8 As shown, the bands of intact genomic DNA are significantly weakened or disappear.

[0063] Lipid residues were detected using a triglyceride assay kit. The results are as follows: Figure 9 As shown, the residual triglyceride content in the decellularized matrix of deep subcutaneous fat on the back of pigs was approximately 3.90 ± 0.42 mg / g dry weight. Combined with the Oil Red O staining results, this indicates that the above preparation method can effectively remove lipid components.

[0064] Example 4: Testing of microstructure, swelling properties and mechanical properties The surface and internal microstructure of the decellularized matrix in the deep subcutaneous fat layer of pig backs were observed using scanning electron microscopy. Results are as follows: Figure 10 As shown, this material exhibits a porous structure and an extracellular matrix fiber network structure, with a porous structure suitable for host cell entry and tissue ingrowth.

[0065] The swelling properties were determined using the PBS water absorption method. The dry weight of the sample was recorded, and the wet weight was measured after the sample had fully absorbed water in PBS. The swelling rate was calculated as "Swelling rate = (Wet weight - Dry weight) / Dry weight × 100%".

[0066] The results are as follows Figure 11 As shown, the swelling rate of the decellularized matrix of deep subcutaneous fat on the back of pigs is approximately 303.64%, indicating that it has good water absorption and tissue fluid adsorption capacity.

[0067] Compression tests were conducted using a mechanical testing device, and the load changes under different compression displacements were recorded to obtain load-displacement response curves. The results are as follows: Figure 12 As shown, the decellularized subcutaneous fat matrix of pig back has detectable compressive mechanical properties and can be used as a supporting matrix for soft tissue repair or tissue integration materials.

[0068] Example 5: Comparison of in vivo tissue responses of decellularized adipose matrix from different sources and screening for preferred sources To compare the in vivo tissue responses of porcine adipose tissue matrix from different anatomical sources, the porcine superficial subcutaneous adipose tissue matrix, deep subcutaneous adipose tissue matrix, and porcine greater omentum adipose tissue matrix prepared in Example 1 were respectively prepared into material forms suitable for subcutaneous injection. Subcutaneous injection was performed in mice under the same conditions of material dry weight, injection volume, injection site, and observation time. Gross observations were performed at 1 week, 2 weeks, 3 weeks, 4 weeks, and 3 months post-injection. Samples were collected at 1 week, 2 weeks, 3 weeks, and 3 months post-injection for HE staining and Masson staining. The results are as follows: Figures 13-17 As shown.

[0069] To reduce the subjectivity of simple morphological observation, tissue sections from 1 week post-injection were subjected to CD86 / CD206 dual immunofluorescence staining. The CD86-positive and CD206-positive areas were used as quantitative indicators of early immune-related tissue responses. Tissue sections from 3 months post-injection were subjected to CD31 / α-SMA dual immunofluorescence staining. The CD31-positive area was used as an indicator of angiogenesis, and the area of ​​double positivity for CD31 and α-SMA was used as an indicator of vascular maturation. The immunofluorescence staining and quantitative results are as follows: Figure 18 and Figure 19 As shown in the figure. "express p < 0.001, “ns” indicates that the difference is not statistically significant.

[0070] Gross observation showed varying degrees of surrounding tissue attachment in the three types of adipose-derived matrix at different time points. HE staining revealed the presence of nucleated host cells in the implanted area and at the material-host tissue interface; Masson staining showed collagen staining signals in and around the material area, and the interface morphology changed over time. These gross and histological results were used to show the temporal morphological changes after material implantation. The immune-related marker signals at 1 week and the angiogenesis-related reactions at 3 months were based on the following quantitative immunofluorescence results.

[0071] The results of CD86 / CD206 dual immunofluorescence one week after injection are as follows: Figure 18 As shown, the CD86-positive area in the decellularized matrix group of porcine greater omentum fat was significantly higher than that in the decellularized matrix group of deep subcutaneous fat in the back of the pig. p < 0.001); the CD206 positive area in the porcine greater omentum fat decellularized matrix group was also significantly higher than that in the porcine back subcutaneous deep fat decellularized matrix group ( pThe difference in CD206 positive area between the superficial subcutaneous fat decellularized matrix group and the deep subcutaneous fat decellularized matrix group of pig back was <0.001. These results indicate that one week after injection, the CD86 and CD206 immune-related signal in the deep subcutaneous fat decellularized matrix of pig back were lower than those in the decellularized omentum fat decellularized matrix of pig back.

[0072] The results of CD31 / α-SMA dual immunofluorescence 3 months after injection are as follows: Figure 19 As shown, the CD31-positive area in the deep subcutaneous fat decellularized matrix group of pig back was significantly higher than that in the superficial subcutaneous fat decellularized matrix group of pig back and the greater omentum fat decellularized matrix group. p < 0.001); the area of ​​double positivity for CD31 and α-SMA in this group was also significantly higher than that in the other two groups ( p <0.001). The above results indicate that, 3 months after injection, the decellularized subcutaneous fat matrix in the back of pigs exhibits strong angiogenesis and vascular maturation-related responses.

[0073] In summary, porcine omental fat decellularized matrix showed higher CD86 and CD206 immune-related marker signals one week after injection, while porcine back subcutaneous deep fat decellularized matrix showed lower signals at the same time point. However, at three months after injection, it exhibited higher angiogenesis and vascular maturation-related indicators than porcine back subcutaneous superficial fat decellularized matrix and porcine omental fat decellularized matrix. Therefore, among the three porcine fat sources compared in this embodiment, porcine back subcutaneous deep fat decellularized matrix showed a relative balance between immune-related marker signals at one week and vascularization-related indicators at three months, thus determining it as the preferred porcine fat decellularized matrix source. The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments and features of the embodiments of the present invention can be combined with each other without conflict.

Claims

1. A method for preparing a decellularized matrix derived from deep subcutaneous adipose tissue of the pig's back, comprising the following steps: 1) Obtain the deep subcutaneous fat tissue from the back of the pig and cut it into pieces; 2) Repeated freeze-thaw treatment of the deep subcutaneous fat tissue on the back of the cut pig; 3) Add buffer solution, homogenize and break up the frozen and thawed deep subcutaneous adipose tissue of the pig back, and collect the precipitate by centrifugation; 4) Mix the precipitate with the degreasing agent and shake to perform degreasing treatment; 5) Remove the defatting reagent and add nuclease for nucleic acid degradation treatment; 6) After removing nucleases, washing, and freeze-drying, a decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back was obtained.

2. The preparation method according to claim 1, characterized in that: In step 1), the deep subcutaneous adipose tissue on the back of the pig is the inner back fat tissue closest to the back skeletal muscles in the subcutaneous adipose tissue located between the skin and the skeletal muscles of the back of the pig.

3. The preparation method according to claim 2, characterized in that: Before cutting, the deep subcutaneous adipose tissue on the back of the pig is cleaned of blood, tissue debris and surface impurities. The size of the cut pieces is 0.5–2 cm. 3 .

4. The preparation method according to claim 1, characterized in that: In step 3), the buffer solution includes phosphate buffer, Tris buffer, HEPES buffer, or physiological saline. The ratio of the buffer solution to the deep subcutaneous adipose tissue on the back of the pig is (1-5) mL: 1g.

5. The preparation method according to claim 1, characterized in that: In step 4), the degreasing agent includes isopropanol, ethanol, acetone, or a combination thereof; The ratio of the defatting reagent to the precipitate is (1-5) mL: 1 g; The temperature for the degreasing treatment is 25–42°C; The degreasing treatment takes 24 to 72 hours.

6. The preparation method according to claim 1, characterized in that: In step 5), the nuclease includes deoxyribonuclease and ribonuclease.

7. A decellularized matrix derived from deep subcutaneous adipose tissue of the back of a pig, prepared by the preparation method according to any one of claims 1 to 6.

8. Application of decellularized matrix derived from deep subcutaneous adipose tissue of pig back in the preparation of soft tissue repair materials, tissue integration promoting materials, or vascularization-inflammation balanced soft tissue integration materials.

9. The application according to claim 8, characterized in that: The decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back includes the decellularized matrix derived from the deep subcutaneous adipose tissue of the pig's back prepared by the preparation method according to any one of claims 1 to 6.

10. A soft tissue repair or tissue integration material, characterized in that: The soft tissue repair or tissue integration material comprises the decellularized matrix derived from the deep subcutaneous adipose tissue of the pig back as described in claim 7, and pharmaceutically acceptable excipients, wherein the material is in the form of a porous sponge, granules, powder, paste, or injectable form.

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