Hydrogel-based topical wound dressing comprising protein extract

Abstract

The present invention relates to a hydrogel-based wound dressing comprising a protein extract, comprising polyethylene glycol, white vaseline, 1, 2-hexanediol, ethylhexylglycerin, sodium hyaluronate, a protein extract, and purified water, and more particularly, to a hydrogel-based wound dressing which is easy to use by maintaining the same viscosity as at normal temperature even at a temperature similar to body temperature. The wound healing rate, the epithelium regeneration rate and the blood vessel formation rate are excellent.

Description

Technical Field

[0001] The present invention relates to a hydrogel-based wound dressing containing a protein extract, and more particularly to a local wound dressing containing polyethylene glycol, white petrolatum, 1,2-hexandiol, ethylhexylglycerin, sodium hyaluronate, a protein extract and purified water, which can maintain almost the same viscosity as at room temperature even at a temperature similar to body temperature and has excellent wound healing effect, and a method for manufacturing the same. Background Art

[0002] Human skin tissue is generally divided into three parts, including the epidermis, which is the outermost layer of the skin, the dermis, which is the lower layer of the epidermis, and the subcutaneous tissue. The epidermis contains epithelial cells, melanocytes, and immune cells differentiated from the basement membrane into multiple layers for the epidermis and the dermis to be closely combined, while the dermis, which is the lower layer of the epidermis, mainly contains fibroblasts and various extracellular matrices secreted by the cells.

[0003] Generally speaking, wound healing is a complex process that proceeds in stages over time. The extracellular matrix can be re-formed through the overlapping of three stages: hemostasis and inflammation, proliferation, and maturation.

[0004] In the early stage of wound formation, blood coagulation occurs due to inflammation and the response of the basal layer. The matrix is ​​a substance that fills the connective fibers of the dermis. It exists in the form of mucous mucopolysaccharides and contains a large amount of water. It is composed of glycosaminoglycans such as hyaluronic acid and chondroitin sulfate. In particular, proteoglycan is a hydrophilic carbohydrate protein complex, which is known to be composed of a jelly-like substance.

[0005] Generally speaking, wound dressings are divided into dry dressings and wet (moisturizing) dressings according to the moisture maintenance of the wound area. As dry dressings, bandages (such as Band-Aids or other bandages) and ointments (such as fusidic acid and Mayinglong, etc.) can be taken as examples. Bandages do not have special therapeutic effects, but only play the role of bandaging wounds, while ointments are added with therapeutic ingredients such as antibiotics or steroids, which promote wound healing and prevent infection.

[0006] However, for dry dressings, as secretions such as blood or exudate dry up and disappear, wound healing cells also disappear, leaving scars, and the scab also falls off when the bandage is removed, resulting in an increased risk of secondary injury. In addition, antibiotics contained in ointments also affect wound healing cells, so their self-healing effect is not significant.

[0007] In order to make up for the shortcomings of dry dressings as mentioned above, wet dressings containing hydrocolloid, alginate, foam and hydrogel can be used. Gelatin in wet dressings is a natural polymer with excellent biocompatibility, biodegradability and high hygroscopicity. It is widely used in many wound dressings because it can provide a moist environment for the skin when a wound occurs. Hydrogel can absorb a large amount of water because it has a three-dimensional hydrophilic polymer network structure with water as the dispersion medium, and is widely used in wound dressings because it has the same flexibility as natural tissue.

[0008] However, the physical properties of hydrogel patches are relatively weak. In particular, the tensile strength of hydrogels made using collagen as the main material is only at the level of 0.01 to 0.05 MPa, and the problem of extremely weak mechanical properties remains. Summary of the invention

[0009] The present invention aims to effectively solve the problems existing in existing wound dressings (wet dressings) and provide a wound dressing that is easy for users to use and has excellent wound recovery effect. Its purpose is to provide a hydrogel-based wound dressing containing a protein extract obtained through a multi-step decellularization process.

[0010] A hydrogel-based local wound dressing according to one embodiment of the present invention comprises polyethylene glycol (PEG), white petrolatum, 1,2-hexandiol, ethylhexylglycerin, sodium hyaluronate, protein extract and purified water.

[0011] The hydrogel-based local wound dressing preferably comprises 11 to 95 wt % of polyethylene glycol, 0.5 to 10 wt % of white petrolatum, 0.1 to 10 wt % of 1,2-hexandiol, 0.01 to 10 wt % of ethylhexylglycerin, 0.001 to 10 wt % of sodium hyaluronate, 0.01 to 10 wt % of protein extract and the remainder of purified water.

[0012] As the polyethylene glycol (PEG) used at this time, a mixture of polyethylene glycol (PEG) having a weight average molecular weight of 400 and polyethylene glycol (PEG) having a weight average molecular weight of 4,000 in a weight ratio of about 2.2:1 is preferably used. Furthermore, within the temperature variation range from the storage temperature of room temperature to the body temperature of the human body, the viscosity of the hydrogel-based local wound dressing in the ointment form hardly changes, so the viscosity measured at 25°C is substantially the same as the viscosity measured at 35°C.

[0013] In addition, hydrogel-based topical wound dressings contain protein extracts including collagen and elastin, and thus have excellent wound healing, re-epithelialization and angiogenesis effects.

[0014] At the same time, the hydrogel-based topical wound dressing according to the present invention may contain the same amount or 0.01 to 5 wt % of one or more selected from the group consisting of chlorhexidine gluconate, chlorhexidine acetate, chlorhexidine hydrochloride, silver sulfadiazine, povidone iodine, benzalkonium chloride, nitrofuracil, ethacridine, hexachlorophene, chlortetracycline, neomycin, penicillin, gentamicin, gentamicin sulfate, acriflavine, ciprofloxacin, benzethonium chloride, fusidic acid, neomycin sulfate, ofloxacin and tobramycin to replace 1,2-hexanediol and ethylhexylglycerol.

[0015] The protein extract is manufactured by: a pretreatment step of preparing and pretreating tissues of mammals other than humans; a first inactivation step of inactivating viruses contained in the pretreated tissues using alcohol; a decellularization step of removing cells from the virus-inactivated tissues; and a second inactivation step of inactivating viruses contained in the decellularized tissues using acid; the deoxyribonucleic acid (DNA) content in the tissues after the second inactivation step is preferably less than 50 ng / mg, and the reduction rate (L) of the elastin content in the tissues from the pretreatment step to the second inactivation step is preferably less than 20%.

[0016] The decellularization step may include: a first decellularization step of removing cells from the virus-inactivated tissue using an alkaline aqueous solution; and a second decellularization step of removing cells by enzymatically treating the tissue that has undergone the first decellularization.

[0017] At this time, the first decellularization step includes: a first decellularization step performed using a mixture of n-propanol (n-PrOH) and sodium hydroxide (NaOH); and a second decellularization step performed using an aqueous sodium hydroxide solution of more than 0.05M but less than 0.2M.

[0018] The tissue is preferably derived from one or more of mammalian blood vessels, ligaments and tendons, and the second decellularization step is preferably performed using deoxyribonuclease (DNase).

[0019] The hydrogel-based local wound dressing containing protein extract according to the present invention will not cause toxicity problems in the human body and has excellent biocompatibility. When the user applies it to the skin tissue with a wound, the viscosity will not decrease due to temperature changes, so it has the advantage of being easy to use.

[0020] Furthermore, since the protein extract is obtained through multiple decellularization and inactivation steps, it has excellent wound healing, re-epithelialization and angiogenesis effects, and can be used as an effective wound dressing for wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 (A) and (B) are graphs illustrating the test results of Test Example 1.

[0022] Figure 2 This is a graph illustrating the test results of Test Example 2.

[0023] Figure 3 (A) and (B) are graphs illustrating the test results of Test Example 3.

[0024] Figure 4 (A) and (B) are graphs illustrating the test results of Test Example 4.

[0025] Figure 5 This is a table showing the test results of Test Example 5 in graph form.

[0026] Figure 6 These are the results of measuring the basic physical properties of the local wound dressing according to Production Example 2, namely, viscosity, storage modulus, and pH.

[0027] Figure 7 It is the result of measuring the cell proliferation rate using Cell Count Kit-8.

[0028] Figure 8 The results are obtained by measuring the cell viability in the hydrogel using a Live / Dead assay kit (Invitrogen).

[0029] Fig. 9 Results of scratch evaluation performed on eluted culture fluid.

[0030] Fig.10 This is a schematic diagram illustrating the acute trauma animal model for performing Test Example 8.

[0031] Figures 11 to 13 These are the results of sorting out the results of wound healing rate (wound healing rate, %), epithelial regeneration rate (re-epitherialization rate, %) or angiogenesis rate (angiogenesis, %) according to Experimental Example 8, respectively. DETAILED DESCRIPTION

[0032] Before describing the preferred embodiments of the present invention in detail, it should be made clear that the terms or words used in the present specification and claims should not be limited to the general or dictionary meanings, but should be interpreted as meanings and concepts that are consistent with the technical ideas of the present invention.

[0033] Throughout this specification, when a certain part is described as “comprising” a certain constituent element, unless otherwise clearly described to the contrary, it does not mean that other constituent elements are excluded but that other constituent elements may be included.

[0034] Throughout the present specification, with respect to “%” used to express the concentration of a specific substance, unless explicitly mentioned otherwise, solid / solid means (weight / weight)%, solid / liquid means (weight / volume)%, and liquid / liquid means (volume / volume)%.

[0035] In addition, throughout the entire content of this specification, "mammals" refers to mammals other than humans. Even if it is simply recorded as "mammals" without mentioning that humans are excluded, it should be understood that it refers to mammals other than humans, and "removing cells" should be understood as a comprehensive meaning that includes the destruction or removal of deoxyribonucleic acid (DNA).

[0036] In addition, unless the context clearly states a specific order, the order of implementation of each step may be different from the order described in the above content. That is, each step may be implemented in the order described, in fact, may be implemented simultaneously, or may be implemented in the reverse order.

[0037] In addition, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by skilled practitioners in the technical field to which the invention belongs. In case of conflict, the description in this specification including the definitions shall prevail.

[0038] A hydrogel-based wound dressing containing a protein extract according to one embodiment of the present invention relates to a composition for effectively healing wounds by applying it to skin tissue with wounds in the form of an ointment, comprising 11 to 95 wt% of polyethylene glycol, 0.5 to 10 wt% of white petrolatum, 0.1 to 10 wt% of 1,2-hexandiol, 0.01 to 10 wt% of ethylhexylglycerin, 0.001 to 10 wt% of sodium hyaluronate, 0.01 to 10 wt% of a protein extract, and the remainder of purified water, thereby providing a local wound dressing that can maintain the same viscosity as at room temperature even at a temperature similar to body temperature and has excellent wound healing, epithelial regeneration and angiogenesis effects.

[0039] Hydrogel refers to a three-dimensional network structure formed by covalent or non-covalent crosslinking of hydrophilic polymers. Due to the hydrophilicity of the constituent material, it can absorb a large amount of water and swell in aqueous solution and aqueous environment, but will not dissolve due to the crosslinked structure. In addition, hydrogels with different forms and properties can be produced according to the constituent components and manufacturing methods, and because they can contain a large amount of water, they have properties between liquid and solid.

[0040] The topical wound dressing of the present invention implemented in an ointment dosage form is different from other existing wound dressings of the white vaseline series in that it is manufactured based on a hydrogel with polyethylene glycol (PEG) as a main component, and therefore has excellent mechanical properties. In particular, as polyethylene glycol (PEG), a mixture of polyethylene glycol (PEG) with a weight average molecular weight of 400 and polyethylene glycol (PEG) with a weight average molecular weight of 4,000 is used in a weight ratio of about 2.2:1, or based on 100wt% of the topical wound dressing, 10 to 95wt% of polyethylene glycol (PEG) with a weight average molecular weight of 400 and 1 to 25wt% of polyethylene glycol (PEG) with a weight average molecular weight of 4,000 are used.

[0041] By using a mixture of polyethylene glycols (PEGs) having different molecular weights as described above, excellent mechanical properties of a local wound dressing used in an ointment dosage form can be ensured. Furthermore, because the viscosity at room temperature storage temperature (approximately 25°C) and the viscosity at the skin temperature (approximately 36°C) at which the local wound dressing is used are almost the same and do not change much but are substantially the same, when the user applies the local wound dressing according to the invention of the present application to a site where a wound has occurred, it will not easily flow and will be fixed around the wound, thereby having the advantage of improving the convenience of the user.

[0042] In addition, in order to further improve the wound recovery effect, the topical wound dressing according to the present invention comprises a protein extract containing collagen and elastin in a range of about 0.01 to 10 wt %. The protein extract is obtained through a multi-step decellularization process, so that cells, crude fat, viruses and other foreign matter in mammalian tissues can be effectively removed, and the loss of elastin during the decellularization process is small, so that excellent effects can be shown in terms of re-epitherialization and angiogenesis.

[0043] In addition, the topical wound dressing according to the present invention may also contain white petrolatum, 1,2-hexanediol, ethylhexylglycerin and sodium hyaluronate, which can act as a contact and preservative agent on the skin or wound site. Preferably, in the mixture of 11-95wt% protein extract and 0.01-10wt% mentioned above, 0.5-10wt% white petrolatum, 0.1-10wt% 1,2-hexanediol, 0.01-10wt% ethylhexylglycerin, 0.001-10wt% sodium hyaluronate and the balance of purified water can be mixed.

[0044] Hyaluronic acid is a type of polysaccharide composed of linearly linked repeating units of N-acetylglucosamine and D-glucuronic acid. It is a biopolymer that was originally isolated from the fluid that fills animal eyeballs. It is now known that it exists in large quantities in animal placentas, synovial fluid, pleural fluid, skin, and rooster combs, and can also be produced from Streptococcus microorganisms such as Streptococcus equi and Streptococcus zooepidemecus.

[0045] The hyaluronic acid has excellent biocompatibility and high viscoelasticity in solution, so it is not only suitable for cosmetic applications such as cosmetic additives, but also widely used in various medical applications such as ophthalmic surgical adjuvants, joint function improvers, drug delivery substances, and eye drops. However, because hyaluronic acid itself can be easily decomposed in vivo or under conditions such as acid and alkali, its use is relatively limited.

[0046] In addition, the hydrogel-based topical wound dressing according to the present invention may contain one or more selected from the group consisting of chlorhexidine gluconate, chlorhexidine acetate, chlorhexidine hydrochloride, silver sulfadiazine, povidone iodine, benzalkonium chloride, nitrofuracil, ethacridine, hexachlorophene, chlortetracycline, neomycin, penicillin, gentamicin, gentamicin sulfate, acriflavine, ciprofloxacin, benzethonium chloride, fusidic acid, neomycin sulfate, ofloxacin and tobramycin in the same amount range as the usable 1,2-hexanediol and ethylhexylglycerol or in the overall composition in the range of about 0.01 to 5 wt %.

[0047] Next, the protein extract components obtained by the multi-step decellularization process will be described in more detail.

[0048] The protein extract contained in the local wound dressing according to the present invention can be obtained by: a preparation step of preparing skin tissue of mammals other than 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 alkaline aqueous solution; a second decellularization step of removing cells by enzymatically treating the tissue that has undergone the first decellularization; and a second inactivation step of inactivating viruses contained in the decellularized tissue using an acid.

[0049] At this time, the deoxyribonucleic acid (DNA) content of the tissue after the second inactivation step can be 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 tissue after the pretreatment step to the second inactivation step is 20% or less.

[0050] The reduction rate (L) is defined by the following formula (1): o refers to the elastin content of the tissue during the preparation step, while L f It refers to the elastin content of tissue that has undergone the second inactivation step.

[0051]

[0052] First, the preparation step is a step of preparing tissue of mammals other than those classified. Mammals are mammals other than humans, such as pigs, horses, cattle, and sheep, and the tissue can be any one of blood vessels, ligaments, and tendons derived from the mammals.

[0053] In order to produce a multi-step decellularized protein extract, mammalian tissue is first collected. For the collected mammalian tissue, in the preparation step, useless tissue and blood attached to the mammalian tissue are removed and washed, dried and cut. The mammalian tissue prepared as described above can be stored in a frozen state and thawed for use when needed. In the case of using a frozen tissue material, the preparation step can be a step of taking out the frozen tissue material and preparing it.

[0054] Next, the mammalian tissue prepared in the preparation step is pretreated in the pretreatment step.

[0055] This step is a step of washing mammalian tissues. If the mammalian tissues are frozen, they can be thawed, washed and cut in this step. In this step, distilled water, purified water and saline can be used as washing water, preferably distilled water. From this step to the second inactivation step, physical stirring can be performed.

[0056] The mammalian tissue prepared by the pretreatment step as described above can be subjected to the first inactivation step, the first decellularization step, the second decellularization step and the second inactivation step to remove cells, crude fat, viruses and other foreign matter that may induce immune and foreign body reactions. By performing decellularization according to the multi-step reaction as described above, the decellularization efficiency and foreign matter removal efficiency in each step can be significantly improved, thereby increasing the amount of tissue processed at one time and shortening the processing time, thereby ultimately achieving the effect of improving the productivity and yield of the decellularized protein extract.

[0057] First, the first inactivation step is a step of inactivating the virus contained in the pre-treated tissue using alcohol.

[0058] The purpose of performing this step is to more effectively remove cells, crude fat and foreign matter in the first and second decellularization steps by first inactivating the virus in the tissue before decellularization of the tissue.

[0059] The alcohol used in this step may be n-propanol. In order to achieve the virus inactivation effect, it is appropriate to use a 50-90% concentration of n-propanol aqueous solution, and more preferably a 65-80% concentration of n-propanol aqueous solution can be used.

[0060] In the first inactivation step, n-propanol is used as the alcohol for virus inactivation. When n-propanol is used for virus inactivation as described above, the tissue can be further softened, and when the tissue is softened, the treatment liquid can better penetrate into the tissue, thereby achieving inactivation and decellularization uniformly on the whole. As a result, the inactivation and decellularization efficiency can be improved and a more uniform tissue can be obtained, and the quality and reliability of the decellularized protein extract finally obtained can also be improved.

[0061] Generally speaking, when ethanol is used for virus extinguishing, it has the characteristics of fixing and solidifying mammalian tissues that need to be decellularized. However, when ethanol is used instead of n-propanol in this step, the hardening of the tissue will lead to a decrease in the engineering efficiency of increasing the permeability of the treatment material by relaxing the tissue in the subsequent steps.

[0062] In addition, isopropanol has a poor virus inactivation effect. Therefore, when isopropanol is used in the first inactivation step, the decellularization efficiency in the first and second decellularization steps will be reduced because sufficient virus inactivation effect cannot be obtained. Therefore, it is appropriate to use n-propanol in the first inactivation step.

[0063] In this step, with respect to 100 parts by weight of the propane aqueous solution, 10 to 25 parts by weight, preferably 20 to 25 parts by weight of the pretreated tissue may be treated.

[0064] The first decellularization step is a step of removing cells by treating the tissue that has been virus-inactivated in the first inactivation step with an alkaline aqueous solution.

[0065] This step includes: a first decellularization step, treating the tissue with a mixture of an alkaline aqueous solution and an alcohol; and a second decellularization step, treating the tissue with an alkaline aqueous solution. It is preferable to use an aqueous sodium hydroxide solution as the alkaline aqueous solution and n-propanol as the alcohol. In the above case, the first decellularization step is a step of treating the tissue with a mixture of n-propanol and sodium hydroxide, and the second decellularization step is a step of treating the tissue with an aqueous sodium hydroxide solution.

[0066] By dividing the decellularization step into two steps as described above, foreign matters such as crude fat can be removed while removing cells in the first decellularization step, so that the decellularization process can be more effectively achieved in the second decellularization step.

[0067] In the first and second decellularization steps, 10 to 25 parts by weight, preferably 20 to 25 parts by weight of tissue can be processed relative to 100 parts by weight of the processing solution, which is approximately twice the existing processing capacity. Because the present invention can effectively achieve decellularization processing through a multi-step method, it can fully process twice the volume of tissue compared to the existing method.

[0068] Specifically, the first decellularization step is a step of treating the virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide. In this step, the crude fat in the tissue will undergo a saponification reaction with sodium hydroxide, thereby separating and removing the crude fat from the tissue. At this time, n-propanol can promote the saponification reaction as described above and condense the products of the saponification reaction, thereby separating and removing the crude fat from the tissue more effectively.

[0069] At this time, the treatment time of the tissue treated with the mixture of n-propanol and sodium hydroxide can be 10 to 48 hours, preferably 15 to 45 hours, more preferably 18 to 28 hours, and the treatment temperature can be 0 to 30° C., preferably 10 to 28° C., more preferably 18 to 25° C. By treating within the temperature range and for the time, sufficient crude fat removal efficiency can be achieved without causing excessive tissue structure damage or elastin loss.

[0070] The mixture of n-propanol and sodium hydroxide can be an aqueous solution with a concentration of n-propanol of 50-95%, preferably 60-90%, more preferably 65-85%, and a sodium hydroxide concentration of more than 0.05 M and less than 0.2 M. The concentration range is a concentration range that can effectively remove foreign matter such as crude fat while minimizing tissue damage in the first decellularization step. In particular, when the concentration of sodium hydroxide is less than the range, the treatment efficiency of the first decellularization step will be reduced, and when it exceeds the range, the tissue structure itself will collapse and dissolve. Therefore, it is appropriate to use n-propanol and sodium hydroxide aqueous solutions within the concentration range.

[0071] The second decellularization step is a step of softening the tissue after the first decellularization step by treating it with an alkaline aqueous solution, thereby performing a decellularization reaction while relaxing the tissue structure. Crude fat or various foreign matter contained in the tissue will act as a physical and chemical barrier in the decellularization reaction, but because the tissue is in a state where crude fat and various foreign matter have been removed in the first decellularization step, the decellularization reaction can be more effectively achieved in the second decellularization reaction.

[0072] In the step, the treatment time of the tissue using the alkaline aqueous solution can be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature can be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C. By treating the tissue within the temperature range for the time, the tissue structure can be fully relaxed and a proper decellularization reaction can be achieved without causing tissue disintegration and loss of elastin.

[0073] At this time, the alkaline aqueous solution used as the treatment solution can be a sodium hydroxide aqueous solution, and the concentration of sodium hydroxide contained in the sodium hydroxide aqueous solution can be more than 0.05 M and less than 0.2 M. When the concentration of sodium hydroxide is less than 0.05 M, the decellularization efficiency in the subsequent second decellularization step may be reduced due to insufficient relaxation of the tissue structure, and when it is more than 0.2 M, part of the tissue in this step may be disintegrated or dissolved due to exceeding the relaxation level, resulting in a significant decrease in the final yield and loss of elastin, so it is appropriate to use the sodium hydroxide aqueous solution of the above concentration.

[0074] After the first decellularization step, a second decellularization step using enzymes will be performed, while a neutralization step and a washing step may be performed between the two steps.

[0075] The neutralization step is a step of treating with an acidic aqueous solution in order to neutralize the sodium hydroxide used in the first decellularization step. The type of the acidic aqueous solution used at this time may include, for example, an acidic aqueous solution containing at least one of hydrochloric acid, sulfuric acid, arsenic acid, and peracetic acid, but is not limited thereto and may be used after appropriately adjusting its concentration according to the working environment or conditions.

[0076] The washing step is performed to prevent the enzyme reactivity from decreasing due to residues when performing enzyme treatment in the subsequent process, i.e., the second decellularization step. As the washing solution used in the washing step, a buffer solution such as phosphate buffered saline (PBS) solution can be used, but it is not limited to this.

[0077] In addition, the second decellularization step is a step of removing cells by enzymatically treating the tissue that has undergone the first decellularization, and is a step of removing deoxyribonucleic acid (DNA) in the tissue that has undergone the first decellularization by treating the tissue that has undergone the first decellularization with a deoxyribonucleic acid (DNA) degrading enzyme.

[0078] In this step, with respect to 100 parts by weight of the treatment solution containing the deoxyribonucleic acid (DNA) degrading enzyme, 10 to 25 parts by weight, preferably 20 to 25 parts by weight of the tissue may be treated.

[0079] In this step, in order to ensure enzyme activity and sufficient deoxyribonucleic acid (DNA) decomposition efficiency, the treatment time of the tissue using the deoxyribonucleic acid (DNA) decomposing enzyme can be 10 to 35 hours, preferably 18 to 30 hours, and the treatment temperature can be 30 to 45°C, preferably 35 to 42°C.

[0080] In the present invention, the concentration of the DNA degrading enzyme contained in the treatment solution can be 0.0001 to 0.005 wt %, which is several to several hundred times lower than the concentration of the DNA degrading enzyme used in general similar technologies. The tissue of the present invention that has undergone the first decellularization can remove foreign matter including crude fat inside the tissue during the first decellularization step and make the structure of the tissue loose. When the DNA degrading enzyme is added in the above state, the DNA degrading enzyme can easily penetrate into the interior of the tissue structure, so even when using a low concentration of the DNA degrading enzyme, at least the same or even better DNA removal efficiency can be ensured, so that the low concentration of the DNA degrading enzyme as described above can be used in the present invention.

[0081] However, if the concentration of the DNA degrading enzyme is less than the above range, the DNA degrading efficiency will decrease, so it is appropriate to use a concentration of 0.0001% or more. If it exceeds the above range, the degree of improvement in the DNA degrading efficiency will be negligible compared to the amount of the added DNA degrading enzyme, resulting in a problem of poor economic efficiency. Therefore, it is appropriate to use a DNA degrading enzyme within the above concentration range.

[0082] The tissue decellularized by the second decellularization step will be additionally subjected to virus removal by the second inactivation step, and a decolorization step and / or a degreasing step may be additionally performed between the two steps. The decolorization step is a step for removing the color of the tissue, which may be performed, for example, by treating the tissue with hydrogen peroxide, and the degreasing step is a step for additionally removing fat contained in the tissue, which may be performed, for example, using a ketone solution, preferably an acetone solution.

[0083] Next, the second inactivation step is performed to additionally inactivate the viruses contained in the tissue after the second decellularization step. This step is a step of inactivating the viruses in the tissue using acid, specifically, a step of treating the tissue with a mixture of organic acid and alcohol.

[0084] As the organic acid used at this time, peracetic acid can be used. Peracetic acid as an oxidant can inactivate the virus by inducing non-specific free radical damage to the virus. The peracetic acid used in this step can be used at a concentration of 0.05 to 1%.

[0085] In addition, the alcohol used in this step can be a lower alcohol with a carbon number of 1 to 4, such as methanol, ethanol, and propanol, preferably ethanol. Ethanol is preferably used because it can inactivate viruses and fix tissue structures to make the tissues more solid.

[0086] Through the above-described process, a protein extract can be finally obtained in which viruses are inactivated, crude fat and deoxyribonucleic acid (DNA) are removed, and the protein extract contains various extracellular matrix components including elastin.

[0087] The content of deoxyribonucleic acid (DNA) contained in the protein extract obtained by the method as described above is less than 50 ng / mg, preferably less than 20 ng / mg, less than 10 ng / mg, and more preferably less than 5 ng / mg, because its deoxyribonucleic acid (DNA) content is extremely low and the induction of various immune foreign body reactions can be minimized.

[0088] In addition, the protein extract has a very low content of elastin in the process from the pretreatment step to the second inactivation step, so that the elastin can be used to achieve effects such as wound healing and scar inhibition. Specifically, the reduction rate (L) of the elastin content of the tissue from the pretreatment step to the second inactivation step is less than 20%. This value is extremely low compared to the case where the reduction rate (L) of the elastin content is as high as 50% or more when the existing decellularization method is applied.

[0089] In addition, 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 or distilled water, and packaging is to prevent contamination of the protein extract during storage or transfer and to improve the convenience of use, and the protein extract may be packaged in a state of being immersed in a preservation solution.

[0090] Sterilization is performed to remove additional contamination that occurs after the second inactivation step, and for sterilization, for example, electron beam irradiation or radiation irradiation may be performed, but the present invention is not limited thereto.

[0091] Processing refers to processing the protein extract into a usable form, for example, it can be applied in methods such as drying, powdering and solubilization. By processing in the manner described above, it can be used in the form of biological ink or injection, but it is not limited to this and can also be used in various other forms.

[0092] In addition, the method for producing the protein extract as described above will be described in more detail below, and the parts that overlap with the above description will be omitted.

[0093] The manufacturing method includes: a preparation step of preparing tissue of mammals other than 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 alkaline aqueous 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.

[0094] At this time, the deoxyribonucleic acid (DNA) content of the tissue after the second inactivation step may be 50 ng / mg or less, and the reduction rate (L) of the elastin content of the tissue after the pretreatment step to the second inactivation step may be 20% or less.

[0095] The reduction rate of the elastin content of the tissue (L) is defined by the following formula (1): L o refers to the elastin content of the tissue during the preparation step, while L f It refers to the elastin content of tissue that has undergone the second inactivation step.

[0096]

[0097] First, the preparation step is a step of preparing tissue of mammals other than those classified. Mammals are mammals other than humans, such as pigs, horses, cattle, and sheep, and the tissue can be any one of blood vessels, ligaments, and tendons derived from the mammals.

[0098] The pretreatment step is a step of washing the mammalian tissue prepared in the preparation step. The tissue can be cut into an appropriate size in this step as needed, and when the frozen tissue is prepared, it can be thawed in this step.

[0099] The first inactivation step is a step of inactivating the virus contained in the pretreated tissue using alcohol. Generally, ethanol is used to inactivate the virus, but n-propanol is used in the present invention. This is because ethanol can firmly fix the tissue and reduce the softening efficiency in the subsequent steps, while n-propanol can improve the softening efficiency in the subsequent steps by softening the tissue.

[0100] The first decellularization step is a step of treating the tissue inactivated by the virus in the first inactivation step with an alkaline aqueous solution to remove cells. This step includes: a first decellularization step of treating the tissue with a mixture of an alkaline aqueous solution and alcohol; and a second decellularization step of treating the tissue with an alkaline aqueous solution.

[0101] Among them, it is preferable to use a sodium hydroxide aqueous solution as the alkaline aqueous solution and n-propanol as the alcohol.

[0102] Specifically, the first decellularization step is a step of treating the virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide, through which crude fat and foreign matter in the tissue can be removed and the tissue can be partially softened. The second decellularization step is a step of treating the tissue with an aqueous sodium hydroxide solution, in which a decellularization reaction can occur while softening the tissue, because foreign matter including crude fat has been removed before, so the effect can be further improved.

[0103] In the first and second decellularization steps, sodium hydroxide may be included at a concentration of more than 0.05 M and less than 0.2 M, which is a preferred concentration range for preventing disintegration or dissolution of tissue while ensuring sufficient saponification and softening reaction by sodium hydroxide.

[0104] Next, after the first decellularization step, a second decellularization step using an enzyme is performed, and a neutralization step and a washing step may be performed between the two steps. The neutralization step is a step of treating with an acidic aqueous solution in order to neutralize the sodium hydroxide used in the first decellularization step, and the washing step is a step performed in order to prevent a decrease in enzyme reactivity due to residues when performing enzyme treatment in the subsequent process, i.e., the second decellularization step.

[0105] The second decellularization step is a step of removing the DNA in the tissue by treating the tissue that has undergone the first decellularization with a DNA degrading enzyme. The tissue that has undergone the first decellularization of the present invention can remove foreign matter including crude fat inside the tissue during the first decellularization step, and make the structure of the tissue loose. When the DNA degrading enzyme is added in the above state, the DNA degrading enzyme can easily penetrate into the interior of the tissue structure, so even when a low concentration of the DNA degrading enzyme is used, at least the same or even better DNA removal efficiency can be ensured, so a low concentration of 0.0001 to 0.005wt% of the DNA degrading enzyme is used in the present invention. When the concentration of the DNA degrading enzyme is less than the above range, the DNA degrading efficiency will be reduced. When it exceeds the above range, the degree of improvement in the DNA degrading efficiency will be negligible compared with the amount of the added DNA degrading enzyme, resulting in poor economic efficiency. Therefore, it is advisable to use the DNA degrading enzyme within the above concentration range.

[0106] The tissue decellularized by the second decellularization step will be additionally subjected to virus removal by the second inactivation step, and a decolorization step and / or a degreasing step may be additionally performed between the two steps. The decolorization step is a step for removing the color of the tissue, which may be performed, for example, by treating the tissue with hydrogen peroxide, and the degreasing step is a step for additionally removing fat contained in the tissue, which may be performed, for example, using a ketone solution, preferably an acetone solution.

[0107] Next, a second inactivation step is performed to additionally inactivate the viruses contained in the tissue that has undergone the second decellularization step. This step is a step of treating the tissue with a mixture of an organic acid and an alcohol, where peracetic acid can be used as the organic acid and ethanol can be used as the alcohol, and by treating the tissue with the mixed solution as described above, the viruses in the tissue can be removed more effectively.

[0108] Through the above-described process, a protein extract can be finally produced in which viruses are inactivated, crude fat and deoxyribonucleic acid (DNA) are removed, and various extracellular matrix components including elastin are contained.

[0109] The content of deoxyribonucleic acid (DNA) contained in the protein extract obtained by the method as described above is less than 50 ng / mg, preferably less than 20 ng / mg, less than 10 ng / mg, and more preferably less than 5 ng / mg, because its deoxyribonucleic acid (DNA) content is extremely low and the induction of various immune foreign body reactions can be minimized.

[0110] In addition, the protein extract has a very low content of elastin in the process from the pretreatment step to the second inactivation step, so that the elastin can be used to achieve effects such as wound healing and scar inhibition. Specifically, the reduction rate (L) of the elastin content of the tissue from the pretreatment step to the second inactivation step is less than 20%. This value is extremely low compared to the case where the reduction rate (L) of the elastin content is as high as 50% or more when the existing decellularization method is applied.

[0111] In addition, after the second inactivation step, additional steps such as washing, packaging, sterilization and processing may be performed. Washing may be performed using, for example, a buffer solution and distilled water, packaging is to prevent contamination of the protein extract during storage or transfer and to improve the convenience of use, and sterilization is performed to remove additional contamination that occurs after the second inactivation step.

[0112] Processing refers to processing the protein extract into a usable form, for example, it can be applied in methods such as drying, powdering and solubilization. By processing in the manner described above, it can be used in the form of biological ink or injection, but it is not limited to this and can also be used in various other forms.

[0113] Next, the specific functions and effects of the present invention will be described through an embodiment of the present invention. However, this is only a preferred example of the present invention, and the scope of the claims of the present invention is not limited by the embodiment.

[0114] [Production Example 1]

[0115] 1. Preparation and preprocessing steps

[0116] First, a pig heart connected to the aorta that had been washed, dried, cut, and frozen was prepared and washed with distilled water after thawing. Next, a raw tissue specimen was prepared by cutting into a size of 50×50 mm.

[0117] 2. First inactivation step

[0118] Next, the first inactivation step was performed by adding 250 g of the raw tissue specimen to 1 L of 70% n-propanol and stirring at 150 RPM for 24 hours at a temperature of 20°C.

[0119] 3-1. First decellularization step (first decellularization step)

[0120] Next, the first decellularization step was performed by preparing a mixed solution containing 70% n-propanol and 0.1M sodium hydroxide in distilled water, mixing the raw tissue specimen in a manner such that 100 parts by weight of the mixed solution contained 23 parts by weight, and then stirring the mixture at a temperature of 20°C and a stirring speed of 150RPM for 24 hours.

[0121] 3-2. First decellularization step (second decellularization step)

[0122] Next, the second decellularization step was performed by mixing 24 parts by weight of the first decellularization step with 100 parts by weight of a 0.1 M sodium hydroxide aqueous solution prepared in advance, and then stirring for 24 hours under the same conditioning conditions as the previous step.

[0123] Next, the tissue specimen that has undergone the second decellularization step is neutralized by immersing it in an acetic acid aqueous solution and stirring it, and then washed with a phosphate buffered saline (PBS) solution.

[0124] 4. Second decellularization step

[0125] Next, the second decellularization step was performed by placing the tissue specimen in a treatment solution containing 0.00011 wt% of deoxyribonuclease (DNase) and stirring at 37° C. at a stirring speed of 150 RPM for 23 hours. The tissue specimen that completed the second decellularization step was decolored by treating it with 3% hydrogen peroxide at 20° C. for 1 hour, and then degreased by treating it with 20% acetone solution at 20° C. for 1 hour.

[0126] 5. Second inactivation step

[0127] Next, a second inactivation step was performed by immersing the tissue specimen in a mixed solution containing 70% n-propanol and 0.2% peracetic acid at 20° C. for 4.5 hours.

[0128] Finally, the protein extract contained in the topical wound dressing of the present invention was prepared by washing the tissue specimens subjected to all the treatment processes using a phosphate buffered saline (PBS) solution and distilled water.

[0129] [Test Example 1]

[0130] The multi-step decellularized protein extract was prepared by the same method as in Example 1. When the first, second, and third decellularization steps were performed in sequence, the tissue specimens immediately after each step were collected and the elastin content contained in each 1 mg tissue specimen was measured and the results are shown in the graph. Figure 1 In addition, the elastin content of the original tissue in the preparation step was measured, and the elastin content after each step was expressed as a percentage relative to the elastin content of the original tissue and is shown in FIG. Figure 1 (B) Chinese.

[0131] Figure 1 (A) and Figure 1 The first process in (B) refers to the first decellularization step, the second process refers to the second decellularization step, and the third process refers to the third decellularization step.

[0132] Regarding the elastin content, the Fastin Elastin Assay kit F2000 (manufacturer: Biocolor) was used to perform the test according to the protocol of the assay kit and the elastin content was measured using a Multimode plate reader Victor Nivo TM (Manufacturer: Perkin Elmer) Elastin was analyzed at an absorbance of 513 nm, and the elastin content was measured by comparison with a standard substance.

[0133] See also Figure 1 The results of (A) and 1(B) confirmed that the elastin content was slightly lost as each process proceeded, but more than 80% of the elastin remained at the end of the final step.

[0134] [Test Example 2]

[0135] The multi-step decellularized protein extract was prepared using the same method as in Manufacturing Example 1, wherein only one of the first, second, and third decellularization steps was omitted, and after collecting the tissue specimens that had undergone the final process, the elastin content contained in each 1 mg of the tissue specimen was measured. Furthermore, the L value was calculated after measuring the elastin content of the original tissue in the preparation step using the same method, and the results are summarized in Table 1.

[0136] In addition, the percentage of elastin content contained in the protein extract obtained after omitting each step was calculated relative to the elastin content of the original tissue in the preparation step, and the results are shown in a graph. Figure 2 middle.

[0137] The elastin content was measured in the same manner as in Test Example 1, and Table 1 and Figure 2 The first process refers to the first decellularization step, the second process refers to the second decellularization step, and the third process refers to the third decellularization step.

[0138]

Table 1

[0139]

[0140] First, referring to Table 1, it can be confirmed that the L value is less than 20% when all processes are executed and when one process is omitted. Figure 2 It can be confirmed that even when a specific process is omitted, the content of elastin does not change too much. Therefore, through the above test results, it can be confirmed that even when one of the first decellularization step, the second decellularization step, and the third decellularization step in the process according to one embodiment of the present invention is omitted, the amount of elastin will not be greatly affected.

[0141] [Test Example 3]

[0142] The experiment was carried out in the same manner as in Experimental Example 1, and the DNA content after each step was measured instead of the elastin content. Regarding the DNA content, the analysis kit Quant-iT TM PicoGreen TM dsDNA Assay kits and dsDNA Reagents (manufacturer: Invitrogen) were used to perform the assay according to the protocol of the assay kit and then the dsDNA was analyzed using the Multimode plate reader Victor Nivo TM (Manufacturer: Perkin Elmer) The deoxyribonucleic acid (DNA) content was measured at an absorbance of 480 to 520 nm.

[0143] The DNA content per mg of tissue after each step is shown in the graph. Figure 3 (A) and the DNA content in the tissue after each step is shown as a percentage relative to the DNA content of the original tissue. Figure 3 (B). At this time, Figure 3 (A) and Figure 3 The first process in (B) refers to the first decellularization step, the second process refers to the second decellularization step, and the third process refers to the third decellularization step.

[0144] See also Figure 3 (A) and Figure 3 (B) It can be confirmed that most of the deoxyribonucleic acid (DNA) can be removed after performing the first decellularization step, the second decellularization step, and all the steps of the third decellularization step.

[0145] [Test Example 4]

[0146] The test was conducted using the same method as in Test Example 2, and the deoxyribonucleic acid (DNA) content of the protein extracts prepared by omitting one step was measured using the same method as in Test Example 3.

[0147] The DNA content per 1 mg of protein extract obtained by omitting each step is shown in the figure. Figure 4 (A), and the DNA content in the tissue obtained by omitting each step is shown as a percentage relative to the DNA content of the original tissue. Figure 4 (B). At this time, Figure 4 (A) and Figure 4 The first process in (B) refers to the first decellularization step, the second process refers to the second decellularization step, and the third process refers to the third decellularization step.

[0148] See also Figure 4 (A) and Figure 4 (B) It was confirmed that deoxyribonucleic acid (DNA) can be effectively removed only when the first decellularization step, the second decellularization step, and all the steps of the third decellularization step are performed. In particular, it was confirmed that the efficiency of deoxyribonucleic acid (DNA) removal will be significantly reduced when the second decellularization step or the second decellularization step is omitted, and it was confirmed through this experiment that deoxyribonucleic acid (DNA) can be removed more effectively by performing all the steps in sequence.

[0149] [Test Example 5]

[0150] A multi-step decellularized protein extract was produced by the same method as in Production Example 1, wherein the tissue was treated while changing the concentration of sodium hydroxide contained in the treatment solution in the first decellularization step to 0.005 M, 0.1 M, 0.2 M, and 0.3 M.

[0151] During the experiment, the state of the tissue during and after the second decellularization step (second process) was photographed and shown in the figure. Figure 5In addition, the deoxyribonucleic acid (DNA) content contained in each 1 mg of the protein extract finally obtained was measured using the same method as in Experimental Example 3, and the content of the protein extract finally obtained was expressed as a percentage value relative to the content of the tissue initially input, and the yield of the protein extract was calculated, and then the photos taken of the hematoxylin & eosin (H&E) staining results were shown in the figure. Figure 5 middle.

[0152] See also Figure 5 It was confirmed that when the concentration of sodium hydroxide (NaOH) was 0.05 M, the yield was good but the deoxyribonucleic acid (DNA) content was high, and there were still unremoved cell nuclei in the tissue, which confirmed that the decellularization efficiency was low.

[0153] In contrast, it was confirmed that when treated with 0.1 M sodium hydroxide (NaOH), the tissue was well maintained in the second decellularization step, and the deoxyribonucleic acid (DNA) content after the final treatment was an extremely low 0.6 ng / mg, while the yield was a very high 20.8%.

[0154] When treated with 0.2M sodium hydroxide (NaOH), although the deoxyribonucleic acid (DNA) content was low, part of the tissue was disintegrated in the first decellularization step, resulting in a final yield of less than half compared to the case of treatment with 0.1M sodium hydroxide (NaOH). When treated with 0.3M sodium hydroxide (NaOH), the tissue could not be obtained because it was almost completely dissolved.

[0155] Therefore, the experimental results confirm that in order to prevent tissue disintegration and dissolution in the first decellularization step, ensure excellent yield and obtain a protein extract in which the deoxyribonucleic acid (DNA) in the tissue is fully removed, the concentration of sodium hydroxide (NaOH) contained in the treatment solution in the first decellularization step, especially the second decellularization step, should be greater than 0.05M and less than 0.2M.

[0156] [Test Example 6]

[0157] The same method as in Manufacturing Example 1 was used to prepare a protein extract for multi-step decellularization, wherein the concentration of sodium hydroxide contained in the treatment solution of the first decellularization step was 0.005 M and 0.1 M, and the concentration of deoxyribonuclease (DNase) was changed to 0.0001 to 0.003 wt %. After the tissue was treated, the deoxyribonucleic acid (DNA) content ratio of each protein extract finally obtained and the yield of the protein extract were calculated and the results are recorded in Table 2.

[0158]

Table 2

[0159]

[0160] Referring to the results in Table 2, it can be confirmed that, when the concentration of sodium hydroxide (NaOH) remains unchanged, the higher the concentration of DNase, the lower the concentration of deoxyribonucleic acid (DNA) in the final protein extract. However, it can also be confirmed that when the concentration of sodium hydroxide (NaOH) is 0.1M compared to 0.05M, even if the concentration of DNase is several or even dozens of times lower, the concentration of deoxyribonucleic acid (DNA) in the final protein extract is lower, and the difference in yield is not obvious.

[0161] It was confirmed that the deoxyribonucleic acid (DNA) removal efficiency was significantly lower when the concentration of sodium hydroxide (NaOH) was 0.05 M compared to 0.1 M, so the concentration of sodium hydroxide (NaOH) in the treatment solution used in the first decellularization step should be greater than 0.05 M.

[0162] [Production Example 2]

[0163] A topical wound dressing was prepared by mixing 55 g of polyethylene glycol (PEG) having a molecular weight of 400, 25 g of polyethylene glycol (PEG) having a molecular weight of 4,000, 0.35 g of the protein extract according to Preparation Example 1, 4 g each of 1,2-hexanediol, ethylhexylglycerin, sodium hyaluronate, and 7.65 g of purified water. Figure 6 The basic physical properties of the topical wound dressing produced by the above method, namely viscosity, storage modulus and pH measurement results, are summarized in the figure.

[0164] For comparison, basic physical properties were measured under the same conditions using wound dressings from other companies that are currently commercialized, namely, Product E and Product T.

[0165] pass Figure 6 The viscosity change of (A) according to the shear rate can confirm that the local wound dressing according to Manufacturing Example 2, similar to other company's products (Product E and Product T) that have been commercialized, shows a typical shear thinning phenomenon in which the shear viscosity decreases as the shear rate increases. Figure 6(B) It was confirmed that the storage modulus value thereof was the highest in all temperature ranges compared with product E and product T. This is considered to be because the local wound dressing according to Production Example 2 contains polyethylene glycol (PEG) having a relatively high molecular weight.

[0166] In particular, through Figure 6 (C) It can be confirmed that when comparing the viscosity value measured at room temperature (25°C) and the viscosity measured at a temperature similar to body temperature, i.e., 35°C, the viscosity of the existing products E and T decreases sharply with the increase of time, but the local wound dressing of Manufacturing Example 2 has almost no viscosity change and can maintain substantially the same viscosity value. It is estimated that this is because a stable hydrogel is formed by mixing polyethylene glycol (PEG) having a low molecular weight and a high molecular weight.

[0167] pass Figure 6 The pH measurement result of (D) can be confirmed to be not much different from the currently commercialized products and is equivalent to the neutral region.

[0168] [Test Example 7]

[0169] In order to confirm the effect of the protein extract used in the topical wound dressing of the present invention, an in vitro test was performed using the test groups shown in Table 3 below.

[0170]

Table 3

[0171]

[0172] (1) Preparation of hydrogel

[0173] As shown in Table 3, atelocollagen was mixed with the protein extract obtained in Preparation Example 1 (0, 0.35, 1 w / v %) and fibroblasts (1×10 5 After that, a hydrogel sample was prepared by gelation at 37°C for 30 minutes.

[0174] (2) Cell counting kit-8 assay (CCK-8 assay)

[0175] In order to measure the cell proliferation rate, a Cell Count Kit-8 (CCK-8, Dojindo, Japan) was used. The hydrogel samples produced were cultured and analyzed on the 0th, 1st, 4th, 7th and 14th days after culture. After sampling a solution in which the CCK-8 solution and the culture medium were mixed at a ratio of 1:10, the absorbance at a wavelength of 450 nm was measured at 37°C for 2 hours. By using the standard curve of the cells, the absorbance measured by the above method was converted into the number of cells, and the results were organized in Figure 7 middle.

[0176] Through the Figure 7 The results confirmed that the G2 and G3 samples containing the protein extract showed a better cell proliferation effect than the G1 sample not containing the protein extract during all test periods (day 1 to day 14).

[0177] (3) Live / dead cell staining (L / D staining)

[0178] To measure the cell survival rate in the hydrogel, the Live / Dead assay kit (Invitrogen) was used to test G1 to G3. After culturing each hydrogel sample, the samples were analyzed on day 0, day 1, day 4, day 7, and day 14, and at each time point, the cells were treated with a culture medium containing 2 μM acetylmethoxycalcein (calcein AM) and 4 μM ethidium iodide homodimer-1 (EthD-1) and observed using a fluorescence microscope (see Figure 8 (A)), and then the cell survival rate was calculated according to the following formula 2 and summarized in Table 8 (B).

[0179] Cell survival rate (%) = [(Live cell number) / (Live cell number+Dead cell number)] × 100 (Formula 2)

[0180] pass Figure 8 The results of (A) and (B) confirmed that the cell survival rate in the hydrogel of the sample containing the protein extract was relatively higher than that in the case of not containing the protein extract.

[0181] (4) Scratch test

[0182] L929 cells were cultured at 2×10 5 After culturing in 6 wells at a concentration of 10 cells / well, a scratch was formed across the center of the well using a pipette tip. At this time, α-modified Eagle's medium (α-MEM) mixed with 2% fetal bovine serum (FBS) was used as the culture medium. In the wells with the scratch, the cells were treated with culture solutions eluted in the manner shown in Table 4 below (each substance was immersed in α-modified Eagle's medium (α-MEM) mixed with 2% fetal bovine serum (FBS) at a concentration of 4 g / 20 ml, and eluted at 37°C and 5% CO2 for 24 hours), and the experiment was completed by culturing the cells on each elution solution. At 0 hours, 12 hours, and 24 hours after culturing, the scratched portion was photographed and the proportion of scratch healing was analyzed (see Fig. 9 (A)), where the wound closure rate of the scratch is determined by the following formula 3 and summarized in Fig. 9 (B) Chinese.

[0183] Wound closure rate (%) = [(wound area at t0) - t final Wound area at t final )) / (Wound area at t0))]×100(Formula 3)

[0184]

Table 4

[0185]

[0186] pass Fig. 9 (A) and (B) can confirm that when the protein extract produced by Manufacturing Example 1 is used, the scratch formed is effectively healed compared with the untreated group, i.e., the control group, and simple collagen, which indicates that the protein extract according to the present invention can effectively act on local wounds.

[0187] [Test Example 8]

[0188] In order to confirm the effect of the local wound dressing according to the present invention, after applying the local wound dressing according to Manufacturing Example 2 to the local wound site formed on the body of a mouse, a vaseline gauze was used to protect it (see Fig.10 ). For comparison, the same local wound was formed, and no measures were taken but only covered with vaseline gauze to serve as a control group (control).

[0189] The wound healing rate (wound healing rate, %), epithelial regeneration rate (re-epitherialization rate, %) and angiogenesis rate (angiogenesis, %) were determined using the following formulas using the acute trauma animal model described above. The photos and results are shown in Figures 11 to 13 shown.

[0190] first, Fig.11 The wound healing rate shown in the figure is the result of visual observation (photographing) of the exposed part of the wound site on the 13th day after the wound site of each treatment group was treated. It was confirmed by observing whether there was blood oozing out when the gauze was in contact, and was calculated by the following formula 4 after graphical analysis of the area of ​​the entire wound site and the area of ​​the remaining part that was not healed.

[0191] Wound healing rate (%) = [(initial defect - remaining defect) / (initial defect)] × 100% (Formula 4)

[0192] Next, samples were collected and histological assays were performed. In order to analyze the epithelial regeneration rate and the neovascularization rate, the central part of the wound was collected ( Fig.12 (A) The diameter of the circular wound was measured and paraffin blocks were prepared, which were then sectioned and subjected to hematoxylin and eosin (H&E) staining.

[0193] The tissues stained with hematoxylin & eosin (H&E) were observed and analyzed, and the length of the overall wound part (defect length) and the length of the epithelial regeneration part (re-epitherilum length) were measured. Based on the measured length, the following formula 5 was used for calculation, and the results are as follows: Fig.13 (B) shown.

[0194] Re-epithelium rate (%) = [(re-epithelial regeneration length) / (initial defect length)] × 100% (Formula 5)

[0195] In addition, in order to confirm the degree of angiogenesis, the tissue stained with hematoxylin & eosin (H&E) was observed and the number of blood vessels per unit area was counted. The counting result of the number of blood vessels per unit area was compared with the number of blood vessels in normal tissue, and the angiogenesis rate (angiogenesis, %) was calculated by the following formula 6, and the result is shown in Figure 14 (B).

[0196] Angiogenesis rate (angiogenesis, %) = [(number of regenerated blood vessels in regenerated area) / (number of blood vessels in normal tissue (skin dermis))] × 100% (Formula 6)

[0197] pass Figures 11 to 13 It can be confirmed that the local wound dressing according to the present invention contains a protein extract obtained through a multi-step decellularization process, and therefore has significant wound healing rate, epithelial regeneration rate and angiogenesis effects in an actual acute wound animal model, as expected in the in vitro test results of Example 7.

[0198] The present invention is not limited to the specific embodiments and descriptions. Personnel with general knowledge in the technical field to which the present invention belongs can make various modifications without departing from the gist of the present invention as claimed in the claims, and the modifications are included in the protection scope of the present invention.

[0199] Industry availability

[0200] The present invention relates to a hydrogel-based wound dressing comprising a protein extract obtained through a multi-step decellularization process, and can provide a hydrogel-based topical wound dressing comprising polyethylene glycol, white petrolatum, 1,2-hexandiol, ethylhexylglycerin, sodium hyaluronate, a protein extract and purified water, thereby having industrial applicability.

Claims

1. A local wound dressing based on hydrogel, Contains polyethylene glycol, white petrolatum, 1,2-hexandiol, ethylhexylglycerin, sodium hyaluronate, protein extract and purified water.

2. The hydrogel-based topical wound dressing according to claim 1, The hydrogel-based local wound dressing comprises 11 to 95 wt % of polyethylene glycol, 0.5 to 10 wt % of white petrolatum, 0.1 to 10 wt % of 1,2-hexandiol, 0.01 to 10 wt % of ethylhexylglycerin, 0.001 to 10 wt % of sodium hyaluronate, 0.01 to 10 wt % of protein extract and the balance of purified water.

3. The hydrogel-based topical wound dressing according to claim 1, characterized in that: The protein extract is obtained by: Pretreatment steps for preparing tissues from mammals other than humans and performing pretreatments; A first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a decellularization step to remove cells from virally inactivated tissue; as well as, A second inactivation step of inactivating viruses contained in the decellularized tissue using acid; To manufacture, The deoxyribonucleic acid (DNA) content of the tissue after the second inactivation step is below 50 ng / mg, The reduction rate (L) of the elastin content of the tissue after the pretreatment step to the second inactivation step is 20% or less.

4. The hydrogel-based topical wound dressing according to claim 3, The decellularization step comprises: A first decellularization step using an alkaline aqueous solution to remove cells from the virus-inactivated tissue; as well as, A second decellularization step is performed to remove cells by enzymatically treating the tissue that has undergone the first decellularization step.

5. The hydrogel-based topical wound dressing according to claim 4, The first decellularization step comprises: A first decellularization step was performed using a mixture of n-propanol (n-PrOH) and sodium hydroxide (NaOH); as well as, The second decellularization step was performed using an aqueous sodium hydroxide solution of more than 0.05M but less than 0.2M.

6. The hydrogel-based topical wound dressing according to claim 3, characterized in that: The tissue is derived from one or more of blood vessels, ligaments, and tendons of mammals.

7. The hydrogel-based topical wound dressing according to claim 4, characterized in that: The second decellularization step is performed using deoxyribonuclease (DNase).

8. The hydrogel-based topical wound dressing according to claim 1, characterized in that: The polyethylene glycol (PEG) is a mixture of polyethylene glycol (PEG) having a weight average molecular weight of 400 and polyethylene glycol (PEG) having a weight average molecular weight of 4,000 in a weight ratio of about 2.2:

1.

9. The hydrogel-based topical wound dressing according to claim 1, characterized in that: The viscosity measured at 25°C is substantially the same as the viscosity measured at 35°C.

10. The hydrogel-based topical wound dressing according to claim 1, characterized in that: The protein extract contains collagen and elastin.

11. The hydrogel-based topical wound dressing according to claim 1, characterized in that: The 1,2-hexanediol and ethylhexylglycerol are replaced by one or more selected from the group consisting of chlorhexidine gluconate, chlorhexidine acetate, chlorhexidine hydrochloride, silver sulfadiazine, povidone iodine, benzalkonium chloride, nitrofuracil, ethacridine, hexachlorophene, chlortetracycline, neomycin, penicillin, gentamicin, gentamicin sulfate, acriflavine, ciprofloxacin, benzethonium chloride, fusidic acid, neomycin sulfate, ofloxacin and tobramycin.

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