KR101806115B1 - Enriched media of human adipose tissue-derived stem cells having skin regeneration or antiwrinkle effect and uses thereof - Google Patents

Abstract

The present invention relates to a composition for improving skin regeneration or wrinkle comprising a stem cell culture concentrate obtained by culturing human adipose-derived stem cells in a serum medium, subculturing in a serum-free medium and then filtering the composition, Wherein the composition is applied locally or injected intradermally to a site requiring removal of wrinkles, a method for improving wrinkles comprising the step of applying the composition to an active ingredient And a skin regeneration or wrinkle improving cosmetic comprising the composition as an active ingredient.

Description

[0001] The present invention relates to a culture concentrate of human adipose-derived stem cells having skin regeneration or wrinkle-improving effect,

The present invention relates to a culture concentrate of human adipose-derived stem cells having skin regeneration or wrinkle-improving effect and, more particularly, to a method for culturing human adipose-derived stem cells in serum-free medium, , A composition for skin regeneration or wrinkle improvement containing a stem cell culture concentrate obtained by filtration as an active ingredient, a skin regeneration method characterized by locally applying or intracutaneously injecting the composition into a wound site, , A skin regeneration or wrinkle remedy comprising the composition as an active ingredient, and a skin regeneration or wrinkle treatment containing the composition as an active ingredient, Improvement cosmetics.

With the recent improvements in medical technology and public health benefits, an aging society is rapidly emerging. As a result, the quality of life is being improved and the desire for the maintenance of youth is increasing. Much research is underway in various areas to delay and prevent appearance, especially the aging that is first manifested in skin.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The outer skin of the skin consists of a stratified squamous epithelium, which acts as a protective barrier against the external environment. The epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is the layer between the epidermis and the subcutaneous tissue. It is divided into the papillary dermis and the reticular dermis. The dermis is composed of blood vessels, collagen, elastin fibers, pores, lipids, sebaceous glands, Sensory nerves, fibroblasts, and macrophages, and they occupy the largest portion of the skin.

The dermis consists mainly of collagen and elastin fibers, which play a role in supporting the skin. Therefore, when such a dermis is troubled, wrinkles occur and skin elasticity is lost and the skin ages. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function to contain a large amount of water, and serves to supply moisture to the dermis. When aging occurs, collagen's water-containing function is deteriorated and wrinkles are increased. Collagen also causes wound healing by filling the wound with continuous collagen production of fibroblasts when a wound is developed.

Stem cells are cells that have the ability to differentiate into all kinds of cells that make up the body, such as nerves, blood, There are two ways to obtain such stem cells. First, stem cells from embryos derived from embryos (embryonic stem cells), and stem cells from adult parts of the body (adult stem cells ). Although there are functional differences, embryonic stem cells and adult stem cells all have the ability to differentiate into different types of cells. Although embryonic stem cells have excellent differentiation potential and long telomeres, they have ethical problems and it is difficult to acquire a large number of cells, whereas adult stem cells can obtain a large number of cells, There is a drawback that the risk of infection or the ability to differentiate is relatively low.

Adult stem cells are very safe for medical applications. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration. Since the immune rejection does not occur because it is in the adult body, autologous transplantation using its own cells is possible. In addition, there is a site-specific differentiation that differentiates itself according to the characteristics of the surrounding tissues. Since it does not cause cancer even if it is injected in undifferentiated state, besides producing cells necessary immediately after transplantation, It has the advantage of self-renewal ability to regenerate and store necessary undifferentiated stem cells. Therefore, the importance of adult stem cells has recently been highlighted, and various studies are under way to reveal its usefulness.

Korean Patent No. 10-0883565 discloses an injectable additive for tissue regeneration using adipogenic stem cells, and Korean Patent Publication No. 2008-0079256 discloses a composition for treating skin defects using mesenchymal stem cells.

The present inventors have found that stem cells derived from human adipose are cultured in a serum medium and then subcultured in a serum-free medium. Then, the resulting stem cell culture concentrate obtained by filtration is used as a skin regeneration or wrinkle The present invention has been completed.

In order to solve the above-mentioned problems, the present invention provides a method for regenerating skin or wrinkles containing stem cells obtained by culturing human adipose-derived stem cells in a serum medium and then subculturing in a serum-free medium, ≪ / RTI >

The present invention also provides a skin regeneration method, characterized in that the composition is applied topically to a wound site or injected intradermally.

The present invention also provides a method for improving wrinkles, characterized in that the composition is topically applied or injected intradermally to a site requiring removal of wrinkles.

The present invention also provides a skin regeneration or wrinkle remedy comprising the composition as an active ingredient.

The present invention also provides a skin regeneration or wrinkle-improving cosmetic comprising the composition as an active ingredient.

Since the culture concentrate of human adipose-derived stem cells of the present invention has an activity of inducing wound healing and generation of type 1 procollagen, it is expected that it can be effectively used for skin regeneration or wrinkle improvement.

Figure 1 shows wound healing effects of AAPE in artificially wounded hairless mice.
FIG. 2 shows the result of analyzing the wound healing effect of AAPE by measuring the area of the wound area remaining in the hairless mouse which was artificially wounded.
Fig. 3 shows the wound healing effect of AAPE in an artificially wounded hairless mouse through histological analysis.
4 shows the effect of AAPE and L-ascorbic acid on the production rate of type 1 procollagen in human dermal fibroblasts. **: Significance test of test group for negative control (Dunnett's t-test, p <0.01).

In order to accomplish the object of the present invention, the present invention provides a method for regenerating or regenerating stem cells comprising culturing human adipose-derived stem cells in a serum medium, subculturing the medium in a serum-free medium, A composition for improving wrinkles is provided.

The stem cell culture concentrate preferably contains

(a) culturing human adipose-derived stem cells in serum medium and then subculturing in serum-free medium;

(b) applying at least one physical stimulus selected from hypoxic culture, ultraviolet irradiation, low frequency treatment, nutrient deficiency and mechanical friction to the subcultured stem cells;

(c) adding to the culture medium one or more vitamins selected from the group consisting of vitamin A, vitamin B, vitamin C and vitamin D, and

(d) filtering the culture solution.

The steps (b) and (c) may be performed in combination with conditions under which the maximum production of human growth factors occurs. The preparation of a stem cell culture concentrate is described in Korean Patent Publication No. 2008-0109725, the entire contents of which are incorporated herein by reference.

In the composition of the present invention, the serum-free medium may preferably be a mixed medium of DMEM (Dulbecco's Modified Eagle's Medium) and Ham's F-12 nutrient mixture (refer to Korean Patent Publication No. 2008-0109725) The mixing ratio may preferably be 1: 1 for DMEM and Ham's F-12 nutrient mixture.

The composition of the present invention may exhibit skin regeneration or wrinkle-improving effects by increasing the production of type 1 procollagen in dermal fibroblasts.

The stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and they are separated from embryonic stem cells and adult tissues isolated from inner cell mass of blastocyst. (Adult stem cells) can be roughly classified into. Adult stem cells are undifferentiated cells having a mutipotency derived from a mammal including a human, preferably a human adult tissue. Examples of the adult stem cells include various cells such as bone marrow, blood, brain, skin, Can be derived from adult cells.

The human adipose-derived stem cells are a kind of adult stem cells isolated from human adipose tissue. The acquisition of adipose tissue can be obtained incidentally during the liposuction process, which is usually performed, and thus it is possible to easily obtain and cultivate a sufficient amount of stem cells, and also has superior safety compared to bone marrow harvesting.

The human adipose-derived stem cells can be cultured by a conventional method using a medium for stem cell culture, for example, a serum medium. Preferably, human adipose-derived stem cells are cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum, and finally cultured in a 1: 1 mixed medium of serum-free DMEM and Ham's F-12 nutrient mixture , The protein content in the obtained culture liquid can be increased. The serum-free medium stage minimizes differentiation by exposing stem cells isolated from adipose tissue to a specific extreme environment, and enables the recovery of the proteins secreted by the stem cells from the culture medium as much as possible.

The present invention also provides a skin regeneration method, characterized in that the composition is applied topically to a wound site or injected intradermally. The dosage form may be, but is not limited to, topical application or intradermal injection.

The present invention also provides a method for improving wrinkles, characterized in that the composition is topically applied or injected intradermally to a site requiring removal of wrinkles. The dosage form may be, but is not limited to, topical application or intradermal injection.

The present invention also provides a skin regeneration or wrinkle remedy comprising the composition as an active ingredient.

The skin regeneration or wrinkle remedy of the present invention can be applied not only to skin for treatment of wounds such as wounds and burns but also to areas where wrinkles are required to be removed.

The pharmaceutically acceptable carriers to be contained in the skin regeneration or wrinkle remedy of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, , Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , But is not limited thereto.

The skin regeneration or wrinkle remedy of the present invention may be formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention pertains, Or may be prepared by penetration into a multi-dose container, and may additionally include a dispersant or stabilizer.

The present invention also provides a skin regeneration or wrinkle-improving cosmetic comprising the composition as an active ingredient.

The components contained in the skin regeneration or wrinkle reduction cosmetic of the present invention may contain, as an active ingredient, components commonly used in cosmetic compositions in addition to the stem cell culture concentrate, and examples thereof include antioxidants, stabilizers, solubilizers, vitamins, Pigments and fragrances. &Lt; RTI ID = 0.0 &gt; [0040] &lt; / RTI &gt;

The skin regenerating or wrinkle-improving cosmetic product of the present invention can be prepared in any formulations conventionally produced in the art, for example, as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, Spray, or the like, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

In the case where the formulation of the present invention is a suspension, a carrier such as 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, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is cleansed, the carrier component may be selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl Aliphatic alcohols, fatty acid glycerides, fatty acid diethanol amides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters, and the like can be used

In addition, the skin regeneration or wrinkle-improving cosmetic composition of the present invention may further contain ingredients for conventional skin regeneration or wrinkle improvement besides the above-mentioned stem cell culture concentrate, and the kinds and contents of these conventional components can be easily selected by those skilled in the art Can be used.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: Obtaining of adipose stem cells

We obtained consent from the patient who underwent subcutaneous fat removal and removed the abandoned fat tissue from the patient through liposuction method. The extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes in a 5% CO ₂ incubator at 37 ° C., and the obtained adipose tissue was centrifuged at about 1200 × g for 5 minutes to form a stroma. Sex blood vessel fraction was obtained. The obtained fractions were washed with PBS (phosphate buffered saline), and the other tissues were removed through a 70 ㎛ nylon cell filter. Cell debris including mononuclear cells were separated by Histopaque-1077 (Sigma, St. Louis, MO, USA) .

Separated mononuclear cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Lonza, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin-streptomycin And cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Then, nonadherent cells were removed to isolate adipose stem cells.

Example  2: Culture of stem cells

1.25 × 10 ⁵ cells of the adipose stem cells obtained in Example 1 were cultured in a medium containing 1000 mg / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate, 10% fetal bovine serum, Was added to DMEM medium containing 1% penicillin-streptomycin and subcultured in a 5% CO ₂ incubator for 30 days at a temperature of about &lt; RTI ID = 0.0 &gt; 90% &lt; / RTI & The culture was removed from the subcultured cultures using a pipette and then washed three times with PBS and incubated with 365 mg / L L-glutamine, 15 mM HEPES, 55 mg / L sodium pyruvate (1.2 × 10 ⁶ cells / dish) in a 1: 1 mixture of DMEM and Ham's F-12 nutrient mixture (DMEM / F-12) for 72 hours under hypoxia conditions.

Example  3: culture concentrate of stem cell and its Fraction  Produce

500 mL of the culture solution finally obtained in Example 2 was centrifuged at 300 x g for 5 minutes to remove the stem cells that had been sunk into the pellet. The resulting supernatant was filtered with a 0.22 mu m syringe filter to remove residual stem cells and unknown macromolecules. The resulting solution was divided in half and concentrated under reduced pressure to obtain 5 mL of a concentrate (hereinafter referred to as AAPE). The resulting concentrate was lyophilized and stored at -70 ° C.

The remaining stem cells and the remaining macromolecules were passed through a membrane having a pore size of 30 kDa. The filtrate, which had not passed through the membrane, was washed three times with PBS to obtain a macromolecule of 30 kDa or more The fractions containing the molecular fractions and the fractions of 30 kDa or less were separated and then the solution containing 30 kDa or less of PBS and washed PBS was passed through a membrane having a pore size of 10 kDa to obtain a concentrated fraction of 10 to 30 kDa and a fraction of 10 kDa or less &Lt; / RTI &gt; The separated 10 to 30 kDa fractions and the 30 kDa or higher fractions were stored at -70 ° C.

Example  4: wound-induced Hairless ( hairless ) From the mouse AAPE Wound healing wound healing ) And skin regeneration regeneration ) effect

Female hairless mice were used to test wound healing effects of AAPE and were performed according to the National Institutes of Health (NIH) Animal Experimental Guidelines. Two circular wounds with a diameter of 8 mm were artificially induced in the dorsal skin of the mice. Then, only the medium (DMEM / F-12, control group) was applied to the left side and the AAPE stock solution collected in Example 3 was applied to the right side. At the same time, the fractions obtained in Example 3 (30 kDa or more and 10 to 30 kDa) were respectively applied to wound areas of mice. The wound healing effect was analyzed by measuring the area of the remaining wound area. In the observation of the external restoration pattern of the dermatitis-induced wound, the control group treated with embryos showed significantly less restoration at the initial observation period than the control group treated with the AAPE stock solution or fraction (FIGS. 1 and 2) . Histologic examination showed that the control group had a large infiltration of inflammatory cells and a flattening of the basal layer of the epidermis. No ridge ridge formation is observed and treatment is incomplete (Figure 3). In the case of AAPE stock treatment, the skin regeneration effect was relatively better during the observation period than the control group. Particularly, as the skin regeneration progressed after the first 3 days, the AAPE stock treated group showed a better effect than the other experimental groups (FIGS. 1 and 2). In addition, histological findings showed that the number of inflammatory cells was significantly reduced compared to the control group, and that the skin regeneration effect was good when the skin layer was relatively thick and the formation of the ridge ridge appeared (Fig. 3).

In the case of the fraction treatment of 30 kDa or more, the skin regeneration effect was relatively superior to the control group during the observation period (FIGS. 1 and 2). Histopathologic findings showed almost no inflammation of epidermal / dermal border, high density of epidermal layer, and complete normalization of network ridge to the dermal layer (Fig. 3). In the case of 10 to 30 kDa fraction treatment, the best skin regeneration effect was obtained in the first 3 days after application (FIGS. 1 and 2). Histopathologic findings showed similar healing effects to those treated with fractions over 30 kDa, but inflammation infiltration was somewhat more frequent than those treated with fractions over 30 kDa (Fig. 3). These results show that AAPE, which is a culture product of adipose stem cells, shows differentially regenerating effect according to the molecular composition of each fraction and has a therapeutic activity of excellent skin regeneration. Furthermore, AAPE whole fractions constituting all of them are differentiated through the first half and the second half Lt; RTI ID = 0.0 &gt; therapeutic activity. &Lt; / RTI &gt; In addition, the AAPE stock solution showed a sufficient regeneration effect as compared with the concentrated fraction. Therefore, it is expected that the whole fraction of AAPE cultured in the adipose stem cell can be usefully used for the symptom improvement and treatment of damaged skin.

Example  5: human dermal fibroblast ( normal human dermal fibroblasts ) To evaluate the promoting effect of type 1 collagen production

2 mL of DMEM mixed with 1% penicillin-streptomycin, an excipient, was added to 20 μg of lyophilized AAPE, followed by vortexing and dissolution. When 0.1 mL of the prepared AAPE solution (10 μg / mL) is added to the total volume of 6 mL by adding vehicle, the stock solution having a final concentration of 0.32 μg / mL is prepared. The stock solution was diluted stepwise with azeotrope 4 to give a total of three concentrations (0.32, 0.08 and 0.02 ㎍ / mL). As a positive control, L-ascorbic acid (Sigma, USA) was dissolved in the excipient to prepare a concentration of 35.22 μg / mL (200 μM), and the excipient was administered as a negative control.

The cell line used in the present invention is normal human dermal fibroblasts (7F3802, Clonetics, USA), and the cell line is cultured in DMEM / DMEM containing complete medium (10% fetal bovine serum and 1% penicillin-streptomycin, F-12), and then used for the experiment in the 5th passage. The human dermal fibroblast cultured in the complete medium was dispensed in a volume of 400 μl into a 48-well plate (1 × 10 ⁵ cells / 400 μl / well), and cultured for 24 hours at 37 ° C. under 5% CO ₂ Lt; / RTI &gt; After completion of the incubation, the culture solution of each well was removed, and 500 μl of D-PBS (Dulbecco's phosphate buffered saline) was added to each well and washed. 800 쨉 l of each preparation of the AAPE and the positive control substance was added to each well, and 800 쨉 l of the excipient was added to the well of the negative control, followed by culturing for 48 hours at 37 째 C under 5% CO ₂ Respectively. After completion of the incubation, the culture broth of each well was recovered and centrifuged at 25 ° C and 3000 rpm for 10 minutes, and then the supernatant was used for quantification of type 1 procollagen type I. One mL of D-PBS was added to each well of the plate from which the culture solution had been removed, washed, and 300 μL of cell lysis buffer was added to each well. The plate was stored in a deep freezer (deep freezer, -70 ° C) for 2 hours, frozen and thawed at room temperature twice to hemolyze the cells. Each cell lysate was recovered and centrifuged at 13,000 rpm for 30 minutes at 4 ° C. The supernatant was taken and used for quantification of total protein.

For quantification of the total protein, the obtained supernatant was added to each well of a 96-well plate in an amount of 40 占 퐇. The standards provided in the BCA Protein Quantitation Kit (Pierce Biotechnology Inc., USA) were diluted stepwise to 250, 125, 50, 25, 5 and 0 μg / mL, and 40 μL each was added to another well. Subsequently, 160 μL of the reagent mixture solution provided in the protein quantitation kit was added to each well containing 40 μL of the supernatant and standard solution, and the mixture was reacted in a 60 ° C. incubator for 30 minutes. After completion of the reaction, the absorbance of each well was measured at 562 nm using an ELISA reader (PowerWave XS, BioTek, Instruments, Ins., USA). The absorbance of each well was substituted into the equation of the standard curve to calculate the total protein amount of the well to which the AAPE, the positive control and the negative control were added.

For quantification of type 1 procollagen, the obtained supernatant was added to each well of a 96 well plate provided in a type 1 procollagen C-peptide EIA kit (Takara Bio Inc., Japan) in an amount of 100 μl. The standard solution provided in the above kit was diluted stepwise to prepare 640, 320, 160, 80, 40, 20, 10 and 0 ng / mL, and then 100 μL of each was added to another well. Then, the reaction was carried out in a 37 ° C incubator for 2 hours. After completion of the reaction, the reaction solution in each well was removed, and 400 μl of PBS was added to each well, followed by washing (repeated 4 times). 100 μl of the TMBZ (tetramethylbenzidine) substrate solution provided in the kit was added to each well, followed by reaction at 30 ° C for 15 minutes. 100 μl of stop solution (1N H ₂ SO ₄ ) was added to each well to complete the reaction, and the absorbance of each well was measured at 450 nm using an ELISA reader. The absorbance was substituted into the equation of the standard curve to calculate the amount of type 1 procollagen in wells to which AAPE, positive control and negative control were added. The amount of corrected type 1 procollagen was substituted into the following equation to calculate the synthesis rate of procollagen type I.

Production rate (%) of type 1 procollagen = A / B x 100

A: The amount of test substance or positive control type 1 type of procollagen.

B: Negative control type 1 type of procollagen.

The production rate of type 1 procollagen obtained in the present invention was determined using a statistical processing program SAS (version 9.1.3, SAS Institute Inc., Cary, NC, USA). (Significance: 5% and 1%, respectively) were performed with Dunnett's t-test to confirm the significance of the test group to the control group. .

As a result, there was no statistically significant difference (0.02 ㎍ / mL; 108.0 ± 0.6%) compared with the negative control group (100.0 ± 0.0%) in the group treated with AAPE at 0.02 ㎍ / mL, (0.08 ㎍ / mL; 114.8 ± 1.2%, 0.32 ㎍ / mL; 142.7 ± 5.5%) in the group treated with 0.08 and 0.32 ㎍ / mL of AAPE. The positive rate of type 1 procollagen production in the positive control group at 35.2 / / mL (200 M M) was significantly increased (35.2 ㎍ / mL; 161.0 ± 9.8%) compared to the negative control group (FIG.

The present invention was conducted to evaluate the promoting effect of AAPE, a test substance, on the generation of type I collagen using normal human dermal fibroblasts. Treatment of AAPE at 0.02, 0.08 and 0.32 ㎍ / mL with human dermal fibroblasts showed a dose - dependent increase in the production of type 1 procollagen. The AAPE administration group at 0.08 and 0.32 ㎍ / mL showed negative Compared with the control group. Therefore, it is considered that the test substance, AAPE, has an effect of promoting the generation of type I collagen.

Claims (8)

delete Culturing human adipose-derived stem cells in a serum medium, and subculturing the medium in serum-free medium;
Applying at least one physical stimulus selected from hypoxic culture, ultraviolet irradiation, low-frequency treatment, nutrient deficiency, and mechanical friction to the subcultured stem cells;
Adding and culturing one or more vitamins selected from vitamin A, vitamin B, vitamin C and vitamin D to the serum medium; And
A composition for treating an early wound within 3 days after a wound containing, as an active ingredient, a stem cell culture concentrate which is a filtrate obtained by filtering the culture solution with a membrane having a pore size of 30 kDa. delete delete 3. The method of claim 2,
Wherein the effective component of the filtrate has a size of 10 to 30 KDa within 3 days after the occurrence of the wound. delete A method for treating an early wound within 3 days after a wound occurs, wherein the composition of claim 2 is locally applied or injected intradermally into a wound site of an animal other than a human. delete

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