Acellular matrix fiber woven scaffold as well as preparation method and application thereof

Abstract

The invention provides an acellular matrix fiber woven scaffold as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The acellular matrix fiber woven scaffold comprises a woven structure unit, wherein the woven structure unit comprises warp yarns and a plurality of acellular matrix fiber weft yarns; the acellular matrix fiber is prepared by cutting an acellular matrix paper-based material at equal intervals; the plurality of acellular matrix fiber wefts are arranged in parallel according to a bionic trend and are interwoven and fixed through the warps, so that a bionic pattern simulating natural tissue fiber arrangement is formed in the weaving structure unit. The acellular matrix fiber scaffold is prepared by innovatively applying a weaving technology, the scaffold can improve the cell loading efficiency, is beneficial to self-repair and reconstruction of damaged tissues, provides suitable survival and growth conditions for cells, and has good application prospects in the field of biomedical engineering, especially in the aspects of tissue repair and regenerative medicine. And huge application potential is shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a decellularized matrix fiber woven scaffold, its preparation method, and its application. Background Technology

[0002] Biomaterials have important applications in tissue repair and regeneration, enabling the replacement and repair of damaged and degenerated tissues. From a performance perspective, biomaterials need to possess a suitable pore structure to provide stable physical structural support for cell and tissue growth; at the same time, they should also have excellent biocompatibility, bioactivity, and mechanical properties.

[0003] Currently, significant progress has been made in the research of tissue regeneration and repair materials, primarily encompassing the following categories: synthetic polymer materials, such as PolyActive® developed by Innoskel; collagen materials, like Integra® Dermal Regeneration Template from IntegraLife Sciences; and decellularized matrix materials, such as MatriStem® from ACell. Specifically, synthetic polymer materials exhibit high mechanical strength and are easy to process and mold; however, their significant drawback lies in their lack of bioactivity, limiting their tissue repair capabilities. While collagen possesses good biocompatibility, its mechanical properties are poor, and its processability is limited. Decellularized matrix materials, on the other hand, exhibit outstanding biocompatibility and bioactivity, capable of creating a physical support environment for cells, transmitting biochemical signals, and providing a dynamic regulatory environment. Therefore, they are suitable for in vivo implantation to promote physiological tissue remodeling.

[0004] However, it is important to note that decellularized matrix materials obtained through direct decellularization of tissue exhibit a dense structure. This dense structure, to some extent, restricts the migration of endogenous cells, thus negatively impacting the tissue's in-situ regeneration and repair capabilities. A global clinical trial led by the University of Pittsburgh, the only one of its kind globally, used decellularized porcine matrix sheet materials (such as the bladder / small intestinal submucosa) combined with physical therapy to repair traumatic muscle defects, but only achieved scar repair, failing to reconstruct functional muscle tissue. This highlights the inherent limitations of using decellularized matrix sheets alone in guiding functional tissue regeneration.

[0005] To improve the structure and properties of decellularized matrix materials, existing technologies are mainly developing in two directions: The first type of process relies on chemical deconstruction and recombination. Numerous studies have introduced diverse techniques such as electrospinning, 3D printing, and hydrogels to construct porous or customized structures. While these processes contribute to improvements in material morphology to some extent, a common problem remains: they typically require the use of acid solutions, enzyme solutions, or organic solvents to dissolve or digest the decellularized matrix. However, this treatment inevitably damages or destroys extracellular matrix components, leading to a significant reduction in the bioactivity of the decellularized matrix material.

[0006] The second type of process attempts to reshape the structure using purely physical methods. For example, some technical solutions disclose a process similar to papermaking, in which decellularized matrix is ​​homogenized, filtered, and dried to produce porous tissue paper (such as patent CN114984320A). This method avoids the damage caused by chemical reagents, but the resulting structure is essentially a two-dimensional random porous sheet, lacking anisotropic structures that guide cell growth and mimic the fiber arrangement of natural tissues. Therefore, it is difficult to achieve complete functional reconstruction when repairing highly ordered tissues such as muscles and tendons. Another technical solution (such as CN118512659A) attempts to spin-cut and twist tubular decellularized tissue into continuous fibers, which are then further woven into tubular scaffolds. While this approach avoids chemical dissolution and achieves fibrous weaving, its process is limited by the specific source of tubular tissue, and its weaving purpose is mainly to construct a macroscopic tubular morphology, rather than mimicking the fiber arrangement of specific tissues (such as muscle bundles) at the microscopic or mesoscopic scale to guide directional cell regeneration. There is still a lack of effective solutions for the repair of large numbers of non-tubular, structurally ordered soft tissue defects (such as volumetric muscle defects and tendon tears).

[0007] Fiber materials have naturally highly interconnected pores and, similar to muscle fibers, have an oriented arrangement structure. The preparation of decellularized matrix into fiber materials has structural adaptability. However, researchers have found that only simple membranous materials, such as the small intestine and bladder membrane, can be prepared into fiber materials by cutting. Simple cutting methods are not suitable for cutting large blocks of muscle or complex structures of other organs and tissues, making them difficult to shape. Therefore, new fiber preparation process strategies are needed.

[0008] Given the above situation, how to completely avoid the use of acids, enzymes, or organic solvents to maximize the preservation of the bioactivity of the decellularized matrix, and how to use simple, widely available technical means to not only improve the porosity of the material, but more importantly, to achieve a biomimetic adaptation of the material's microstructure to the natural tissue fiber arrangement, and construct a three-dimensional scaffold structure that can actively guide the directional migration, arrangement, and functionalization of cells, thereby truly improving the regeneration effect of functional tissues, has become a key technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention proposes a decellularized matrix fiber woven scaffold, its preparation method, and its application. This invention uses decellularized matrix paper-based material as raw material and employs a cutting process to obtain decellularized matrix fibers. Subsequently, the fibers are longitudinally arranged in the weft portion to simulate the biomimetic structure of muscle bundles. Furthermore, a warp-weft orthogonal weaving method, with warp threads interlaced and fixed, provides stability to the scaffold, thereby obtaining a decellularized matrix fiber woven scaffold material. This invention, on the one hand, maximizes the preservation of the bioactivity of the decellularized matrix fibers; on the other hand, it innovatively integrates fiber weaving technology with the biomimetic concept of the material's natural structure, opening up new avenues for the application of decellularized matrix materials in tissue repair and related fields.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a decellularized matrix fiber woven scaffold, comprising a woven structural unit, wherein the woven structural unit includes warp threads and multiple decellularized matrix fiber weft threads; The decellularized matrix fibers are made by cutting decellularized matrix paper-based material at equal intervals; The multiple decellularized matrix fiber weft threads are arranged in parallel according to a biomimetic direction and fixed by the interlacing of the warp threads, thereby forming a biomimetic pattern in the weaving structure unit that simulates the arrangement of natural tissue fibers.

[0011] Furthermore, the width of the decellularized matrix fibers is 1-2000 μm and the length is 1-100000 μm.

[0012] Furthermore, the weaving method of the warp and weft threads is any one of the following: orthogonal weave, plain weave, twill weave, or satin weave.

[0013] Furthermore, the biomimetic orientation is an arrangement direction that mimics the orientation of skeletal muscle bundles, tendon fiber bundles, nerve bundles, or vascular smooth muscle cells.

[0014] Furthermore, the support is formed by stacking two or more layers of the woven structural units to form a three-dimensional structure.

[0015] This invention also proposes a method for preparing the above-mentioned decellularized matrix fiber woven scaffold, comprising the following steps: The decellularized matrix paper-based material is cut at equal intervals to obtain decellularized matrix fibers; Multiple decellularized matrix fibers are arranged in parallel according to a predetermined biomimetic orientation, serving as weft lines; The warp threads are interlaced to fix the weft threads, forming a single-layer woven structure unit with a biomimetic pattern; At least one layer of the single-layer woven structural unit is stacked to obtain the decellularized matrix fiber woven scaffold.

[0016] Furthermore, equidistant cutting ensures the consistency of fiber materials in size, facilitating subsequent fiber arrangement and weaving.

[0017] Furthermore, the decellularized matrix paper-based material is prepared by homogenizing, filtering, and drying the decellularized matrix material; more specifically, the preparation method of the decellularized matrix paper-based material includes the following steps: (1) Preparation of decellularized matrix material: Select fresh tissue, perform strict disinfection and sterilization treatment on it, and then perform decellularization treatment on the tissue. Through this treatment process, remove the cellular components in the tissue and retain only the extracellular matrix to obtain the decellularized matrix material; (2) Preparation of decellularized matrix paper-based material: Crush the decellularized matrix material obtained in step (1), and then homogenize and stir it to form a uniform homogenate. Place the homogenate on a flat-bottomed filter screen, filter the homogenate, and let it stand and dry. After the water has completely evaporated, the decellularized matrix paper-based material is obtained. This process aims to transform the decellularized matrix material into a paper-based form for further processing.

[0018] Furthermore, the arrangement of the weft threads aims to mimic the natural structure of muscle tissue and other similar structures to improve the adaptability of the scaffold to biological tissues, forming a single-layer woven structural unit with a biomimetic pattern. This unit not only provides structural stability to the entire scaffold but also creates a certain porosity inside the scaffold through the gaps formed by the interlacing of warp and weft threads. These pores are of great significance for cell growth, migration, and nutrient transport.

[0019] Furthermore, at least one layer of the aforementioned single-layer woven structural unit is stacked in an orderly manner along the Z-axis. This stacking method allows the sheets (single-layer woven structural units) to overlap and form a three-dimensional structure, ultimately obtaining a decellularized matrix fiber woven scaffold. This three-dimensional structure can be a conventional geometric structure such as a cube or cone, which offers advantages such as ease of manufacturing and structural stability in certain common biomedical applications. Alternatively, it can be a specific structure tailored to the actual site of injury, such as wounds from war or tumor resection. This structure, customized for the actual injury site, aims to maximize the satisfaction of different types of wound repair needs, improve the adaptability of the decellularized matrix fiber woven scaffold to damaged tissue, thereby providing a more ideal support framework for tissue repair and facilitating the efficient repair and regeneration of damaged tissue.

[0020] The present invention also proposes an application of the above-mentioned decellularized matrix fiber woven scaffold in the preparation of tissue engineering scaffold materials.

[0021] Furthermore, the tissue is skeletal muscle, tendon, ligament, skin, or fascia.

[0022] The decellularized matrix fiber scaffold has two main applications in practice. First, it can be directly used for in vivo transplantation, leveraging its structural and performance advantages to provide physical support and a suitable microenvironment for damaged tissues, promoting tissue repair and regeneration. Second, drugs can be attached to the decellularized matrix fiber scaffold through specific loading methods such as immersion, cell loading, or hydrogel coating before transplantation. Immersion allows drugs to fully penetrate the scaffold and be slowly released in vivo; cell loading utilizes cells as drug carriers, achieving targeted drug delivery as cells colonize and migrate on the scaffold; hydrogel coating encapsulates the drug and enhances the scaffold's biocompatibility with surrounding tissues, allowing the drug to exert its effects continuously in specific areas. These drug loading methods further expand the application scope of decellularized matrix fiber scaffolds in tissue engineering, improving their adaptability to different disease treatments and tissue repair needs.

[0023] The present invention also proposes a tissue repair product comprising the above-mentioned decellularized matrix fiber scaffold and an active ingredient loaded thereon; the active ingredient is selected from one or more of cells, growth factors, drugs or biocompatible hydrogels.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention innovatively utilizes weaving technology to prepare decellularized matrix fiber scaffolds. In the subsequent processing of the decellularized matrix material, no chemical reagents such as acids, enzymes, or organic solvents that could damage the material are used. This process is gentle and can retain the inherent components and bioactivity of the natural decellularized matrix to the greatest extent. Compared with traditional decellularized matrix scaffold materials, this invention can improve cell loading efficiency, providing a more favorable attachment environment for cells, enabling the scaffold to support more cells per unit volume or area, thereby promoting the endogenous regeneration process of tissues and aiding in the self-repair and reconstruction of damaged tissues. Furthermore, the decellularized matrix fibers provided by this invention have an accelerated degradation rate, releasing various bioactive factors during degradation, which can accelerate the tissue repair process. This scaffold can create a microenvironment highly similar to that of real decellularized matrix, providing suitable conditions for cell survival and growth, demonstrating enormous application potential in the field of biomedical engineering, especially in tissue repair and regenerative medicine.

[0025] (2) Existing pure decellularized matrix materials have some inherent defects. Their structure is relatively dense, with relatively low mechanical strength and poor material controllability, making it impossible to accurately meet the diverse needs of different tissue repair scenarios. The woven scaffold structure constructed in this invention has unique advantages. It has abundant pores, providing more attachment sites for cells, improving cell loading efficiency, and does not hinder cell metabolism or nutrient diffusion, thus creating more favorable conditions for cell growth and tissue repair.

[0026] (3) This invention possesses high flexibility and controllability. On the one hand, by selecting decellularized matrix materials from different tissue sources and choosing materials with tissue specificity, combined with precise adjustment of key parameters such as the thickness and mechanical properties of the paper-based material, as well as fiber diameter, length, and weft spacing, decellularized matrix fiber scaffolds that can be precisely prepared with adjustable dimensions, pore size, and mechanical strength. On the other hand, based on the diversity of actual clinical needs, this invention can flexibly adjust the scaffold structure to better suit various specific application scenarios. This high adaptability effectively expands the scope of application of this invention in the biomedical field, demonstrating great application potential in other related fields such as wound repair and tissue regeneration.

[0027] (4) The decellularized matrix paper-based material prepared by the present invention is processed by a precise equidistant cutting process. The equidistant cutting can ensure the consistency of fiber material in size, and ensure the efficiency of weaving and the regularity of the fabric.

[0028] (5) The single-layer fiber woven sheet of the present invention utilizes the principle of weaving technology, and obtains the fiber woven sheet by interlacing warp and weft threads. It aims to simulate the natural structure of muscle tissue to improve the compatibility of the scaffold with biological tissue. The gaps formed by the interlacing of warp and weft threads create a certain porosity inside the scaffold, and these pores are of great significance for cell growth, migration and nutrient transport.

[0029] (6) The monolayer fiber-woven sheets prepared in this invention are stacked in an orderly manner to form a three-dimensional structure, ultimately obtaining the decellularized matrix fiber-woven scaffold mentioned in this invention. This three-dimensional structure can better fill wounds and provide a highly interconnected porous structure. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1This document describes the preparation and characterization of decellularized muscle matrix fibers. A is a schematic diagram of the preparation process; B shows the hematoxylin-eosin and nuclear staining results of porcine skeletal muscle before and after decellularization in step (1); C and D are quantitative analyses of the number of cell nuclei and DNA content in the material before and after decellularization (n=5); E shows macroscopic images and micrographs of decellularized matrix fibers cut from decellularized matrix paper-based materials with thicknesses ranging from 100 μm to 500 μm; F shows the width distribution test results of decellularized matrix fibers of different thicknesses (n=5); G shows the thickness of decellularized matrix fibers of different thicknesses (n=5); H shows the tensile stress-strain curves of decellularized matrix fibers of different thicknesses; I shows the breaking strength of different decellularized matrix fibers (n=3); J shows macroscopic photographs of the bending (500 μm fiber), knotting (500 μm fiber), twisting (100 μm fiber), bonding (500 μm fiber), and entanglement (500 μm fiber) behaviors of the decellularized matrix fibers.

[0031] Figure 2 This document describes the preparation and physicochemical characterization of the fiber scaffold. A shows a schematic diagram of the preparation process of the decellularized matrix fabric (fiber scaffold); B shows macroscopic and electron microscopic images (top and side views) of fiber scaffolds prepared from decellularized matrix fibers of different thicknesses and a decellularized block from Comparative Example 1; C shows the thickness measurements of different fiber scaffold samples (n=5); D shows the results of toluidine blue permeability measurements of different fiber scaffolds and decellularized block samples; E shows the quantitative statistical analysis results of absorbance of different fiber scaffolds and decellularized block samples (n=3); F and G show the tensile stress-strain curves and corresponding fracture strength statistics of different fiber scaffolds and decellularized block samples (n=3); H shows a schematic diagram of the fiber scaffold being processed into a large structure (ns = no significant difference in the figure). P<0.01; P<0.001; P<0.0001).

[0032] Figure 3 The behavioral changes of L6 cells after culturing on fibrous scaffolds and decellularized blocks for 1, 3, and 5 days are shown. In the figures, A represents the distribution of DiD-labeled red fluorescently labeled L6 cells on the materials; red represents DiD-labeled cells, and black represents the scaffold material. B shows the quantitative analysis of DiD fluorescence (n=3). C shows the quantitative analysis of cell viability using the CCK-8 assay (n=3). D shows the live / dead staining results of L6 cells; green represents live cells, and red represents dead cells. E shows the quantitative analysis of cell viability (n=3). F shows the distribution of L6 cells on fibrous scaffolds and decellularized blocks as shown by FITC-phalloidin (green) and nucleus (blue) staining. G shows the quantitative analysis results of phalloidin fluorescence intensity (n=3).

[0033] Figure 4Tissue reactions at 2 and 4 weeks after subcutaneous implantation of fibrous scaffolds and decellularized blocks in rats. A shows a schematic diagram of subcutaneous scaffold implantation and tissue sampling at different time points; B and C show macroscopic views and weight measurements of the fibrous scaffolds and decellularized blocks removed at 2 and 4 weeks (n=3); D and E show nuclear staining indicating cell infiltration within the implant and corresponding quantitative analysis (n=3); F and I show hematoxylin and eosin staining indicating tissue ingrowth into the implant and quantitative analysis of the ingrowth area (n=3); J and K show hematoxylin and eosin staining indicating microvascular ingrowth and related quantitative analysis (n=3); L and M show α-SMA immunofluorescence (green) indicating the functional vascular smooth muscle layer and corresponding quantitative analysis (n=3).

[0034] Figure 5 This study aimed to promote muscle regeneration in rat defects after implantation of fibrous scaffolds and decellularized blocks. A shows a schematic diagram of scaffold implantation in the tibialis anterior muscle of rats and the experimental timeline; B shows macroscopic images of the implant and harvested tissue at different time points; C shows the muscle mass ratio (defective side / normal side) at 2 months post-implantation (n=3); D and E show collagen deposition and its quantitative analysis using Masson's trichrome staining (n=3). Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] This invention provides a decellularized matrix fiber woven scaffold, comprising a woven structural unit, wherein the woven structural unit includes warp threads and multiple decellularized matrix fiber weft threads; Decellularized matrix fibers are made by cutting decellularized matrix paper-based material at equal intervals; Multiple decellularized matrix fiber weft threads are arranged in parallel according to a biomimetic direction and fixed by interlacing warp threads, thereby forming a biomimetic pattern in the woven structural unit that simulates the arrangement of natural tissue fibers.

[0041] In a preferred embodiment of the present invention, the width of the decellularized matrix fibers is 1-2000 μm and the length is 1-100000 μm.

[0042] In a preferred embodiment of the present invention, the weaving method of the warp and weft is any one of the following: orthogonal weave, plain weave, twill weave, or satin weave.

[0043] In a preferred embodiment of the present invention, the biomimetic orientation is an arrangement direction that simulates the orientation of skeletal muscle bundles, tendon fiber bundles, nerve bundles, or vascular smooth muscle cells.

[0044] In a preferred embodiment of the present invention, the support is formed by stacking two or more layers of woven structural units to form a three-dimensional structure.

[0045] This invention also proposes a method for preparing the above-mentioned decellularized matrix fiber woven scaffold, comprising the following steps: The decellularized matrix paper-based material is cut at equal intervals to obtain decellularized matrix fibers; Multiple decellularized matrix fibers are arranged in parallel according to a predetermined biomimetic orientation, serving as weft lines; The warp threads are interlaced to fix the weft threads, forming a single-layer weaving structure unit with a biomimetic pattern; At least one single-layer woven structural unit is stacked to obtain a decellularized matrix fiber woven scaffold.

[0046] In a preferred embodiment of the present invention, equidistant cutting can ensure the consistency of fiber material in size, providing convenience for subsequent fiber arrangement and weaving.

[0047] In a preferred embodiment of the present invention, the decellularized matrix paper-based material is prepared by homogenizing, filtering and drying the decellularized matrix material; more specifically, the preparation method of the decellularized matrix paper-based material includes the following steps: (1) Preparation of decellularized matrix material: Select fresh tissue, perform strict disinfection and sterilization treatment on it, and then perform decellularization treatment on the tissue. Through this treatment process, remove the cellular components in the tissue and retain only the extracellular matrix to obtain the decellularized matrix material; (2) Preparation of decellularized matrix paper-based material: Crush the decellularized matrix material obtained in step (1), and then homogenize and stir it (for 1-60 min) to form a uniform homogenate. Place the homogenate on a flat-bottomed filter screen (500 mesh size), and let the filtered homogenate stand and dry for 1-120 h. After the water has completely evaporated, the decellularized matrix paper-based material is obtained. Traditionally, the processing of decellularized materials requires the use of acids, enzymes to dissolve them, or the addition of organic solvents, which damages their composition and activity. The decellularized matrix material processing method employed in this invention does not use any acids, enzymes, or organic solvents, thus preserving the original components of the decellularized matrix to the greatest extent possible and maintaining its biological activity. This process aims to transform the decellularized matrix material into a paper-based form for further processing.

[0048] Furthermore, in step (1) of the preparation method for the decellularized matrix paper-based material, the decellularization treatment specifically includes the following steps: Fresh tissue is placed in a 0.1% peracetic acid solution and stirred at 60 r / min for 2 hours for sterilization. After sterilization, the tissue is washed with water to remove residual peracetic acid solution from the surface. The tissue is then washed in a 1% sodium dodecyl sulfate solution at a stirring rate of 50 r / min for 1 day, with the sodium dodecyl sulfate solution being replaced every 8 hours to ensure the stability and continuity of the washing effect and to fully remove cell-related impurities from the tissue. The washed tissue is then washed once with water at a stirring rate of 80 r / min for 3 days, with the washing water being replaced every 3 hours to further remove residual sodium dodecyl sulfate solution and other impurities from the tissue surface. The tissue was placed in a Tris-HCl solution containing DNase and RNase (50 units / mL of DNase and 1 unit / mL of RNase) for enzymatic washing. The washing process was carried out at a constant temperature of 37°C with a stirring rate of 70 rpm for 10 hours. Through the action of enzymes, the DNA and RNA components in the tissue were effectively degraded, further achieving the purpose of decellularization. After the enzymatic washing, the tissue was washed a second time with water for 3 days. This step ensured that residual enzymes and other impurities in the tissue were fully removed, and the decellularization process was completed. The fresh tissue was derived from any human or animal tissue or organ; animals included one or more of pigs, cattle, sheep, dogs, horses, rats, and rabbits; fresh tissue specifically included brain, heart, liver, spleen, lungs, kidneys, muscles, skin, fat, amnion, heart valves, submucosa of the small intestine, muscles, blood vessels, tendons, ligaments, cartilage, etc.

[0049] In a preferred embodiment of the present invention, at least one layer of single-layer woven structural units are stacked in an orderly manner along the Z-axis. This stacking method allows the thin sheets (single-layer woven structural units) to overlap and form a three-dimensional structure, ultimately obtaining a decellularized matrix fiber woven scaffold. This three-dimensional structure can be a conventional geometric structure such as a cube or a cone.

[0050] This invention also proposes an application of the above-mentioned decellularized matrix fiber woven scaffold in the preparation of tissue engineering scaffold materials.

[0051] In a preferred embodiment of the present invention, the tissue is skeletal muscle, tendon, ligament, skin, or fascia.

[0052] This invention also proposes a tissue repair product comprising the aforementioned decellularized matrix fiber scaffold and an active ingredient loaded thereon; the active ingredient is selected from one or more of cells, growth factors, drugs, or biocompatible hydrogels.

[0053] All materials used in the embodiments of this invention were commercially available. Specifically, DMEM culture medium was purchased from Solarbio Science & Technology Co., Ltd., and the rat skeletal muscle myoblast cell line (L6) was purchased from Tianjin Ruigent Trading Co., Ltd. Fresh porcine skeletal muscle was purchased from Tianjin Yingbin Ershang Co., Ltd., etc.

[0054] The technical solution of the present invention will be further illustrated by the following embodiments.

[0055] Example 1 A method for preparing a sheet-like decellularized matrix fiber scaffold for porcine skeletal muscle includes the following steps: (1) Fresh porcine skeletal muscle was placed in a 0.1% peracetic acid solution and stirred at 60 r / min for 2 hours for sterilization. After sterilization, the tissue was washed with water to remove residual peracetic acid solution from the surface. The tissue was then washed in a 1% sodium dodecyl sulfate solution at 50 r / min for 1 day, with the sodium dodecyl sulfate solution being changed every 8 hours to ensure the stability and continuity of the washing effect and to fully remove cell-related impurities from the tissue. The washed tissue was then washed once with water at 80 r / min for 3 days, with the washing water being changed every 3 hours to further remove residual sodium dodecyl sulfate solution and other impurities from the tissue surface. The tissue was placed in a Tris-HCl solution containing DNase and RNase (50 units / mL of DNase and 1 unit / mL of RNase) for enzymatic washing. The washing process was carried out at a constant temperature of 37°C with a stirring rate of 70 rpm for 10 hours. Through the action of enzymes, the DNA and RNA components in the tissue were effectively degraded, further achieving the purpose of decellularization. After the enzymatic washing, the tissue was washed a second time with water (the steps were the same as the first water wash) for 3 days. This second water wash ensured that residual enzymes and other impurities in the tissue were fully removed, and the decellularization process was completed.

[0056] (2) The decellularized matrix material obtained in step (1) is crushed and homogenized for 5 minutes. The homogenate is placed in a 500-mesh flat-bottom filter to remove water. The filtered homogenate is then allowed to stand and dry for 12 hours to obtain decellularized matrix paper-based materials with thicknesses of 100 μm, 200 μm, 300 μm and 500 μm respectively. (3) Cut the decellularized matrix paper-based materials of different thicknesses obtained in step (2) at equal intervals to obtain decellularized matrix fibers of different thicknesses with a width of 700 μm and a length of 5 cm. (4) Using decellularized matrix fibers of different thicknesses as raw materials, multiple decellularized matrix fibers are arranged in parallel according to the direction of muscle bundles as weft lines; (5) The warp threads are fixed with the weft threads in an interlacing manner to form a single-layer woven structure unit with a biomimetic pattern, which is a decellularized matrix fabric (i.e., different fiber scaffolds were prepared by using decellularized matrix fibers of different thicknesses as raw materials in Example 1).

[0057] Comparative Example 1 A method for preparing sheet-like decellularized matrix blocks of porcine skeletal muscle includes the following steps: (1) Decellularization: Fresh porcine skeletal muscle was placed in a 0.1% peracetic acid solution and stirred at 60 r / min for 2 hours for sterilization. After sterilization, the tissue was washed with water to remove residual peracetic acid solution from the surface. The tissue was then washed in a 1% sodium dodecyl sulfate solution at 50 r / min for 1 day, with the sodium dodecyl sulfate solution being changed every 8 hours to ensure the stability and continuity of the washing effect and to fully remove cell-related impurities from the tissue. The washed tissue was then washed once with water at 80 r / min for 3 days, with the washing water being changed every 3 hours to further remove residual sodium dodecyl sulfate solution and other impurities from the tissue surface. The tissue was then enzymatically washed in a Tris-HCl solution containing DNase and RNase. This solution contains 50 units / mL of DNase and 1 unit / mL of RNase. The enzyme washing process is carried out at a constant temperature of 37°C, with a stirring rate of 70 rpm, for 10 hours. Through the action of the enzymes, the DNA and RNA components in the tissue are effectively degraded, further achieving the purpose of decellularization. After the enzyme washing is completed, the tissue is washed a second time with water for 3 days. This step ensures that residual enzymes and other impurities in the tissue are fully removed, and the decellularization process is completed.

[0058] (2) Preparation of decellularized matrix block material: The decellularized matrix material is cut at equal intervals (700 μm in diameter and 5 cm in length), then frozen in a freezer at -80°C for 2 hours, and finally dried in a freeze dryer for 24 hours to obtain decellularized matrix block (decellularized block).

[0059] Figure 1This document describes the preparation and characterization of decellularized muscle matrix fibers. A is a schematic diagram of the preparation process; B shows the hematoxylin-eosin and nuclear staining results of porcine skeletal muscle before and after decellularization in step (1); C and D are quantitative analyses of the number of cell nuclei and DNA content in the material before and after decellularization (n=5); E shows macroscopic images and micrographs of decellularized matrix fibers cut from decellularized matrix paper-based materials with thicknesses ranging from 100 μm to 500 μm; F shows the width distribution test results of decellularized matrix fibers of different thicknesses (n=5); G shows the thickness of decellularized matrix fibers of different thicknesses (n=5); H shows the tensile stress-strain curves of decellularized matrix fibers of different thicknesses; I shows the breaking strength of different decellularized matrix fibers (n=3); J shows macroscopic photographs of the bending (500 μm fiber), knotting (500 μm fiber), twisting (100 μm fiber), bonding (500 μm fiber), and entanglement (500 μm fiber) behaviors of the decellularized matrix fibers.

[0060] Depend on Figure 1 It can be seen that by decellularizing, homogenizing, drying, and slicing pig muscle tissue, decellularized muscle matrix fibers are finally obtained. Figure 1 (A). The decellularization process employed a strategy combining sodium dodecyl sulfate and enzymatic digestion to obtain decellularized muscle matrix. Hematoxylin and eosin staining confirmed the disappearance of cell nuclei after decellularization, while retaining abundant extracellular matrix components. Nuclear staining further revealed extensive removal of cell nuclei, and statistical analysis showed a significant reduction in both the number of cell nuclei and DNA content in the decellularized muscle tissue. Figure 1 (BD).

[0061] The decellularized matrix paper-based materials of varying thicknesses were uniform in thickness and pale yellow in color. These membranes were then cut into decellularized matrix fibers using a cutting device. Macroscopic images showed that, viewed from above, the fiber width was uniform; scanning electron microscopy further indicated a relatively dense surface morphology. From a side view, the fiber thickness was controllable, and electron microscopy revealed a characteristically rough morphology in its sidewall structure. Quantitative analysis confirmed that the prepared decellularized matrix fibers had a uniform width of 0.72 ± 0.04 mm. Figure 1 (E and F). Quantitative analysis showed that the thickness of decellularized matrix fibers in the muscle was relatively uniform, at 0.11±0.02 mm, 0.21±0.03 mm, 0.31±0.02 mm and 0.53±0.02 mm, respectively. Figure 1 (G).

[0062] Tensile mechanical tests showed that the breaking strength increased with increasing fiber thickness. Since 100 μm decellularized matrix fibers had the lowest mechanical strength and were easily broken, decellularized matrix fibers in the 200-500 μm range were selected for subsequent experiments. Figure 1(H and I). To assess processing adaptability, a series of operations were performed on the fibers, including bending into loops, knotting, twisting, bonding two fibers together, and winding onto a spool; none of these operations resulted in structural damage. Figure 1 (J). These results demonstrate that the decellularized matrix fibers obtained in Example 1 can successfully transform muscle into uniform, mechanically strong, and processable decellularized muscle matrix fibers.

[0063] Figure 2 This document describes the preparation and physicochemical characterization of the fiber scaffold. A shows a schematic diagram of the preparation process of the decellularized matrix fabric (fiber scaffold); B shows macroscopic and electron microscopic images (top and side views) of fiber scaffolds prepared from decellularized matrix fibers of different thicknesses and a decellularized block from Comparative Example 1; C shows the thickness measurements of different fiber scaffold samples (n=5); D shows the results of toluidine blue permeability measurements of different fiber scaffolds and decellularized block samples; E shows the quantitative statistical analysis results of absorbance of different fiber scaffolds and decellularized block samples (n=3); F and G show the tensile stress-strain curves and corresponding fracture strength statistics of different fiber scaffolds and decellularized block samples (n=3); H shows a schematic diagram of the fiber scaffold being processed into a large structure (ns = no significant difference in the figure). P<0.01; P<0.001; P<0.0001).

[0064] Combination Figure 2 As can be seen, after the decellularized muscle matrix membrane is cut into fibers and woven to form a woven scaffold (fiber scaffold), macroscopic images show that the fiber scaffold is characterized by an interlaced arrangement of decellularized muscle matrix fibers, with sparser transverse fibers and denser longitudinal fibers. In contrast, the dense block structure of the decellularized muscle matrix is ​​relatively compact. Electron microscopy shows that the surface and cross-section of the fiber scaffold exhibit a porous, interwoven fiber structure, and the porosity becomes more pronounced with increasing fiber thickness. In contrast, the surface and cross-sectional morphology of the decellularized block is relatively dense. Figure 2 (B) The overall thickness of the resulting fiber scaffold increases accordingly with the thickness of its constituent fibers. Figure 2 (C)

[0065] Toluidine blue permeation assays confirmed that the fibrous scaffold exhibited significant permeability, while decellularized blocks prevented solution permeation. Figure 2 (D). Quantitative analysis further showed that the greater the fiber thickness in the fiber scaffold, the stronger the permeability (D). Figure 2 (E). Tensile mechanical tests showed that all fiber scaffold samples had lower mechanical strength compared to decellularized blocks. Within the fiber scaffold group, the breaking strength gradually increased with increasing fiber thickness. Figure 2 (F, G).

[0066] Furthermore, the fiber scaffold exhibits excellent processability, allowing for the fabrication of large specimens, such as fiber scaffolds up to 10 cm long (strips) and 2 cm wide (strips), thus enabling the creation of sizable structures. These structures can be further shaped by methods such as stacking two layers, bending into loops and triangles, or cutting into letter shapes ('NKU') to meet specific processing requirements. Figure 2 (H). These results indicate that the fiber scaffold prepared by weaving in Example 1 has a suitable porous structure and adjustable mechanical properties.

[0067] Application Example 1 A method for preparing a block-shaped porcine skeletal muscle decellularized matrix fiber scaffold loaded with L6 includes the following steps: After sterilizing the fiber scaffold (decellularized matrix paper-based material with a thickness of 200 μm) obtained in Example 1, it was immersed in DMEM medium (Duborough modified Eagle medium) supplemented with 10% fetal bovine serum, and L6 cells were cultured at 5 × 10⁻⁶ cells / day. 4 / cm 2 The cells were densely seeded onto the scaffold material and cultured until they adhered. Then, 0.5 mL of DMEM medium was added, and the cells were cultured for another 5 days to obtain block-shaped porcine skeletal muscle decellularized matrix fiber scaffolds loaded with L6 cells. The decellularized blocks obtained in Comparative Example 1 were treated in the same way and used as a control group.

[0068] Figure 3 The behavioral changes of L6 cells after culturing on fibrous scaffolds and decellularized blocks for 1, 3, and 5 days are shown. In the figures, A represents the distribution of DiD-labeled red fluorescently labeled L6 cells on the materials; red represents DiD-labeled cells, and black represents the scaffold material. B shows the quantitative analysis of DiD fluorescence (n=3). C shows the quantitative analysis of cell viability using the CCK-8 assay (n=3). D shows the live / dead staining results of L6 cells; green represents live cells, and red represents dead cells. E shows the quantitative analysis of cell viability (n=3). F shows the distribution of L6 cells on fibrous scaffolds and decellularized blocks as shown by FITC-phalloidin (green) and nucleus (blue) staining. G shows the quantitative analysis results of phalloidin fluorescence intensity (n=3).

[0069] DiD-labeled L6 cells were seeded onto fibrous scaffolds and decellularized blocks, respectively, to assess their cell compatibility and migration behavior. Figure 3 As can be seen, cells were observed inside the fibrous scaffold on day 1 post-inoculation, and the fluorescence intensity increased over time, indicating continuous inward migration of cells. In contrast, cells on the decellularized block were confined to the surface, with only a slight increase in fluorescence. Figure 3 (A, B). CCK-8 assay showed comparable cell proliferation on both materials, with no significant difference between the groups. Figure 3(C). Live / dead staining showed that both scaffolds were dominated by green fluorescence (live cells), with the cell number increasing significantly over time, while red fluorescence (dead cells) was extremely rare. Figure 3 (D). Quantitative analysis confirmed that from day 1 to day 5, cell viability on both the fibrous scaffold and the decellularized blocks remained above 90%, with no statistically significant difference between groups. Figure 3 E). F-actin / nuclear staining further indicated good cell morphology spreading on both materials. Fluorescence-based quantitative analysis showed that the cell spreading area increased over time, with no significant difference again between the fibrous scaffold and the decellularized block. Figure 3 (F, G). In summary, these results indicate that the fibrous scaffold not only supports cell survival and proliferation but also promotes three-dimensional cell migration.

[0070] Application Example 2 A method for treating a bulk muscle loss model in porcine skeletal muscle using a decellularized matrix fiber scaffold, comprising the fiber scaffold (200 μm thick decellularized matrix paper-based material) obtained in Example 1 as the experimental group, which was implanted into the tibialis anterior muscle defect site in rats, specifically as follows: Experimental rats (SD) were anesthetized with isoflurane to ensure they were painless and calm during the surgery. After the anesthesia took effect, the hair at the surgical site was carefully shaved to fully expose the area. The skin and fascia were then precisely cut layer by layer with scissors. After fully exposing the tibialis anterior muscle, a defect measuring 1 × 0.5 × 0.5 cm was precisely created on the muscle. This defect size was designed to simulate a specific degree of muscle damage to assess the repair effect of the block-shaped decellularized matrix fiber scaffold.

[0071] The fiber-woven scaffold obtained in Example 1 was implanted into the tibialis anterior muscle defect model, ensuring a tight fit between the scaffold and the defect area to provide effective support and a repair environment for the damaged tissue. The scaffold was then properly sutured and fixed to ensure it was securely positioned at the defect site, preventing postoperative displacement or detachment that could affect the repair process. After scaffold implantation and fixation, the fascia and skin tissue at the injured site were sutured according to the tissue layers to restore the integrity of the rat's body surface. During suturing, attention should be paid to the uniformity of stitch spacing and pressure to promote good wound healing. Finally, the surgical wound was thoroughly disinfected with povidone-iodine to reduce the risk of postoperative infection, thus completing the entire surgical procedure. The decellularized block obtained in Comparative Example 1 was treated in the same way and served as a control group.

[0072] Figure 4Tissue reactions at 2 and 4 weeks after subcutaneous implantation of fibrous scaffolds and decellularized blocks in rats. A shows a schematic diagram of subcutaneous scaffold implantation and tissue sampling at different time points; B and C show macroscopic views and weight measurements of the fibrous scaffolds and decellularized blocks removed at 2 and 4 weeks (n=3); D and E show nuclear staining indicating cell infiltration within the implant and corresponding quantitative analysis (n=3); F and I show hematoxylin and eosin staining indicating tissue ingrowth into the implant and quantitative analysis of the ingrowth area (n=3); J and K show hematoxylin and eosin staining indicating microvascular ingrowth and related quantitative analysis (n=3); L and M show α-SMA immunofluorescence (green) indicating the functional vascular smooth muscle layer and corresponding quantitative analysis (n=3).

[0073] Fiber scaffolds and decellularized blocks were subcutaneously implanted into rats, and tissue samples were collected at 2 and 4 weeks post-implantation to assess tissue infiltration, angiogenesis, and macrophage polarization. Figure 4 (A). At 2 weeks, both the fibrous scaffold and the decellularized mass were encapsulated by host tissue. By 4 weeks, both materials showed a reduction in volume. Figure 4 (Medium B). Quantitative analysis showed no significant difference in weight between the two groups. Figure 4 (C). Nuclear staining showed extensive nuclear infiltration within the fibrous scaffold at both 2 and 4 weeks, while the decellularized blocks contained fewer nuclei compared to the fibrous scaffold. Figure 4 (D). Quantitative analysis confirmed that the number of infiltrating cells in the fibrous scaffold was significantly higher than that in the decellularized block (D). Figure 4 (E). Hematoxylin and eosin staining showed that by 2 weeks, cells and ECM had occupied most of the fibrous scaffold, while areas without cell infiltration remained in the decellularized block. At 4 weeks, degradation of the fibrous scaffold with volume shrinkage was observed, while the decellularized block retained residual material and persistent uninfiltrated areas. Quantitative analysis showed that the percentage of uninfiltrated areas in the decellularized block was significantly greater than that in the fibrous scaffold (E). Figure 4 (F–I).

[0074] Next, vascularization was assessed. Hematoxylin and eosin staining showed that the fibrous scaffold supported extensive microvascular formation at 2 weeks, with a trend towards increasing vascularization over time. In contrast, the decellularized block showed very little vascularization at 2 weeks and only limited capillaries at 4 weeks. Quantitative results confirmed that at both time points, the number of vessels in the fibrous scaffold was significantly higher than that in the decellularized block. Figure 4 Immunostaining with α-smooth muscle actin revealed neovascularization within the fibrous scaffold at 2 weeks, which was absent in the decellularized block. By 4 weeks, the fibrous scaffold showed increased microvessels, significantly more numerous than the decellularized block group. Quantitative analysis confirmed these observations. Figure 4 (Middle L, M).

[0075] Figure 5This study aimed to promote muscle regeneration in rat defects after implantation of fibrous scaffolds and decellularized blocks. A shows a schematic diagram of scaffold implantation in the tibialis anterior muscle of rats and the experimental timeline; B shows macroscopic images of the implant and harvested tissue at different time points; C shows the muscle mass ratio (defective side / normal side) at 2 months post-implantation (n=3); D and E show collagen deposition and its quantitative analysis using Masson's trichrome staining (n=3).

[0076] A fibrous scaffold (1.0 × 0.5 × 0.5 cm) and decellularized blocks were implanted in a rat model of volumetric muscle loss in the anterior tibialis muscle for 2 and 8 weeks to assess their ability to promote in situ muscle regeneration. Figure 5 (Category A) At 2 weeks, both the fibrous scaffold and the decellularized mass were encapsulated by host tissue. The untreated group showed muscle atrophy, while the autologous graft group showed better integration of the graft with the surrounding muscle. By 8 weeks, both the fibrous scaffold and the decellularized mass had completely degraded, and there was no clear boundary between the wound and the adjacent muscle tissue. Figure 5 (Medium B). The mass ratio (damaged side / normal side) measured at 8 weeks showed that the fibrous scaffold group was significantly higher than the decellularized block group and the untreated group, but still lower than the autologous graft group. Figure 5 (C)

[0077] Masson's trichrome staining showed that at 2 weeks, there was a large amount of collagen deposition at the implantation site and around the autologous graft in both the fibrous scaffold group and the decellularized block group. Quantitative analysis confirmed that the amount of collagen deposition in the fibrous scaffold group was significantly higher than that in the autologous graft group and the untreated group. At 8 weeks, only a very small amount of collagen was observed in the fibrous scaffold group and the autologous graft group, while abundant collagen remained in the defect area of ​​the decellularized block group. The collagen content in the decellularized block group was significantly higher than that in the fibrous scaffold group and the untreated group. Figure 5 D, E).

[0078] The test results above demonstrate that the scaffold obtained through a specific combination of processes in this invention not only mimics natural tissue in its physical structure but, more importantly, achieves a breakthrough in biological function. Experiments have shown that this scaffold significantly promotes directional cell growth along the biomimetic fiber pathway, enhances the orderly arrangement of the cytoskeleton, and ultimately guides the formation of regenerated muscle fibers with correct orientation and function in animal models, effectively reducing scar tissue formation. This precise biomimetic guidance of cell behavior and tissue remodeling while preserving complete biological activity is unattainable with existing technologies.

[0079] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A decellularized matrix fiber woven scaffold, characterized in that, It includes a weaving structure unit, which comprises warp threads and multiple decellularized matrix fiber weft threads; The decellularized matrix fiber weft is made by cutting decellularized matrix paper-based material at equal intervals; The multiple decellularized matrix fiber weft threads are arranged in parallel according to a biomimetic direction and fixed by the interlacing of the warp threads, thereby forming a biomimetic pattern in the weaving structure unit that simulates the arrangement of natural tissue fibers.

2. The decellularized matrix fiber woven scaffold according to claim 1, characterized in that, The decellularized matrix fibers have a width of 1-2000 μm and a length of 1-100000 μm.

3. The decellularized matrix fiber woven scaffold according to claim 1, characterized in that, The weaving method of the warp and weft threads is any one of the following: orthogonal, plain weave, twill weave, or satin weave.

4. The decellularized matrix fiber woven scaffold according to claim 1, characterized in that, The biomimetic orientation is the arrangement direction that mimics the orientation of skeletal muscle bundles, tendon fiber bundles, nerve bundles, or vascular smooth muscle cells.

5. The decellularized matrix fiber woven scaffold according to claim 1, characterized in that, The decellularized matrix fiber woven scaffold is formed by stacking two or more layers of the woven structural units to form a three-dimensional structure.

6. A method for preparing a decellularized matrix fiber woven scaffold according to any one of claims 1-5, characterized in that, Includes the following steps: The decellularized matrix paper-based material is cut at equal intervals to obtain decellularized matrix fibers; Multiple decellularized matrix fibers are arranged in parallel according to a predetermined biomimetic orientation, serving as weft lines; The warp threads are interlaced to fix the weft threads, forming a single-layer woven structure unit with a biomimetic pattern; At least one layer of the single-layer woven structural unit is stacked to obtain the decellularized matrix fiber woven scaffold.

7. The method for preparing the decellularized matrix fiber woven scaffold according to claim 6, characterized in that, The decellularized matrix paper-based material is prepared by homogenizing, filtering, and drying the decellularized matrix material.

8. The use of the decellularized matrix fiber woven scaffold as described in any one of claims 1-5 in the preparation of tissue engineering scaffold materials.

9. The application according to claim 8, characterized in that, The tissues are skeletal muscle, tendons, ligaments, skin, or fascia.

10. A tissue repair product, characterized in that, It comprises a decellularized matrix fiber woven scaffold as described in any one of claims 1-5, and an active ingredient loaded thereon.

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