KR20130052441A - Composition and kit comprising recombinant human bone morphogenetic protein and twist transcription factor for skin repair as active ingredient - Google Patents

Abstract

PURPOSE: A composition and a kit containing recombinant human morphogenetic protein and twist transcription factor as active ingredients are provided to regenerate skin tissues alternately using recombinant human morphogenetic protein and twist transcription factor. CONSTITUTION: A composition for regenerating skin tissues contains recombinant human morphogenetic protein and twist transcription factor type 2 as an active ingredient. The recombinant human morphogenetic protein is selected from a group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, and a mixture thereof. The composition is used for a pharmaceutical product or a cosmetic composition for skin tissue regeneration. A kit for skin tissue regeneration contains the skin tissue regeneration protein and twist transcription factor type 2.

Description

Composition and kit comprising recombinant human Bone Morphogenetic Protein and twist transcription factor for skin repair as active ingredient}

The present invention relates to a composition and kit for regenerating defective skin tissue using a combination of recombinant human bone morphogenetic protein and twisted transcription factors.

Recombinant human osteoblastic protein type 2 (BMP-2) has long been used for the purpose of regenerating missing bone tissue. BMP-2 is a representative bone tissue growth factor. Therefore, its mechanisms of intracellular action, bone formation mechanisms and clinical applications have been discovered by many scholars.

Growth factors are proteins that are expressed and synthesized in stem cell genes during the growth of body tissues, such as embryonic, neonatal and puberty. This produces signals that allow the surrounding stem cells to proliferate and differentiate into tissues or organs.

In particular, human bone forming protein type 2 is a growth factor that acts on proliferation of osteoblasts as well as mesenchymal stem cells among 20 kinds of human bone forming proteins.

Human bone morphogenetic proteins attach to bone morphogenetic protein receptor type 1 and type 2 receptors on the cell membranes of stem cells and activate phosphatase in the cell to induce intracellular signaling pathways, SMAD, and transfer them to the nucleus of cells. . Therefore, the Twist-2 transcription factor gene, bone morphogenetic protein gene, and vascular growth factor (VEGF) are expressed in genes, thereby synthesizing each protein.

Mesenchymal stem cells expressing a large number of genes due to bone formation factors synthesize bone-forming proteins by themselves and release them out of the cell, while also stimulating other stem cells around them. In addition, Twist-2 transcript factor expressed and synthesized from the twist transcript factor gene acts as a cancer by preventing the formation of bone morphogenic proteins into bone cells only through the mutual regulation of cells in the surrounding heterologous tissue layer and cells normally distributed by location. Do not do it. At the same time, vascular epithelial growth factor (osteoblast-derived vascular epithelial growth factor, Osteoblast drived VEGF-alpha) is released to allow the growth of blood vessels to supply the nutrition and oxygen necessary for tissue regeneration.

In addition, the osteogenic proteins attached to the isolated osteogenic protein receptor type 1 and type 2 causes the stem cells to divide and proliferate. Cell membranes modified by the binding of osteogenic proteins and receptors are transformed into gastric limbs and migrate to the defective tissues. At this time, the stem cells sensitized to the osteoblastic protein can be used to proliferate epidermal keratinocytes, which are growth cells of epidermal cells of the skin, to proliferate fibroblasts of the dermal layer, to proliferate fibroblasts of ligaments, to proliferate osteoblasts of bone tissue, or to cartilage. It is known that it is possible to proliferate chondrocytes of cells or neurofibrillary cells of neural tissues.

BMP-2 also causes the surrounding mesenchymal stem cells, which are necessary for healing, to migrate to the defect site chemotactically and differentiate into various tissues. BMP-2 grows mesenchymal stem cells into bone tissue in patent WO 1993/000050, and BMP-2 grows mesenchymal stem cells into bone tissue in patent WO 1993/000050. In order to induce the growth of neural tissues, patent WO 1996/039169 describes BMP-2 for tissue regeneration with various carriers or scaffolds for inducing tendon or ligament regeneration.

In vertebrate embryos, the ectoderm, which is destined to become the epithelium of the skin and the hair, feathers, or scales that are the appendages of the skin, is directly affected by mesenchymal stem cells. Twist-2, a transcription factor protein, is expressed in the Twist-2 gene of mesenchymal stem cells, and this protein causes interaction between epithelial stem cells and mesenchymal stem cells to normally generate skin and skin appendages composed of the dermis and epithelium. .

When adult skin is not damaged due to large skin damage or defects, Fibroblast Growth Factor (FGF) and FGF2 and FGF7 are known to be effective in dermal tissue regeneration. However, these growth factors are not known to express the Twist-2 transcription factor, which makes it difficult to induce a balanced epithelial regeneration of the epithelial cells during intimate interaction between mesenchymal stem cells and fibroblasts.

On the other hand, BMP-2, although its mechanism of action is unknown, induces differentiation and proliferation of mesenchymal stem cells and fibroblasts. This Twist-2 transcription factor appears to be in the early stages of differentiation of mesenchymal stem cells in the dermis, interacting with epithelial stem cells and generating skin (Scaal et al .; Development growth differentiation, 2008 Volume: 50, Issue: 9, Pages: 743-754). In this paper, a study related to the mechanism of regeneration of neoplastic tissues in Derongongong, known to regenerate the cut tail naturally. There has been no report on the effects of bone growth protein growth factor and twist transcript factor on damaged skin tissue regeneration in animals that are not naturally regenerated.

Clinicians have long desired to restore their original skin tissue in case of damaged epidermal tissue, dermal tissue, congenital facial deformities, burns, diabetic skin ulcers, cancer, inflammation, or physicochemical causes. The present invention is expected to make great progress in this field, and also in the regeneration of skin tissues such as skin aging prevention, wrinkle improvement, and skin care, it is also a future problem to improve the regeneration completeness and integrity of the skin.

In the skin tissue regeneration test of Jo et al. (Biomaterial, 2010), animal experiments showed that the BMP-only group differentiated and proliferated into bone tissue within 2 weeks. In order to prevent the growth of heterogeneous bone tissue in the skin, it is intended to promote fibroblast growth of normal dermis by using a twist transcription factor.

The amino acid sequence of the twist 2 type is as follows.

In the present invention, the skin regeneration ability of bone forming protein type 2 and twisted transcription factor type 2 in skin defects including epithelial cells and dermal tissue in a circular shape of 15 mm diameter, which is a critical defect size that does not naturally heal in rats, etc. As a result, no differentiated growth of bone tissue was found and complete regeneration of the dermis and epidermal layer was observed in the test group compared to the control group.

Skin tissue consists of epidermal keratinocytes, mercer cells, melanocytes, langerhans cells, keratinocytes, and dermal or subcutaneous tissues are fibroblasts, vascular endocrine cells, vascular smooth muscle cells, sebaceous gland cells, fat cells, and glandular cells. It consists of follicular cells, hair root cells, and mesenchymal cells. Gingival tissue has the same type of cells and composition as multi-layered epidermal cells of skin tissue. However, because glands, sebaceous glands and hair are not needed in the oral cavity, unlike dermal tissue, gingival cells, sebaceous gland cells, and glandular cells, follicular cells and hair root cells are not found in the gingival tissue.

To evaluate the effects of human bone morphogenetic proteins (BMPs) and twisted transcription factors on skin tissue regeneration, we used gingival regeneration in gingival defects due to extraction. In adult extraction and gingival tissue, there is a limit to natural healing, so in large defects such as molars, the gingival tissue of other parts must be removed and transplanted. This has the side effect of making defects in other normal areas. We evaluated the effect of gingival tissue regeneration in molars with limited natural healing using human bone morphogenetic proteins and twisted transcription factors. These combinations have a direct effect on skin regeneration.

 In the present invention, human bone morphogenetic protein directly regenerates dermal tissue by directly proliferating fibroblasts and epithelial cells of the dermis, while Twist-2 transcription factor has integrity with surrounding skin tissues through interaction between epithelial cells and dermal cells. The present invention has been completed by discovering that the growth of fibroblasts of epidermis and epidermal keratinocytes of epithelium is not differentiated into other heterologous bone cells.

Recombinanat human Bone Morphogenetic Protein using the differentiation and proliferation of human osteoblastic proteins into various cells and the ability to differentiate and proliferate into normal cells according to the coordination of twisted transcription factors between cells of various tissues. : rhBMP) and a combination of a human twisted transcription factor in combination with a composition for regenerating skin tissues that have been deleted.

As growth factors that can be used in the present invention, recombinant human Bone Morphogenetic Protein (hereinafter referred to as "BMP") has been developed and utilized to date, and these BMPs have almost equivalent bone formation functions. Such BMPs include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, and BMP-13. All have been developed and can be used in the present invention TGF-β supergroup, which is a co-duplex of amino acids constituting BMP (a conjugate made by twisting amino acids of B2 and amino acids of B4), fibroblast differentiation factor or protein such as GDF , Growth factor selected from one or two of platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and transformative growth factor, keratinocyte growth factor 2 (KGF2) and MP52 protein and the recombinant protein of the growth factor. Can also be used in the present invention have.

In the present invention, the cell proliferation effect was confirmed by MTT assay test with gastric recombinant human bone morphogenic protein, the wound healing effect was confirmed in the test of making a full-thickness wound on the back of the rat, and the regeneration of the gingival tissues histologically identical to the skin In the evaluation of the effect on the regeneration of gingival tissue by recombinant human bone morphogenetic protein and twisted transcription factor, it was found that their skin tissue regeneration ability is extremely excellent.

Accordingly, the present invention provides a composition for use in skin tissue regeneration that contains recombinant human bone morphogenetic proteins (BMPs) and twisted transcription factors as essential ingredients.

Still another object of the present invention is to provide a kit for regenerating skin tissue containing recombinant human bone morphogenetic proteins (BMPs) and twisted transcription factors as essential components.

Next, Examples and Experimental Examples relating to the use for skin regeneration are illustrated.

Example 1

This example illustrates a method for producing BMP-2 (rhBMP-2) by a recombinant DNA technique, which is a known method. Other growth factors used in the present invention are all known, and these known growth factors are used in the present invention.

1. Production of rhBMP-2:

1) Cloning of Human BMP-2 gene:

To obtain human BMP-2 gene, total cellular RNA was extracted from U2OS cells using Trizol (Gibco BRL, NY, USA) solution and reverse transcription reaction was performed. Polymerase chain reaction (PCR) was performed using 5'-AATTTACAGCTTCTAGCGACACCCACAACCCT-3 '. PCR products were isolated and inserted into pGEM-T vector (Promega, USA) and cloned using E. coli (DH5α) cells.

2) High-density cell culture:

The fermentation tank (KoBioTec, Incheon, Korea) was used for the cultivation at a high density using the method of Fatemeh et al. Liposomes were prepared by adding sterilized nutrient media (Glucose 33.3 g / L, peptone 10 g / L, yeastextract 5 g / L, MgSO4 1 g / L, CaCl2 0.048 g / L, ZnSO40.0176 g / L, CuSO4 0.008 g / Lt; / RTI > for 24 hours.

3) Protein purification:

The suspension was stored in -80 ° C deep freezer (Nihon Freezer, Japan). After thawing the frozen suspension at refrigeration temperature, the cells were crushed by pressing and centrifuged for 45 minutes at 5,500xg, 4 ℃. When the mature rhBMP-2, which has undergone regeneration, has an intrinsic tertiary structure, the N-terminus has a heparin-binding site.

2. Biochemical properties of purified rhBMP-2:

1) Purity and identification test of rhBMP-2 stock solution: SDS-PAGE:

As a result, the rhBMP-2 stock solution showed the same moving distance as the standard solution, and showed a purity of 95% or more (FIG. 1A).

2) HPLC (High performance Liquid Chromatography) analysis:

The purified rhBMP-2 dimer was dissolved in 0.1% trifluoroacetic acid (TFA) at a concentration of 1 ug / ul to detect proteins in each fraction of C4 reversed-phase HPLC column (4.6 mm x 50 mm, 300 µm, 5um particle size; Gracevydac, CA, USA). And monitored. The test results of the rhBMP-2 stock solution showed the same retention time as that of the standard solution, and the rhBMP-2 stock solution should show the same retention time as the standard solution.

3. Production and purification of rhBMP-2 protein:

And then subjected to affinity chromatography on a heparin column. As a result, as shown in FIG. 1A, most monomers and a small amount of dimer were eluted from the 0.3M NaCl fraction and the dimers eluted from the 0.5M NaCl fraction. Purified rhBMP-2 monomers and monomers were identified by DS-PAGE analysis. The molecular weight of the monomer was about 14 kDa, and the dimer had the same size band as the monomer in the non - reducing condition because the two monomers were connected by the disulfide bond. The size of the dimer was about 28 kDa.

4. In vitro test of purified rhBMP-2:

As a result of rhBMP-2 application to myoblasts, alkaline phosphatase, a representative protein of osteoblasts, was secreted after 3 days of culturing (Fig. 1c), and the cell morphology was transformed into osteoblasts under microscope (Fig. 1d). Therefore, rhBMP-2 confirmed its function as an osteogenic protein.

Next, the present invention will be described in more detail as a test example.

Test Example 1

In vitro test of purified rhBMP-2:

As a result of rhBMP-2 application to myoblasts, alkaline phosphatase, a representative protein of osteoblasts, was secreted after 3 days of culturing (Fig. 1c), and the cell morphology was transformed into osteoblasts under microscope (Fig. 1d). Therefore, rhBMP-2 was confirmed to function as a osteogenic protein.

Test Example 2

Effect of rhBMP-2 on Fibroblasts:

As in Test Example 1, MTT assay was first performed to examine the cell proliferation effect of rhBMP-2, which was confirmed as bioaccumulative activity. Briefly, the method divides 2 x 10 ³ fibroblast NIH3T3 cells into 96 wells and 0.1, 0.25, 0.5, 1.0, 10, 25, 50, 100 ng / ml of the purified rhBMP-2 as described above. Treat for 48 hours at concentration. Thereafter, semi-removing medium and MTT solution was put into one-tenth volume of culture medium 37 ℃ for 90 min, CO ₂ Incubator was incubated. Remove the MTT solution, add 200 μl of DMSO, mix well, and incubate in 37 ° C and CO ₂ incubator for about 5 minutes. Then, the value was measured at 570 nm of ELISA.

The growth rate of cells can be indirectly confirmed by increasing the mitochondria's effect on MTT without directly counting the number of cells. NIH3T3 cells were dispensed into 96 wells at 2 x 10 ⁸ cells and the proliferation effect of rhBMP-2 cells was examined at various concentrations. rhBMP-2 concentration was treated at 0, 0.1, 0.25, 0.5, 1.0, 10, 25, 50, 100 ng / ml. To determine the effect of rhBMP-2 on cell proliferation, serum starvation was performed after incubation in 0.1% FBS DMEM. MTT assay was performed 48 hours after treatment for each rhBMP-2 concentration (FIG. 2A). The rhBMP-2 concentration showed little cell proliferation effect at 0.1 ng / ml and showed the highest activity at 10 ng / ml. At 25, 50 and 100 ng / ml or more, the cell proliferation effect did not increase significantly at 10 ng / ml. Therefore, rhBMP-2 showed a proliferative effect of fibroblasts in a concentration-dependent manner, and the effect of skin regeneration was confirmed by cytological findings. Therefore, it was found that rhBMP-2 directly induces the proliferation of fibroblasts, which mainly constitute the dermal layer of skin tissue.

Test Example 3

Effect of rhBMP-2 on keratinocytes of epithelium:

Wound-healing assay was performed to analyze the effect of rhBMP-2 on skin regeneration using human keratinocyte cells.

By dividing the cell by a 12 well plate for each well ⁵ x 10 5 cell (500,000 pieces) were cultured for 48 hours in serum-free RPMI1640 medium.

To artificially create a wound, a plastic tip was used to draw a line across the cell monolayer to make the wound the same size in each well at the same location. In addition, the culture medium was exchanged with RPMI1640 medium containing 0.5 FBS, and then treated with rhBMP-2 according to concentrations of 0, 10, 50, and 100 ng / ml.

Cell migration to the wound surface was observed over time with a microscope.

As shown in FIG. 2B, the wound surface at time 0 was incubated for 12 hours and 48 hours, and was regenerated into neoplastic cells depending on the concentration dependency of rhBMP-2. Thus, it was demonstrated that rhBMP-2 acts directly on epithelial cells and has a proliferative effect.

Test Example 4

Effects of Recombinant Human Bone Protein 2 and Twist Transcription Factor 2 on the Skin Wound Healing in Rats:

As shown in FIG. 3a, a full-thickness wound including epithelial cells and dermal tissue was formed in a circular diameter of 15 mm, which is the size of a critical defect that does not naturally heal in the rat's back. The neglected control group, the sham control group covered with the collagen sponge, the test group to which 12.5 μg BMP-2 solution and the same amount of twist transfer factor type 2 were applied to the collagen sponge, and 25 μg BMP-2 solution and the same amount of the twist transfer factor to the collagen sponge The microscopic findings (Figs. 3b, 3c, and 3d) and histoscopic microscopic findings on the 18th day at the regenerated site divided into the test group to which Type 2 was applied were magnified X40 (Fig. 3e) and X100. (FIG. 3f) observed at magnification.

Visually, control or BMP-2 and twisted transcript factor 2 were found to have a regeneration effect in the 12.5 μg treatment group, but histologic examination showed that the BMP-2 and twisted transcript factor was only in the 25 μg treatment group. .

In the photo of the control group, the incomplete production of Dermis and Epidermis can be seen, and in the Sham control, the regeneration of Epidermis is almost absent. Able to know.

On the other hand, in the group treated with BMP-2 on the collagen sheet at the same time, the regeneration of Dermis and Epidermis can be confirmed.When BMP-2 was treated in two groups in a dose-dependent manner, the BMP-2 and twisted transcription factors were 12.5 μg. Epidermis regeneration is not complete in the group treated with, but in the group treated with 25 μg of BMP-2 and twisted transcription factor, it can be seen that the regeneration of Epidermis is near the normal state.

Test Example 5

Effect of recombinant human bone morphogenetic protein type 2 and twisted transcription factor on gingival regeneration of the ankle in clinical trials:

The purpose of this study was to evaluate the effect of recombinant human bone morphogenetic protein type 2 and twisted transcription factor on the regeneration of gingival tissues histologically identical to skin. In adult extraction and gingival tissue, natural healing is limited, so in large defects such as molars, gingival in other areas should be removed and implanted. This has the side effect of making defects in other normal areas. We evaluated the effect of gingival tissue regeneration in molars with limited natural healing using human bone morphogenetic protein type II.

In adults, implantation of PTFE shielding membrane, rhBMP-2 (concentration 0.75mg / ml rhBMP-2), and twist transfer factor (concentration 0.75mg / ml Twist2) solution was implanted. This is shown in FIG. 4. In the visual comparison, rhBMP-2 and twisted type 2 transcription factors and PTFE shielding membranes were applied to prevent invasion of external bacteria to prevent infection and to grow to the site where the surrounding fibroblasts and epithelial cells were deleted (FIG. 4). After 3 weeks, the screen was removed and observed. New gingival tissue was regenerated, and after 8 weeks, it was matured into hard gingiva.

According to the present invention, it was found that the skin tissue regeneration effect is excellent when the recombinant human bone morphogenetic protein and the twisted transcription factor are used in combination, and the epithelial tissue or congenital facial malformation, burn, diabetes, which are deleted using the present invention. Skin damage caused by sexual skin ulcers, cancer or inflammation, or physicochemical causes. In the regeneration of skin tissues such as skin aging prevention, wrinkle improvement, and skin care, the regeneration completeness and integrity of the skin can be improved.

Figure 1a is a SDS-PAGE analysis after dimerization and purification of rhBMP-2, where U is non-reducing rhBMP-2, R is reduced rhBMP-2, Dimer is a dimer fraction, Monomer is a monomer fraction,
1B is a graph showing that pure rhBMP-2 was purified in HPLC profile,
Figure 1c is a result of the enzyme test showing osteoblast activity by the injection of purified rhBMP-2 dimer into C2C12 cells, myoblasts, alkaline phosphatase in a concentration-dependent 3 days culture,
Figure 1d is a cell experiment showing the change in osteoblasts after the injection of rhBMP-2 into myoblasts C2C12 cells,
1E is a lyophilized electron micrograph (500-fold) containing rhBMP-2 in a calcium phosphate scaffold,
1f is a lyophilized electron micrograph (3000-fold) containing rhBMP-2 in a calcium phosphate scaffold,
Figure 2a is a growth stimulus distribution in proportion to the BMP-2 dose in fibroblasts,
Figure 2b is a growth healing analysis proportional to the rhBMP-2 dose in human keratinocytes,
Figure 3a shows a full-thickness wound containing epithelial cells and dermal tissue in a 15 mm diameter circle, the size of a critical defect that does not heal naturally in the rat's back. Neglected control group, sham control group covered with collagen sponge, test group applied 12.5μg BMP-2 and 12.5μg Twist-2 solution to collagen sponge and test applied 25μg BMP-2 and 25μg Twist-2 solution to collagen sponge Test methods classified into groups,
FIG. 3b shows gross findings at day 9 of skin defects in test and control groups,
Figure 3c shows the visual observation of healing at 13 days in the skin defects of the test group and the control group,
3D shows gross findings at day 18 of skin defects in the test and control groups,
FIG. 3E shows the histologic microscopic findings (50 magnification) at 18 days of skin defects in the test and control groups.
3F shows the histologic microscopic findings (100-fold magnification) at day 18 in the skin defects of the test and control groups.
4 is a visual examination of the oral cavity after 3 and 8 weeks of healing by applying skin tissue regeneration protein to the gingival tissue of the gingiva.
5 is a collagen sponge scaffold of 3 mm thickness,
6A shows a lyophilized gel-like scaffold filled in a syringe,
6B shows a scaffold gelled with water for injection in a syringe,
7a is a collagen membrane,
7b is a PTFE sheet,
8 is a double shielding membrane composed of a PTFE sheet and a collagen shielding membrane having affinity for skin tissue regeneration protein or stem cells,
9 is a shielding film reinforced with a synthetic polymer mesh to maintain shape,
10 is a manufacturing procedure of the skin regeneration shape maintenance shielding film,
11 shows the wound area at day 12 in skin wound regeneration using rhBMP-2, twisted transcription factor type 2 and hMSC,
12 is a hydrogel for wound treatment using skin tissue regeneration protein.

As confirmed in the above experimental examples, recombinant human bone morphogenetic proteins and twisted transcription factor type 2 (these combinations are referred to as skin tissue regenerative proteins, rhBMP) have excellent skin tissue regeneration effects. It is confirmed.

Therefore, the skin tissue regeneration protein identified by the present invention can be used in the clinic to prepare a composition or kit for skin tissue regeneration.

Skin tissue regeneration protein used in the present invention may vary depending on the degree of symptoms or skin damage, but can generally be used at a concentration of 0.01mg / ml ~ 10mg / ml. Twist transcription factor used in the present invention may vary depending on the degree of symptoms or skin damage, but can generally be used at a concentration of 0.01mg / ml ~ 10mg / ml.

In the present invention, in addition to the skin tissue regeneration protein, TGF-β super group, fibroblast growth factor, epithelial cell growth factor, platelet-derived growth factor, insulin-like growth factor, transforming growth factor, keratinocyte growth factor 2 (KGF2) and The growth factor may be further included in MP52 to further increase the effect. These proteins are known to be effective in skin tissue regeneration, and may be used together with the skin tissue regeneration proteins (rhBMPs) of the present invention.

The skin defect tissue may be accompanied by pain, inflammation, or complex defects in the skin tissue. In the present invention, other growth factors or extracellular matrix components, anti-inflammatory analgesics, antibacterial agents, and adrenal cortex hormones, in addition to skin tissue regeneration proteins, may be caused. It may also contain one or more drugs selected from.

Examples of such drugs include phenylpropionic acid derivatives including ketoprofen, flubiprofen, fenoprofen, and ibuprofen; NSAIDs of oxycam derivatives including pyroxicam, tenoxicam, and meloxicam; Diclofenac; And anti-inflammatory drugs selected from indomethacin (indomethacin), metronidazole, Amoxicillin, Amoxicillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, Other cephalosporin, Lincosamide (Clindamycincycline) Antibacterial agents selected from hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride, and corticosteroid drugs selected from methylprednisolone, hydrocortisone acetate.

In the present invention can also provide a composition for improving skin conditions comprising a skin tissue regeneration protein and twisted transcription factor as an active ingredient.

The composition for skin regeneration of the present invention includes an effective amount of skin tissue regenerating protein, an effective amount of twist transcription factor, and a pharmaceutically acceptable carrier or excipient. Carriers and excipients which may be used in the present invention include dextrose lactose, sucrose, sorbitol, starch, acacia rubber, calcium phosphate, sodium alginate, gelatin, sodium CMC, starch, calcium phosphate, polyvinylpyrrolidone (PVP), water , Alcohol, glycerin, vegetable oil and the like. In addition to these components, the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like. Such ingredients are ingredients commonly used in the medical field, cosmetics, and the like, and may further contain ingredients commonly used in this field.

Formulation form of the present invention can be formulated in the form of liquids, suspensions, emulsifiers, extracts, powders, granules, tablets or capsules, as a skin solvent, liquids, suspensions, coalescing agents, pastes, gels, creams It may be formulated into a lotion, powder, surfactant-containing cleansing agent, oil, spray, and the like.

Next, the present invention will be described in more detail with reference to examples of the composition of the present invention.

Example 2

A patch for patch regeneration comprising skin tissue regenerating protein and twisted transcription factor as an active ingredient was prepared (FIG. 13).

ingredient Content (% by weight) Skin Tissue Regeneration Protein Type 2 0.005 Twisted transcription factor 0.005 Polyurethane foam 99.90 Sum 100.00

The patch containing the said component was manufactured by the normal manufacturing method of a patch.

Example 3

In Experimental Examples 4 to 5, it was shown that skin tissue regeneration protein type 2 and twisted transcription factors directly proliferate fibroblasts, which are the main components of the dermis, and keranocytes, which are growth cells of the epithelial cell layer, and have an excellent effect on healing skin wounds. Using this property, it is a composition characterized in that it is wrinkle improvement, skin elasticity improvement, skin aging prevention, hair loss prevention or hair growth, skin moisturization improvement, removal of blotch or acne treatment as prepared in a liquid composition as shown in Table 2 The lyophilized growth factor was solutionized to prepare a lotion.

ingredient Content (% by weight) Skin Tissue Regeneration Protein Type 2 0.005 Twisted transcription factor 0.005 glycerin 10.0 1,3- Butylene glycol 5.0 Allantoin 0.1 DL - Panthenol 0.3 Hydroxyethyl  cellulose 0.1 Sodium hyaluroseate 0.2 Carboxyvinyl polymer 0.2 Octyldodes -16 0.4 Purified water Suitable amount system 100

The lotion agent was manufactured by the conventional manufacturing method of lotion agent.

The present invention also provides a kit containing a skin tissue regenerating protein having a skin tissue regeneration effect of the present invention and a twisted transcription factor as an active ingredient, and comprising a scaffold and a shielding membrane.

 In the present invention, the skin tissue regeneration protein and twisted transcription factor having the skin tissue regeneration effect configured for the skin tissue regeneration kit is contained in a gel-like scaffold, filled in a syringe or a vial, and then lyophilized and stored in a vial or a syringe. It can be provided as a kit, and when used, it can be injected into the defect site by injecting water for injection.

The material of the shielding film that can be used in the present invention is PCL, PLA, PLGA, PGA synthetic absorbent polymer material; Natural absorbent polymers of alginic acid, chitosan, collagen, hyaluronic acid, cellulose and mixtures thereof; poly (vinylidene fluoride), poly (tetrafluoroethylene), poly (vinyl alcohol), poly (hydroxyalkanoate), poly (ethylene terephthalate), poly (butylene terephthalate), poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N- isopropylacrylamide), poly (dimethyl siloxane), polydioxanone, and polypyrrole, poly (glycolic acid), poly (lactic acids), poly (ethylene oxides), poly (lactide-co-glycolides), poly (s-caprolactone), polyanhydrides, It is selected from the group consisting of metal thin films of polyphosphazenes, poly (ortho-esters and polyimides); titanium and stainless steel.

The shielding membrane may have pores of less than 0.01 ~ 30㎛ prevent neighboring bacteria or cells from passing through or growing inside. Externally exposed areas prevent the invasion of bacteria and the escape of body fluids from defective tissues or organs. On the other hand, the shielding film installed inside the tissue or organ shields other adjacent tissues or organs from interfering with each other. This allows the tissue or organ to be regenerated to be integral with the surrounding normal tissue or organ.

The thickness of the shielding film is 0.01-0.5 mm. If it is thinner than 0.01mm, it is easily deformed to the external magnetic pole, and if it is thicker than 0.5mm, it is difficult to mold.

In the present invention, the shielding film for the skin tissue regeneration kit may be composed of a single layer or a multilayer of 2 to 5 layers. In order to contain the human bone morphogenic protein for skin tissue regeneration in the shielding membrane, a plurality of shielding membrane layers may be formed by adding a shielding membrane layer of hydrophilic material. The outer layer may be composed of a layer containing physicochemical stability to block external stimuli and a growth factor inside.

In the present invention, the shielding film constituted for the kit for skin tissue regeneration may have non-porous sheets or pores. Preferably, the pores of the shielding film may be used as a shielding film as well as a shielding film made by compressing a collagen sponge or a shielding film made of nanofibers having pores of 0.01-30 μm or more. Bacteria do not penetrate the sheet or film that has no pores in the shielding membrane to prevent infection, and nanofibers or compressed sponges return to their original unfolded shape even if they are deformed to conform to the curved shape, but the sheet or film is deformed. It has the advantage of being maintained.

In the present invention, the shielding film configured for the skin tissue regeneration kit may be reinforced with a reinforcing layer to increase the strength of the shielding film.

To this end, the material of the reinforcing layer may be a metal selected from polycaprolactone or polylactic acid, polyglycolic acid and copolymers of lactic acid and glycoic acid, titanium and stainless steel. Preferably, in order to maintain the shape of the shielding film, a synthetic polymer layer or a metal layer may be formed between the plurality of shielding film layers or under a single layer. This layer may consist of plastic polycaprolactone or polylactic acid, polyglycolic acid and copolymers of the three materials or metals of titanium or stainless steel. The shape may be a mesh layer, and the thickness may be 0.01 to 0.5 mm, and may be formed to be extended in the outer direction from the center of the shielding film.

In the skin tissue regeneration kit of the present invention, a shielding membrane may be variously configured. For example,

1) It can be two kinds of shielding membranes, which are collagen membranes pressed and crosslinked with collagen sponges and PTFE sheets kept in a molded state.

2) A double shielding membrane may be composed of a PTFE sheet for external bacterial and physicochemical defense and a collagen shielding membrane having affinity for growth factors or stem cells.

3) A shielding film composed of a reinforced metal or a synthetic polymer mesh may be configured to maintain the shape of the shielding film. The shielding film may be a mesh fixed between different shielding layers or a mesh fixed to a lower layer of the shielding layer. The mesh may be configured to extend in an outward direction from the center of the shielding film. The synthetic polymer mesh may be molded by injection molding or sheet and bonded to the shielding film by thermocompression bonding.

In the case of extensive skin damage, a three-dimensional image of skin tissue is acquired by computer tomography (CT) or a three-dimensional optical analyzer, and a three-dimensional shape in which the skin tissue is reproduced is acquired by a simulation program. Using this three-dimensional shape and simulation program, we design a shield that maintains shape during skin regeneration, and a laser photopolymerization shield using polyepoxy polymer, a thermoplastic shield using polycarborolactone (PCL) polymer, or PEEK resin or PTFE. An engineering polymer block, such as a resin, is produced in a computer milling center to form a shield.

The material of the scaffold that can be used in the present invention is a biodegradable polymer material of PCL, PLA, PLGA, PGA; Natural polymers of chitosan, alginic acid, collagen, hyaluronic acid, chondroitin, glucosamine, cellulose; Complexes thereof; And a material selected from the group consisting of synthetic bone, allogeneic bone, xenograft, demineralized bone or cadaveric derived dermis.

The shape of the scaffold that can be used in the present invention is a sheet, porous sheet, sponge, jelly, gel, lyophilized, microspheres, tubular (tube), lattice or lattice structure containing collagen It may be a grid.

In the present invention, a single layer of the scaffold may be used, and if necessary, the scaffold may be composed of a multilayer of two or more layers and five or less layers.

In the present invention, when using a multi-layer scaffold, it can be used in the form of being separated from each other, bonded or glued.

In the present invention, the growth factor may also be prepared by incorporating the growth factor into a gel-like scaffold and filling the syringe or vial with lyophilization.

In the present invention, it is also possible to use a scaffold added with a reinforcing layer for maintaining the shape of the scaffold. At this time, the material of the reinforcing layer may be a material selected from biodegradable polymer materials of PCL, PLA, PLGA, PGA.

In the present invention, it is also possible to use a scaffold having a lattice solid shape and a collagen sponge filled inside the lattice. As the lattice-like material, it is possible to use PCL, PLA, PLGA, PGA synthetic polymer material, a mixture of synthetic polymer material and calcium phosphate ceramic, or a material selected from titanium or stainless steel metal.

More preferably, the skin tissue cells have different growth rates so that they grow or regenerate in the order of epithelial cells, dermal cells, ligament cells and nerve cells. Therefore, when the skin consisting of the epidermis and the dermis becomes defective, the epithelium is regenerated first rather than the dermis, and the epithelium grows first before the regenerated dermis is regenerated, resulting in a dent scar. Alternatively, when the bone tissue and the skin are damaged at the same time, the skin tissue first occupies a bone tissue defect, and thus the bone tissue cannot be completely regenerated. At the same time, the skin that is being regenerated has a property to maintain a constant thickness, so that an inner groove is formed in accordance with the appearance of the missing bone tissue. Therefore, when two or more tissues or organs are damaged, the growth rate is faster so that the scaffold of the first healing tissue or organ is larger and the scaffold of the later healing tissue or organ is less porous and growing first. The tissue or organ is regenerated first to a certain thickness. In addition, the extinction or absorption of the material should be controlled as much as the rate at which new tissues or organs are completed, and the slow regeneration site will allow the material to retain its shape until regeneration is complete. The materials used as scaffolds have different shape retention periods in the body, so that the collagen sponge is maintained for less than 3 weeks, the PGA polymer for less than 6 months, the PLA polymer for less than 12 months, and the PCL polymer for less than 24 months. The retention period can be controlled by synthesizing a polymer having various retention periods into a copolymer. As such, various shapes suitable for the shape of the defect area are essential for the shielding film constituted in the skin tissue regeneration kit of the present invention.

As various configurations of the scaffold, a double scaffold consisting of a polymer (PLGA) sponge of collagen and glycolic acid and a collagen sponge can be constructed.

1) Calcium phosphate synthetic bone particles and collagen solution may be composed of a scaffold gel.

2) The scaffold injected into the lattice layer by PCL can be constructed to maintain the shape in a wide range of scaffolds.

(1) A tubular scaffold made of a polymer (PLGA) sponge of lactic acid and glycolic acid can be constructed for nerve or blood vessel regeneration.

(2) If two or more tissues or organs are missing, a scaffold consisting of a collagen scaffold that is first biodegraded for the first regenerated tissue or organ and a PLGA sponge that is slower biodegraded for the later regenerated tissue or organ Can be.

(3) If two or more tissues or organs are missing, a sketch consisting of a larger, first biodegradable pore sponge for the first regenerating tissue or organ, and a smaller, slower, biodegradable sponge for later regenerating tissue or organ. Folds can be constructed.

(4) PLGA sponge layer for the last reclaimed site with one layer of shielding membrane and a scaffold consisting of 1 to 5 types, blocks of calcium phosphate and collagen for the next regenerated site, and the shape of the scaffold in a wide range of areas. A scaffold may be constructed that consists of a lattice layer of PCL to maintain and a collagen sponge for the tissue or organ that is regenerated fastest. In addition, the damaged tissue or organ may be up to five skin, muscles, ligaments, bones, nerves, etc. The scaffold may also be composed of five layers accordingly.

(5) A scaffold composed of collagen filled inside the lattice geometry of the PCL to maintain its shape in the case of severe external pressure and a collagen sponge for shock absorption may be constructed.

In the present invention, a growth factor constituted for a tissue or organ regeneration kit is contained in a gel-like scaffold, filled in a syringe or a vial, and lyophilized to be stored in a vial or a syringe to provide a kit. Water can be injected into the defects and gelled.

As an example for the skin tissue regeneration kit in the present invention, in preparing a gel scaffold, hyaluronic acid solution (0.1 ~ 10wt / wt%) contained in the skin tissue regeneration protein 1mcg ~ 2 mg / ml is preferred Preferably 0.5 mg / ml in a syringe or vial and lyophilized. Immediately before use in the clinic, water for injection or saline is inhaled or injected into a syringe or vial to gel and inject into the defect.

Tissue or organ cells have different growth rates and grow or regenerate in the order of epithelial cells, dermal cells, bone cells, ligament cells and nerve cells. Therefore, when the skin consisting of the epidermis and the dermis becomes defective, the epithelium is regenerated first rather than the dermis, and the epithelium grows first before the regenerated dermis is regenerated, resulting in a dent scar. Alternatively, when the bone tissue and the skin are damaged at the same time, the skin tissue first occupies a bone tissue defect, and thus the bone tissue cannot be completely regenerated. At the same time, the skin that is being regenerated has a property to maintain a constant thickness, so that an inner groove is formed in accordance with the appearance of the missing bone tissue. Therefore, when two or more tissues or organs are damaged, the growth rate is faster so that the scaffold of the first healing tissue or organ is larger and the scaffold of the later healing tissue or organ is less porous and growing first. The tissue or organ is regenerated first to a certain thickness. In addition, the extinction or absorption of the material should be controlled as much as the rate at which new tissues or organs are completed, and the slow regeneration site will allow the material to retain its shape until regeneration is complete. The materials used as scaffolds have different shape retention periods in the body, so that the collagen sponge is maintained for less than 3 weeks, the PGA polymer for less than 6 months, the PLA polymer for less than 12 months, and the PCL polymer for less than 24 months. The retention period can be controlled by synthesizing a polymer having various retention periods into a copolymer.

The skin dispute kit provided by the present invention may be a single layer or multiple scaffolds may be separated from each other, bonded or glued together. Preferably, in the case where two or more tissues or organs are to be regenerated, the scaffold is preferably made of multiple layers. Each layer can be a different polymeric material. The layer corresponding to the tissue or organ that is regenerated first may be first degraded and replaced with a neoplastic tissue or organ. The layers are divided according to the order in which they are reproduced, so that the scaffold layer of tissue or organ to be regenerated later is maintained in shape.

In the case where two or more kinds of tissues or organs are to be regenerated at the same time, even through the same polymer material, the distribution of the through pores may be different from each other. The more penetrating pores, the more likely it is to take advantage of the properties that are first created by the new tissue or organ. The layer corresponding to the tissue or organ that is regenerated first has a lot of penetrating pores so that it can be regenerated first, and the scaffold layer of the cell layer that is regenerated in a lower order can be configured to have fewer penetrating pores so that the shape retention of the scaffold can be improved. have.

If a defect in one type of tissue or organ (e.g. dermis or bone tissue or organ alone) is so large that the volume of the scaffold is too large to maintain shape, an additional layer is provided to facilitate shape deformation and maintain the deformed shape. May be

In the present invention, a reinforcing layer for maintaining the shape of the scaffold configured for the skin tissue regeneration kit may be added, and the material may be selected from biodegradable polymer materials of PCL, PLA, PLGA, and PGA.

In the present invention, the scaffold configured for the kit for skin tissue regeneration is lattice solid and collagen sponge may be filled in the lattice.

In the present invention, the material for the regeneration regeneration kit for skin tissues may be selected from a mixture or metal of synthetic polymer material, synthetic polymer material and calcium phosphate ceramic. Preferably, when damaged tissues or organs are exposed to strong external forces, a mixture of PCL, PLA, PLGA, PGA synthetic polymers or synthetic polymers and calcium phosphate ceramics, or lattice-shaped scaffolds made of titanium or stainless steel metal, A polymeric sponge filled inside the lattice can be constructed with other scaffold layers.

In the present invention, the skin tissue regenerating protein is composed of the scaffold while contained in the shielding membrane, the skin tissue regenerating protein is composed of the shielding membrane contained in the scaffold, or the tissue composed of skin tissue regenerating protein is contained in the scaffold and the shielding membrane. Alternatively, the skin tissue regeneration protein, the scaffold, and the shielding membrane may be configured together to be provided as a kit for organ regeneration to complement the cell proliferation effect of the skin tissue regeneration protein, the function of the membrane and the role of the scaffold.

In the existing publications, studies on scaffolds that maintain the space and shape in which tissues of damaged tissues or organs are grown at the defect site in tissue or organ regeneration using growth factors have been mainly conducted, and recently, growth factors have been scaffolded. Studies using only barriers have been started, and these studies have an experimental approach rather than studies based on clinical efficacy or side effects prevention. The present invention is based on the results of a case using a growth factor in the clinic, the conditions to be achieved for tissue or organ regeneration that is integral with the surrounding normal tissue or organ of the defect site should be used at the same time growth factor, scaffold and shielding membrane Is that.

The scaffold layer serves as a carrier of skin tissue regenerating proteins such as BMP-2, twisted transcription factors and stem cells, and maintains a three-dimensional space with penetrating pores for peripheral normal blood vessels to grow inside.

In order to apply skin regenerating proteins and twisted transcription factors to scaffolds or barriers, these materials must be hydrophilic. Therefore, in order to directly hydrate or use skin regeneration protein and twisted transcription factors in scaffolds or shielding membranes, there are matters to be considered in the material composition and composition.

In applying the shielding membrane to a defective tissue or organ, it is preferable that the shielding membrane positioned at the open site uses a hydrophobic polymer to prevent bacterial invasion. In order to apply the skin regeneration protein, a hydrophilic shielding layer must be added to the lower layer.

Also, in applying or containing skin regeneration protein and twisted transcription factor to the scaffold, the material for maintaining the shape is hydrophobic synthetic polymer or calcium phosphate salt crystal that chelates with growth factor protein to inhibit the release. Alternatively, natural polymers should be filled inside or contained outside the scaffold.

There are four ways to apply skin tissue regeneration protein and twisted transcription factor to scaffold and shield.

Generally, a method of hydrating a skin tissue regenerating protein and a twisted transcription factor directly on a scaffold and a barrier layer is used. However, due to poor operability, skin tissue regenerating proteins and twisted transcription factors may be contained in the scaffold, in the shield, or in the scaffold and the shield. The method of incorporating the skin tissue regeneration protein and the twisted transcription factor is a method of directly hydrating a solution of the effective drug concentration of the skin tissue regeneration protein and the twisted transcription factor in the target scaffold or shielding membrane and then freeze-dried.

In order to apply skin regenerating proteins and twisted transcription factors to scaffolds or barriers, these materials must be hydrophilic. Therefore, in order to directly hydrate or use skin regeneration protein and twisted transcription factors in scaffolds or shielding membranes, there are matters to be considered in the material composition and composition.

In applying the shielding film to the defective tissues, it is preferable that the shielding film placed at the open site uses a hydrophobic polymer to prevent bacterial invasion. To apply skin regeneration protein and twisted transcription factor, a hydrophilic shielding layer should be added to the lower layer.

Also, in applying or containing skin regeneration protein and twisted transcription factor to the scaffold, the morphological materials are hydrophobic synthetic polymers or calcium phosphate crystals that chelate the skin tissue regeneration protein and twisted transcription factor to inhibit their release. Therefore, the hydrophilic synthetic or natural polymer should be filled inside or contained outside the scaffold.

There are four ways to apply skin tissue regeneration protein and twisted transcription factor to scaffold and shield. Generally, a method of hydrating a skin tissue regenerating protein and a twisted transcription factor directly on a scaffold and a barrier layer is used. However, due to poor operability, skin tissue regenerating proteins and twisted transcription factors may be contained in the scaffold, in the shield, or in the scaffold and the shield. The method of incorporating the skin tissue regeneration protein and the twisted transcription factor is a method of directly hydrating a solution of the effective drug concentration of the skin tissue regeneration protein and the twisted transcription factor in the target scaffold or shielding membrane and then freeze-dried.

Example 4

Manufacture of Scaffolds

In skin regeneration, a scaffold composed mainly of collagen sponge was prepared as follows (FIG. 5).

Bovine ligaments and pig skin subcutaneous tissues are removed, ground to small size, and the collagen component is extracted using a surfactant, a defatting agent and a bactericide. Pepsin treatment is performed at pH 3 to separate the collagen fiber bundles to obtain fine collagen fibers. It is lyophilized to purify pure collagen to make a collagen solution with a weight ratio of 3 to 5%. The collagen solution and the crosslinker are added to the mold and lyophilized. Freeze-drying conditions are shown in Table 3 below.

Temperature
(° C) -30 -20 -10 0 5 10 15 20 time
(time) 3 One One One One One One 9

In order to increase the efficiency of application to skin defects, the skin tissue regenerating protein and twisted transcription factor may be contained in gel scaffolds, filled in syringes or vials, and then lyophilized to be stored in vials or syringes to constitute kits. At the time of use, water for injection can be injected and gelled to be injected into the defect site.

Example 5

Preparation of Gel Scaffolds

Hyaluronic acid solution (0.1 ~ 10wt / wt%) containing skin tissue regeneration protein at 0.5 mg / ml, filled in a syringe or vial and lyophilized (Fig. 6a). Immediately before use in the clinic, water for injection or saline is inhaled or injected into a syringe or vial to gel and inject into the defect (FIG. 6B).

Example 6

Preparation of the shielding film:

1) Two types of shielding membranes were prepared: a collagen membrane (FIG. 7A) and a PTFE sheet (FIG. 7B) cross-bonded by pressing a 3 mm thick collagen sponge (FIG. 5) to 0.3 mm.

2) A double shielding membrane (FIG. 8) may be composed of a PTFE sheet 1 for protecting external bacteria and physicochemical defenses and a collagen shielding membrane 2 having affinity for recombinant human bone morphogenic proteins or stem cells.

3) In order to maintain the shape of the shielding film, the synthetic polymer mesh 3 is fixed between the two shielding layers 4 and 5, or the shielding film is fixed to the lower layer of the shielding film (FIG. 9). It may be configured to extend in the outward direction from the center of the. The synthetic polymer mesh can be molded by injection molding or sheet and bonded to the shielding film 4 by thermocompression bonding.

Example 7

Preparation of Skin Regeneration Shape Retaining Shielding Film:

If the skin damage is extensive, a three-dimensional image of the skin tissue is obtained by CT (computer tomography) or a three-dimensional optical analyzer, etc., and a three-dimensional shape in which the skin tissue is to be reproduced is obtained by a simulation program (FIG. 10). Using this three-dimensional shape and simulation program, we design a shield that maintains shape during skin regeneration, and a laser photopolymerization shield using polyepoxy polymer, a thermoplastic shield using polycarborolactone (PCL) polymer, or PEEK resin or PTFE. An engineering polymer block, such as a resin, is produced in a computer milling center to form a shield.

Into the volume of the depleted skin tissue, a scaffold such as collagen, rhBMP-2, and a twisted transcription factor are added, and a skin regeneration shape maintenance membrane is fixed.

Test Example 6

Skin tissue regeneration effect using collagen sponge scaffold and human adipose stem cells with skin regeneration protein and twisted transcription factor:

To identify and culture stem cells (hMSC) from the fat removed from human liposuction, and to examine the skin tissue regeneration response according to skin tissue regeneration protein (reBMP-2) and twisted transcription factors, skin tissue regeneration in mice with immunodeficiency experiments Was observed. 12-day wound healing effect in rats with 8-mm-diameter full-thickness wounds using collagen sponges (test group) with rhBMP-2, twisted transcription factor type 2 and hMSC and no sponges (control group) Was compared.

The control group was transplanted with collagen sponge but the test group was transplanted with collagen sponge with BMP-2 and twist type 2 without hMSC. Type 2 and hMSCs were tested in groups implanted with collagen sponges.

As a result, 8% of the wounds were left in the group containing both BMP-2 and Twist Transcription Factor 2 and hMSC in the collagen sponge, and only 12% of the patients who received only BMP-2 and Twist Transcription Factor 2 were implanted. 25% and 32% wounds remained in the control group (FIG. 11).

Therefore, BMP-2 and twisted transcript factor type 2 and stem cells were excellent for the regeneration of skin tissues. Especially, the combination of BMP-2 and twisted transcript factor type 2 and stem cells was found to be the most effective. Could.

In addition, in Test Example 4, it was found that the skin regeneration ability was excellent in the skin tissue regeneration result using stem cell, skin tissue regeneration protein, twist transcription factor type 2 and scaffold.

Claims (20)

A composition for use in regenerating defective skin tissue containing human bone forming protein and twisted transcription factor type 2 as active ingredients.
The method of claim 1 wherein the recombinant human bone morphogenetic protein is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP- 12, BMP-13 and a composition for use in skin tissue regeneration of a skin tissue regeneration protein in combination of a twisted transcript factor type 2 and a recombinant human bone forming protein selected from the group consisting of co-conjugates of the amino acids constituting these BMPs.
The composition for use in skin tissue regeneration according to claim 1 or 2, wherein the composition for regenerating defective skin tissue is for medicine or cosmetics.
According to claim 3, TGF-β super group, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and the like, other than skin tissue regeneration protein and twist transcript factor type 2 as active ingredients, fibroblast differentiation factor and GDF A composition for regenerating skin tissue further comprising one or two or more selected from the group consisting of a transforming growth factor, keratinocyte growth factor 2 (KGF2) and MP52 protein, and a genetically modified protein of the growth factors.
The method according to claim 3 or 4, further comprising ketoprofen, flurbiprofen, fenoprofen, and ibuprofen, in addition to skin tissue regeneration protein and twisted transcription factor type 2. Phenyl propionic acid derivatives; Oxycam derivatives such as pyroxicam, tenoxicam and meloxicam; Anti-inflammatory drugs selected from diclofenac and indomethacin, metronidazole, Amoxicillin, Amoxicillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, (Ecephaphaloscinin) ), Tetracycline hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride selected from the group consisting of antimicrobial agent, methyl prednisolone, hydrocortisone acetate further comprises a composition for skin tissue regeneration.
The method according to claim 3 to 5, wherein the composition for skin tissue regeneration is to improve the skin condition, wrinkle improvement, skin elasticity improvement, skin aging prevention, hair loss prevention or hair growth promotion, skin moisturization improvement, removal of blotch or acne treatment Skin tissue regeneration composition.
A skin tissue regeneration kit comprising the skin tissue regeneration protein of claim 1 or 2 and a twist transcription factor type 2 as essential components.
According to claim 7, TGF-β super group, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and the like, other than skin tissue regeneration protein and twist transcript factor type 2 as active ingredients, fibroblast differentiation factor and GDF Transgenic growth factor, keratinocyte growth factor 2 (KGF2) and MP52 protein and genetically modified protein of said growth factor; Stem cells capable of differentiating into neurons, astrocytes, adipocytes, chondrocytes, osteoblasts and insulin secreting pancreatic beta cells; Pluripotent stem cells, pluripotent stem cells, pluripotent neural stem cells, pluripotent pancreatic stem cells, extracted pulp cells, periodontal ligament cells; And fat cells collected by liposuction; One component selected from the group consisting of TGF-β supergroup, fibroblast growth factor, epithelial cell growth factor, platelet derived growth factor, insulin-like growth factor, transformative growth factor, keratinocyte growth factor 2 (KGF2) and MP52 Skin tissue regeneration kit containing more.
The method according to claim 7 to 8, further comprising ketoprofen, flurbiprofen, fenoprofen, ibuprofen in addition to skin tissue regeneration protein and twisted transcription factor type 2 Phenyl propionic acid derivatives; Oxycam derivatives such as pyroxicam, tenoxicam and meloxicam; Anti-inflammatory drugs selected from diclofenac and indomethacin, metronidazole, Amoxicillin, Amoxicillin-clavulanate, Ampicillin-sulbactam, Ampicillin, Piperacillin, Benzathine penicillin, Cephalosporin, Cefazolin, (Ecephaphaloscinin) ), Tetracycline hydrochloride, cetylpyridinium chloride, chlorhexidine hydrochloride selected from the group consisting of antibacterial agent, methyl prednisolone, hydrocortisone acetate kit for skin tissue regeneration further containing.
The skin tissue regeneration kit according to claim 7, wherein the skin tissue regeneration protein and twist transcript factor type 2 are selected from the group consisting of a scaffold and a shielding membrane.
9. The scaffold according to claim 7 or 8, comprising skin tissue regenerating protein and twisted transcript factor 2 in the scaffold, skin tissue regenerating protein and twisted transcript factor 2 in the shielding membrane, or skin tissue regeneration. Skin tissue regeneration kit comprising protein and twist transcription factor 2 in the scaffold and shielding membrane, respectively.
The material of the scaffold according to claim 9 or 10, wherein the scaffold is made of PCL, PLA, PLGA, PGA polymer, chitosan, alginic acid, collagen, hyaluronic acid, chondroitin, glucosamine, cellulose, and the like. A kit for skin regeneration, which is a scaffold selected from the group consisting of a complex of substances and a carcass-derived dermis.
11. The scaffold is shaped like a sheet, gel, aerosol, jelly, porous sheet, lyophilized, sponge, microsphere, tubular (tubular), lattice or lattice structure. A kit for skin regeneration that is a scaffold selected from the group consisting of lattice-like collagen contained therein.
The skin tissue regeneration kit according to claim 7, wherein the skin tissue regeneration protein and twist transcript factor type 2 are contained in a gel-like scaffold and filled in a syringe or a vial and then lyophilized.
The method of claim 9, wherein the material of the shielding film is PCL, PLA, PLGA, PGA synthetic absorbent polymer material; Natural absorbent polymeric material of alginic acid, chitosan, collagen, hyaluronic acid, cellulose, or a mixture thereof; poly (vinylidene fluoride), poly (tetrafluoroethylene), poly (vinyl alcohol), poly (hydroxyalkanoate), poly (ethylene terephthalate), poly (butylene terephthalate), poly (methyl methacrylate), poly (hydroxyethyl methacrylate), poly (N- isopropylacrylamide), poly (dimethyl siloxane), polydioxanone, and polypyrrole, poly (glycolic acid), poly (lactic acids), poly (ethylene oxides), poly (lactide-co-glycolides), poly (s-caprolactone), polyanhydrides, A kit for skin regeneration selected from the group consisting of polyphosphazenes, poly (ortho-esters or polyimides synthetic polymer material) and metal films of titanium and stainless steel.
The tissue regeneration kit according to claim 9, wherein the shielding film is composed of a single layer or a multilayer of 2 to 5 layers.
The tissue regeneration kit according to claim 9, wherein the shielding membrane is a non-porous sheet or a membrane having pores.
The kit for regenerating skin tissue using a shielding membrane according to claim 9, wherein the extensively damaged skin tissue is designed and manufactured with a shielding membrane for maintaining the skin form to be regenerated.
The kit for skin tissue regeneration according to claim 9, wherein the shielding film is reinforced with a reinforcing layer to increase strength.
19. The kit of claim 18, wherein the material of the reinforcing layer is selected from the group consisting of polycaprolactone or polylactic acid, polyglycolic acid and copolymers of lactic acid and glycoic acid, titanium and stainless steel.

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