WO2024096401A1 - Pharmaceutical composition for wound healing or skin tissue regeneration, containing extracellular matrix and kelp extract - Google Patents

Abstract

One aspect relates to a pharmaceutical composition for wound healing or skin tissue regeneration, the pharmaceutical composition containing an extracellular matrix and a kelp extract. According to one aspect, the extracellular matrix and the kelp extract have excellent biodegradation, swelling, protein release, cell proliferation, and biocompatibility, reduce the expression of MMP, and can convert the phenotype of macrophages from the M1 type to the M2 type, and thus can effectively reduce the time required for wound healing. Therefore, the pharmaceutical composition can be effectively used for wound healing and/or skin tissue regeneration.

Description

Pharmaceutical composition for wound treatment or skin tissue regeneration containing extracellular matrix and kelp extract

It relates to a pharmaceutical composition for wound treatment or skin tissue regeneration containing extracellular matrix and kelp extract.

Biomaterials for wound healing must meet various requirements such as non-toxicity, biocompatibility, wound exudate absorption, moisture permeability, microbial resistance, and adhesion. Currently, much research is being conducted to develop ideal biomaterials that satisfy all of these. .

Decellularized ECM (extracellular matrix), one of the biomaterials, is rich in growth factors, chemokines, and cytokines, and the nuclear, cellular, and cytoplasmic components that can stimulate immune responses are removed through the decellularization process, making it effective in wound healing. It is known that it can be used. However, in order to use decellularized ECM as a scaffold for wound healing, a crosslinker is required. In particular, ECM derived from liver tissue has a low proportion of soluble collagen, so a strong crosslinker is required. However, these cross-linking agents have problems such as cytotoxicity or low biocompatibility, so there is a need to develop cross-linking agents with excellent biocompatibility.

Meanwhile, seaweed is rich in minerals and bioactive molecules such as fucoidan and is known to have various biomedical properties such as antioxidant, anti-inflammatory, anti-tumor, anti-coagulant, collagen matrix formation and neovascularization. Accordingly, a study was conducted to use kelp, one of the seaweeds, as a cross-linking agent for the liver-derived ECM. As a result, although the cross-linking ability of kelp was excellent, there was a problem of low biocompatibility due to the high pH of the entire scaffold. Accordingly, the above problem was solved by introducing p-coumaric acid (p-Cou), one of the phenolic acids, into the scaffold, and ultimately a biomaterial for wound healing with excellent biocompatibility was developed (see Figure 1).

One aspect is to provide a pharmaceutical composition for wound treatment or skin tissue regeneration, comprising an extracellular matrix and Kelp extract.

Another aspect is to provide a topical skin preparation for wound treatment or skin tissue regeneration, comprising an extracellular matrix and Kelp extract.

Another aspect is to provide a cosmetic composition for improving wounds or regenerating skin tissue, including an extracellular matrix and kelp extract.

Another aspect is to provide a method of treating wounds or regenerating skin tissue, comprising treating a subject with an extracellular matrix and a Kelp extract.

Another aspect is to provide the use of the extracellular matrix and Kelp extract for the manufacture of drugs for wound treatment or skin tissue regeneration.

One aspect provides a pharmaceutical composition for wound treatment or skin tissue regeneration, comprising an extracellular matrix and Kelp extract.

The term “extracellular matrix” refers to a collection of biopolymers that fill the space within tissues or outside cells, including collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, and cytokines. and growth factors.

In one aspect, the extracellular matrix may be derived from a mammal. The mammal may be a pig, cow, sheep, goat, or primate, and specifically, the mammal may be a pig.

Additionally, in one aspect, the extracellular matrix may be derived from fibroblasts, stem cells, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, or cardiomyocytes, and specifically, The extracellular matrix may be derived from hepatocytes.

The term "kelp" refers to seaweed belonging to the Kelp family of the Kelp order of the Kelp family. It attaches to rocks and reproduces through spores, and has thick leaves of about 2 to 3 mm. In one aspect, the kelp can be used as a crosslinker for the extracellular matrix.

According to one aspect, the pharmaceutical composition containing the extracellular matrix and kelp extract is excellent in biodegradation, swelling, protein release, cell proliferation, and biocompatibility, and contains growth factors. It contains excellent wound healing effects. In addition, the composition can reduce the expression of MMPs and change the phenotype of macrophages from type M1 to type M2, effectively shortening the time required for wound healing. Therefore, the composition can be usefully used for wound treatment and/or skin tissue regeneration.

In one aspect, the extracellular matrix and kelp extract may be decellularized.

The term “decellularization” refers to the process of removing cellular components, such as nuclei, cell membranes, and nucleic acids, without damaging the original components of the tissue or cell. The decellularization may be performed by treating the cells with a solution containing one or more selected from the group consisting of cell lysis solvent, stock solution, cationic surfactant, anionic surfactant, nonionic surfactant, RNase, and DNase.

According to one aspect, the extracellular matrix and kelp extract have cellular components such as nuclei, cell membranes, and nucleic acids removed through the decellularization, so that when treated in an individual, stimulation of the individual's immune response can be minimized.

In one aspect, the concentration ratio of the extracellular matrix and kelp extract may be 1:0.75 to 1:1.25.

According to one aspect, as the content of the kelp extract increases, the mechanical strength of the entire composition increases, and pores are formed uniformly in a small size, thereby increasing the wound exudate inhibition ability, and the concentration ratio of the extracellular matrix and the kelp extract When 1:0.75 to 1:1.25, it is effective for wound healing and/or skin tissue regeneration.

In one aspect, the composition may further include an acidic drug, and the acidic drug may be p-coumaric acid.

The p-coumaric acid is an acidic drug of the polyphenol group, and can stably control the pH of the entire composition by reducing the high pH of the entire composition as the content of the kelp extract increases. In addition, p-coumaric acid is known to have anti-inflammatory, antioxidant and antibacterial effects, and by further including p-coumaric acid, the wound healing and/or skin tissue regeneration effects can be further improved.

In one aspect, the composition may have an antibacterial effect, and the antibacterial effect may be against gram-positive bacteria and gram-negative bacteria.

The gram-positive bacteria include bacteria of the genus Staphylococcus , bacteria of the Streptococcus genus, bacteria of the genus Enterococcus , bacteria of the genus Klebsiell , bacteria of the genus Corynebacterium , and clostrites. It may be one or more selected from the group consisting of Clostridium genus bacteria, Listeria genus bacteria, and Bacillus genus bacteria. Specifically, the Gram-positive bacteria are Staphylococcus genus bacteria and/or Bacillus. It may be a bacterium of the genus ( Bacillus ).

In addition, the Gram-negative bacteria include Escherichia species, Salmonella , Pseudomonas , Neisseria , Chlamydia , and Yersinia . ) may be one or more selected from the group consisting of bacteria of the genus, and specifically, the gram-negative bacteria may be bacteria of the genus Escherichia and/or bacteria of the genus Salmonella .

In one aspect, the composition may inhibit the expression of matrix metalloproteinases (MMPs).

The MMP is a proteolytic enzyme that can decompose extracellular matrix proteins, and is known to destroy newly formed ECM and delay wound healing time when activated for a long period of time.

According to one aspect, the composition can effectively shorten the time required for wound healing and/or skin tissue regeneration by inhibiting the expression of MMP.

In one aspect, the composition may convert the phenotype of macrophages from type M1 to type M2.

The macrophage is a type of white blood cell, derived from hematopoietic stem cells in the bone marrow, and performs phagocytosis by engulfing and decomposing cell debris, foreign substances, microorganisms, cancer cells, abnormal proteins, etc. ) function, and is known to play an important role in not only non-specific defense mechanisms (innate immunity) but also in initiating specific defense mechanisms (adaptive immunity) through interactions with other immune cells such as lymphocytes.

Meanwhile, the macrophages can be divided into two subtypes: M1 type (inflammatory/offensive type) and M2 type (anti-inflammatory/repair type). M1 type macrophages cause an inflammatory response, remove bacteria, and perform anticancer actions. M2-type macrophages suppress inflammatory responses, perform wound healing, and tissue repair, and can exhibit conflicting actions.

According to one aspect, the composition can change the phenotype of macrophages from type M1 to type M2 by reducing the expression of CD68, an M1 type macrophage marker, and the concentration of nitrate, thereby promoting wound healing and/or skin tissue regeneration. is more effective.

In one aspect, the “wound” means that the normal continuity of the skin structure is disrupted due to physical damage to the skin. Additionally, in one aspect, the wound may be one or more selected from the group consisting of a wound, abrasion, laceration, stab wound, and ulcer.

In one aspect, the “treatment” means that a wound is healed in a shorter time compared to natural healing following administration of a pharmaceutical composition according to one aspect. The treatment may include improvement and/or alleviation of the wound, and the treatment may include treatment of the wound and/or disease related to the wound. Additionally, the treatment may promote healing and/or regeneration of the damaged tissue while minimizing scarring and/or complications of diseases associated with the wound.

In one aspect, “administering” means introducing a substance into a subject in an appropriate manner. The pharmaceutical composition according to one aspect may be administered through any general route capable of reaching the target in vivo. The administration route of the composition is not particularly limited, but may be administered orally or parenterally. Specifically, it can be administered parenterally, and more specifically, it can be applied by applying it to the skin (i.e., transdermal administration).

The “improvement” may include lowering or alleviating the severity of the injury.

In one aspect, the “skin tissue regeneration” is a process of recovering tissue from damage, and the skin tissue regeneration may be one or more selected from the group consisting of epidermal regeneration, reproduction of glands or hair follicles, and microvascular formation in dermal tissue. there is.

The pharmaceutical composition may be provided in any formulation suitable for topical application. For example, it may be a solution for external use on the skin, a suspension, an emulsion, a gel, a patch, or a spray, but is not limited thereto. The formulation can be easily prepared according to conventional methods in the field, and contains surfactants, excipients, wetting agents, emulsification accelerators, suspending agents, salts or buffers for adjusting osmotic pressure, colorants, flavorings, stabilizers, preservatives, preservatives or Other commonly used supplements can be used appropriately.

The pharmaceutical composition may further include appropriate carriers, excipients, or diluents commonly used in its preparation. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof. Alternatively, the carrier may be gauze, bandage, band, film, adhesive patch, or non-adhesive patch, and can be used as a wound dressing by using these carriers.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the drug's effect. It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The administration may be administered once a day, or may be administered several times. For example, it may be administered every other day, or it may be administered once a week.

The pharmaceutical composition may be provided by mixing with a conventionally known pharmaceutical composition for wound treatment or skin tissue regeneration or a newly developed pharmaceutical composition for wound treatment or skin tissue regeneration. When the pharmaceutical composition further includes a pharmaceutical composition for wound treatment or skin tissue regeneration, it is important to mix in an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. .

Additionally, in one aspect, the pharmaceutical composition may be administered alone or in combination with other drugs for wound treatment or skin tissue regeneration. That is, the pharmaceutical composition may be administered in combination with a known composition having a wound treatment or skin tissue regeneration effect or another drug for wound treatment or skin tissue regeneration, and may be administered simultaneously, separately, or sequentially. Or it can be administered in multiple doses. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect provides a topical skin preparation for wound treatment or skin tissue regeneration, comprising an extracellular matrix and Kelp extract.

The terms “extracellular matrix,” “kelp,” “wound,” “treatment,” “skin tissue regeneration,” etc. may be within the scope described above.

According to one aspect, the external skin preparation containing the extracellular matrix and kelp extract is excellent in biodegradation, swelling, protein release, cell proliferation, and biocompatibility, and contains growth factors. It has excellent wound healing effects. In addition, the topical skin agent can reduce the expression of MMPs and change the phenotype of macrophages from type M1 to type M2, effectively shortening the time required for wound healing. Therefore, the skin topical agent can be useful for wound treatment and/or skin tissue regeneration.

The “external skin preparation” is an inclusive concept that generally includes all materials used for external skin use. The skin external preparation contains a cosmetically or dermatologically acceptable medium or base and can be formulated for topical application to the skin. . These are all formulations suitable for topical application, for example, solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and non-ionic forms. It may be provided in the form of an ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be used in the form of foam or in the form of an aerosol composition further containing compressed propellant. These compositions can be prepared according to conventional methods in the art.

In addition, the skin external agent may contain fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic types. Cosmetic, such as emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid exosomes or any other ingredients commonly used in cosmetics. Alternatively, it may contain adjuvants commonly used in the field of dermatology. The auxiliaries can be introduced in amounts commonly used in the fields of cosmetology or dermatology.

Another aspect provides a cosmetic composition for improving wounds or regenerating skin tissue, comprising an extracellular matrix and kelp extract.

The terms “extracellular matrix,” “kelp,” “wound,” “improvement,” “skin tissue regeneration,” etc. may be within the above-mentioned scope.

According to one aspect, the cosmetic composition containing the extracellular matrix and kelp extract is excellent in biodegradation, swelling, protein release, cell proliferation, and biocompatibility, and contains growth factors. It contains excellent wound improvement effect. In addition, the composition can reduce the expression of MMPs and change the phenotype of macrophages from type M1 to type M2, effectively shortening the time required for wound healing. Therefore, the cosmetic composition can be usefully used for wound improvement and/or skin tissue regeneration.

The cosmetic composition includes solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen-cream, sun oil, suspension, and emulsion. , paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray.

In addition, the cosmetic composition may additionally include one or more cosmetically acceptable carriers that are mixed with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, Chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto.

Cosmetically acceptable carriers included in the cosmetic composition vary depending on the formulation. When the formulation of the cosmetic composition is ointment, paste, cream or gel, the carrier ingredients include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide. Or a mixture thereof can be used.

When the formulation of the cosmetic composition is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, or mixtures thereof may be used as carrier ingredients, and especially in the case of spray, additional May contain propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, and propylene glycol. , 1,3-butyl glycol oil can be used, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. You can.

When the formulation of the cosmetic composition is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the cosmetic composition is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier ingredients. It can be.

When the formulation of the cosmetic composition is a pack (peel-off pack, wash-off pack, or sheet mask pack), polyvinyl alcohol, kaolin, talc, zinc oxide, or titanium dioxide may be used as a carrier ingredient. there is.

Another aspect provides a method of wound healing or skin tissue regeneration comprising treating a subject with an extracellular matrix and Kelp extract.

The terms “extracellular matrix,” “kelp,” “wound,” “treatment,” “skin tissue regeneration,” etc. may be within the scope described above.

According to one aspect, the extracellular matrix and kelp extract are excellent in biodegradation, swelling ability, protein release ability, cell proliferation ability, and biocompatibility, reduce the expression of MMPs, and The phenotype of phagocytes can be converted from M1 type to M2 type, effectively shortening the time required for wound healing, and can be useful for wound treatment and/or skin tissue regeneration.

The “individual” refers to a human or non-human organism, such as a non-human mammal such as a cow, monkey, bird, cat, mouse, rat, hamster, pig, dog, rabbit, sheep, horse, etc., which may cause injury or injury. It can be used on individuals with damaged skin tissue.

Another aspect is to provide the use of the extracellular matrix and Kelp extract for the manufacture of drugs for wound treatment or skin tissue regeneration.

The terms “wound”, “treatment”, “skin tissue regeneration”, “extracellular matrix”, “kelp”, etc. may be within the above-described scope.

According to one aspect, the extracellular matrix and kelp extract are excellent in biodegradation, swelling ability, protein release ability, cell proliferation ability, and biocompatibility, reduce the expression of MMPs, and The phenotype of phagocytes can be converted from M1 type to M2 type, effectively shortening the time required for wound healing, and can be useful for wound treatment and/or skin tissue regeneration.

Figure 1 shows a schematic diagram of the wound healing effect mechanism of the EK-20@Cou scaffold.

Figure 2 shows a schematic diagram of the decellularization process of porcine liver-derived ECM.

Figure 3 shows optical and H&E staining images of porcine liver-derived ECM before and after decellularization.

Figure 4A shows DNA quantification data before and after decellularization of porcine liver-derived ECM.

Figure 4b shows gel electrophoresis results before and after decellularization of porcine liver-derived ECM.

Figure 5 shows a schematic diagram of the kelp decellularization process.

Figure 6a shows H&E staining images before and after kelp decellularization.

Figure 6b shows SEM images before and after kelp decellularization.

Figure 7a shows the DNA content before and after kelp decellularization.

Figure 7b shows protein content before and after kelp decellularization.

Figure 8 shows the results of FTIR spectrum analysis for decellularized kelp.

Figure 9 shows a schematic diagram of the ECM-kelp (EK) scaffold fabrication process.

Figure 10 shows an optical image of the EK scaffold.

Figure 11a shows optical images of EK scaffolds (EK-5, EK-10, EK-15, and EK-20) with different kelp concentrations.

Figure 11b shows the stress/strain curves of EK-5, EK-10, EK-15 and EK-20.

Figure 12 shows SEM images of EK-5, EK-10, EK-15 and EK-20 surfaces.

Figure 13 shows the pore size distribution of EK-5, EK-10, EK-15 and EK-20.

Figure 14A shows the ECM protein (GAGs, elastin and soluble collagen) content of EK-20.

Figure 14b shows the growth factor (VEGF and FGF) content of EK-20.

Figure 15a shows the biodegradability of EK-5, EK-10, EK-15 and EK-20 measured in PBS.

Figure 15b shows the biodegradability of EK-5, EK-10, EK-15 and EK-20 measured in PBS and FBS.

Figure 16A shows the swelling capacity of EK-5, EK-10, EK-15 and EK-20 measured in PBS.

Figure 16B shows the swelling capacity of EK-5, EK-10, EK-15 and EK-20 measured in PBS and FBS.

Figure 17 shows total protein release for EK-5, EK-10, EK-15 and EK-20.

Figure 18 shows pH changes for EK-5, EK-10, EK-15 and EK-20.

Figure 19 shows the pH change of EK-20, EK-25, EK-30, EK-35, EK-40 and EK-45.

Figure 20a shows the results of indirect analysis of rBMSC viability through MTT analysis of EK-5, EK-10, EK-15 and EK-20.

Figure 20b shows the results of direct analysis of rBMSC viability through Almer blue analysis of EK-5, EK-10, EK-15, and EK-20.

Figure 20C shows confocal images of rBMSC proliferation in EK-5, EK-10, EK-15 and EK-20.

Figure 21 shows the pH change of EK-20 and EK-20(PW).

Figure 22a shows the stress/strain curve of EK-20 and EK-20(PW).

Figure 22B shows the residual protein amount of EK-20 and EK-20(PW).

Figure 23 shows the pH change measured after loading 2, 3, 4, and 10 mg of p-coumaric acid (p-Cou) in EK-20.

Figure 24 shows the pH change of EK-20 and EK-20@Cou.

Figure 25 shows the FT-IR spectroscopy results of EK-20 and EK-20@Cou.

Figure 26 shows the p-Cou emission amount of EK-20@Cou.

Figure 27 shows the p-Cou release mechanism of EK-20@Cou.

Figure 28 shows the total antioxidant capacity of EK-20@Cou and EK-20(PW).

Figure 29a shows the inhibition zone image of EK-20@Cou and EK-20(PW) against Gram-positive bacteria ( Bacillus subtilis and Staphylococcus aureus ) and Gram-negative bacteria ( Escherichia coli and Salmonella typhimurium ).

Figure 29b shows the size of the inhibition zone of EK-20@Cou and EK-20(PW) against Gram-positive bacteria ( Bacillus subtilis and Staphylococcus aureus ) and Gram-negative bacteria ( Escherichia coli and Salmonella typhimurium ).

Figure 30 shows the results of rBMSC viability analysis through MTT analysis of EK-20@Cou and EK-20(PW).

Figure 31 shows confocal images of rBMSC proliferation in EK-20@Cou and EK-20(PW).

Figure 32 shows images and quantitative wound closure data of dorsal wounds in rats treated with EK-20@Cou or EK-20(PW).

Figure 33 shows a schematic diagram of the wound healing process of EK-20@Cou.

Figure 34 shows H&E staining images and Mason's Trichrome staining images of rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 35a shows new epidermal thickness data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 35b shows granulation tissue thickness data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 35C shows epithelialization score data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 36 shows DAB staining images for fibronectin, collagen-1 and α-SMA in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 37A shows fibronectin expression data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 37b shows collagen-1 expression data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 37c shows α-SMA expression data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 38a shows the MS prediction results for the binding potential of p-Cou in the MMP active site and the interaction between amino acid residues and p-Cou atoms in the protein active site.

Figure 38b shows the MS prediction results for the binding potential of p-Cou in the protein active site and the interaction between amino acid residues and p-Cou atoms in the protein active site.

Figure 38c shows MS prediction results for the free binding energy (-ΔG) of p-Cou compared to the control drug in the protein active site.

Figure 39A shows the inhibitory effect of EK-20@Cou and EK-20(PW) on MMP-2 and MMP-12.

Figure 39B shows MMP-2 and MMP-12 expression data in rat wounds treated with EK-20@Cou or EK-20(PW).

Figure 40 shows MS prediction results for the binding potential of p-Cou in the NF-κB active site and the interaction between amino acid residues and p-Cou atoms in the protein active site.

Figure 41A shows NF-κB staining images in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 41B shows NF-κB expression data in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 42A shows CD68 staining images in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 42B shows CD68 expression data in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 43 shows nitrate concentration in rat wound samples treated with EK-20@Cou or EK-20(PW).

Figure 44 shows a schematic diagram of the wound healing process of EK-20@Cou.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Materials and Methods

1-1. Liver-derived ECM preparation

Fresh pig liver was obtained from Sajo Industrial Co., Ltd. immediately after slaughter, placed in an icebox and transferred to a laboratory freezer (-80°C). After thawing the pig liver at room temperature, liver slices of approximately 5 mm ² were obtained using a blade and stored in demineralized water (DW). After cutting, the liver slices were washed until the blood disappeared, and then the liver slices were immersed in 1% SDS solution and rotated at 200 rpm for 12 hours. Afterwards, the SDS (sodium dodecyl sulfate) (Bio-Rad Laboratories, California, USA) concentration was maintained at 0.5% for 48 hours, and the SDS concentration was changed every 8 hours, reducing the concentration to 0.1% until the section became completely white. I ordered it. Then, the white ^ECM was treated with 1% Triton To completely remove residual substances from the ECM prepared as above, it was washed several times with PBS, and the decellularized liver tissue was frozen and lyophilized. Afterwards, a cryogenic grinder (Retsch, Germany) was used to obtain the powder form, and the ECM powder was stored at -80°C.

1-2. Histological analysis of decellularized ECM.

Histological analysis was performed to evaluate the degree of decellularization of ECM. Both native and decellularized ECM samples were fixed overnight using 4% paraformaldehyde (PFA) solution. Afterwards, the samples were washed with DW for 1 hour and dehydrated using ethanol solution and xylene (for alcohol separation) before embedding in paraffin. Next, the paraffin block was cut to a thickness of 5 μm using a microtome (Leica Biosystem, Germany), and the sections were left at 60°C for 1 hour to remove paraffin, followed by H&E staining. Stained tissue sections were visualized using a BX53 light microscope (Olympus), images were captured with an Olympus DP72 camera, and images were analyzed using Cellsens software.

1-3. Quantification of genetic material (DNA) in decellularized ECM

Total genetic material (DNA) of native and decellularized ECM samples was quantified according to standard protocols. First, 50 mg of sample was dissolved in 0.5 mL lysis buffer (0.5 M EDTA, 5 M NaCl, 1 M Tris-HCl, and 20% SDS) with 20 μL of proteinase K (Thermo Fisher Scientific, USA) at 20 mg mL ^-1 ) and cultured overnight at 55°C. Afterwards, the mixture was centrifuged at 10,000 rpm for 10 minutes to extract the genetic material from the soluble portion. Next, the obtained material was treated with 400 μL of 6M NaCl (MilliporeSigma, USA) to remove proteins. Afterwards, DNA was precipitated using cold isopropyl alcohol, washed twice with 70% ethanol, and dissolved in TE buffer (10mM Tris HCl, pH 8.0, 1mM EDTA). Afterwards, the total DNA content was quantified using a Nanodrop spectrophotometer (Implen, Germany) and normalized to initial weight, and the genetic material content of native and decellularized and ECM tissue samples was analyzed by agarose (1% MilliporeSigma, USA) gel electrolysis. It was analyzed by Youngdong.

1-4. ECM digestion

ECM particles were dispersed in 0.01 M HCl solution (pH 2) and 10% (w/w) 3,200 units mg ^-1 pepsin (Millipore Sigma, USA) for 72 to 96 hours at room temperature. Afterwards, 0.1% (v/v) cold 0.1 M NaOH (MilliporeSigma, USA) and 10X PBS were used to inactivate pepsin, and HCl solution was added to neutralize the high pH. The total concentration was adjusted to 20 mg mL ^-1 PBS and stored at -80°C until use.

1-5. Kelp decellularization

To extract cell contents from brown algae samples (kelp), optimized decellularization was performed. Kelp ( Saccharina japonica ) was obtained from a seawater harvested fish farm (WESC, Wando County, Jeolla, South Korea). For decellularization, bleach and sodium hydroxide solutions were used to separate the vasculature from the surrounding soft tissue without detergent. Specifically, 100 g of wet kelp samples were boiled with bleach (20% v/v, NaClO) and alkaline solution (6% w/v, NaOH) at 60 to 70°C for 3 hours. Accordingly, it was confirmed that the sample was decellularized and became transparent. The sample was washed with DW several times, incubated with DW at 4°C overnight to completely remove residual bleach and alkaline solution, and then lyophilized. The lyophilized samples were ground with a cryogenic grinder (Retsch, Germany) under liquid nitrogen and stored at 4°C until use.

1-6. Characterization of decellularized kelp

Histological staining and biochemical analysis were performed to confirm decellularization in kelp sections. For this purpose, the native and decellularized samples were cut into square shapes and fixed with 4% PFA, and DNA quantification was performed to compare the total content of genetic material (DNA) in the native and decellularized samples.

1-7. scanning electron microscope (SEM) imaging

Since the ultrastructure in native tissue and decellularized tissue was not the same, SEM was performed to confirm decellularization of kelp. For this purpose, each sample was dried in a freeze dryer and cut under liquid nitrogen using a knife. Afterwards, the sample was placed in the SEM sample holder and sputter-coated with platinum. JEOL JSM-6701F (Tokyo, Japan) was used for SEM analysis with energy dispersive spectrophotometer (EDS), and an acceleration voltage of 10 kV was used to obtain SEM images.

1-8. Chemical characterization using FT-IR spectroscopy

The chemical composition of decellularized kelp samples was analyzed using Fourier transform infrared spectroscopy Nicolet spectrometer (Nicolet Ia10, Thermo Scientific). FT-IR spectra were analyzed with OMNIC version 7.3 software at a resolution of approximately 8 cm ^-1 and a wavelength range of 650 to 4000 cm ^-1 .

1-9. Protein quantification

The Bradford Assay kit was used to measure the amount of protein in native kelp samples and decellularized kelp samples, and bovine serum albumin (BSA) (Biosera, France) was used as a standard to obtain a calibration curve. Additionally, the protein concentration of the sample was determined using a protein assay kit. A decrease in protein concentration in decellularized kelp samples was considered as a complete decellularization process.

1-10. ECM-kelp (EK) scaffold fabrication

For the fabrication of the ECM-kelp scaffold, the ECM was set to a constant concentration of 20 mg mL ^-1 . To evaluate the crosslinking effect of kelp concentration, the ECM:kelp ratio was increased from 1:0.25 to 1:1, and when 5 mg mL ^-1 (1:0.25) of kelp was added, the mechanical properties of ECM were changed. It was observed that A small amount of kelp also served as a natural non-toxic cross-linking agent in the ECM-based scaffold, and the concentration of kelp was increased from 5 mg mL ^-1 to 20 mg mL ^-1 while keeping the ECM concentration constant. All samples were thoroughly mixed with a homogenizer, then the freeze-thaw process was performed twice (30 minutes each) and frozen at -80°C to prepare an elastic scaffold. The cross-linked scaffold was frozen. It was dried using a drying technique. The composition of the prepared ECM-kelp scaffold is shown in Table 1 below.

	ECM (mg mL -1 )	Kelp (mg mL -1 )	ECM-kelp ratio
EK-5	20	5	1:0.25
EK-10	20	10	1:0.50
EK-15	20	15	1:0.75
EK-20	20	20	1:1

1-11. Morphological characteristics and histogram analysis

The microstructure of the freeze-dried scaffold was observed using SEM (JEOL JSM-6701F, Tokyo, Japan). The cross section and surface of the sample were cut into small pieces under liquid nitrogen and sputter coated with platinum. Pore size distribution was analyzed using Image J software.

1-12. ECM protein and growth factor content in EK scaffolds

Representatively, the contents of ECM proteins and growth factors were measured within the EK-20 scaffold. Sulfated glycosaminoglycan (sGAG) and elastin contents within the scaffold were measured using the Blyscan sGAG assay kit (Biocolor, UK) and Fastin elastin assay kit (Biocolor, UK), respectively. Total soluble collagen in the folds was detected using the Sirius red total collagen detection kit (Chondrex, USA), and each assay was performed according to the manufacturer's instructions for each kit. Additionally, to measure the content of growth factors, EK-20 was homogenized with heparin buffer and dialyzed in a dialysis cassette. Afterwards, the cassette was placed in a beaker containing deionized water (DI water) and stirred at 4°C for 96 hours using a magnetic bar, and the deionized water was frequently replaced during this process. In the obtained extract, the amount of growth factors VEGF and FGF was measured using Eliza kits (R&D systems, USA) according to the manufacturer's instructions.

1-13. Protein release assay

Protein release analysis was performed for each EK construct using the Bradford Assay kit according to the manufacturer's instructions. EK samples were cultured in FBS-free cell growth alpha-MEM medium for 1, 3, and 7 days. The extracted medium was used to identify proteins released from each scaffold at different time points.

1-14. Analysis of biodegradation and swelling characteristics

Experiments were performed to analyze the biodegradation and swelling properties of the EK scaffold. First, the weight of each scaffold was measured, and the scaffold was immersed in 5 mL of PBS (pH 7.4) and kept at 37°C in an incubator with continuous stirring at 80 rpm. For use in measuring biodegradation and swelling properties, a surrogate model for modeling physiological conditions was also used. As a culture medium for the model, a medium containing PBS, 10% FBS, 100 U cm ^-3 penicillin, and 100 ug cm ^-3 streptomycin was used. Afterwards, the weight of the swollen scaffold was recorded and dried in a freeze dryer. Next, the weight of the dried scaffold was recorded, and the decomposition rate was measured over 14 days. Swelling analysis was performed for 24 hours, and the degree of decomposition (%) and swelling rate were calculated using the following equations 1 and 2, respectively:

[Equation 1]

Resolution (%) = W _i -W _d /W _i *100

(W _i = initial weight, W _d = dry weight)

[Equation 2]

Swelling rate = W _s /W _i .

(W _s = swelling weight, W _i = initial weight)

1-15. pH adjustment

Initially, the pH of all EK scaffolds (especially EK-20) was measured to be high, so a prolonged wash (PW) was performed to adjust the pH of the scaffolds. Specifically, each EK composition was washed with DW for 12 hours, and the water was changed every 3 hours.

1-16. Mechanical strength analysis

The mechanical strength of the samples was measured using a universal testing machine (R&B Unitech TM, Korea) (width 2 mm x height 10 mm, thickness 0.5 mm). The loading rate of cells was 5 mm min ^-1 , and the sample was pulled outward using a paper frame.

1-17. Indirect analysis of cytotoxicity of EK scaffolds using MTT

After washing the scaffold for a long time, it was sterilized with ethylene oxide (EO) and cultured in cell growth medium for rBMSC (alpha-MEM) for 1, 3, and 7 days to prevent the generation of debris in the scaffold. For this purpose, the medium was collected using a 0.22 μm syringe filter (Millipore, Germany). A density of 10 The next day, to form formazan crystals in living cells, MTT solution was added and cultured for 4 hours. Afterwards, formazan was dissolved in DMSO, and the absorbance was measured at 570 nm with a 650 nm reference filter, and the cell viability was calculated using Equation 3 below:

[Equation 3]

Cell viability (%) = 100*OD _570e /OD _570b .

(OD _570e = average value of optical density (OD) measured in the extracted sample medium,

OD _570b = average value of optical density (OD) measured in the control group)

1-18. Direct cell proliferation assay (Almer blue assay)

For direct cell growth analysis, long-washed EK scaffolds were placed in a 24-well agarose-coated plate for 12 h in the medium required for rBMSC culture, and each scaffold was seeded with 10 × ¹⁰ cells per well. The agarose-coated plate was set to attach only to the scaffold, and the medium was changed every other day. Afterwards, Almer blue (including non-fluorescent resazurin blue) was added at a concentration of 1:10. Since the Almer blue is non-toxic to cells, the experiment can be restarted for several periods (1, 3, 5, 6, and 7 days) and cell growth on the scaffold is possible. It was confirmed that resazurin was produced when cultured for 4 hours with the Almer blue solution. Next, 100 μL of the medium was aspirated into a 96-well uncoated plate, and the fluorescence intensity was measured using a fluorescence spectrophotometer. To continue the analysis, the medium containing Almer blue solution was reduced and fresh medium was added.

1-19. pH adjustment by drug loading

Basically, the pH of each EK scaffold (especially EK-20) was basic, so a long-term washing was initially performed to adjust the pH of the EK scaffold. However, long-term washing takes a long time and there is a possibility that ECM proteins and growth factors important for cell development may be lost, making it difficult to adjust pH. To overcome this problem, we loaded them with p-coumaric acid (p-Cou), an acidic drug that belongs to the polyphenol group and has anti-inflammatory, antioxidant and antibacterial effects and can improve the healing process. In addition, EK-20 was selected, which maintains a uniform microstructure even after prolonged washing and shows higher cell proliferation compared to other groups in direct and indirect cell survival analysis. For drug loading, various doses (2, 3, 4, and 10 mg per scaffold) of p-Cou were added to the ECM/kelp slurry, which was homogenized to modify EK-20 into EK-20@Cou. In addition, in order to control the amount of drug added, the pH when loaded with various doses of p-Cou and a selected dose (4 mg per scaffold) was compared in scaffolds that were washed for a long time and scaffolds that were not washed. .

1-20. Release analysis of p-coumaric acid from EK-20@Cou

To analyze the release characteristics of p-coumaric acid, EK-20@Cou was first immersed in 5 mL of PBS (pH 7.4) at physiological temperature (37°C) and stirred at 20 rpm. Thereafter, 1 mL of sample was withdrawn at different time intervals, filtered through a 0.22 syringe filter (Millipore, Germany), and a total of 1 mL of fresh PBS was added. To confirm the release of p-Cou, which belongs to the polyphenol group, experiments were performed according to established protocols, using 7.5% Na ₂ CO ₃ and FC reagent (Folin-Ciocaltenus reagent, Sigma Aldrich) for both standards and samples. analyzed. The absorbance at 760 nm of each sample extract and standard was measured in triplicate against a blank (water) using a nanodrop spectrophotometer (Implen, Germany). Using the standard calibration curve of p-Cou, the p-Cou release concentration on specific days (continued for 10 days) was measured.

1-21. Total antioxidant capacity analysis

The total radical scavenging capacity of long-washed EK-20(PW) and p-Cou-loaded EK-20@Cou scaffolds was measured using a total antioxidant capacity assay kit (Sigma Aldrich, USA) according to the manufacturer's instructions. First, the scaffold was soaked in PBS, and then 1 mL of PBS was replaced with fresh PBS at specific times. Additionally, sets of standards were prepared at various concentrations, and both samples and standards were treated with 100 μL of Cu ²⁺ working solution. The incubation time was set to 90 minutes at room temperature in the dark, and the absorbance at 517 nm was measured using a spectrophotometer. Radical scavenging activity was calculated using equation 4:

[Equation 4]

S _a /S _v = C.

(S _a = Trolox equivalent of an unknown sample well (nmole) from the standard curve,

S _v = sample volume added to the well (μL),

C = antioxidant concentration in sample (nmole μL ^-1 or mM Trolox equivalents)

1-22. Antibacterial effect analysis

To analyze the antibacterial ability of EK-20(PW) and EK-20@Cou, a disk diffusion protocol was used. First, two Gram-positive bacterial strains ( Bacillus subtilis NCCP11101 and Staphylococcus aureus NCCP14402) and two Gram-negative bacterial strains ( Escherichia coli NCCP14762 and Salmonella typhimurium NCCP16207) were stored in Lysogeny broth for 4 hours. The temperature was maintained at 37°C, and a lawn was prepared by inoculating the strain on a Muller-Hinton agar plate. Amikacin AK disks (30 μg, Liofilchem, Via Scozia, Italy) were used as positive controls, and sterile blotting paper disks were used as negative controls. After preparing the sample by cutting the disk into 6 mm, the cut sample was cultured on an agar plate containing bacteria at 37°C for 18 to 24 hours. The antibacterial effect due to drug release was measured based on the inhibition zones of the plate compared to the positive control, and images were obtained using a digital microscope (Sony, Thailand).

1-23. Biocompatibility analysis

For biocompatibility analysis of EK-20, EK-20(PW) and EK-20@Cou samples, indirect MTT analysis was performed according to the above-mentioned protocol.

1-24. Visual analysis of cell proliferation using confocal staining

Three sets of EK-20, EK-20(PW) and EK-20@Cou samples were sterilized with ethylene oxide (EO) and cultured in rBMSC cell growth medium for 7 days at 5% CO ₂ and 37°C. Additionally, ¹⁰ Afterwards, 100% filtered medium from the scaffold was added and the treatment was continued for 1, 3 and 7 days. After completing the treatment step, cells were fixed with 4% PFA (Sigma-Aldrich) for 15 minutes. Next, cells were washed with PBS for 15 minutes (3 × 5 minutes) and permeabilized by incubation with 0.5% Triton X-100 (Sigma-Aldrich) for 10 minutes. The process was continued for 60 minutes using blocking reagent and 2.5% bovine serum albumin (BSA). Afterwards, the cytoskeleton and nucleus were stained with 25 μg mL ^-1 FITC (fluorescein isothiocyanate)-conjugated phalloidin (Sigma-Aldrich, USA) overnight in the dark at 4°C, and HOECHST-33342 (2'-[4-ethoxyphenyl ]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole-tri hydrochloride trihydrate (1 μg mL ^-1 ); Next, after washing with PBS for 15 minutes and adding mounting solution, images were acquired using a confocal fluorescence microscope (Olympus, FV10i-W, Tokyo, Japan) and analyzed using FV10i-ASW 2.0 Viewer.

1-25. In vivo Experiment

For the in vivo experiment, a total of 24 male rats (9 weeks old, weight 200-250 g, Rattus norvegicus ) were used. The rats were purchased from Dayoon (animal center, Korea) and followed the standard protocol of the Animal Ethics Committee of Soonchunhyang University (approval number SCH22). -0026). Rats were randomly divided into control, positive control, EK-20(PW), and EK-20@Cou groups, respectively. The control group received no treatment, and the positive control group was treated with a commercial wound care material (MatriDerm®, DERMAL MATRIX, Medskin Solutions, Germany). The experiment was conducted for a total of 2 weeks using 12 animals (3 animals per group) each week. On the day of surgery, Isoflurane (Piramal Critical Care, Schelden Circle, Bethlehem, PA) was used to anesthetize the rat, and a biopsy punch (circular shape, Kai Industries Co. Ltd., Japan) was used to cut the rat's dorsal skin into 7 mm thick sections. It was excised. The resection area was treated with povidone iodine solution.

1-26. Wound healing analysis

At days 0, 7, and 14, wound contraction images were captured with a digital camera, and the degree of wound closure was quantified using Equation 5 below:

[Equation 5]

Percent closure (%) = (initial wound area - wound area on day n/initial wound area)*100.

On the day of sample extraction, an excessive amount of diethyl ether (Daejung, South Korea) was used to euthanize the mice. The extracted samples were fixed with formaldehyde (10%, Duksan Pure Chemicals, Korea) for 2 days at room temperature, and tissue samples were extracted from each group and stored at -80°C until further experiments.

1-27. Histological staining

Samples were fixed, washed with DW, and then dehydrated with 50%, 70%, 80%, 90%, 95%, and 100% ethanol solutions. Serial xylene (100%) was used as a clarifying agent to remove residual alcohol, and embedding was performed with paraffin wax (Leica Biosystem, Germany). Samples were cut to a thickness of 5 ± 2 μm using a microtome (Leica Biosystem, Germany), and the sections were deparaffinized and stained using Masson's Trichrome method and H&E method. Additionally, immunohistochemical staining was performed using a DAB staining kit (3'-Diaminobenzidine, Agilent Technologies, US). Briefly deparaffinized and rehydrated slides were incubated with 3% BSA for 1 hour at ambient temperature. Blocked samples were treated overnight at 4°C with primary antibodies (Anti-CD68, Anti-fibronectin, Anti-collagen-1, Anti-alpha smooth muscle actin, and Anti NF-κB), followed by secondary antibodies conjugated with HRP. was treated for 1 hour. Antigen-antibody reactions were measured using DAM chromogen, and samples were viewed using an optical microscope (BX53 Olympus) and photographed using a camera (DP72 Olympus). Afterwards, the samples were analyzed using Cellsens software, and the stained samples were quantified immunohistochemically using Image J software.

1-28. In Silico Molecular Simulation (MS)

Crystalline 3D structures of proteins and receptors were retrieved from the PDB (Protein Data Bank) database (/www.rcsb.org), and the energy of each protein structure was minimized using Swiss-PDB Viewer version (v) 4.1.1. . Removal of heteroatoms and water molecules was performed with Discovery Studio Visualizer (DSV, Biovia, v 2021, Accelrys, San Diego, CA). The active site of each protein was determined based on the literature, missing hydrogen atoms and residues were added to the protein, and grid box parameters were plotted using the Autodock tool (ADT, v. 4.2, Scripps Research Institute, La Jolla, CA). Optimized. The chemical structure of p.cou was obtained from the PubChem database (https:/pubchem.ncbi.nlm.nih.gov/) and converted to PDB format using the open source chemistry toolbox Open Babel version 2.3.1 (www.openbabel.org). converted. Conformational search and geometry optimization were performed in Avogadro 1.2.0 (http:/avogadro.cc/). In addition, to minimize the total energy of the ligand (p.Cou), Merck molecular force field 94 (MMFF94) was applied, and Gasteiger was selected as the charge calculation method. The rotatable bond of the ligand was converted to a non-rotatable one for rigid docking to minimize standard errors. Additionally, co-crystalized ligands or known inhibitory drugs were used as control ligands. Cocrystallized ligands were prepared with DSV, known inhibitory drugs were searched in the ZINC database (https:/zinc12.docking.org/), and control ligands were processed similarly to p.Cou. AutoDock Vina (v. 1.2.0) was used to perform the MS study, and the Lamarckian Genetic Algorithm (LGA) was used to find the most suitable conformational space for the ligand with a population size of 150. The generation number was set to 27,000, and the evaluation number was set to a maximum of 2,500,000. The stabilized complex conformation was predicted by the free binding energy ( -ΔG ) and the optimal binding pose and position. After MS, results were analyzed using DSV, PyMOL molecular graphics system (v. 2.5.2, https:/pymol.org) and LigPlot+ (v. 1.4.5, https://www.ebi.ac.uk/thornton-srv/software / LigPlus/) and analyzed and visualized. The ligand efficiency (LE) index was calculated using Equation 6 below:

[Equation 6]

LE = (-Δ G )/MW.

(LE = ligand efficiency, ( -ΔG ) = free binding energy, MW = molecular weight)

1-29. MS followed by biochemical and enzyme-linked immunosorbent assay (ELISA)

The MS prediction results were further verified through experiments. For in vitro investigation, both EK-20(PW) and EK-20@Cou scaffolds were incubated in PBS (5 mL, pH 7.4) at 37°C with agitation at 20 rpm. Thereafter, 1 mL of solution was withdrawn every day, and 1 mL of fresh PBS was added. Sample extracts were filtered through a 0.22 μm syringe filter. Afterwards, the extract was applied to the MMP-2 (Matrix metalloproteinases-2) and MMP-12 inhibition screening kit (Abcam, UK) to measure the inhibition rate of MMP-2 and MMP-12. The levels of active MMP-2 and MMP-12 in the wound area were verified using biopsy tissue samples from the wound area. Additionally, 100 mg of tissue sample was homogenized in 1 mL of cold PBS, and the clear supernatant was separated using centrifugation (15,000 rpm, 10 minutes, 4°C). Afterwards, the concentration of active enzyme was measured in the extracted tissue homogenate using the Rat MMP-2 ELISA kit (Abcam, UK) and the rat MMP-12 ELISA kit (Elabscience Biotechnology Inc. USA). All experiments were performed in triplicate according to each manufacturer's instructions.

1-30. statistical analysis

All data were expressed as mean ± standard deviation (SD). T-test was performed for comparison between two groups, and analysis of variance (ANOVA) was performed for more than two groups. Graph Pad Prism (Version 7) was used for statistical analysis, and Microsoft Excel (2019) was used for data calculations. Additionally, the spectral data was analyzed and plotted using Sigma plot (Version 10). The significance level was expressed as ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and ns = not significant, and the experiment was performed three times.

Example 2: Experimental results

2-1. Liver-derived ECM extraction and decellularization

In order to use liver-derived ECM as a biomaterial for wound healing, liver-derived ECM was first extracted from pigs using a surfactant, then cut into uniform sizes and decellularized (see Figure 2). Afterwards, it was confirmed with the naked eye that the liver ECM had turned white that the decellularization process had been performed, and it was confirmed through H&E staining that there were no cellular components in the liver ECM (see Figure 3). In addition, as a result of quantifying genetic material (DNA) in decellularized liver ECM, the nuclear content of decellularized ECM was measured to be significantly lower than that of native liver tissue, confirming that DNA was effectively removed (Figures 4a and 4b reference). Additionally, the decellularized ECM was digested with pepsin to prepare a hydrogel concentration (20 mg mL ^-1 ), but the consistency of the hydrogel was not suitable for solid form.

2-2. Kelp decellularization

To use as a cross-linking agent for liver-derived ECM, cell contents were extracted from brown algae kelp and decellularized (see Figure 5). Decolorization of kelp began within 2 hours of decellularization, and decellularization was completed within 3 hours. To confirm the decellularization process, H&E staining and SEM analysis were performed on native kelp and decellularized kelp. As a result of H&E staining, the presence of cell nuclei (hematoxylin, purple-blue) and cytoplasm (eosin, pink) were confirmed in the leaves of native kelp, but neither cell nuclei nor cytoplasm were detected in decellularized kelp (see Figure 6a) ). In addition, as a result of SEM analysis, unlike the native kelp image, there were no salt molecules in the decellularized kelp image, confirming that decellularization was completed (see Figure 6b).

Then, to further verify whether decellularization was complete, DNA and protein levels were measured and compared in Natebee kelp and decellularized kelp. As a result, the DNA content of decellularized kelp was measured at 16.8 ± 1.2 ng DNA mg ^-1 tissue, which was significantly lower than the DNA content of native kelp (306 ± 112 ng DNA mg ^-1 tissue), which is the minimum requirement for decellularization. (Less than 50 ng DNA mg ^-1 tissue) was confirmed to be satisfied (see Figure 7a). In addition, the protein content of decellularized kelp was measured at 6.56 ± 0.5 μg protein mg ^-1 tissue, which was significantly lower than the protein content of native kelp, which was 17.43 ± 1.9 μg protein mg ^-1 tissue (see Figure 7b).

Additionally, to analyze the composition of the decellularized kelp, FTIR spectrum analysis was performed on the decellularized kelp. As a result of the analysis, the absorption bands at 1671.06 cm ^-1 (S=O) and 880.06 cm ^-1 (C-0-S) were classified as fucoidan, and cellulose was classified as 3360.02 cm ^-1 (-OH) and 2973.86 cm Peaks were identified at ^-1 (-CH), 1452.96 cm ^-1 (C=O), 1048.40 cm ^-1 (COC) and 880.05 cm ^-1 (β-glycosidic bond). Additionally, pectin was identified as a peak at 2885.56 cm ^-1 (CH) (see Figure 8). Through the above analysis, it was confirmed that cellulose and pectin maintained the structural integrity of kelp even after the decellularization process.

2-3. ECM-kelp (EK) scaffold fabrication

ECM-derived hydrogels can be composed of 3D structural networks that absorb biological fluids or water due to the presence of hydrophilic groups. On the other hand, natural ECM-derived hydrogel possesses natural ECM and has excellent biocompatibility, but has the disadvantage of poor mechanical strength. Therefore, to overcome the above drawbacks, an ECM-kelp (EK) scaffold was prepared using decellularized kelp as a natural cross-linker for ECM.

First, decellularized kelp powder was mixed with a specific concentration of ECM in a homogenizer at different ratios, and an elastic scaffold was prepared through a freeze-thaw process (see Figure 9). At this time, due to the freezing process of water molecules, the decellularized kelp molecules are attracted to the hydrophilic groups in the liver-derived ECM, resulting in hydrogen bonding between the decellularized kelp molecules and the hydrophilic molecules in the liver-derived ECM. Because of this, the decellularized kelp molecules can act as a cross-linker within the ECM, and a three-dimensional scaffold can be formed (see Figure 10). In addition, it was confirmed that as the kelp content increased, the mechanical strength of the scaffold increased (see Figures 11a and 11b).

Meanwhile, while observing the microstructure of the scaffold using SEM, it was confirmed that the pore size within the scaffold changes depending on the concentration of kelp (see Figure 12). Specifically, it was confirmed that as the concentration of kelp decreased, pores were created irregularly with various sizes ranging from 10 to 55 μm, and as the concentration of kelp increased, pores were created uniformly with a size of 10 to 28 μm (see Figure 13). Since the smaller the pore size, the more effective it is in suppressing wound exudate, it was confirmed that adding kelp at a high concentration to manufacture the scaffold was more effective in wound healing.

(1) Measurement of contents of ECM proteins and growth factors

To determine whether the ECM-kelp (EK) scaffold contained ECM proteins and growth factors even after decellularization, the contents of ECM proteins (GAGs, elastin, and soluble collagen) and growth factors (VEGF and FGF) were measured. As a result, approximately 10,000 μg mg ^-1 of sulfated glycosaminoglycans (GAGs) and 40,000 μg mg ^-1 of elastin were detected in the EK scaffold, while soluble collagen was detected at a somewhat lower concentration of 0.5 μg mg ^-1 (see Figure 14a). In the case of growth factors, VEGF and FGF were detected in large quantities at 1363.07 pg mg ^-1 tissue and 1422.16 pg mg ^-1 tissue, respectively (see Figure 14b).

Through the above experiment, it was confirmed that the EK scaffold contains a large amount of ECM proteins and growth factors even after the decellularization process and can be effectively used for wound healing and tissue regeneration.

(2) Analysis of biodegradation and swelling characteristics

In the case of wound dressings, biodegradability is essential because it eliminates the need for dressing replacement and is effective in regenerating new tissue. Accordingly, an experiment was performed to confirm whether the EK scaffold was biodegradable.

As a result, the weight of all EK scaffolds (EK-5, EK-10, EK-15, and EK-20) decreased by 25 to 30% after one week while submerged in PBS, confirming that they are all biodegradable. did. Additionally, after two weeks, the weight decreased by 43.07% and 48.67% for EK-5 and EK-10, respectively, but the weight decreased by 61.49% and 71.61% for EK-15 and EK-20, respectively, indicating biodegradability. It was confirmed that it was superior to this (see Figure 15a). Meanwhile, in order to mimic physiological conditions, FBS (fetal bovine serum) was added to PBS and the weight was measured. Similar to the above, the biodegradability of EK-15 and EK-20 was measured to be superior (Figure 15b) reference). This phenomenon can be interpreted as because EK-20 releases more proteins when it comes into contact with fluid.

Then, to analyze the swelling ability of the EK scaffold, the EK scaffold was placed in a culture medium containing PBS or a culture medium containing PBS and FBS, and the swelling capacity was measured. As a result, all EK scaffolds showed swelling ability, but it was confirmed that EK-20 had the best swelling ability (see Figures 16a and 16b).

(3) Total protein release analysis

The EK scaffold is composed of decellularized liver-derived ECM and decellularized kelp and contains a large amount of protein. To measure protein release over time from the EK scaffold, total protein release analysis was performed.

As a result, the protein release rate was slow for 3 days, but the release rate increased over time, and for the EK-15 and EK-20 scaffolds, a higher amount of protein was released after 7 days compared to the other scaffolds. was released (see Figure 17). In the case of the EK-5 scaffold, protein release was measured significantly slower than that of other scaffolds. This phenomenon can be interpreted as being correlated with biodegradability, as protein release may increase as the scaffold decomposes.

(4) pH change analysis

To analyze the pH change of the EK scaffold, the pH of EK-5, EK-10, EK-15, and EK-20 was measured daily for 2 weeks. As a result, all scaffolds except EK-20 were maintained close to the optimal pH (7.8), but EK-20 showed that the pH initially rose rapidly and stabilized over time (see Figure 18).

To further confirm the effect of the concentration of kelp in the EK scaffold on pH, scaffolds were manufactured with a higher concentration of kelp (EK-25, EK-30, EK-35, EK-40, EK-45, and EK-50), pH was measured for 2 weeks. As a result, it was confirmed that the pH of the scaffold increases as the kelp concentration increases, and that when the kelp concentration is above 30 mg mL ^-1 (EK-30), the pH gradually increases, but the difference between scaffolds is minimal ( 19). This phenomenon can be interpreted as being due to the release of minerals or residual decellularizing agent (especially NaOH) from the decellularized kelp.

(5) pH adjustment

In the above experiments, the biodegradability, swelling ability, and total protein release ability of the EK-20 scaffold were determined to be the best. To further confirm the superiority of the EK-20 scaffold, cell viability was compared directly and indirectly by comparing cell proliferation ability using rBMSC cells. As a result, it was confirmed that the EK-20 scaffold exhibited the best cell proliferation ability compared to other scaffolds (see Figures 20a to 20c). For this reason, EK-20 was selected as a wound healing scaffold, but EK-20 had the problem of high initial pH.

To solve this problem, we attempted to adjust the pH of EK-20 by prolonged washing (PW). As a result of measuring the pH change of EK-20 (PW) for 3 days after washing, it was confirmed that the high initial pH of the scaffold decreased as expected (see Figure 21). However, EK-20 (PW) showed a problem of decreased mechanical strength and protein content compared to the unwashed scaffold (EK-20) (see Figures 22a and 22b).

In order to reduce the loss of growth factors and proteins and time consumption due to such long-term washing, we attempted to adjust the pH by loading the acidic drug p-coumaric acid (p-Cou) on the EK-20 scaffold. The p-Cou is an acidic drug that can not only reduce high pH, but is also known to have various bioactive effects such as anti-inflammatory, antimicrobial, and antioxidant. First, p-Cou was treated at 2, 3, 4, and 10 mg per scaffold, respectively, and then the pH change was measured to select 4 mg/scaffold as the most appropriate drug dose (see Figure 23). Subsequently, the pH changes of the p-Cou-loaded EK-20 scaffold (EK-20@Cou) and the long-term washed EK-20 scaffold (EK-20(PW)) were compared and analyzed, and the results showed that the two scaffolds were similar. It was confirmed that a pH change pattern was observed (see Figure 24).

Through the above experiment, it was possible to minimize the decrease in proteins and growth factors due to long-term washing by reducing the high pH of EK-20, which shows the most suitable characteristics for wound healing, through loading of acidic drugs rather than long-term washing.

(6) FTIR spectrum analysis

Analysis of the FTIR spectra of liver-derived ECM showed representative peaks of the stretching frequencies of NH and OH at approximately 3,400 cm ^-1 , and representative peaks of amide I and amide II at approximately 1,650 cm ^-1 (primary amine) and 1,550 cm -1 , respectively. Displayed in cm ^-1 (secondary amine) (see Figure 25). For polysaccharides COC and CCO, a stretch was identified between 1,200 cm ^-1 and 1,000 cm ^-1 , with oscillations for C=C aromatics after p-Cou loading, with amide peaks corresponding to liver-derived ECM and kelp ECM; It was identified at 1,604 cm ^-1 .

(7) Release of p-coumaric acid from EK-20@Cou

Measurement of the cumulative release of p-Cou from the EK-20@Cou scaffold showed an initial burst release, with approximately 15% of p-Cou released after 4 hours of incubation, and approximately 30% of p-Cou over 3 days. was found to be released (see Figure 26). This initial burst release of p-Cou is useful in reducing the high initial pH of the EK scaffold and can provide anti-inflammatory and antibacterial effects to the scaffold, making it useful for wound healing.

Thereafter, in order to identify the release mechanism of p-Cou from the scaffold, a kinetic study was performed based on the cumulative drug release amount (see Table 2 below). As a result, it was confirmed that the drug release profile was closest to the Korsmeyer-Peppas model, indicating a Quasi-Fickian diffusion release mechanism (see Figure 27).

Models	R 2	AIC	BIC		RMES
0	0.743	70.36	71.33	4.58
1st	0.635	76.65	77.62	5.95
2nd	0.510	92.72	93.69	11.63
Korsmeyer-Peppas	0.958	46.03	47.00	1.66
Hixson-Crowell	0.674	73.99	74.96	5.33
Higuchi	0.837	113.46	114.43	27.61

(AIC: Akaike information criterion, BIC: Bayesian information criterion; RMSE: Root-mean-squared error.)

(8) Antioxidant activity analysis before and after drug loading

p-Cou is a type of phenol with hydroxy groups and is known to have excellent antioxidant properties. As a result of comparing the total antioxidant capacity of the p-Cou-loaded EK-20@Cou scaffold and EK-20(PW) scaffold, the antioxidant capacity of EK-20@Cou scaffold was higher than that of EK-20(PW). It was confirmed that it was significantly high (see Figure 28). The antioxidant capacity of EK-20@Cou was found to increase for the first 4 days and then gradually decrease over time.

Through the above experiment, it was confirmed that the EK-20@Cou scaffold has excellent antioxidant activity and can be used more effectively for wound healing.

2-4. Antibacterial activity analysis

Bacterial infection can easily occur in the wound environment, and if 90% of the wound is infected, the wound can become chronic. Therefore, in order to be used as an effective wound healing material, antibacterial activity is essential, so an experiment was performed to measure the antibacterial effect of the EK-20 scaffold.

Specifically, as a result of analyzing the antibacterial effect of EK-20@Cou and EK-20(PW) against Gram-positive bacteria ( Bacillus subtilis and Staphylococcus aureus ) and Gram-negative bacteria ( Escherichia coli and Salmonella typhimurium ), EK-20@Cou scan It was confirmed that Fold exhibited a strong antibacterial effect against all tested bacteria (see Figures 29a and 29b).

Through the above experiment, it was confirmed that the EK-20@Cou scaffold has excellent antibacterial effect and can be effectively used for wound healing.

2-5. Biocompatibility analysis of EK-20@Cou scaffold

To measure the biocompatibility and cell viability of EK-20, EK-20(PW) and EK-20@Cou, MTT-based colorimetric assay was performed. As a result, the EK-20 scaffold showed cytotoxicity (cell viability less than 70%), but cell viability was measured similarly for EK-20(PW) and EK-20@Cou, and cell growth patterns over time were also observed. appeared (see Figure 30).

In addition, after confocal staining of cells treated with EK-20, EK-20(PW), and EK-20@Cou, cell proliferation was visually confirmed on days 1, 3, and 7. As a result, it was confirmed that significantly higher cell proliferation was induced in the EK-20(PW) and EK-20@Cou treatment groups compared to the other groups (see Figure 31).

Through the above experiment, it was confirmed that the cell proliferation of the EK-20@Cou scaffold was measured similarly to the scaffold not loaded with p-Cou (EK-20(PW)), indicating that cell growth by loading of p-Cou It was confirmed that this was not disturbed.

2-6. Wound healing analysis

To confirm the wound healing effect of EK-20@Cou, an in vivo experiment was performed using a full-thickness wound model. To prepare a full-thickness wound model, a full-thickness wound with a diameter of 7 ± 1 mm was created on the back of a rat, and the wound healing effect of EK-20@Cou was tested as a control group, positive control group (group treated with commercially available collagen-based wound healing material). and EK-20 (PW) treatment group. After 7 days of experiment, the skin crust was removed, and neo-epidermis was formed within 14 days.

As a result of the experiment, after 7 days, the control group and the positive control group showed wound closure of 6.75% and 16%, respectively, but the EK-20(PW) treatment group and the EK-20@Cou treatment group showed wound closure of 25.99% and 47%, respectively. showed. Additionally, after 14 days, the control group and positive control group showed wound closure of 26.82% and 67.12%, respectively, while the EK-20(PW) treated group and EK-20@Cou treated group showed wound closure of 82.11% and 98.75%, respectively. It was visible (see Figure 32). Afterwards, the wound healing mechanism of EK-20@Cou was schematically drawn (see Figure 33).

Then, to evaluate the wound healing effect at the tissue level, H&E staining and Masson's Trichrome staining were performed (see Figure 34), and then the neoepidermal thickness, granulation tissue thickness, and epithelial formation score of each group were compared.

As a result, it was confirmed that the neoepidermal thickness, granulation tissue thickness, and epithelial formation score of the EK-20@Cou treatment group were significantly higher than those of the other groups, and the EK-20(PW) treatment group and EK-20@Cou treatment group In the case of , unlike the control group, it was confirmed that the wound was completely closed 14 days after injury (see FIGS. 35a to 35c). In addition, as a result of Masson's Trichrome staining analysis showing collagen deposition, the degree of collagen deposition was higher in the EK-20(PW) treated group and the EK-20@Cou treated group compared to the control group, confirming that wound healing occurred effectively. there was.

Additionally, to confirm the progress of wound healing, immunohistochemical staining for fibronectin, collagen-1, and α-SMA markers was performed on the wound area (see Figure 36). As a result, it was confirmed that the EK-20@Cou treated group had significantly higher contents of fibronectin, collagen-1, and α-SMA markers compared to the control and EK-20(PW) treated groups (see Figures 37a to 37c).

Through the above experiment, it was confirmed that the EK-20@Cou scaffold had the best wound healing effect in vivo .

2-7. Wound healing mechanism analysis

To analyze the mechanism of the excellent wound healing effect of the EK-20@Cou scaffold, an in-silico MS study was performed along with biochemical and histological studies. MMP-2, MMP-3, MMP-8, MMP-12, NF-κB, TNF-α, STAT-1, IL-4R, and IL-12 as target proteins/receptors that play important roles in the wound healing process during MS. 1β was selected. In addition, p-Cou and known inhibitors of each protein were used as ligands, and inhibitors for each protein or receptor were selected as controls while considering binding energy, binding distance, junction type, position, and pose in prediction (table below) 3).

Target [proteins/receptors]	Catalytic site	Ligand	LE a index	Interacting residue	Distance [A˚]	Category	Type	Control ligand
MMP-2 (PDB ID: 1ck7)	X, Y & Z = (51.93, 89.15 &147.59)	p-Coumaric acid	0.05	VAL 400	5.15	Hydrophobic	Pi-alkyl	Marimastat
				TYR 425	5.24	Hydrophobic	Pi-pi T-shaped
				THR 428	3.05	H-bond	Conventional
				HIS 403	4.44	Hydrophobic	Pi-pi stacked
MMP-3 (PDB ID: 1b3d)	X, Y & Z = (-18.45, 28.25 & 1.56)	p-Coumaric acid	0.05	LEU 164	4.74	Hydrophobic	Pi-alkyl	PGV-25727
				ALA 165	2.47	H-bond	Conventional
				ALA 165	5.28	Hydrophobic	Pi-alkyl
				LEU 164	4.74	Hydrophobic	Pi-alkyl
				VAL 198	5.36	Hydrophobic	Pi-alkyl
				HIS 201	4.84	Hydrophobic	Pi-pi stacked
				GLU 202	4.08	Electrostatic	Pi-anion
				Zn 301	4.93	Electrostatic	Pi-cation
MMP-8 (PDB ID: 1a85)	X, Y & Z = (25.74, 56.76 & 53.26)	p-Coumaric acid	0.05	HIS 197	4.06	Hydrophobic	Pi-pi stacked	Q27451034
				HIS 197 (Zn ligating)	3.77	Electrostatic	Pi-cation
				VAL 194	4.76	Hydrophobic	Pi-alkyl
				ALA 220	2.63	H-bond	Conventional
				ARG 222	2.61	H-bond	Conventional
MMP-12 (PDB ID: 1jiz)	X, Y & Z = (4.74, 5.75 & 69.59)	p-Coumaric acid	0.05	HIS 119	4.11	Hydrophobic	Pi-pi stacked	CGS27023A
				HIS 119 (Zn ligating)	4.70	Electrostatic	Pi-cation
				TYR 141	5.30	Hydrophobic	Pi-pi T-shaped
ALA 83	2.06	H-bond	Conventional
NFκ-b (PDB ID: 3do7)	X, Y & Z = (4.74, 5.75 & 69.59)	p-Coumaric acid	0.04	HIS 140	5.75	Hydrophobic	Pi-pi T-shaped	PBS-1086
				TYR 55	4.56	Hydrophobic	Pi-pi T-shaped
				GLU 58	2.91	H-bond	Conventional
				LYS 221	3.77	Hydrophobic	Pi-alkyl
PRO 222	3.56	H-bond	C-H bond
TNF-α (PDB ID: 2az5)	X, Y & Z = (-14.22, 70.99 & 27.50)	p-Coumaric acid	0.03	GLY 121	2.41	H-bond	Conventional	Dimethylamine spacer linked trifluoromethylphenyl indole and dimethyl chromone
STAT-1 (PDB ID: 1bf5)	X, Y & Z = (67.94, 62.26 & 88.47)	p-Coumaric acid	0.02	THR 419	3.98	Hydrophobic	Pi-sigma	Niclosamide
				ARG 378	4.61	Hydrophobic	Pi-alkyl
				ILE 425	1.87	H-bond	Conventional
IL-4R (PDB ID: 1iar)	X, Y & Z = (22.70, 17.95 & -17.59)	p-Coumaric acid	0.01	TYR 127	4.96	Hydrophobic	Pi-pi T-shaped	N/A b
				ASP 67	3.88	Electrostatic	Pi-Anion
IL-1β (PDB ID: 6y8m)	X, Y & Z = (9.19, 26.99 & -5.84)	p-Coumaric acid	0.02	PHE 46	3.91	Hydrophobic	Pi-pi stacked	Abrasin
LEU 6	5.29	Hydrophobic	Pi-alkyl
a LE = Δ G /molecular weight of the ligand. p-Coumaric acid had a binding affinity of 8.3 kcal mol -1 and a LE index of 0.05.

As a result of the analysis, p-Cou showed the highest binding affinity with matrix metalloproteinases (MMPs) (see Figures 38a to 38c). Specifically, the binding energy was measured at -8.10 ± 0.10 in MMP-2, and -7.65 ± 0.05 in MMP-12, which was the highest value. The catalytic activity of the MMP enzyme is known to mainly depend on Zn ²⁺ , and through the above analysis, it was confirmed that p-Cou forms an electrostatic bond with Zn ²⁺ and the ligation residue of MMP to inactivate proteolytic activity. .

In addition, as a result of in vitro experiments, it was confirmed that the expression levels of MMP-2 and MMP-12 were significantly reduced in the EK-20@Cou treated group (see Figures 39a and 39b). MMPs are activated during the inflammatory phase of the wound healing process, but when activated for a long period of time, they are known to destroy newly formed ECM and delay wound healing time. Therefore, EK-20@Cou can effectively shorten wound healing time by inhibiting the expression of MMP.

Additionally, an experiment was performed to determine whether the EK-20@Cou scaffold could regulate NF-κB signaling (see Figure 40). As a result of measuring NF-κB expression, NF-κB expression significantly increased at week 1 in both the EK-20 (PW) and EK-20@Cou treated groups, while NF-κB expression significantly decreased at week 2. This was confirmed (see FIGS. 41a and 41b).

Additionally, to confirm whether the EK-20@Cou scaffold induces phenotypic conversion of macrophages, the expression of CD68, a macrophage marker, and nitrate concentration were measured. As a result, CD68 expression in the EK-20@Cou treatment group was significantly lower than that of the other groups (see Figures 42a and 42b), and nitrate concentration was 0.275 ± 0.013 nmol μL ^-1 and 0.026 ± 1 after 1 and 2 weeks, respectively. It was confirmed that it was significantly lower than other groups at 0.001 nmol μL ^-1 (see Figure 43). From the above results, it can be confirmed that p-Cou released from the EK-20@Cou scaffold to the wound site regulates the activity of immune cells and switches the phenotype of macrophages from M1 type to M2 type.

From the above experiments, it was confirmed that the EK-20@Cou scaffold can be effectively used for wound healing, and a schematic diagram of the wound healing mechanism of the EK-20@Cou scaffold is shown in Figure 44 below.

The description of the present invention stated above is for illustrative purposes, and a person skilled in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (15)

1. A pharmaceutical composition for wound treatment or skin tissue regeneration, comprising an extracellular matrix and kelp extract.
2. The pharmaceutical composition according to claim 1, wherein the extracellular matrix and kelp extract are decellularized.
3. The pharmaceutical composition according to claim 1, wherein the extracellular matrix is derived from liver cells.
4. The pharmaceutical composition according to claim 1, wherein the concentration ratio of the extracellular matrix and the kelp extract is 1:0.75 to 1:1.25.
5. The pharmaceutical composition of claim 1, further comprising an acidic drug.
6. The pharmaceutical composition of claim 5, wherein the acidic drug is p-coumaric acid.
7. The pharmaceutical composition according to claim 1, wherein the composition has an antibacterial effect.
8. The pharmaceutical composition according to claim 7, wherein the antibacterial effect is against Gram-positive bacteria and Gram-negative bacteria.
9. The method of claim 8, wherein the Gram-positive bacteria are bacteria of the genus Staphylococcus , bacteria of the genus Streptococcus , bacteria of the genus Enterococcus , bacteria of the genus Klebsiell , and Corynebacterium . At least one selected from the group consisting of bacteria of the genus, Clostridium , Listeria , and Bacillus ,

   The gram-negative bacteria include Escherichia species , Salmonella , Pseudomonas , Neisseria , Chlamydia , and Yersinia . A pharmaceutical composition comprising at least one selected from the group consisting of bacteria.
10. The pharmaceutical composition according to claim 1, wherein the composition inhibits the expression of matrix metalloproteinases (MMPs).
11. The pharmaceutical composition according to claim 1, wherein the composition converts the phenotype of macrophages from type M1 to type M2.
12. The pharmaceutical composition of claim 1, wherein the wound is at least one selected from the group consisting of a wound, abrasion, laceration, stab wound, and ulcer.
13. The pharmaceutical composition according to claim 1, wherein the skin tissue regeneration is at least one selected from the group consisting of epidermal regeneration, reproduction of glands or hair follicles, and microvascular formation in dermal tissue.
14. An external skin preparation for wound treatment or skin tissue regeneration containing extracellular matrix and kelp extract.
15. A cosmetic composition for improving wounds or regenerating skin tissue, comprising an extracellular matrix and kelp extract.

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