Absorbable composite hemostatic agent for internal use and manufacturing method therefor

Abstract

The present invention relates to an absorbable composite hemostatic agent for internal use, having a multi-layered structure in which a porous layer and a non-porous layer are laminated, and the present invention may have a two-layer structure in which a porous blood-absorbing layer and a non-porous protective layer are laminated, or a three-layer structure in which a contact layer, a porous blood-absorbing layer, and a non-porous protective layer are laminated. The present invention combines blood absorption, physical hemostasis, and chemical hemostasis mechanisms to provide rapid, stable, and effective bleeding control, is made of biocompatible materials to prevent immune reactions, secondary infections, etc., and is naturally degraded and absorbed after a certain period of time following use.

Description

Absorbable intracellular complex hemostatic agent and method for manufacturing the same

[0001] The present invention relates to an absorbable in vivo composite hemostatic agent, and more specifically, to an absorbable in vivo composite hemostatic agent that can stably adhere to a bleeding site, rapidly absorb blood, provide a physical hemostatic effect, and provide a rapid hemostatic effect by assisting in the formation of platelet plugs, and a method for manufacturing the same.

[0002] In healthy individuals, when bleeding occurs, natural hemostasis is achieved through the complex interaction of platelets and plasma factors, thereby preventing loss of bodily function, shock, or disability caused by excessive bleeding. However, when excessive internal bleeding occurs due to unexpected accidents, surgical procedures, diseases, or disorders, natural hemostasis driven by physiological factors does not take place. This leads to secondary side effects caused by blood loss and can obstruct the medical team's view during surgery, making treatment difficult; consequently, additional measures such as blood transfusion are required. Since excessive bleeding can lead to death, it is crucial to take rapid early action to prevent blood loss in cases of bleeding caused by serious factors such as accidents, surgeries, or diseases.

[0003] Hemostasis methods primarily involve chemical methods, such as the use of hemostatic agents, or mechanical methods that physically block the site of bleeding to prevent blood loss. Chemical hemostasis involves supplying hemostatic factors, such as thrombin and fibrinogen, from an external source to achieve hemostasis. Mechanical hemostasis methods include attaching hemostatic agents in the form of patches—such as sponges, films, fabrics, or non-woven materials—to the bleeding site, or using syringes containing biocompatible materials to inject the biocompatible fluid onto the site. Since these methods utilize biocompatible materials such as cellulose, polysaccharides, collagen, and gelatin, they can be absorbed into the body after a certain period and excreted through metabolism.

[0004] However, in the case of hemostatic products currently commercialized in the market, problems still exist such as delamination occurring because they do not adhere sufficiently to the injured area, secondary infection occurring due to a structure that is easily contaminated, or insufficient hemostatic effect.

[0005] The present invention aims to provide an absorbable in vivo composite hemostatic agent and a method for manufacturing the same, which can stably attach to a bleeding site, rapidly absorb blood, provide a physical hemostatic effect, and provide a rapid hemostatic effect by assisting in the formation of platelet plugs.

[0006] One embodiment of the present invention for achieving the above-described purpose relates to a composite hemostatic agent comprising: a blood-absorbing layer in the form of a porous sponge; and a non-porous protective layer laminated over the blood-absorbing layer; wherein the protective layer comprises a decellularized tissue supplement and polyethylene glycol, and the blood-absorbing layer comprises a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract.

[0007] The above protective layer may further include at least one from the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials.

[0008] The above blood absorption layer may further include at least one from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin.

[0009] The above natural material may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

[0010] The above-mentioned decellularized tissue supplement can be manufactured through the following steps: a preparation step of preparing a mammalian tissue excluding humans; a pretreatment step of pretreating the tissue; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0011] The DNA content of the tissue that has undergone the above pretreatment step to the second inactivation step may be 50 ng / mg or less.

[0012] In addition, the degradation time using 100 U / ml of collagenase may be at least 90 hours.

[0013] The above first decellularization step may include a first decellularization step performed using a mixture of n-PrOH and NaOH; and a second decellularization step performed using NaOH of 0.05M or more and less than 0.5M.

[0014] The above decellularized protein extract can be prepared by undergoing a pretreatment step of preparing and pretreating mammalian tissue excluding humans; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a decellularization step of removing cells from the virus-inactivated tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0015] The DNA content of the tissue that has undergone the second inactivation step above may be 50 ng / mg or less.

[0016] In addition, the reduction rate (L) of the elastin content of the tissue that has undergone the above pretreatment step to the second inactivation step may be 20% or less.

[0017] The above decellularization step may include a first decellularization step of removing cells from virus-inactivated tissue using a base aqueous solution; and a second decellularization step of removing cells by enzymatically treating the first decellularized tissue.

[0018] The above first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0019] The above first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0020] The method for manufacturing the above-mentioned composite hemostatic agent may include: a protective layer manufacturing step of applying a protective layer raw material mixture to a tray and drying it to manufacture a protective layer; and a blood absorption layer manufacturing step of laminating a blood absorption layer raw material mixture onto the protective layer and then freeze-drying it.

[0021] The above protective layer raw material mixture can be manufactured through the following steps: Step A1, mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; Step A2, preparing polyethylene glycol by dissolving it in water or melting it by applying heat; and Step A3, mixing the materials prepared through Steps A1 and A2.

[0022] The above blood absorption layer raw material mixture can be prepared through Step B1, which involves mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; and Step B2, which involves adding gelatin, thrombin, and a decellularized protein extract to the solution obtained through Step B1 and stirring.

[0023] Another embodiment of the present invention relates to a composite hemostatic agent comprising: a blood-absorbing layer in the form of a porous sponge; a non-porous protective layer laminated over the blood-absorbing layer; and a contact layer formed on the underside of the blood-absorbing layer; wherein the protective layer comprises a decellularized tissue supplement and polyethylene glycol, the blood-absorbing layer comprises a decellularized tissue supplement and gelatin, and the contact layer comprises thrombin and a decellularized protein extract.

[0024] The above protective layer may further include at least one from the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials.

[0025] The above blood absorption layer may further include at least one from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin.

[0026] The above contact layer may further include at least one of the group consisting of decellularized tissue supplements, calcium chloride, natural materials, and polyvinylpyrrolidone.

[0027] The above natural material may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

[0028] The above-mentioned decellularized tissue supplement can be manufactured through the following steps: a preparation step of preparing a mammalian tissue excluding humans; a pretreatment step of pretreating the tissue; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0029] The DNA content of the tissue that has undergone the above pretreatment step to the second inactivation step may be 50 ng / mg or less.

[0030] In addition, the degradation time using 100 U / ml of collagenase may be at least 90 hours.

[0031] The above first decellularization step may include a first decellularization step performed using a mixture of n-PrOH and NaOH; and a second decellularization step performed using NaOH of 0.05M or more and less than 0.5M.

[0032] The above decellularized protein extract can be prepared by undergoing a pretreatment step of preparing and pretreating mammalian tissue excluding humans; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a decellularization step of removing cells from the virus-inactivated tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0033] The DNA content of the tissue that has undergone the second inactivation step above may be 50 ng / mg or less.

[0034] The reduction rate (L) of the elastin content of the tissue that has undergone the above pretreatment step to the second inactivation step may be 20% or less.

[0035] The above decellularization step may include a first decellularization step of removing cells from virus-inactivated tissue using a base aqueous solution; and a second decellularization step of removing cells by enzymatically treating the first decellularized tissue.

[0036] The above first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0037] The method for manufacturing the above-mentioned composite hemostatic agent may include: a protective layer manufacturing step of applying a protective layer raw material mixture to a tray and drying to manufacture a protective layer; a blood absorption layer manufacturing step of laminating a blood absorption layer raw material mixture onto the protective layer and freeze-drying it; and a contact layer manufacturing step of applying a contact layer raw material mixture onto the blood absorption layer and drying to manufacture a contact layer.

[0038] The above protective layer raw material mixture can be manufactured through the following steps: Step A1, mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; Step A2, preparing polyethylene glycol by dissolving it in water or melting it by applying heat; and Step A3, mixing the materials prepared through Steps A1 and A2.

[0039] The above blood absorption layer raw material mixture can be prepared through a C1 step of mixing a decellularized tissue supplement and an acidic aqueous solution and then grinding; and a C2 step of adding gelatin to the solution obtained through the C1 step and stirring.

[0040] The above contact layer raw material mixture can be prepared through Step D1, which involves adding thrombin and decellularized protein extract to an alcohol solvent and stirring.

[0041] The application of the contact layer raw material mixture can be performed through any one of the dipping method, the spray method, and the 3D printing method.

[0042] The absorbable in vivo composite hemostatic agent of the present invention can provide a physical hemostatic effect by stably attaching to the bleeding site, rapidly absorbing blood, and thereby expanding to restrict blood flow.

[0043] In addition, it can provide a rapid hemostatic effect by assisting in the formation of platelet plugs, and it is formed with a multi-layer structure in which porous and non-porous layers are stacked to prevent secondary infection from the outside. Furthermore, since it has excellent flexibility and adhesion, it effectively responds to tissue movement, so it has the advantage of maintaining stable adhesion.

[0044] FIGS. 1(A) and FIGS. 1(B) are drawings illustrating various embodiments of the absorbable in vivo composite hemostatic agent of the present invention.

[0045] Figure 2 is a photograph showing the results of analyzing an H&E staining photograph taken according to Experimental Example 1.

[0046] Figure 3 is a photograph showing the results of analyzing an H&E staining photograph taken according to Experimental Example 2.

[0047] Figure 4 is a graph showing the tensile strength with respect to strain of Experimental Example 3.

[0048] Figure 5 is a photograph showing the results of analyzing an H&E staining photograph taken according to Experimental Example 4.

[0049] Figures 6(A) and 6(B) are graphs showing the experimental results of Experimental Example 5.

[0050] Figure 7 is a graph showing the experimental results of Experimental Example 6.

[0051] Figures 8(A) and 8(B) are graphs showing the experimental results of Experimental Example 7.

[0052] Figures 9(A) and 9(B) are graphs showing the experimental results of Experimental Example 8.

[0053] Figure 10 is a table showing the experimental results of Experimental Example 9.

[0054] Figure 11 is a photograph of the composite hemostatic agent of Example 3.

[0055] Figure 12 is a scanning electron microscope image of the composite hemostatic agent of Example 3.

[0056] Figures 13(A) and 13(B) are the results of the absorption time and absorption power experiments of Experimental Example 12, respectively.

[0057] Figures 14(A) and 14(B) are initial photographs of the absorption time experiment of Experimental Example 12.

[0058] Figure 15 shows the results of the hemostasis time experiment of Experimental Example 13.

[0059] Before describing the preferred embodiments of the present invention in detail below, it should be noted that the terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0060] Throughout this specification, when a part is described as "comprising" a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0061] Throughout this specification, "%" used to indicate the concentration of a specific substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0062] The term "mammal" as used in the specification refers to mammals excluding humans; therefore, even if the term "mammal" is used without mentioning the exclusion of humans, it must be understood as referring to mammals excluding humans, and the expression "cells are removed" must be understood in a comprehensive sense that includes the destruction or removal of DNA.

[0063] In each step, identification codes are used for convenience of explanation and do not describe the order of the steps; unless the context clearly indicates a specific order, the steps may be performed differently from the specified order. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the reverse order.

[0064] Hereinafter, embodiments of the present invention are examined. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art may implement various modified forms of the contents described herein within the scope of the present invention.

[0065] The present invention relates to an absorbable in vivo composite hemostatic agent having a multi-matrix structure in which porous and non-porous layers are laminated, and a method for manufacturing the same. By applying a combination of physical and chemical hemostatic methods, it provides a more effective and rapid hemostatic function. Furthermore, since it is made of a biocompatible material, it is absorbed into the body after a predetermined period has elapsed following application and can be naturally excreted through metabolism.

[0066] The absorbable in vivo composite hemostatic agent of the present invention may be formed into a two-layer structure as shown in FIG. 1(A) or into a three-layer structure as shown in FIG. 1(B).

[0067] First, with reference to FIG. 1(A), an absorbable in vivo composite hemostatic agent with a two-layer structure according to one embodiment of the present invention will be described.

[0068] An absorbable in vivo composite hemostatic agent according to one embodiment of the present invention comprises: a blood absorption layer (200) in the form of a porous sponge; and a non-porous protective layer (100) laminated over the blood absorption layer (200).

[0069] The blood absorption layer (200) is directly attached to the bleeding site, i.e., the application site, to perform the function of absorbing blood or body fluids, and the protective layer (100) is integrally provided thereon to perform the function of stopping bleeding by preventing the movement of blood, body fluids, and air, and to prevent secondary infection and contamination by the outside.

[0070] In addition, the protective layer (100), in addition to the previously described hemostasis and infection prevention functions, performs an anti-adhesion function to prevent adhesion between the composite hemostatic agent attached to the application site and surrounding organs, and has flexible and soft physical characteristics so that it can be stably attached to the application site even with tissue movement.

[0071] This protective layer (100) comprises a decellularized tissue supplement and polyethylene glycol, specifically 9 to 73 wt% of a decellularized tissue supplement and 27 to 91 wt% of polyethylene glycol.

[0072] The decellularized tissue supplement is manufactured by decellularizing mammalian-derived tissue in multiple stages and is a mixture containing collagen and elastin in a weight ratio of approximately 6:4. It not only has excellent biocompatibility but can also provide effects such as wound healing and scar suppression through elastin. The specific manufacturing process and characteristics of this decellularized tissue supplement will be described later.

[0073] Polyethylene glycol is a material with excellent biocompatibility and has been applied in various medical fields in the past. The weight-average molecular weight of the polyethylene glycol used in the present invention may be 100,000 to 700,000 g / mol. If the molecular weight is lower than this, the strength of the protective layer (100) is weakened, and the protective function is reduced. If it is higher than this, problems such as reduced workability and reduced quality may occur due to increased viscosity during the manufacture of the protective layer (100).

[0074] If the protective layer (100) contains only a decellularized tissue supplement or its content is insufficient, there is a problem in that the protective layer (100) does not peel off well from the tray after the protective layer (100) is formed by applying the raw material mixture of the protective layer (100) to the tray during the manufacturing process, and the protective layer (100) lacks flexibility and does not bond homogeneously with the absorbent layer. On the other hand, if polyethylene glycol is used only or its content is insufficient in the protective layer (100), when the absorbent layer is laminated on top of the protective layer (100) during the manufacturing process, the protective layer (100) may be dissolved by the absorbent layer solution, and the shape and structure may not be maintained. Therefore, it is preferable to use the decellularized tissue supplement and polyethylene glycol together in the protective layer (100) in the weight ratio described above.

[0075] In particular, the protective layer (100) is provided in the form of a film including a decellularized tissue supplement and polyethylene glycol, thereby reinforcing the strength of the blood absorption layer (200) formed with a relatively low strength, helping to stably maintain the state where the composite hemostatic agent is attached to the application area, and additionally providing functions such as hemostasis and prevention of adhesion, thus providing a more effective and rapid hemostatic function.

[0076] The protective layer (100) may further include at least one of the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials. These additional components can ensure safety and stability for the application area or surrounding organs by changing physical properties such as strength and flexibility of the protective layer (100).

[0077] The natural material may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

[0078] Poloxamer is a copolymer having a structure of polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO), has a weight-average molecular weight of about 12,700 to 17,400 g / mol, and may have an HLB value of 12 to 30, and it is preferable to use one in which the polyethylene oxide content in the poloxamer is 50 to 85 wt%.

[0079] Polyvinyl alcohol, glycerin, and natural materials are biocompatible materials, and among these, the natural materials may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

[0080] If one or more of these additional components are included, such as poloxamer, polyvinyl alcohol, and glycerin, they may be included to replace a portion of polyethylene glycol, and within the total content of the protective layer (100), poloxamer may be included in an amount of 1 to 10 wt%, polyvinyl alcohol in an amount of 0.1 to 10 wt%, and glycerin in an amount of 0.1 to 5 wt%. For example, if only poloxamer is included as an additional component, 9 to 73 wt% of decellularized tissue supplement, 26 to 90 wt% of polyethylene glycol, and 1 to 10 wt% of poloxamer may be included.

[0081] In addition, if a natural material is included as an additional component, it is included to partially replace the decellularized tissue supplement, and the natural material may be included in the total content of the protective layer (100) at 1 to 10 wt%. That is, the protective layer (100) may contain 8 to 72 wt% of the decellularized tissue supplement, 27 to 91 wt% of polyethylene glycol, and 1 to 10 wt% of the natural material.

[0082] In addition, when all additional ingredients are included, it may contain 8-72 wt% decellularized tissue supplement, 25-89 wt% polyethylene glycol, 1-10 wt% poloxamer, 0.1-10 wt% polyvinyl alcohol, 0.1-5 wt% glycerin, and 1-10 wt% natural material.

[0083] The blood absorption layer (200) comprises a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract, and in addition to the functions described above, physically supports the entire structure, rapidly absorbs blood or body fluids, and expands through such absorption to restrict blood flow, thereby primarily providing a physical hemostatic effect.

[0084] The blood absorption layer (200) is a porous sheet with a structure comprising a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract, specifically comprising 10 to 73 wt% of decellularized tissue supplement, 10 to 75 wt% of gelatin, 0.1 to 10 wt% of thrombin, and 0.1 to 7 wt% of a decellularized protein extract.

[0085] The decellularized tissue supplement is a biocompatible material manufactured by decellularizing mammalian-derived tissue in multiple stages, and together with gelatin and decellularized protein extract, forms the basic framework of the blood absorption layer (200), enabling effective blood absorption. In addition, it can provide an additional hemostatic function in addition to the hemostatic function by thrombin. The decellularized tissue supplement and decellularized protein extract will be described later.

[0086] Thrombin induces a blood coagulation reaction by hydrolyzing fibrinogen in the blood into insoluble fibrin, thereby providing a hemostatic effect.

[0087] The decellularized protein extract is collagen obtained by multi-stage decellularization of one or more tissues derived from mammals other than humans, such as skin, cartilage, ligaments, tendons, myocardial fascia, pericardium, small intestine, bladder, liver, heart, lungs, pancreas, spleen, kidney, stomach, bronchi, uterus, placenta, and nerves. It is added to control the strength or degradation period of the blood absorption layer (200) and to improve adhesion to the application site where the composite hemostatic agent is applied, and provides an environment very suitable for cell behavior to promote the formation of new tissue at the hemostatic site.

[0088] The blood absorption layer (200) may additionally include at least one from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin, in addition to the components described above. Here, the natural material may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA), just as in the case of the protective layer (100) described above.

[0089] These additional components are added to control the physical properties or hemostatic performance of the blood absorption layer (200), and in particular, calcium chloride among them can promote hemostasis by being involved in the production of thrombin and fibrin during the physiological blood coagulation mechanism.

[0090] If additional ingredients are included, calcium chloride may be included in a weight ratio of 0.01 to 3 wt%, poloxamer in a weight ratio of 0.1 to 10 wt%, natural material in a weight ratio of 1 to 20 wt%, polyethylene glycol in a weight ratio of 1 to 15 wt%, polyvinylpyrrolidone in a weight ratio of 1 to 15 wt%, and glycerin in a weight ratio of 0.1 to 10 wt%. For example, if all additional components are included in the blood absorption layer (200), it may include 10-73 wt% decellularized tissue supplement, 10-75 wt% gelatin, 0.1-10 wt% thrombin, 0.1-7 wt% decellularized protein extract, 0.01-3 wt% calcium chloride, 0.1-10 wt% poloxamer, 1-20 wt% natural material, 1-15 wt% polyethylene glycol, 1-15 wt% polyvinylpyrrolidone, and 0.1-10 wt% glycerin.

[0091] A method for manufacturing a composite hemostatic agent according to the present embodiment comprises: a protective layer manufacturing step of manufacturing a protective layer (100) by applying a protective layer raw material mixture to a tray and drying it; and a blood absorption layer manufacturing step of stacking a blood absorption layer raw material mixture on the protective layer (100) and then freeze-drying it.

[0092] The above protective layer manufacturing step is a step of manufacturing a protective layer (100) by applying a protective layer raw material mixture to a tray and drying it, the application method is not particularly limited, and drying can be performed at 50 to 80°C for 10 to 60 minutes.

[0093] A protective layer raw material mixture for manufacturing a protective layer (100) can be manufactured through the following steps: A1, mixing a decellularized tissue supplement and an acidic aqueous solution and then grinding; A2, preparing polyethylene glycol by dissolving it in water or melting it by applying heat; and A3, mixing the materials prepared through steps A1 and A2.

[0094] Step A1 is a step performed to liquefy the decellularized tissue filler. Due to the characteristic of the decellularized tissue filler that dissolves only in acidic aqueous solutions, it is preferable to prepare the decellularized tissue filler by dissolving it in an acidic aqueous solution with a pH of 2.3 to 3.2. Furthermore, for faster dissolution, it is more preferable to mix the decellularized tissue filler with the acidic aqueous solution and then grind it using a blender. At this time, it is preferable to mix the decellularized tissue filler with the acidic aqueous solution such that the concentration of the decellularized tissue filler is 0.75 to 10 wt%.

[0095] Step A2 is a step performed to liquefy polyethylene glycol, which may be a step of preparing polyethylene glycol by dissolving it in water or melting it by applying heat. In the case of dissolving polyethylene glycol in water, it may be prepared by dissolving it so that the concentration of polyethylene glycol is 1 to 20 wt%.

[0096] Step A3 is a step of mixing the liquefied decellularized tissue supplement prepared in the previously described step with the liquefied polyethylene glycol, and stirring may be performed to ensure uniform mixing.

[0097] In addition, if the protective layer (100) contains one or more of the previously mentioned additional components (poloxamer, polyvinyl alcohol, glycerin, and natural materials), the additional components may be dissolved in water or dissolved in the same way as the liquidization method of decellularized tissue supplements, and then ground with a blender to prepare each additional component in advance, and then mixed in step A3.

[0098] The above blood absorption layer manufacturing step includes a process of laminating a blood absorption layer raw material mixture onto a protective layer (100) and then freeze-drying it. Since the protective layer (100) is already formed in the form of a film, when the blood absorption layer raw material mixture is laminated onto it and then freeze-dried, the blood absorption layer (200) dries and bonds with the protective layer (100), and simultaneously a plurality of pores are formed, changing into a porous structure.

[0099] Since rapid freezing during freeze-drying can cause problems such as crystal formation, it is advisable to perform freeze-drying by slowly lowering the temperature to the freezing point of water.

[0100] A blood absorption layer raw material mixture can be prepared through Step B1, which involves mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; and Step B2, which involves adding gelatin, thrombin, and a decellularized protein extract to the solution obtained through Step B1 and stirring.

[0101] Step B1 is the step of liquefying the decellularized tissue supplement, and can be carried out in the same manner as Step A1.

[0102] Step B2 may be a step of adding gelatin, thrombin, and decellularized protein extract to the liquidized decellularized tissue supplement obtained through Step B1 and stirring to mix uniformly. It is preferable that the mixing ratio of each component mixed in this step be set so that the composition within the blood absorption layer (200) of the final completed composite hemostatic agent is the same as described.

[0103] If the blood absorption layer (200) contains at least one additional component selected from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin, it may be mixed together in step B2. The additional component may be introduced as is, introduced after being prepared in an aqueous solution mixture state, or introduced after being prepared by dissolving and then grinding with a blender, similar to the liquefaction method of decellularized tissue supplements, and then mixed in step B2 after being pretreated in various ways depending on the characteristics of the additional component.

[0104] The composite hemostatic agent manufactured through these steps can be packaged and stored after being removed from the tray.

[0105] The above relates to an absorbable in vivo composite hemostatic agent with a two-layer structure illustrated in FIG. 1(A), and below, an absorbable in vivo composite hemostatic agent with a three-layer structure will be described with reference to FIG. 1(B).

[0106] An absorbable in vivo composite hemostatic agent according to another embodiment of the present invention may comprise a blood absorption layer (200) in the form of a porous sponge; a non-porous protective layer (100) laminated over the blood absorption layer (200); and a contact layer (300) formed on the lower surface of the blood absorption layer (200).

[0107] The blood absorption layer (200) and the protective layer (100) perform the same or similar functions as described above with reference to FIG. 1(A). That is, the blood absorption layer (200) provides blood and body fluid absorption and hemostatic effects, and the protective layer (100) provides functions to prevent infection or contamination and to prevent adhesion.

[0108] Specifically, the protective layer (100) comprises a decellularized tissue supplement and polyethylene glycol as described with reference to FIG. 1(A), specifically comprising 9 to 73 wt% of the decellularized tissue supplement and 27 to 91 wt% of the polyethylene glycol. In addition, at least one of the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials may be further included. Since the protective layer (100) is identical to that described in the previous example, a redundant description is omitted.

[0109] The blood absorption layer (200) is a porous sheet comprising a decellularized tissue supplement and gelatin, and, if necessary, may further include at least one from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin. The decellularized tissue supplement will be described later, and the natural material may be at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

[0110] In this embodiment, since thrombin, which is the core of the hemostatic function, is included in the contact layer (300), the main function of the blood absorption layer (200) is to absorb blood and body fluids and maintain the shape of the complex hemostatic agent, and it can also provide a slight hemostatic effect.

[0111] The blood absorption layer (200) may contain 12 to 85 wt% of decellularized tissue supplement and 15 to 88 wt% of gelatin, and if additional components described above are included, they may be included in place of at least a portion of one or more of the two. If additional components are included, the preferred addition content for each component may be 0.01 to 3 wt% for calcium chloride, 0.1 to 10 wt% for poloxamer, 1 to 20 wt% for natural materials, 1 to 15 wt% for polyethylene glycol, 1 to 15 wt% for polyvinylpyrrolidone, and 0.1 to 10 wt% for glycerin. In addition, when all additional components are included, the blood absorption layer (200) may contain 10 to 82 wt% decellularized tissue supplement, 14 to 86 wt% gelatin, 0.01 to 3 wt% calcium chloride, 0.1 to 10 wt% poloxamer, 1 to 20 wt% natural material, 1 to 15 wt% polyethylene glycol, 1 to 15 wt% polyvinylpyrrolidone, and 0.1 to 10 wt% glycerin.

[0112] The above contact layer (300) improves the adhesive performance of the application area to which the absorbable internal composite hemostatic agent is attached, thereby allowing the composite hemostatic agent to adhere more stably and strongly to the application area, and can provide a hemostatic effect by including thrombin. In particular, since thrombin is concentratedly distributed in the contact layer (300) rather than the blood absorption layer (200) and is configured to come into contact with the affected area, more efficient hemostatic performance can be provided.

[0113] The contact layer (300) may include thrombin and a decellularized protein extract, and, if necessary, may additionally include at least one selected from the group consisting of a decellularized tissue supplement, calcium chloride, a natural material, and polyvinylpyrrolidone. The decellularized tissue supplement will be described later, and the natural material will be omitted as it is the same as previously described.

[0114] Specifically, when the contact layer (300) is composed of thrombin and decellularized protein extract, it may contain 30 to 90 wt% thrombin and 10 to 70 wt% decellularized protein extract. If additional components are included, they may be included to replace a portion of one or more of the components among thrombin or decellularized protein extract. In this case, the decellularized tissue supplement may be included in an amount of 1 to 5 wt%, calcium chloride in an amount of 0.01 to 3 wt%, natural material in an amount of 1 to 5 wt%, and polyvinylpyrrolidone in an amount of 1 to 15 wt%. Specifically, the contact layer (300) may contain 26 to 86 wt% thrombin, 10 to 70 wt% decellularized protein extract, 1 to 5 wt% decellularized tissue supplement, 0.01 to 3 wt% calcium chloride, 1 to 5 wt% natural material, and 1 to 15 wt% polyvinylpyrrolidone.

[0115] The method for manufacturing a composite hemostatic agent according to the present embodiment may include: a protective layer manufacturing step of manufacturing a protective layer (100) by applying a protective layer raw material mixture to a tray and drying it; a blood absorption layer manufacturing step of manufacturing a blood absorption layer by laminating a blood absorption layer raw material mixture on the protective layer (100) and then freeze-drying it; and a contact layer manufacturing step of manufacturing a contact layer (300) by applying a contact layer raw material mixture to the blood absorption layer (200) and drying it.

[0116] The step of manufacturing the protective layer described above is identical to the embodiment of the present invention described above, so a redundant description is omitted.

[0117] The above blood absorption layer manufacturing step is a step of stacking a blood absorption layer raw material mixture on a protective layer (100) and then freeze-drying it. When the blood absorption layer raw material mixture is stacked on the protective layer (100) and freeze-dried, the blood absorption layer (200) is dried and formed into a porous structure, and at the same time bonded with the protective layer (100).

[0118] In the freeze-drying process for manufacturing the blood absorption layer (200), if rapid freezing occurs, problems such as the formation of crystals may occur, so it is preferable to perform freeze-drying by slowly lowering the temperature to the freezing point of water.

[0119] The above blood absorption layer raw material mixture can be manufactured through a C1 step of mixing a decellularized tissue supplement and an acidic aqueous solution and then grinding; and a C2 step of adding gelatin to the solution obtained through the C1 step and stirring.

[0120] Step C1 is the step of liquefying the decellularized tissue supplement, and can be carried out in the same manner as Step A1.

[0121] Step C2 is the step of adding gelatin to the liquefied decellularized tissue supplement obtained through Step C1 and stirring to mix uniformly.

[0122] In addition, if the blood absorption layer (200) further includes at least one additional component from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin, it may be mixed in step C2, and the mixing ratio of each component mixed in this step may be set so that the composition within the blood absorption layer (200) of the final completed composite hemostatic agent satisfies the composition ratio described above. In addition, gelatin or additional components may be mixed in step B2 after being pretreated in various ways depending on the characteristics of these components, such as being added as is, being prepared first in the form of an aqueous mixture and then added, or being prepared by dissolving and then grinding with a blender, similar to the liquefaction method of decellularized tissue supplements.

[0123] The above contact layer manufacturing step is a step of manufacturing a contact layer (300) by applying a contact layer raw material mixture onto a blood absorption layer (200) and drying it. The application of the contact layer raw material mixture can be performed through any one of the dipping method, the spray method, and the 3D printing method, and the contact layer (300) finally obtained through the drying step is formed in the form of a thin coating layer on the blood absorption layer (200).

[0124] The contact layer raw material mixture can be prepared through Step D1, which involves adding thrombin and decellularized protein extracts to an alcohol solvent and stirring.

[0125] At this stage, the total solute concentration of the contact layer raw material mixture can be mixed with an alcohol solvent to be 1 to 90 wt%, and the weight ratio between each solute can be set and mixed to satisfy the weight ratio described above.

[0126] Additionally, the contact layer raw material may further include at least one additional component selected from the group consisting of decellularized tissue supplement, calcium chloride, natural materials, and polyvinylpyrrolidone. In this case, these additional components may be mixed together in step D1 or added and mixed after step D1. The amount of these additional components added may be appropriately measured and added to satisfy the weight ratio in the contact layer (300) after drying described in the preceding example.

[0127] Using alcohol as a solvent for the contact layer raw material mixture offers the advantage of enabling rapid drying and the formation of a coating layer. Here, ethanol or isopropyl alcohol may be used as the alcohol solvent, but is not limited thereto; any alcohol-based substance may be used as long as it has excellent volatility and, preferably, is safe for the body.

[0128] The composite hemostatic agent manufactured through these steps can be packaged and stored after being removed from the tray.

[0129] Below, the decellularized tissue supplement and decellularized protein extract, which are raw materials of the composite hemostatic agent described above with reference to FIG. 1(A) and FIG. 1(B), will be described in detail.

[0130] The above-mentioned decellularized tissue supplement and decellularized protein extract are each obtained by multi-stage decellularization treatment of mammalian tissues other than humans, and may be derived from different tissues.

[0131] Specifically, the decellularized tissue supplement can be obtained by multi-stage decellularization of one or more mammalian tissues including skin, cartilage, ligaments, tendons, myocardial fascia, pericardium, small intestine, bladder, liver, heart, lungs, pancreas, spleen, kidney, stomach, bronchi, uterus, placenta, and nerves, and the decellularized protein extract can be obtained by multi-stage decellularization of one or more mammalian tissues including blood vessels, ligaments, and tendons.

[0132] First, the decellularized tissue supplement is a multi-stage decellularized tissue supplement manufactured through the following steps: a preparation step of preparing mammalian tissue excluding humans; a pretreatment step of pre-treating the tissue; a first inactivation step of inactivating viruses contained in the pre-treated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0133] At this time, the DNA content of the tissue that has undergone the pretreatment step to the second inactivation step may be 50 ng / mg or less. In addition, the degradation time using 100 U / ml of collagenase may be at least 90 hours, and preferably 90 to 750 hours.

[0134] As such, the decellularized tissue supplement of the present invention has a low DNA content, so it hardly induces an immune reaction or foreign body reaction, and since the degradation time using collagenase can be controlled from a short time to a long time as described above, the tissue supplement of the present invention can be customized to have different degradation times depending on the part of the human body to which it is applied.

[0135] The above preparation step is a step of preparing tissues of mammals other than humans. Here, mammals are mammals other than humans, such as pigs, horses, cattle, sheep, etc., and the said tissues may be one or more of mammalian-derived skin, cartilage, ligaments, tendons, myocardial fascia, pericardium, small intestine, bladder, liver, heart, lungs, pancreas, spleen, kidneys, stomach, bronchi, uterus, placenta, and nerves.

[0136] To manufacture decellularized tissue supplements, mammalian tissue is first harvested. During the preparation stage, the harvested mammalian tissue is prepared by removing useless tissue and blood attached to it, and then washing, drying, and cutting it. The prepared mammalian tissue can be stored in a frozen state and thawed for use when required. When using frozen tissue raw materials, the preparation stage may involve removing the frozen tissue raw materials and preparing them.

[0137] Next, the mammalian tissue prepared in the preparation stage is pretreated through the pretreatment stage.

[0138] This step is for washing mammalian tissues, and if the mammalian tissues are frozen, they can be thawed, washed, and cut through this step. Distilled water, purified water, saline solution, etc., may be used as the washing water in this step, and preferably, distilled water may be used.

[0139] In addition, physical stirring may be performed from this stage to the second inactivation stage, and it is preferable that the physical stirring be performed at a stirring speed of less than 150 rpm. This is because if the physical stirring speed is 150 rpm or higher, the tissue may be damaged and become difficult to use.

[0140] Mammalian tissues prepared through this pretreatment step undergo a first inactivation step, a first decellularization step, a second decellularization step, and a second inactivation step, through which cells, crude fats, viruses, and other foreign substances that may induce immune and foreign body reactions can be removed.

[0141] As decellularization is achieved through such a multi-stage reaction, the decellularization efficiency and foreign substance removal efficiency at each stage are significantly improved. Consequently, the amount of tissue processed at once increases, and the processing time is shortened, resulting in improved productivity and yield of the decellularized tissue supplement. Furthermore, by slightly altering the multi-stage reaction conditions, the degradation time can be adjusted from tens to hundreds of hours.

[0142] After the pretreatment step, a first inactivation step is performed, and an additional step to remove the epidermis or basement membrane may be performed between the two steps. This step can be performed by enzymatically treating the tissue or physically separating it.

[0143] The first inactivation step described above is a step of inactivating viruses contained in pre-treated tissue using alcohol.

[0144] This step is performed to more effectively remove cells, crude fat, and foreign substances during the primary and secondary decellularization steps by first inactivating viruses within the tissue before decellularizing the tissue.

[0145] The alcohol used in this step may be n-propanol, and to obtain a virus inactivation effect, it is preferable to use an aqueous solution of n-propanol at a concentration of 50 to 90 percent, and more preferably, an aqueous solution of n-propanol at a concentration of 65 to 80 percent may be used.

[0146] Generally, ethanol is used for virus inactivation, but in this invention, n-propanol is used as the alcohol for virus inactivation in the first inactivation step. When n-propanol is used for virus inactivation in this way, the tissue becomes softer, and as a result, it acquires strength, elasticity, and texture similar to connective tissue such as actual human skin.

[0147] In addition, if the tissue is soft, the treatment solution penetrates better into the tissue, resulting in the effect of performing virus inactivation and decellularization uniformly throughout. Consequently, the efficiency of inactivation and decellularization treatments is improved, more uniform tissue can be obtained, and the quality and reliability of the finally obtained decellularized tissue supplement can be enhanced.

[0148] Ethanol has the characteristic of fixing and hardening tissues. However, if ethanol is used at this stage, the tissue becomes hard, leading to a problem where the difference in appearance or texture between the tissue and human connective tissue increases, and the process efficiency of improving the penetration of the treatment material is reduced by loosening the tissue in subsequent stages. Additionally, since iso-propanol has a low virus inactivation effect, if iso-propanol is used in the first inactivation stage, a sufficient virus inactivation effect is not obtained, resulting in reduced decellularization efficiency in the subsequent first and second decellularization stages; therefore, it is preferable to use n-propanol.

[0149] At this stage, the pretreated tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the aqueous propanol solution.

[0150] The above first decellularization step is a step of removing cells by treating the virus-inactivated tissue, which has undergone the first inactivation step, with an aqueous base solution.

[0151] This step comprises a first decellularization step of treating tissue using a mixture of an aqueous base solution and an alcohol; and a second decellularization step of treating tissue using an aqueous base solution. Here, the aqueous base solution is an aqueous sodium hydroxide solution, and it is preferable to use n-propanol as the alcohol; in this case, the first decellularization step is a step performed by treating the tissue with a mixture of n-propanol and sodium hydroxide, and the second decellularization step is a step performed by treating the tissue with an aqueous sodium hydroxide solution.

[0152] When the decellularization step is performed in two stages as described above, the first decellularization stage removes not only cells but also foreign substances such as crude fat, allowing for a more effective decellularization process in the second stage.

[0153] Furthermore, at this stage, physical strengths such as tensile strength and suture strength, as well as the degradation rate, can be controlled depending on the processing conditions. In particular, this invention does not use a separate crosslinking agent throughout the entire process. Since tensile strength and suture strength can be controlled by partially modifying processing conditions without using a crosslinking agent, there is an advantage in being able to manufacture a tissue filler with desired strength and degradation rate in a simpler and more efficient manner.

[0154] In each of the first and second decellularization steps, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the treatment solution. This is close to approximately twice the conventional treatment capacity, and since effective decellularization is possible through the multi-stage method of this invention, it has the effect of sufficiently treating twice the amount of tissue compared to the conventional method.

[0155] Specifically, the first decellularization step involves treating virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide. In this step, crude fat within the tissue undergoes a saponification reaction with sodium hydroxide, thereby separating and removing the crude fat from the tissue. During this process, n-propanol promotes this saponification reaction and acts to aggregate the products of the reaction, allowing the crude fat to be separated and removed from the tissue more quickly and efficiently.

[0156] At this time, the treatment time for treating the tissue with a mixture of n-propanol and sodium hydroxide may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C, and when treated for the above time at this temperature range, sufficient crude fat removal efficiency can be obtained without significant damage to the tissue structure.

[0157] The above mixture of n-propanol and sodium hydroxide may have an n-propanol concentration of 50 to 95%, preferably 60 to 90%, more preferably 65 to 85%, and may be an aqueous solution of sodium hydroxide with a concentration of 0.05 M or higher and less than 0.5 M. These concentration ranges are for minimizing tissue damage and removing foreign substances such as crude fat during the first decellularization step. In particular, if the concentration of sodium hydroxide is below the above range, the treatment efficiency of the first decellularization step decreases, and if it exceeds the above range, there is a problem of the tissue structure itself collapsing and dissolving. Therefore, it is preferable to use an aqueous solution of n-propanol and sodium hydroxide within the above concentration ranges.

[0158] The aforementioned second decellularization step involves treating the tissue that has undergone the first decellularization step with an aqueous alkaline solution to soften the tissue, thereby loosening its structure while simultaneously allowing the decellularization reaction to occur. Crude fat and various foreign substances contained in the tissue act as physical and chemical barriers to the decellularization reaction; however, since the tissue has already undergone the first decellularization step to remove crude fat and various foreign substances, the decellularization reaction can be carried out more effectively in the second decellularization step.

[0159] At this stage, the treatment time for treating the tissue in an aqueous alkaline solution may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C, and when treated for the above time at this temperature range, the tissue structure is sufficiently loosely deformed without collapse of the tissue structure or loss of elastin, and an appropriate decellularization reaction can be achieved.

[0160] The aqueous base solution used as the treatment solution at this time may be an aqueous sodium hydroxide solution, and the concentration of sodium hydroxide contained in the aqueous sodium hydroxide solution may be 0.05M or higher and less than 0.5M. If the concentration of sodium hydroxide is less than 0.05M, the tissue structure is not sufficiently loosened, which causes a problem of reduced decellularization efficiency in the secondary decellularization step described later; and if it is 0.5M or higher, some tissues may disintegrate or dissolve beyond the level of loosening in this step, which may significantly reduce the final yield, so it is desirable to use an aqueous sodium hydroxide solution of the aforementioned concentration.

[0161] After the first decellularization step, a second decellularization step using enzymes is performed, and between these two steps, a neutralization step and a washing step may be performed.

[0162] The neutralization step is a step of treating the sodium hydroxide used in the first decellularization step with an acidic aqueous solution to neutralize it. The type of acidic aqueous solution used at this time may include, for example, an acidic aqueous solution containing at least one of hydrochloric acid, sulfuric acid, acetic acid, and peracetic acid, but is not limited thereto, and the concentrations thereof may also be appropriately adjusted to suit the working environment or conditions.

[0163] The washing step is a step performed to prevent a decrease in enzyme reactivity due to residues when enzymatically treating in the subsequent secondary decellularization step. A buffer solution may be used as the washing solution in the washing step, for example, a PBS (Phosphate buffered saline) solution may be used, but is not limited thereto.

[0164] Meanwhile, the above-mentioned second decellularization step is a step of removing cells by enzymatically treating the first decellularized tissue, and a step of removing DNA within the first decellularized tissue by treating the first decellularized tissue with a DNA degrading enzyme.

[0165] At this stage, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of a treatment solution containing DNA degrading enzymes. This is a treatment capacity nearly double that of the conventional method, as previously explained, and is achievable because the treatment efficiency is increased by the multi-stage decellularization process of the present invention.

[0166] In this step, to obtain enzyme activity and sufficient DNA degradation efficiency, the treatment time for treating the tissue with a DNA degrading enzyme may be 10 to 35 hours, preferably 18 to 30 hours, and the treatment temperature may be 30 to 45°C, preferably 35 to 42°C.

[0167] In this invention, the concentration of DNA degrading enzyme included in the treatment solution may be less than 0.03 wt%, which is at least several times lower than the concentration of DNA degrading enzymes used conventionally. In the primary decellularized tissue of this invention, foreign substances including crude fat within the tissue are removed and the tissue structure is loosened as the primary decellularization step is performed. When DNA degrading enzyme is introduced in this state, the DNA degrading enzyme can penetrate very easily into the tissue structure. Accordingly, even when using a low concentration of DNA degrading enzyme, at least the same or superior DNA removal efficiency can be secured; therefore, in this invention, such a low concentration of DNA degrading enzyme can be used.

[0168] However, when the concentration of DNA degrading enzyme is 0.03 wt% or higher, the improvement in DNA degradation efficiency is extremely minimal compared to the amount of additional DNA degrading enzyme, making it uneconomical; therefore, it is preferable to use DNA degrading enzyme within the concentration range described above.

[0169] The tissue decellularized through the above second decellularization step undergoes a second inactivation step to remove additional viruses, and between these two steps, an additional decolorization step and / or degreasing step may be performed.

[0170] The decolorization step is a step for removing the color of the tissue, which can be performed, for example, by treating the tissue with hydrogen peroxide, and the degreasing step is a step for further removing lipids contained in the tissue, which can be performed, for example, using a ketone solution, preferably an acetone solution.

[0171] Next, a second inactivation step is performed to further inactivate the viruses contained within the tissue that has undergone the second decellularization step. This step involves inactivating the viruses within the tissue using acid, specifically treating the tissue with a mixture of organic acid and alcohol.

[0172] The organic acid used at this stage may be peracetic acid, which acts as an oxidizing agent and can inactivate viruses by causing non-specific free radical damage. The peracetic acid used in this step can be used at a concentration of 0.05 to 1%.

[0173] In addition, the alcohol used in this step may be a lower alcohol having 1 to 4 carbon atoms, such as methanol, ethanol, or propanol, and preferably ethanol. It is desirable to use ethanol because it performs the function of inactivating viruses and fixing tissue structures to make the tissues firm.

[0174] Through these steps, the virus is finally inactivated, crude fat and DNA are removed, and a tissue supplement containing various extracellular matrix components can be obtained.

[0175] The DNA content contained in the tissue supplement obtained in this way is 50 ng / mg or less, which is a very low DNA content, so the induction of immunity or various foreign body reactions can be minimized.

[0176] After the second inactivation step mentioned above, additional steps such as washing, processing, packaging, and sterilization may be performed.

[0177] Meanwhile, the decellularized protein extract is prepared through the following steps: a preparation step of preparing mammalian tissue excluding humans; a pretreatment step of pretreating the tissue; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0178] At this time, the DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less. In addition, the reduction rate (L) of the elastin content of the decellularized protein extract that has undergone the pretreatment step to the second inactivation step is 20% or less.

[0179] Here, the reduction rate (L) is defined by the following formula (1), where L0 represents the elastin content of the tissue during the preparation stage, and L f represents the elastin content of the decellularized protein extract that has undergone the second inactivation step.

[0180]

[0181] First, the above preparation step is a step of preparing tissues of mammals other than humans. Here, mammals are mammals other than humans, such as pigs, horses, cattle, sheep, etc., and the tissues may be one or more of blood vessels, ligaments, and tendons derived from these mammals.

[0182] To prepare decellularized protein extracts, mammalian tissue is first collected. During the preparation stage, the collected mammalian tissue is prepared by removing useless tissue and blood attached to it, and then washing, drying, and cutting it. The prepared mammalian tissue can be stored in a frozen state and thawed for use when required. When using frozen tissue raw materials, the preparation stage may involve removing the frozen tissue raw materials and preparing them.

[0183] Next, the mammalian tissue prepared in the preparation stage is pretreated through the pretreatment stage.

[0184] This step is for washing mammalian tissue; if the mammalian tissue is frozen, it can be thawed, washed, and cut through this step. In this step, distilled water, purified water, saline solution, etc., may be used as the washing water, and preferably, distilled water may be used. Physical stirring may be performed from this step to the second inactivation step.

[0185] Mammalian tissues prepared through this pretreatment step undergo a first inactivation step, a first decellularization step, a second decellularization step, and a second inactivation step, through which cells, crude fats, viruses, and other foreign substances that may induce immune and foreign body reactions can be removed. By decellularizing through such a multi-step process, the decellularization efficiency and foreign substance removal efficiency at each step are significantly improved, thereby increasing the amount of tissue processed at once and shortening the processing time, which consequently results in improved productivity and yield of decellularized protein extracts.

[0186] First, the first inactivation step is a step of inactivating viruses contained in pre-treated tissue using alcohol.

[0187] This step is performed to more effectively remove cells, crude fat, and foreign substances during the primary and secondary decellularization steps by first inactivating viruses within the tissue before decellularizing the tissue.

[0188] The alcohol used in this step may be n-propanol, and to obtain a virus inactivation effect, it is preferable to use an aqueous solution of n-propanol at a concentration of 50 to 90 percent, and more preferably, an aqueous solution of n-propanol at a concentration of 65 to 80 percent may be used.

[0189] In the present invention, n-propanol is used as the alcohol for virus inactivation in the first inactivation step. When n-propanol is used for virus inactivation in this manner, the tissue becomes softer; since soft tissue allows the treatment solution to penetrate the tissue more effectively, the effect of performing virus inactivation and decellularization uniformly throughout is obtained. Consequently, the efficiency of inactivation and decellularization treatment is improved, a more uniform tissue can be obtained, and the quality and reliability of the finally obtained decellularized protein extract can be enhanced.

[0190] Generally, when ethanol is used for virus inactivation, it has the characteristic of fixing and hardening mammalian tissue to be decellularized; however, if ethanol is used instead of n-propanol in this step of the present invention, the tissue becomes hardened, which is undesirable because it reduces the efficiency of the process that loosens the tissue in subsequent steps to improve the penetration power of the treatment material.

[0191] In addition, since iso-propanol has a low viral inactivation effect, if iso-propanol is used in the first inactivation step, a sufficient viral inactivation effect is not obtained, and the decellularization efficiency decreases in the subsequent first and second decellularization steps, so it is preferable to use n-propanol in the first inactivation step.

[0192] At this stage, the pretreated tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the aqueous propanol solution.

[0193] The above first decellularization step is a step of removing cells by treating the virus-inactivated tissue, which has undergone the first inactivation step, with an aqueous base solution.

[0194] This step comprises a first decellularization step of treating tissue using a mixture of an aqueous base solution and an alcohol; and a second decellularization step of treating tissue using an aqueous base solution. Here, the aqueous base solution is an aqueous sodium hydroxide solution, and it is preferable to use n-propanol as the alcohol; in this case, the first decellularization step is a step performed by treating the tissue with a mixture of n-propanol and sodium hydroxide, and the second decellularization step is a step performed by treating the tissue with an aqueous sodium hydroxide solution.

[0195] When the decellularization step is performed in two stages as described above, the first decellularization stage removes not only cells but also foreign substances such as crude fat, allowing for a more effective decellularization process in the second stage.

[0196] In each of the first and second decellularization steps, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the treatment solution. This is close to approximately twice the conventional treatment capacity, and since effective decellularization treatment is possible through the multi-stage method of the present invention, it has the effect of sufficiently treating twice the amount of tissue compared to the conventional method.

[0197] Specifically, the first decellularization step involves treating virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide. In this step, crude fat within the tissue undergoes a saponification reaction with sodium hydroxide, thereby separating and removing the crude fat from the tissue. During this process, n-propanol promotes this saponification reaction and acts to aggregate the products of the reaction, allowing the crude fat to be separated and removed from the tissue more quickly and efficiently.

[0198] At this time, the treatment time for treating the tissue with a mixture of n-propanol and sodium hydroxide may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C. When treated for the above time within this temperature range, sufficient crude fat removal efficiency can be obtained without significant damage to the tissue structure or loss of elastin.

[0199] The above mixture of n-propanol and sodium hydroxide may have an n-propanol concentration of 50 to 95%, preferably 60 to 90%, more preferably 65 to 85%, and may be an aqueous solution with a sodium hydroxide concentration greater than 0.05 M and less than 0.2 M. These concentration ranges are ranges in which tissue damage can be minimized in the first decellularization step and foreign substances such as crude fat can be effectively removed at the same time. In particular, if the concentration of sodium hydroxide is below the above range, the processing efficiency of the first decellularization step decreases, and if it exceeds the above range, there is a problem of the tissue structure itself collapsing and dissolving; therefore, it is preferable to use an aqueous solution of n-propanol and sodium hydroxide within the above concentration ranges.

[0200] The aforementioned second decellularization step involves treating the tissue that has undergone the first decellularization step with an aqueous alkaline solution to soften the tissue, thereby loosening its structure while simultaneously allowing the decellularization reaction to occur. Crude fat and various foreign substances contained in the tissue act as physical and chemical barriers to the decellularization reaction; however, since the tissue has already undergone the first decellularization step to remove crude fat and various foreign substances, the decellularization reaction can be carried out more effectively in the second decellularization step.

[0201] At this stage, the treatment time for treating the tissue in an aqueous alkaline solution may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C, and when treated for the above time at this temperature range, the tissue structure is sufficiently loosely deformed without collapse of the tissue structure or loss of elastin, and an appropriate decellularization reaction can be achieved.

[0202] The aqueous base solution used as the treatment solution at this time may be an aqueous sodium hydroxide solution, and the concentration of sodium hydroxide contained in the aqueous sodium hydroxide solution may be greater than 0.05 M and less than 0.2 M. If the concentration of sodium hydroxide is 0.05 M or less, the tissue structure is not sufficiently loosened, which causes a problem of reduced decellularization efficiency in the secondary decellularization step described later; and if it is 0.2 M or more, some tissues may disintegrate or dissolve beyond the level of loosening in this step, resulting in a significant decrease in the final yield and loss of elastin, so it is desirable to use an aqueous sodium hydroxide solution of the aforementioned concentration.

[0203] After the first decellularization step, a second decellularization step using enzymes is performed, and between these two steps, a neutralization step and a washing step may be performed.

[0204] The neutralization step is a step of treating the sodium hydroxide used in the first decellularization step with an acidic aqueous solution to neutralize it. The type of acidic aqueous solution used at this time may include, for example, an acidic aqueous solution containing at least one of hydrochloric acid, sulfuric acid, acetic acid, and peracetic acid, but is not limited thereto, and the concentrations thereof may also be appropriately adjusted to suit the working environment or conditions.

[0205] The washing step is a step performed to prevent a decrease in enzyme reactivity due to residues when enzymatically treating in the subsequent secondary decellularization step. A buffer solution may be used as the washing solution in the washing step, for example, a PBS (Phosphate buffered saline) solution may be used, but is not limited thereto.

[0206] Meanwhile, the above-mentioned second decellularization step is a step of removing cells by enzymatically treating the first decellularized tissue, and a step of removing DNA within the first decellularized tissue by treating the first decellularized tissue with a DNA degrading enzyme.

[0207] At this stage, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of a treatment solution containing DNA degrading enzymes. This is a treatment capacity nearly double that of the conventional method, as previously explained, and is achievable because the treatment efficiency is increased by the multi-stage decellularization process of the present invention.

[0208] In this step, to obtain enzyme activity and sufficient DNA degradation efficiency, the treatment time for treating the tissue with a DNA degrading enzyme may be 10 to 35 hours, preferably 18 to 30 hours, and the treatment temperature may be 30 to 45°C, preferably 35 to 42°C.

[0209] In the present invention, the concentration of DNA degrading enzyme included in the treatment solution is 0.0001 to 0.005 wt%, which is several to hundreds of times lower than the concentration of DNA degrading enzyme typically used in conventional similar technologies. In the primary decellularized tissue of the present invention, foreign substances including crude fat within the tissue are removed and the tissue structure becomes loose as the primary decellularization step is performed. When DNA degrading enzyme is introduced in this state, the DNA degrading enzyme can penetrate very easily into the tissue structure. Accordingly, since DNA removal efficiency that is at least the same or superior can be secured even when using a low concentration of DNA degrading enzyme, the present invention allows for the use of such a low concentration of DNA degrading enzyme.

[0210] However, if the concentration of the DNA degrading enzyme is below the above range, the DNA degradation efficiency decreases, so it is desirable to use it at a concentration of 0.0001% or higher. If it exceeds the above range, the improvement in DNA degradation efficiency is extremely minimal compared to the amount of additional DNA degrading enzyme, making it uneconomical. Therefore, it is desirable to use the DNA degrading enzyme within the concentration range described above.

[0211] The tissue decellularized through the above second decellularization step undergoes a second inactivation step to further remove viruses, and between these two steps, a decolorization step and / or a degreasing step may be additionally performed. The decolorization step is a step to remove the color of the tissue, which can be performed, for example, by treating the tissue with hydrogen peroxide, and the degreasing step is a step to further remove lipids contained in the tissue, which can be performed, for example, using a ketone solution, preferably an acetone solution.

[0212] Next, a second inactivation step is performed to further inactivate the viruses contained within the tissue that has undergone the second decellularization step. This step involves inactivating the viruses within the tissue using acid, specifically treating the tissue with a mixture of organic acid and alcohol.

[0213] The organic acid used at this stage may be peracetic acid, which acts as an oxidizing agent and can inactivate viruses by causing non-specific free radical damage. The peracetic acid used in this step can be used at a concentration of 0.05 to 1%.

[0214] In addition, the alcohol used in this step may be a lower alcohol having 1 to 4 carbon atoms, such as methanol, ethanol, or propanol, and preferably ethanol. It is desirable to use ethanol because it performs the function of inactivating viruses and fixing tissue structures to make the tissues rigid.

[0215] Through these steps, the virus is finally inactivated, crude fat and DNA are removed, and a decellularized protein extract containing various extracellular matrix components, including elastin, can be obtained.

[0216] The DNA content contained in the decellularized protein extract obtained in this way is 50 ng / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less, so that the DNA content is very low, so that the induction of immunity or various foreign body reactions can be minimized.

[0217] In addition, the above-mentioned decellularized protein extract has the advantage of having a very low elastin content reduced through the pretreatment step to the second inactivation step, thereby providing effects such as wound healing and scar suppression by elastin. Specifically, the reduction rate (L) of the elastin content of the tissue that has undergone the pretreatment step to the second inactivation step is 20% or less. This numerical value is very low compared to the case where the reduction rate (L) of the elastin content is very high, at 50% or more, when a conventional decellularization method is applied.

[0218] Meanwhile, after the second inactivation step, additional steps such as washing, packaging, sterilization, and processing may be performed. Washing may be performed using a buffer solution, distilled water, etc., and packaging is performed to prevent contamination and facilitate handling when the decellularized protein extract is stored or transported.

[0219] Hereinafter, the specific operation and effects of the present invention will be explained through one embodiment of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited according to the embodiment.

[0220] [Preparation Example 1]

[0221] 1. Preparation and Preprocessing Steps

[0222] Pork skin that had been washed, dried, cut, and frozen was prepared, thawed, washed with distilled water, and then immersed in an enzyme treatment solution containing 0.1 wt% lipase and 1 M NaCl at 25°C for 2 hours to prepare a raw tissue specimen (pork dermis) with the epidermis and basement membrane removed.

[0223] 2. First inactivation step

[0224] Next, 230g of raw tissue specimen was added to 1L of 70% n-propanol and stirred for 24 hours at a temperature of 20℃ and a stirring speed of 50RPM to perform the first inactivation step.

[0225] 3-1. Primary Decellularization Stage (First Decellularization Stage)

[0226] Next, a mixed solution containing n-propanol at a concentration of 70% and sodium hydroxide at a concentration of 0.1M was prepared in distilled water, and the raw tissue specimen was mixed in such a way that 22 parts by weight were included in 100 parts by weight of the mixed solution, and then the first decellularization step was performed by stirring at a temperature of 20℃ and a stirring speed of 50 RPM for 24 hours.

[0227] 3-2. Primary Decellularization Stage (Second Decellularization Stage)

[0228] Next, 24 parts by weight of the tissue specimen that had undergone the first decellularization step were mixed with 100 parts by weight of a pre-prepared 0.1M aqueous sodium hydroxide solution, and then the second decellularization step was performed by stirring for 24 hours under the same conditions as the previous step.

[0229] The tissue specimen that had undergone the second decellularization step was subsequently immersed in an aqueous acetic acid solution and stirred to neutralize it, after which it was washed with a PBS solution.

[0230] 4. Secondary decellularization stage

[0231] Next, the tissue specimen was placed in a treatment solution containing DNase at a concentration of 0.003 wt%, and a second decellularization step was performed by stirring at a stirring speed of 50 RPM for 22 hours at a temperature of 37°C. The tissue specimen, after the second decellularization step was completed, was decolorized by treating it in a 3% hydrogen peroxide solution at 20°C for 1 hour, and degreased by treating it in a 20% aqueous acetone solution at 20°C for 1 hour.

[0232] 5. Second inactivation step

[0233] Next, the tissue specimen was immersed in a mixed solution containing 70% n-propanol and 0.2% peracetic acid in distilled water, and treated at 20°C for 4 and a half hours to perform a second inactivation step.

[0234] Finally, the tissue specimen that had undergone all of the above processing steps was washed with PBS solution and distilled water to prepare the decellularized tissue supplement of Example 1.

[0235] [Experimental Example 1]

[0236] Tissue supplements of Comparative Examples 1 to 4 were prepared by using the same method as in Preparation Example 1, but without performing a second decellularization step, and by varying the concentration of sodium hydroxide in the first decellularization step. Then, H&E stained photographs of Example 1 and Comparative Examples 1 to 4 were taken and shown in FIG. 2, and the DNA and crude fat content were measured and the results were listed in Table 1.

[0237] DNA content was determined by performing the experiment according to the protocol of the analysis kit using the analysis kit Quant-iT™ PicoGreen™ dsDNA Assay kits and dsDNA Reagents (Manufacturer: Invitrogen), and then measuring the DNA content at absorbances of 480–520 mm using a Multimode plate reader Victor Nivo™ (Manufacturer: Perkin Elmer).

[0238] Crude fat content was measured using the Sohxlet extraction method from ISO 1444 Methods of test for Meat and meat products-Part 5. Determination of free fat content.

[0239]

[0240] Referring to Table 1 and Figure 2, it can be seen that when both the first and second decellularization steps are performed, as in Example 1, no cell nuclei are observed, and the DNA and crude fat content are very low. On the other hand, when the second decellularization step is not performed, as in Comparative Examples 1 to 4, decellularization proceeds to a certain level as the concentration of sodium hydroxide contained in the treatment solution increases, but overall decellularization does not proceed completely, so a large number of cell nuclei are found (refer to the parts indicated by arrows in the photos of Comparative Examples 1 to 4 in Figure 1), and the DNA content is also confirmed to be high. Therefore, considering the above experimental results, it can be confirmed that both the first and second decellularization processes are necessary.

[0241] [Experimental Example 2]

[0242] Tissue fillers were prepared using the same method as in the preparation example, but with varying concentrations of sodium hydroxide during the first decellularization step. After preparing the tissue fillers, H&E stained photographs of each tissue filler were taken and shown in Figure 3. Collagen degradation time, tensile strength, suture strength, DNA, and crude fat content were measured and listed in Table 2.

[0243] Collagenase degradation time was determined by preparing 0.5 x 0.5 cm tissue filler specimens, treating each sample with 1 ml of collagenase diluted to 100 U / ml, and measuring the time required for the tissue filler specimen to be completely degraded. Three experiments were performed for each tissue filler, and the average value was determined as the collagenase degradation time.

[0244] Tensile strength was measured using the test method based on the ASTM D638 standard. Specimens were cut to fit a Dog-bone type 5, and measurements were taken by clamping the specimens in jigs spaced 2.54 cm apart and tensile-stretching them at a speed of 10 mm / min. The test was performed using five specimens prepared for each tissue filler.

[0245] Suture strength was measured in accordance with the ISO 7198 standard. Specimens were prepared by cutting them to a size of 2 x 4 cm. After inserting the suture thread vertically through the end of the specimen at a position 2 mm away, the specimen and suture thread were clamped in each jig and pulled at a speed of 50 to 200 mm / min to measure the strength. Five specimens were prepared for each tissue filler to measure the suture strength, and the average of the measured values ​​was determined as the suture strength of the corresponding tissue filler.

[0246]

[0247] Referring to the experimental results in Table 2 and Figure 3 above, it can be seen that mechanical properties, suture strength, and resistance to collagenase degradation decrease as the concentration of sodium hydroxide increases during the primary decellularization stage. Additionally, it can be seen that the DNA content decreases with increasing sodium hydroxide concentration, thereby increasing decellularization efficiency, and that the DNA content in all experimental groups is 50 ng / mg or less. Generally, different degradation periods and mechanical properties are required depending on the site where the tissue supplement is implanted. From the experimental results above, it was confirmed that tensile strength, suture strength, and collagenase degradation time can be controlled by varying the NaOH concentration during the primary decellularization stage. Thus, it was confirmed that the tissue supplement according to this invention can be manufactured as a customized product for implantation sites requiring different properties and degradation periods.

[0248] In addition, no tissue breakdown was observed when the concentration of sodium hydroxide was 0.05–0.2 M, but tissue breakdown was observed when 0.5 M was used. Therefore, from the above experimental results, it was confirmed that in order to obtain a tissue supplement with appropriate strength and degradation time and sufficient decellularization, the concentration of sodium hydroxide in the first decellularization step should be 0.05 M or higher and less than 0.5 M.

[0249] [Experimental Example 3]

[0250] A tissue supplement was prepared using the same method as in the preparation example, but using ethanol as the alcohol in the first decellularization step to prepare the tissue supplement of Comparative Example 5.

[0251] Subsequently, the tensile stress according to strain of the tissue filler of Example 1 and Comparative Example 5 was measured, the results are shown in FIG. 4, and the modulus (slope) values ​​were calculated and listed in Table 3.

[0252]

[0253] Referring to the results in Table 3 and Figure 4 above, it was confirmed that Example 1 exhibited higher tensile strength and strain than Comparative Example 5, indicating superior mechanical properties. Additionally, regarding the slope indicating softness, Example 1 was gentler than Comparative Example 5, confirming that Example 1 is a softer material, i.e., a material more similar to the human body. Therefore, based on the experimental results, it was confirmed that n-propanol is preferable to be used as the alcohol included in the treatment solution during the first decellularization step.

[0254] [Experimental Example 4]

[0255] Tissue supplements were prepared using the same method as in the preparation example, but the concentration of sodium hydroxide used in the first decellularization step was changed to 0.05 M, and the stirring speed in the entire process was changed to 50 RPM and 150 RPM, respectively, to prepare the tissue supplements of Example 2 and Comparative Example 6.

[0256] Afterwards, H&E stained photographs of the two tissue fillers were taken and shown in Fig. 5, and collagenase degradation time, tensile strength, DNA content, and crude fat content were measured in the same manner as in Experimental Example 2, and the results were listed in Table 4.

[0257]

[0258] Referring to Table 4 and Figure 5, it was confirmed that tissue damage worsened as the stirring speed increased, and consequently, mechanical properties decreased, degradation time decreased, and DNA removal efficiency actually declined. Therefore, based on the experimental results, it was confirmed that high stirring speeds cause physical tissue damage during the manufacturing process of tissue supplements, and specifically, it was confirmed that it is desirable to perform the stirring speed at less than 150 RPM throughout the entire process.

[0259] [Preparation Example 2]

[0260] 1. Preparation and Preprocessing Steps

[0261] A porcine heart aorta that had been washed, dried, cut, and frozen was prepared, and after thawing, washed with distilled water and cut into 50×50 mm pieces to prepare raw tissue specimens.

[0262] 2. First inactivation step

[0263] Next, 250g of raw tissue specimens were added to 1L of 70% n-propanol and stirred at a temperature of 20℃ and a stirring speed of 150RPM for 24 hours to perform the first inactivation step.

[0264] 3-1. Primary Decellularization Stage (First Decellularization Stage)

[0265] Next, a mixed solution containing 70% n-propanol and 0.1M sodium hydroxide in distilled water was prepared, and the raw tissue specimen was mixed in such a way that 23 parts by weight were included in 100 parts by weight of the mixed solution, and then the first decellularization step was performed by stirring at a temperature of 20℃ and a stirring speed of 150 RPM for 24 hours.

[0266] 3-2. Primary Decellularization Stage (Second Decellularization Stage)

[0267] Next, 24 parts by weight of the tissue specimen that had undergone the first decellularization step were mixed with 100 parts by weight of a pre-prepared 0.1M aqueous sodium hydroxide solution, and then the second decellularization step was performed by stirring for 24 hours under the same conditions as the previous step.

[0268] The tissue specimen that had undergone the second decellularization step was subsequently immersed in an aqueous acetic acid solution and stirred to neutralize it, after which it was washed with a PBS solution.

[0269] 4. Secondary decellularization stage

[0270] Next, the tissue specimen was placed in a treatment solution containing DNase at a concentration of 0.00011 wt%, and a second decellularization step was performed by stirring at a stirring speed of 150 RPM for 23 hours at a temperature of 37°C. The tissue specimen, after the second decellularization step was completed, was decolorized by treating it in a 3% hydrogen peroxide solution at 20°C for 1 hour, and degreased by treating it in a 20% aqueous acetone solution at 20°C for 1 hour.

[0271] 5. Second inactivation step

[0272] Next, the tissue specimen was immersed in a mixed solution containing 70% n-propanol and 0.2% peracetic acid in distilled water, and treated at 20°C for 4 and a half hours to perform a second inactivation step.

[0273] Finally, the tissue specimen that had undergone all of the above processing steps was washed with PBS solution and distilled water to prepare a decellularized protein extract according to one embodiment of the present invention.

[0274] [Experimental Example 5]

[0275] A decellularized protein extract was prepared using the same method as in Preparation Example 2, but the first, second, and second decellularization steps were performed sequentially. A tissue specimen was collected immediately after each step was performed, and the elastin content per 1 mg of tissue specimen was measured, and the results are shown in Fig. 6(A). In addition, the elastin content of the original tissue was measured during the preparation step, and the elastin content at the time each step was performed was expressed as a percentage relative to the elastin content of the original tissue, as shown in Fig. 6(B).

[0276] In FIGS. 6(A) and FIGS. 6(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0277] The elastin content was determined by performing the experiment according to the protocol of the analysis kit using the Fastin Elastin Assay kit F2000 (Manufacturer: Biocolor), analyzing the elastin at an absorbance of 513 mm using a Multimode plate reader Victor Nivo (Manufacturer: Perkin Elmer), and measuring the elastin content by comparing it with a standard material.

[0278] Referring to the results in Figures 6(A) and 6(B), it can be seen that the elastin content is slightly lost as each process proceeds, but at the time the final process is completed, more than 80% of the elastin remains.

[0279] [Experimental Example 6]

[0280] A decellularized protein extract was prepared using the same method as in Preparation Example 2, but with the first, second, and second decellularization steps each omitted by only one step, and a decellularized protein extract specimen was collected after going through the final process. The elastin content per 1 mg of the decellularized protein extract specimen was measured, and the elastin content of the original tissue was measured in the same way during the preparation stage. Then, the L value according to Formula (1) was calculated and the results were listed in Table 5.

[0281] In addition, regarding the elastin content of the original tissue in the preparation stage, the elastin content contained in the decellularized protein extract obtained by omitting each step was calculated as a percentage, and the results are shown in Fig. 7.

[0282] The elastin content was measured in the same manner as in Experimental Example 5, and in Table 5 and Figure 7, Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0283]

[0284] First, looking at Table 5, it can be seen that the L value is 20% or less in both cases where all processes are performed and when one process is omitted, and referring to Figure 7, it can be seen that the elastin content does not change significantly even if a specific process is omitted. Therefore, from the above experimental results, it was confirmed that the elastin content is not significantly affected even if any one of the first decellularization step, the second decellularization step, and the second decellularization step is omitted in the process according to one embodiment of the invention.

[0285] [Experimental Example 7]

[0286] The experiment was conducted in the same manner as in Experimental Example 5, but DNA content was measured instead of elastin content after each step. DNA content was measured using the analysis kit Quant-iT™ PicoGreen™ dsDNA Assay kits and dsDNA Reagents (Manufacturer: Invitrogen) according to the analysis kit protocol, and then the DNA content was measured at an absorbance of 480–520 mm using a Multimode plate reader Victor Nivo™ (Manufacturer: Perkin Elmer).

[0287] The DNA content per 1 mg of tissue after each step is shown in Fig. 8(A), and the DNA content of the tissue after each step relative to the DNA content of the original tissue is shown as a percentage value in Fig. 8(B). In Fig. 8(A) and Fig. 8(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0288] Referring to Figures 8(A) and 8(B), it can be seen that most of the DNA is removed after undergoing the first decellularization step, the second decellularization step, and the second decellularization step.

[0289] [Experimental Example 8]

[0290] The experiment was conducted in the same manner as Experimental Example 6, but with one step omitted for each, and the DNA content of the tissue matrix prepared was measured in the same manner as Experimental Example 7.

[0291] The DNA content per 1 mg of tissue matrix obtained by omitting each step is shown in Fig. 9(A), and the DNA content of the tissue obtained by omitting each step relative to the DNA content of the original tissue is shown as a percentage value in Fig. 9(B). In this case, in Figs. 9(A) and 9(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0292] Referring to Figures 9(A) and 9(B), it can be confirmed that DNA is reliably removed only after undergoing the first decellularization step, the second decellularization step, and the second decellularization step. In particular, it can be seen that the efficiency of DNA removal drops significantly when the second decellularization step or the second decellularization step is omitted; therefore, through the above experiment, it was confirmed that sequentially undergoing each step is more effective for DNA removal.

[0293] [Experimental Example 9]

[0294] A decellularized protein extract was prepared using the same method as in Preparation Example 2, but the tissue was treated by varying the concentration of sodium hydroxide included in the treatment solution to 0.005M, 0.1M, 0.2M, and 0.3M during the first decellularization step.

[0295] During the experiment, the condition of the sample during and after the second decellularization step (second process) was photographed and is shown in Fig. 10. In addition, the DNA content contained in 1 mg of the finally obtained decellularized protein extract was measured using the same method as in Experimental Example 3, and the yield of the decellularized protein extract was calculated by expressing the content of the finally obtained decellularized protein extract as a percentage value relative to the content of the initially introduced tissue, and the H&E stained photograph is shown in Fig. 10.

[0296] Referring to Figure 10 above, when the concentration of NaOH is 0.05M, the yield is good, but the DNA content is high and it is confirmed that there are cell nuclei that were not removed within the tissue, indicating that the decellularization efficiency is low.

[0297] On the other hand, when treated with 0.1M NaOH, it can be seen that the tissue structure is well maintained in the second decellularization step, the DNA content after final treatment is very low at 0.6 ng / mg, and the yield is high at 20.8%.

[0298] When treated with 0.2M NaOH, the DNA content was low, but some of the tissue was disintegrated during the first decellularization step, resulting in a final yield that was less than half of that of the case treated with 0.1M NaOH, and when treated with 0.3M NaOH, the tissue was almost dissolved, making it impossible to obtain tissue.

[0299] Therefore, from the above experimental results, it was confirmed that in order to obtain a decellularized protein extract with excellent yield and sufficient removal of DNA from the tissue, where tissue disintegration or lysis does not occur during the first decellularization step, the concentration of NaOH included in the treatment solution during the first decellularization step, particularly during the second decellularization step, is greater than 0.05M and less than 0.2M.

[0300] [Experimental Example 10]

[0301] A decellularized protein extract was prepared using the same method as in Preparation Example 2, but when the concentration of sodium hydroxide included in the treatment solution in the first decellularization step was 0.005M and 0.1M, the tissue was treated while varying the concentration of Dnase to 0.0001~0.003wt%, and then the DNA content ratio and yield of each tissue matrix obtained at the end were calculated and the results are listed in Table 6.

[0302]

[0303] Referring to the results in Table 6 above, it can be seen that when the concentration of NaOH is constant, the DNA concentration in the final decellularized protein extract decreases as the concentration of DNase increases. However, when the NaOH concentration was 0.1M, the DNA concentration in the final decellularized protein extract was actually lower despite treatment with DNase at a concentration several to tens of times lower than when it was 0.05M, and it was confirmed that the difference in yield was not significant. Experimental results showed that when the NaOH concentration was 0.05M, the DNA removal efficiency was significantly lower compared to when it was 0.1M, indicating that it is desirable for the concentration of NaOH in the treatment solution used in the first decellularization step to exceed 0.05M.

[0304] [Preparation Example 3]

[0305] First, the decellularized tissue supplement of Example 1 was mixed with an acidic aqueous solution with a pH of 2.9 and then ground into a liquid using a blender. Polyethylene glycol was heated to 45°C to melt, and the liquidized decellularized tissue supplement and polyethylene glycol were mixed to prepare a protective layer raw material mixture. Subsequently, a tray was prepared, the protective layer raw material mixture was thinly coated onto it, and then dried at 70°C for 30 minutes to produce a protective layer.

[0306] Next, a blood absorption layer raw material mixture was laminated onto a protective layer, and freeze-drying was performed by lowering the temperature from 15°C to -25°C in increments of 5°C and maintaining it for about 15 to 30 minutes after lowering, and then raising the temperature again to 25°C in increments of 5°C and maintaining it for 20 to 30 minutes after raising. The blood absorption layer raw material mixture used at this time was prepared by mixing the decellularized tissue supplement of Example 1 with an acidic aqueous solution with a pH of 2.9, grinding it with a blender to liquefy it, adding gelatin, thrombin, the decellularized protein extract of Preparation Example 2, calcium chloride, and poloxamer to the mixture, and stirring to prepare the composite hemostatic agent of Example 3.

[0307] The composite hemostatic agent of Example 3 consists of two layers, a protective layer and a blood absorption layer, wherein the protective layer comprises 45 wt% decellularized tissue filler and 55 wt% polyethylene glycol, and the blood absorption layer comprises 30 wt% decellularized tissue filler, 60 wt% gelatin, 3 wt% thrombin, 2 wt% decellularized protein extract, 0.5 wt% calcium chloride, and 4.5 wt% poloxamer.

[0308] A photograph of the composite hemostatic agent of Example 3 manufactured here was taken and is shown in FIG. 11. The left photograph of FIG. 11 is a photograph of the individually packaged product, and the right photograph is a photograph of the composite hemostatic agent taken out of the packaging container.

[0309] [Experimental Example 11]

[0310] Scanning electron microscope images of the composite hemostatic agent of Example 3 were taken and are shown in FIG. 12. Referring to FIG. 12, it can be seen that the protective layer has a closed structure and the blood absorption layer has a porous structure.

[0311] [Experimental Example 12]

[0312] To evaluate the absorption performance of the composite hemostatic agent of Example 3, the absorption time and absorption capacity were measured. For Comparative Example 7, a conventional commercially available hemostatic agent product was used.

[0313] The absorption time was evaluated by preparing two trays, cutting the composite hemostatic agent sample of Example 3 into the same size (20×20 mm) and the composite hemostatic agent sample of Comparative Example 7 into the other tray, dispensing 100 µl of blood onto the upper layer of each sample, and visually observing the time it took for the dispensed blood to be completely absorbed by the composite hemostatic agent. The graph of the experimental results is shown in Fig. 13(A).

[0314] Additionally, in the above experiment, the weight of the initial composite hemostatic agent and the weight after absorption were measured, and the percentage of the weight after absorption compared to the initial weight was defined as the absorption capacity, and the absorption capacity graph was shown in Fig. 13(B).

[0315] In addition, an initial photograph of the experiment of Comparative Example 7 is shown in FIG. 14(A), and an initial photograph of the experiment of Example 3 is shown in FIG. 14(B). As shown in FIG. 14(A) and FIG. 14(B), in the case of Comparative Example 7, the blood aggregated into a round shape and absorption occurred slowly, whereas in Example 3, absorption occurred immediately after the blood was dispensed.

[0316] Referring to FIG. 13(A) and FIG. 13(B), Comparative Example 7 took more than 10 minutes to absorb, whereas Example 3 took 2 to 3 seconds to absorb, confirming that Example 3 could be absorbed more quickly than a commercial product and that its absorption capacity was also about 3 times better.

[0317] [Experimental Example 13]

[0318] The hemostatic performance of Example 3 and Comparative Example 7 was evaluated using the Lee-White method, and the results are shown in FIG. 15.

[0319] First, a test tube was prepared and 100 mg of the complex hemostatic agent sample was placed in it. Then, 900 μL of blood and 240 mM CaCl solution were pipetted into an Ep tube for 15 seconds to prepare the blood sample. Next, the test tube containing the complex hemostatic agent sample was laid flat, 1 mL of the blood sample was dispensed into it, and it was placed in a 37°C water bath. The tube was tilted 30 degrees every 15 seconds to observe whether coagulation occurred. The time until the blood stopped flowing even when tilted up to 180 degrees was measured and recorded.

[0320] In addition, the experimental results obtained by performing the above experiment without using a separate complex hemostatic agent—namely, the hemostatic time when blood alone was used as the hemostatic time of the control group—were used to calculate the percentage of the hemostatic time of the complex hemostatic agent relative to the control group, and the results are shown in Fig. 15. In addition, photographs of the final results of each experiment, namely the samples after coagulation was completed, are attached to Fig. 15.

[0321] Referring to Figure 15, it was confirmed that the hemostasis time was reduced to less than half when using a combined hemostatic agent compared to the blood coagulation time alone, and it was confirmed that the hemostasis of the product of Example 3 proceeded about four times faster than that of a commercial product.

[0322] Therefore, as a result of this experiment, it was confirmed that the hemostatic performance of the composite hemostatic agent according to this embodiment is significantly superior to that of commercial products.

[0323] The absorbable in vivo composite hemostatic agent according to the present invention comprises a blood-absorbing layer in the form of a porous sponge; and a non-porous protective layer laminated over the blood-absorbing layer. Since the protective layer comprises a decellularized tissue supplement and polyethylene glycol, and the blood-absorbing layer comprises a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract, it can stably adhere to a bleeding site to rapidly absorb blood, and thereby expand to restrict blood flow, thereby providing a physical hemostatic effect. In addition, it can provide a rapid hemostatic effect by assisting in the formation of platelet plugs, and since it is formed as a multi-layered structure in which a porous layer and a non-porous layer are laminated, it can prevent secondary infection from the outside. Furthermore, because it has excellent flexibility and adhesion, it effectively responds to tissue movement, and thus has the advantage of maintaining stable adhesion, it has industrial applicability.

Claims

1. A blood-absorbing layer in the form of a porous sponge; and A non-porous protective layer laminated over the blood absorption layer; comprising The above protective layer comprises a decellularized tissue supplement and polyethylene glycol, and The above blood absorption layer is a complex hemostatic agent comprising a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract.

2. In Paragraph 1, The above protective layer is a composite hemostatic agent comprising at least one additionally from the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials.

3. In Paragraph 1, The above blood absorption layer is a composite hemostatic agent comprising at least one more selected from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin.

4. In Paragraph 2 or 3, A composite hemostatic agent characterized in that the above natural material is at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

5. In Paragraph 1, The above-mentioned decellularized tissue supplement is, Preparatory stage for preparing mammalian tissues excluding humans; A pretreatment step for pretreating the above organization; A first inactivation step of inactivating viruses contained in pre-treated tissue using alcohol; Primary decellularization step of removing cells from virus-inactivated tissue using an aqueous base solution; A secondary decellularization step of enzymatically treating the primary decellularized tissue to remove cells; and It is manufactured through a second inactivation step in which viruses contained in decellularized tissue are inactivated using acid; and A composite hemostatic agent characterized by the DNA content of the tissue that has undergone the above-mentioned pretreatment step to the second inactivation step being 50 ng / mg or less.

6. In Paragraph 5, The degradation time using 100 U / ml collagenase is at least 90 hours, and A composite hemostatic agent characterized by comprising: a first decellularization step performed using a mixture of n-PrOH and NaOH; and a second decellularization step performed using NaOH with a concentration of 0.05M or more and less than 0.5M.

7. In Paragraph 1, The above-mentioned decellularized protein extract comprises a pretreatment step of preparing and pretreating mammalian tissue excluding humans; A first inactivation step of inactivating viruses contained in pre-treated tissue using alcohol; A decellularization step for removing cells from virus-inactivated tissue; and It is manufactured through a second inactivation step in which viruses contained in decellularized tissue are inactivated using acid; and The DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, and A composite hemostatic agent characterized by a reduction rate (L) of the elastin content of the tissue that has undergone the above-mentioned pretreatment step to the second inactivation step being 20% ​​or less.

8. In Paragraph 7, The above decellularization step is, A primary decellularization step of removing cells from virus-inactivated tissue using an aqueous base solution; and A secondary decellularization step comprising enzymatically treating the primary decellularized tissue to remove cells; and A composite hemostatic agent characterized by comprising: a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution having a concentration of more than 0.05 M and less than 0.2 M.

9. In a method for manufacturing a complex hemostatic agent, A protective layer manufacturing step of applying a protective layer raw material mixture to a tray and drying it to manufacture a protective layer; and A blood absorption layer manufacturing step comprising laminating a blood absorption layer raw material mixture onto the protective layer and then freeze-drying; The above protective layer raw material mixture comprises a decellularized tissue supplement and polyethylene glycol, and A method for manufacturing a composite hemostatic agent, wherein the above blood absorption layer raw material mixture comprises a decellularized tissue supplement, gelatin, thrombin, and a decellularized protein extract.

10. In Paragraph 9, The above protective layer raw material mixture is, Step A1, mixing decellularized tissue supplement with an acidic aqueous solution and then grinding; Step A2, which involves preparing by dissolving polyethylene glycol in water or melting it by applying heat; and A method for manufacturing a composite hemostatic agent characterized by being manufactured through a step A3 of mixing materials prepared through steps A1 and A2.

11. In Paragraph 9, The above blood absorption layer raw material mixture is, Step B1, which involves mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; and A method for manufacturing a composite hemostatic agent characterized by being prepared through a B2 step in which gelatin, thrombin, and decellularized protein extracts are added to and stirred in a solution obtained through a B1 step.

12. Porous sponge-type blood absorption layer; A non-porous protective layer laminated over the blood absorption layer; and A contact layer formed on the lower surface of the blood absorption layer; comprising The above protective layer comprises a decellularized tissue supplement and polyethylene glycol, and The above blood absorption layer comprises a decellularized tissue supplement and gelatin, and The above contact layer is a composite hemostatic agent comprising thrombin and a decellularized protein extract.

13. In Paragraph 12, The above protective layer is a composite hemostatic agent comprising at least one additionally from the group consisting of poloxamer, polyvinyl alcohol, glycerin, and natural materials.

14. In Paragraph 12, The above blood absorption layer is a composite hemostatic agent comprising at least one more selected from the group consisting of calcium chloride, poloxamer, natural materials, polyethylene glycol, polyvinylpyrrolidone, and glycerin.

15. In Paragraph 12, The above contact layer is a composite hemostatic agent comprising at least one of the group consisting of decellularized tissue supplements, calcium chloride, natural materials, and polyvinylpyrrolidone.

16. In any one of paragraphs 13 through 15, A composite hemostatic agent characterized in that the above natural material is at least one selected from the group consisting of alginate, carboxymethyl cellulose (CMC), and hyaluronic acid (HA).

17. In a method for manufacturing a complex hemostatic agent, A protective layer manufacturing step of applying a protective layer raw material mixture to a tray and drying it to manufacture a protective layer; A blood absorption layer manufacturing step of laminating a blood absorption layer raw material mixture onto the protective layer and then freeze-drying; and A contact layer manufacturing step comprising applying a contact layer raw material mixture onto a blood absorption layer and drying it to manufacture a contact layer; The above protective layer raw material mixture comprises a decellularized tissue supplement and polyethylene glycol, and The above blood absorption layer raw material mixture comprises a decellularized tissue supplement and gelatin, and A method for manufacturing a composite hemostatic agent, wherein the above contact layer raw material mixture comprises thrombin and a decellularized protein extract.

18. In Paragraph 17, The above protective layer raw material mixture is, Step A1, mixing decellularized tissue supplement with an acidic aqueous solution and then grinding; Step A2, which involves preparing by dissolving polyethylene glycol in water or melting it by applying heat; and A method for manufacturing a composite hemostatic agent characterized by being manufactured through a step A3 of mixing materials prepared through steps A1 and A2.

19. In Paragraph 17, The above blood absorption layer raw material mixture is, Step C1, which involves mixing a decellularized tissue supplement with an acidic aqueous solution and then grinding it; and A method for manufacturing a composite hemostatic agent characterized by being manufactured through a C2 step in which gelatin is added to a solution obtained through a C1 step and stirred.

20. In Paragraph 17, The above contact layer raw material mixture is, A method for manufacturing a composite hemostatic agent characterized by being prepared through step D1, which involves adding thrombin and decellularized protein extracts to an alcohol solvent and stirring.

21. In Paragraph 17, A method for manufacturing a composite hemostatic agent, characterized in that the application of the contact layer raw material mixture is performed through any one of the dipping method, the spray method, and the 3D printing method.

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