KR20040028515A - Dermal substitute consisting of amnion and biodegradable polymer, the preparation method and the use thereof - Google Patents

Abstract

PURPOSE: Provided is an implantable derma substitute formed of amnion and bio-degradable materials, which has anti-inflammatory effect and an effect of stimulating wound-healing by various growth and differentiation factors. CONSTITUTION: The derma substitute has a composite structure obtained by adhering, incorporating or inserting a bio-compatible amnion separated from a placenta and a bio-degradable polymer substrate each other. Particularly, the bio-degradable polymer is selected from the group consisting of collagen, gelatin, hyaluronic acid or a derivative thereof, chitin, chitosan, PLGA(poly(D,L-lactic-co-glycolic acid)), PGA(polyglycolic acid) and PLA(poly lactic acid).

Description

Dermal substitute consisting of amnion and biodegradable polymer, the preparation method and the use etc.

The present invention relates to a dermal substitute consisting of a biodegradable polymer such as collagen and amnion, which is a biological material separated from the placenta, a method for producing the same, and a use thereof.

In general, when skin damage is limited to the epidermis, such as a mild burn within 2 degrees, skin tissue can be regenerated, but in the case of wounds such as deep burns, it is not possible to regenerate autologous skin. It is customary. However, until transplantation, the affected area must also be covered and protected for some time. Therefore, the wound dressing should be able to block the external environment such as bacteria and protect the human body while maintaining the permeability of appropriate moisture. To date, the clinic has used synthetic polymer membranes such as urethane polymers and poly-L-leucine as wound coatings to protect these damaged areas. However, these materials have no biological function. There is no choice but to be. Therefore, in order to regenerate the skin more aggressively, there are those designed to drop off when the skin tissue is regenerated by making a composite of a suitable water-permeable polymer membrane and a biocompatible structure. Among them, a relatively successful biobrane released in the early 1980s was biobrane. It has a double structure consisting of a nylon weave coated with collagen and a fine porous silicone membrane. Because it has good adhesion to the skin and elasticity, it adheres well to the wound surface and antibiotics applied routinely through the silicone membrane are delivered to the affected area. It has the advantage that it can be widely used in burns within 2 degrees or in the same degree of peeling.

In the case of severe wounds above 2 degrees, the dermal replacement is currently covered with a partial layer of epidermis or about 2 weeks after transplantation, and the epidermal replacement is essential. It is made by using sponge or gel form or using biodegradable polymer.

When collagen sponges are used for whole skin defect wounds, fibroblasts and capillaries penetrate into the sponges and proliferate at the wound site, and collagen fibers are formed by the synthesis of autologous collagen, thereby constructing dermal tissue. The collagen sponge is then decomposed and absorbed in an extremely pure manner. Thus, this dermal tissue becomes a complete self-dermal tissue. Because the material itself is turned into living dermal tissue, the sponge is also called a dermal substitute and has already been commercialized in some and contains various research and clinical reports.

The prior art related to such dermal substitutes are disclosed as follows. Korean Patent Application Publication No. 2000-0013701 discloses a porous collagen dual structure wound protective film for dermal regeneration induction consisting of a hard layer composed of collagen, laminin and hyaluronic acid and a porous layer, and Korean Application Publication No. 2000-0007983 The present invention discloses a biological tissue comprising a reinforced collagen layer made of a collagen of sponges and meshes and a use thereof. The biotissue equivalent of Korean Patent Application Publication No. 94-1379 is a fiber in the collagen layer of gel state. Dermal replacement consisting of a dermal layer composed of collagen lattice form shrunk into blasts is disclosed.

In addition, currently commercialized dermal substitutes include Integra ^TM of the United States and Terudermis ^TM of Japan, which are licensed as medical supplies from the FDA and the US Food and Drug Administration. It consists of a collagen layer extracted from the skin of cows and cows. In this case, the upper layer is composed of a silicone membrane to prevent evaporation and infection of body fluids, and the lower layer is composed of collagen and the like to induce the regeneration of new blood vessels and connective tissue. It is broken down and absorbed by the human body.

Treatment of severe burns with such a commercially available dermal replacement generally requires two transplant surgery procedures. The first step is to implant the dermal replacement into the wound and form a new dermis within two to three weeks. Step 2 is a step of skin graft surgery for epidermal layer formation on the formed neoplasia. In the case of the conventional two-time transplantation, long-term hospitalization and pain of the patient are inevitable, and as a method of reducing the transplantation, the replacement of the dermal replacement with the one-time transplantation method is performed by performing autoepidermal or cultured epidermal transplantation. While groping, the existing dermal substitute has a disadvantage that the engraftment rate is limited during autologous epidermal transplantation because there is no part corresponding to the basement membrane of the skin. Therefore, there is a need for a membrane of ingredients that can replace the basement membrane of the skin as an alternative.

In addition, the existing dermal substitutes use silicone membranes for the temporary epidermis until the second partial layer implantation, but this is only for protection from bodily fluid loss and infection, and more aggressive wound regeneration considering the patient's pain and hospitalization period. This should be planned.

Therefore, while increasing the engraftment rate for epidermal transplantation, more effective wound healing effect through anti-inflammatory and wound healing promoting effect, and biocompatibility that can be treated by one transplant surgery by autologous or cultured epidermal transplantation at the same time as dermal replacement The development of dermal substitutes has long been scarce.

The research team of the present inventors have already manufactured a collagen yarn in the form of a mesh (mesh) and inserted it into the collagen sponge to produce a reinforced collagen sponge with a greatly improved tensile strength (Korean Patent Application Publication No. 2000-0007983) In further research, the present inventors have used the biocompatible amnion to prevent wound infection, loss protection of water, protein, etc., and the anti-inflammatory properties of the amniotic membrane itself, and wound growth by various growth and differentiation factors. In order to promote epidermal cell migration, we confirmed more aggressive wound healing effect. We also developed a dermal substitute that can be transplanted once by attaching amnion to collagen scaffold as an alternative to traditional two-stage transplantation. Biosynthetic skin using amnion as a basement membrane for the treatment of widespread severe burns or ulcers The present invention was completed by developing.

Therefore, an object of the present invention is biodegradability of the biosynthetic skin using the amnion as a basement membrane component and a dermal substitute for transplantation, which has an effect of promoting wound healing by anti-inflammatory, various growth and differentiation factors, using amnion and biodegradable material having excellent biocompatibility. It is to provide a polymer substrate.

1A is a scanning electron micrograph of a porous collagen sponge cross section,

Figure 1b is a scanning electron micrograph of the bottom surface (window surface) of the porous collagen sponge,

Figure 2a is a scanning electron micrograph of the amnion and collagen sponge attached portion in the amnion-collagen sponge composite structure according to Example 1 of the present invention,

Figure 2b is a scanning electron micrograph of the amnion and collagen sponge attached portion in the amnion-collagen sponge composite structure according to Example 2 of the present invention,

3A is a photograph of H & E staining at 1 month after implanting only collagen sponge into intra-stroma of rabbit cornea,

Figure 3b is a picture of inflammatory cells infiltrated around the implant of Figure 3a,

3C is a photograph of H & E staining at 1 month after implantation of the composite structure of the amnion-collagen sponge of the present invention into the stroma of the rabbit cornea,

Figure 4a is a H & E stained photograph of a week after transplanting the collagen sponge on the back of the nude mouse,

4B is a photograph of H & E staining 1 week after transplanting the collagen sponge immersed in the amniotic membrane extract of the present invention to a nude mouse, etc.

FIG. 5A is a H & E staining photograph 10 days after implanting a commercially available artificial dermis into a full-thickness skin defect of 2 cm diameter on the back of a guinea pig.

5B is a 10-day H & E staining photograph after implanting the amnion-collagen sponge composite structure of the present invention into a full-thickness skin defect having a diameter of 2 cm on the back of the guinea pig,

FIG. 6 is a photograph of 3, 7 and 30 days after implanting the collagen sponge and the amnion-collagen sponge complex structure on the rabbit corneal limbus damaged with N-heptanol,

7A is a photograph of H & E staining on the 7th day after transplanting only the collagen sponge after inducing skin defects of the entire layer on the back of a New Zealand white rabbit,

7B is a photograph of H & E staining at 7 days after autologous epidermal transplantation with collagen sponge transplantation after inducing skin defects on the back of New Zealand white rabbits.

FIG. 7C is a 7-day H & E staining photo of the New Zealand white rabbit after inducing a skin defect on the back of the rabbit and implanting the amnion-collagen sponge complex structure of the present invention and simultaneously performing an epidermal transplantation.

8 is a reference absorbance graph according to the standard EGF amount,

Figure 9a is an immunohistochemical staining picture for EGF-R of the amniotic membrane,

9b is an immunohistochemical staining picture for collagen type IV of amnion,

10 is an immunohistochemical staining photograph of cytokine keratin (cytokeratin) of the artificial artificial skin prepared by culturing the epidermal cells and fibroblasts of the skin in the amnion-collagen sponge complex structure of the present invention.

In order to achieve the above object, the present invention provides a dermal replacement characterized in that the biocompatible amnion separated from the placenta and the biodegradable polymer structure is attached, incorporated or inserted to have a complex structure, and a method for producing the same.

In addition, the biodegradable polymer is collagen, gelatin, hyaluronic acid (hyaluronic acid) and its derivatives, chitin, chitosan, alginate, fibronectin (fibronetin), dextran natural materials and PLGA (poly (D, L-lactic-) co-glycolic acid)), polyglycolic acid (PGA), poly (lactic acid) and similar copolymers, poly ε-caprolactone, polyanhydride, polyortho It includes one or more selected from the group consisting of a synthetic material of polyester (polyorthoesters), polyurethane (polyurethane), the structure thereof is preferably in the form of a sponge, film, fibrous or the like.

The amniotic membrane is made of an extract of a sheet-like structure or a mesh-like structure or an amniotic membrane using a double ring, an insert, or a silicone ring, and is then cross-linked to a biodegradable polymer matrix. Attachment, incorporation or insertion.

As used herein, the terms "attach" and "insert" refer to a state in which amnion and biodegradable polymer structures are physically adjacent to each other or interposed between structures in a layered state, and "incorporation" means amnion or Any one or both of the biodegradable polymer structures are mixed in the liquid phase relative to the counter structure, for example, in a state of being bonded to each other.

In order to prepare the dermal substitute of the present invention, a method of attaching amnion during the process of manufacturing a sponge using a biodegradable polymer, and attaching amnion after manufacturing a biodegradable polymer sponge using a conventional crosslinking method to slow biodegradability And dipping the biodegradable polymer sponge in the amniotic membrane extract, and the process is as follows.

Hereinafter, the present invention will be described in detail.

The present invention is to improve the therapeutic effect by using the properties of the amniotic membrane itself by mutual attachment, incorporation or insertion of the amniotic membrane separated from the placenta into a biodegradable polymer substrate that is conventionally used as an artificial substrate. It has its features.

Biodegradable polymers that can be used in the present invention should act as a skeleton that cells adhere well to form a three-dimensional tissue structure, and should be immunologically inert so as not to cause an inflammatory or foreign body reaction after transplantation. It must be able to actively react with surrounding tissues to induce the growth of cells present in the surroundings, and also because of foreign body reactions to the implants, and before the role of the transplanted cells and tissues is finished, The decomposition rate should be used so that the material does not decompose, and after a certain period of time it should not decompose itself and remain as a foreign substance.

Therefore, any biodegradable polymer material applied to the preparation of the biodegradable polymer matrix according to the present invention is applicable to any polymer that can be decomposed in vivo. In a preferred embodiment of the present invention, collagen, a structural protein in vivo, was used as a biodegradable polymer material.

In order to prepare a dermal substitute having a composite structure by attaching amnion structures to each other during a collagen sponge manufacturing process, for example, the present invention may be used to convert collagen fibers into an acid solution, preferably acetic acid or pepsin, at a pH of 3 to 4 and a concentration of 0.3 to A first step prepared by dissolving at 1%; A second step of applying to the mold to which the amniotic membrane is attached; Provided is a method for preparing a dermal substitute having a complex structure comprising a biodegradable polymer and amnion comprising a third step of freeze-drying for at least 36 hours after freezing in a freezer at -196 ° C to 0 ° C.

In addition, the present invention provides a dermal substitute having a composite structure by the amnion structure is attached to each other during the collagen sponge manufacturing process of the present invention by the above production method.

In addition, to increase the biodegradability and tensile strength, the dermis consisting of amnion-collagen sponge composite structure prepared by crosslinking the collagen and amnion using the conventional crosslinking method to the amnion-collagen sponge composite structure prepared above It includes a method of making a substitute.

The conventional crosslinking method is 0.25% glutaraldehyde (GAD) treatment, 33% 1,3-carbodiimide and 6mM hydroxysuccinimide (N-hydroxysuccinimide) dissolved in 90% acetone solution treatment , Solution treatment in which 33 mM 1,3-carbodiimide and 6 mM hydroxysuccinimide are dissolved in 40% alcohol, UV and gamma irradiation treatment, and other chemical crosslinking.

In addition, in order to prepare a dermal substitute having a composite structure in which the amnion structures are attached to each other after the collagen sponge manufacturing process, for example, the present invention may be used to convert collagen fibers into an acid solution, preferably acetic acid or pepsin, pH 3 to 4, A first step of dissolving at a concentration of 0.3 to 1%; A second step of applying to a mold (eg 12-well plate); A third step of lyophilizing at least 36 hours after freezing in a freezer at -196 ° C to 0 ° C; Putting the obtained collagen sponge in a vacuum oven to maintain vacuum for 2 hours at room temperature and 24 hours at 110 ° C, and then removing the vacuum at 30 ° C to perform DHT dehydrothermal crosslinking; A fifth step of crosslinking the collagen again using the conventional crosslinking treatment method; Then a sixth step of coating the collagen sponge or amnion crosslinked with 0.01% to 0.05% collagen solution; A seventh step of attaching the collagen sponge and the amnion; It provides a method for producing a dermal substitute having a complex structure consisting of a biodegradable polymer and amnion, comprising the step consisting of an eighth step of freezing and drying for at least 36 hours after freezing in an cryogenic freezer of -196 ℃ to 0 ℃ do.

At this time, the pore size of the collagen sponge is prepared to 40 to 150㎛, preferably 60 to 120㎛.

Through the above manufacturing method, the present invention provides a dermal replacement having a composite structure by the amnion structure is attached to each other after the collagen sponge manufacturing process.

In order to prepare the biodegradable polymer sponge by immersing it in the amniotic membrane extract, for example, the present invention is prepared by dissolving collagen fibers in an acid solution, preferably acetic acid or pepsin, at a pH of 3 to 4 and a concentration of 0.3 to 1%. First step; A second step of applying to a mold (eg 12-well plate); A third step of lyophilizing at least 36 hours after freezing in a freezer at -196 ° C to 0 ° C; A fourth step of performing the DHT crosslinking by putting the obtained collagen sponge in a vacuum oven and maintaining the vacuum at room temperature for 2 hours and at 110 ° C for 24 hours and then removing the vacuum at 30 ° C; A fifth step of crosslinking the collagen again using the conventional crosslinking treatment method; A sixth step of preparing amnion extract; A seventh step of dipping the collagen sponge in the amniotic membrane extract for at least 12 hours; It provides a method for producing a dermal substitute having a complex structure consisting of a biodegradable polymer and amnion, comprising the step consisting of an eighth step of freezing and drying for at least 36 hours after freezing in an cryogenic freezer of -196 ℃ to 0 ℃ do.

The preparation of the amniotic membrane extract of step 6 may be prepared by crushing and homogenizing the rapidly frozen amniotic membrane and then centrifuging to obtain a supernatant, followed by filtration.

The amniotic membrane-collagen sponge dermal substitute of the present invention prepared by the above method exhibits an anti-inflammatory effect by inhibiting inflammatory cell infiltration when transplanted into the corneal strom of rabbits, and when the collagen sponge is transplanted alone, and also nude mice and the like. When the collagen sponge immersed in the amniotic membrane extract in the subcutaneous tissue is also less inflammatory reaction, and after the transplantation, the degradation of collagen is delayed and durable.

As a test example of the present invention, when the commercially available Teruthemis and the amnion-collagen sponge composite structure of the present invention were implanted and adhered to the back of the whole layer skin-deficient guinea pig, angiogenesis and fibroblast proliferation effect were shown. Amnion-collagen sponge dermal replacement showed better effect on re-epithelialization.

In addition, after the burn to the corneal limbus of the rabbit, when the amnion-collagen sponge dermal substitute of the present invention is implanted, unlike the collagen sponge structure has an anti-inflammatory effect of inhibiting the infiltration of inflammatory cells around the implant.

In addition, the freezing or lyophilization treatment of the amnion used in the preparation of the dermal substitute of the present invention does not significantly reduce the amount of epidermal growth factor, thereby promoting wound healing.

The dermal replacement amnion-collagen sponge composite structure obtained by the method of the present invention has a higher engraftment rate than the conventional dermal replacement by using amnion instead of a silicone membrane, and is implanted in the wound tissue which is characteristic of the amnion itself. It can be usefully used as a dressing for wound healing by showing anti-inflammatory effect and promoting wound healing.

In another aspect, the present invention provides a dermal replacement, characterized in that the cells are cultured in the amnion-biodegradable polymer structure prepared by the method for preparing the dermal replacement.

Cells cultured in the polymer structure may be used, such as fibroblasts, keratinocytes, chondrocytes, bone cells, muscle cells, corneal stem cells.

The present invention also provides a artificial artificial skin prepared by culturing cells on the amnion-biodegradable polymer structure, and then culturing the cells on the amniotic membrane of artificial dermis and a method of manufacturing the same.

The cultured cells can be used fibroblasts, keratinocytes, chondrocytes, bone cells, muscle cells, corneal stem cells, and the like.

By using the amnion as a basement membrane component of bioartificial skin, it is possible to overcome the problem that there is no basement membrane component of a conventionally used dermal substitute.

According to the role of the amnion itself as the basement membrane of the amnion itself, it is possible to provide a dermal substitute that can reduce pain and reduce the patient's pain by creating an environment in which a single transplant can be performed as an alternative to the existing two-stage transplantation.

In addition, by culturing human fibroblasts in the artificial dermal substitute prepared according to the present invention, partial layer transplantation or autologous and homogenous culture of patients for the development of bioartificial skin that can be used when the wound is large and the cell migration around the wound is difficult It can be used as a support including the basement membrane for epidermal cell transplantation. Therefore, the dermal substitute of the present invention can be usefully used as a substrate for manufacturing various artificial organs such as artificial skin, artificial cornea, artificial cartilage, artificial bone, artificial muscle, and the like.

As an example of such an application range, a stem cell line or adult cell line, which is transplanted with autologous cells of a patient on the amniotic membrane of the present invention after culturing fibroblasts in the biodegradable polymer scaffold of the present invention or isolated from the human body. ) Or as a substrate for culturing immortalized cell lines.

In another test example of the present invention, when the amnion-collagen sponge composite structure and the self-dermal epidermis of the present invention are implanted in a New Zealand white rabbit, the engraftment rate is superior to that of using only the collagen sponge or the case where the collagen sponge and the self-epidermal graft are transplanted. It does not have an inflammatory reaction and is excellent in the formation and proliferation of fibroblasts and blood vessels, and shows an excellent function as a dermal substitute.

Hereinafter, the present invention will be described in more detail based on the following reference examples, examples and test examples, but the content of the present invention is not limited to the following reference examples, examples and test examples.

Reference Example 1 Preparation Example 1 of Amniotic Membrane

Amniotic membranes are classified as infectious wastes under the domestic law, and in order to ensure their safety, serological screening for placental mothers was performed in accordance with commonly applied tissue bank regulations, and the amnion was separated from the placenta only in normal cases. Refrigerated and stored in saline. After transportation, the separated amniotic membrane was added to about 400 ml of sterile saline solution (0.9% NaCl), washed four times with a shaker for 10 minutes, soaked in fresh sterile saline solution (0.9% NaCl), and stored in the refrigerator overnight. To hydrate the sponge layer of the amniotic membrane and then remove it. It was again washed four times for 10 minutes in sterile saline (0.9% NaCl). The washed amniotic membrane was used in the form of a sheet, or in the form of a mesh, which is a conventional medical device, or artificially perforated mesh, and used when attached to a collagen sponge. .

Reference Example 2 Preparation Example 2 of Amnion

The amnion extract was ground using a mortar and pestle after rapidly freezing the amnion washed in Reference Example 1 in liquid nitrogen. The pulverized product was homogenized using a homogenizer, followed by centrifugation (6000 rpm, 30 minutes) to obtain a supernatant, which was filtered using an ultrafiltration membrane filter (Centrikon) to prepare amnion extract.

Example 1 Amnion-Collagen Sponge Composite Preparation Example 1 (Adhesion of Amnion during Collagen Sponge Preparation)

Collagen (Matrixen-ASP, Bioland) was dissolved in acetic acid or pepsin to prepare a solution of pH 3.0, concentration 0.5%, and stirred for 5 minutes at 1500rpm with a homogenizer (homogenizer). The solution in the form of a cream was applied to a 12-well plate, which is a mold with amnion prepared in Reference Example 1, 1.5 ml, and freeze-dried for at least 36 hours after freezing in a cryogenic freezer of -196 ℃ to 0 ℃. Then, the amnion-collagen sponge composite prepared to increase the biodegradability and tensile strength was treated with 0.25% glutaraldehyde (GAD), which is a conventional crosslinking method, to cross-link collagen and amnion to prepare amnion-collagen sponge composite. It was.

<Example 2> Collagen Sponge-Amnion Composite Preparation Example 2 (Amnion adhesion after crosslinking treatment of sponge)

Collagen (Matrixen-ASP, Bioland) was dissolved in acetic acid or pepsin to prepare a solution of pH 3.0, concentration 0.5%, and stirred for 5 minutes at 1500rpm with a homogenizer. 1.5 ml of the solution in the form of a cream was applied to a 12-well plate, and then frozen in an cryogenic freezer of -196 ℃ to 0 ℃, and then lyophilized for at least 36 hours. The lyophilized porous sponge was placed in a vacuum oven, and vacuum was removed at room temperature for 2 hours or more to remove a small amount of water from the sponge, and the temperature was raised to 110 ° C. to maintain a vacuum for 24 hours. After 24 hours, the temperature was lowered to 30 ° C., and the vacuum was removed to form a covalent bond in the collagen molecule, thereby performing DHT crosslinking. This stabilizes the structure and suppresses the change in structure at the time of crosslinking. After the second cross-linking process using a conventional cross-linking method of 0.25% glutaraldehyde (GAD) or 1,3-carbodiimide (CAD) and the like to cross-link the collagen surface of the collagen sponge with 0.05% collagen solution After the coating, the amniotic membrane of Reference Example 1 was attached to one or more sides, and then frozen in an cryogenic freezer at -196 ° C to 0 ° C and lyophilized for at least 36 hours.

1A and 1B are scanning electron micrographs of a bottom surface and a cross section of a collagen sponge prepared according to the prior art, and a pore size is about 60 μm to 120 μm.

In addition, the attached form of the amnion and the sponge of the composite structure of the amnion-collagen sponge of the present invention is shown in Figures 2a and 2b, Figure 2a is a method of Example 1, Figure 2b is prepared by Example 2 There was no significant difference in the attached form.

Experimental Example 1 Observation of Anti-inflammatory Effect of Amnion-Collagen Sponge Composite Structure: Intrastromal Transplantation Test of Rabbit Cornea

In order to confirm the degree of inflammatory cells through animal transplantation of the prepared amnion-collagen sponge complex structure, by implanting the collagen sponge and the amnion-collagen sponge complex structure of Example 2 of the present invention in the intrastroma of rabbit cornea The anti-inflammatory effect of the amniotic membrane was confirmed, and tissues were collected at 1 month after transplantation, and then cut into 5 μm thickness through fixation, washing, paraffin embedding, and H & E (Hematoxyline & eosin) staining. The anti-inflammatory effect was observed under the microscope.

Figure 3a is a picture of the entire tissue implanted only in the stromal cornea, it was confirmed that the infiltration of inflammatory cells around such implants continued on day 30 in Figure 3b, the amnion-collagen sponge of the present invention as shown in Figure 3c When the complex structure was implanted, it was confirmed that the anti-inflammatory effect by inhibiting the infiltration of inflammatory cells.

Experimental Example 2 Observation of Anti-inflammatory Effect of Amnion-Collagen Sponge Complex: Subcutaneous Transplantation Experiment in Nude Mouse

To confirm the anti-inflammatory effect of the amniotic membrane, a subcutaneous transplantation test was performed on the collagen sponge and the collagen sponge immersed in the amniotic extract on the back of nude mice. Observed.

After immersing the collagen sponge in the amnion extract prepared in Reference Example 2 for 24 hours, it was transplanted to a nude mouse to observe the anti-inflammatory effect of the amnion extract, and only the collagen sponge was transplanted as a control.

Figure 4a is only a collagen sponge implant, Figure 4b is a collagen sponge immersed in the amniotic membrane extract, the collagen sponge immersed in the amniotic membrane extract is less inflammatory reaction, and even after a week of implantation without decomposing the original form of the sponge It was kept as it is.

Experimental Example 3 Observation of Skin Wound Healing Effect of Amniotic-Collagen Sponge Composite Structure: Dermal Substituted Transplantation Experiment of a Skin-Defected Guinea Pig

In order to confirm the wound healing effect of the amnion-collagen sponge composite structure manufactured on skin defects, 100 mg of ketamine hydrochloride was injected into the guinea pig (SAMTACO), weighing 350-400 g, by intramuscular injection. After performing general anesthesia by administering / kg, hairs such as guinea pigs were removed with an electric epilator, washed with soap and water, and then again coated with 10% povidone-iodine and then sterilized with 70% ethanol. . Make two round 2cm-diameter wounds on the back of the guinea pig and transplant one of Terudermis ^TM (Terumo, Japan), currently commercially available as artificial dermis, as the control group, and the other as the test group. After implanting the amnion-collagen sponge composite structure of Example 1 of the invention was covered with a polyurethane film (polyurethane film) and fixed with an elastic bandage. Tissue sections were made 10 days after transplantation, H & E (Hematoxyline & eosin) staining, and wound healing was observed through the stained cell morphology.

5A and 5B are biopsy photographs of the tenth day after transplanting the complex structure of the amnion-collagen sponge of the present invention, which is a control group, Teruthemis and a test group, to the entire skin defect site, respectively, and on the 10th day after the dermal replacement implantation. Angiogenesis and fibroblast proliferation were observed from the lower part of the dermis, and both groups showed increased collagen synthesis.

In addition, in the control group of FIG. 5A, migration of keratinocytes under the silicon membrane was not observed, whereas in the test group of FIG. 5B using amnion, thin skin and migration of keratinocytes from around the wound were observed. It was confirmed that it is faster than the control group.

Experimental Example 4 Observation of Corneal Wound Healing Effect of Amnion-Collagen Sponge Composite Structure

The corneal limbus of rabbits was damaged with N-heptanol to allow neovascularization of the cornea to grow, followed by implantation of the collagen sponge and the amnion-collagen sponge complex structure of the present invention, respectively, for 3 days, 7 days, and 30 days. On the day, the effect of collagen sponge and amnion-collagen sponge composite structure was compared.

6a, 6b, and 6c are photographs of the 3rd, 7th, and 30th days after transplanting only the collagen sponge, and FIGS. 6d, 6e, and 6f are 3, 7 after implanting the amnion-collagen sponge composite structure of the present invention. As a photograph of the day and 30 days, the complex structure of the amnion-collagen sponge was inhibited in the complex structure of the amnion-collagen sponge 30 days after transplantation, and the collagen sponge of the complex structure was less degraded.

Test 5 Observation of the engraftment effect on autologous epidermal transplantation of amnion-collagen sponge composite structure

The effect of increasing the engraftment rate of the amniotic membrane on epidermal transplantation and the wound healing effect on the simultaneous execution of the manufactured artificial dermis and autologous epidermal transplantation were performed by performing the epidermal transplantation at the same time as the transplantation of the amnion-collagen sponge composite structure of the present invention.

First, three round-cut wounds of 4 cm diameter were created on the back of a New Zealand White Rabbit (Samtaco) weighing 2.4 kg, and only one collagen sponge was implanted on one side and 0.25 using dermatome. mm epidermal grafts were performed, and the other side was implanted with the amnion-collagen sponge composite structure of Example 2 of the present invention and then subjected to pressure dressing after the same thickness autografts.

In addition, only the collagen sponge was transplanted without performing the above autograft as a control group, and three mice per group were compared.

Tissues were collected 7 and 14 days after transplantation and stained with H & E (Hematoxyline & eosin), and wound healing was observed. Wound healing was assessed under the light microscope under observing the degree of engraftment, inflammatory cells, neovascularization, and fibroblast proliferation. The wound healing was divided into four groups: +, ++, +++, and ++++.

Figure 7a is only a sponge implant as a control, Figure 7b is a self-epidermal graft on the site where the sponge is implanted, Figure 7c is a self-epidermal graft on the amnion-collagen sponge complex structure of the present invention, histology As a result, the epidermal layer of the amnion-collagen sponge was partially detached when the collagen sponge without amnion was transplanted in the group with autologous epithelial transplantation. The amnion of the site was confirmed to be biodegradable without an inflammatory response. In addition, as the epidermal transplantation of the complex structure of the present invention at the same time, the infiltration of peripheral fibroblasts and vascular endothelial cells into the complex structure proceeded faster and more actively than the control group that did not have autologous epidermal transplantation. Table 1 below.

Work Control collagen sponge only transplant Collagen Sponge / Self Cuticle Transplantation Amnion- Collagen Sponge Skin Graftance (Split-thickness) 7 - + ++++ 14 - +++ Inflammation 7 ++ ++ ++ 14 + + + Newvascularization 7 + + +++ 14 ++ ++ ++++ Fibroblastproliferation 7 + ++ +++ 14 ++ +++ ++++ Inflammation (number of infiltrated leukocytes): 0-10 (+), 10-100 (++), 100-500 (+++),> 500 (++++) neovascularization (endothelium) Number of lumens alongside cells): 0-10 (+), 10-20 (++), 20-30 (+++),> 30 (++++) Fibroblast proliferation (proliferated fiber) Blast number): 0-100 (+), 100-500 (++), 500-1000 (+++),> 1000 (++++)

Test Example 6 Determination of Epidermal Growth Factor in Amniotic Membrane

The amniotic membrane was collected within 12 hours from the placenta obtained by caesarean section. The harvested amnion was microbially cultured in TSA medium (Tryptic soybean casein digest medium), and the culture was incubated at a temperature of 37 ° C. for at least 7 days. Discard microorganisms after incubation, discard the amnion without microorganisms in about 400 ml of sterile saline, wash 4 times in a shaker for 10 minutes, and soak them in fresh sterile saline (0.9% NaCl). After storing overnight in a refrigerated state, the sponge layer of the amniotic membrane was hydrated and then removed. It was again washed four times for 10 minutes in sterile saline (0.9% NaCl).

The washed amniotic membrane was frozen and lyophilized, weighed and extracted in PBS to obtain amnion extract. The extract was centrifuged at 15,000 rpm for 5 minutes and the supernatant was taken to quantify epidermal growth factor (EGF, Goma Biotech). EGF quantification was performed using an EGF detection kit (Asan Pharmaceutical), and the amount of EGF of the samples was determined based on the absorbance (540 nm) value according to the standard EGF amount of FIG. 8, and the results are shown in Table 2 below.

Untreated group (pg / mg) Freezing (pg / mg) Lyophilization (pg / mg) Sample 1 3.02 2.2 2.31 Sample 2 2.53 2.27 2.54 Sample 3 3.89 3.4 3.7

The results of Table 2 show that the amount of epidermal growth factor is not significantly reduced and preserved even in the processing of the desired product such as amnion freezing and lyophilization. The epidermal growth factor of the amniotic membrane can be expected to promote the healing of wounds.

Experimental Example 7 Immunohistochemical Staining for Bioactive Factors in Amniotic Membrane

EGF-R antibody (DAKO, K0675) and collagen to identify localization of epidermal growth factor receptor EGF-R and collagen type IV, the major constituents of basal lamina, among bioactive factors Immunohistochemical staining was performed by immuno-peroxidase method using a type IV antibody (NeoMarkers, MS-747-S1).

In FIG. 9A, the dark brown portion is a portion stained for EGF-R, and the dark brown portion of FIG. 9B is a portion stained for collagen type IV. As a result of staining, a basement membrane with epithelial cells in the amnion is obtained. Strong staining was observed around.

<Example 3> Preparation of full-layered artificial skin using amnion-collagen sponge composite structure .

3-1. Manufacture of artificial dermis

The dermal replacement of the amnion-collagen sponge of Example 2 was prepared in the form of a circular disk with a diameter of 3 cm and placed in a culture dish with a diameter of 3.5 cm. About 3 × 10 ⁶ dermal cells were inoculated into 1 ml of 10% FBS / DMEM inoculated into the prepared dermal substitutes, and 2 ml of medium was added after 5 hours in 37 ° C. and 5% CO ₂ incubator. The day medium was changed and then incubated for one week while the medium was changed every two days.

3-2. Manufacture of Full-Layered Artificial Skin

Artificial skin was prepared by double culturing keratinocytes on the amniotic membrane of the artificial dermis prepared in 3-1 above.

A culture plate insert (millicell, Millipore) with a 3 μm pore polycarbonate membrane was placed in a 6-well plate, cultured, shrunk artificial dermis was placed on the insert, and keratinocytes were inoculated thereon. . 5 x 10 ⁵ keratinocytes were inoculated onto the artificial dermis per insert having a diameter of 30 mm, and the insert was filled with 2 ml of serum-free medium for keratinocytes and 3 ml of the medium outside. When the tissue thus produced is immersed in the medium for one week, keratinocytes grow and fill the surface. The medium in the insert was then removed and the medium only filled out of the insert. This means that all the nutrients and growth factors are delivered to the epidermal cell layer by diffusion through artificial dermis, so it can be regarded as an environment similar to the biological epidermis. Thus, the growth of keratinocytes in multiple layers by culturing for two weeks at the interface between the atmospheric phase and the liquid medium. And differentiated. Figure 10 is an immunohistochemical staining picture of the whole layer of artificial artificial skin of the amniotic membrane of the present invention prepared as described above, in order to confirm the cytokeratin expressed during differentiation of keratinocytes, cytokeratin antibody (Biomedical Technologies) Immunohistochemical staining was performed using the immuno-peroxidase method, and staining of the entire keratinocyte layer was observed.

According to the present invention, compared with the dermal substitute composed of the conventional silicone film and collagen sponge, the use of amnion instead of the silicone film is more biocompatible, and the inflammatory response due to anti-inflammatory action, which is one of the characteristics of the amniotic membrane, and thus Durability can be secured by delaying biodegradation rate. In addition, it promotes wound healing by various growth factors and differentiation factors of the amnion itself, and also provides an environment in which one transplantation operation is possible as an alternative method of transplantation that is applied twice over the existing dermal replacement depending on the role of the amnion itself. By making it possible to provide a dermal substitute that can reduce pain and reduce cost of the patient. Furthermore, by culturing human fibroblasts in the artificial dermis prepared by the present invention, partial layer transplantation or autologous and allogeneic cultivation of a patient for the development of a bioartificial skin that can be used when the wound is large and cell migration around the wound is difficult It can be used as a support including the basement membrane for the epidermal cell transplantation. Therefore, the present invention consisting of amnion and biodegradable substrate can be usefully used as a substrate for the preparation of artificial organs using various tissue engineering such as artificial skin, artificial cornea, artificial cartilage, artificial muscle.

Claims (9)

A dermal substitute characterized by having a complex structure in which a biocompatible amnion separated from the placenta and a biodegradable polymer matrix are attached, incorporated or inserted into each other. The dermal substitute of claim 1, wherein the biodegradable polymer is selected from the group consisting of collagen, gelatin, hyaluronic acid and derivatives thereof, chitin, chitosan, PLGA, PGA and PLA. A biocompatible amniotic membrane separated from the placenta and a biodegradable polymer matrix are attached to each other, incorporated or inserted to have a complex structure. The method of claim 3, wherein the biodegradable polymer is at least one selected from the group consisting of collagen, gelatin, hyaluronic acid and derivatives thereof, chitin, chitosan, PLGA, PGA and PLA. The method of claim 3, wherein the amnion is a membrane-like structure, a mesh-like structure or an extract form using doubling or inserts. The method of claim 3, wherein the amnion is attached or inserted into one or more sides during or after collagen sponge production. A method for producing a dermal replacement, characterized in that the cells are cultured in the amnion-biodegradable polymer structure prepared by the method for preparing the dermal replacement of claim 3. The method of manufacturing bioartificial skin, wherein the cells are cultured in the amnion-biodegradable polymer structure prepared by the method for preparing the dermal substitute of claim 3, and then the cells are double-cultured on the amnion. The method of claim 7 or 8, wherein the cells are at least one selected from the group consisting of fibroblasts, keratinocytes, chondrocytes, bone cells, muscle cells and corneal stem cells.