Biological scaffold and its manufacturing method

Abstract

The biological scaffold of the present invention comprises a body, a biological material layer, and optionally a tissue adhesive layer. The body has at least an unreconstituted collagen matrix. The biological material layer is coated on at least one surface of the body, and the tissue adhesive layer is disposed on at least another surface of the body. When the biological scaffold is attached to tissue through the tissue adhesive layer, a plurality of cells migrate from the tissue into either the adhesive layer or the biological material layer, thereby performing tissue repair / regeneration.

Description

[Technical field]

[0001] The present invention provides a biological scaffold, and a method for its manufacture, which is comprised of a non-reconstituted collagen matrix and a biological material, and optionally a tissue adhesive layer. More specifically, such a non-reconstituted collagen matrix and such a biological scaffold are useful as a dressing for promoting wound healing. [Background technology]

[0002] Tissue engineering refers to the reconstruction or repair of organs and tissues through in vitro culture or construction techniques using synthetic or bioactive materials. Tissue engineering involves multiple fields such as biology, material 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," consists of the following steps. First, cells are taken from the human body and cultured in vitro until they reach a sufficient number. Then, the cells are placed in an artificial scaffold and allowed to grow. Sometimes, some chemicals or biological factors need to be added to promote cell differentiation. Finally, the artificial tissue is placed into the patient. Thus, the three elements of tissue regeneration are cells, biological scaffolds, and growth factors.

[0003] Among them, the most basic structural and functional unit of the human body is the cell. Between the cells, there exists the extracellular matrix formed by the cells themselves. This is a substance that contains structural components such as protein fibrils / bundles (e.g., collagen), crystalline inorganic minerals, etc.

[0004] Tissues consist of many cells with similar morphology and function, which are connected to each other through the extracellular matrix. Among them, they are divided into different tissues according to different cell types, such as epithelial tissue, connective tissue, muscle tissue, and nervous tissue. Among them, connective tissue is composed of a large number of extracellular matrices and cells scattered therein. The extracellular matrix contains matrix, fibrils, tissue fluid, etc. Collagen is the most important structural protein in animal connective tissues and the most important component in the extracellular matrix. Animal connective tissues contain 20-30% collagen in addition to 60-70% water. Therefore, collagen is a protein widely present in animals, while connective tissues are widely distributed in the human body and found in almost all organs.

[0005] Collagen exists mainly in the form of insoluble fibrils and accounts for about 33% of the proteins in human tissues. In other words, collagen accounts for one-third of the proteins in the human body and is a very important structural protein of connective tissues. It plays the role of a "mattress" and "cement" that protects and connects various tissues to support the structure of the human body. Therefore, collagen is often used as a material for bioprostheses.

[0006] The most basic unit of collagen is tropocollagen, a molecule composed of three polypeptide chains. The three polypeptide chains are tightly bound in parallel by interchain hydrogen bonds to form a stable triple helix structure. The triple helix collagen unit is called tropocollagen and is about 1.5 nm in diameter and about 280 nm in length. Under physiological conditions, multiple tropocollagen molecules self-aggregate into collagen microfibril structures, with each molecule offset by a distance of about 67 nm. The microfibrils can then aggregate into fibrils of various diameters, usually about 50 nm. In the body, the fibrils can stack into different quaternary structures to form reticular fibrils, such as the matrix beneath epidermal cells. These fibrils can further aggregate into fibers such as ligaments and tendons, which contain collagen fibrils regularly arranged in one direction.

[0007] Animal collagens discovered so far can be classified into at least 28 types. Except for hyaline cartilage, which is mainly type II collagen, most connective tissues mainly contain type I collagen, accounting for about 90% of the total collagen content, and it is also the most widely used collagen biomaterial. Its main structural protein provides connective tissue with the support, protection, and various mechanical properties required for connective tissue, such as tension, tensile 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.

[0008] The method of preparing a conventional collagen matrix, such as the method of preparing a porous collagen matrix in Chinese Patent No. 476642, is to reconstitute the collagen matrix at 30-45°C under similar physiological conditions. A porous collagen matrix can be obtained by freezing and lyophilizing such a matrix. Figure 3 shows an electron micrograph of a conventional porous collagen matrix at 100x magnification. Its appearance is loose and irregularly flaky, with a structure that does not provide better tensile strength and anti-cell contractility, which are required to optimize proper cell migration and proliferation. In addition, the surface properties of collagen also have a negative effect on cell infiltration.

[0009] Another prior art such as US Patent No. 5,997,895 "Dural / meningeal repair product using collagen matrix" provides a method for repairing and promoting meningeal growth. A tested and biocompatible collagen source without pathogenic virus residues can be used. A cross-linking reaction is used in the collagen matrix to form a porous collagen structure with a pore size of 10-500 μm, which can repair the damaged meninges and promote meningeal growth. However, since collagen is harvested from the dermal layer of animal skin such as pig skin, if the hair follicles are not completely removed during the processing of pig skin, the overall structure of the subsequent collagen matrix preparation will be collapsed. It will also affect the purity of the prepared collagen, which may cause bacterial infection and immune reaction or immune rejection in clinical use.

[0010] Taiwan Patent No. 098125217, entitled "Preparation of high purity collagen", 2 ~2m 2 The present invention provides a method for preparing collagen, comprising: providing a connective tissue having a surface area of ​​100 mm; expanding the volume of the connective tissue by at least 50% using a substantially salt-free first acid solution having a pH value of 1-6 to form a swollen connective tissue; washing the swollen connective tissue to remove non-collagenous material to form a collagen matrix; and extracting collagen from the collagen matrix using an extraction solution to create a collagen-containing solution. In the aforementioned technology, the solution is collagen, and the porous collagen matrix formed through conventional methods does not provide better tensile strength and anti-cell contractility required to optimize proper cell infiltration and proliferation.

[0011] Another U.S. Patent Publication No. US8067149B2, entitled "Acellular dermal matrix and method of use thereof for grafting," provides a method for extracting an acellular tissue matrix that includes removing hair follicles and extracting with the surfactant SDS. However, this extracts a different acellular tissue matrix than the highly pure porous collagen matrix of the present invention.

[0012] The current general situation of biological scaffolds is as follows:

[0013] Currently, most of the biological scaffolds on the market are made of reconstituted collagen matrix, and related products have been disclosed in many prior patents. For example, in U.S. Pat. No. 4,193,813, collagen is first dissolved in acetic acid, cross-linked with glutaraldehyde, and 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. U.S. Pat. No. 4,412,947 teaches a method in which high-purity insoluble collagen particles are suspended in an acetic acid solution, frozen to −65° C. at a cooling rate of about 0.3-0.4° C. / min, and freeze-dried to form a porous sheet matrix. U.S. Pat. No. 4,522,753 teaches mixing collagen and chondroitin sulfate to form a copolymer, cross-linking with glutaraldehyde, and freeze-drying to obtain a porous matrix with a pore size of about 20-180 μm that can be used as an artificial skin matrix. US Patent No. 4,970,298 discloses that an acid-soluble collagen solution or a mixed solution containing a solution of acid-soluble collagen, hyaluronic acid, and fibronectin can be crosslinked by carbodiimide and dehydrothermal crosslinking methods. After freezing at various temperatures, a porous matrix is ​​prepared using lyophilization. The freezing temperature is -30 to -50°C, and the pore size of the resulting matrix is ​​about 50 to 250 μm, and the pore size of the collagen matrix containing hyaluronic acid or fibronectin is 100 to 150 μm. The preparation method of the collagen matrix disclosed in the above patent mainly uses acidic or alkaline collagen crosslinked at high temperature or with a chemical crosslinking agent, and freeze-dries to form pores to obtain a porous matrix. Matrices prepared by these methods have low pore uniformity and the pore size is difficult to control. In addition, most of the chemical crosslinking agents used are toxic. Reconstituted collagen microfibrils or fibrils are less ideal for clinical applications since they provide the matrix with limited support, protection, and variable mechanical properties such as tension, tensile strength, and viscoelasticity.

[0014] Unreconstituted collagen matrices have high industrial value for clinical applications, and a process for producing unreconstituted collagen matrices is disclosed in U.S. Pat. No. 8,198,408, previously filed by the present inventor, entitled "Method for preparing porous collagen matrices."

[0015] In the method used in the prior art, such as Chinese Patent No. 150746 entitled "Preparation of a porous collagen matrix", collagen is first placed in a neutral solution at 30-45°C for a certain period of time to reorganize collagen microfibrils, resulting in a gelatinous collagen matrix. The gelatinous collagen matrix is ​​frozen to a suitable low temperature with a suitable cooling rate, and then freeze-dried under reduced pressure to obtain a porous collagen matrix. In Chinese Patent No. I284665 entitled "Process for preparing porous collagen matrix from connective tissue", the connective tissue, which is essentially treated in situ, is subjected to a porous treatment step after a washing step to remove impurities and non-collagen from the connective tissue, and the porous treatment step is to obtain a collagen matrix with porous properties. This in situ treatment omits the traditional procedure of shredding or chopping the connective tissue, and the process does not require the use of a cross-linking agent. This method is more convenient to prepare collagen matrix by omitting two or three treatment steps, but does not describe the porous structure and its bearing stress and tension. Summary of the Invention [Problem to be solved by the invention]

[0016] Generally, when there is a wound on the surface of the human body, gauze, bandages, patches, medical tapes, etc. are used to cover and wrap the wound in order to prevent infection by external bacteria. However, the above-mentioned external dressings can only suppress external bacteria and do not directly help the wound heal itself.

[0017] In summary, to solve the problem of preventing wounds from being infected by external bacteria and obtaining highly pure porous collagen, the present invention provides a biological scaffold comprising a non-reconstituted collagen matrix for wound dressing. [Means for solving the problem]

[0018] The present invention provides a biological scaffold comprising at least one non-reconstituted collagen matrix and at least one biological material uniformly covering at least one surface or both the outer and inner surfaces of the non-reconstituted collagen matrix. Also disclosed in the present invention is a method for preparing the non-reconstituted collagen matrix and the biological scaffold, which has higher strength than conventional collagen matrices. Furthermore, the biological scaffold provided by the present invention can be uniformly coated with a biological material layer, which can facilitate cell infiltration into the non-reconstituted collagen matrix. In addition, the hair follicle tissue in the non-reconstituted collagen matrix can be destroyed without destroying the main structure of dermal collagen, which results in a non-reconstituted collagen matrix with higher strength than matrices made by conventional techniques. In addition, the biological scaffold reduces the immune response caused by hair follicles and favors cell growth, making it more suitable for further use in surgery, human organ and tissue repair, regenerative medicine, skin grafting, or aesthetic medicine. It may also be used as a surgical repair mesh fabric, a dental repair mesh fabric, a human skin substitute, an artificial meninges substitute, an orthopedic repair substitute, or a wound dressing substitute.

[0019] The biological scaffold provided by the present invention comprises at least a non-reconstituted collagen matrix and at least one biological material. The non-reconstituted collagen matrix system has a plurality of collagen bundle fibrils and a plurality of pores formed by the collagen bundle fibrils. The biological material is coated on at least one outer surface of the body and / or the outer surface of the collagen bundle fibrils. Since the plurality of pores are formed by the collagen bundle fibrils, i.e., the collagen fibrils form the inner walls of the pores, coating the surface of the collagen fibrils can be equivalent to coating the inner walls of the pores.

[0020] A method for preparing a non-reconstituted collagen matrix can include obtaining a skin sheet from an animal skin tissue, performing a depilation step in which the skin sheet is immersed in a high ionic strength salt solution and then a proteolytic enzyme is added to the salt solution to produce a depilated skin sheet, and performing a swelling step in which the depilated skin sheet is immersed in a first acidic solution for a sufficient time for the depilated skin sheet to form a swollen skin sheet layer to form a non-reconstituted collagen matrix, the non-reconstituted collagen matrix having a plurality of intertwined overlapping bundled collagen fibrils and a plurality of pores formed by the bundled collagen fibrils.

[0021] Optionally, the method of preparing the unreconstituted collagen matrix can further comprise a disinfection step, a delipidation step, a deantigenization step, and / or a strengthening step.

[0022] A method of preparing a biological scaffold can include providing an unreconstituted collagen matrix as described herein and contacting the unreconstituted collagen matrix with a solution containing a biological material under a reduced pressure environment to coat the biological material on a first exterior surface and within the plurality of pores of the unreconstituted collagen matrix.

[0023] In conclusion, the preparation method of the present invention can further include a disinfection step before or after any step, in which the sheet or skin sheet is washed with a disinfectant solution. Most preferably, the disinfectant solution is a peracetic acid solution.

[0024] In one embodiment of the present invention, the preparation method provided by the present invention further comprises the following steps: First, take out a non-reconstituted collagen matrix coated with biological material; Then, inject cell culture medium into the pores, and then inject a solution containing cells, so that the cells enter the pores and attach to the biological scaffold; Preferably, the cells can be stem cells, satellite cells, progenitor cells, or tissue cells.

[0025] In one embodiment of the method of preparing a non-reconstituted collagen matrix according to the present invention, the animal skin tissue is pigskin tissue, bovine skin tissue, or sheepskin tissue.

[0026] In one embodiment of the method for preparing a non-reconstituted collagen matrix according to the present invention, the high ionic strength salt solution is a salt solution in which anions and cations are bound by ionic bonds. Preferably, the ionic strength of the high ionic strength salt solution is greater than 0.15N. Most preferably, the ionic strength of the high ionic strength salt solution is between 0.5N and 10N. Most preferably, the high ionic strength salt solution is a solution containing sodium chloride, ammonium sulfate, and a mixture of the above compounds. This high salt condition stabilizes the structure of the non-reconstituted collagen matrix, making it not swell and extruded into the hair follicle, so that the enzyme can smoothly enter the hair follicle for reaction.

[0027] In one embodiment of the method for preparing a non-reconstituted collagen matrix according to the present invention, the proteolytic enzyme is capable of working in a high ionic strength salt solution. Preferably, the proteolytic enzyme is selected from the group consisting of serine proteases, threonine proteases, cysteine ​​proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and combinations of the above enzymes. Preferably, the cysteine ​​enzyme is selected from the group consisting of papaya enzymes (e.g., papain), pineapple enzymes (e.g., bromelain), and mixtures of the above enzymes. Most preferably, the metalloprotease is dispase.

[0028] In one embodiment of the method for preparing a non-reconstituted collagen matrix according to the present invention, a defatting step is further included before or after the depilation step. The defatting step is a saponification step, an organic solvent treatment, or a combination thereof. In the saponification step, an alkaline substance is used to contact the surface of the skin sheet. Preferably, the alkaline substance is a strongly alkaline particle having a fine particle size. 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.

[0029] In one embodiment of the method of preparing a non-reconstituted collagen matrix according to the invention, the depilation step or any subsequent step further comprises performing a shaking or agitation step. Preferably, the shaking or agitation step is an ultrasonic vibration treatment.

[0030] In one embodiment of the method of preparing a non-reconstituted collagen matrix of the invention, the depilation step or any subsequent step further comprises applying heat, optimally to a temperature of 25°C to 40°C.

[0031] In one embodiment of the method of preparing a non-reconstituted collagen matrix according to the invention, the optional step after the depilatory step further comprises a cleaning step in which the sheet or dermal sheet is immersed in a cleaning solution or passed through a disinfecting gas. Preferably, the cleaning solution is a phosphate or aqueous solution. Preferably, the disinfecting gas is ozone.

[0032] In one embodiment of the method of preparing a non-reconstituted collagen matrix according to the present invention, the first acidic solution is a weak acid solution.

[0033] In one embodiment of the method for preparing a non-reconstituted collagen matrix according to the present invention, the method further comprises a de-antigenizing step after the swelling step. The de-antigenizing step is to immerse the sheet or the skin sheet in a second acidic solution. The second acidic solution contains pepsin. Most preferably, the second acidic solution is a weak acidic solution.

[0034] In one embodiment of the method for preparing a non-reconstituted collagen matrix according to the present invention, a strengthening step is further included after the swelling step: the sheet or skin sheet is immersed in an additive, preferably a crosslinker solution.

[0035] In one embodiment of the invention, the crosslinker is a transglutaminase or acts through a functional group, one of the functional groups of the crosslinker being amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photocrosslinker, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoids and derivatives thereof, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(suberate), ... sulfosuccinimidyl), N-hydroxy-succinimide 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.

[0036] In one embodiment of the invention, the walls of the plurality of pores in the body have an intertwined, overlapping structure of collagen bundle fibrils.

[0037] In one embodiment of the invention, the biological scaffold comprises at least a body comprising a non-reconstituted collagen matrix, a biological material coated on at least a first surface of the body, and a tissue adhesive layer on another surface of the body opposite or adjacent to the first surface or on two or more surfaces other than the first surface, the tissue adhesive layer comprising a tissue adhesive. When the biological scaffold is attached to the tissue by the tissue adhesive layer, a plurality of cells migrate from the tissue adhesive layer to the biological material layer, resulting in tissue remodeling.

[0038] In one embodiment of the invention, the biological material is selected from the group consisting of cell adhesion materials, tissue repair materials, cell guidance materials, growth factor materials, antimicrobial materials, and mixtures of the above materials.

[0039] Preferably, the cell adhesive material may be a saccharide material, a peptide, a protein, a phospholipid, or a combination thereof.

[0040] Preferably, the saccharide material may be a glycosaminoglycan material.

[0041] 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.

[0042] Preferably, the tissue repair material can be a biomaterial, an extracellular matrix, a nutrient, and combinations thereof.

[0043] Preferably, the biomaterial 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, and the like, and combinations thereof.

[0044] Preferably, the extracellular matrix is ​​selected from the group consisting of collagen, gelatin, elastin, glycosaminoglycans, proteoglycans, glycoproteins, fibronectin, laminin, aggrecan, metalloproteases, and the like, and combinations thereof.

[0045] Preferably, the nutrients are selected from the group consisting of vitamins, minerals, proteins, lipids, carbohydrates, amino acids, peptides, nucleotides, deoxynucleotides, medicinal herbs, and the like, and combinations thereof.

[0046] Preferably, the cell-inducing material is selected from the group consisting of molecules such as vitamins, minerals, chemicals, drugs, medicinal herbs, metabolites, intermediary metabolites, sugars, peptides, proteins, phospholipids, or combinations thereof.

[0047] Preferably, the antimicrobial material is an antibiotic, an antimicrobial protein, or an antimicrobial peptide.

[0048] 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), epidermal growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF), granulocyte macrophage colony stimulating factor (GMCSF), and the like. ), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic proteins (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factors (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and combinations thereof.

[0049] In one embodiment of the present invention, the biological scaffold provided by the present invention further comprises a cell culture medium within the pores.

[0050] Preferably, it further comprises cells, the cells being attached within any one of the pores of the biological scaffold.

[0051] Preferably, the cells may be stem cells, satellite cells, progenitor cells, or tissue cells.

[0052] In one embodiment of the present invention, the reduced pressure environment is generated by a vacuum pull operation.

[0053] In one embodiment of the invention, the tissue adhesive layer comprises a crosslinker that is or has at least a functional group of transglutaminase, one matrix component, the unreconstituted collagen matrix itself or additional matrix molecules that are crosslinked with the crosslinker, and a quenching agent that is used to react with the functional groups of the crosslinker or with the remaining functional groups of the crosslinker to reduce cytotoxicity.

[0054] 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, keratin, 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.

[0055] Preferably, the quenching agent 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, saccharides, oligosaccharides, polysaccharides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotides, azides, photocrosslinking compounds, and mono- or heterobifunctional groups, and combinations thereof.

[0056] Preferably, the tissue is skin tissue. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic cross-sectional view of a non-reconstituted biological scaffold in one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic cross-sectional view showing the main body of a non-reconstituted biological scaffold in one embodiment of the present invention. [Diagram 3] Electron microscope images of a conventional reconstituted porous collagen matrix at 100x magnification are shown. [Figure 4] 1 shows a flow chart for the preparation of a non-reconstituted collagen matrix of the present invention. [Diagram 5] 1 shows another flow chart for the preparation of a non-reconstituted collagen matrix of the present invention. [Figure 6] 6A-6C show electron microscopy images of unreconstituted collagen matrices prepared by the methods of the invention. [Figure 7] 7A-7B show electron microscope images of a biological scaffold from one embodiment of the present invention. [Figure 7C] FIG. 1 shows Alcian blue staining showing good distribution of hyaluronic acid within the biological scaffold. [Figure 8] FIG. 1 is a comparison of shear stress testing of a biological scaffold from one embodiment of the present invention and a biological scaffold from the prior art. [Figure 9] 9A and 9B show the storage and loss moduli of a non-reconstituted porous collagen matrix of the present invention and a reconstituted porous collagen matrix from the prior art. [Figure 10A] FIG. 2 is a comparative diagram showing wound healing between the biological scaffold of the present invention and a reconstituted porous collagen-chondroitin sulfate matrix prepared by a conventional method. [Figure 10B] 1 is a graph showing wound healing under various treatments. [Figure 11] A pair of microscopy images showing a comparison of wound healing status 28 days after surgery: (A) normal skin control, (B) healed matrix of the PCHM group, (C) healed matrix of the Integra group. [Figure 12] 1 is a flow chart showing a method for preparing a biological scaffold in one embodiment of the present invention. [Figure 13] 13A and 13B are a schematic cross-sectional view and a partially enlarged view showing a biological scaffold according to another embodiment of the present invention. [Figure 14] 14A-14C are comparative diagrams of water absorption capabilities of biological scaffolds having different degrees of biological material coating in one embodiment of the present invention. [Figure 15] 15A to 15D are diagrams showing the antibacterial action of biological scaffolds covered with different antibacterial materials in one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Referring to Fig. 1, the present invention provides a non-reconstituted material biological scaffold 1, which includes a body 10, a biological material layer 20, and a tissue adhesive layer 30. The body 10 includes at least one non-reconstituted collagen matrix 10a. The biological material layer 20 and the tissue adhesive layer 30 are coated on one surface of the body and an adjacent or opposite surface of the body, respectively. When the biological scaffold is attached to a tissue 2 by the tissue adhesive layer, a plurality of cells are chemically attracted by the biological material and migrate from the tissue adhesive layer to the biological material layer, thereby reconstructing the tissue 2.

[0059] Please refer to Fig. 2, which shows a schematic cross-sectional view of a body 10 of a biological scaffold according to one embodiment of the present invention. As shown in the figure, the body 10 is preferably a non-reconstituted collagen matrix 10a, which is composed of a plurality of cylindrical collagen fibrils and includes a plurality of pores 10b structured by the collagen fibrils. The inner surfaces of the pores 10b are coated with a biological material 10c.

[0060] 4, for the wound dressing prepared from the biological scaffold with the non-reconstituted collagen matrix of the present invention, first, as shown in step S100, prepare animal tissue or animal skin tissue such as pig skin tissue, cow skin tissue, or sheep skin tissue in an environment below 15°C, remove the subcutaneous fat of the animal skin tissue with a sharp object, and cut the tissue into a size of 28±1 cm×70±1 cm. Next, depilate the animal skin tissue, scrape off the remaining oil and moisture thereon, and cut each skin tissue into 4 pieces each with a size of 13±1 cm×34±1 cm. Then, peel off the skin layer of the pig skin by machine to obtain at least one skin sheet, and rinse with pure water. Most preferably, the skin layer is taken from pig skin.

[0061] Furthermore, as shown in step S102, the skin sheet is immersed in a high ionic strength salt solution, and after adding a protease, the salt solution is heated to 25 to 40°C, thereby performing a hair removal treatment step. Then, ultrasonic vibration treatment is performed for 2 hours. A phosphate buffer solution is used as a cleaning solution for cleaning the skin sheet after the hair removal treatment. The high ionic strength salt solution is a salt solution in which anions and cations are ionic bonded. The ionic strength of the high ionic strength salt solution is greater than 0.15N. Most preferably, the ionic strength of the high ionic strength salt solution is between 0.5N and 10N. Most preferably, the high ionic strength salt solution is a solution containing sodium chloride, ammonium sulfate, or a mixture of the above compounds.

[0062] In one embodiment, the high ionic strength salt solution is a phosphate buffer solution containing sodium chloride. This high salt condition stabilizes the structure of the unreconstituted collagen matrix, preventing the tissue from swelling and being extruded into the hair follicle, allowing the enzyme to easily enter the hair follicle for reaction.

[0063] In one embodiment, the proteolytic enzyme operates in a high ionic strength salt solution. Most preferably, the proteolytic enzyme is selected from the group consisting of serine proteases, threonine proteases, cysteine ​​proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and combinations of the above enzymes. Most preferably, the cysteine ​​proteases are selected from the group consisting of papaya enzymes, pineapple enzymes, and mixtures of the above enzymes. Most preferably, the metalloprotease is dispase.

[0064] Referring to Table 1 below, when the skin sheets were soaked in papain (papaya enzyme) in a phosphate buffer solution containing about 5 mM or 0.16% (w / w) L-cysteine, and the concentration of papain was greater than 0.6 mg / mL (Groups B, C, and D), the pig hair was completely removed. When the skin sheets were soaked in bromelain (pineapple enzyme) in a phosphate buffer solution, and the concentration of bromelain was 0.2 mg / mL and 0.6 mg / mL (Groups A and B), some pig hair was still removed. When the concentration of bromelain was 1.2 mg / mL, the pig hair was completely removed. The pig hair follicles could also be completely removed with dispase greater than 0.2 U / mL. In the pigs in the control group (phosphate buffer only), all the pig hair was still intact.

[0065] [Table 1]

[0066] Referring to FIG. 4, as shown in step S104, the skin sheet after the hair removal treatment is immersed in a first acidic solution, and the first acidic solution is heated to 33-37°C by ultrasonic vibration treatment to perform a swelling treatment step. The ultrasonic vibration treatment time is continuous for 24 hours, so that the volume of the skin sheet increases by at least 50 percent. Preferably, the first acidic solution is a weak acid solution 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.

[0067] As shown in step S105, a washing step is performed to remove non-collagenous materials from the cortical sheet to obtain a purified collagen matrix, which is then frozen at −20° C. and −80° C. and freeze-dried or lyophilized to obtain a purified unreconstituted porous collagen matrix.

[0068] 5, which illustrates another embodiment of the method for preparing a non-reconstituted collagen matrix of the present invention, a disinfection treatment is performed by washing the sheet or skin sheet with a disinfectant solution before or after performing any step, as shown in step S101a. Most preferably, the disinfectant solution is a peracetic acid solution.

[0069] As shown in step S102a, the preparation method of the present invention further includes a degreasing step before or after the depilation step S102. The degreasing step is a saponification step, an organic solvent treatment, or a combination thereof. The saponification step is to contact the sheet or the skin sheet with an alkaline substance, and when only one surface is oily, it can be contacted with one specific surface. Most preferably, the alkaline substance is in the form of granules of strongly basic particles. Most preferably, the strongly basic particles are sodium hydroxide particles. The organic solvent treatment is an alcohol treatment, a hexane treatment, or a chloroform treatment. Nevertheless, an optimal treatment amount and treatment time are required to prevent the denaturation of collagen in the matrix.

[0070] As shown in step S104a, the preparation method of the present invention further includes a de-antigenizing step after the swelling step S104. Most preferably, the de-antigenizing step is to immerse the sheet or skin sheet in a second acidic solution containing pepsin. The second acidic solution containing pepsin is heated to 33°C to 37°C. Then, ultrasonic vibration treatment is performed for 4 hours. The solution remaining in the sheet or skin sheet is washed away with pure water. The sheet or skin sheet is treated again with potassium dihydrogen phosphate solution at 25°C. Preferably, the second acidic solution is a weak acid solution 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.

[0071] As shown in step S104b, the preparation method of the present invention further includes a strengthening step after the swelling step, in which a plurality of sheets or skin sheets are stacked and immersed in an additive, preferably a crosslinking agent solution. The crosslinking agent solution is heated to 33°C-37°C and subjected to ultrasonic vibration treatment for 24 hours to complete the crosslinking reaction. The solution remaining on the sheets or skin sheets is rinsed with pure water. Most preferably, one functional group of the crosslinker is selected from the group consisting of amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photocrosslinker, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoids and derivatives thereof, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, N-hydroxyphenyl, ... -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 ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, psoralen, vinyl sulfone, and combinations thereof.

[0072] Reference is now made to Figures 6A-6C, which show the appearance of an unreconstituted collagen matrix prepared according to the above method. Figure 6A is a scanning electron microscope image of an unreconstituted collagen matrix in one embodiment of the invention, magnified 100 times. Figure 6B is a scanning electron microscope image of an unreconstituted collagen matrix in one embodiment of the invention, magnified 400 times. Figure 6C is a scanning electron microscope image of an unreconstituted collagen matrix in one embodiment of the invention, magnified 10,000 times.

[0073] Compared with the porous collagen matrix prepared by the prior art (see FIG. 3), the non-reconstituted porous collagen matrix prepared by the present invention is composed of a plurality of intertwined aggregated collagen fibril bundles, and has a plurality of pores with deep groove walls. The aforementioned collagen fibril bundles are composed of procollagen molecules and have a diameter of less than 100 nm, but the present invention is not limited thereto. The appearance of the pores is a random radius opening, and the size of the pores is controlled within a narrow range. Thus, the collagen matrix prepared by the method described herein has relatively uniform pores.

[0074] Please refer to Figures 7A-7C, which show the appearance of a biological scaffold made from a non-reconstituted collagen matrix prepared according to the above method. Figure 7A is a scanning electron microscope image of a biological scaffold in one embodiment of the present invention at 100 times magnification. Figure 7B is a scanning electron microscope image of a biological scaffold in one embodiment of the present invention at 400 times magnification. Figure 7C is an optical microscope image of a biological scaffold with hyaluronic acid pre-activated by CNBr and cross-linked to a non-reconstituted collagen matrix in one embodiment of the present invention. Alcian blue was used to stain the hyaluronic acid derivatives present in the scaffold after sectioning of the histology sample. The image shows good distribution of the hyaluronic acid derivatives in the biological scaffold.

[0075] Refer to FIG. 8, which shows the shear stress test of the non-reconstituted porous collagen matrix according to the embodiment of the present invention (line B) and the conventional porous collagen matrix obtained by the prior art (line A). The results show that the shear stress of the porous collagen matrix obtained by the prior art is 63.56e-03MPa at a temperature of 20°C, and the shear stress of the non-reconstituted porous collagen matrix of the present invention is 189.07MPa. Therefore, it is demonstrated that under the same test conditions, the shear strength of the non-reconstituted porous collagen matrix of the present invention is about three orders of magnitude higher than that of the conventional porous collagen matrix. Since the hair follicles can be completely removed first, the non-reconstituted porous collagen matrix certainly has better structural strength and tensile strength than the conventional porous collagen matrix. In summary, the present invention provides a process for preparing a non-reconstituted porous collagen matrix that can destroy hair follicle tissue without destroying the original crosslinks of collagen in the dermis layer, thereby preparing a porous matrix with higher structural strength than the conventional technology.

[0076] See Figures 9A and 9B, which show the change in storage and loss modulus along with the change in oscillatory strain monitored by the rheometer. Storage modulus is a measure of the ability of the matrix to elastically store deformation energy. It is directly related to the degree of crosslinking, the higher the degree of crosslinking the greater the storage modulus. Figure 9A shows data for a non-reconstituted porous collagen matrix of the present invention, while Figure 9B shows data for a conventional reconstituted porous collagen matrix. Microstructure means that there are forces between the molecules or particles within the material. A greater force is required to break the microstructure than is holding it together. When the applied force is smaller than the molecular or interparticle forces, G' will be greater than G". The material has the ability to store energy and should be able to return to some degree to its original configuration before the mechanical force is applied. The material behaves as an elastic solid, which is not the same as ideal, since some of the mechanical energy is dissipated. The results in Figures 9A and 9B show that the storage modulus of the unreconstituted porous collagen matrix is ​​higher than that of the reconstituted porous collagen matrix prepared by conventional techniques. Most of the native crosslinks in the unreconstituted porous collagen matrix are preserved, while most of the native crosslinks are lost in the conventional reconstituted porous collagen matrix.

[0077] Furthermore, since the non-reconstituted collagen matrix 10a itself is not a hydrophilic material, the body 10 may further include a hydrophilic biomaterial 10c coated on the outer surface of the collagen fibrils. That is, the inner walls of these pores 10b are also coated with a hydrophilic biological material 10c (shown in FIG. 2) to promote cell infiltration into the non-reconstituted porous collagen matrix 10a and then migration therein and adhesion / attachment. Preferably, the aforementioned biological material may be a growth factor or a glycosaminoglycan such as hyaluronic acid (HA), chondroitin sulfate (CS), or heparan (Hep). More preferably, the hydrophilic biological material 10c is hyaluronic acid, although the present invention is not limited thereto.

[0078] Referring again to FIG. 1, in a preferred embodiment of the present invention, the growth factor material layer 20 coated on one side of the 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), epidermal growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF), and granulocyte colony stimulating factor (GCSF). The inhibitor is selected from the group consisting of macrophage colony stimulating factor (GMCSF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic proteins (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factors (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and combinations thereof.

[0079] In addition, tissue adhesives used in clinical medicine and tissue engineering can achieve the effects of controlling drug release, regulating the degradation of biocompatible materials, or promoting vascular conjugation through their adhesive properties. However, because the tissue adhesives of the prior art are made with crosslinking agents such as glutaraldehyde (GA), glyoxal, formaldehyde, or sodium tripolyphosphate (TPP), and crosslinked with carrier materials such as gelatin, chitosan, or collagen, the crosslinking agents are cytotoxic and easily induce strong and persistent immune responses, making the tissue adhesives cytotoxic.

[0080] Therefore, in the present invention, a tissue adhesive layer 30 with better bond strength and low cytotoxicity is used, which includes at least one crosslinking agent, at least one matrix component, and a quenching agent. The crosslinking agent is transglutaminase or has a functional group, and the matrix component is used for crosslinking with the crosslinking agent. The quenching agent is used to react with the functional group of the crosslinking agent to reduce cytotoxicity.

[0081] In a preferred embodiment, said crosslinker is a transglutaminase or one of its functional groups is an amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photocrosslinker, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoids and their derivatives, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate. In some embodiments, the reactive compound is selected from the group consisting of reactive compounds containing 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.

[0082] 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, keratin, 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.

[0083] More preferably, the matrix component can be prepared from any naturally produced collagen or its functional variant and / or hyaluronic acid. Moreover, it has been shown that hyaluronic acid, especially high molecular weight hyaluronic acid (greater than 5 kDa), can effectively promote angiogenesis and further promote wound healing. For example, the molecular weight of hyaluronic acid can be 50 kDa to 5,000 kDa, 70 kDa to 1,500 kDa, 200 kDa to 1,500 kDa, 500 kDa to 1,500 kDa, or 700 kDa to 1,500 kDa. When cells grow in a matrix of collagen or hyaluronic acid, they have good viability. Thus, in the matrix component of the tissue adhesive layer 30 used in the present invention, the concentration of hyaluronic acid is 0.001 mg / mL to 100 mg / mL, and the concentration of collagen is 0.001 mg / mL to 100 mg / mL. Preferably, the collagen concentration is 0.1 mg / mL to 100 mg / mL, and the hyaluronic acid concentration is 0.01 mg / mL to 35 mg / mL. More preferably, the collagen concentration is 3 mg / mL to 75 mg / mL (e.g., 6 mg / mL or 9 mg / mL), and the hyaluronic acid concentration is 0.2 mg / mL to 20 mg / mL.

[0084] In a preferred embodiment, the quenching agent 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, saccharides, oligosaccharides, polysaccharides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotides, azides, photocrosslinking compounds, monofunctional or heterobifunctional groups, and combinations thereof. More preferably, the quenching agent used in the present invention includes spermine, protamine, 1-6 hexanediamine, and polylysine of different groups with various molecular weights. The average molecular weights of polylysine are 3.4 kDa, 20 kDa, 99 kDa, 212 kDa, and 225 kDa, respectively.

[0085] FIG. 10A is a comparative diagram showing the healing of full-thickness wounds on the dorsal skin of guinea pigs implanted with Integra® artificial skin or PCMH. Integra® artificial skin, which serves as a control, is a reconstituted porous collagen-chondroitin sulfate matrix prepared by a conventional method by dehydrothermal crosslinking. PCHM is a biological scaffold of the present invention, which is composed of a non-reconstituted porous collagen matrix and hyaluronic acid coated on the outer and inner surfaces of the matrix. The stable porous structure of PCHM allows better infiltration of skin cells, up to four times faster than the control group. Thus, it was demonstrated that the healing rate of wounds filled with PCHM was much faster (about 80%) than the Integra group. The quality of healing, as demonstrated by histology of the regenerative matrix in the PCHM group, was also much better than the control group at 4 months after surgery.

[0086] Referring to Figure 11, on the 28th day after surgery, compared with normal skin, the biological scaffold of PCHM degraded within 2 weeks, and the healed matrix of the PCHM group was essentially replaced by neomatrix as shown in the tissue structure in Figure 11B. As shown in Figure 11C, debris of the Integra artificial skin was observed, which hindered the regeneration process of wound healing.

[0087] Referring to FIG. 12, as shown in steps S106 to S109, the non-reconstituted collagen matrix is ​​contacted with a solution containing a biological substance to generate a reduced pressure environment in the non-reconstituted collagen matrix. In one embodiment, one end of the non-reconstituted collagen matrix is ​​placed in a solution containing at least one biological substance. As shown in step S108, a reduced pressure environment is generated at the other end of the non-reconstituted collagen matrix, which allows the biological substance to be absorbed into the structure of the non-reconstituted collagen matrix and the biological substance to be uniformly coated on the outer and inner surfaces of the collagen fibrils in the bundles (also considered as being uniformly coated on the inner walls of the pores). It should be noted that since the pore size of the non-reconstituted collagen matrix is ​​extremely small, it is impossible to avoid the remaining air by simply immersing the non-reconstituted collagen matrix in a solution. Therefore, in the present invention, the siphon principle is used to indirectly remove the air, which allows the biological substance to completely cover the outer surface of the collagen fibrils in the bundles (also considered as being the inner walls of the pores). In addition, the aforementioned reduced pressure environment is preferably generated by a vacuum drawing operation.

[0088] The biological material is selected from the group consisting of cell adhesive materials, tissue repair materials, cell guidance materials, growth factor materials, antimicrobial materials, and combinations thereof. Preferably, the cell adhesive materials are sugars, peptides, proteins, phospholipids, or combinations thereof. Preferably, the sugar materials are glycosaminoglycan materials. Preferably, the glycosaminoglycan materials are 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. Preferably, the biological 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-co-glycolic acid) PLGA, silk protein, and fibroin, but the present invention is not limited thereto.

[0089] Preferably, the cell-inducing material is selected from the group consisting of molecules such as vitamins, minerals, growth factors, chemicals, drugs, medicinal herbs, metabolites, intermediates, sugars, peptides, proteins, phospholipids, or combinations thereof.

[0090] 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), epidermal growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF), granulocyte macrophage colony stimulating factor (GMCSF), and the like. ), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic proteins (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factors (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and combinations thereof.

[0091] Preferably, the antimicrobial material is an antibiotic, an antimicrobial protein, an antimicrobial peptide, or a combination thereof.

[0092] For the detailed structure of the non-reconstituted collagen matrix, please refer to Fig. 2, which is a schematic cross-sectional view of a biological scaffold according to one embodiment of the present invention. As shown above, the non-reconstituted collagen matrix 10a is composed of a plurality of collagen fibrils bundled with a plurality of pores 10b. The biological material 10c is absorbed into the non-reconstituted collagen matrix 10a and coated on the outer surface of the collagen fibrils bundled by the siphon principle. That is, the inner walls of the pores 10b are all coated with the biological material 10c, which is more conducive for cells to enter the pores and attach to the biological material for subsequent applications.

[0093] In a preferred embodiment of the present invention, although not shown in FIG. 2, cells can be introduced into the biological scaffold after the biological material is coated on the outer surface of the bundled collagen fibrils. Injecting a cell culture medium or a solution containing cells into the pores 10b allows the cells to enter the pores of the non-reconstituted collagen matrix. That is, in a preferred embodiment of the present invention, the biological scaffold provided by the present invention further comprises cells as shown in FIG. 13A. Referring to FIG. 13B, which is a partial enlarged view of the dotted circle in FIG. 13A, the cells 30 are attached to the biological material 10c in the pores 10b. Preferably, the cells may be stem cells, satellite cells, progenitor cells, progenitor cells, or tissue cells, and the tissue cells may include skin tissue, cartilage tissue, or adipose tissue, etc.

[0094] Table 2 below shows the comparison of the number of attached cells between the non-reconstituted collagen matrix without biological material and the non-reconstituted collagen matrix with biological material in different fields of view. As can be seen from Table 2, the non-reconstituted collagen matrix itself is a hydrophobic material, which is not conducive for cells to attach to the pores. However, the present invention shows that the surface of the non-reconstituted collagen matrix is ​​further coated with a cell adhesion material, which is then beneficial for cells to enter these pores and attach to the biological scaffold. [Table 2]

[0095] Next, the water absorption rates of non-reconstituted collagen matrix without biological material (A1), non-reconstituted collagen matrix coated with hyaluronic acid (once) (A2), and non-reconstituted collagen matrix coated with hyaluronic acid (five times) (A3) were further compared. See Figure 14A-C. The three figures show the water absorption conditions of A1, A2, and A3, respectively. Therefore, it was demonstrated that the non-reconstituted collagen matrix coated with more layers of biological material has better water absorption ability.

[0096] The effect of the biological scaffold used in the wound dressing provided by the present invention was confirmed. See Figures 10B and 11. Figure 10B compares the wound healing time of various biological scaffolds, and Figure 11 compares the wound healing state of various biological scaffolds.

[0097] First, FIG. 10B shows various treatments of wounds, which were divided into groups A, B, C, D, and E to observe the relationship between wound area and healing time. In them, the initial wound area was 100% and was measured every two days until the wound was completely healed. A represents an untreated wound, B represents a wound treated with a porous collagen matrix (PCM), C represents a wound treated with a combination of a porous collagen matrix and CNBr-activated hyaluronic acid (aH) (PCM-aH), D represents a wound treated with PCM-aH and additional 5 mg / mL hyaluronic acid, and E represents a wound treated with Integra® artificial skin. As shown in the figure, the wounds in groups B, C, and D all healed in about 30 days, while the wound treated with artificial skin (group E) took 44 days to heal. Group A healed slightly faster, but exhibited scar contracture and other symptoms.

[0098] As shown in Figure 10B, after 12 days, the fibroblast proliferation status of group C (wounds treated with PCM-aH) certainly exceeded that of group E (wounds treated with artificial skin). Therefore, it was shown that the combination of the main body 10 and biological material described above can effectively promote cell proliferation without causing scar contracture, and can heal wounds more quickly than the artificial skin of the prior art.

[0099] Next, in another embodiment, as shown in Figures 15A to 15D, the biological scaffold was coated with various antibacterial materials, such as the antibacterial peptide pexiganan in Figures 15A and 15B and neomycin in Figures 15C and 15D. The results showed that the antibacterial effect was concentration-dependent, and the biological scaffold coated with the antibacterial material had good antibacterial effect.

[0100] In summary, the objective of the present invention is to provide a biological scaffold that can be used for wound dressing. In one embodiment, one side of the body is coated with a layer of biological material, and another side is coated with a tissue adhesive layer, which allows cells to migrate within the body based on the effect of concentration gradient. In addition, the body contains a hydrophilic biological material, which can further facilitate cell migration and attachment. Therefore, in addition to effectively blocking the entry of foreign bacteria to prevent wound infection, the biological scaffold provided by the present invention can also be used to accelerate cell growth and promote wound healing speed.

[0101] Furthermore, in the prior art, it is known to add finely divided collagen to biological materials (such as hyaluronic acid) and crosslink them to solidify them into a structure, but when collagen and biological materials are intertwined, the outside of the biological scaffold is not completely hydrophilic. In addition, when a reconstituted collagen scaffold (as shown in FIG. 3) is first prepared and then the method provided by the present invention is carried out, the support capacity of the reconstituted collagen matrix is ​​insufficient to withstand the external pressure exerted by the vacuum step required for the subsequent coating of the scaffold with biological materials (see FIG. 9 and the related description above). As mentioned above, the siphon principle is applied so that the biological materials are completely absorbed or introduced into the non-reconstituted collagen matrix and can be uniformly coated on the surface, so that the surface of the biological scaffold is favorable for cell infiltration and attachment. The biological scaffold can be widely applied in surgery, human organ and tissue repair, regenerative medicine, skin grafting, or cosmetic medicine, and can also be used as surgical repair mesh fabric, dental repair mesh fabric, human skin substitute, artificial meninges substitute, orthopedic repair substitute, or wound dressing substitute.

[0102] The above detailed description is a specific description of certain possible embodiments of the present invention, but these embodiments are not intended to limit the patent scope of the present invention. Any equivalent implementation or variation that does not depart from the technical spirit of the present invention should be included in the patent scope of the present invention. [Explanation of symbols]

[0103] 1 Biological scaffolds 10 Main unit 10a Unreconstituted collagen matrix 10b Pore 10c Biological substances 20 Biological material layer 30 Tissue adhesive layer 2 organization S100-S105 Method for preparing a non-reconstituted collagen matrix S100-S104b Method for preparing unreconstituted collagen matrix S106~S109 Preparation of biological scaffolds

Claims

1. A biological scaffold comprising: a body comprising an unreconstituted collagen matrix; the unreconstituted collagen matrix having a plurality of intertwined, overlapping collagen bundles and a plurality of pores formed by the collagen bundles; a biological material coating a first exterior surface of one of the bodies and an interior of a plurality of pores; Equipped with the unreconstituted collagen matrix comprising: Obtaining a layer sheet of connective tissue; performing a depilation step, in which the layer sheet is immersed in a high ionic strength salt solution and then a proteolytic enzyme is added to the salt solution to produce a depilated layer sheet; performing a swelling step in which the depilated layer sheet is immersed in a first acidic solution for a time sufficient for the depilated layer sheet to form a swollen layer sheet, thereby forming a non-reconstituted collagen matrix; The method is prepared by a method comprising the steps of: The high ionic strength is between 0.5N and 10N; The biological material is absorbed into the structure of the non-reconstituted collagen matrix by the siphon principle under reduced pressure, and is uniformly coated on the outer and inner surfaces of the collagen bundle fibrils; The reduced pressure environment is generated by a vacuum pulling operation.

2. 2. The biological scaffold of claim 1, wherein the biological material is selected from the group consisting of a cell adhesion material, a tissue repair material, a cell guidance material, a growth factor material, an antibacterial material, and combinations thereof.

3. the antimicrobial material is an antibiotic, an antimicrobial protein, an antimicrobial peptide, or a combination thereof; the cell adhesive material is a saccharide, a peptide, a protein, a phospholipid, or a combination thereof; The growth factor materials include 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 (NG F), hepatocyte growth factor (HGF), colony stimulating factor (CSF), stem cell factor (SCF), epidermal cell growth factor (KGF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF), granulocyte macrophage colony stimulating factor (GMCSF) , glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic proteins (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factors (TGF-α, TGF-β), interleukins (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and combinations thereof; the cell-inducing material is selected from the group consisting of vitamins, minerals, chemicals, drugs, medicinal herbs, metabolites, intermediate metabolites, sugars, peptides, proteins, phospholipids, and combinations thereof; the tissue repair material is a biomaterial, an extracellular matrix, a nutrient, or a combination thereof; the biomaterial is gelatin, hyaluronic acid, elastin, glycosaminoglycan, chitosan, alginate, polyglutamic acid (γ-PGA), polylysine, poly(lactic-co-glycolic acid) (PLGA), silk fibroin, polyamino acids, cellulose, or a combination thereof; the extracellular matrix is ​​selected from the group consisting of gelatin, elastin, glycosaminoglycans, proteoglycans, glycoproteins, fibronectin, laminin, aggrecan, metalloproteases, and combinations thereof; The nutrients are selected from the group consisting of vitamins, minerals, proteins, lipids, carbohydrates, amino acids, peptides, nucleotides, deoxynucleotides, medicinal herbs, and combinations thereof. The biological scaffold according to claim 2 .

4. 4. The biological scaffold according to claim 1, further comprising cells within a plurality of pores of the body, the cells being stem cells, satellite cells, progenitor cells, precursor cells, or tissue cells.

5. 10. The biological scaffold of claim 1, further comprising a tissue adhesive layer coated on a surface adjacent to or opposite to one surface coated with the biological material layer of the body.

6. The tissue adhesive layer comprises: a crosslinker having a functional or bifunctional group; matrix molecules that are crosslinked with the crosslinker; a quenching agent for reacting with excess functional or difunctional groups of the crosslinker that have not reacted with the matrix molecules; Including, The functional group of the crosslinker is selected from the group consisting of amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photocrosslinker, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoid, hydroxymethylphosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, N-hydroxy-succinimidyl is selected from the group consisting of 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; the matrix molecule is selected from the group consisting of hyaluronic acid, gelatin, silk protein, fibroin, fibronectin, elastin, tenascin, laminin, vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratin, 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, and combinations thereof; the quenching agent is an amino acid, an oligopeptide, a polypeptide, a protein, an amine, a diamine, an oligoamine, a polyamine, a carbonyl compound, a glycol compound, a carboxyl compound, a dicarboxylate, an oligocarboxylate, a polycarboxylate, a sulfhydryl compound, an oligosulfhydryl compound, a polysulfhydryl compound, a hydroxyl compound, an oligohydroxyl compound, a polyhydroxyl compound, a saccharide, an oligosaccharide, a polysaccharide, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), an oligonucleotide, an azide, or a photocrosslinking compound; the functional groups of the quenching agent are selected from the group consisting of monofunctional groups or heterobifunctional groups, and combinations thereof; The biological scaffold of claim 5, wherein the synthetic polymer is an acrylate, polylactic acid, polyglycolic acid, or polylactic-glycolic acid.

7. 2. The biological scaffold of claim 1, wherein the connective tissue layer sheet is a dermis layer sheet obtained from animal skin tissue.

8. preparing an unreconstituted collagen matrix, wherein an unreconstituted collagen matrix is ​​formed having a plurality of intertwined overlapping collagen bundles and a plurality of pores formed by said collagen bundles; a decompression step, in which a biological material is absorbed into the structure of the non-reconstituted collagen matrix by the siphon principle under reduced pressure, and is uniformly coated on the outer and inner surfaces of the collagen bundle fibrils; Including, The reduced pressure environment is generated by a vacuum drawing operation, The step of preparing the unreconstituted collagen matrix comprises obtaining a layer of connective tissue; a depilation step, in which the layer sheet is immersed in a high ionic strength salt solution and then a proteolytic enzyme is added to the salt solution to produce a depilated layer sheet; performing a swelling step in which the depilated layer sheet is immersed in a first acidic solution for a time sufficient for the depilated layer sheet to form a swollen layer sheet, thereby forming a reconstituted collagen matrix; The method for preparing a biological scaffold, wherein the high ionic strength is between 0.5N and 10N.

9. The connective tissue layer sheet is a dermis layer sheet obtained from animal skin tissue, the salt solution is a solution containing sodium chloride, ammonium sulfate, or a mixture thereof; 9. The method of claim 8, 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.

10. The method described in claim 8 or 9, further comprising a disinfection step or a degreasing step.

11. The method according to claim 9, further comprising a de-antigenizing step, comprising immersing the dermal layer sheet in a second acidic solution containing pepsin, after the swelling step.

12. The method of claim 9, further comprising a strengthening step comprising immersing the swollen dermis sheet layer in an additive.

13. injecting a cell culture medium into a plurality of pores of said unreconstituted collagen matrix; injecting cells into the plurality of pores such that the cells adhere to the biological material; The method of claim 8 or 9, further comprising:

14. 10. The method of claim 8 or 9, wherein the biological material is selected from the group consisting of a cell adhesive material, a tissue repair material, a cell guidance material, a growth factor material, an antimicrobial material, and combinations thereof.

15. The method of claim 8 or 9, further comprising contacting one outer surface of the unreconstituted collagen matrix with a solution containing a biological material, and coating an outer surface of the unreconstituted collagen matrix adjacent to or opposite the one outer surface coated with the biological material with a tissue adhesive layer.

16. 16. The method of claim 15, wherein the tissue adhesive layer comprises a crosslinking agent having functional or bifunctional groups, matrix molecules to be crosslinked with the crosslinking agent, and a quenching agent for reacting with excess functional or bifunctional groups of the crosslinking agent that have not reacted with the matrix molecules.

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