KR20190111864A - Pharmaceutical composition for wound healing or tissue regeneration, preparation method thereof, and use thereof - Google Patents

Abstract

Provided are: a pharmaceutical composition for wound healing or tissue regeneration comprising a complex containing an extracellular matrix and polyvinyl alcohol according to an aspect and antibiotics carried on the complex; a manufacturing method thereof; and a method using the same. According to the same, the pharmaceutical composition provides biomimetic microenvironment and deliver antibiotics by a single structure, thereby being able to be used for healing skin infection wounds, tissue regeneration and preventing bacterial infection.

Description

Pharmaceutical compositions for wound healing or tissue regeneration, methods for preparing the same, and methods using the same {Pharmaceutical composition for wound healing or tissue regeneration, preparation method according,

It relates to a pharmaceutical composition for wound treatment or tissue regeneration, a method for preparing the same, and a method using the same.

Wounds are a form of injury that can be divided into open wounds in which the skin is torn, pierced, or cut, closed wounds such as bruises, bruises, etc., caused by dull force. In the case of a wound, it can be treated only by washing, but it requires treatment such as disinfection, suture and dressing. In particular, bacteria may be infected or infected wounds may require antibiotic treatment. Treatment with antimicrobial activity that inhibits bacterial infections can greatly help the wound healing process.

One of the key factors in the progression of skin wound treatment is the interaction between extracellular matrix (ECM) and growth factors. ECM-growth factor interactions are bidirectional, occurring either directly or indirectly. For example, ECM can trap or bind growth factors to prevent growth factors from breaking down. In addition, cell attachment to ECM can improve the growth factor production mechanism of the cell. In addition, the ECM was able to be applied to various types of three-dimensional scaffolds based on excellent physical properties (Korea Patent Registration 10-0715505).

Accordingly, there is a need to develop antimicrobial wound treatment compositions that prevent bacterial infection and provide a supportive ECM microenvironment for wound healing, bacterial invasion and regeneration of skin tissue.

Provided are pharmaceutical compositions for wound healing or tissue regeneration.

It provides a method of preparing a pharmaceutical composition for wound healing or tissue regeneration.

Provided are a wound treatment or tissue regeneration method using a pharmaceutical composition for wound treatment or tissue regeneration.

One aspect includes a complex comprising an extracellular matrix (ECM) and poly (vinyl alcohol) (PVA); And

It provides a pharmaceutical composition for wound treatment or tissue regeneration comprising an antibiotic supported on the complex.

The term "extracellular matrix" (ECM) is a complex collection of biopolymers that fills the space inside or outside a tissue. Cellular proteins such as fibrous proteins, complex proteins such as proteoglycans, and cell adhesion proteins such as fibronectin and laminin. It can be composed of various kinds of molecules synthesized by and secreted and accumulated besides cells. Therefore, the extracellular matrix may vary in composition depending on the type of cell or the degree of differentiation of the cell.

The ECM may be decellularized from the cell. The cells can be cells derived from cells or tissues cultured in vitro. Decellularization refers to the process of separating the original ECM of a tissue from a cell. The decellularization may be to lyse and kill cells in tissue without damaging the ECM components and to obtain a natural ECM scaffold with the same physical and biochemical functions of natural tissue. The decellularization can be accomplished by decellularization-inducing treatments such as physical, chemical, and enzymatic treatments, whereby ECM can be monitored to maintain the structural chemical robustness of the original tissue. Examples of decellularization treatments include contacting a cell with a solution comprising one selected from the group consisting of a cell lysis solvent, a stock solution, a cationic surfactant, an anionic surfactant, a nonionic surfactant, RNase A, and DNase I. Can be. The decellularization solvent may be a mixture of 0.25% Triton X-100 and 50 mM ammonium hydroxide (NH ₄ OH) in PBS. The decellularized extracellular matrix may be an extracellular matrix remaining after cellular components such as nuclei, membranes, and nucleic acids are removed from tissues or cells.

The ECM may be derived from a cell selected from the group consisting of fibroblasts, stem cells, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, neurons, cardiomyocytes, and spinal intervertebral disc cells. The ECM may be fibroblast-derived cells. The fibroblasts may be human fibroblasts. The fibroblasts may be fibroblasts of the lung. For example, the fibroblasts may be human lung fibroblasts.

The PVA is a water soluble synthetic polymer. The PVA has a molecular weight of about 50,000 to about 300,000; About 75,000 to about 275,000; About 100,000 to about 250,000; About 110,000 to about 225,000; About 120,000 to about 200,000; About 130,000 to about 190,000; Or about 140,000 to about 190,000.

The PVA may be physically or chemically crosslinked. The PVA can be a mixture of PVA with a copolymer or other polymer including PVA.

The ECM and PVA may be physically or chemically bound. The ECM may be surrounded, buried, embedded or embedded in PVA. For example, the ECM may be located at the surface layer of the PVA.

The antibiotic is ciprofloxacin (ciprofloxacin), gentamicin (gentamicin), cefadroxil (lin), lincomycin, amikacin (amikacin), kanamycin (kanamycin), netylmicin (netilmicin), t-obramycin (t-obramycin), streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate / ethylsuccinate / ethylsuccinate Septatelactobionate / stearate, penicillin G, penicillin V, methicillin, nafcillin, oxacillin, oxacillin, cloxacillin, dicloxacillin (dicloxacillin), ampicillin, amoxicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, piperacillin, cefalal Cephalothin, cefazolin, cefaclor, cefamandol, cefamandole, cefoxitin, cefuiroxime, cefonicid, cefmetazole , Cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftri Ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, i-mipenem, aztreo Aztreonam, fleroxacin, nalidixic acid, norfloxacin, ofloxacin, enoxacin, lomefloxacin, synoxacin cinoxacin, doxycycline, m-inocycline, tetracycline, vancomycin, and teicoplanin planin) may be any one or more selected from the group consisting of.

The amount of antibiotic in the pharmaceutical composition may be about 1% to about 20%.

The pharmaceutical composition may be one having a bacterial infection prevention effect. The pharmaceutical composition may have antimicrobial activity against Gram negative bacteria. The Gram-negative bacteria from the group consisting of Escherichia species bacteria, Pseudomonas bacteria, Neisseria bacteria, Chlamydia bacteria, and Yersinia bacteria Can be selected. For example, the Gram negative bacteria is Escherichia coli or Pseudomonas aeruginosa. The pharmaceutical composition may have antimicrobial activity against Gram positive bacteria. The Gram-positive bacteria are Staphylococcus bacteria, Streptococcus bacteria, Enterococcus bacteria, Klebsiell bacteria, Corynebacterium bacteria, Clos It may be selected from the group consisting of bacteria of the genus Trisdium (Clostridium), bacteria of Listeria, and bacteria of Bacillus. For example, the Gram-positive bacteria are Staphylococcus epidermidis and Staphylococcus aureus.

The wound may be a wound, abrasion, laceration, stab wound, ulcer, or a combination thereof. The wound may be on a skin, for example an external skin that is directly exposed to air.

The tissue regeneration may be epidermal regeneration, reproduction of glands or hair follicles, microvascular formation of dermal tissue, or a combination thereof.

The pharmaceutical composition may be in the form of a gel. The pharmaceutical composition may be a hydrogel. The pharmaceutical composition may be in wet form or dry form. The pharmaceutical composition may be a skin patch or film.

Another aspect includes obtaining an extracellular matrix (ECM) from a cell;

Crosslinking the resulting mixture comprising ECM and polyvinyl alcohol (PVA) to form a complex; And

It provides a method for preparing a pharmaceutical composition for wound treatment or tissue regeneration comprising the step of supporting an antibiotic in the complex.

The method includes obtaining ECM from the cells.

The cells and ECM are as described above.

Obtaining ECM from the cells may be to decellularize the cells to obtain ECM. The decellularization is as described above.

The method includes crosslinking the obtained mixture comprising ECM and PVA to form a complex.

Crosslinking the mixture may be physically or chemically crosslinking. Crosslinking the mixture may be to perform freezing and thawing. Refrigeration is about -10 ° C to about -70 ° C, about -15 ° C to about -60 ° C, about -20 ° C to about -50 ° C, about -20 ° C to about -40 ° C, or about -20 ° C to about- It may be carried out at a temperature of 30 ℃. Freezing is for a time sufficient to freeze the mixture, for example about 1 second to about 12 hours, about 1 minute to about 11 hours, about 30 minutes to about 10 hours, about 1 hour to about 9 hours, about 2 hours. To about 8 hours, about 3 hours to about 7 hours, about 4 hours to about 6 hours, or about 5 hours to about 6 hours. The thawing is about 4 ° C. to about 60 ° C., about 10 ° C. to about 55 ° C., about 15 ° C. to about 50 ° C., about 20 ° C. to about 45 ° C., about 25 ° C. to about 40 ° C., about 25 ° C. to about 35 ° C. Or at room temperature. Thawing may be performed for a time sufficient to thaw the frozen mixture, for example from about 1 second to about 6 hours, from about 1 minute to about 5 hours, from about 30 minutes to about 4 hours, from about 1 hour to about 4 hours, about For 2 hours to about 4 hours, or about 3 hours. The freezing and thawing is once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, or fifteen times. The above may be performed.

The method includes supporting antibiotics in the complex.

The antibiotic is as described above.

The antibiotic may be diluted in an acidic solution. The acidic solution may be hydrochloric acid. The acidic solution may be about 0.1 N hydrochloric acid solution. The antibiotic is about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 7 mg / ml, about 10 mg / ml, about 12 mg / ml , About 15 mg / ml, about 17 mg / ml, about 20 mg / ml, about 25 mg / ml, or about 30 mg / ml.

The supporting step may be performed by mixing and incubating the complex with an antibiotic. The incubation may be about 4 ° C. to about 50 ° C., about 10 ° C. to about 45 ° C., about 15 ° C. to about 40 ° C., about 20 ° C. to about 40 ° C., about 25 ° C. to about 40 ° C., or about 30 ° C. to about 40 It may be carried out at ℃. For example, the incubation is performed at about 37 ° C. The incubation is about 1 second to about 2 days, about 1 minute to about 1 day, about 5 minutes to about 18 hours, about 10 minutes to about 12 hours, about 15 minutes to about 9 hours, about 20 minutes to about 6 hours. , About 25 minutes to about 3 hours, or about 30 minutes to about 1 hour.

Another aspect provides a method of treating wounds or tissues, comprising administering a pharmaceutical composition according to one aspect to a subject.

The subject may be a mammal including humans, mouse rats, dogs, cats, horses, cattle, pigs, and goats.

The administration can be to provide and attach or implant the composition to the wound site.

Complex comprising an extracellular matrix and polyvinyl alcohol according to one aspect; And a pharmaceutical composition for wound treatment or tissue regeneration comprising the antibiotic supported on the complex, a method for preparing the same, and a method using the same, provide a biomimetic microenvironment by delivering a single structure and deliver antibiotics to prevent skin infection wounds. It can be used to heal and prevent tissue regeneration and bacterial infection.

1 is a schematic of (i) a method of preparing a PVA / hFDM membrane containing hFDM, (ii) an optical microscope image of hFDM, and (iii) an optical microscope image of the original plate bottom after separation of the PVA / hFDM membrane (arrow: Residual hFDM remaining on the plate), (iv) immunofluorescence staining (fibronectin) image of hFDM on PVA / hFDM membrane, and (v) immunofluorescence staining image of cross section cut PVA / hFDM membrane (red dotted line: PVA and hFDM Interface).
Figure 2a is a graph showing the content (%) of ciprofloxacin supported on the PVA / hFDM hydrogel according to the concentration of ciprofloxacin, Figure 2b is a graph showing the release (%) of ciprofloxacin over time (incubation time).
Figure 3a is an image of agar culture medium cultured bacteria using agar medium diffusion test method, Figure 3b is a graph showing the diameter (mm) of the inhibitory site.
Figure 4a is a photograph showing the wound healing level over time (day), Figure 4b is a graph showing the wound size (%) over time (day), and Figure 4c shows the viability of bacterial bacteria (CFU) according to the treatment group It is a graph.
5A is an H & E stained image of a wound site according to the treatment group, FIG. 5B is an enlarged image of a specific site of FIG. 5A, FIG. 5C is a graph of the thickness (μm) of the regenerated epidermis according to the treatment group, and FIG. 5D is FIG. 5B 5E is a magnified image of a specific region of, and FIG. 5E is a graph showing the density (micron / 3 × 10 ⁵ μm ² ) of microvascular formation in the dermal layer according to the treatment group (**: p <0.01 and ***: p <0.001) .

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

Example 1. Preparation and Characterization of Antibiotic-Supported PVA / hFDM Membrane

1. Preparation of antibiotic-supported PVA / hFDM membrane

1) Preparation of extracellular matrix

Human lung fibroblast WI-38 cell line (ATCC CCL-75) was seeded in 24-well plates in an amount of 2 × 10 ⁴ cells / cm 2. Dulbecco's modified Eagle's medium with 10% (v / v) fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin added to the inoculated plates : DMEM) was added, and cultured for about 7 days while replacing the medium at regular culture conditions (5% CO ₂ , 37 ° C) at intervals of about 2 to 3 days. The cultured cells were washed with phosphate-buffered saline (PBS). Then, 0.25% (v / v) Triton-X 100 and 50 mM NH ₄ OH (Sigma) were added to the washed cells, followed by 50 U / ml of DNase I (Invitrogen) and 2.5 μl / ml of RNase A ( Invitrogen) was added and incubated at 37 ° C. for 2 hours to decellularize the cells. The decellularized extracellular matrix (ECM) was washed with PBS to obtain a human lung fibroblast-derived matrix (hFDM).

The obtained hFDM was confirmed by light microscopy ((ii) of FIG. 1), and hFDM was used immediately or stored at about 4 ° C. in the presence of PBS for up to about 2 weeks.

2) PVA / hFDM Membrane Manufacturing

Hydrogel solution was prepared by adding 500 μl of 7% (w / v) water soluble polyvinyl alcohol (PVA) (molecular weight about 146,000 to about 186,000, catalog number 363065, Sigma) to hFDM prepared as described in 1.1). Ready. The prepared hydrogel solution was stored at about −20 ° C. for about 24 hours and then thawed at room temperature for about 1 hour to physically crosslink the hydrogel. The physically crosslinked PVA / hFDM hydrogel was carefully stripped off the plate with tweezers and transferred to a new plate.

After stripping off the PVA / hFDM membrane, it was confirmed by optical microscopy that hFDM was almost removed from the original plate (Fig. 1 (iii)). PVA / hFDM was immunostained using anti-fibronectin antibody (catalog number SC-8422, Santa Cruz Biotechnology) as primary antibody and Alexa Fluor 488-conjugated anti-mouse IgG antibody as secondary antibody It was. Fibronectin-specific immunostained PVA / hFDM was observed by confocal laser microscopy (Fluo View FV1000, Olympus) (FIG. 1 (iv)). Furthermore, PVA / hFDM was cut | disconnected with the surgical knife, and the cross section was observed with the confocal laser microscope (FIG. 1 (v)).

As shown in (iv) and (v) of FIG. 1, hFDM was attached to the PVA hydrogel while still maintaining its original fiber structure, and it was confirmed that the hFDM was located on the surface of the PVA hydrogel.

3) Preparation of antibiotic-supported PVA / hFDM membrane and release of ciprofloxacin

PVA / hFDM membranes were prepared as described in 1.2). Then, ciprofloxacin (Sigma) was added to 0.1 N hydrochloric acid to prepare a ciprofloxacin solution. The concentration of ciproflocincin solution was prepared at 1 mg / ml, 10 mg / ml, or 20 mg / ml. The prepared 2 ml ciprofloxacin solution was added to PVA / hFDM and incubated at about 37 ° C. for about 30 minutes to prepare a PVA / hFDM membrane (PVA / cipro / hFDM) structures carrying ciprofloxacin and washed with PBS. The weight difference of ciprofloxacin solution before and after loading was calculated to calculate the content of supported ciprofloxacin, and the results are shown in FIG. 2A. In FIG. 2A, no statistical significance was observed between groups relative to the initial drug concentrations (1 mg / ml, 10 mg / ml and 20 mg / ml).

In addition, to investigate the release of supported ciprofloxacin, PBS was added to the PVA / cipro / hFDM construct and incubated at about 37 ° C. for a period of time. The concentration of released ciprofloxacin was then measured spectrophotometrically at a wavelength of 355 nm and the results are shown in FIG. 2B.

As shown in FIG. 2A, the loading efficiency of ciprofloxacin in the PVA / hFDM membrane was calculated to be about 8% from the initial loading amount. This loading efficiency was similarly observed for other concentrations of ciprofloxacin. On the other hand, as shown in Figure 2b, the release of ciprofloxacin was found to be dependent on the initial concentration. Lower concentrations of ciprofloxacin initially used resulted in a slower release profile, particularly slow release of ciprofloxacin at the initial time (0.2 hours).

2. Identification of PVA / hFDM Membrane Characteristics

1) Evaluation of antimicrobial activity

The antimicrobial activity of the PVA / hFDM membrane carrying the antibiotic prepared as described in 1.3) was evaluated.

E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were prepared as Gram-negative bacteria, and Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus as Gram-positive bacteria. ( Staphylococcus aureus ) (ATCC 25923) was prepared.

Prepared bacteria were incubated aerobicly in Mueller-Hinton broth (Difco Laboratories, Detroit, MI) at 35 ° C. for about 18 hours. Agar medium diffusion assay was used to evaluate antimicrobial activity and was performed according to the guidelines of the Clinical and Laboratory Standards Institute. Briefly, sterile swabs were immersed in a bacterial suspension adapted to turbidity of 0.5 McFarland standard units and inoculated in Muller-Hinton agar medium. The agar medium was then dried for about 5 minutes. PVA with 0.1 N hydrochloric acid was used as a control. Sensitive dishes were incubated at about 35 ° C. for about 16-18 hours and then the average diameter of the inhibition zone around the disc was measured. Images of cultured agar medium and graphs of diameters of inhibition sites are shown in FIGS. 3A and 3B, respectively (A: Escherichia coli, B: P. aeruginosa, C: S. epidermis, D: S. aureus). Reus) (***: p <0.001).

As shown in FIGS. 3A and 3B, the construct containing ciprofloxacin showed antimicrobial activity in all strains tested. Constructs containing ciprofloxacin were similar in killing capacity to all strains tested, whereas constructs not containing ciflofloxacin had little bacterial killing ability. It also shows that the presence of hFDM on PVA hydrogel did not negatively affect the diffusion and antimicrobial activity of ciprofloxacin. For E. coli, there was some bacterial growth inhibition in the control group, but the levels were significantly lower compared to PVA / cipro and PVA / cipro / hFDM.

2) Evaluation of infection prevention and wound healing effect

In animal models, it was determined whether PVA / cipro / hFDM prevented infection and induced wound healing.

Female BALB / c mice were prepared for 6 to 8 weeks, and after being approved by the Kyung Hee University Experimental Animal Steering Committee, the prepared mice were used for animal experiments.

To induce a single epidermal wound, the back of the mouse was wounded and 10 ⁷ colony forming units (CFU) / 100 μl of S. aureus infected the bacteria via gauze. The gauze was removed 6 hours after bacterial infection and treated with PVA (with 0.1 N hydrochloric acid), PVA / cipro, or PVA / cipro / hFDM, with regular replacement of the gauze at the wound site every 3-4 days. Mice infected but not treated were used as controls. In each group, three animals were used and sacrificed on day 21.

Bacterial samples were collected from the wound site of mice 6 hours after bacterial infection and the viability of the collected bacteria was calculated by colony forming unit assay. The progress of wound healing was analyzed by photo observation, and the level of wound healing was expressed as a percentage of wound size relative to the original wound area. A photograph of wound healing over time is shown in FIG. 4A, the wound size (%) is shown in FIG. 4B, and the viability of the bacteria is shown in FIG. 4C (*: p <0.05, **: p <0.01, and ***). : p <0.001).

As shown in FIGS. 4A and 4B, in the PVA / cipro treated group and the PVA / cipro / hFDM treated group, the wound infection and the treatment of the wound progressed faster than the control group. On day 3, pus deposits appeared in the wounds in the control group without Pyprofloxacin and the PVA treated group. Treatment groups containing ciprofloxacin have been shown to be treated faster than treatment groups without ciprofloxacin, and the presence of hFDM appears to increase treatment speed to some extent. On the other hand, the PVA treatment group showed little treatment effect.

As shown in FIG. 4C, no bacterial activity was observed in the PVA / cipro treated group and the PVA / cipro / hFDM treated group, and the bacterial colonies were significantly reduced compared to the control and PVA treated groups.

3) Histological Analysis

In animal models treated as described in 2.2), morphological analysis of the tissues in each group was performed.

Three weeks after the start of treatment in a bacterial infected animal model, tissue samples were collected from each treatment group and the collected tissue samples were fixed in 10% (v / v) formalin. The immobilized tissue sample was placed in paraffin and then made bisected and very thin tissue sections across the wound site. Hematoxylin & Eosin (H & E) staining was performed to evaluate the morphological features of epidermal and dermal areas. Epidermal thickness of regenerated tissue was measured using microscopic images of H & E staining taken from three replicates per group and three random epidermal regions in each replicate.

H & E staining images of the epidermal region are shown in FIG. 5A. In FIG. 5A, the red arrow indicates the wound position, and the black dotted line is a portion corresponding to the enlarged image of FIG. 5B. FIG. 5B is an enlarged image of FIG. 5A, with white cut marks (†) and double cut marks (‡) respectively representing epidermis and dermis layers, yellow triangles showing regenerated glands, and red dotted rectangles corresponding to enlarged images of FIG. 5D Site. Evaluation for epidermal regeneration was measured by the thickness of the new epidermis (μm) and the results are shown in FIG. 5C.

FIG. 5D is an enlarged image of FIG. 5B, with yellow arrows indicating microvessels in the regenerated dermal layer. FIG. In addition, the density of the micro-vessels in the dermal layer was shown in Figure 5e (**: p <0.01 and ***: p <0.001). In FIG. 5E, the y-axis legend is the density of the microvessels (open / 3x10 ⁵ ), and the density of the microvessels was measured on each microscope slide by selecting three regions from the regenerated epidermal layer with the highest microvessel density and averaging the values. Positive signals from blood vessels are confirmed by the presence of red blood cells inside the distinct lumen structure.

As shown in FIGS. 5A-5C, after 3 weeks of treatment, re-epithelialization was seen in the control and PVA / cipro / hFDM treated groups, and the PVA treated group showed incomplete treatment. The PVA / cipro / hFDM treated group showed much better tissue regeneration compared to the other treated groups, and glands and hair follicles were reproduced. In contrast, in the other treatment groups, the glands and hair follicles did not regenerate. In addition, the shape and thickness of the new epidermis at the wound site in the PVA / cipro / hFDM treated group is characterized by similarity to that of normal skin.

As shown in FIG. 5D and FIG. 5E, angiogenesis was observed in regenerated dermal tissue throughout the control and treatment groups. The microvascular density of the PVA / cipro treated group and PVA / cipro / hFDM treated group had significantly higher values than the control and PVA treated groups. The density of the PVA / cipro / hFDM treated group was at the same level as normal skin, but the microvascular of normal skin was relatively larger and stronger.

Thus, animal experiments confirmed that the PVA / cipro / hFDM construct has a tissue regeneration effect in a bacterial infected wound model.

Claims (20)

A complex comprising an extracellular matrix (ECM) and poly (vinyl alcohol) (PVA); And
A pharmaceutical composition for wound treatment or skin tissue regeneration comprising an antibiotic supported on the complex. The pharmaceutical composition of claim 1, wherein the ECM is decellularized from a cell. The method of claim 2, wherein the cells are selected from the group consisting of fibroblasts, stem cells, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, neurons, cardiomyocytes, and spinal intervertebral disc cells. Composition. The pharmaceutical composition of claim 1, wherein the PVA is physically or chemically crosslinked. The pharmaceutical composition of claim 1, wherein the ECM and PVA are physically or chemically bound. The pharmaceutical composition of claim 5, wherein the ECM is located at the surface layer of the PVA. The method of claim 1, wherein the antibiotic is ciprofloxacin, gentamicin, gentamicin, cefadroxil, lincomycin, amikacin, kanamycin, kanamycin, netylmicin, t-obramycin, streptomycin, azithromycin, clarithromycin, erythromycin, erythromycin, erythromycin estolate / ethylsuccinate (ethylsuccinate) / gluceptatellactobionate / stearate, penicillin G, penicillin V, methicillin, nafcillin, oxacillin, oxacillin, cloxacillin , Dicloxacillin, ampicillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, azlocillin, piperasilin Piperacillin, cephalothin, cefazolin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuiroxime, cefuiroxime, cefonicid Cefmetazole, cefotetan, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, cefti Ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, i-mifenem ( i-mipenem, aztreonam, fleroxacin, nalidixic acid, norfloxacin, ofloxacin, enoxacin, romefloxacin (lomefloxacin), cinoxacin, doxycycline, m-inocycline, tetracycline, vancomycin, Teicoplanin A pharmaceutical composition is at least one selected from the group consisting of (teicoplanin). The pharmaceutical composition of claim 1, wherein the amount of antibiotic in the pharmaceutical composition is 1% (w / v) to 20% (w / v). The pharmaceutical composition of claim 1, which has the effect of preventing bacterial infection. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition has antimicrobial activity against Gram negative bacteria. The method of claim 10, wherein the Gram-negative bacteria are Escherichia species bacteria, Pseudomonas bacteria, Neisseria bacteria, Chlamydia bacteria, and Yersinia genes. Pharmaceutical composition is selected from the group consisting of bacteria. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition has antimicrobial activity against Gram positive bacteria. The method of claim 12, wherein the Gram-positive bacteria are Staphylococcus bacteria, Streptococcus bacteria, Enterococcus bacteria, Klebsiell bacteria, Corynebacterium (Corynebacterium) ) Genus bacteria, Clostridium bacteria, Listeria bacteria, and Bacillus bacteria. The pharmaceutical composition of claim 1, wherein the wound is a wound, abrasion, laceration, stab wound, ulcer, or a combination thereof. The pharmaceutical composition of claim 1, wherein the skin tissue regeneration is epidermal regeneration, reproduction of glands or hair follicles, microvascular formation of dermal tissue, or a combination thereof. The pharmaceutical composition of claim 1, which is in the form of a gel. Obtaining an extracellular matrix (ECM) from the cell;
Crosslinking the resulting mixture of ECM and polyvinyl alcohol (PVA) to form a complex; And
Method of preparing a pharmaceutical composition for wound treatment or skin tissue regeneration comprising the step of supporting an antibiotic in the complex. The method of claim 17, wherein obtaining the ECM from the cells is to decellularize the cells to obtain ECM. The method of claim 17, wherein the crosslinking is performed repeatedly one or more times of freezing and thawing. A method of treating wounds or regenerating skin tissue, comprising administering the pharmaceutical composition of any one of claims 1 to 16 to a mammal other than a human.

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