Bioscaffold and method for preparing the same

Abstract

A method for preparing a non-reconstructed collagen matrix is ​​provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application with an application date of December 30, 2019, application number 201980087040.5, and invention name “Biological scaffold and method for preparing the same”. Technical Field

[0003] The present invention provides a bioscaffold and a method for preparing the same, wherein the bioscaffold comprises a non-reconstructed collagen matrix and a biomaterial, and an optional tissue adhesive layer. More specifically, the non-reconstructed collagen matrix and the bioscaffold can be used as a dressing for promoting wound healing. Background Art

[0004] Tissue engineering refers to the reconstruction or repair of organs and tissues by using in vitro culture or construction techniques of synthetic or biologically active substances. Tissue engineering involves multiple disciplines such as biology, materials science, and engineering, and has been able to reconstruct tissues such as bone, cartilage, skin, kidney, liver, digestive tract, cornea, muscle, and breast. The basic method of tissue engineering, also known as "regenerative medicine," includes the following steps: first, cells are taken from the human body and cultured in vitro until a sufficient number is reached; then, the cells are placed in an artificial scaffold and allowed to grow; sometimes even some chemicals or biological factors need to be added to promote cell differentiation; and finally, the artificial tissue is transplanted to the patient. Therefore, the three elements of tissue regeneration are cells, biological scaffolds, and growth factors.

[0005] Among them, the most basic structural and functional unit of the human body is the cell. Existing between cells is the extracellular matrix, which is produced by the cells themselves. It is a substance that includes structural components, such as protein fibrils / bundles (such as collagen), crystalline inorganic minerals, etc.

[0006] Tissues are composed of many cells with similar morphology and function, which are bound together by extracellular matrix. Among them, according to different cell types, they are divided into different tissues, such as epithelial tissue, connective tissue, muscle tissue and nervous tissue, etc. Among them, connective tissue is composed of a large amount of extracellular matrix and cells scattered in it. The extracellular matrix contains matrix, fibrils and tissue fluid, etc. Collagen is the most important structural protein in animal connective tissue and the most important component in the extracellular matrix. In animal connective tissue, in addition to 60-70% of water, it also contains 20-30% of collagen. Therefore, collagen is a protein widely present in animals, and connective tissue is widely distributed in the human body and exists in almost all organs.

[0007] Collagen exists mainly in the form of insoluble fibrils, accounting for about 33% of the protein in human tissue. In other words, collagen accounts for one-third of the protein in the human body and is a very important structural protein in connective tissue. It plays the role of a "mattress" and "cement", protecting and connecting various tissues to support the human body structure. Therefore, collagen is often used as a material for bioprostheses.

[0008] The most basic unit of collagen is tropocollagen, which is a molecule composed of three polypeptide chains. The three polypeptide chains are closely bound together in parallel by interchain hydrogen bonds to form a stable triple helix structure. The collagen unit of the triple helix chain is called tropocollagen, with a diameter of about 1.5nm and a length of about 280nm. Under physiological conditions, multiple tropocollagen molecules self-aggregate into collagen microfibril structures, with each molecule staggered at a distance of about 67nm. The microfibrils can then be aggregated into fibrils of different diameters, usually about 50nm. In vivo, the fibrils are stacked into different quaternary structures, which can form reticular fibrils, such as the matrix under epidermal cells. These fibrils can be further aggregated into fibers, such as ligaments and tendons, which contain collagen fibrils arranged regularly in a single direction.

[0009] So far, the animal collagen discovered can be divided into at least 28 types. Except for hyaline cartilage, which is mainly type II collagen, most connective tissues mainly contain type I collagen, which accounts for about 90% of the total collagen content and is also the most widely used collagen biomaterial. Its main structural protein provides connective tissue with the support, protection and various mechanical properties required by connective tissue, such as tension, tension, strength and viscoelasticity. In terms of biochemical properties, it can promote the coagulation cascade and catalyze the formation of blood clots. The functions of collagen include controlling molecular permeability, promoting wound healing and tissue repair, and regulating the physiological functions of cells and tissues.

[0010] The method for preparing a conventional collagen matrix, such as the method for preparing a porous collagen matrix in Taiwan Patent No. 476642, is to reconstitute the collagen matrix under physiological conditions at 30-45° C. The porous collagen matrix can be obtained by freezing and freeze-drying the matrix. Figure 3 A 100× electron micrograph of a conventional porous collagen matrix is ​​shown. Its appearance has a structure that presents a loose and irregular flake-like appearance and does not provide good tension and resistance to cell contraction, both of which are required to optimize proper cell migration and proliferation. In addition, due to the surface properties of collagen, it is not conducive to cell entry.

[0011] Another conventional technology in U.S. Pat. No. 5,997,895, "Dura mater / meningeal repair product using collagen matrix", provides a method for repairing and promoting the growth of meninges. Collagen from a proven biocompatible source that does not contain pathogenic virus residues can be used. A cross-linking reaction is used for a collagen matrix and a porous collagen structure is formed with a porous diameter of 10-500 μm, which allows damaged meninges to be repaired and promotes the growth of meninges. However, since collagen is taken from the dermis of animal skin (such as pig skin, etc.), when the hair follicles are not completely removed during the processing of pig skin, the overall structure of the subsequent preparation of the collagen matrix will be destroyed. It will also affect the purity of the prepared collagen, which can cause bacterial infection and immune response, or cause immune rejection in clinical use.

[0012] Taiwan Patent No. 098125217 entitled "Preparation of High-Purity Collagen" provides a method for preparing collagen, comprising: providing a surface area of ​​20 mm 2 Up to 2m 2 connective tissue; using a first acid solution to expand the volume of the connective tissue by at least 50% to form expanded connective tissue, wherein the pH value of the acid solution is 1-6 and the acid solution is substantially free of salt; washing the expanded connective tissue to remove non-collagenous substances to form a collagen matrix; and then using an extraction solution to extract collagen from the collagen matrix to prepare a solution containing collagen. In the above technology, the solution is collagen, and the porous collagen matrix formed by conventional methods cannot provide good tension and resistance to cell contraction, both of which are required to optimize appropriate cell infiltration and proliferation.

[0013] Another U.S. invention patent publication No. US8067149B2 entitled "Acellular dermal matrix and method of use thereof for transplantation" provides a method for extracting acellular tissue matrix, which includes removing hair follicles and extracting with a surfactant SDS. However, it extracts acellular tissue matrix, which is different from the high-purity porous collagen of the present invention.

[0014] The current overall status of bioscaffolds is as follows.

[0015] At present, most biological scaffolds on the market are made of reconstructed collagen matrix, and its related products have been disclosed in multiple previous patents. For example, in U.S. Patent No. 4,193,813, collagen is first dissolved in acetic acid, cross-linked with glutaraldehyde, and then frozen at low temperature for a long time (e.g., 20 hours). After thawing, water is mechanically removed to form a sponge-like matrix, and the pore size of the matrix obtained by this method is about 80-1400 μm. The method taught by U.S. Patent No. 4,412,947 includes suspending high-purity insoluble collagen particles in acetic acid solution, freezing to -65 ° C at a cooling rate of about 0.3 to 0.4 ° C per minute, and freeze-drying to form a porous sheet substrate. U.S. Patent No. 4,522,753 teaches that collagen and chondroitin sulfate are mixed into copolymers, cross-linked by glutaraldehyde, and freeze-dried to obtain a porous matrix with a pore size of about 20 to 180 μm, which can be used as an artificial skin substrate. U.S. Patent No. 4,970,298 discloses that a mixture solution containing an acid-soluble collagen solution or a solution of an acid-soluble collagen, hyaluronic acid and fibronectin, etc. can be cross-linked by carbodiimide and dehydration heat method. After freezing at different temperatures, freeze-drying is used to prepare a porous matrix. The freezing temperature is -30 to -50°C, and the pore size of the obtained matrix is ​​about 50 to 250 μm, and the collagen matrix containing hyaluronic acid or fibronectin has a pore size of 100-150 μm. The method for preparing a collagen matrix disclosed in the above patent mainly uses acidic or alkaline collagen, cross-linked at high temperature or cross-linked by a chemical cross-linking agent and then freeze-dried to produce pores to obtain a porous matrix. The matrix prepared by these methods has poor pore uniformity and is difficult to control the pore size. In addition, most of the chemical cross-linking agents used are toxic. Reconstructed collagen microfibrils or fibrils provide limited support, protection and various mechanical properties such as tension, tensile force, strength and viscoelasticity to the matrix and are therefore not ideal for clinical applications.

[0016] The non-remodeled collagen matrix has high industrial value for clinical application, and the inventor's previously applied US Pat. No. 8,198,408 entitled “Method for Preparing Porous Collagen Matrix” discloses a method for preparing the non-remodeled collagen matrix.

[0017] Conventionally, the methods used in the prior art, such as the Taiwan invention patent No. 150746 entitled "Preparation of porous collagen matrix", initially introduce collagen into a neutral solution at 30 to 45°C for a period of time to allow the collagen microfibrils to be reconstructed to obtain a gel-like collagen matrix. The gel-like collagen matrix is ​​frozen to an appropriate lower temperature at an appropriate temperature cooling rate, and then freeze-dried under reduced pressure to obtain a porous collagen matrix. In Taiwan patent No. I284665 entitled "Method for preparing porous collagen matrix from connective tissue", a porous treatment step is performed on the connective tissue that has been substantially treated in situ after the cleaning step to remove impurities and non-collagen from the connective tissue, wherein the porous treatment step is to obtain a collagen matrix with porous properties. The in situ treatment omits the conventional procedure of tearing or shredding the connective tissue, and the method does not require the use of a cross-linking agent. This method is more convenient for preparing a collagen matrix by omitting 2 or 3 processing steps, but does not record the porous structure and its bearing stress and tension.

[0018] Generally speaking, if there is a wound on the human body surface, gauze, bandages, patches and medical tapes are used to cover and bandage the wound to prevent external bacterial infection. However, the above external dressings can only inhibit external bacteria but have no direct help to the wound itself.

[0019] In summary, in order to solve the problem of preventing wounds from being infected by external bacteria and obtaining high-purity porous collagen, the present invention provides a biological scaffold having a non-reconstructed collagen matrix for wound dressing. Summary of the invention

[0020] The present invention provides a bioscaffold, including at least one non-reconstructed collagen matrix and at least one biomaterial, the biomaterial uniformly covers at least one surface or both the outer surface and the inner surface of the non-reconstructed collagen matrix. In addition, the present invention also discloses a method for preparing the aforementioned non-reconstructed collagen matrix and a bioscaffold, which has a higher strength than a traditional collagen matrix. In addition, since the bioscaffold provided by the present invention can be uniformly coated with a biomaterial layer, it is conducive to infiltrating cells into the non-reconstructed collagen matrix. In addition, the hair follicle tissue in the non-reconstructed collagen matrix can be destroyed without destroying the main structure of the dermal collagen, thereby making the non-reconstructed collagen matrix have a higher strength than the matrix prepared by conventional techniques. It also reduces the immune response caused by the hair follicles and helps cell growth, making the bioscaffold more suitable for further application in surgery, repair of human organs and tissues, regenerative medicine, skin transplantation or cosmetic medicine. It can also be used as a surgical repair mesh fabric, a dental repair mesh fabric, a human skin substitute, an artificial meningeal substitute, an orthopedic repair substitute or a wound dressing substitute.

[0021] The bioscaffold provided by the present invention comprises at least a non-reconstructed collagen matrix and at least one biomaterial. The non-reconstructed collagen matrix system has a plurality of bundled collagen fibrils and a plurality of holes formed by the bundled collagen fibrils. The biomaterial is coated on at least one outer surface of the main body and / or the outer surface of the bundled collagen fibrils. Since the plurality of holes are formed by the bundled collagen fibrils, that is, the collagen fibrils form the inner wall of the hole, the surface coated with the collagen fibrils can be equivalent to the inner wall of the coated hole.

[0022] The method for preparing a non-reconstructed collagen matrix may include: obtaining a dermis layer sheet from animal skin tissue; performing a depilatory step, the depilatory step including immersing the dermis layer sheet in a high ionic strength salt solution, and then adding a proteolytic enzyme to the salt solution, thereby producing a depilated dermis layer sheet; and performing a swelling step, the swelling step including immersing the depilated dermis layer sheet in a first acidic solution for a sufficient period of time to allow the depilated dermis layer sheet to form a swollen dermis layer, thereby forming a non-reconstructed collagen matrix, the non-reconstructed collagen matrix having a plurality of interwoven and overlapping bundles of collagen fibrils and a plurality of pores formed by the bundles of collagen fibrils.

[0023] Optionally, the method for preparing a non-reconstructed collagen matrix may further include a sterilization step, a defatting step, a deantigenization step and / or a strengthening step.

[0024] The method for preparing a bioscaffold may include: providing the non-reconstructed collagen matrix described herein; contacting the non-reconstructed collagen matrix with a solution containing a biomaterial under reduced pressure, thereby coating the biomaterial on a first outer surface of the non-reconstructed collagen matrix and inside a plurality of pores.

[0025] In summary, the preparation method of the present invention may further include: performing a disinfection step before or after any step, and washing the sheet or dermis sheet with a disinfection solution. Most preferably, the disinfection solution is a peracetic acid solution.

[0026] In an embodiment of the present invention, the preparation method provided by the present invention further comprises the following steps: first, taking out the non-reconstructed collagen matrix coated with the biomaterial; then, injecting the cell culture medium into the well; and then, injecting a solution containing cells so that the cells enter the well and adhere to the bioscaffold. Preferably, the cells can be stem cells, satellite cells, precursor cells or tissue cells.

[0027] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, the animal skin tissue is pig skin, cow skin or sheep skin tissue.

[0028] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, the salt solution containing high ionic strength is a salt solution in which anions and cations are ionically bonded. Preferably, the ionic strength of the salt solution containing high ionic strength is greater than 0.15N. Most preferably, the ionic strength of the salt solution containing high ionic strength is between 0.5N-10N. Most preferably, the salt solution containing high ionic strength is a solution containing sodium chloride, ammonium sulfate and a mixture of the above compounds. The high salt condition stabilizes the structure of the non-reconstructed collagen matrix, does not cause it to swell and squeeze into the hair follicles, and allows the enzyme to smoothly enter the hair follicles for reaction.

[0029] In one embodiment of the method for preparing non-reconstructed collagen matrix according to the present invention, proteolytic enzyme can act on a saline solution containing high ionic strength. Preferably, proteolytic enzyme is selected from the group consisting of: serine protease, threonine protease, cysteine ​​protease, aspartic protease, glutamic protease, metalloprotease and the combination of the above enzymes. Preferably, cysteine ​​enzyme is selected from the group consisting of: papaya enzyme (e.g., papaya enzyme), pineapple enzyme (e.g., bromelain) and a mixture of the above enzymes. Most preferably, metalloprotease is a neutral protease.

[0030] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, a degreasing step is further included before or after the depilatory treatment step. The degreasing step is a saponification treatment, an organic solvent treatment or a combination thereof. In the saponification step, an alkaline substance is used to contact the surface of the dermis layer. Preferably, the alkaline substance is in granular form and is a strongly alkaline particle. Most preferably, the strongly alkaline particle is a sodium hydroxide particle. The organic solvent treatment is an alcohol treatment, a hexane treatment or a chloroform treatment.

[0031] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, the depilatory treatment step or any subsequent step further comprises a vibration or agitation step. Preferably, the vibration or agitation step is an ultrasonic vibration treatment.

[0032] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, the depilatory treatment step or any subsequent step further comprises heating treatment. Optionally, the heating treatment is heating to 25°C to 40°C.

[0033] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, any step after the depilatory treatment step further includes a cleaning step, in which the sheet or dermis sheet is immersed in a cleaning solution or passed through a sterilizing gas. Preferably, the cleaning solution is a phosphate solution or an aqueous solution. Preferably, the sterilizing gas is ozone.

[0034] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, the first acidic solution is a weak acidic solution.

[0035] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, after the swelling treatment step, a de-antigen treatment step is also included. The de-antigen treatment step is to immerse the sheet or dermis sheet in a second acidic solution. The second acidic solution contains pepsin. Most preferably, the second acidic solution is a weakly acidic solution.

[0036] In one embodiment of the method for preparing a non-reconstructed collagen matrix according to the present invention, after the swelling treatment step, a strengthening treatment step is further included. The sheet or dermis layer sheet is immersed in an additive. Preferably, the additive is a cross-linking agent solution.

[0037] In one embodiment of the invention, the cross-linking agent is transglutaminase or acts through a functional group, wherein one functional group of the cross-linking agent is selected from the group consisting of: amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidate, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyl dithiol, hydrazide, photoreactive, carbodiimide, diaziridine, aziridine, acryloyl, arylate, thiol, gardenia, riboflavin, flavonoids and their derivatives, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidocaproic acid reactive Ester, disuccinimidyl suberate, bis(sulfosuccinimidyl suberate), N-hydroxy-succinimidyl ester (NHS-ester), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, pentafluorophenyl ester (PFP-ester), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, psoralen, vinyl sulfone, and combinations thereof.

[0038] In an embodiment of the present invention, the pore walls of the plurality of pores in the body include a structure in which bundles of collagen fibrils are interwoven and overlapped.

[0039] In one embodiment of the present invention, the bioscaffold comprises at least: a main body, the main body comprises a non-reconstructed collagen matrix; a biomaterial, the biomaterial is coated on at least the first surface of the main body; and a tissue adhesive layer, the tissue adhesive layer is on another surface of the main body opposite to the first surface, or on another surface adjacent to the first surface, or on two or more surfaces outside the first surface, wherein the tissue adhesive layer comprises a tissue adhesive. Wherein, when the bioscaffold is adhered to the tissue through the tissue adhesive layer, a plurality of cells move from the tissue adhesive layer to the biomaterial layer to reconstruct the tissue.

[0040] In one embodiment of the present invention, the biomaterial is selected from the group consisting of: cell attachment materials, tissue repair materials, cell induction materials, growth factor materials, antibacterial materials and mixtures thereof.

[0041] Preferably, the cell attachment material may be a sugar material, a peptide, a protein, a phospholipid or a mixture thereof.

[0042] Preferably, the sugar material may be a glycosaminoglycan material.

[0043] Preferably, the glycosaminoglycan material is selected from the group consisting of chondroitin, chondroitin sulfate, heparin, heparan sulfate, heparan sulfate proteoglycan, keratan, keratan sulfate, dermatan sulfate, carrageenan, hyaluronic acid, and combinations thereof.

[0044] Preferably, the tissue repair material may be a biocompatible material, an extracellular matrix, nutrients, and combinations thereof.

[0045] Preferably, the biocompatible material is selected from the group consisting of collagen, gelatin, elastin, glycosaminoglycans, chitosan, alginate, polyglutamic acid (γ-PGA), polylysine, poly(lactic-co-glycolic acid) (PLGA), silk fibroin, the like, and combinations thereof.

[0046] Preferably, the extracellular matrix is ​​selected from the group consisting of collagen, gelatin, elastin, glycosaminoglycans, proteoglycans, glycoproteins, fibronectin, laminin, aggrecan, metalloproteinases, etc. and combinations thereof.

[0047] Preferably, the nutrients are selected from the group consisting of vitamins, minerals, proteins, lipids, carbohydrates, amino acids, peptides, nucleotides, deoxynucleotides, herbs, etc. and combinations thereof.

[0048] Preferably, the cell inducing material is selected from the group consisting of molecules such as vitamins, minerals, compounds, drugs, herbs, metabolites, intermediate metabolites, sugars, peptides, proteins, phospholipids or combinations thereof.

[0049] Preferably, the antimicrobial material is an antibiotic, an antimicrobial protein or an antimicrobial peptide.

[0050] Preferably, the growth factor material is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF) , granulocyte macrophage colony stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial monocyte activation polypeptide, epithelial neutrophil activation peptide, erythropoietin, bone morphogenetic protein (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF) and their combinations.

[0051] In one embodiment of the present invention, the bioscaffold provided by the present invention further comprises a cell culture medium in the pores.

[0052] Preferably, it further comprises cells, wherein the cells adhere to any pore of the bioscaffold.

[0053] Preferably, the cell may be a stem cell, a satellite cell, a precursor cell or a tissue cell.

[0054] In one embodiment of the present invention, the reduced pressure environment is accomplished by evacuating the environment.

[0055] In one embodiment of the present invention, the tissue adhesive layer comprises: a cross-linking agent, which is a transglutaminase or at least contains a functional group; a matrix component, other than the non-reconstructed collagen matrix itself or the matrix molecule, which is cross-linked with the cross-linking agent; and a quencher, which is used to react with the functional groups of the cross-linking agent or with the remaining functional groups of the cross-linking agent to reduce cytotoxicity.

[0056] Preferably, the matrix molecule is selected from the group consisting of collagen, hyaluronic acid, gelatin, silk fibroin, fibronectin, elastin, tenascin, laminin, vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, aggrecan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen, fibrin, fibrinogen, thrombin, polyglutamic acid, polylysine, polyamino acids, synthetic polymers (e.g., acrylates, polylactic acid, polyglycolic acid, polylactic-glycolic acid), derivatives thereof, and combinations thereof.

[0057] Preferably, the quencher is selected from the group consisting of amino acids, oligopeptides, polypeptides, proteins, amines, diamines, oligoamines, polyamines, carbonyl compounds, glycol compounds, carboxyl compounds, dicarboxylates, oligocarboxylates, polycarboxylates, sulfhydryl compounds, oligosulfhydryl compounds, polysulfhydryl compounds, hydroxyl compounds, oligohydroxyl compounds, polyhydroxyl compounds, sugars, oligosaccharides, polysaccharides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotides, azide compounds, photocrosslinking compounds, and groups consisting of monofunctional groups or heterobifunctional groups, and combinations thereof.

[0058] Preferably, the tissue is skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a schematic cross-sectional view showing a non-remodeled bioscaffold according to an embodiment of the present invention.

[0060] Figure 2 is a schematic cross-sectional view showing the main body of a non-reconstructable bioscaffold according to an embodiment of the present invention.

[0061] Figure 3 Electron microscopy image at 100× magnification showing a conventional reconstructed porous collagen matrix.

[0062] Figure 4 A flow chart showing the present invention for preparing a non-remodeled collagen matrix.

[0063] Figure 5 Another flow chart showing the present invention for preparing a non-remodeled collagen matrix.

[0064] Figures 6A to 6C An electron microscope image showing a non-remodeled collagen matrix prepared by the method of the present invention.

[0065] Figures 7A to 7B Electron microscope image showing a bioscaffold according to an embodiment of the present invention.

[0066] Figure 7C It showed that HA indicated by Alcian blue staining was well distributed inside the bioscaffold.

[0067] Figure 8 A graph showing a comparison of shear stress between a bioscaffold according to an embodiment of the present invention and a bioscaffold of conventional technology.

[0068] Fig. 9A and 9B The storage modulus and loss modulus of the non-reconstructed porous collagen matrix of the present invention and the reconstructed porous collagen matrix of the conventional technology are shown.

[0069] Fig. 10A The figure shows the comparison of the wound healing effect of the bioscaffold of the present invention and the reconstructed porous collagen-chondroitin sulfate matrix prepared by a conventional method.

[0070] Fig. 10B are graphs showing wound healing under different treatments.

[0071] Fig.11 A set of microscopic images showing the comparison of wound healing conditions at 28 days after surgery. (A) Normal skin control, (B) healing matrix of the PCHM group, and (C) healing matrix of the Integra group.

[0072] Fig.12 is a flow chart showing a method for preparing a bioscaffold in an embodiment of the present invention.

[0073] Figures 13A to 13B 1 is a schematic cross-sectional view and a partial enlarged view thereof showing a bioscaffold according to another embodiment of the present invention.

[0074] Figures 14A to 14C 4 is a comparative diagram of the water absorption capacity of bioscaffolds coated with different degrees of biomaterials according to an embodiment of the present invention.

[0075] Figures 15A to 15D The antibacterial effect of the bioscaffolds covered with different antibacterial materials according to the embodiments of the present invention is shown. DETAILED DESCRIPTION

[0076] refer to Figure 1 The present invention provides a non-reconstructive material bioscaffold 1, which includes: a main body 10, a biomaterial layer 20, and a tissue adhesive layer 30. The main body 10 includes at least one non-reconstructive collagen matrix 10a. The biomaterial layer 20 and the tissue adhesive layer 30 are coated on one surface of the main body and the adjacent or opposite surface of the main body, respectively. When the above-mentioned bioscaffold is adhered to the tissue 2 through the tissue adhesive layer, a plurality of cells are chemically attracted by the biomaterial and move from the tissue adhesive layer to the biomaterial layer, and the tissue 2 is reconstructed.

[0077] refer to Figure 2 , Figure 2 A schematic cross-sectional view of a main body 10 of a bioscaffold according to an embodiment of the present invention is shown. As shown in the figure, the main body 10 is preferably a non-reconstructed collagen matrix 10a, and the collagen matrix 10a is composed of a plurality of columnar collagen fibrils and includes a plurality of pores 10b composed of collagen fibrils. The biomaterial 10c is coated on the inner surface of the pores 10b.

[0078] refer to Figure 4 The wound dressing is prepared from the bioscaffold having the non-reconstructed collagen matrix of the present invention, comprising: first, as shown in step S100, providing animal tissue or animal skin tissue, such as pig skin, cow skin or sheep skin tissue, in an environment below 15°C, removing subcutaneous fat of the animal skin tissue by a grinder, and cutting the tissue into a size of 28±1cm×70±1cm; then, depilating the animal skin tissue, scraping off the grease and moisture remaining thereon, and then cutting each piece of skin tissue into 4 pieces, each piece having a size of 13±1cm×34±1cm; then, peeling the dermis layer of the pig skin by a machine to obtain at least one dermis layer sheet, and then rinsing with pure water. Most preferably, the dermis layer is taken from pig skin.

[0079] In addition, as shown in step S102, the depilatory treatment step is performed by immersing the dermis layer sheet in a salt solution containing high ionic strength, and after adding a proteolytic enzyme, the salt solution is heated to 25-40°C. Then, an ultrasonic oscillation treatment is performed for two hours. A phosphate buffer solution is used as a cleaning solution to purify the dermis layer sheet after the depilatory treatment. The salt solution containing high ionic strength is a salt solution in which anions and cations are ionically bonded. The ionic strength of the salt solution containing high ionic strength is greater than 0.15N. Most preferably, the ionic strength of the salt solution containing high ionic strength is between 0.5N and 10N. Most preferably, the salt solution containing high ionic strength is a solution containing sodium chloride, ammonium sulfate, or a mixture of the above compounds.

[0080] In one embodiment, the salt solution containing high ionic strength is a phosphate buffer solution containing sodium chloride. The high salt condition stabilizes the structure of the non-remodeled collagen matrix and prevents the tissue from swelling and squeezing into the hair follicles, so that the enzyme can smoothly enter the hair follicles for reaction.

[0081] In one embodiment, the proteolytic enzyme acts on a salt solution containing high ionic strength. Most preferably, the proteolytic enzyme is selected from the group consisting of: serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, glutamic proteases, metalloproteases and combinations of the above enzymes. Most preferably, the cysteine ​​protease is selected from the group consisting of: papaya enzymes, pineapple enzymes and mixtures of the above enzymes. Most preferably, the metalloprotease is a neutral protease.

[0082] Referring to Table 1 below, when the dermis slices were immersed in a phosphate buffer solution of papain (an enzyme from papaya) containing ~5mM or 0.16% (w / w) L-cysteine, and the concentration of papain was higher than 0.6mg / mL (Groups B, C and D)), the pig hair was completely removed. When the dermis slices were immersed in a phosphate buffer solution of bromelain (an enzyme from pineapple), and when the bromelain concentration was 0.2mg / mL and 0.6mg / mL (Groups A and B), there were still pig hairs that did not fall off. When the concentration of bromelain was 1.2mg / mL, the pig hair was completely removed. Neutral protease exceeding 0.2U / mL can also completely remove pig hair follicles. For the pigs in the control group (phosphate buffer only), all pig hair did not fall off.

[0083] Table 1. Dermal slices immersed in phosphate buffered saline containing papain, bromelain or neutral protease (Groups A-D)

[0084] refer to Figure 4 As shown in step S104, a swelling treatment step is performed, in which the depilated dermis layer is immersed in a first acidic solution, and the first acidic solution is heated to 33-37° C. and ultrasonic oscillation treatment is performed at the same time. The ultrasonic oscillation treatment time lasts for 24 hours to expand the volume of the dermis layer by at least 50%. Preferably, the first acidic solution is a weak acid solution, and the weak acid solution is selected from the group consisting of: formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, hydrochloric acid and mixtures thereof. Most preferably, the first acidic solution is an acetic acid solution.

[0085] As shown in step S105, a cleaning step is performed to remove non-collagenous materials from the dermis and obtain a purified collagen matrix. The purified collagen matrix is ​​then frozen at -20°C and -80°C and subjected to freeze drying or lyophilization to obtain a purified non-reconstructed porous collagen matrix.

[0086] refer to Figure 5 , Figure 5Another embodiment of the method for preparing the non-reconstructed collagen matrix of the present invention is shown, comprising: as shown in step S101a, performing a disinfection treatment before or after any step, and washing the sheet or dermis sheet with a disinfection solution during the disinfection treatment. Most preferably, the disinfection solution is a peracetic acid solution.

[0087] As shown in step S102a, the preparation method of the present invention further includes: a degreasing step before or after the depilatory treatment step S102. The degreasing step is a saponification treatment, an organic solvent treatment or a combination thereof. The saponification step is to contact the sheet or dermis sheet with an alkaline substance, and if only one surface has grease, the one specific surface can be contacted. Most preferably, the alkaline substance is a strongly alkaline particle in a granular form. Most preferably, the strongly alkaline particle is a sodium hydroxide particle. The organic solvent treatment is an alcohol treatment, a hexane treatment or a chloroform treatment. However, an optimal amount and treatment time period are required to prevent the denaturation of collagen in the matrix.

[0088] As shown in step S104a, the preparation method of the present invention further includes: a de-antigen treatment step after the swelling treatment step S104. Most preferably, the de-antigen treatment step is to immerse the sheet or dermis layer sheet in a second acidic solution containing pepsin. The second acidic solution containing pepsin is heated to between 33°C and 37°C. Then an ultrasonic oscillation treatment is performed for 4 hours. The solution remaining on the sheet or dermis layer sheet is washed off with pure water. The sheet or dermis layer sheet is treated again with potassium dihydrogen phosphate at 25°C. Preferably, the second acidic solution is a weak acid solution, and the weak acid solution is selected from the group consisting of: formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, hydrochloric acid and mixtures thereof. Most preferably, the second acidic solution is an acetic acid solution.

[0089] As shown in step S104b, the preparation method of the present invention further includes a strengthening treatment step after the swelling treatment step, wherein a plurality of the sheets or dermis sheets are laminated and immersed in an additive (preferably a crosslinker solution). The crosslinker solution is heated to between 33°C and 37°C, and subjected to ultrasonic oscillation treatment for 24 hours to complete the crosslinking reaction. The residual solution on the sheet or dermis sheet is rinsed off with pure water. Most preferably, a functional group of the crosslinker is selected from the group consisting of: amine, sulfhydryl, carbonyl, ethylene glycol, hydroxyl, carboxyl, azide, imidate, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridine dithiol, hydrazide, photoreaction, carbodiimide, diaziridine, aziridine, acryloyl, arylate, thiol, gardenia, riboflavin, flavonoids and their derivatives, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidocaproic acid active ester, disuccinimidyl suberate, disuccinimidyl suberate, (sulfosuccinimide) esters, N-hydroxy-succinimide esters (NHS-esters), sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, pentafluorophenyl esters (PFP-esters), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, chlorambucil-N-hydroxysuccinimide esters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, psoralen, vinyl sulfones, and combinations thereof.

[0090] refer to Figures 6A to 6C , which show the appearance of a non-remodeled collagen matrix prepared according to the above method. Fig. 6A is a 100× magnified scanning electron microscope image of a non-remodeled collagen matrix according to an embodiment of the present invention. Figure 6B is a 400× magnified scanning electron microscope image of a non-remodeled collagen matrix according to an embodiment of the present invention. Figure 6C is a scanning electron microscopic image at 10000× magnification of a non-remodeled collagen matrix according to an embodiment of the present invention.

[0091] Compared with the porous collagen matrix prepared by the prior art (see Figure 3 ), the non-reconstructed porous collagen matrix prepared by the present invention is composed of a plurality of interlaced and aggregated collagen fibril bundles, and has a plurality of holes with depth and groove walls. The above-mentioned collagen fibril bundles are composed of original collagen molecules and have a diameter of less than 100nm, but the present invention is not intended to be limited thereto. The appearance of the hole is a random radius opening, and the pore size is controlled in a narrow range. Therefore, the collagen matrix prepared by the method described herein has a relatively uniform pore size.

[0092] Please refer to Figures 7A to 7C, which show the appearance of a bioscaffold made from a non-remodeled collagen matrix prepared according to the method described above. Fig. 7A is a 100× magnified scanning electron microscope image of a bioscaffold according to an embodiment of the present invention. Figure 7B is a 400× magnified scanning electron microscope image of a bioscaffold according to an embodiment of the present invention. Figure 7C : is an image of an optical micrograph of a bioscaffold in which hyaluronic acid in an embodiment of the present invention is pre-activated by CNBr and cross-linked with a non-reconstructed collagen matrix. After sectioning the histological sample, the presence of hyaluronic acid derivatives inside the scaffold was stained with Alcian blue. The image shows that the hyaluronic acid derivatives are well distributed inside the bioscaffold.

[0093] refer to Figure 8 , Figure 8 The shear stress test of the non-reconstructed porous collagen matrix (line B) according to the embodiment of the present invention and the conventional porous collagen matrix (line A) obtained by the prior art is shown. From the results, it can be seen that the shear stress of the porous collagen matrix obtained by the prior art is 63.56e-03MPa at a temperature of 20°C, while the shear stress of the non-reconstructed porous collagen matrix of the present invention is 189.07MPa. It is thus proved that under the same test conditions, the shear strength of the non-reconstructed porous collagen matrix of the present invention is about 3 orders of magnitude higher than the shear strength of the porous collagen matrix of the prior art. Since the hair follicles can be completely removed at the beginning, the non-reconstructed porous collagen matrix is ​​indeed superior to the conventional porous collagen matrix in terms of structural strength and tension. In summary, the present invention provides a method for preparing a non-reconstructed porous collagen matrix, which can destroy the hair follicle tissue without destroying the natural cross-linking of collagen in the dermis, and the structural strength of the porous matrix prepared thereby is higher than the structural strength of the porous matrix prepared by the prior art.

[0094] refer to Fig. 9A and 9B , they show the changes in storage modulus and loss modulus monitored by a rheometer as a function of oscillatory strain. The storage modulus is an indicator of the ability of a matrix to store deformation energy in an elastic manner. This is directly related to the degree of cross-linking, with the higher the degree of cross-linking, the greater the storage modulus. Fig. 9A is the data of the non-reconstructed porous collagen matrix of the present invention, and Fig. 9BData for a conventional remodeled porous collagen matrix are shown. Microstructure implies the presence of forces between molecules or particles in the material. To destroy the microstructure, a force greater than the force that maintains the microstructure is required. When the applied force is less than the molecular or inter-particle forces, G' is greater than G"; the material has some ability to store energy and should be able to return to some degree to the initial configuration prior to the application of the mechanical force. The material behaves as an elastic solid, although not an ideal one because some mechanical energy is dissipated. Fig. 9A and 9B The results in show that the storage modulus of the non-reconstructed porous collagen matrix is ​​higher than that of the reconstructed porous collagen matrix prepared by the prior art. Most of the native crosslinks in the non-reconstructed porous collagen matrix are retained, while most of the native crosslinks in the conventional reconstructed porous collagen matrix are lost.

[0095] In addition, since the non-reconstructed collagen matrix 10a itself is not a hydrophilic material, the main body 10 may further include a hydrophilic biomaterial 10c coated on the outer surface of the collagen fibrils. That is, the inner walls of the holes 10b are also coated with the hydrophilic biomaterial 10c (such as Figure 2 The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c. The hydrophilic biomaterial 10c is preferably a hydrophilic biomaterial 10c.

[0096] Refer to Figure 1In a preferred embodiment of the present invention, the growth factor material layer 20 coated on one surface of the main body 10 is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (GCSF), Cell colony stimulating factor (MCSF), granulocyte macrophage colony stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial monocyte activation polypeptide, epithelial neutrophil activation peptide, erythropoietin, bone morphogenetic protein (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF) and combinations thereof.

[0097] In addition, although tissue adhesives used in clinical medicine and tissue engineering can achieve the effects of controlling drug release, regulating the decomposition of biocompatible materials or promoting vascular bonding by virtue of their adhesive properties, conventional tissue adhesives are made of cross-linking agents such as glutaraldehyde (GA), glyoxal, formaldehyde or sodium tripolyphosphate (TPP) and cross-linked with carrier materials such as gelatin, chitosan or collagen, the cross-linking agents are cytotoxic and easily cause strong and persistent immune responses, making the tissue adhesives cytotoxic.

[0098] Therefore, in the present invention, a tissue adhesive layer 30 having good bonding strength and low cytotoxicity is used, which comprises at least one cross-linking agent, at least one matrix component and a quencher. The cross-linking agent is transglutaminase or has a functional group, and the matrix component is used for cross-linking with the cross-linking agent. The quencher is used to react with the functional group of the cross-linking agent to reduce cytotoxicity.

[0099] In a preferred embodiment, the cross-linking agent is transglutaminase, or one of its functional groups is selected from the group consisting of reactive compounds including amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyl dithiol, hydrazide, photoreactive, carbodiimide, diaziridine, aziridine, acryloyl, arylate, thiol, gardenia, riboflavin, flavonoids and their derivatives, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidocaproic acid active ester, octane Disuccinimidyl diacid esters, bis(sulfosuccinimidyl) suberate, N-hydroxy-succinimidyl esters (NHS-esters), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, pentafluorophenyl ester (PFP-ester), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, psoralen, vinyl sulfone, and combinations thereof.

[0100] In a preferred embodiment, the matrix component is selected from the group consisting of collagen, hyaluronic acid, gelatin, silk fibroin, fibronectin, elastin, tenascin, laminin, vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, aggrecan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen, fibrin, fibrinogen, thrombin, polyglutamic acid, polylysine, polyamino acids, synthetic polymers (e.g., acrylates, polylactic acid, polyglycolic acid, polylactic-glycolic acid), their derivatives and their combinations.

[0101] More preferably, matrix component can be prepared by any naturally occurring collagen or its functional variant and / or hyaluronic acid. In addition, it has been shown that hyaluronic acid (particularly high molecular weight hyaluronic acid (i.e. greater than 5kDa)) can effectively promote angiogenesis and then promote wound healing. For example, the hyaluronic molecular weight can be 50kDa to 5000kDa, 70kDa to 1500kDa, 200kDa to 1500kDa, 500kDa to 1500kDa or 700kDa to 1500kDa. When cell grows in collagen or hyaluronic matrix, cell has good survival ability. Therefore, in the matrix component of the tissue adhesive layer 30 used in the present invention, the concentration of hyaluronic acid is 0.001mg / mL to 100mg / mL, and the concentration of collagen is 0.001mg / mL to 100mg / mL. Preferably, the concentration of collagen is 0.1mg / mL to 100mg / mL, and the concentration of hyaluronic acid is 0.01mg / mL to 35mg / mL. More preferably, the concentration of collagen is 3mg / mL to 75mg / mL (such as 6mg / mL or 9mg / mL), and the concentration of hyaluronic acid is 0.2mg / mL to 20mg / mL.

[0102] In a preferred embodiment, the quencher is selected from the group consisting of amino acids, oligopeptides, polypeptides, proteins, amines, diamines, oligoamines, polyamines, carbonyl compounds, glycol compounds, carboxyl compounds, dicarboxylates, oligocarboxylates, polycarboxylates, sulfhydryl compounds, oligosulfhydryl compounds, polysulfhydryl compounds, hydroxyl compounds, oligohydroxyl compounds, polyhydroxyl compounds, sugars, oligosaccharides, polysaccharides, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), oligonucleotides, azides, photocrosslinking compounds, monofunctional groups or heterobifunctional groups, and combinations thereof. More preferably, the quencher used in the present invention includes spermine, protamine, 1-6 hexanediamine and polylysine, which have different groups with different molecular weights. The average molecular weights of polylysine are 3.4kDa, 20kDa, 99kDa, 212kDa and 225kDa, respectively.

[0103] Fig. 10A It shows that implantation Comparison of healing of full-thickness wounds in the dorsal skin of guinea pigs with artificial skin or PCMH. Artificial skin is a reconstructed porous collagen-chondroitin sulfate matrix prepared by a conventional method of dehydrothermal cross-linking. PCHM is a bioscaffold of the present invention, consisting of a non-reconstructed porous collagen matrix and hyaluronic acid coated on the outer and inner surfaces of the matrix. The stable porous structure of PCHM allows dermal cells to infiltrate up to 4 times better than the dermal cells of the control group. Therefore, the healing rate of wounds filled with PCHM was shown to be much faster than that of the wounds in the Integra group (about 80%). At the fourth month after surgery, the histology of the regenerated matrix in the PCHM group also showed much better healing quality than the control group.

[0104] refer to Fig.11 , on postoperative day 28, the bioscaffolds of PCHM degraded within two weeks compared with normal skin, and the healing matrix of the PCHM group was essentially replaced by new matrix, e.g. Fig.11 B indicates histology. Fig.11 As shown in C, some fragments of the Integra artificial skin were observed, which interfered with the regeneration process of wound healing.

[0105] refer to Fig.12 , as shown in step S106 to step S109, the non-reconstructed collagen matrix is ​​contacted with a solution containing a biomaterial, and the non-reconstructed collagen matrix is ​​provided in a reduced pressure environment. In one embodiment, one end of the non-reconstructed collagen matrix is ​​placed in a solution containing at least one biomaterial. A reduced pressure environment is established at the other end of the non-reconstructed collagen matrix so that the biomaterial can be absorbed into the structure of the non-reconstructed collagen matrix to allow the biomaterial to be evenly coated on the outer surface and inner surface of the bundled collagen fibrils (it can also be considered to be evenly coated on the inner wall of the hole), as shown in step S108. It must be noted that due to the extremely small porous diameter of the non-reconstructed collagen matrix, it is impossible to avoid air residue by simply immersing the non-reconstructed collagen matrix in the solution. Therefore, in the present invention, the siphon principle is used to indirectly remove air so that the biomaterial can completely cover the outer surface of the bundled collagen fibrils (it can also be considered the inner wall of the hole). In addition, the above-mentioned reduced pressure environment is preferably carried out by vacuuming.

[0106] The above-mentioned biomaterial is selected from the group consisting of: cell attachment material, tissue repair material, cell induction material, growth factor material, antibacterial material and their combination. Preferably, the above-mentioned cell attachment material is sugar, peptide, protein, phospholipid or their combination. Preferably, the sugar material is a glycosaminoglycan material. Preferably, the glycosaminoglycan material is selected from the group consisting of: chondroitin, chondroitin sulfate, heparin, heparan sulfate, heparan sulfate proteoglycan, keratan, keratan sulfate, dermatan sulfate, carrageenan, hyaluronic acid and their combination. Preferably, the biocompatible material used in the present invention is selected from the group consisting of: collagen, elastin, glycosaminoglycan, chitosan, alginate, polyglutamic acid (γ-PGA), polylysine, poly (lactic acid-co-glycolic acid) (PLGA), silk protein and fibroin, but the present invention is not intended to be limited thereto.

[0107] Preferably, the cell inducing material is selected from the group consisting of molecules such as vitamins, minerals, growth factors, compounds, drugs, herbs, metabolites, intermediate metabolites, sugars, peptides, proteins, phospholipids or combinations thereof.

[0108] Preferably, the growth factor material is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF ), granulocyte macrophage colony stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial monocyte activation peptide, epithelial neutrophil activation peptide, erythropoietin, bone morphogenetic protein (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and combinations thereof.

[0109] Preferably, the antimicrobial material is an antibiotic, an antimicrobial protein, an antimicrobial peptide or a combination thereof.

[0110] For detailed structure of non-remodeled collagen matrix please refer to Figure 2 , Figure 21 is a schematic cross-sectional view of a bioscaffold according to an embodiment of the present invention. As shown above, the non-reconstructed collagen matrix 10a is composed of a plurality of bundled collagen fibrils including a plurality of pores 10b. The biomaterial 10c is absorbed into the non-reconstructed collagen matrix 10a by the siphon principle and is coated on the outer surface of the bundled collagen fibrils. That is, the inner wall of the pore 10b is completely coated with the biomaterial 10c, which is more conducive to the cells entering the pores and adhering to the biomaterial for subsequent applications.

[0111] In a preferred embodiment of the present invention, although Figure 2 Not shown, after the biomaterial is coated on the outer surface of the bundled collagen fibrils, cells can be introduced into the bioscaffold. Cell culture medium or a solution containing cells is injected into the hole 10b so that the cells enter the pores of the non-reconstructed collagen matrix. That is, in a preferred embodiment of the present invention, the bioscaffold provided by the present invention also includes cells, such as Fig.13A Reference Fig. 13B , Fig. 13B yes Fig.13A A partial enlarged view of the dotted circle in the middle, where cells 30 are attached to the biomaterial 10c in the hole 10b. Preferably, the cells may be stem cells, satellite cells, precursor cells, progenitor cells or tissue cells, and the tissue cells may further include skin tissue, cartilage tissue or adipose tissue, and the like.

[0112] Table 2 below shows a comparison of the number of attached cells in different fields of view between a non-reconstructed collagen matrix without biomaterials and a non-reconstructed collagen matrix with biomaterials. As can be seen from Table 2, the non-reconstructed collagen matrix itself is a hydrophobic material, which is not conducive to cell attachment to the pores. However, when the present invention also coats the surface of the non-reconstructed collagen matrix with a cell attachment material, it is shown to be conducive to subsequent cells entering these pores and attaching to the bioscaffold.

[0113] Table 2

[0114]

[0115] Next, the water absorption of the non-reconstructed collagen matrix without biomaterial (A1), the non-reconstructed collagen matrix coated (once) with hyaluronic acid (A2), and the non-reconstructed collagen matrix coated (five times) with hyaluronic acid (A3) were further compared. FIG. 14A to FIG. 14C These three figures show the water absorption conditions of A1, A2 and A3 respectively. Therefore, it is proved that the non-reconstructed collagen matrix coated with more layers of biomaterials has better water absorption capacity.

[0116] The effect of the biological scaffold used in wound dressing provided by the present invention is confirmed. Fig. 10B and Fig.11 . Fig. 10B The wound healing time of different bioscaffolds was compared. Fig.11 The wound healing profiles of different bioscaffolds were compared.

[0117] first, Fig. 10B The different treatments of wounds are shown, which are divided into groups A, B, C, D and E to observe the relationship between wound area and healing time. Among them, the initial wound area is 100%, and the wound area is measured every two days until the wound is completely healed. A represents untreated wounds, B represents wounds treated with porous collagen matrix (PCM), C represents wounds treated with CNBr activated hyaluronic acid (aH) combined with porous collagen matrix (PCM-aH), D represents wounds treated with PCM-aH with additional 5 mg / mL hyaluronic acid, and E represents wounds treated with Wounds treated with artificial skin. As shown in the figure, the wounds of Groups B, C, and D all healed in about 30 days, but the wound treated with artificial skin (Group E) took 44 days to heal. Although Group A healed slightly faster, it showed wound contraction and other conditions.

[0118] like Fig. 10B As shown in the figure, after 12 days, the proliferation of fibroblasts in group C (wounds treated with PCM-aH) was indeed higher than that in group E (wounds treated with artificial skin). This proves that the above-mentioned main body 10 combined with biomaterials can effectively promote cell proliferation without causing wound contraction, and can make wounds heal faster than artificial skin of conventional technology.

[0119] Then, in another embodiment, Figures 15A-15D As shown, bioscaffolds are coated with different antimicrobial materials, e.g. Fig.15A and 15B The antimicrobial peptide Pexiganan is coated with Fig. 15C and 15D The results showed that the antibacterial effect was concentration-dependent, and the bioscaffold coated with antibacterial materials had a good antibacterial effect.

[0120] In summary, the object of the present invention is to provide a bioscaffold that can be used for wound dressings. In one embodiment, one side of the main body is coated with a biomaterial layer, and the other side is coated with a tissue adhesive layer, which allows cells to move within the main body based on the effect of concentration gradient. In addition, since the main body contains hydrophilic biomaterials, it can also help the movement and attachment of cells. Therefore, in addition to effectively blocking foreign bacterial invasion to prevent wound infection, the bioscaffold provided by the present invention can also be used to accelerate cell growth and promote wound healing speed.

[0121] In addition, although it is known in the conventional art to add chopped collagen to a biomaterial (e.g., hyaluronic acid) and then cross-link them to condense into a structure, the exterior of the bioscaffold is not completely hydrophilic due to the interweaving of the collagen and the biomaterial. Figure 3 As shown), and then the method provided by the present invention is carried out, the supporting force of the reconstructed collagen matrix will be insufficient (see Figure 9 and the above related description) to withstand the external pressure applied by the vacuum step, which is required for the subsequent coating of the scaffold with biomaterials. As described above, the application of the siphon principle allows the biomaterial to be completely absorbed or introduced into the non-reconstructed collagen matrix and evenly coat the surface, so that the surface of the bioscaffold is conducive to cell infiltration and attachment. The bioscaffold can be widely used in surgical operations, repair of human organs and tissues, regenerative medicine, skin transplantation or cosmetic medicine, and can also be used for surgical repair mesh fabrics, dental repair mesh fabrics, human skin substitutes, artificial meninges substitutes, orthopedic repair substitutes or wound dressing substitutes.

[0122] The above detailed description is a specific description of some feasible embodiments of the present invention, but these embodiments are not intended to limit the scope of the invention. Any equivalent implementation or change that does not depart from the technical spirit of the present invention should be included in the scope of the invention. All publications cited herein are incorporated herein by reference in their entirety.

[0123]

Explanation of symbols

[0124] 1Biological scaffold

[0125] 10 Main Body

[0126] 10a Non-remodeled collagen matrix

[0127] 10b hole

[0128] 10c Biomaterials

[0129] 20 biomaterial layers

[0130] 30 tissue adhesive layers

[0131] 2 Organization

[0132] S100-S105 Method for preparing non-reconstructed collagen matrix

[0133] S100-S104b Method for preparing non-reconstructed collagen matrix

[0134] Preparation of S106-S109 bioscaffolds

Claims

1. A method for preparing a non-reconstructed collagen matrix, comprising: Dermal sheets were obtained from animal skin tissue; performing a depilatory step, the depilatory step comprising immersing the dermis layer sheet in a saline solution and then adding a proteolytic enzyme to the saline solution, thereby producing a depilated dermis layer sheet; performing a swelling step, the swelling step comprising immersing the depilated dermis layer sheet in a first acidic solution for a sufficient period of time to expand the volume of the depilated dermis layer sheet by at least fifty percent to form a swollen dermis layer, thereby forming the non-reconstructed collagen matrix, the non-reconstructed collagen matrix having a plurality of interwoven and overlapping bundles of collagen fibrils and a plurality of pores formed by the bundles of collagen fibrils; and performing a de-antigenization step, the de-antigenization step comprising immersing the non-reconstructed collagen matrix in a second acidic solution containing pepsin, The ionic strength of the salt solution is greater than 0.15N.

2. The method of claim 1, wherein the ionic strength of the salt solution is between 0.5N and 10N.

3. The method of claim 1, wherein the salt solution is a phosphate solution containing sodium chloride, ammonium sulfate or a mixture thereof.

4. The method of claim 1, wherein the salt solution is a phosphate buffer solution containing sodium chloride.

5. The method of claim 1, wherein the proteolytic enzyme is selected from the group consisting of serine proteases, threonine proteases, cysteine ​​proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and mixtures thereof.

6. The method of claim 5, wherein the cysteine ​​protease is selected from the group consisting of papain, bromelain and a mixture thereof; and the metalloprotease is a neutral protease.

7. The method of any one of claims 1 to 6, further comprising a sterilization step.

8. The method according to any one of claims 1 to 6, further comprising a degreasing step before or after the depilatory step.

9. The method of any one of claims 1 to 6, further comprising a strengthening step after the swelling step, the strengthening step comprising immersing the swollen dermis sheet in an additive.

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