Fish-derived acellular dermal matrix and preparation method therefor

Abstract

The present invention relates to: a fish-derived acellular dermal matrix having improved tissue-to-tissue adhesion problems while ensuring biocompatibility by removing cellular components from fish-derived by-products; and a preparation method therefor.

Description

Fish-derived acellular dermal matrix and method for producing the same

[0001] The present invention relates to a fish-derived acellular dermal matrix and a method for producing the same.

[0002] Acellular dermal matrices (ADMs) have been used to reconstruct skin defects and injuries, or as tissue engineering scaffolds. These acellular dermal matrices are typically derived from mammalian skin tissues, such as human, porcine, or bovine. By removing cellular components, they minimize immune rejection upon transplantation and promote cell attachment and tissue regeneration. However, mammalian acellular dermal matrices present limitations, including the risk of potential pathogen infection, the potential for zoonotic disease transmission, religious and ethical concerns, and limited raw material supply. Furthermore, their manufacturing process is expensive, and their use is restricted in certain countries and regions.

[0003] Accordingly, interest in acellular dermal matrices derived from marine organisms, particularly fish, as a safer and more abundant source is growing. Compared to mammals, fish have the advantages of a lower risk of zoonotic infections, abundant byproduct resources, and relatively low cost. However, fish skin tissues differ structurally and biochemically from those of humans or mammals, requiring an appropriate decellularization process that effectively removes cellular debris and antigenicity while preserving key matrix components such as collagen. Furthermore, fish skin tissues exhibit lower inter-tissue bonding strength compared to those derived from humans or mammals, leading to swelling during crosslinking, making sheet-type production difficult.

[0004] Therefore, there is an urgent need to develop a fish-derived acellular dermal matrix that removes cellular components from fish-derived byproducts to ensure biocompatibility while improving inter-tissue bonding.

[0005] The purpose of the present invention is to provide a fish-derived acellular dermal matrix and a method for manufacturing the same, which are derived to solve the above-described problems, and which secure biocompatibility by removing cellular components from fish-derived by-products while improving the problem of inter-tissue bonding.

[0006] In order to achieve the above object, a method for producing a fish-derived acellular dermal matrix according to one embodiment of the present invention comprises (i) a step of decellularizing a fish-derived by-product, (ii) a step of freeze-drying the decellularized fish-derived by-product in step (i), (iii) a step of pulverizing the freeze-dried fish-derived by-product in step (ii), (iv) a step of mixing a biocompatible polymer with the powder of the fish-derived by-product pulverized in step (iii) and adding a chemical cross-linking agent to perform a primary cross-linking, (v) a step of heat-drying or freeze-drying the primary cross-linked product obtained in step (iv), (vi) a step of adding a chemical cross-linking agent and an ionic cross-linking agent to the heat-dried or freeze-dried primary cross-linked product to perform a secondary cross-linking at least once, and (vii) a step of washing and then drying the secondary cross-linked product obtained in step (vi).

[0007] According to one embodiment of the present invention, a fish-derived acellular dermal matrix comprises a fish-derived by-product powder and a biocompatible polymer, and is manufactured by two stages of cross-linking.

[0008] Here, the fish-derived by-product powder may be a freeze-dried and pulverized decellularized fish-derived by-product.

[0009] According to one embodiment of the present invention, a fish-derived acellular dermal matrix and a method for producing the same can secure biocompatibility by removing cellular components from fish-derived by-products.

[0010] Additionally, the tensile strength can be improved while reducing moisture absorption through the application of biocompatible polymers and two cross-linking processes.

[0011] Figure 1 is a process diagram illustrating a method for manufacturing a fish-derived acellular dermal matrix according to an embodiment of the present invention.

[0012] Figure 2 is a photograph showing salmon skin.

[0013] Figure 3 is a photograph showing freeze-dried fish-derived by-products.

[0014] Figure 4 is a photograph showing a fish-derived by-product powder according to Example 2.

[0015] Figure 5 is an SEM photograph showing the results according to Experimental Example 1.

[0016] Figure 6 is a photograph showing the results according to Experimental Example 2.

[0017] Figure 7 is a graph showing the results according to Experimental Example 3.

[0018] Figure 8 is a graph showing the results according to Experimental Example 4.

[0019] FIG. 9 is a photograph showing the shape of a fish-derived acellular dermal matrix manufactured in the same manner as in Example 3, except that it does not include a biocompatible polymer.

[0020] FIG. 10a is a photograph showing the shape of a sheet-type fish-derived acellular dermal matrix according to Example 3 and Comparative Examples 1 to 7, and FIG. 10b is a photograph showing the shape of a sponge-type fish-derived acellular dermal matrix according to Example 4 and Comparative Examples 8 to 13.

[0021] Figure 11 is a photograph showing the results according to Experimental Example 7.

[0022] Figures 12a, 12b and 13 are graphs showing the results according to Experimental Example 8.

[0023] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0024] In addition, the terms used in this specification are terms used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0025] Throughout this specification, '%' used to indicate the concentration of a particular substance is %(w / w) for solid / solid, %(w / v) for solid / liquid, and %(v / v) for liquid / liquid, unless otherwise stated.

[0026] The fish-derived acellular dermal matrix according to the present invention can be applied as a tissue supplement for rotator cuff injury, a repair material for a defect after breast conservation surgery, a soft tissue supplement after cervical spine surgery, and for skin soft tissue wound reconstruction.

[0027]

[0028] Hereinafter, a method for manufacturing a fish-derived acellular dermal matrix according to one embodiment of the present invention will be described in detail with reference to drawings.

[0029] Figure 1 is a process diagram illustrating a method for manufacturing a fish-derived acellular dermal matrix according to one embodiment of the present invention.

[0030] Referring to Figure 1, first, fish-derived by-products are decellularized (S10).

[0031] Decellularization of fish-derived by-products may be performed by a step of washing with a delipidating solution, a step of immersing in a hypertonic solution, a step of immersing in an alkaline solution, and a step of washing. Here, salmon skin may be used as the fish-derived by-product, but is not limited thereto.

[0032] The step of washing with a degreasing solution may involve washing the fish-derived by-products with a degreasing solution having a concentration of 50 to 100% at 4 to 40°C for 1 to 24 hours. If the concentration of the degreasing solution is less than 50%, the fat removal rate may be as low as 20% or less, which is not preferable. If the washing time is less than 1 hour, the fat removal rate may be as low as 20% or less, and if it exceeds 24 hours, the fat removal rate may not be significantly greater than the increase in time. If the washing temperature is less than 4°C, the fat removal rate may be very low as 10% or less, and if it exceeds 40°C, skin tissue may be denatured.

[0033] When the above-described desirable concentration of the degreasing solution and the desirable temperature and time conditions for washing are all satisfied, the fat removal rate can be very excellent, at 50% or more.

[0034] The delipidating solution may contain a polar solvent, wherein the polar solvent is an alcohol, preferably one or more selected from isopropyl alcohol (IPA), ethanol, methanol, butanol, octanol and water, preferably isopropyl alcohol.

[0035] The step of immersing in a high-temperature solution can be performed by immersing in 20 to 30 parts by weight of a high-temperature solution for 2 to 24 hours.

[0036] In this embodiment, “hypotonic” means a state in which the inside has a higher osmotic pressure than the outside when comparing the osmotic pressures of two solutions separated by a semipermeable membrane, that is, a state in which the concentration inside the membrane is higher than the concentration outside, and thus the net movement of water is directed toward the inside of the membrane. The rate of this passive diffusion phenomenon of water, osmosis, increases or decreases in proportion to the concentration gradient between the two solutions, and when the concentration difference disappears, the net movement of water may disappear.

[0037] In this embodiment, the term "hypotonic solution" is used in a treatment to affect cell lysis, and may refer to a solution having an osmolarity of about 300 mOsm / L or higher. For example, a hypertonic solution permeates the cell membrane through osmosis and enters the cell, causing the cells of the tissue to swell and rupture, thereby eliminating any remaining cells. If the concentrations of solutions across a membrane are different, water will move from a place of lower concentration to a place of higher concentration, and the rate of this passive diffusion of water, called osmosis, may be proportional to the concentration gradient between the two solutions.

[0038] In this embodiment, "osmolarity" refers to the expression of osmotic concentration per volume of solution (mOsm / L), and the osmolarity of plasma and other body fluids can be 270 to 300 mOsm / L. If the osmolarity of a solution is less than about 270 mOsm / L, it can be defined as a hypotonic solution, if it is in the range of about 270 to 300 mOsm / L, it can be defined as an isotonic solution, and if it is more than about 300 mOsm / L, it can be defined as a hypertonic solution.

[0039] The hypertonic solution of the present embodiment has an osmolarity of 300 mOsm / L or more, and may preferably include at least one selected from 5 to 20 mM Tris-HCl, 0.1 to 1.5% EDTA (ethylenediaminetetraacetic acid), 0.01 to 1 M NaOH (sodium hydroxide), and 0.05 to 1 M NaCl (sodium chloride).

[0040] For example, NaCl in the hypertonic solution can be applied at a concentration of 0.05 to 1 M, but if the hypertonic solution is outside the above-mentioned range, the skin of the fish-derived by-product may not be separated well.

[0041] Meanwhile, the high-temperature solution may further include a surfactant, and the surfactant may include an ionic surfactant, a non-ionic surfactant, or a mixture thereof, and preferably an ionic surfactant. For example, ionic surfactants include glutamate-based surfactants such as alkyl benzene sulfonate, alkyl sulfate, alkyl ether sulfate, alkoxylated amide, olefin sulfonate, alkyl xylene sulfonate, dialkyl sulfosuccinate, fatty acid ester sulfonate, alcohol sulfate, glycerol fatty acid ester, isethionate-based surfactants, phosphate-based surfactants such as lauryl phosphate or laureth-1-phosphate, taurate-based surfactants, and alkoxylated alcohols. At least one selected from the group consisting of alcohol, alkyl carboxylate, hydroxyalkyl, quaternary ammonium salt and ethoxylated alkyl, preferably sodium dodecyl sulfate (SDS), sodium laureth sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium myreth sulfate (SMES), dioctyl sodium sulfosuccinate (DSS), perfluorooctanesulfonate (PFOS),It may include at least one selected from the group consisting of PFBS (Perfluorobutanesulfonate), PFNA (perfluorononanoate), PFOA (perfluorooctanoate), sodium stearate, sodium lauroyl sarcosinate, CTAB (cetrimonium bromide), CPC (cetylpyridinium chloride), BAC (benzalkonium chloride), BZT (benzethonium chloride), DDAC (dimethyldioctadecylammonium chloride), and DODAB (dioctadecyldimethylammonium bromide), and more preferably, SDS (sodium dodecyl sulfate), but is not limited thereto.

[0042] In the past, non-ionic surfactants or high-concentration ionic surfactants were used to dissolve cell membranes to remove cellular components other than proteins from tissues. However, there were problems in that residual protein denaturation within the support was induced, the microstructure of the extracellular matrix was destroyed, and growth factors were excessively removed. Therefore, in the present embodiment, the problems described above were solved by applying a low-concentration ionic surfactant. Specifically, a low-concentration alkyl sulfate, more preferably a low-concentration SDS (sodium dodecyl sulfate) can be used as the ionic surfactant. For example, the concentration of SDS (sodium dodecyl sulfate) may be 0.1 to 0.5% (w / v), preferably 0.2 to 0.4% (w / v) in a hypertonic solution, but is not limited thereto.

[0043] The step of immersing in an alkaline solution may be immersed in one or more alkaline solutions selected from NaOH, KOH, Ca(OH)2, and NH4OH for 2 to 3 hours.

[0044] The washing step can remove fat, epidermis, and cells remaining on the salmon skin with PBS.

[0045] Additionally, after the washing step, a decolorizing step and a washing step can be further performed by stirring with a solution containing H2O2.

[0046]

[0047] Then, the decellularized fish-derived by-product is freeze-dried (S20).

[0048] At step S10, decellularized fish-derived by-products can be freeze-dried at -40°C or lower for 1 to 18 hours.

[0049]

[0050] Next, the freeze-dried fish-derived by-product is crushed (S30).

[0051] In step S20, the freeze-dried fish by-product can be ground to a diameter of 10 to 1000 μm using one or more grinders selected from a cutting mill, a food processor, an agate grinder, a freeze grinder, a micronizer, a vibrating micro mill, a jaw crusher, a mortar grinder, a planetary mill, a disk mill, a ball mill, a knife mill, and a variable speed rotor mill. At this time, it is preferable to set the grinder to a rotation speed of 500 to 3000 rpm to facilitate grinding in a short period of time. In addition, the particle size can be uniformly sieved using a sieve having a mesh size of 10 to 1000 μm. Therefore, not only can the degree of cross-linking be maintained uniformly in the first and second cross-linking steps described below, but also the possibility of decomposition and damage can be reduced when manufacturing in the form of a sheet or sponge, and manufacturing stability can be improved.

[0052]

[0053] Afterwards, a biocompatible polymer is mixed with the fish-derived by-product powder and a chemical cross-linking agent is added to perform primary cross-linking (S40).

[0054] In step S30, 100 parts by weight of the powder of fish-derived by-products is mixed with 1 to 50 parts by weight of a biocompatible polymer, and a chemical cross-linking agent is added at a concentration of 0.1 to 50 mM to perform primary cross-linking at 4 to 40°C for 30 seconds to 60 minutes.

[0055] In this example, since the fish-derived by-product powder is not pure collagen, it has weak inter-tissue binding force and is difficult to maintain shape when used alone. Therefore, in the present invention, the binding force problem of the fish-derived by-product was solved by applying a biocompatible polymer together.

[0056] As the biocompatible polymer, one or more selected from hyaluronic acid, gelatin, carrageenan, polylactic acid, polycaprolactone, polyglycolic acid, and alginate may be used. If the biocompatible polymer is mixed in an amount of less than 1 part by weight, crosslinking of the fish-derived byproduct may be difficult, and the binding force may be weak, making it difficult to maintain the shape, and the moisture absorption may increase.

[0057] The chemical cross-linking agent may include one or more selected from glutaraldehyde, Genipin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0058]

[0059] Then, the primary cross-linked product is heat-dried or freeze-dried (S50).

[0060] The primary cross-linked product obtained in step S40 can be heat-dried at 30 to 40°C for 1 to 18 hours until the moisture content becomes 0.5 to 52 wt%, or freeze-dried at -40°C or lower for 1 to 18 hours.

[0061] In the present invention, when freeze-drying is performed, a sponge type can be formed.

[0062] In the present invention, when heat drying is performed, a sheet type can be formed.

[0063] The primary cross-linked product of the present invention can be heat-dried or freeze-dried in a mold. This improves physical properties and tensile strength, making it easier for practitioners to use during procedures, and can also serve as a physical barrier.

[0064] In the case of heat drying, it can be carried out at 30 to 40°C for 1 to 18 hours, for example, 6 hours or more, for example, 10 hours or more, for example, 10 to 24 hours, preferably 16 to 18 hours, until the moisture content in the mold becomes 0.5 to 52 wt%.

[0065] If the above heat drying temperature is less than 30°C, drying may take an excessive amount of time, and if it exceeds 40°C, the tissue may become deformed, resulting in a decrease in amino acid content as well as a decrease in tensile strength and hydration degree.

[0066] Additionally, by performing heat drying or freeze drying through a mold, the structure can be made dense and can also function as a physical barrier.

[0067]

[0068] Afterwards, a chemical cross-linking agent and an ionic cross-linking agent are added to the heat-dried or freeze-dried primary cross-linked product to perform secondary cross-linking at least once (S60).

[0069] In step S50, a chemical cross-linking agent at a concentration of 0.1 to 50 mM and an ionic cross-linking agent at a concentration of 9 to 500 mM are added to 100 parts by weight of the heat-dried or freeze-dried primary cross-linked product, and secondary cross-linking is performed at 4 to 40°C for 30 minutes to 24 hours. In this embodiment, it is preferable to mix and use the chemical cross-linking agent and the ionic cross-linking agent at a weight ratio of 1: 0.1 to 2900, and as the ionic cross-linking agent, one or more selected from calcium chloride (CaCl2), zinc chloride (ZnCl2), iron chloride (FeCl3), and aluminum chloride (AlCl3) may be used.

[0070]

[0071] Finally, the secondary cross-linked product is washed and dried (S70).

[0072] The secondary cross-linked product obtained in step S60 can be washed several times with purified water and then dried at -40°C or lower for 1 to 20 hours, for example, freeze-dried.

[0073]

[0074] Hereinafter, a fish-derived acellular dermal matrix according to another embodiment of the present invention will be described in detail. Any description that overlaps with the method for manufacturing the fish-derived acellular dermal matrix will be omitted.

[0075] The fish-derived acellular dermal matrix may be of sheet or sponge type, and may contain 100 parts by weight of fish-derived by-product powder and 1 to 50 parts by weight of a biocompatible polymer, and may be manufactured by two cross-linking processes. The first cross-linking process may be cross-linking by a chemical cross-linking agent, and the second cross-linking process may be cross-linking by a chemical cross-linking agent and an ionic cross-linking agent.

[0076] Meanwhile, the fish-derived by-product powder may be freeze-dried and ground decellularized fish-derived by-product, and the average diameter of the powder may be in the range of 10 to 1000 μm. Such fish-derived by-product powder may have a total amino acid content of 300 mg / g or more and a hydroxyproline content of 6% or more. In addition, in the case of sheet type, the water absorption rate may be 130% or less and the tensile strength may be 0.4 MPa or more, and in the case of sponge type, the water absorption rate may be 600% or less.

[0077]

[0078] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0079]

[0080] Example 1. Decellularization of fish-derived by-products

[0081] As shown in Fig. 2, salmon skin was obtained, and the fat and scale tissue attached to the salmon skin tissue was removed using forceps, and washed three times with sterile water. Then, it was washed with 50-100% isopropyl alcohol (IPA) and stirred in 20-30 parts by weight of a solution containing 1 M NaCl for 2-3 hours. Then, the salmon skin was stirred in 20-35 parts by weight of a solution containing 0.1 M to 1 M NaOH for 2-3 hours at 4°C. After washing with PBS to remove the fat, epidermis, and cells remaining in the salmon skin, it was left overnight, and then decolorized by stirring in 20-30 parts by weight of a solution containing H2O2 for 1-3 hours. After that, it was washed with PBS for 1 hour to overnight to decellularize fish-derived by-products.

[0082]

[0083] Example 2. Preparation of fish-derived by-product powder

[0084] In Example 1, the decellularized fish by-product was freeze-dried at -25 to -85°C for 24 hours or more as shown in Fig. 3, and then ground to a diameter ranging from 10 to 1000 μm. Then, the by-product was filtered using a sieve having a mesh size of 10 to 1000 μm to produce a fish by-product powder having a uniform particle size as shown in Fig. 4.

[0085]

[0086] Example 3. Preparation of sheet-type fish-derived acellular dermal matrix

[0087] 100 parts by weight of the fish-derived by-product powder according to Example 2 was mixed with 1 to 50 parts by weight of alginate, and EDC-NHS at a concentration of 0.1 to 50 mM (purified water solvent) was added, followed by primary cross-linking at 4 to 40°C for 30 seconds to 60 minutes, and then heat-dried in a mold at 30 to 40°C for more than 6 hours. Then, EDC-NHS at a concentration of 0.1 to 50 mM and calcium chloride at a concentration of 9 to 500 mM were added, followed by secondary cross-linking once at 4 to 40°C for 30 minutes to 24 hours, washing with purified water, and drying at -40°C or lower for more than 5 hours to produce a sheet-type fish-derived acellular dermal matrix.

[0088]

[0089] Example 4. Preparation of sponge-type fish-derived acellular dermal matrix

[0090] 100 parts by weight of the fish-derived by-product powder according to Example 2 was mixed with 1 to 50 parts by weight of alginate, and 0.1 to 50 mM of EDC-NHS (purified water solvent) was added, followed by primary cross-linking at 4 to 40°C for 30 seconds to 60 minutes, freezing in a mold at -20°C or lower for 1 hour or longer, and freeze-drying for 16 hours or longer. Then, 0.1 to 50 mM of EDC-NHS and 9 to 500 mM of calcium chloride were added, followed by secondary cross-linking once at 4 to 40°C for 30 minutes to 24 hours, washing with purified water, and drying at -40°C or lower for 5 hours or longer to produce a sponge-type fish-derived acellular dermal matrix.

[0091]

[0092] Comparative Examples 1 to 7. Preparation of sheet-type fish-derived acellular dermal matrix

[0093] A sheet-type acellular dermal matrix derived from fish was manufactured in the same manner as in Example 3, except that the first or second cross-linking was performed under the conditions described in Table 1 below and the heat-drying or freeze-drying (S50) step was omitted.

[0094] Classification1st crosslinking2nd crosslinkingComparative example 8XEDC-NHS+CaCl₂ 1 timeComparative example 9XCaCl₂ 1 timeComparative example 10XEDC-NHS 1 timeComparative example 11XEDC-NHS+CaCl₂ 1 timeComparative example 12EDC-NHSCaCl₂ 1 timeComparative example 13EDC-NHSEDC-NHS 1 time

[0095]

[0096] Comparative Example 8. Manufacturing of a sponge-type fish-derived acellular dermal matrix

[0097] A sponge-type acellular dermal matrix derived from fish was manufactured in the same manner as in Example 4, except that alginate was not applied, secondary cross-linking was performed under the conditions described in Table 2 below, and the heat-drying or freeze-drying (S50) step was omitted.

[0098]

[0099] Comparative Examples 9 to 12. Preparation of sponge-type fish-derived acellular dermal matrix

[0100] A sponge-type acellular dermal matrix derived from fish was manufactured in the same manner as in Example 4, except that the first or second cross-linking was performed under the conditions described in Table 2 below and the heat-drying or freeze-drying (S50) step was omitted.

[0101] Classification1st crosslinking2nd crosslinkingComparative example 8XEDC-NHS+CaCl₂ 1 timeComparative example 9XCaCl₂ 1 timeComparative example 10XEDC-NHS 1 timeComparative example 11XEDC-NHS+CaCl₂ 1 timeComparative example 12EDC-NHSCaCl₂ 1 timeComparative example 13EDC-NHSEDC-NHS 1 time

[0102]

[0103] Experimental Example 1. Confirmation of the structure and morphology of decellularized fish-derived byproducts.

[0104] To confirm the structure and morphology of the decellularized fish-derived by-product manufactured in Example 1, SEM images were taken, and the results are shown in Fig. 5.

[0105] Referring to Figure 5, a layered and porous structure were confirmed, confirming that most cells within the tissue had been removed. Furthermore, a three-dimensional network of reticular structures was confirmed to have formed in the dermis and superficial areas.

[0106]

[0107] Experimental Example 2. Decellularization Evaluation (H&E Staining)

[0108] The decellularized fish-derived by-products prepared in Example 1 above were subjected to a histological examination using H&E staining to evaluate decellularization.

[0109] Paraffin blocks were cut into 4 μm thick sections and dried to prepare paraffin sections. Afterwards, for deparaffinization, the sections were treated with xylene three times for 5 minutes each, 100% ethanol three times for 2 minutes each, 90% ethanol once for 1 minute each, 80% ethanol once for 1 minute each, and 70% ethanol once for 1 minute each, and then washed in running water for 10 minutes. After reacting with hematoxylin staining solution for 10 minutes, they were washed in running water for 3 minutes, and then reacted with eosin staining solution for 10 minutes, and then washed in running water until no eosin staining solution appeared. After reacting 10 times for 1 second in 70% ethanol, 10 times for 1 second in 80% ethanol, 10 times for 1 second in 90% ethanol, 2 times for 1 minute in 100% ethanol, and 3 times for 3 minutes in xylene, the sample was mounted with a mounting solution and then photographed with an optical microscope (Olympus BX51, H&E staining) and a scanning electron microscope (Hitachi S-4700, Japan), and the results are shown in Fig. 6.

[0110] Referring to Figure 6, it was confirmed that most of the cells within the tissue were removed.

[0111]

[0112] Experimental Example 3. Quality Assessment by Decellularization

[0113] In order to confirm the changes in the residual amounts of amino acids, hydroxyfolic acid, crude fiber, and DNA due to decellularization of fish-derived by-products, measurements were performed as follows, and the results are shown in Figure 7.

[0114] (a) Amino acid content

[0115] Measurement was performed using an amino acid auto-analyzer (S433D, Sykam GmbH Co., Germany).

[0116] (b) Hydroxyproline content

[0117] Quantification was performed through color change by oxidation reaction (Chloramine-T method) after hydrochloric acid hydrolysis.

[0118] (c) Crude fat content

[0119] The weight of residual insoluble crude fat after acid-alkali hydrolysis was measured using the AOAC method.

[0120] (d) DNA residue

[0121] After extraction using a DNA Extraction kit, measurement was performed using UV absorbance measurement.

[0122] Referring to Figure 7, amino acids and hydroxyproline were identified in Example 1, indicating that even after decellularization, the cells contained a large amount of collagen ECM, which is beneficial for tissue regeneration. Furthermore, the low level of DNA residues indicated a low risk of immune rejection.

[0123]

[0124] Experimental Example 4. Safety Evaluation (Cell Viability)

[0125] To evaluate safety through cell viability, a CCK-8 kit was used, and the results are shown in Figure 8.

[0126] The sheet-type acellular dermal matrix (ADM) derived from fish according to Example 3 was extracted at a ratio of 0.2 g / mL in culture medium (Media) for 24 hours. The extracted solution was prepared as a stock solution, a 2-fold dilution, and a 4-fold dilution, respectively. The culture medium itself was used as a negative control, and DMSO solution was used as a positive control.

[0127] Each prepared sample was dispensed at 200 μL / well into L929 cells cultured in a 96-well plate and cultured for 48 hours. After the culture was completed, cell viability was evaluated using a CCK-8 kit.

[0128] Referring to Figure 8, it was confirmed that the cell viability was over 80% and that there was no cytotoxicity due to the crosslinking agent.

[0129]

[0130] Experimental Example 5. Shape Evaluation with and without Biocompatible Polymers

[0131] The shape of a fish-derived acellular dermal matrix prepared in the same manner as in Example 3, except that it did not include a biocompatible polymer, was visually evaluated.

[0132] Referring to Fig. 9, it was confirmed that the volume expanded compared to Example 3 when only the fish-derived by-product powder was applied. Therefore, it was found that when a biocompatible polymer is included, the volume expansion is minimized during crosslinking and the sheet shape can be maintained.

[0133]

[0134] Experimental Example 6. Shape Evaluation According to Crosslinking Conditions

[0135] The shape of the fish-derived acellular dermal matrix according to Examples 3 and 4 and Comparative Examples 1 to 13 was visually evaluated.

[0136] Referring to Fig. 10a, in the case of Comparative Examples 1 and 2, CaCl2 clumped and crosslinked through the Alginate crosslinking reaction, making sheet implementation difficult, and in the case of Comparative Example 3, it was confirmed that the volume expanded after the first EDC-NHS crosslinking and the second crosslinking was not applied, resulting in the disintegration during washing. In the case of Comparative Examples 4 to 7, it was confirmed that sheet implementation was possible in the same manner as in Example 3.

[0137] Referring to Fig. 10b, in the case of Comparative Example 8, since Alginate was not included, it was difficult to implement a sponge due to insufficient shape maintenance during the secondary crosslinking, and in the case of Comparative Examples 9 to 11, it was also difficult to implement a sponge through secondary crosslinking using EDC-NHS or CaCl2 alone without the primary crosslinking, and in the case of Comparative Examples 12 and 13, it was confirmed that sponge implementation was possible in the same manner as in Example 4. Therefore, it was found that ionic crosslinking of Alginate was more advantageous in maintaining shape than chemical crosslinking of collagen in the sponge formulation.

[0138]

[0139] Experimental Example 7. Evaluation of Hydration Degree According to Crosslinking Conditions

[0140] The fish-derived acellular dermal matrix (size 1X1 cm) according to Example 3 and Comparative Examples 4, 5, and 6 was hydrated in physiological saline solution for 1 minute, and then the shrinkage and expansion were visually evaluated.

[0141] Referring to Figure 11, EDC-NHS or CaCl2 In Comparative Examples 4 and 5, where single crosslinking was performed, swelling occurred upon hydration, and in Comparative Example 6, where mixed crosslinking of EDC-NHS and CaCl2 was performed without primary crosslinking, it was confirmed that volume expansion occurred due to a decrease in crosslinking efficiency, resulting in shrinkage upon hydration. On the other hand, in Example 3, where primary and secondary crosslinking were performed, no changes in shrinkage or expansion were observed upon crosslinking.

[0142]

[0143] Experimental Example 8. Evaluation of Physical Properties According to Crosslinking Conditions

[0144] To determine the physical properties of the sheet-type fish-derived acellular dermal matrix according to the cross-linking conditions, water absorption and tensile strength were measured, and the results are shown in Figs. 12 and 13.

[0145] The water absorption capacity was determined by measuring the initial weight of the acellular dermal matrix derived from fish, injecting 1 ml of saline solution for the sheet type and 3 ml for the sponge type, hydrating for 1 minute, measuring the weight after hydration, and calculating the value using the following equation (1).

[0146] Water absorption rate = [(weight after hydration - initial weight) / initial weight] * 100 (%)… … … … … Equation (1)

[0147] Referring to Fig. 12a, the moisture absorption capacity of Example 3 was lower than that of Comparative Examples 4 to 6, and it was confirmed that the moisture absorption capacity gradually decreased from Comparative Example 6 to Comparative Example 4. Therefore, it was found that Example 3 had the lowest moisture absorption rate and the best crosslinking efficiency and mechanical stability.

[0148] Referring to Fig. 12b, it was confirmed that the water absorption capacity of Example 4 was lower than that of Comparative Example 12. This is due to the difference in the crosslinking rate of collagen and alginate in the acellular dermal matrix derived from fish. As the crosslinking rate increases, the network becomes denser and the pores decrease, which leads to a decrease in water absorption. As a result, it was found that Example 4 had the best crosslinking efficiency and structural stability.

[0149] Referring to Figure 13, the tensile strength of Example 3 was found to be higher than that of Comparative Examples 4 to 7, and there was no significant difference in the tensile strength when single crosslinking and mixed crosslinking were performed once, but it was confirmed that Comparative Example 7, in which mixed crosslinking was repeated three times, was found to be higher than that of Comparative Examples 4 to 6.

[0150]

[0151] Although exemplary embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0152] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

Claims

1. (i) A step of decellularizing fish-derived by-products; (ii) a step of freeze-drying the decellularized fish-derived by-product from step (i) above; (iii) a step of grinding the freeze-dried fish-derived by-product from step (ii) above; (iv) A step of mixing a biocompatible polymer with the fish-derived byproduct powder ground in step (iii) above and adding a chemical crosslinking agent to perform primary crosslinking; (v) a step of heat-drying or freeze-drying the primary crosslinking product obtained in step (iv) above; (vi) a step of adding a chemical crosslinking agent and an ionic crosslinking agent to the heat-dried or freeze-dried primary crosslinking product to perform secondary crosslinking at least once; and (vii) a step of washing and drying the secondary crosslinking product obtained in step (vi) above; a method for preparing a fish-derived acellular dermal matrix.

2. In paragraph 1, A method for preparing a fish-derived acellular dermal matrix, wherein in steps (iv) and (vi) above, the chemical crosslinking agent comprises one or more selected from glutaraldehyde, genipin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

3. In paragraph 1, A method for preparing a fish-derived cell-free dermal matrix, wherein the ionic crosslinking agent in step (vi) comprises one or more selected from calcium chloride (CaCl2), zinc chloride (ZnCl2), iron chloride (FeCl3) and aluminum chloride (AlCl3).

4. In paragraph 1, A method for preparing a fish-derived acellular dermal matrix, wherein in step (vi) above, the chemical crosslinking agent and the ionic crosslinking agent are mixed in a weight ratio of 1:0.1 to 2900.

5. Containing fish-derived byproduct powder and biocompatible polymer, Fish-derived acellular dermal matrix produced by two crosslinking steps.

6. In paragraph 5, The above fish-derived byproduct powder is a fish-derived acellular dermal matrix obtained by freeze-drying and grinding decellularized fish-derived byproducts.

7. In Paragraph 5, The above fish-derived by-product powder is a fish-derived acellular dermal matrix having a total amino acid content of 300 mg / g or more and a hydroxyproline content of 6% or more.

8. In paragraph 5, The above fish-derived acellular dermal matrix is ​​a fish-derived acellular dermal matrix of sheet type or sponge type.

9. In paragraph 8, The above sheet type is a fish-derived acellular dermal matrix having a moisture absorption rate of 130% or less and a tensile strength of 0.4 MPa or more.

10. In paragraph 8, The above sponge type is a fish-derived acellular dermal matrix with a water absorption rate of 600% or less.

Images