KR20130083596A - Method for preparing dermal substitute for treatment of wound containing collagen and hialuronic acid and dermal substitute prepared therefrom - Google Patents

Abstract

PURPOSE: A method for preparing a dermal substitute containing collagen and hyaluronic acid for wound healing and the dermal substitute are provided to effectively substitute dermis with excellent biocompatibility by comprising large and uniform pores and connection paths and to overcome the limit of a conventional two-step surgery through a hyaluronic acid regeneration effect and a moisturizing effect. CONSTITUTION: A method for preparing a dermal substitute for wound healing comprises the steps of: reacting non-antigenic collagen or collagen derivatives with hyaluronic acid and forming a collagen-hyaluronic acid complex by ionic bond; homogenizing the collagen-hyaluronic acid complex and preparing a dispersion; naturally precipitating the dispersion and freeze-drying the collagen-hyaluronic acid complex; and physically or chemically crosslinking the freeze-dried collagen-hyaluronic acid complex.

Description

TECHNICAL FIELD OF PREPARING DERMAL SUBSTITUTE FOR TREATMENT OF WOUND CONTAINING COLLAGEN AND HIALURONIC ACID AND DERMAL SUBSTITUTE PREPARED THEREFROM}

The present invention relates to a method for the preparation of dermal replacements for the treatment of wounds containing collagen and hyaluronic acid and to the dermal replacements prepared therefrom.

In general, if skin damage is limited to the epidermis, such as a mild burn within 2 degrees, skin tissue can be regenerated without special measures.However, if the dermis is damaged, such as a deep burn, regeneration is difficult. Implanting. In this case, in order to cover and protect the affected area until skin graft, wound dressings (wound dressings) that can block external environments such as bacteria and the like while maintaining adequate permeability are used.

In the past, synthetic polymer membranes such as urethane polymers and leucine polymers (poly-L-leucine) have been used as wound dressings, but they have problems in which they are not suitable for living bodies. To improve this, wound dressings designed to be removed when skin tissue is regenerated by making a film combining a suitable water-permeable polymer film and a biocompatible structure have been developed, and Biobrane ^TM developed in the early 1980s is a representative example. Can be. BioBrain has a double structure consisting of a nylon woven body coated with collagen and a fine porous silicone membrane. Because it has good adhesion to the skin and elasticity, it adheres to the wound surface and the antibiotic applied regularly through the silicone membrane is affected. It has the advantage that it can be transmitted to, it is widely used in burns of less than 2 degrees or the same degree of skin peeling.

On the other hand, dermal substitutes are currently used for severe wounds of 2 degrees or more. Dermal replacements have been used to treat wounds by implanting them in the wound and then covering them with subcutaneous epidermal pieces about two weeks later, or with cultured epidermal cells. Dermal replacements are made by using collagen in the form of sponges or gels or by using biodegradable polymers. When the dermal replacements are used in whole skin defect wounds, fibroblasts and capillaries penetrate into the replacements at the wound site and proliferate, Collagen fibers are formed by the synthesis of autologous collagen, and dermal tissue is constructed. Subsequently, the substitute is decomposed and absorbed, and the formed dermis-like tissue becomes complete magnetic dermal tissue. As such, substances that play a role in changing the material itself into biological dermal tissue are called dermal substitutes.

Current commercialized dermal substitutes include Integra ^TM in the US and Terudermis ^{TM in} Japan, imported and licensed as medical devices from the US FDA and the FDA. The dermal substitute consists of a synthetic polymer of silicon membrane and a collagen layer extracted from bovine skin, the upper layer of silicon membrane prevents evaporation and infection of body fluids, and the lower layer of collagen induces regeneration of new blood vessels and connective tissue. It is broken down and absorbed in the human body. However, the treatment of severe burns using the dermal substitute is performed at least twice, including the step of forming a new dermis within 2 to 3 weeks by implanting the dermal substitute in a wound and performing a skin graft surgery to form an epidermal layer on the formed new dermis. Since there is a need for a transplant procedure over a long period, long-term hospitalization and pain of the patient are inevitable.

In order to improve this, we have been seeking to replace a single surgical procedure by performing autologous or cultured epidermal transplantation at the same time as transplantation of dermal substitutes. Necrosis of the autologous epidermis attached to the dermal substitute is induced due to the proliferation of fibroblasts and the delay of capillary formation for the formation of the growth rate is limited.

In addition, in the case of conventional dermal replacement, silicone membrane is used for temporary epidermis until second partial transplantation, 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 promoted. Therefore, while increasing the engraftment rate for epidermal transplantation, it is possible to heal wounds more effectively through anti-inflammatory and wound healing promoting effect, and it can be treated by one-step surgery by autologous epidermal transplantation or culture epidermal transplantation with the transplantation of dermal substitute. There is a need for development of biocompatible dermal replacements.

In order to enable one-stage surgery with autologous epidermal transplantation, it is important to prepare a porous dermal replacement. Preferred dermal replacements have a high rate of absorption that allows for transport of material by diffusion during the period prior to neovascularization through the dermal replacement so that autologous epidermal grafts applied at the same time as the dermal replacement can survive. It may have a thickness that is sufficient to maintain the state, and may have a pore size to allow the surrounding cells to enter, proliferate, and differentiate to form a new dermis after application to a damaged dermal region, and to migrated fibroblasts. In order to facilitate the formation of neovascularization for smooth nutrition, it may have a connection path through which blood vessels and substances can be transported between adjacent pores.

As such, the lyophilization method has been most frequently used to ensure a uniform distribution of pores of 40 to 150 μm and a through passageway between adjacent pores. The freeze-drying method may freeze water in an aqueous solution through freezing to form crystals, and sublimate (dry) the frozen ice crystals without going through a liquid state under vacuum to obtain a porous form. As a freeze-drying method currently used to obtain a dermal substitute having an excellent porous structure, there may be mentioned a method of controlling the temperature to be frozen and a method of preparing a collagen solution to be frozen using an acetic acid solution. For example, as a method of controlling the temperature condition to be frozen, there are a rapid freezing method and a cooling rate control freezing method, which is usually frozen under ultra low temperature (below -180 ° C) using liquid nitrogen to obtain collagen solution. Although it can be induced to freeze uniformly by minimizing the rate of freezing, it is difficult to secure pores above a certain size, and there are disadvantages in that the size of pores that can be formed is restricted. In addition, in order to overcome this problem, a method of obtaining a uniform pore structure while maximizing the size of pores while slowly freezing by controlling a constant cooling temperature when freezing a collagen solution has been studied. There is a limit to obtaining a dermal replacement with uniform pores.

Therefore, the present inventors are studying to prepare a dermal substitute capable of one-step surgery by adjusting the size of the pores, and make a complex by combining collagen and hyaluronic acid, and from the freeze-drying using natural sedimentation induction, excellent porous structure It has been found that dermal replacements can be prepared and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for the preparation of dermal substitutes having a dualized porous microstructure, which is capable of one-step surgery that can be used simultaneously with autologous epidermal implantation in the treatment of burns or wounds of more than two degrees deep.

Another object of the present invention is to provide a dermal replacement for wound treatment prepared by the above method.

In order to achieve the above object, the present invention comprises the steps of 1) reacting non-antigenic collagen or collagen derivatives with hyaluronic acid to form an ion-bonded collagen-hyaluronic acid complex; 2) preparing a dispersion by homogenizing the collagen-hyaluronic acid complex; 3) natural precipitation of the dispersion, followed by lyophilization; And 4) physically or chemically crosslinking the lyophilized collagen-hyaluronic acid complex.

In order to achieve the above another object, the present invention provides a dermal replacement for wound treatment prepared by the method.

The dermal substitute prepared by the method of the present invention has a large and uniform sized pore and air gap connecting passage, and is excellent in biocompatibility and dermal replacement effect, and the conventional two-stage surgery due to the regeneration effect of hyaluronic acid and excellent moisturizing power In addition to overcoming the limitations, the engraftment effect of the autologous epidermis implanted by the dualized porous microstructure can be enhanced, so that the treatment can be performed in one step of surgery, and thus can be usefully used for the treatment of burns or wounds.

1 is a photograph of a dermal substitute prepared by Example 1 of the present invention.
Figure 2a is a scanning electron micrograph of the dermal substitute surface prepared by Example 1 of the present invention, Figure 2b is a scanning electron micrograph of the dermal substitute surface prepared by Comparative Example 1.
Figure 3a is a whole cross-section of the dermal substitute prepared by Example 1, Figure 3b is a top view and Figure 3c is a scanning electron micrograph of the bottom.
Figure 4a is a whole cross-section of the dermal substitute prepared by Comparative Example 1, Figure 4b is a top view and Figure 4c is a scanning electron micrograph of the bottom.
Figure 5 shows the cytotoxicity of the negative control group (5a), positive control group (5b), collagen-hyaluronic acid complex (5c), collagen derivatives-hyaluronic acid complex (5d) for cytotoxicity experiments under the microscope The photograph was observed.
FIG. 6 shows the collagen-hyaluronic acid complex (FIG. 6a) prepared using the non-derivatized collagen of Example 1 and the collagen derivative-hyaluronic acid complex of FIG. Tissue stain slide photo.
Figure 7 shows the dermal replacement of Example 2 in the pig that induced the wound more than 2 degrees deep in the same day 5 days (7a), 7 days after surgery (7b), 14 days after surgery (7c) , And 21 days after surgery (7d) H & E biopsy slide observation picture.
FIG. 8 shows the dermal replacement of Example 3 in which pigs induced deep wounds of 2 degrees or more deeply at the same time with autologous skin at 5 days (7a), 7 days after surgery (7b), and 14 days after surgery (7c). , And 21 days after surgery (7d) H & E biopsy slide observation picture.

The present invention comprises the steps of: 1) reacting non-antigenic collagen or collagen derivatives with hyaluronic acid to form ion-bonded collagen-hyaluronic acid complexes; 2) preparing a dispersion by homogenizing the collagen-hyaluronic acid complex; 3) natural precipitation of the dispersion, followed by lyophilization; And 4) physically or chemically crosslinking the lyophilized collagen-hyaluronic acid complex.

Step 1 of the method according to the invention is a step of reacting non-antigenic collagen or collagen derivatives with hyaluronic acid to form an ionically bonded collagen-hyaluronic acid complex.

In the method of the present invention, it is preferable to use non-antigenic collagen (for example, Matrixen-PSC, Bioland, Korea) as a non-antigenic collagen. While conventional dermal replacements use collagen extracted from cow skin or tendon, there is a risk of infection of transmissible spongiform encephalopathy (TSE), whereas collagen used in the method of the present invention is essentially TSE infection. Extracted using non-hazardous pig skin, and both ends of the antigenic collagen molecule are removed during the extraction process, resulting in non-antigenic activity, and have been proven to be inactivated against viruses that are at risk of being transmitted through pigs. Do.

In one embodiment, the collagen may be derivatized. In one embodiment, the derivatized collagen may be methylated collagen. Derivatization of collagen can be achieved by substituting a methyl group with a negatively charged carboxyl group, such as aspartic acid and glutamic acid, among the various amino acids constituting collagen, and when derivatizing collagen, Molecules change to a positive charge-rich state to prevent sedimentation by reaching the isoelectric point at neutral conditions of physiological pH, and negatively charged by abundant positive charges with the advantage of utilizing dissolved collagen at physiological pH conditions. Can improve the binding of ions and macromolecules.

In one embodiment of the invention, the methylated collagen is prepared by adding a large amount of acetone to 0.1-4%, preferably 0.5% of collagen dissolved in 0.1-100 mM, preferably 1 mM hydrochloric acid solution. Recovering the precipitate; Dissolving the recovered collagen in anhydrous methanol containing 1 to 0.001 M, preferably 0.1 M hydrochloric acid, and then reacting with stirring for 1 to 30 ° C., preferably 4 ° C., under sterile conditions for 1 to 6 days; The dispersion is placed in a dialysis membrane and dialyzed at 2 to 25 ° C., preferably 4 ° C., using purified water, followed by lyophilization of the dialyzed solution.

The reaction of the non-antigenic collagen or collagen derivative obtained above with hyaluronic acid can be carried out by preparing each as a solution. The solution may contain, but is not limited to, non-antigenic collagen or collagen derivatives and hyaluronic acid in concentrations of 0.5% to 3% by weight, respectively. In addition, the non-antigenic collagen or collagen derivative and hyaluronic acid used in the reaction may be used in a weight ratio of 50:50 to 90:10, but is not limited thereto.

Step 2 of the method according to the invention is a step of homogenizing the collagen-hyaluronic acid complex to prepare a dispersion. The homogenization is a step for facilitating ionic bonding between collagen and hyaluronic acid, and may be performed using a conventional method known in the art, for example, a homogenizer.

Step 3 of the method according to the invention is a step of spontaneous precipitation of the dispersion obtained in step 2, followed by lyophilization. In one embodiment, the dispersion obtained in step 2 is poured into a mold of a high thermal conductivity metal material (e.g., platinum, tin, copper, aluminum, iron, lead, tungsten, etc.) to achieve a given size and thickness. It may then be allowed to spontaneously settle for 1 to 24 hours at a constant temperature, such as 1 to 10 ° C. Through the process of inducing the natural sedimentation, the concentration gradient of the dispersion can be made, and then through the freezing process it can form a binary porous microstructure having an upper dense layer and a lower dense layer. The pre-cooled dispersion is frozen for 24 hours to -50 ℃ at a uniform cooling rate, and then subjected to initial drying sequentially to -40 ℃, -30 ℃, -20 ℃, -10 ℃, 0 ℃ while performing a vacuum After 48 hours, it may be sequentially dried for 24 hours to 10 ℃, 20 ℃. The freezing and drying manner can be carried out using various methods known in the art.

Step 4 of the method according to the invention is a step of physically or chemically crosslinking the lyophilized collagen-hyaluronic acid complex. The crosslinking reaction is carried out to impart robustness to maintain morphology after hydration and to maintain degradation resistance in the body so that the lyophilized complex can serve as a dermal substitute. The crosslinking reaction may be accomplished through various methods known in the art, for example, physical or chemical crosslinking methods. Physical crosslinking includes vacuum heating (Dehydrothermal treatment), for example, it may be carried out by heating to 90 ℃ ~ 150 ℃ under vacuum, then maintained for 10 hours to 48 hours. On the other hand, the chemical crosslinking method includes a method using a crosslinking agent. For example, as a crosslinking agent, EDC (N-Ethyl-N '-(3-dimethylaminopropyl) which is removed during the washing process without being bound to the collagen sponge after participating in the reaction. carbodiimide hydrochloride) and formaldehyde, paraformaldehyde, glutaraldehyde, hexamethylene diisocyanate (1,6-diisocyanatohexane), epoxy resin (polyepoxy: PEC) and the like.

Dermal substitutes subjected to step 4 may be further subjected to a sterilization process. In one embodiment, the dermal replacement is of suitable size (eg, 6 mm (1 or 2 mm), 8 mm (1 or 2 mm), 3 * 3 cm, 5 * 5 cm, 5 * 7 cm, 10 * 10 cm, 10 * 15 cm, 21 * 30 cm) can be cut and packaged and sterilized (eg, ethylene oxide sterilization or gamma and electron beam irradiation, etc.).

On the other hand, in the method of the present invention, in order to increase the wound healing effect, step 1 may further include beta glucan known to exhibit an immune enhancing and wound healing effect. The beta glucan may be used in an amount of 0.1 to 2% by weight, preferably 0.5% by weight based on the concentration of the collagen or collagen derivative solution.

Furthermore, the present invention provides a dermal replacement for wound treatment prepared by the method described above. The dermal substitute prepared according to the method of the present invention is characterized by having a dualized porous microstructure composed of a top dense layer and a bottom dense layer. The dermal replacement may be prepared in various sizes depending on the intended use, but may preferably have a thickness of 0.5 to 5 mm, most preferably 1 to 3 mm. In addition, the dermal substitute is characterized in that the content of hyaluronic acid in the dermal substitute is 50 to 90% based on the amount of hyaluronic acid used in the reaction.

The dermal substitute according to the present invention not only has larger and uniformly sized pores and space connecting passages, but also can increase the engraftment effect with the transplanted autologous epidermis due to the dualized porous microstructures. Surgery can be useful for treating wounds of more than two degrees deep.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the embodiments are only illustrated to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example  1: Preparation of dermal substitutes (1)

Dermal substitutes were prepared as follows using collagen and hyaluronic acid.

<1-1> Preparation of Collagen-Haluronic Acid Complex

Specifically, 100 mL of non-antigenic collagen solution (Matrixen-PSC, Bioland, South Korea) in which 0.5% collagen was dissolved in 1 mM hydrochloric acid was purchased from Bioland (Korea). On the other hand, hyaluronic acid was dissolved in purified water to prepare a 0.5% by weight hyaluronic acid solution.

The collagen solution and the hyaluronic acid solution prepared above were mixed at a capacity corresponding to a ratio of 50:50 based on the weight percent of each solution to induce ion bonding. The collagen-hyaluronic acid complex aggregates precipitated after ion bonding were homogenized using a homogenizer to evenly disperse the aggregates. The prepared collagen-hyaluronic acid dispersion is poured into a rectangular parallelepiped mold made of a high thermal conductivity metal material (copper or stainless steel) to a thickness of the desired dermal substitute (for example, 1 mm or 2 mm), and then the mold is cooled. The freezing was mounted on a shelf of a freeze dryer capable of adjusting the speed and temperature. At this time, the temperature of the shelf was cooled to 1 to 2 ° C. in advance, and in order to induce a dual porous microstructure of the dermal substitute, the dispersion solution poured into the mold was precooled to the temperature for 7 hours to naturally settle the dispersion solution. Induced the concentration gradient of the dispersion. After the preliminary cooling, the cooling rate was adjusted step by step and then frozen at -50 ° C for 24 hours, and vacuum was performed for 48 hours to prepare a lyophilized sponge.

<1-2> Crosslinking and Sterilization of Collagen-Haluronic Acid Complex

When the collagen-hyaluronic acid composite sponge prepared in the above <1-1> is put into water, the form of the sponge collapses (it becomes a dispersion state), and thus cannot serve as a dermal substitute of a porous structure, and thus, as a dermal substitute. In order to maintain the shape and enhance the decomposition resistance in the body, the crosslinking agent was chemically crosslinked using a crosslinking agent, N-Ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

Specifically, 50 mg of the collagen-hyaluronic acid composite sponge prepared in Example <1-1> was added to 50 mM 2-morpholinoethane sulfonic acid (MES), 24 mM EDC and 5 mM NHS (N-) in 20 ml of 70% ethanol. It was added to a pH 5.5 solution containing hydroxysuccinimide) and shaken at room temperature for 4 to 24 hours. Then, washed twice with 0.1 M Na ₂ HPO ₄ (pH 9.1) solution for 1 hour, 2 hours with 1 M NaCl solution, 24 hours with 2 M NaCl solution, and finally several times with distilled water to remain. One crosslinking reagent and sodium chloride were completely removed. The crosslinked sponge was secondary lyophilized for drying. Then, use a suitable size (8 mm (1 or 2 mm)) 10 * 10cm) and cut and then sterilized using ethylene oxide gas. The prepared dermal substitute is shown in FIG. 1.

Example  2: Preparation of Dermal Substitutes (2)

Instead of using the collagen used in Example 1, except that using the derivatized collagen through the following method, the procedure of Example 1 was repeated to prepare a dermal replacement. Collagen derivatization was carried out not only to solve the aggregation phenomenon at the neutral pH of collagen, but also to improve the stabilization of the pH, as well as to improve the degree of ionic bonds with hyaluronic acid. A methyl group was bound to the carboxyl groups of phosphorous aspartic acid and glutamic acid to induce a richer positively charged state.

Specifically, 100 ml of a non-antigenic collagen solution (Matrixen-PSC, Bioland, South Korea) in which 0.5% collagen is dissolved in 1 mM hydrochloric acid was added to 2 L of acetone to precipitate collagen, and the precipitated collagen was recovered and recovered again to 0.1. 1 L of methanol containing M hydrochloric acid was dissolved in the solution, followed by stirring for 1 to 6 days at 4 ° C. under sterile conditions. Then, the resultant was placed in a dialysis membrane (molecular cut-off 12000-14000 dalton), subjected to repeated dialysis several times using purified water, and then lyophilized to obtain derivatized collagen (methylated collagen). The derivatized collagen was stored at -20 ° C until use.

Example  3: Preparation of dermal substitutes (2006.01)

In order to enhance the effect of the dermal substitute, beta glucan (β-D-Glucan, Sigma Co., Ltd.) exhibiting immuno-enhancing and wound healing effects was included in the collagen-hyaluronic acid complex prepared in Example <1-1>. Specifically, in preparing the hyaluronic acid solution in Example 2, beta-glucan was added to 0.5% by weight, and then mixed with the collagen derivative solution to prepare a collagen derivative-hyaluronic acid-betaglucan complex, The process was carried out in the same way.

Comparative example  1: Preparation of dermal replacement

In Example <1-1>, the dermal substitute was prepared by repeating the procedure of Example 1, except that the dispersion was immediately frozen instead of freezing the dispersion through natural sedimentation induction.

Experimental Example  1: Observation of the microstructure of the dermal substitute

The porous microstructures of the dermal substitutes of Example 1 and the dermal substitutes of Comparative Example 1 according to the present invention were observed using a scanning electron microscope (HITACHI S-2500) at 50 and 200 times magnifications. The scanning electron micrographs are shown in FIGS. 2A and 2B, respectively.

As a result, the dermal substitutes of Example 1 (FIG. 2a), which were freeze-dried and then freeze-dried according to the method of the present invention, were freeze-dried, the pore sizes of the dermal substitutes of Comparative Example 1 It was confirmed that it was more than twice as large and uniform and secured a through passage of air space. In contrast, the dermal substitutes of Comparative Example 1 (FIG. 2B), which were freeze-dried immediately after dispersion, were distributed in a non-uniform and dense state, such as a thin thread, so that the pore structure was too narrow for cell migration and proliferation or neovascularization. It was found to have.

In addition, as a result of observing the cross-section of the dermal substitutes of Example 1 and Comparative Example 1, according to the method of the present invention, the dispersion was frozen by a method through natural sedimentation induction, followed by the lyophilization process. In case of 3), it showed dual microporous microstructure that clearly distinguished the difference between air-side and pan-side of the cross-section, and dense layer and dense by natural sedimentation of dispersion from top to bottom. It has been observed to show a dual structured porous microstructure with a layered structure, while maintaining a perforated connection structure of the airspace. In contrast, in the case of the dermal substitute of Comparative Example 1 prepared by freeze-drying the dispersion (FIG. 4), the air-side and the pan-side showed a unitary porous microstructure with no boundary. It was.

These structural differences demonstrate that the dermal substitutes of the present invention are very advantageous for the migration and proliferation of cells or for the generation of neovascularization.

Experimental Example  2: Characterization of dermal substitutes

The dermal substitutes of Example 1 prepared using collagen and the dermal substitutes of Example 2 prepared using derivatized collagen were compared. Specifically, the safety and histocompatibility of the cytotoxicity test, the measurement of tensile strength and elongation rate, the hyaluronic acid content test, and the subcutaneous transplantation test of animals were performed as follows.

<2-1> Cytotoxicity Test

The cytotoxicity test of the collagen-hyaluronic acid complex dermal substitutes of Examples 1 and 2 and the collagen derivative-hyaluronic acid complex dermal substitutes to evaluate the cell proliferation inhibition and other toxic effects due to collagen derivatization and residual crosslinking reagents Was carried out.

Specifically, 0.5 g of each sample was placed in 20 mL of serum-free MEM medium at 37 ° C. for 24 hours to elute the residue of the sample. 2 mL of each elution medium was added to NCTC clone 929 cells grown at least 80% in 6-well plates, followed by incubation for 24 hours and 48 hours at 37 ° C., 5% CO ₂ incubator, respectively. Grade comparison (0 (no inhibition) ~ 4 (severe)) was performed, and MTT analysis was used to compare the inhibition rate of proliferation compared to the negative control. Polyethylene (HDPE) was used as the negative control group, and MEM medium containing 10% dimethyl sulfoxide (DMSO) was used as the positive control group.

The results are shown in Table 1 and FIG. 5.

sample Cytotoxicity score Growth inhibition rate
(%) 24 hours 48 hours Negative control group 0 0 100 Positive control group 3 3 33 Collagen-hyaluronic acid complex 0 0 93 Collagen derivatives-hyaluronic acid complex 0 0 92

As shown in Table 1 and FIG. 5, the positive control group had a cytotoxic score of 3, whereas the complexes of the present invention had a cytotoxic score of 0. This shows that there are no potential degradation products that cause cytotoxicity in the complexes of the present invention, making them suitable for use in vivo.

<2-2> The tensile strength  And elongation measurements

In order to examine the mechanical properties of the dermal substitutes prepared in Examples 1 and 2, tensile strength and elongation were measured by using a universal measuring instrument (LF plus, LLOYD Instrument). The measuring method is to cut a sample of the same thickness (2 mm) into a width of 10mm × length of 30mm, position the distance between the grips to 10mm, and set the tensile speed to 30mm / min to determine the maximum load when the specimen is cut. The elongation was determined by measuring and elongating the length until cutting. The measurement results are shown in Table 2 below.

Name of sample Load when cut (N) Elongation (%) Example 1 6 ± 2.7 18 ± 5.2 Example 2 9 ± 3.5 23 ± 2.3

As shown in Table 2 above, the dermal replacement of Example 2 using derivatized collagen was shown to be superior to the dermal replacement of Example 1 with both the maximum load and elongation when the specimen was cut.

<2-3> Hyaluronic Acid Content Experiment

The combined amounts of hyaluronic acid were quantitatively analyzed for collagen-hyaluronic acid complex dermal replacements and collagen derivative-hyaluronic acid complex dermal replacements prepared in Examples 1 and 2. The quantitative method was tested using the Korean Pharmacopoeia 'sodium hyaluronate assay', and the measurement results are shown in Table 3 below.

Name of sample Hyaluronic acid content (%) Collagen-hyaluronic acid complex 29.3 ± 0.34 Collagen derivatives-hyaluronic acid complex 44.08 ± 0.59

As a result of the measurement, the hyaluronic acid complex (Example 2) using the collagen derivative was found to have a hyaluronic acid content of 15% or more higher than the hyaluronic acid complex (Example 1) using the collagen without derivatization. . This shows that by derivatizing collagen (methylated collagen), the positive charge is enriched, allowing more hyaluronic acid to ionize. The results also show that, based on the hyaluronic acid content used in the reaction, 58.6% of the collagen-hyaluronic acid complex remained in the prepared dermal substitutes, and 88.2% of the collagen derivative-hyaluronic acid complex remained.

<2-4> Guinea pig  Subcutaneous transplant experiment

In order to confirm biocompatibility and in vivo degradation resistance of the dermal substitutes prepared in Examples 1 and 2, subcutaneous transplantation experiments using guinea pigs were performed as follows.

First, each dermal replacement was cut into 8 mm diameter disc types using a biopsy punch and used in the experiment. The animals for the transplant experiments used guinea pigs. The general anesthesia guinea pig was epilated using the hair removal machine and the razor blade to remove hair from the entire back area, and was cut into two left and right centers around the spine of the guinea pig to make a graft, and the dermal replacement of the present invention was implanted. . After transplantation, the suture was closed using nylon sutures, and after 4 weeks, the guinea pigs were sacrificed. Tissue biopsy of the transplanted area was fixed in 4% formalin, and then H & E tissue staining was performed for biocompatibility of inflammation and foreign body reaction, dermal degradation of dermal replacement, and distribution and density of fibroblasts. Confirmed.

As a result of histology, as shown in FIG. 6, no cells related to inflammation and foreign body reactions were observed in both dermal replacements, and the inflow and proliferation of fibroblasts into the dermal replacements were observed and transplanted 4 weeks after transplantation. It was found that more than 40% of each dermal replacement remained.

Experimental Example  3: dermal replacement Deep 2 degrees  Effectiveness Evaluation Using Damaged Animal Model

The dermal substitutes prepared in Examples 1 to 3 are most similar to the skin of the human body to determine whether a one-step operation simultaneously with autologous epidermal transplantation is possible in treating skin damaged by burns and wounds of more than 2 degrees deep. Effectiveness evaluation was performed using known pigs.

The pigs used in the experiment were micro pigs (Micro pig, Medikinetics Co., Gyeonggi-do, Korea) weighing about 20 kg, and after sleeping anesthesia, the hair on the back was removed using a hair removal machine and a razor blade. In order to cause burns or wounds of more than 2 degrees in depth, the epidermis and dermis of more than 2 mm in thickness were removed by using dermatome. At this time, the autologous epidermis (about 0.5mm thick) for the first stage surgery was obtained and stored in sterile saline solution and refrigerated until transplanted with the dermal substitute. After inducing a wound of more than 2 degrees deep, hemostasis was performed carefully, and the dermal substitutes of Examples 1 to 3 of 3 cm x 3 cm and 1 mm thick were immersed in sterile physiological saline to hydrate and then damaged. Attached. Then, the previously prepared porcine epidermis of the pig was covered with sutures to completely adhere and adhere, and then covered with a foam dressing to protect the implanted area, and then a permeable adhesive cloth was attached again. Surgery was completed using a mesh and compression bandage.

Observation period after transplantation was 5, 7, 14, and 21 days. Tissue biopsies were performed using 6 mm biopsy punches on days 5 and 14, and the entire graft site was sacrificed on day 7 and 21 after pigs were sacrificed. Biopsy was performed for H & E tissue staining.

The evaluation method was visual observation and histological observation during each transplantation period. The engraftment of grafted autologous epidermis, inflammatory reaction, foreign body reaction and neovascularization were evaluated by microscopic observation.

Experimental results are shown in FIGS. 7 and 8. FIG. 7 shows H & E histology slides observed at Day 5 (FIG. 7A), Day 7 (FIG. 7B), Day 14 (FIG. 7C) and Day 21 (FIG. 7D) after implantation using the dermal replacement of Example 2. FIG. Figure 8 is a photograph of the observation of the H & E histology slide over the same period using the dermal substitute of Example 3.

As a result of the above experiment, the histological results of each observation period, the transplanted autologous epidermis was not necrotic and engraftd with each dermal substitute, and fibroblasts and neovascularization into each dermal substitute actively progressed from the 5th day of transplantation. It was observed that as the transplantation time increased, the autologous dermis structure was gradually formed. In addition, due to the large and uniform pores of the dermal substitute of the present invention and the penetrating sufficient connecting passage of the void space, the cells were uniformly distributed into the dermal substitute, and the nutrient supply and the material transport were made by the formation of neovascularization. The autologous epidermis was not necrotic, and the autologous epidermis transplanted under the influence of dual microporous structure, which is a characteristic of the dermal substitute of the present invention, effectively and stably engrafts with the dermal substitute to treat skin defects of more than 2 degrees deep. It was confirmed that there was sufficient effect.

Claims (15)

1) reacting the non-antigenic collagen or collagen derivative with hyaluronic acid to form an ionically bonded collagen-hyaluronic acid complex;
2) preparing a dispersion by homogenizing the collagen-hyaluronic acid complex;
3) natural precipitation of the dispersion, followed by lyophilization; And
4) physically or chemically crosslinking the lyophilized collagen-hyaluronic acid complex
Including, method for producing a dermal replacement for wound treatment.
The method of claim 1, wherein the collagen derivative is methylated collagen prepared by replacing a carboxyl group of aspartic acid and glutamic acid of non-antigenic collagen with a methyl group.
The method of claim 1, wherein the non-antigenic collagen or collagen derivative and hyaluronic acid of step 1 are used in a solution of 0.5 wt% to 3 wt%, respectively.
The method of claim 1, wherein the non-antigenic collagen or collagen derivative of step 1 and hyaluronic acid are mixed in a weight ratio of 50:50 to 90:10.
The method of claim 1, wherein the natural sedimentation of step 3 is carried out for 1 to 24 hours in a temperature range of 1 ~ 10 ℃, method for producing a dermal replacement for wound treatment.
According to claim 1, wherein the physical crosslinking of step 4 is heated to 90 ℃ ~ 150 ℃ under vacuum, characterized in that it is carried out by maintaining for 10 hours to 48 hours, the method of producing a dermal replacement for wound treatment.
The method of claim 1, wherein the chemical crosslinking of step 4 comprises N-Ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and formaldehyde, paraformaldehyde, glutaraldehyde, hexamethylene diisocyanate (1,6). -diisocyanatohexane) and an epoxy resin (polyepoxy: PEC), characterized in that it is carried out using a crosslinking agent selected from the group consisting of, the method for producing a dermal replacement for wound treatment.
The method of claim 1, further comprising sterilizing the dermal substitute after step 4.
The method of claim 1, wherein beta glucan is further included in the reaction of Step 1.
A dermal replacement for wound treatment, prepared by the method of claim 1.
11. The dermal replacement for wound treatment according to claim 10, wherein the dermal replacement has a dualized porous microstructure consisting of a top dense layer and a bottom dense layer.
The dermal replacement for wound treatment according to claim 10, wherein the dermal replacement has a thickness of 0.5 to 5 mm.
The dermal replacement for wound treatment according to claim 10, wherein the content of hyaluronic acid in the dermal replacement is 50 to 90% based on the amount of hyaluronic acid used in the reaction.
11. The dermal replacement for wound treatment according to claim 10, wherein the dermal replacement is used in combination with the epidermis.
11. The dermal replacement for wound treatment according to claim 10, wherein the dermal replacement is used for the treatment of wounds of more than 2 degrees deep.

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