Composite nanoweb material comprising decellularized extracellular matrix, manufacturing method therefor, and therapeutic material including same

Abstract

One embodiment of the present invention relates to a composite nanoweb material including a decellularized extracellular matrix and a therapeutic material including same and, more specifically, provides a multiscale nanofiber composite in which a decellularized extracellular matrix and a biocompatible polymer are mixed in various ratios and formed into micro- and nano-sized fibrous structures through an electrospinning process. In addition, another embodiment of the present invention relates to a method for manufacturing a composite nanoweb material and, more specifically, provides a multiscale nanofiber composite having micro- and nano-sized fibrous structures formed through an electrospinning process and a composite crosslinking process.

Description

Composite nanoweb material containing decellularized extracellular matrix, method of manufacturing the same, and therapeutic material containing the same

[0001] The present invention relates to a composite nanoweb material comprising a decellularized extracellular matrix and a therapeutic material comprising the same. More specifically, it relates to a multiscale nanofiber composite in which micro and nano-sized fiber structures are formed through an electrospinning process by mixing a decellularized extracellular matrix and a biocompatible polymer in various ratios.

[0002] Furthermore, the present invention relates to a method for manufacturing a composite nanoweb material, and more specifically, to a method for manufacturing a nanofiber composite by mixing a decellularized extracellular matrix and a biocompatible polymer through an electrospinning process and a composite crosslinking process.

[0003] The Extracellular Matrix (ECM) provides an essential physiological environment in vivo that supports cells and regulates intercellular interactions. The ECM performs various physiological functions, such as cell adhesion, signal transduction, and growth factor binding, which are primarily determined by its nano and micro-scale structures. The nanostructures of the ECM facilitate interactions with cells, while the microstructures provide a physical environment, such as mechanical support, to help maintain tissue function. As such, the ECM is considered a crucial biomaterial for regenerative medicine, tissue engineering, and the development of various medical devices.

[0004] Decellularized Extracellular Matrix (dECM) is pure ECM remaining after cellular components have been removed, and it is a material that minimizes immune responses and maximizes biocompatibility. As a result, dECM has great potential to promote tissue regeneration and has very high potential for application, particularly in the fields of regenerative medicine and medical devices. dECM-based materials have properties that promote cell adhesion, growth, and differentiation, so they can be used in various fields such as wound dressings, artificial blood vessels, and cartilage regeneration.

[0005] The applicant's Patent No. 10-2024-0108874 (Patent Document 1) describes a method for manufacturing decellularized biomaterials. Through the process of Patent Document 1, the destruction of growth factors including collagen and GAGs was minimized, and undesirable components such as DNA were removed as much as possible. Subsequently, a water-soluble decellularized biomaterial was obtained through a solubilization step. Active research is being conducted to develop processes applying such dECM, composite materials manufactured through such processes, and products utilizing these composite materials. Meanwhile, in order to apply dECM to a process, reproducibility must be guaranteed in terms of various physical properties, such as the composition and characteristics of the raw materials, so that process conditions can be consistently set in the future. Therefore, the process for manufacturing nanocomposites using dECM obtained through the process of Patent Document 1 can be considered optimized accordingly.

[0006] Electrospinning is a technique that uses high voltage to draw out a polymer solution into nano-sized fibers, making it highly useful for manufacturing biocompatible nanofibers. Conventional electrospinning techniques have primarily been limited to mimicking the structural characteristics of ECMs using biocompatible polymers, and this method had limitations in fully realizing the inherent physiological functions of ECMs. Furthermore, dECM alone has weak mechanical properties, making it very difficult to form stable and functional nanofiber structures; additionally, dECM's low solubility limits the ability to obtain uniform nanofibers through electrospinning. Previous studies addressed these issues by mixing minute amounts of dECM with other polymers or coating them on surfaces, but these methods failed to maximize the functionality of dECM.

[0007] Accordingly, in this invention, a biocompatible polymer was mixed and used to improve the radioactivity and mechanical strength of dECM. By using a biocompatible polymer that has the property of slowly degrading in the body and excellent mechanical stability, and mixing it with dECM in an appropriate ratio, a composite material was developed that maximizes the advantages of each material, enhances the physical stability of the nanofiber structure, and maintains the biocompatibility of dECM.

[0008] In addition, in the present invention, dECM and a biocompatible polymer are mixed and electrospun, and a complex cross-linking reaction is carried out to improve the mechanical strength of the fiber, thereby preventing shrinkage or deformation. Through the cross-linking reaction, the mechanical properties of dECM are improved, so the nanofiber composite can be maintained stably even in a moist environment.

[0009]

[0010] [Prior Art Literature]

[0011] (Patent Document 0001) Republic of Korea Published Patent No. 10-2024-0108874

[0012]

[0013] The present invention has been devised to solve the aforementioned problems, and one embodiment of the present invention provides a nanofiber composite.

[0014] In addition, another embodiment of the present invention provides a mat-type therapeutic material.

[0015] In addition, another embodiment of the present invention provides a tubular therapeutic material.

[0016] Another embodiment of the present invention provides a method for manufacturing a nanofiber composite.

[0017] In addition, another embodiment of the present invention provides a method for manufacturing a mat-type therapeutic material.

[0018] In addition, another embodiment of the present invention provides a method for manufacturing a tubular therapeutic material.

[0019] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0020]

[0021] As a technical means for achieving the aforementioned technical problem, one aspect of the present invention provides a nanofiber composite comprising a decellularized extracellular matrix (dECM) and a biocompatible polymer, wherein the nanofiber composite has a first region and a second region, wherein the first region represents a nanoweb structure in the form of a net formed between nanofibers, and the second region represents nanofibers in the form of a non-net.

[0022] The above biocompatible polymer may be at least one polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(lactide-co-caprolactone) (PLCL), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PVL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, dextran, and silk.

[0023] The nanoweb structure of the first region above may be derived from a monovalent cation or a monovalent anion upon radiation.

[0024] As a result of Scanning Electron Microscopy with Energy Dispersive X-Ray Analysis (SEM-EDX), the first region and the second region may commonly contain the elements C, O, and N.

[0025] In the first region above, the elemental content of N may be 4 to 35 parts by weight relative to 100 parts by weight of individual fibers of the nanoweb structure.

[0026] In the second region above, the elemental content of N may be 6 to 30 parts by weight relative to 100 parts by weight of nanofiber.

[0027] The ratio of the average diameter of individual fibers of the nanoweb structure of the first region to the average diameter of nanofibers in the second region may be 1:6 to 50.

[0028] The average diameter of individual fibers of the nanoweb structure in the first region above may be 20 to 50 nm.

[0029] The average diameter of the nanofibers in the second region above may be 0.3 to 1.0 μm.

[0030] The average pore size of the above nanofiber composite may be 0.1 to 10 μm.

[0031] The tensile strength of the above nanofiber composite may be 0.5 to 10 MPa.

[0032] The weight ratio of the decellularized extracellular matrix and the biocompatible polymer may be 0.2 to 9.8 : 9.8 to 0.2.

[0033]

[0034] To achieve the above technical problem, another aspect of the present invention provides a mat-type therapeutic material comprising a structure formed by molding a nanofiber composite into a plate shape.

[0035]

[0036] To achieve the above technical problem, another aspect of the present invention provides a tubular therapeutic material comprising a structure formed by molding a nanofiber composite into a tubular shape.

[0037]

[0038] As a technical means for achieving the aforementioned technical problem, one aspect of the present invention provides a method for manufacturing a nanofiber composite, comprising the steps of: mixing a decellularized extracellular matrix and a biocompatible polymer in a solvent; electrospinning the mixture to produce a nanofiber sheet; and drying or washing the nanofiber sheet.

[0039] In addition, one aspect of the present invention provides a method for manufacturing a nanofiber composite, comprising the steps of: preparing a first solution in which a decellularized extracellular matrix is ​​dissolved in a solvent; preparing a second solution in which a biocompatible polymer is dissolved in a solvent; electrospinning the first or second solution to produce a first nanofiber sheet; electrospinning the second or first solution onto the first nanofiber sheet to produce a second nanofiber sheet; and drying or washing the laminated first and second nanofiber sheets.

[0040] Prior to the drying or washing step, the method may include the step of crosslinking the nanofiber composite with a crosslinking agent.

[0041] The above biocompatible polymer may be at least one polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(lactide-co-caprolactone) (PLCL), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PVL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, dextran, and silk.

[0042] The above solvent may comprise one or more selected from the group consisting of trifluoroacetic acid, dimethylformamide, dimethyl sulfoxide, trifluoroethylene, acetone, hexafluoroisopropanol, dichloromethane, tetrahydrofuran, ethanol, water, phosphate-buffered saline (PBS), acetic acid, and formic acid.

[0043] The weight ratio of the decellularized extracellular matrix and the biocompatible polymer in the above nanofiber composite may be 0.2 to 9.8 : 9.8 to 0.2.

[0044] The above electrospinning can be performed under conditions where the voltage is 5 to 100 kV, the radiation distance is 3 to 50 cm, and the fluid velocity is 1 to 100 ml / hr.

[0045] The average thickness of the electrospun nanofiber sheet may be 5 to 400 μm.

[0046] The solvent of the above crosslinking agent may include ethanol.

[0047] With respect to 100 parts by weight of the solvent, the content of the ethanol may be 40 to 99.9 parts by weight.

[0048] The above crosslinking agent may include one or more selected from the group consisting of glutaraldehyde, EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide), thrombin, genipin, Ru / SPS, hexamethylene diisocyanate, isophorone diisocyanate, ethylene glycol diglycidyl ether, glyceraldehyde, 1,4-butanediol diglycidyl ether (BDDGE), and tannic acid.

[0049] The tensile strength of the above nanofiber composite may be 0.5 to 10 MPa.

[0050] The average pore size of the above nanofiber composite may be 0.1 to 10 μm.

[0051]

[0052] To achieve the above technical objective, another aspect of the present invention provides a method for manufacturing a mat-type therapeutic material comprising the steps of: manufacturing a nanofiber composite; and forming the nanofiber composite into a sheet or plate shape to obtain a mat-type therapeutic material.

[0053]

[0054] To achieve the above technical objective, another aspect of the present invention provides a method for manufacturing a tubular therapeutic material, comprising the step of manufacturing a nanofiber composite; wherein the step of manufacturing the nanofiber composite is performed in a cylindrical collector.

[0055]

[0056] To achieve the above technical objective, another aspect of the present invention provides a method for manufacturing a nanofiber composite, comprising the steps of: mixing a decellularized extracellular matrix with a solvent; electrospinning the mixture to produce a nanofiber sheet; and drying or washing the nanofiber sheet.

[0057]

[0058] To achieve the above technical problem, another aspect of the present invention provides a nanofiber composite comprising a decellularized extracellular matrix (dECM) prepared according to the above method.

[0059]

[0060] According to one embodiment of the present invention, the nanofiber composite of the present invention maximizes mechanical stability and biocompatibility simultaneously by mixing PCL and dECM in different ratios.

[0061] In addition, according to one embodiment of the present invention, the nanofiber structure is modified by adjusting the ratio of PCL and dECM, and as the dECM content increases, a nano-web structure resembling a net is formed between the fibers. The nano-web structure plays an important role in promoting cell attachment and growth, which can provide an ideal environment for various tissue regeneration and medical device applications.

[0062] According to one embodiment of the present invention, electrospinning was performed by mixing a biocompatible polymer with dECM, which minimizes the destruction of growth factors and removes undesirable components such as DNA as much as possible, and by carrying out a cross-linking reaction based on an appropriate solvent to improve the mechanical strength of the fiber and prevent shrinkage or deformation.

[0063] In addition, according to one embodiment of the present invention, the mechanical properties of dECM are improved through a cross-linking reaction, and the nanofiber composite of the present invention can be maintained stably even in a moist environment.

[0064] In addition, according to one embodiment of the present invention, the method for manufacturing a nanofiber composite of the present invention is an optimal method for manufacturing nanofibers that maximize not only biocompatibility but also mechanical stability.

[0065] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims.

[0066]

[0067] Figure 1 shows the results of analyzing the molecular weight of a pig skin-derived dECM sample of Example 0 according to one embodiment of the present invention.

[0068] FIG. 2 shows the results of measuring the viscosity of the electrospinning solutions of Examples 1-1 to 1-6 and Comparative Example 1 according to one embodiment of the present invention.

[0069] FIG. 3 is an image of a nanofiber composite of an example and a comparative example according to one embodiment of the present invention analyzed by a field emission scanning electron microscope (FE-SEM).

[0070] FIG. 4 is an image of a nanofiber composite according to an embodiment of the present invention analyzed by a field emission scanning electron microscope (FE-SEM), where FIG. 4(a) shows an enlarged image of the nanofiber composite of Example 1-3 and FIG. 4(b) shows an enlarged image of the nanofiber composite of Example 1-4.

[0071] FIG. 5 shows the results of analyzing the elemental mass contained in the nanoweb fibers of the first region and the non-net-shaped fibers of the second region using scanning electron microscope-energy dispersive X-ray analysis (SEM-EDX) for Comparative Example 1 and Examples 1-1 to 1-5 according to one embodiment of the present invention.

[0072] FIG. 6 shows the results of analyzing the diameter sizes of individual fibers (Nano fibers) of the nano web structure in the first region and fibers (Micro fibers) in the second region of Comparative Example 1 and Examples 1-1 to 1-6 according to one embodiment of the present invention.

[0073] FIG. 7 shows the thickness of the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-6 according to one embodiment of the present invention.

[0074] FIG. 8 shows the tensile strength results measured using a universal testing machine (UTM) for nanofiber composites of examples and comparative examples according to one embodiment of the present invention, FIG. 8 (a) shows the maximum load of Examples 1-1 to 1-5 and Comparative Example 1, and FIG. 8 (b) shows the elongation at the maximum load of Examples 1-1 to 1-5 and Comparative Example 1.

[0075] FIG. 9 shows the results of residual solvent analysis using gas chromatography-mass spectrometry (GC-MS) of Examples 1-5 according to one embodiment of the present invention.

[0076] FIG. 10 shows the results of analyzing cell adhesion for the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 according to one embodiment of the present invention.

[0077] FIG. 11 shows the cell growth rate of the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 according to one embodiment of the present invention.

[0078] FIG. 12 is the result of observing living and dead cells with a fluorescence microscope after staining and culturing cells on the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 according to one embodiment of the present invention.

[0079] FIG. 13 is an image of a nanofiber composite of an example and a comparative example according to one embodiment of the present invention analyzed by a field emission scanning electron microscope (FE-SEM), FIG. 13 (a) shows an image of the nanofiber composite of Comparative Example 2 and Examples 2-1 to 2-4, and FIG. 13 (b) shows an enlarged image of the nanofiber composite of Example 2-3.

[0080] FIG. 14 shows the thickness of the nanofiber composites of Examples 2-1 to 2-4 and Comparative Example 2 according to one embodiment of the present invention.

[0081] FIG. 15 shows the tensile strength results measured using a universal testing machine (UTM) for nanofiber composites of an example and a comparative example according to one embodiment of the present invention, FIG. 15 (a) shows the maximum load of Examples 2-1 to 2-4 and Comparative Example 2, and FIG. 15 (b) shows the elongation at the maximum load of Examples 2-1 to 2-4 and Comparative Example 2.

[0082] FIG. 16 shows the results of measuring the pore size of the nanofiber composites of Examples 2-1 to 2-4 according to one embodiment of the present invention.

[0083] Figure 17 is an FE-SEM image of a nanofiber composite prepared with a PCL:dECM (90:10) ratio before and after crosslinking (comparison of PCL:dECM (2024) and PCL:dECM (2025)).

[0084] Figure 18 is a photograph showing the results of a comparison of the solubility of KITECH dECM and commercially available dECM (Company D, Company C) in different solvents (PBS, HFIP).

[0085] Figure 19 shows the results of residual solvent analysis using gas chromatography-mass spectrometry (GC-MS).

[0086] Figure 20 shows FE-SEM images of a 100% dECM nanofiber composite before (left) and after (right) crosslinking.

[0087] Figure 21 shows an FE-SEM image of nanofibers prepared with 100% dECM using TFE solvent.

[0088] Figure 22 shows FE-SEM images of nanofiber composites with PCL 100% and PCL:dECM ratios (97:3, 95:5, 90:10, 80:20, 70:30).

[0089] Figure 23 is a graph showing the cell adhesion (%) of nanofiber composites before and after crosslinking at 4 hours, according to PCL:dECM ratios (100:0, 97:3, 95:5, 90:10).

[0090] Figure 24 is a graph showing the quantitative analysis of the cell growth rate of NIH3T3 cells over time (2, 6, 10, and 14 days) according to the PCL:dECM ratio and whether or not crosslinking was performed.

[0091] Figure 25 shows live / dead fluorescence microscopy images according to the PCL:dECM ratio and whether or not crosslinking occurs.

[0092] Figure 26 is an FE-SEM image of a nanofiber composite prepared with a PCL / dECM 90 / 10 ratio using TFE solvent.

[0093] Figure 27 is an FE-SEM image of a nanofiber composite prepared using TFE solvent with PCL:dECM ratios (100:0, 90:10, 80:20, 70:30, 50:50) and 100% dECM.

[0094] Figure 28 is an FE-SEM image of nanofiber composites prepared using water (DW) solvent according to PVA:dECM ratios (100:0, 90:10, 80:20).

[0095]

[0096] The present invention will be described in more detail below. However, the present invention may be implemented in various different forms and is not limited by the embodiments described herein, and is defined only by the claims set forth below.

[0097] The present invention will be described below with reference to the attached drawings. However, the present invention may be implemented in various different forms and is therefore not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0098] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0099] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0100] Unless otherwise specified in this specification, "microfiber" may mean a fiber having a diameter in micrometers of a number from a two-digit integer to a first decimal place that is easy to describe in micrometer units from the perspective of a person skilled in the art, and "nanofiber" may mean a fiber having a diameter in nanometers of a number from a two-digit integer to a first decimal place that is easy to describe in nanometer units from the perspective of a person skilled in the art.

[0101]

[0102] First Embodiment: Nanofiber Composite and Applications thereof

[0103]

[0104] The first aspect of the present invention is,

[0105] A nanofiber composite comprising a decellularized extracellular matrix (dECM) and a biocompatible polymer, wherein the nanofiber composite has a first region and a second region, wherein the first region represents a nanoweb structure in the form of a net formed between nanofibers, and the second region represents nanofibers in the form of a non-net.

[0106]

[0107] Hereinafter, a nanofiber composite according to the first aspect of the present invention will be described in detail.

[0108]

[0109] In one embodiment of the present invention, the dECM is pure ECM remaining after removing cellular components from ECM, and may be a material that minimizes immune responses and maximizes biocompatibility. As a result, dECM has great potential to promote tissue regeneration and has very high potential for application, particularly in the fields of regenerative medicine and medical devices. dECM-based materials have properties that promote cell adhesion, growth, and differentiation, so they can be used in various fields such as wound dressings, artificial blood vessels, and cartilage regeneration.

[0110] In one embodiment of the present invention, the dECM may be obtained by decellularizing biological tissue derived from mammals, and examples of mammals may be tissues isolated from pigs, cattle, horses, rabbits, dogs, cats, sheep, goats, humans, non-human primates, guinea pigs, or rodents, but are not limited thereto. Preferably, it may be derived from pigs. The biological tissue may be selected from the liver, heart, kidney, stomach, small intestine, large intestine, spleen, bladder, lungs, skin, cornea, adipose tissue, ovary, brain, and placenta, but is not particularly limited thereto.

[0111] In one embodiment of the present invention, the biocompatible polymer may be at least one polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(lactide-co-caprolactone) (PLCL), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, dextran, and silk. In a preferred embodiment of the present invention In this case, the biocompatible polymer may be polycaprolactone (PCL). PCL has high mechanical strength and can form stable nanofibers, and by mixing PCL with dECM, a nanofiber composite that simultaneously maximizes mechanical stability and biocompatibility can be manufactured.

[0112] In one embodiment of the present invention, the nanoweb structure of the first region may be derived from a monovalent cation or a monovalent anion during spinning. In a preferred embodiment of the present invention, the monovalent cation may be a Na ion and the monovalent anion may be a Cl ion. Na in the spinning solution mixed with dECM and a biocompatible polymer + , Cl -The presence of the same ions can cause the polymer chains to become charged. This can lead to the formation of bonded or branched structures among the polymer fibers, thereby creating a nanoweb structure. The Na ions and Cl ions may originate from dECM.

[0113] In one embodiment of the present invention, based on the results of Scanning Electron Microscopy with Energy Dispersive X-Ray Analysis (SEM-EDX), the first region and the second region may commonly contain the elements C, O, and N. The first region and the second region may further contain the elements Cl, Na, and S.

[0114] In one embodiment of the present invention, the nanofiber composite may contain nitrogen (N) element based on Scanning Electron Microscopy with Energy Dispersive X-Ray Analysis (SEM-EDX) results. The elemental content of nitrogen (N) may be 4 to 35 parts by weight relative to 100 parts by weight of the nanofiber composite. In a preferred embodiment of the present invention, the elemental content of nitrogen (N) may be 5 to 30 parts by weight, and more preferably 5 to 20 parts by weight. The nitrogen (N) element is derived from dECM and is essential for the inherent physiological functions of dECM. If the content is below the above range, the dECM content is insufficient, which may reduce the biocompatibility effect of regulating the cell microenvironment and promoting tissue regeneration. If the above range is exceeded, this means that the dECM content becomes excessively high, which may reduce electrospinning properties or decrease the mechanical properties of the composite, making it difficult to maintain a stable structure.

[0115] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may contain carbon (C) based on EDS analysis results. The elemental content of carbon (C) may be 35 to 60 parts by weight relative to 100 parts by weight of the dECM. In a preferred embodiment of the present invention, the elemental content of carbon (C) may be 40 to 58 parts by weight. If the content is below the above range, the content of proteins and organic frameworks, which are the main components of the dECM, is insufficient, making it difficult to secure the basic physical properties of the material. If the content exceeds the above range, this may indicate a decrease in the purity of the dECM or the inclusion of unwanted residues during the manufacturing process, which may have a negative impact on biocompatibility.

[0116] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may contain oxygen (O). The elemental content of oxygen (O) may be 20 to 38 parts by weight relative to 100 parts by weight of the dECM. In a preferred embodiment of the present invention, the elemental content of oxygen (O) may be 22 to 35 parts by weight. If the range is less than the above range, the proportion of peptide bonds and hydrophilic groups constituting the dECM decreases, which may lead to reduced biocompatibility and solvent solubility. If the range is exceeded, it may indicate that excessive oxidation of the dECM has occurred, or that impurities such as metal oxides of Fe, Mo, etc., are included, thereby reducing the purity of the material.

[0117] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may contain sodium (Na). The elemental content of sodium (Na) may be 1 to 10 parts by weight relative to 100 parts by weight of the dECM. In a preferred embodiment of the present invention, the elemental content of sodium (Na) may be 1.5 to 9.5 parts by weight. The sodium (Na) is a monovalent cation derived from PBS, etc., used in the dECM manufacturing process, and can play an essential role in inducing the formation of a nanoweb structure, which is a feature of the present invention, by imparting a charge to the polymer chain during electrospinning. If the amount is below the above range, the formation of the nanoweb structure may be insufficient or the solubility of the dECM may be reduced due to a lack of such monovalent cations. If the amount exceeds the above range, excessive salt may remain, causing cytotoxicity or negatively affecting the physical properties of the material.

[0118] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may contain chlorine (Cl). The elemental content of chlorine (Cl) may be 1 to 10 parts by weight relative to 100 parts by weight of the dECM. In a preferred embodiment of the present invention, the elemental content of chlorine (Cl) may be 3.0 to 9.0 parts by weight. The chlorine (Cl) is a monovalent anion derived during the dECM manufacturing process and, together with sodium (Na), can contribute to inducing the formation of a nanoweb structure during electrospinning. If the amount is below the above range, the formation of the nanoweb structure may be insufficient due to a lack of such monovalent anions, and if the amount exceeds the above range, excessive chloride may remain, causing cytotoxicity or negatively affecting the physical properties of the material.

[0119] In one embodiment of the present invention, the elemental content of N in the first region may be 4 to 35 parts by weight. In a preferred embodiment of the present invention, the elemental content of N may be 5 to 27 parts by weight, and more preferably 5 to 20 parts by weight. If the range is less than the above range, it may cause problems in exercising the main functions of the ECM, and effects related to the functions of the ECM, such as regulating the cell microenvironment, promoting tissue regeneration, and improving biocompatibility, may be reduced. If the range is exceeded, the viscosity of the electrospinning solution may become very low and the radioactivity may be significantly reduced, and the physical properties of the nanofiber composite may be reduced.

[0120] In one embodiment of the present invention, the elemental content of N in the second region may be 6 to 30 parts by weight. In a preferred embodiment of the present invention, the elemental content of N may be 6 to 20 parts by weight, and more preferably 8 to 17 parts by weight. If the range is less than the above range, the mechanical strength of the material is not sufficiently secured, so the cell support role may be limited, and if the range is exceeded, cell growth and attachment may be inhibited.

[0121] In one embodiment of the present invention, the ratio of the average diameter of individual fibers of the nanoweb structure of the first region to the average diameter of nanofibers of the second region may be 1:6 to 50.

[0122] In one embodiment of the present invention, the average diameter of individual fibers of the nanoweb structure in the first region may be 20 to 50 nm. In a preferred embodiment of the present invention, the average diameter of individual fibers of the nanoweb structure may be 30 to 40 nm. If the range is less than the above range, related effects such as cell adhesion, proliferation, and differentiation induction, which are functions that may occur by promoting interaction with cells, may be reduced, and the activation of cell signaling may be inhibited. If the range is exceeded, the characteristics of the nanostructure may be weakened.

[0123] In one embodiment of the present invention, the average diameter of the nanofibers in the second region may be 0.3 to 1.0 μm. In a preferred embodiment of the present invention, the average diameter of the nanofibers may be 0.4 to 0.8 μm. If the range is less than the above range, the mechanical strength of the nanofiber composite may be reduced, there may be difficulties in maintaining the shape, and interlayer movement of cells may not be free. If the range is exceeded, interaction with cells may be reduced, and it may have a negative effect on cell adhesion, proliferation, and induction of cell differentiation.

[0124] In one embodiment of the present invention, the average pore size of the nanofiber composite may be 0.1 to 10 μm. In a preferred embodiment of the present invention, the average pore size may be 0.2 to 6 μm. If the range is less than the above range, the function of the nanofiber composite as a nutrient supply and metabolite excretion pathway may be impaired, which may have a negative effect on cell adhesion, proliferation, and differentiation induction; if the range is exceeded, the volume of the nanofiber composite may increase unnecessarily, or the required level of strength may not be secured.

[0125] In one embodiment of the present invention, the tensile strength of the nanofiber composite may be 0.5 to 10 MPa. If it is below the above range, it may not provide the mechanical support required as a medical material, and if it exceeds the above range, the flexibility of the composite may decrease and the integration with the tissue may be impaired.

[0126] In one embodiment of the present invention, the weight ratio of the decellularized extracellular matrix and the biocompatible polymer may be 0.2 to 9.8 : 9.8 to 0.2. In a preferred embodiment of the present invention, the weight ratio may be 0.3 to 9.7 : 9.7 to 0.3, or 0.5 to 9.5 : 9.5 to 0.5, and more preferably 1 to 9 : 9 to 1. As the weight ratio of the biocompatible polymer increases, the ions in the spinning solution decrease, making it difficult to form a nanoweb structure formed in the first region of the nanofiber composite, and the effects related to the functions of the ECM, such as controlling the cell microenvironment, promoting tissue regeneration, and improving biocompatibility, may be reduced. As the weight ratio of the biocompatible polymer decreases, the electrospinning radioactivity decreases significantly, and the physical properties of the nanofiber may become very weak.

[0127] Meanwhile, although the above paragraph mentioned that electrospinning radioactivity may be significantly reduced as the weight ratio of the biocompatible polymer decreases, the decellularized extracellular matrix (dECM) according to the present invention may exceptionally possess the characteristic of being electrospinning even alone. Generally, dECM has low solubility in organic solvents used in the electrospinning process, making it difficult to prepare a stable spinning solution; consequently, it is known that it is very difficult to manufacture uniform nanofibers using 100% dECM alone. However, the dECM used in one embodiment of the present invention is manufactured through the applicant's proprietary manufacturing process (see Patent Document 1) and exhibits high solubility stability even in organic solvents. Based on this excellent solvent solubility, in one embodiment of the present invention, a nanofiber structure can be manufactured by performing stable electrospinning with only 100% dECM without mixing a separate biocompatible polymer.

[0128] In one embodiment of the present invention, the crosslinking agent used in the manufacture of the nanofiber composite may comprise one or more selected from the group consisting of chemical crosslinking agents such as glutaraldehyde, EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide), Ru / SPS, hexamethylene diisocyanate, isophorone diisocyanate, ethylene glycol diglycidyl ether, glyceraldehyde, and 1,4-butanediol diglycidyl ether (BDDGE), enzymatic substances such as thrombin, and crosslinking agents derived from natural substances such as genipin. In a preferred embodiment of the present invention, the crosslinking agent may comprise EDC / NHS. The solvent of the crosslinking agent must be a solvent that minimizes the dissolution of the ECM, and in a preferred embodiment of the present invention, the solvent of the crosslinking agent may comprise ethanol. Decellularized extracellular matrix exhibits high solubility in water and high sensitivity to humidity, making it preferable to perform cross-linking using a solvent other than water. When cross-linking is performed after spinning, the nanoweb structure of the present invention can be preserved while maintaining its original nanoweb shape. The preserved nanoweb structure can facilitate interactions with cells and influence cell adhesion, proliferation, and the induction of cell differentiation. Furthermore, the nanoweb structure can activate cell signaling and alter cell behavior by regulating interactions with the intracellular and surrounding environments.

[0129] In one embodiment of the present invention, the nanofiber composite may have a residual solvent content measured by gas chromatography-mass spectrometry (GC-MS) of 500 ppm or less by weight, 400 ppm or less, 200 ppm or less, 50 ppm or less, or 2.0 ppm or less. If the above range is exceeded, there is a problem that the residual solvent is not highly biocompatible, so it may not be suitable for use as a medical material.

[0130] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may have a DNA content of less than 20 ng / mg, preferably 10 ng / mg or less. If the above-described range is exceeded, the degree of decellularization is insufficient, which increases the likelihood of adverse effects when applied to the body in the future and may also act as an impurity in forming a nanomaterial composite. As described above, by using a decellularized extracellular matrix according to the method presented in Patent Document 1 of the inventors, it is possible to manufacture a nanomaterial composite by uniformly adjusting the quality of the raw material.

[0131] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may include glycosaminoglycans (GAGs). GAGs are one of the major components of the ECM and play an important role in maintaining the functional properties of tissues and enhancing cell affinity. The dECM used in the present invention may be characterized by being manufactured such that the loss of these GAG ​​components is minimized during the decellularization process. As the GAG ​​components are effectively preserved in this manner, the dECM according to one embodiment of the present invention may contain GAGs in the range of 1 μg / mg or more, preferably 5 μg / mg to 15 μg / mg, and more preferably 8 μg / mg to 12 μg / mg, but is not particularly limited thereto.

[0132] In one embodiment of the present invention, the decellularized extracellular matrix (dECM) may include collagen. Collagen is a major component of the ECM and plays an essential role in maintaining the structural stability of tissues and supporting cell adhesion and proliferation. The dECM used in the present invention may be characterized by being manufactured such that the loss or denaturation of this key collagen component is minimized during the decellularization process. As the collagen component is effectively preserved in this way, the dECM according to one embodiment of the present invention may contain collagen in the range of 500 μg / mg or more, preferably 800 μg / mg to 1500 μg / mg, and more preferably 1000 μg / mg to 1400 μg / mg, but is not particularly limited thereto.

[0133] In one embodiment of the present invention, the decellularized extracellular matrix can be characterized through gel permeation chromatography (GPC). Indicators that can be derived through this include Mw, Mn, Mp, Mz, and molecular weight distribution (PDI). Mw represents the weight-average molecular weight, which is calculated by dividing the average molecular weight by the number of molecules within the sample, as the higher the molecular weight of the polymer, the greater the proportion it occupies. Mn represents the number-average molecular weight, which is the average molecular weight divided by the total number of molecules; it is heavily influenced by molecules with small molecular weights and is one of the most fundamental average molecular weight values ​​in GPC. Mn can be usefully utilized to evaluate properties such as the mechanical properties of the material. Mp represents the peak molecular weight, which is the molecular weight exhibiting the highest peak in the GPC chromatogram; simply put, it may represent the molecular weight of the molecule most abundant in the sample. Mz represents the zeta-average molecular weight, which is an average molecular weight that reflects the influence of the polymer portion much more than Mw and Mn. Mz is significantly affected, especially in the presence of superpolymers, and can provide in-depth information about the polymer distribution within the sample. PDI represents the molecular weight distribution and is generally the value obtained by dividing Mw by Mn (PDI = Mw / Mn). PDI indicates how wide the size distribution of molecules within the sample is; a value closer to 1 indicates a single molecular weight distribution (monodiscency), while a value greater than 1 indicates a state where multiple molecular weights are mixed (polydispersity).

[0134] In one embodiment of the present invention, the decellularized extracellular matrix may have at least three peak molecular weight values ​​when measured by gel permeation chromatography (GPC). More specifically, it may include a first peak detected in the range of Mp value 1000 or less, a second peak detected in the range of Mp value 5000 to 50000, and a third peak detected in the range of Mp value 150000 to 600000. Since a decellularized extracellular matrix satisfying multiple peak molecular weight values ​​within the above-described range is utilized as a raw material, the physical properties of the nanofiber composite of the present invention, specifically the net-shaped nanoweb structure and the non-net-shaped structural region, can be considered to be realized simultaneously in a single manufacturing process.

[0135] A nanofiber composite according to one embodiment of the present invention secures basic mechanical strength of the composite through regions composed of relatively large-diameter fibers of a non-net structure, while being suitable for application as a channel for nutrient supply and metabolic product excretion. In particular, it can promote cell migration by providing a channel for cell movement during the tissue regeneration process. Considering that if the structure consists only of regions composed of nano-sized fibers, it becomes difficult for cells to move freely between layers when the cell size is generally around tens of microns, a form in which nano and micro are appropriately mixed can be efficient for tissue regeneration. Furthermore, by including a nanoweb structure with a net structure, it is expected to promote interaction with actual cells, thereby influencing cell adhesion, proliferation, and the induction of cell differentiation. This can be more effective in activating cell signaling and changing cell behavior by regulating interactions with the intracellular and surrounding environments. In other words, since the present invention allows two regions to be manufactured through a single manufacturing process, it is efficient in terms of the manufacturing method, and the heterogeneity of each manufactured region can be minimized. These characteristics can be a significant advantage when utilized in biocompatible medical devices, such as the mat-type or tubular therapeutic materials described below.

[0136]

[0137] The second aspect of the present invention is,

[0138] A mat-type therapeutic material is provided that includes a structure formed by molding a nanofiber composite into a plate shape.

[0139]

[0140] Detailed explanations have been omitted for parts that overlap with the first aspect of the present invention; however, the content described in the first aspect of the present invention may be applied equally even if such explanations are omitted in the second aspect.

[0141]

[0142] Hereinafter, a mat-type therapeutic material according to the second aspect of the present invention will be described in detail.

[0143]

[0144] In one embodiment of the present invention, the mat-type therapeutic material may be manufactured by laminating sheet-type nanofiber composites and may include a structure formed by molding the laminated nanofiber composites into a sheet or plate shape. Specifically, it may be composed of a laminated structure and may include a shape in which the nanofiber composite is laminated or composited with a functional material such as a nonwoven fabric or foam. Due to the high mechanical strength and biocompatibility of the nanofiber composite, the mat-type therapeutic material can contribute to the reconstruction of biological tissues and the restoration of damaged areas. The mat-type therapeutic material can maintain a structure in the form of appropriate pores and a support, allowing for rapid blood absorption and adhesion, and can maximize hemostatic effects by promoting blood coagulation. Furthermore, it exhibits a wound healing effect on surrounding tissues and is biodegradable, making it suitable for internal application within the human body. The mat-type therapeutic material has a soft texture and an even surface shape, rapidly absorbs blood and exudate from the wound site, and possesses fixation capabilities for the wound site as well as excellent hemostatic performance.

[0145]

[0146] The third aspect of this invention is,

[0147] A tubular therapeutic material is provided that includes a structure formed by molding a nanofiber composite into a tubular shape.

[0148]

[0149] Detailed explanations have been omitted for parts that overlap with the first and second aspects of the present invention; however, the explanations provided for the first and second aspects of the present invention may be applied equally to the third aspect even if such explanations are omitted.

[0150]

[0151] Below, a tubular therapeutic material according to the third aspect of the present invention will be described in detail.

[0152]

[0153] In one embodiment of the present invention, the tubular therapeutic material may be capable of inhibiting inflammation and restenosis of the luminal tissue due to the nanofiber composite containing an extracellular matrix.

[0154]

[0155] Second embodiment: Manufacturing method

[0156]

[0157] The first aspect of the present invention is,

[0158] A method for manufacturing a nanofiber composite comprises the steps of: mixing a decellularized extracellular matrix and a biocompatible polymer in a solvent; electrospinning the mixture to produce a nanofiber sheet; crosslinking the nanofiber sheet with a crosslinking agent; and drying or washing the crosslinked nanofiber sheet.

[0159]

[0160] Hereinafter, a method for manufacturing a nanofiber composite according to the first aspect of the present invention will be described in detail.

[0161]

[0162] In one embodiment of the present invention, the dECM is pure ECM remaining after removing cellular components from ECM, and may be a material that minimizes immune responses and maximizes biocompatibility. As a result, dECM has great potential to promote tissue regeneration and has very high potential for application, particularly in the fields of regenerative medicine and medical devices. dECM-based materials have properties that promote cell adhesion, growth, and differentiation, so they can be used in various fields such as wound dressings, artificial blood vessels, and cartilage regeneration.

[0163] In one embodiment of the present invention, the dECM may be obtained by decellularizing biological tissue derived from mammals, and examples of mammals may be tissues isolated from pigs, cattle, horses, rabbits, dogs, cats, sheep, goats, humans, non-human primates, guinea pigs, or rodents, but are not limited thereto. Preferably, it may be derived from pigs. The biological tissue may be selected from the liver, heart, kidney, stomach, small intestine, large intestine, spleen, bladder, lungs, skin, cornea, adipose tissue, ovary, brain, and placenta, but is not particularly limited thereto.

[0164] In one embodiment of the present invention, the step of mixing a decellularized extracellular matrix and a biocompatible polymer in the solvent; wherein the biocompatible polymer is polyvinyl alcohol (PVA), poly(lactide-co-caprolactone) (PLCL), polyethylene oxide (PEO), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PVL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, It may be at least one polymer selected from the group consisting of dextran and silk. In one preferred embodiment of the present invention, the biocompatible polymer may be polycaprolactone (PCL). PCL has high mechanical strength and can form stable nanofibers, and by mixing PCL and dECM, a nanofiber composite that simultaneously maximizes mechanical stability and biocompatibility can be manufactured.

[0165] A method for manufacturing a nanofiber composite according to one embodiment of the present invention may include a step of mixing a decellularized extracellular matrix and a biocompatible polymer in a solvent; however, the decellularized extracellular matrix (dECM) used in the present invention has the characteristic that it can be electrospun alone without mixing with such a biocompatible polymer due to its inherent physical properties. Typically, dECM has low solubility in organic solvents used in the electrospinning process, making it difficult to prepare a stable spinning solution and very difficult to form uniform nanofibers using 100% dECM alone. In contrast, the dECM according to the present invention is obtained through a proprietary manufacturing process disclosed in the applicant's prior art (Patent Document 1) and exhibits excellent solubility stability even in organic solvents. Based on these characteristics, the manufacturing method of the present invention may include a step of preparing a spinning solution by dissolving the dECM alone in a solvent instead of the step of 'mixing the dECM and the biocompatible polymer in a solvent.' Accordingly, the manufacturing method of the present invention may include a process of manufacturing a nanofiber sheet by electrospinning a 100% dECM solution, which is attributed to the excellence of the dECM raw material used in the present invention.

[0166] In one embodiment of the present invention, the nanofiber composite may have a first region and a second region, wherein the first region represents a nanoweb structure in the form of a net formed between nanofibers, and the second region represents nanofibers in a non-net form. The nanoweb structure of the nanofiber composite can be preserved while maintaining its nanoweb shape when crosslinking is performed after electrospinning according to the method for manufacturing the nanofiber composite of the present invention.

[0167] A nanofiber composite according to one embodiment of the present invention secures basic mechanical strength of the composite through regions composed of relatively large-diameter fibers of a non-net structure, while being suitable for application as a channel for nutrient supply and metabolic product excretion. In particular, it can promote cell migration by providing a channel for cell movement during the tissue regeneration process. Considering that if the structure consists only of regions composed of nano-sized fibers, it becomes difficult for cells to move freely between layers when the cell size is generally around tens of microns, a form in which nano and micro are appropriately mixed can be efficient for tissue regeneration. Furthermore, by including a nanoweb structure with a net structure, it is expected to promote interaction with actual cells, thereby influencing cell adhesion, proliferation, and the induction of cell differentiation. This can be more effective in activating cell signaling and changing cell behavior by regulating interactions with the intracellular and surrounding environments. In other words, since the present invention allows two regions to be manufactured through a single manufacturing process, it is efficient in terms of the manufacturing method, and the heterogeneity of each manufactured region can be minimized. These characteristics can be a significant advantage when utilized in biocompatible medical devices, such as the mat-type or tubular therapeutic materials described below.

[0168] In one embodiment of the present invention, the nanoweb structure of the first region may be derived from a monovalent cation or a monovalent anion during spinning. In a preferred embodiment of the present invention, the monovalent cation may be a Na ion and the monovalent anion may be a Cl ion. Na in the spinning solution mixed with dECM and a biocompatible polymer + , Cl - The presence of the same ions can cause the polymer chains to become charged. This can lead to the formation of bonded or branched structures among the polymer fibers, thereby creating a nanoweb structure. The Na ions and Cl ions may originate from dECM.

[0169] In one embodiment of the present invention, the step of mixing a decellularized extracellular matrix and a biocompatible polymer in the solvent; wherein the solvent may comprise one or more selected from the group consisting of trifluoroacetic acid, dimethylformamide, 2,2,2-trifluoroethanol (2,2,2-Trifluoroethanol, TFE), dimethyl sulfoxide, trifluoroethylene, acetone, hexafluoroisopropanol, dichloromethane, tetrahydrofuran, ethanol, acetic acid, and formic acid. In a preferred embodiment of the present invention, the solvent may be hexafluoroisopropanol (HFIP) or 2,2,2-trifluoroethanol (2,2,2-Trifluoroethanol, TFE). In addition, the above-mentioned solvent may be used by mixing the aforementioned solvent type with a suitable buffer solution. For example, the buffer solution may be phosphate-buffered saline (PBS), but this is merely a non-limiting example, and any buffer solution known in the art may be utilized.

[0170] In one embodiment of the present invention, in the step of mixing a decellularized extracellular matrix and a biocompatible polymer in the solvent, the weight ratio of the decellularized extracellular matrix and the biocompatible polymer may be 0.2 to 9.8 : 9.8 to 0.2. As the weight ratio of the biocompatible polymer increases, the ions in the spinning solution decrease, making it difficult to form a nanoweb structure, and the effects related to the functions of the ECM, such as controlling the cell microenvironment, promoting tissue regeneration, and improving biocompatibility, may be reduced. As the weight ratio of the biocompatible polymer decreases, the electrospinning radioactivity decreases significantly, and the physical properties of the nanofiber may become very weak.

[0171] In one embodiment of the present invention, in the step of manufacturing a nanofiber sheet by electrospinning the mixture, the electrospinning may be performed under conditions where the voltage is 5 to 100 kV, the spinning distance is 3 to 50 cm, and the fluid velocity is 1 to 100 ml / hr. In a preferred embodiment of the present invention, the voltage may be 5 to 50 kV, and more preferably 10 to 25 kV. In a preferred embodiment of the present invention, the fluid velocity may be 1 to 50 ml / hr, and more preferably 10 to 25 ml / hr. If the above-described range is exceeded, it may be difficult to form nanofibers or the nanofibers may be formed too thickly.

[0172] In one embodiment of the present invention, in the step of manufacturing a nanofiber sheet by electrospinning the mixture, the average thickness of the nanofiber sheet may be 5 to 400 μm. In a preferred embodiment of the present invention, the average thickness may be 5 to 100 μm, more preferably 5 to 50 μm, and even more preferably 6 to 9 μm. If the thickness is less than the above range, it may be difficult to secure mechanical strength, and if it exceeds the above range, it may be difficult to utilize it as a tubular and mat-type therapeutic material.

[0173] In one embodiment of the present invention, the "step of manufacturing a nanofiber sheet by electrospinning the mixture" may refer to a method of preparing a single solution by pre-mixing dECM and a biocompatible polymer as described above and then spinning it. However, the manufacturing method of the present invention is not limited thereto, and as an alternative embodiment, a method may be adopted in which a first spinning solution in which dECM is dissolved and a second spinning solution in which a biocompatible polymer is dissolved are prepared separately, and then the first and second spinning solutions are spun simultaneously using a dual-nozzle or multi-nozzle electrospinning system. Through this co-electrospinning process, nanofibers derived from each component can be deposited together on a collector to form the nanofiber composite sheet of the present invention. In addition, conventional techniques in the art for simultaneously spinning multiple spinning solutions, such as a method of forming core-shell structured fibers using a co-axial nozzle, may be applied to this step.

[0174] In one embodiment of the present invention, in the step of crosslinking the nanofiber sheet with a crosslinking agent; the solvent of the crosslinking agent must be a solvent in which the ECM is not dissolved as much as possible, and in a preferred embodiment of the present invention, the solvent of the crosslinking agent may include ethanol. The decellularized extracellular matrix has high solubility in water and high sensitivity to humidity, and it may be preferable to proceed with crosslinking using a solvent other than water.

[0175] In one embodiment of the present invention, the step of crosslinking the nanofiber sheet with a crosslinking agent may include the step of manufacturing a nanofiber sheet by electrospinning the mixture; and the step of drying the electrospun nanofiber sheet immediately thereafter, but preferably, the drying step may be minimized or omitted. Through this configuration, the residual amount of the solvent used in the electrospinning solution is prevented from increasing due to strong attachment or adsorption during the drying of the nanofiber sheet, thereby minimizing the content of residual solvent in the final product after crosslinking. Considering that the biocompatibility of the nanofiber composite may decrease as the residual amount of, for example, organic solvents used in the electrospinning step increases in the final product, the nanofiber composite, this configuration can be seen as having a methodological advantage that is differentiated from the prior art.

[0176] In one embodiment of the present invention, the content of the ethanol may be 40 to 99.9 parts by weight relative to 100 parts by weight of the solvent. In a preferred embodiment of the present invention, the content of the ethanol may be 45 to 99.5 parts by weight, and more preferably 47 to 80 parts by weight. If the above-described range is exceeded, shrinkage of the nanofiber composite may occur due to the rapid evaporation and dehydration effect of the ethanol, making it difficult to maintain the shape; if the above-described range is below, the dECM may dissolve due to excessive moisture, and the crosslinking strength may decrease, weakening the bonding between fibers.

[0177] In one embodiment of the present invention, in the step of crosslinking the nanofiber sheet with a crosslinking agent; the crosslinking agent may comprise one or more selected from the group consisting of chemical crosslinking agents such as glutaraldehyde, EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide), Ru / SPS, hexamethylene diisocyanate, isophorone diisocyanate, ethylene glycol diglycidyl ether, glyceraldehyde, 1,4-butanediol diglycidyl ether (BDDGE), enzymatic substances such as thrombin, and crosslinking agents derived from natural substances such as tannic acid and genipin. In a preferred embodiment of the present invention, the crosslinking agent may comprise EDC / NHS.

[0178] In one embodiment of the present invention, the tensile strength of the nanofiber composite may be 0.5 to 10 MPa. In a preferred embodiment of the present invention, the tensile strength may be 1.0 to 7.5 MPa, or 1.5 to 3.0 MPa. If the strength is below the above range, it may not provide the mechanical support required for medical materials, and if the strength exceeds the above range, the flexibility of the composite may decrease and the integration with tissue may be impaired.

[0179] In one embodiment of the present invention, the average pore size of the nanofiber composite may be 0.1 to 10 μm. In a preferred embodiment of the present invention, the average pore size may be 0.2 to 0.6 μm, and more preferably 0.3 to 0.5 μm. If the range is less than the above range, the function of the nanofiber composite as a nutrient supply and metabolite excretion pathway may be impaired, which may have a negative effect on cell adhesion, proliferation, and differentiation induction; if the range is exceeded, the volume of the nanofiber composite may increase unnecessarily, or the required level of strength may not be secured.

[0180] The nanofiber composite obtained by the method according to one embodiment of the present invention may have a residual solvent content measured by gas chromatography-mass spectrometry (GC-MS) of 500 ppm or less, 400 ppm or less, 200 ppm or less, 50 ppm or less, or 2.0 ppm or less on a weight basis. If the above range is exceeded, there is a problem that the residual solvent is not highly biocompatible, so it may not be suitable for use as a medical material.

[0181] The decellularized extracellular matrix (dECM) obtained by the method according to one embodiment of the present invention may have a DNA content of less than 20 ng / mg, preferably 10 ng / mg or less. If the above-described range is exceeded, the degree of decellularization is insufficient, which increases the likelihood of adverse effects when applied to the body in the future and may also act as an impurity in forming a nanomaterial composite. As described above, by using a decellularized extracellular matrix obtained by the method presented in Patent Document 1 of the inventors, it is possible to manufacture a nanomaterial composite by uniformly adjusting the quality of the raw material.

[0182] The decellularized extracellular matrix obtained by the method according to one embodiment of the present invention can be characterized through gel permeation chromatography (GPC). Indicators that can be derived through this include Mw, Mn, Mp, Mz, and molecular weight distribution (PDI). Mw represents the weight-average molecular weight, which is calculated by dividing the average molecular weight by the number of all molecules within the sample, as the higher the molecular weight of the polymer, the greater the proportion. Mn represents the number-average molecular weight, which is the average molecular weight divided by the total number of molecules; it is heavily influenced by molecules with small molecular weights and is one of the most fundamental average molecular weight values ​​in GPC. Mn can be usefully utilized to evaluate properties such as the mechanical properties of the material. Mp represents the peak molecular weight, which is the molecular weight exhibiting the highest peak in the GPC chromatogram; simply put, it may represent the molecular weight of the molecule most abundant in the sample. Mz represents the zeta-average molecular weight, which is an average molecular weight that reflects the influence of the polymer portion much more than Mw and Mn. Mz is significantly affected, especially in the presence of superpolymers, and can provide in-depth information about the polymer distribution within the sample. PDI represents the molecular weight distribution and is generally the value obtained by dividing Mw by Mn (PDI = Mw / Mn). PDI indicates how wide the size distribution of molecules within the sample is; a value closer to 1 indicates a single molecular weight distribution (monodiscency), while a value greater than 1 indicates a state where multiple molecular weights are mixed (polydispersity).

[0183] The decellularized extracellular matrix obtained by the method according to one embodiment of the present invention may have at least three peak molecular weight values ​​when measured by gel permeation chromatography (GPC). More specifically, it may include a first peak detected in the range of Mp value 1000 or less, a second peak detected in the range of Mp value 5000 to 50000, and a third peak detected in the range of Mp value 150000 to 600000. Since a decellularized extracellular matrix satisfying multiple peak molecular weight values ​​within the above-described ranges is utilized as a raw material, the physical properties of the nanofiber composite of the present invention, specifically the net-shaped nanoweb structure and the non-net-shaped structural region, can be considered to be realized simultaneously in a single manufacturing process.

[0184]

[0185] The second aspect of the present invention is,

[0186] A method for manufacturing a mat-type therapeutic material is provided, comprising the steps of: manufacturing a nanofiber composite; and forming the nanofiber composite into a sheet or plate shape to obtain a mat-type therapeutic material.

[0187]

[0188] Detailed explanations have been omitted for parts that overlap with the first aspect of the present invention; however, the content described in the first aspect of the present invention may be applied equally even if such explanations are omitted in the second aspect.

[0189]

[0190] Hereinafter, a method for manufacturing a mat-type therapeutic material according to the second aspect of the present invention will be described in detail.

[0191]

[0192] In one embodiment of the present invention, the mat-type therapeutic material may be manufactured by laminating sheet-type nanofiber composites and may include a structure formed by molding the laminated nanofiber composites into a sheet or plate shape. Specifically, it may be composed of a laminated structure and may include a shape in which the nanofiber composite is laminated or composited with a functional material such as a nonwoven fabric or foam. Due to the high mechanical strength and biocompatibility of the nanofiber composite, the mat-type therapeutic material can contribute to the reconstruction of biological tissues and the restoration of damaged areas. The mat-type therapeutic material can maintain a structure in the form of appropriate pores and a support, allowing for rapid blood absorption and adhesion, and can maximize hemostatic effects by promoting blood coagulation. Furthermore, it exhibits a wound healing effect on surrounding tissues and is biodegradable, making it suitable for internal application within the human body. The mat-type therapeutic material has a soft texture and an even surface shape, rapidly absorbs blood and exudate from the wound site, and possesses fixation capabilities for the wound site as well as excellent hemostatic performance.

[0193] In one embodiment of the present invention, the step of forming the laminated nanofiber composite into a sheet or plate shape and then leaving it as is or adjusting the thickness through compression may be further included. Additionally, the step of sterilizing the obtained mat-type therapeutic material may be further included.

[0194]

[0195] The third aspect of this invention is,

[0196] A method for manufacturing a tubular therapeutic material is provided, comprising the step of manufacturing a nanofiber composite; wherein the step of manufacturing the nanofiber composite is performed in a cylindrical collector.

[0197]

[0198] Detailed explanations have been omitted for parts that overlap with the first and second aspects of the present invention; however, the explanations provided for the first and second aspects of the present invention may be applied equally to the third aspect even if such explanations are omitted.

[0199]

[0200] Hereinafter, a method for manufacturing a tubular therapeutic material according to the third aspect of the present invention will be described in detail.

[0201]

[0202] In one embodiment of the present invention, the tubular therapeutic material may be capable of inhibiting inflammation and restenosis of the luminal tissue due to the nanofiber composite containing an extracellular matrix, and may enable stable drug release control.

[0203]

[0204] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0205]

[0206] Example 0: Preparation of Decellularized ECM

[0207] Experiments were conducted using ECM manufactured according to the method disclosed in Korean Patent Publication No. 10-2024-0108874. The detailed manufacturing process is as follows.

[0208] Pig skin samples were collected from the slaughterhouse, stored at -20℃, and transported to the laboratory. Blood was removed by centrifugation with 100 mL of heparin (50 IU / mL) for 1 hour, and the remaining blood and solvent were washed with PBS (phosphate buffered saline) for 10 minutes. 1.0% antibiotics (penicillin, streptomycin) were added to 0.9% physiological saline to remove odors for 30 minutes, sterilize, and then wash. Specifically, 100 μL was removed from a total volume of 100 mL of physiological saline (a mixture of 900 μL NaCl and 99.1 mL distilled water (DW)), and 100 μL of antibiotics (penicillin, streptomycin) was added.

[0209] Then, 100 mL of cleanser was added to the pig skin. Sodium dodecyl sulfate (SDS) and Triton X-100 were mixed in a 1:1 ratio and placed in a beaker. The washed sample was then placed in the beaker, and cells were removed using a rotary shaker at 4°C at a speed of 50-60. After decellularization treatment, the skin was washed with PBS for 10 minutes. Once washing was complete, the skin was placed in PBS containing 1.0% antibiotics and stored at 4°C. All detergents used in the experiment were dissolved in PBS. Due to the use of PBS, monovalent ions such as Na and Cl may remain in the dECM, and the said dECM was utilized in subsequent processes.

[0210]

[0211] Example 1-1: Nanofiber composite prepared by mixing PCL and dECM in a 9:1 ratio

[0212] Polycaprolactone (PCL) was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 16 w / v% by stirring, and dECM was also dissolved in HFIP at a concentration of 16 w / v%. Subsequently, PCL and dECM were uniformly mixed by vortexing in a 90:10 volume ratio, and electrospinning was performed with a flow rate of 15 ml / hr, a voltage of 16 kV, a nozzle tip to collector distance (TCD) of 16 cm, and a duration of 3 minutes. Immediately after electrospinning was completed, the sheets were immersed in an NHS / EDC 100 / 100 mM solution in 95% EtOH and crosslinked for at least 3 hours. The crosslinked sheets were washed three times with ethanol and air-dried.

[0213]

[0214] Example 1-2: Nanofiber composite prepared by mixing PCL and dECM in an 8:2 ratio

[0215] PCL and dECM were mixed in a volume ratio of 80:20, and the manufacturing process of the nanofiber composite was the same as in Example 1-1.

[0216]

[0217] Examples 1-3: Nanofiber composite prepared by mixing PCL and dECM in a 7:3 ratio

[0218] PCL and dECM were mixed in a volume ratio of 70:30, and the manufacturing process of the nanofiber composite was the same as in Example 1-1.

[0219]

[0220] Examples 1-4: Nanofiber composite prepared by mixing PCL and dECM in a 5:5 ratio

[0221] PCL and dECM were mixed in a 50:50 volume ratio, and the manufacturing process of the nanofiber composite was the same as in Example 1-1.

[0222]

[0223] Examples 1-5: Nanofiber composite prepared by mixing PCL and dECM in a 3:7 ratio

[0224] PCL and dECM were mixed in a volume ratio of 30:70, and the manufacturing process of the nanofiber composite was the same as in Example 1-1.

[0225]

[0226] Examples 1-6: Nanofiber composite prepared by mixing PCL and dECM in a 1:9 ratio

[0227] PCL and dECM were mixed in a volume ratio of 10:90, and the manufacturing process of the nanofiber composite was the same as in Example 1-1.

[0228]

[0229] Comparative Example 1: Nanofiber composite prepared with PCL

[0230] Polycaprolactone (PCL) was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 16 w / v% by stirring, and electrospinning was performed with a flow rate of 15 ml / hr, a voltage of 16 kV, a TCD (nozzle tip to collector distance) of 16 cm, and a time of 3 minutes.

[0231]

[0232] Example 2-1: Nanofiber composite using PCL and dECM mixed in a 3:7 ratio and 95% EtOH as the solvent for NHS / EDC

[0233] Experiments were conducted using ECM manufactured according to the method disclosed in Korean Patent Publication No. 10-2024-0108874.

[0234] Polycaprolactone (PCL) was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 16 w / v% by stirring, and dECM was also dissolved in HFIP at a concentration of 16 w / v%. Subsequently, PCL and dECM were uniformly mixed by vortexing in a volume ratio of 30:70, and electrospinning was performed with a flow rate of 15 ml / hr, a voltage of 16 kV, a nozzle tip to collector distance (TCD) of 16 cm, and a duration of 3 minutes. Immediately after the completion of electrospinning without any drying time, the sample was immersed in an NHS / EDC 100 / 100 mM solution in 95% EtOH and crosslinked for at least 3 hours. Minimizing the drying time and immersing the sample in the solution is expected to facilitate the release of HFIP solvent components. The crosslinked sheet was washed three times with ethanol and then air-dried.

[0235]

[0236] Example 2-2: Nanofiber composite using PCL and dECM mixed in a 3:7 ratio and 76% EtOH as the solvent for NHS / EDC

[0237] The electrospun sheet was immersed in an NHS / EDC 100 / 100mM solution in 76% EtOH (24% DW) and crosslinked for more than 3 hours, and the manufacturing process of the nanofiber composite was prepared in the same manner as in Example 2-1.

[0238]

[0239] Examples 2-3: Nanofiber composite using PCL and dECM mixed in a 3:7 ratio and 63% EtOH as the solvent for NHS / EDC

[0240] The electrospun sheet was immersed in an NHS / EDC 100 / 100mM solution in 63% EtOH (37% DW) and crosslinked for more than 3 hours, and the manufacturing process of the nanofiber composite was prepared in the same manner as in Example 2-1.

[0241]

[0242] Examples 2-4: Nanofiber composite using PCL and dECM mixed in a 3:7 ratio and 47.5% EtOH as the solvent for NHS / EDC

[0243] The electrospun sheet was immersed in an NHS / EDC 100 / 100mM solution in 47.5% EtOH (52.5% DW) to crosslink it for more than 3 hours, and the manufacturing process of the nanofiber composite was the same as in Example 2-1.

[0244]

[0245] Comparative Example 2: Uncrosslinked nanofiber composite prepared by mixing PCL and dECM in a 3:7 ratio

[0246] Polycaprolactone (PCL) was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 16 w / v% by stirring, and then dECM was also dissolved in HFIP at a concentration of 16 w / v%. Afterward, PCL and dECM were uniformly mixed by vortexing in a volume ratio of 30:70, and electrospinning was performed with a flow rate of 15 ml / hr, a voltage of 16 kV, a TCD (nozzle tip to collector distance) of 16 cm, and a time of 3 minutes.

[0247]

[0248] Experimental Example 0-1: Analysis of Molecular Weight of ECM Using Gel Permeation Chromatography (GPC)

[0249] The molecular weight of the porcine skin-derived ECM sample of Example 0 was analyzed using gel permeation chromatography (GPC). The GPC analysis was performed using a Waters instrument with a TSK gel column and a hexafluoro-2-propanol mobile phase. The flow rate was set to 0.3 mL / min and the column temperature to 40°C. Poly(methyl methacrylate) and hexylbenzene were used as standard substances for analysis, and the results of the GPC analysis of the porcine skin ECM are shown in Figure 1 and Table 1. According to Figure 1, each maximum MP value (355417, 18948, 618) appears as three peaks, which indicates that three or more substances with different molecular weights are mixed within the ECM sample.

[0250] SampleName MnMwMPMzMz+1PolydispersitySkin ECM2493783823983554175901758709201.5339601206011894837420523482.1463354976185856371.483

[0251] Experimental Example 0-2: Characterization of dECM used in electrospinning

[0252] (1) Experimental method

[0253] The quality of the dECM manufactured in this technology was evaluated under the same conditions by referring to the quality inspection items and specifications of commercially available dECM products. The evaluation items included the degree of decellularization, preservation of ECM components, cell affinity, endotoxin, and sterility status.

[0254] (2) Experimental results

[0255] Table 2 below shows the quality evaluation results of dECM.

[0256] Item (Test) Standard (Specification) Result (Result) AppearanceWhite powderPassCellular residue removalDNA contents: < 50 (ng / mg)3.16 ± 0.75ECM preservationGAG contents: 1 ~ 10 (µg / mg)11.26 ± 0.56Collagen contents: 0.5 ~ 1.5 (mg / mg)1.281 ± 0.023Cell proliferationCell morphology: No difference relative to the controlPassEndotoxinMTT assay: >80% relative to the controlPassEndotoxin assay: < 5 EU / mgPassSterilityNot detected of bacteria and fungiPass (Negative)Not detected of mycoplasmaPass (Negative)

[0257] In all items performed according to the quality standards of commercially available dECM, the dECM of this technology met or exceeded the standard values. The DNA residue was low at 3.16 ± 0.75 ng / mg, confirming that sufficient decellularization had occurred. The GAG ​​and collagen content was maintained within the standard range, confirming that the original components of the ECM were not damaged. In the cell proliferation test, a survival rate of over 80% was observed compared to the control group, confirming good cell affinity. Both endotoxin and sterilization items received negative results, confirming that biological safety has been secured. As a result of evaluation based on the quality specifications of commercially available products, the dECM manufactured by the applicant received a passing grade in all test items, verifying that it is a material that secures commercial-level quality and biological safety.

[0258]

[0259] Experimental Example 1: Confirmation of Decellularization Performance of Porcine Skin dECM

[0260] For DNA analysis of dECM in Example 0, the GenJETGenomic DNA Purification Kit and PicogreendsDNA Assay Kit (Thermo, USA) were used; for collagen analysis, a hydroxyproline assay kit (BioVisionResearch Products, USA) was used; and for glycosaminoglycan (GAG) analysis, a dimethyl methylene blue assay (Sigma, USA) was used. The results are shown in Table 3.

[0261] ComponentsKITECH dECMDNA (ng / mg)3.28 ± 0.4Collagen (µg / mg)1227.9 ± 25GAG (µg / mg)9.84 ± 0.5

[0262] The manufactured dECM showed excellent results in decellularization performance. It was confirmed that there were almost no cellular residues remaining, as the DNA content was very low at 3.28 ng / mg. Additionally, it was found that structural stability was effectively preserved, as the collagen content was maintained at a high level of 1227.9 µg / mg. The GAG ​​content also showed an appropriate level of 9.84 µg / mg, contributing to the maintenance of the functional properties of the tissue. These analytical results support the fact that the dECM manufactured by the applicant possesses superior biocompatibility compared to products from other companies. Furthermore, in processes utilizing dECM, reproducibility must be guaranteed in terms of various physical properties, such as the composition and characteristics of the raw materials, in order to consistently set future process conditions; therefore, the process of forming a nanocomposite using the ECM that has undergone the aforementioned decellularization process can be considered optimized accordingly.

[0263]

[0264] Experimental Example 1-1: Confirmation of dECM Manufacturing Reproducibility

[0265] (1) Experimental method

[0266] To verify the stability and reproducibility of the dECM manufacturing process, Skin dECM DNA & preservation analysis was performed. The DNA residue, collagen content, and GAGs content were analyzed for each dECM batch manufactured at different times (January 2024, April 2024, and April 2025).

[0267]

[0268] (2) Experimental results

[0269] Table 4 below shows the results of the component analysis of dECM by manufacturing batch.

[0270] SampleDNA (ng / mg)Collagen (μg / mg)GAGs (μg / mg)Tissue Collection 0h (Before Treatment) 11 24.5 48 09.16 8.04 20 24.01 1st 3.28 ± 0.1 12 27.93 ± 13.6 29.84 ± 0.3 20 24.01 2nd 3.16 ± 0.6 21 33.07 ± 7.7 210.38 ± 0.4 320 24.04 3rd 3.16 ± 0.75 12 80.98 ± 22.5 911.26 ± 0.56 20 25.04

[0271]

[0272] Residual DNA content was maintained at a level of 3 ng / mg across all manufacturing batches, confirming the consistency of high decellularization efficiency. It was confirmed that structural proteins were stably preserved and the biological functional components of the ECM were maintained, with collagen and GAG content showing almost no variation between the first and third manufacturing batches. The results for the first and second batches were obtained by re-measuring the same tissue sample over different storage periods, while the third batch involved analysis after manufacturing dECM from a new tissue sample. Since the variation in results was minimal, it was confirmed that the stability and reproducibility of the manufacturing process were ensured.

[0273]

[0274] Experimental Example 1-2: dECM Solubility Comparison Experiment

[0275] (1) Experimental method

[0276] The solubility of commercially available dECM products (Company D, Company C) and KITECH dECM manufactured in this technology was compared. Each sample was dispersed in PBS and HFIP solvents, respectively, to observe the dissolution process, and the occurrence of precipitates and changes in homogeneity were evaluated.

[0277]

[0278] (2) Experimental results

[0279] Figure 18 is a photograph showing the results of a comparison of the solubility of KITECH dECM and commercially available dECM (Company D, Company C) in different solvents (PBS, HFIP).

[0280] As shown in Figure 18, KITECH dECM exhibited high solubility in both PBS and HFIP and maintained a homogeneous and transparent state during dissolution. In contrast, samples from companies D and C showed precipitation and aggregation, confirming that they had low solubility and poor dispersion homogeneity. These differences stemmed from KITECH’s proprietary dECM manufacturing process, which was confirmed to have simultaneously secured water solubility and organic solvent solubility by effectively removing residual impurities while preventing excessive deformation of the protein structure during the process.

[0281]

[0282] Experimental Example 1-3: Elemental Analysis by Energy Dispersive X-ray Analysis (EDX)

[0283] (1) Experimental method

[0284] The elemental composition of commercially available dECM materials (Company D, Company C) and the KITECH dECM of this technology was compared and analyzed using EDX (Energy Dispersive X-ray Analysis).

[0285] (2) Experimental results

[0286] Table 5 below shows the EDX analysis results (Weight %) of KITECH dECM and commercially available dECM products.

[0287] ElementKITECH skin dECMD skin dECMC skin dECMC41.062.653.0O34.336.544.6N7.5S0.50.3Na8.90.2Cl7.9Fe0.41.1Mo1.3Weight (%)100100100

[0288] As a result of EDS analysis, nitrogen (N), a major element of protein components, was detected in KITECH dECM. Nitrogen (N) was not detected in other companies' dECM, which suggests that the protein content may be insufficient or removed. Additionally, impurities such as iron (Fe) and molybdenum (Mo) were detected in other companies' dECM, which is presumed to be contamination caused by blade wear during powdering in the manufacturing process.

[0289]

[0290] Experimental Example 2: Measurement of the viscosity of an electrospinning solution using a viscometer

[0291] The viscosity of the spinning solutions of Comparative Example 1 and Examples 1-1 to 1-6 dissolved for electrospinning was measured using a viscometer (Brookfield, DV2TLV), and the results are shown in Figure 2.

[0292] As the proportion of PCL increases, the viscosity of the electrospinning solution rises sharply, which significantly affects fiber formation and physical properties during the electrospinning process. At low PCL ratios, the solution viscosity is low, making it highly likely unsuitable for electrospinning; however, stable fiber formation can be expected when the PCL ratio is at an appropriate level (50:50 or higher).

[0293]

[0294] Experimental Example 3: Field Emission Scanning Electron Microscope (FE-SEM) Analysis of Nanofiber Composites

[0295] To verify the actual surface morphology of the sample, it was coated with gold for 150 seconds using an ion coater (E-1045). The coated sample was observed at magnifications of 1,000 and 5,000 using a field emission scanning electron microscope (FE-SEM, SU8010), and the results are shown in Figures 3 and 4. Figure 3 shows images of the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-6, Figure 4 (a) shows an enlarged image of the nanofiber composite of Example 1-3, and Figure 4 (b) shows an enlarged image of the nanofiber composite of Example 1-4.

[0296] In Comparative Example 1, no nanoweb structure was found, and it was confirmed that a nanoweb structure is formed starting from Example 1-1 even when dECM is contained at 10%. In addition, it can be confirmed that as the dECM content increases, a nanoweb structure in the form of a spiderweb net is formed more frequently.

[0297]

[0298] Experimental Example 3-1: Confirmation of Reproducibility (SEM)

[0299] (1) Experimental method

[0300] A mixed solution was prepared at a PCL:dECM (90:10, w / w) ratio using dECM prepared in 2024 and 2025, respectively. Nanofibers were prepared under the same electrospinning conditions, and the fiber structure before and after crosslinking was analyzed using FE-SEM.

[0301]

[0302] (2) Experimental results

[0303] Figure 17 shows FE-SEM images of nanofiber composites prepared with a PCL:dECM (90:10) ratio before and after crosslinking (comparison of PCL:dECM (2024) and PCL:dECM (2025)). It was confirmed that the dECM sample prepared in 2025 also exhibited excellent spinnability and fiber formation properties, similar to the sample prepared in 2024. There were no significant differences in fiber diameter distribution and surface morphology before and after crosslinking, and it was confirmed that a uniform fiber network was maintained during electrospinning. A spider-web-shaped nanoweb structure was observed in the SEM image after crosslinking. Both prepared samples showed identical electrospinning results, confirming that quality consistency between production lots of dECM was ensured.

[0304]

[0305] Experimental Example 4: Elemental Mass Analysis of Nanofiber Composites Using Scanning Electron Microscopy-Energy Dispersive X-ray Analysis (SEM-EDX)

[0306] For Comparative Example 1 and Examples 1-1 to 1-5, the elemental mass ratios of the nanoweb fibers of the first region and the non-net-shaped fibers (microfibers) of the second region were determined using an SEM (SU-8010, Hitachi, Ltd., Japan) embedded with an EDX (energy dispersive X-ray) to analyze the elemental masses contained therein, and this is shown in Fig. 5.

[0307] The elemental mass analysis results of Comparative Example 1 and Examples 1-1 to 1-5 confirmed that the nanowebs were mixed in two fibers of different diameters, confirming that the nanowebs were not formed by phase separation. Additionally, the presence of ions such as NaCl in the solution causes the polymer chains to be charged, which leads to the formation of a structure where the polymer fibers are bonded or branched, thereby creating a nanoweb structure. It appears that the nanoweb structure was formed because ions such as Na and Cl are present in the dECM itself.

[0308]

[0309] Experimental Example 4-1: EDX Elemental Analysis

[0310] (1) Experimental method

[0311] The elemental composition of dECM raw material powders manufactured in 2024 and 2025 was analyzed using EDX (energy dispersive X-ray analysis).

[0312] (2) Experimental results

[0313] Table 6 below shows the EDX analysis results (Weight %) of dECM raw materials.

[0314] ElementKITECH dECM (2024)KITECH dECM (2025)C41.055.6O34.322.6N7.515.0S0.5Na8.91.9Cl7.93.9Fe1.0MoWeight (%)100100

[0315] EDS analysis of the dECM produced in 2025 confirmed that nitrogen (N), a protein constituent element, was the same as that of the 2024 sample, confirming that the protein components were maintained stably. It was also confirmed that Na and Cl, essential elements for nanoweb formation, were detected.

[0316]

[0317] Experimental Example 5: Analysis of Fiber Diameter Size of Nanofiber Composite

[0318] Using the Image J program, the diameter sizes of individual fibers (nano fibers) of the nano web structure in the first region and fibers (micro fibers) in the second region of Comparative Example 1 and Examples 1-1 to 1-6 were analyzed in SEM images, and the results are shown in Fig. 6.

[0319] The fiber diameter of Comparative Example 1 is approximately 1.7 µm, and as in Examples 1-1 to 1-6, it can be confirmed that when dECM is included, the microfiber diameter is reduced to 500 nm. In addition, it can be seen that when dECM is present, a nanoweb of 30 to 40 nm is formed together.

[0320]

[0321] Experimental Example 6: Measurement of the thickness of a nanofiber composite using a thickness gauge

[0322] The thickness of the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-6 was measured using a thickness gauge, and this is shown in FIG. 7.

[0323] When comparing the thickness of the sheets after 30 minutes of electrospinning, the thickness of Comparative Example 1 (PCL 100%) and the thickness of Example 1-1 (PCL 90:ECM 10) were similar. This indicates a tendency for the radioactivity of the two samples to be similar, whereas the radioactivity of Examples 1-2 to 1-5 is relatively lower.

[0324]

[0325] Experimental Example 7: Measurement of Tensile Strength of Nanofiber Composites Using a Universal Testing Machine (UTM)

[0326] The tensile strength of Comparative Example 1 and Examples 1-1 to 1-5 was measured using a universal testing machine (UTM, INSTRON 5967, USA) with sample sizes of 1 cm in width and 2 cm in length, and the results are shown in Fig. 8.

[0327] As a result of measuring tensile strength according to PCL:dECM content, it was confirmed that tensile strength and elongation decreased as the dECM content increased. It appears that physical properties can be secured to some extent only when PCL is mixed in, rather than when dECM is 100%.

[0328]

[0329] Experimental Example 8: Results of residual solvent analysis using gas chromatography-mass spectrometry (GC-MS)

[0330] The residual HFIP solvent of Examples 1-5 was analyzed using HS-GC-MS. The HS-GC-MS analysis was performed using an Agilent instrument and a DB-624 UI column, with He set as the mobile phase. Detection was performed using SIM mode to identify specific m / z values ​​(51, 99, 129, 149) of the HFIP solvent. The components were identified through the NIST / Wiley library, and the analysis results are shown in Figure 9 and Table 7. It was confirmed that approximately 1.2 ppm of HFIP was detected in the nanofiber composite of Examples 1-5. Since 1.2 ppm is a trace amount, it is considered safe for applying the nanofiber composite to medical devices.

[0331] No. Sample Name Target Component Unit Result Value 1 dEC MH exafluoroisopropyl alcohol mg / kg 1.2

[0332] Experimental Example 8-1: Analysis of Residual Organic Solvents (GC-MS)

[0333] (1) Experimental method

[0334] Residual HFIP solvent in nanofiber composites was analyzed using HS-GC-MS. In this experiment, PCL90:ECM10 specimens (crosslinked) fabricated with a larger size than conventional specimens (thickness approximately 60–80 μm) were used as samples. HS-GC-MS analysis was performed using an Agilent instrument and a DB-624 UI column, with He set as the mobile phase.

[0335] (2) Experimental results

[0336] The measurement results (HS-GC-MS) are shown in Fig. 19 and Table 7 below. Fig. 19 shows the chromatogram (90 °C) of the standard solution, where the target component (hexafluoroisopropyl alcohol) peak was identified at 13.453 min and the internal standard substance (tetrahydrofuran, IS) peak was identified at 7.570 min.

[0337] Table 8 and Figure 19 below are a summary of the measured HFIP results for PCL90:ECM10 (dECM) samples.

[0338] No. Sample Name Target Component Unit Result Value 1 dEC MH exafluoroisopropyl alcohol mg / kg Not Detected

[0339] In the EDC / NHS (in EtOH) crosslinked samples after electrospinning PCL90:ECM10 (thickness: 60 ~ 80 μm), residual HFIP was confirmed to be 'undetectable,' with a detailed measurement of 0.4 ppm. This is a lower level than that of the nanofiber composites of Examples 1-5 (approx. 1.2 ppm), reconfirming that the residual solvent is present in very small amounts through the manufacturing method and washing step of the present invention.

[0340]

[0341] Experimental Example 9: Observation of cell adhesion to nanofiber composites

[0342] NIH3T3 cells 10 4 After dispensing at a rate of 100 μl per cell, cell adhesion of the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 was analyzed against the 100% control cell count using the MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) reagent after 4 hours, and the results are shown in Fig. 10 (a). In addition, the results of the reproduction experiment are shown in Fig. 10 (b).

[0343] In Fig. 10 (a), Comparative Example 1 showed 80% adhesion, whereas Examples 1-2 to 1-5 showed high cell adhesion of about 100%, and it was confirmed that cell adhesion improved as the dECM content increased. Similarly, in Fig. 10 (b), it was also confirmed that cell adhesion improved as the dECM content increased.

[0344]

[0345] Experimental Example 10: Observation of cell growth rate for nanofiber composites

[0346] NIH3T3 cells 10 4 Cell growth rates for the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 were confirmed by dispensing cells / 100μl and quantifying them with the MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), and the results are shown in Fig. 11. Fig. 11 (a) shows the results of confirming cell growth rates after growing cells for 3, 7, 10, and 14 days, and Fig. 11 (b) shows the results of a replication experiment in which cell growth rates were confirmed after growing cells for 7 days.

[0347] As shown in the analysis of cell growth rates up to day 14 in Fig. 11 (a), it was confirmed that the cell growth rate increased as the dECM content increased compared to Comparative Example 1, and the highest growth rate was observed in Example 1-1 (dECM 10%). This is judged to be because the mechanical properties of PCL were maintained up to day 14, resulting in a higher growth rate. As shown in the analysis of cell growth rates up to day 7 in Fig. 11 (b), the highest growth rates were observed in Examples 1-1 and 1-2.

[0348]

[0349] Experimental Example 11: Live / Dead cell observation of nanofiber composite

[0350] 10 NIH3T3 cells in the nanofiber composites of Comparative Example 1 and Examples 1-1 to 1-5 4 Cells were seeded at 100 μl and cultured for 3, 7, 10, and 14 days. Afterward, the cells were stained with the LIVE / DEAD™ Viability / Cytotoxicity Kit for Mammalian Cells (Thermo Fisher Scientific, USA), and living cells (green) and dead cells (red) were observed using a fluorescence microscope, as shown in Fig. 12. Fig. 12 (a) shows the cell morphology by date measured at x2 magnification relative to the objective lens, and Fig. 12 (b) shows the cell morphology by date measured at x20 magnification relative to the objective lens.

[0351] Live / dead analysis results up to day 14 confirmed that cells grew well in Examples 1-1 to 1-6, and on day 3, more cell morphology was observed in samples 1-1 to 1-5 compared to Comparative Example 1, and in particular, it was confirmed that cell growth was faster in Examples 1-2 to 1-4.

[0352]

[0353] Experimental Example 12: Field Emission Scanning Electron Microscope (FE-SEM) Analysis of Nanofiber Composites

[0354] To verify the actual surface morphology of the sample, it was coated with gold for 150 seconds using an ion coater (E-1045). The coated sample was observed at magnifications of 1,000 and 5,000 using a field emission scanning electron microscope (FE-SEM, SU8010), and the results are shown in Fig. 13. Fig. 13 (a) is an image of the nanofiber composites of Comparative Example 2 and Examples 2-1 to 2-4, and (b) is an enlarged image of the nanofiber composite of Example 2-3.

[0355] The EDC / NHS reaction crosslinks the carboxyl groups (-COOH) and amino groups (-NH2) of dECM, such as collagen and gelatin, in which EDC reacts with the carboxyl groups to form an activated ester intermediate, and NHS acts as a stabilizing agent to stabilize the ester intermediate formed by EDC, which then reacts with the amino groups to form an amide bond (-CONH-). Looking at the results according to the ratio of ethanol and distilled water in the 100 / 100 mM solvent, it can be confirmed that the nanoweb formed in Comparative Example 2 is preserved to some extent even after crosslinking.

[0356]

[0357] Experimental Example 13: Measurement of the thickness of a nanofiber composite using a thickness gauge

[0358] The thickness of the nanofiber composites of Examples 2-1 to 2-4 and Comparative Example 2 was measured using a thickness gauge, and the results are shown in Fig. 14. As a result of the thickness measurement, the thickness of the nanofibers in Examples 2-1 to 2-4, which underwent the EDC / NHS crosslinking reaction, decreased overall compared to Comparative Example 2. As the ethanol content decreased and the distilled water content increased, the thickness tended to become progressively thinner, and the thinnest thickness was observed in Example 2-4, which used 47.5% ethanol. This phenomenon may be due to the possibility that some of the water content affected the dECM during the crosslinking reaction and dissolved.

[0359]

[0360] Experimental Example 14: Measurement of Tensile Strength of Nanofiber Composites Using a Universal Testing Machine (UTM)

[0361] Tensile strength was measured using a universal testing machine (UTM, INSTRON 5967, USA) with a sample size of 1 cm in width and 2 cm in length, and the results are shown in FIG. 15. FIG. 15 (a) shows the maximum load of Examples 2-1 to 2-4 and Comparative Example 2, and (b) shows the elongation at the maximum load of Examples 2-1 to 2-4 and Comparative Example 2.

[0362] Examples 2-1 to 2-4, in which the EDC / NHS crosslinking reaction was performed, showed higher strength compared to Comparative Example 2. In particular, Example 2-1, which used 95% ethanol (EtOH), showed the highest tensile strength, exhibiting a maximum tensile strength of approximately 2.9 MPa. As the proportion of ethanol decreased, the tensile strength showed a tendency to gradually decrease, and at an ethanol content of 47.5%, it decreased to approximately 2.1 MPa. These results indicate the effect of the crosslinking reaction on the mechanical properties of the fiber depending on the ethanol concentration, and it is determined that stronger crosslinking bonds were formed at higher ethanol concentrations.

[0363] In terms of elongation, there was a tendency for it to increase as the ethanol content decreased. In particular, the highest elongation was observed when the ethanol content was 63% and 47.5%, which showed an elongation of over 60%. This is interpreted as the flexibility of the fiber being added at low ethanol concentrations, and it means that the elongation characteristics of the fiber were improved even if the mechanical strength was somewhat lower.

[0364]

[0365] Experimental Example 15: Measurement of pore size of nanofiber composite using a Pore Measurement Instrument (PMI)

[0366] The largest pore size (µm) and average pore size (µm) of Examples 2-1 to 2-4 were measured using a capillary flow meter (PMI), and these results are shown in FIG. 16.

[0367] In Example 2-1, EDC / NHS crosslinking was performed more effectively with a 95% ethanol ratio, which strengthened the bonding between fibers, resulting in higher fiber density and smaller pore size. On the other hand, as the proportion of distilled water increased, the crosslinking power decreased, weakening the bonding between fibers, and consequently, it appears that the pore size increased significantly. Additionally, when crosslinked by immersion in a solution with a high water ratio, the electrospun sheet absorbed more water, causing the gaps between fibers to widen and potentially resulting in a larger measured pore size.

[0368] However, in the case of composite nanofibers with a high dECM ratio, immersion in 95% EtOH during crosslinking caused shrinkage due to the rapid evaporation and dehydration effects of EtOH, and it was difficult to maintain the shape of the thin nanofibers. Therefore, the distilled water content was slightly increased during crosslinking, and in this case, the shrinkage phenomenon could be improved. In terms of physical properties, crosslinking in 95% EtOH appears to be the best, but when the dECM content is high, crosslinking with a slightly lower EtOH ratio is advantageous in terms of shape retention.

[0369]

[0370] Experimental Example 16-1: dECM alone radiation

[0371] (1) Experimental method

[0372] The material was dissolved in HFIP at a concentration of 16 w / v% dECM by stirring. Electrospinning was performed under the following conditions: Flow rate: 15 ml / hr, Voltage: 16 kV, TCD: 16 cm, Time: 3 min. Immediately after electrospinning was completed, the material was immersed in an NHS / EDC 100 / 100 mM solution in 95% EtOH for at least 3 hours to crosslink. The crosslinked sheets were washed three times with ethanol and then air-dried.

[0373]

[0374] (2) Experimental results

[0375] Figure 20 shows FE-SEM images of a 100% dECM nanofiber composite before (left) and after (right) crosslinking.

[0376] As a result of electrospinning dECM dissolved in HFIP solvent at a concentration of 16 w / v%, it was confirmed that stable spinning is possible using only pure dECM. In the specimen without crosslinking after electrospinning (Fig. 20, left), a uniform nanofiber structure was formed, and the fiber diameter was homogeneous and observed to be intertwined. In the specimen after EDC / NHS crosslinking treatment (Fig. 20, right), the bonding between fibers was strengthened to form a dense structure, and a stable mat structure was confirmed as the fibers were observed to be fused together. Through this, it was confirmed that nanofiber sheets can be manufactured using only 100% dECM material, and that structural stability is secured through the crosslinking process. This indicates that single spinning conditions can be satisfied because the dECM developed by the applicant was introduced.

[0377]

[0378] Experimental Example 16-2: dECM 100% Spinning (TFE Solvent Conditions)

[0379] (1) Experimental method

[0380] It was dissolved by stirring in TFE based on 16 w / v% dECM. The electrospinning was carried out under the following conditions: Flow rate: 15 ml / hr, Voltage: 16 kV, TCD: 16 cm, Time: 3 min.

[0381] (2) Experimental results

[0382] Figure 21 shows an FE-SEM image of nanofibers prepared with 100% dECM using TFE solvent.

[0383] As a result of electrospinning dECM dissolved in TFE solvent at a concentration of 16 w / v%, it was confirmed that stable spinning is possible using only pure dECM. The spun specimens were formed in the shape of uniform nanofibers, and it was confirmed that they exhibited a continuous and dense structure with well-maintained inter-fiber entanglement.

[0384]

[0385] Experimental Example 17-1: Radioactivity Confirmation by PCL / dECM Ratio

[0386] (1) Experimental method

[0387] Previously, the dECM content started at 10%, but radioactivity and cell regeneration efficacy were confirmed at lower ranges. From a commercialization perspective, it is advantageous if better effects are achieved using the minimum amount. To verify the actual surface morphology of the sample, it was coated with gold for 150 seconds using an ion coater (E-1045). The coated sample was observed at magnifications of 1,000 and 5,000 using a field emission scanning electron microscope (FE-SEM, SU8010).

[0388]

[0389] (2) Experimental results

[0390] Figure 22 shows FE-SEM images of nanofiber composites with PCL 100% and PCL:dECM ratios (97:3, 95:5, 90:10, 80:20, 70:30).

[0391] Additional radiation was carried out by mixing 3% and 5% of dECM, and as shown in Figure 22, stable radioactivity was confirmed, and it was also confirmed that a net-shaped nanoweb structure was formed at least partially.

[0392]

[0393] Experimental Example 17-2: Observation of cell adhesion before and after crosslinking

[0394] (1) Experimental method

[0395] NIH3T3 cells 10 4After dispensing at a rate of cells / 100μl, adhesion was analyzed after 4 hours using the MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) reagent relative to the number of 100% control cells.

[0396] (2) Experimental results

[0397] Figure 23 is a graph showing the cell adhesion (%) of nanofiber composites before and after crosslinking at 4 hours, according to PCL:dECM ratios (100:0, 97:3, 95:5, 90:10).

[0398] Regarding adhesion results, the non-crosslinked samples showed higher adhesion. On the other hand, the cell growth rate (to be discussed later) tended to be more favorable for the crosslinked samples.

[0399]

[0400] Experimental Example 17-3: Observation of Cell Growth Rate

[0401] (1) Experimental method

[0402] NIH3T3 cells 10 4 Cells / 100 μl were dispensed onto nanofiber composite specimens containing 3%, 5%, and 10% dECM, pre-crosslinking (PCL:dECM 97:3, 95:5, 90:10) and post-crosslinking (PCL:dECM 97:3, 95:5, 90:10), respectively. After culturing for 2, 6, 10, and 14 days, the cell growth rate was quantitatively determined using the MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega).

[0403] (2) Experimental results

[0404] Figure 24 is a graph showing the quantitative analysis of the cell growth rate of NIH3T3 cells over time (2, 6, 10, and 14 days) according to the PCL:dECM ratio and whether or not crosslinking was performed.

[0405] As shown in Fig. 24, when compared to the specimen of Comparative Example 1 consisting only of PCL 100%, all experimental groups containing dECM (97:3, 95:5, 90:10) showed significantly superior cell proliferation rates over time, regardless of crosslinking status and the composition ratio of dECM. Overall, a similar pattern of cell number increase was observed in all conditions containing dECM compared to the PCL 100% specimen, and it was confirmed that stable proliferation was maintained until day 14.

[0406] In addition, when comparing samples before and after crosslinking, the crosslinked specimens (after crosslinking) in all groups including 3%, 5%, and 10% dECM showed a higher cell growth rate during long-term culture (10 days, 14 days).

[0407] In particular, among the crosslinked specimens, the best cell growth rates were observed at 3% and 5% dECM content compared to 10%. This demonstrates that even with a low content of dECM, the cell proliferation effect can be maximized while complementing the physical properties of PCL.

[0408]

[0409] Experimental Example 17-4: Live / dead observation

[0410] (1) Experimental method

[0411] NIH3T3 cells 10 4 Cells were inoculated into specimens with PCL:dECM ratios (100:0, 97:3, 95:5, 90:10) and before / after crosslinking at a rate of 100 μl / cells / 100 μl, and cultured for 0, 2, 6, 10, and 14 days. Afterward, the cells were stained with the LIVE / DEAD™ Viability / Cytotoxicity Kit for Mammalian Cells (Thermo Fisher Scientific, USA), and living cells (green) and dead cells (red) were observed under a fluorescence microscope.

[0412] (2) Experimental results

[0413] Figure 25 shows live / dead fluorescence microscopy images according to the PCL:dECM ratio and whether or not crosslinking occurs.

[0414] As shown in Figure 25, the results of the Live / dead analysis up to day 14 of culture showed that a significantly larger number of living cells (green) were observed in all groups containing dECM (97:3, 95:5, 90:10 before / after cross-linking) compared to the PCL 100% (100:0) specimen.

[0415] In particular, more cell morphology was observed in the dECM content 3% (97:3) and 5% (95:5) samples (both before and after crosslinking) than in the dECM 10% (90:10) sample, which is consistent with the cell growth rate results of Experimental Example 17-3 above. Since almost no dead cells (red) were observed in most specimens, it was confirmed that the nanofiber composite of the present invention provides excellent cell viability.

[0416]

[0417] Experimental Example 18: Preparation of Nanofiber Composites with Different PCL / dECM Ratios Using TFE Solvent

[0418] (1) Experimental method

[0419] Trifluoroethylene (TFE) was used instead of HFIP as the electrospinning solvent. PCL and dECM were prepared in ratios of 100:0, 90:10, 80:20, 70:30, and 50:50 (w / w), and 100% dECM specimens were also prepared using TFE as the solvent, as in Example 16-2 above. The electrospinning conditions were performed under the same conditions as in Example 1-1 above, and the surface morphology of the prepared specimens was observed using FE-SEM.

[0420] (2) Experimental results

[0421] Figure 26 is an FE-SEM image of a nanofiber composite prepared with a PCL / dECM 90 / 10 ratio using TFE solvent. Figure 27 is an FE-SEM image of a nanofiber composite prepared with PCL:dECM ratios (100:0, 90:10, 80:20, 70:30, 50:50) and 100% dECM using TFE solvent.

[0422] As shown in FIGS. 26 and 27, excellent radioactivity was confirmed even when using TFE solvent, and it was confirmed that a spiderweb (nanoweb) structure was formed in specimens containing dECM (90:10, 80:20, 70:30, 50:50). This result is similar to that obtained when using HFIP solvent, demonstrating that the nanoweb structure of the present invention is not limited to a specific solvent (HFIP) and can be realized even when using TFE solvent.

[0423]

[0424] Experimental Example 19: Preparation of Nanofiber Composites with Different Ratios of PVA Polymer and dECM

[0425] (1) Experimental method

[0426] In this experimental example, polyvinyl alcohol (PVA) was used instead of polycaprolactone (PCL) as the biocompatible polymer, and water (DW) was used instead of organic solvents (HFIP, TFE) as the electrospinning solvent.

[0427] Water (DW)-based mixed solutions were prepared by preparing PVA and dECM in ratios of 100:0, 90:10, and 80:20 (w / w). Electrospinning conditions were performed under conditions similar to those of the above examples, and the surface morphology of the prepared specimens was observed using FE-SEM.

[0428] (2) Experimental results

[0429] Figure 28 is an FE-SEM image of nanofiber composites prepared using water (DW) solvent according to PVA:dECM ratios (100:0, 90:10, 80:20).

[0430] As shown in Fig. 28, stable electrospinning was confirmed under conditions where the dECM ratio was 10% (90:10) and 20% (80:20), just like with the PVA 100% (100:0) specimen. This demonstrates that the dECM material of the present invention provides excellent spinnability even in aqueous solvents and hydrophilic polymer systems.

[0431] In particular, in the case of the specimen with 20% dECM content (80:20) (Fig. 28, right), the possibility of forming a fine nanoweb (spiderweb) structure between the fibers was confirmed. This suggests that the multiscale nanofiber composite structure of the present invention can be realized not only in hydrophobic polymer and organic solvent systems such as PCL, but also in hydrophilic polymer and aqueous solvent systems such as PVA.

[0432]

[0433] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0434] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

[0435]

Claims

1. A nanofiber composite comprising a decellularized extracellular matrix (dECM) and a biocompatible polymer, wherein The above nanofiber composite has a first region and a second region, The first region above represents a nanoweb structure in the form of a net formed between nanofibers, and A nanofiber composite characterized in that the second region above represents a non-net-shaped nanofiber.

2. In Paragraph 1, A nanofiber composite characterized in that the above-mentioned biocompatible polymer is at least one polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(lactide-co-caprolactone) (PLCL), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, dextran, and silk.

3. In Paragraph 1, A nanofiber composite characterized in that the nanoweb structure of the first region is derived from a monovalent cation or a monovalent anion upon spinning.

4. In Paragraph 1, Scanning Electron Microscopy with Energy Dispersive X-Ray Analysis (SEM-EDX) results, A nanofiber composite characterized in that the first region and the second region commonly contain C, O, and N elements.

5. In Paragraph 4, A nanofiber composite characterized in that, relative to 100 parts by weight of individual fibers of a nanoweb structure in the first region, the elemental content of N is 4 to 35 parts by weight.

6. In Paragraph 4, A nanofiber composite characterized in that, in the second region above, the elemental content of N is 6 to 30 parts by weight relative to 100 parts by weight of nanofiber.

7. In Paragraph 1, A nanofiber composite characterized in that the ratio of the average diameter of individual fibers of the nanoweb structure of the first region to the average diameter of nanofibers in the second region is 1:6 to 50.

8. In Paragraph 1, A nanofiber composite characterized in that the average diameter of individual fibers of the nanoweb structure in the first region is 20 to 50 nm.

9. In Paragraph 1, A nanofiber composite characterized in that the average diameter of the nanofibers in the second region is 0.3 to 1.0 μm.

10. In Paragraph 1, A nanofiber composite characterized by having an average pore size of 0.1 to 10 μm.

11. In Paragraph 1, A nanofiber composite characterized by having a tensile strength of 0.5 to 10 MPa.

12. In Paragraph 1, A nanofiber composite characterized by the weight ratio of the decellularized extracellular matrix and the biocompatible polymer being 0.2 to 9.8 : 9.8 to 0.

2.

13. A mat-type therapeutic material comprising a structure formed by molding the nanofiber composite according to paragraph 1 into a plate shape.

14. A tubular therapeutic material comprising a structure formed by molding the nanofiber composite according to claim 1 into a tubular shape.

15. A method for manufacturing a nanofiber composite, A step of mixing a decellularized extracellular matrix and a biocompatible polymer in a solvent; A step of manufacturing a nanofiber sheet by electrospinning the above mixture; and A method for manufacturing a nanofiber composite, comprising the step of drying or washing the nanofiber sheet.

16. A method for manufacturing a nanofiber composite, A step of preparing a first solution in which the decellularized extracellular matrix is ​​dissolved in a solvent; A step of preparing a second solution in which a biocompatible polymer is dissolved in a solvent; A step of manufacturing a first nanofiber sheet by electrospinning the first or second solution; A step of manufacturing a second nanofiber sheet by electrospinning the second or first solution onto a first nanofiber sheet; and A method for manufacturing a nanofiber composite, comprising the step of drying or washing the laminated first and second nanofiber sheets.

17. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, comprising the step of crosslinking the nanofiber composite with a crosslinking agent prior to the drying or washing step.

18. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, characterized in that the above-mentioned biocompatible polymer is at least one polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(lactide-co-caprolactone) (PLCL), polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PL), polyvalerolactone (PL), polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polydioxanone, collagen, gelatin, chitosan, hyaluronic acid, dextran, and silk.

19. In Paragraph 15 or 16, A method for preparing a nanofiber composite, characterized in that the solvent comprises one or more selected from the group consisting of trifluoroacetic acid, dimethylformamide, dimethyl sulfoxide, trifluoroethylene, acetone, hexafluoroisopropanol, 2,2,2-trifluoroethanol (2,2,2-Trifluoroethanol, TFE), dichloromethane, tetrahydrofuran, ethanol, water, acetic acid, and formic acid.

20. In Paragraph 15 or 16, In the above nanofiber composite, A method for manufacturing a nanofiber composite, characterized in that the weight ratio of the decellularized extracellular matrix and the biocompatible polymer is 0.2 to 9.8 : 9.8 to 0.

2.

21. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, characterized in that the electrospinning is performed under conditions where the voltage is 5 to 100 kV, the spinning distance is 3 to 50 cm, and the fluid velocity is 1 to 100 ml / hr.

22. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, characterized in that the average thickness of the electrospun nanofiber sheet is 5 to 400 μm.

23. In Paragraph 17, A method for manufacturing a nanofiber composite, characterized in that the solvent of the crosslinking agent comprises ethanol.

24. In Paragraph 23, A method for manufacturing a nanofiber composite, characterized in that the content of the ethanol is 40 to 99.9 parts by weight relative to 100 parts by weight of the solvent.

25. In Paragraph 17, A method for preparing a nanofiber composite, characterized in that the crosslinking agent comprises one or more selected from the group consisting of glutaraldehyde, EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide), thrombin, genipin, Ru / SPS, hexamethylene diisocyanate, isophorone diisocyanate, ethylene glycol diglycidyl ether, glyceraldehyde, 1,4-butanediol diglycidyl ether (BDDGE), and tannic acid.

26. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, characterized in that the tensile strength of the nanofiber composite is 0.5 to 10 MPa.

27. In Paragraph 15 or 16, A method for manufacturing a nanofiber composite, characterized in that the average pore size of the nanofiber composite is 0.1 to 10 μm.

28. A step of manufacturing a nanofiber composite in accordance with paragraph 15 or 16; and A step of forming the above nanofiber composite into a sheet or plate shape to obtain a mat-type therapeutic material; A method for manufacturing a mat-type therapeutic material including 29. A step of manufacturing a nanofiber composite in accordance with paragraph 15 or 16; Includes, A method for manufacturing a tubular therapeutic material, characterized in that the step of manufacturing the above nanofiber composite is performed in a cylindrical collector.

30. A method for manufacturing a nanofiber composite, A step of mixing decellularized extracellular matrix with a solvent; A step of manufacturing a nanofiber sheet by electrospinning the above mixture; and A method for manufacturing a nanofiber composite, comprising the step of drying or washing the nanofiber sheet.

31. A nanofiber composite comprising a decellularized extracellular matrix (dECM) prepared according to the method of claim 30.

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