KR101916759B1 - The Method of High-yield and High-purity Manufacturing of Allo-collagen Composition Extracted From Human origin - Google Patents

Abstract

The present invention relates to a method for producing a human-derived allogeneic collagen extract composition, and more particularly, to a method for producing a human-derived allogeneic collagen extract composition which is superior in biocompatibility to heterogeneous collagen and which extracts allogeneic collagen which can be used for medical materials such as pain relief and tissue replenishment ≪ / RTI >
The human-derived allogeneic collagen extract composition according to the present invention is superior in biocompatibility to heterogeneous collagen, and can produce collagen with high purity and high concentration. The collagen can be efficiently separated and extracted by extracting collagen from the noncellular allogenic dermis from which non-collagen substances are removed without directly extracting collagen from the skin tissue.
In addition, non-collagen substances such as cytoplasm, DNA, and fat can be excluded from the extraction process, and high purity and high concentration of collagen can be purified.
Through the lyophilization process, it is possible to produce high purity and high concentration homogeneous collagen compositions and to manufacture various formulations.

Description

Technical Field [0001] The present invention relates to a method for producing high-purity high-purity homologous collagen and a method for producing the same,

The present invention relates to a human-derived allogeneic collagen extraction composition and a method for producing the same. More particularly, the present invention relates to a human-derived allogeneic collagen extraction composition and a method for preparing the same, which are superior in biocompatibility to heterogeneous collagen and capable of extracting the same kind of collagen which can be used for medical materials such as pain relief and tissue replenishment . The present invention also relates to a method for preparing a high-purity, high-concentration homologous collagen composition by excluding non-collagen materials from the initial stage of homologous collagen extraction. The present invention relates to a homogeneous collagen capable of preparing a high purity high-concentration homologous collagen composition through a freeze-drying process and producing various formulations.

Collagen is a natural polymer, a structure for support and proliferation of cells distributed in almost all tissues in vivo. It accounts for approximately one-third of the total protein of mammals and is the main protein component of the extra-cellular matrix. It is highly contained in soft tissues such as skin and tendon as well as hard tissues such as bones and teeth have. In addition, it can form cells and organ organs by forming and maintaining the morphology of organs and tissues, and can help to regenerate collagen to the original tissue by supplying collagen to the injured area of the living body.

Such collagen can be obtained not only from human skin but also from skin of animals such as goats, buffalo, pigs, and chickens. Collagen can be obtained through the skin of various animals, but these collagens are mutually different due to species and genetic differences.

Extracted collagen is now used in medical devices for replacement or reinforcement of damaged tissues due to burns, trauma, various diseases, and the like. Wound dressings, hemostatic agents, biomaterials for tissue restoration, and collagen-supplemented tissues for use in collagen, and its application range and product development are increasing. Recently, Regenseal, a domestic pig-derived collagen, has been used as an adjunct to osteoarthritis treatment, and it has been reported that collagen is effective for pain relief, inflammation reduction and cartilage regeneration.

Generally used collagen is mostly heterologous collagen extracted from animal tissues such as pigs and cows, and it has an antigenic telo-peptide and induces an immune rejection reaction. To prevent this, we make atelocollagen using pepsin, but we can not completely prevent immune rejection. In addition, in order to prevent the transfer of animal-derived virus, the step of inactivating the virus and the step of removing microorganisms must be included. In addition, if residual process remains, such collagen can cause various side effects when commercialized. Therefore, it is important to secure safety and efficacy for collagen extraction.

Due to structural differences, the collagen extraction methods differ depending on the object to be obtained because of differences in species and genetic differences between the structures ( Sadaf Quereshi et al./ Rec Res Sci Tech 2 (2010) 28-31).

In the conventional process of extracting collagen, the collected tissues are washed, the impurities such as fats and the like are removed and cut, and then the collagen is extracted by treatment with an acid, an alkali, or an enzyme.

Korean Patent Registration No. 10-1105603 proposes a method of treating collagen with high purity through acid dissolution, salt precipitation, filtration, secondary acid dissolution, secondary salt precipitation, phase separation, concentration and acid dissolution in animal tissues have. Specifically, a method of separating collagen from a tissue using a hydrochloric acid solution, an enzyme, or a phosphoric acid solution, and recovering enzyme-isolated collagen using a salt is used. However, such a collagen extraction method is not a method of extracting collagen from the skin of the same species, and is complicated and difficult to commercialize because it is complicated and has poor purity in a process using several steps of acid and base treatment and enzyme treatment. As a result, this method has the limitation that the reagent used for the pretreatment may remain or the impurities may not be completely removed, so that the toxicity and the risk of the human body can not be completely eliminated.

However, when the collagen is extracted from the acellular allogeneic dermis, which has already been removed from the immunogen and the non - collagenous material, the initial pretreatment process is omitted and higher purity collagen can be obtained. Further, in the present invention, by extracting collagen from a cell-free allogeneic dermis, biocompatibility can be enhanced and human-derived collagen can be utilized as a medical material.

It is an object of the present invention to provide a method for producing collagen, which comprises the step of treating pepper powder with an acidic solution of pepsin to extract collagen.

In order to accomplish the above object, the present invention provides a method for producing a polyphenol compound, which comprises stirring a dermis powder derived from a human body with a mixed solution of pepsin and an acidic solution at 200 to 300 rpm and dissolving the same, stirring the solution at a rate of 15,000 to 17,000 x g at 3 to 6 An extraction step for obtaining a supernatant from which collagen was extracted by centrifugation for 30 minutes,

The supernatant was filled in a 50 kDa cellulose ultrafiltration membrane, and a solution of 1 M sodium chloride dissolved in 1 M acetic acid was dialyzed at 4 째 C for 24 hours using an external dialysis solution, and the external dialysis solution was replaced every 12 hours to precipitate collagen Dialyzing the external fluid of the dialysis solution with 1M acetic acid for 24 hours and then exchanging the external dialysis fluid for 12 hours to purify the collagen and the solution in which the collagen has been precipitated is frozen at -80 to -70 ° C for more than 2 hours And a step of freeze-drying under reduced pressure so as to have a water residual rate of 10% or less.

The method for preparing the dermis powder comprises the steps of: removing the skin layer from the skin tissue; Removing cells of the dermal layer from the skin tissue from which the skin layer has been removed; And lyophilizing the dermal layer where the cells have been removed.

The acidic solution may be citric acid, acetic acid, citric acid, or a combination thereof.

The acidic solution may have a pH of 4 to 6.5 and the acidic solution may have a concentration of 0.5 to 2M.

The particle size powder of the dermis powder may be 250 m or less.

The content ratio of pepsin and dermis powder may be dermis powder: pepsin = 1 g: 250 to 350 mg.

The method for producing collagen comprises: precipitating extracted collagen; Neutralizing the precipitated collagen; And then lyophilizing the neutralized collagen.

The dermal layer may be treated with a Triton X-100 solution at a concentration of 0.5-1.5% (v / v) at 25-35 ° C for 95-105 minutes.

The stirring is carried out for 24 to 120 hours, and the stirring temperature may be 4 to 10 ° C.

In order to accomplish the object of the present invention, there is provided a method for preparing a daphnia powder, comprising the steps of stirring and dissolving a dipalphe powder in a mixed solution of pepsin and an acidic solution at 200 to 300 rpm, stirring the solution at 15,000 to 17,000 x g for 30 minutes The supernatant was filled into a 50 kDa cellulose ultrafiltration membrane and dialyzed against 1 M acetic acid and 1 M sodium chloride solution at 4 ° C. for 24 hours. Exchanging with 1M acetic acid for 24 hours to precipitate collagen, freezing the solution in which collagen has been precipitated by freezing at -70 to -80 占 폚 for 2 hours or longer, and then lyophilized under reduced pressure so as to have a water residual rate of 10% Freeze-dried collagen into a syringe, injecting saline into other syringes so that the collagen concentration is 3% (w / v) to 6% (w / v), mixing the freeze-dried collagen into saline Mixing the dissolved solution with 2% (w / v) hyaluronic acid and then treating with 4% (v / v) crosslinking agent BDDE (1,4-butanediol diglycidyl ether) to prepare a mixed solution, Injecting the mixture into a mold, allowing the cross-linking reaction to proceed for 5 hours at 37 ° C, and lyophilizing the mixed solution after completion of the cross-linking reaction.

By extracting collagen from human-derived allogeneic dermis by the method proposed in the present invention, it is possible to extract collagen with high purity and high concentration, which is excellent in biocompatibility. The extracted collagen can be controlled in concentration and can be applied to various formulations.

In addition, it is possible to omit the steps of preventing the generation of such an immune response and extracting viruses and microorganisms by extracting collagen from human skin.

1 is a table showing the composition of the collagen extraction solution of the present invention.
2 is a table showing the yield of collagen according to sodium chloride concentration during dialysis of the present invention.
FIG. 3 is a photograph of human collagen extracted according to the extraction time of the present invention.
4 is a graph showing the yield of collagen according to the extraction time of the present invention.
FIG. 5 is a photograph showing a state in which the separated and purified collagen of the present invention is injected into a syringe mixed with physiological saline at a concentration of 3% and 6%.
6 is a photograph showing a cylindrical scaffold made by lyophilizing a collagen solution in a round mold according to the present invention.
FIG. 7 is an electrophoresis image showing that the collagen extracted by the method of the present invention has undergone electrophoresis and then subjected to Western blotting to confirm that the collagen is type 1 collagen.
FIG. 8 is a photograph of the electrophoresis result of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) on the extracted collagen of the present invention and staining with Coomasie Brilliant Blue.
Fig. 9 is a quantitative curve of hydroxyproline, which is a standard substance used for the determination of collagen.
FIG. 10 shows the results of the experiment on the content of collagen contained in extracted collagen, that is, the purity of collagen, as a result of quantitative analysis of collagen.

In the present invention, a method of using acid and pepsin at the same time is applied to a granulated cell-free allogeneic dermis in a widely generalized method in which an acid, a base or a salt is used or an enzyme such as pepsin or tryptase is used for extraction To provide extracted human collagen.

The method for producing collagen of the present invention comprises the steps of: preparing and pulverizing an acellular dermis tissue; treating the acellular dermis powder with a predetermined level of non-collagenous material removed, acid and pepsin, extracting collagen, lyophilization, , Followed by sterilization by gamma-ray sterilization.

More specifically, the method for producing collagen from human body comprises:

(i) removing the epidermal layer from the skin tissue;

(ii) removing cells of the dermal layer in the skin tissue from which the skin layer has been removed;

(iii) lyophilizing the cell-free allogeneic dermis layer;

(iv) granulating the dried cell-free allogenic dermal layer;

(v) extracting collagen by treating an acid solution containing pepsin in allogeneic dermis particles;

(vi) purifying the collagen by a salt precipitation method;

(vii) neutralizing the collagen;

(viii) lyophilizing the purified collagen;

(ix) adjusting the concentration of lyophilized collagen using physiological saline.

The 'acellular dermis tissue' in the present invention refers to a dermis layer in which cells capable of causing an immune rejection reaction are removed by chemically treating the dermis, and collagen and elastin are main components.

In the embodiment of the present invention, the acid solution used for collagen extraction may be an acid which can dissolve collagen, preferably citric acid, acetic acid, and citric acid, more preferably acetic acid. . The concentration of acetic acid is preferably 0.1 to 2 M, and the pH is 4 to 6.5. At this time, it is preferable that the extraction can be performed by stirring for 24 to 120 hours, and the temperature range of stirring is 4 to 10 ° C, and it is preferable to proceed at a low temperature to minimize the damage of the collagen structure. The extraction efficiency can be increased by replacing with fresh solution every 24 hours.

Pepsin is a proteolytic enzyme that is used to easily break down cross-linking in collagen. Pepsin can be easily purchased in pure form and can be applied to acidic solutions in which monomer molecules can be easily dissolved. Limited proteolysis using pepsin is very useful for obtaining large amounts of various collagens in the form of single molecules from human tissue.

It is preferable to use a filtration membrane with a molecular weight cut-off (MWCO) of 50 kDa or more in the course of purifying the collagen extract using the acid solution and pepsin. This means that 300 kDa of collagen is retained during dialysis and 35 kDa to remove the pepsin.

When performing the salt precipitation method, the collagen mass obtained by subjecting the collagen extracted from human tissue to cell-free treatment and filtration and then subjecting to salt precipitation instead of neutral precipitation can be subjected to compression concentration, so that high-purity high-concentration collagen can be produced . Since proteins have the property of aggregating at a specific salt concentration, even in the case of collagen, it is possible to increase the purity of collagen by discarding the non-aggregated impurities through aggregation through the salt.

According to the collagen extraction method of the present invention, collagen can be separated and extracted by an efficient process by extracting collagen from a noncellular allogenic dermis from which non-collagen substances are removed without directly extracting collagen from the donated skin tissue. In addition, by extracting human-derived collagen from a cell-free allogeneic dermis, non-collagen substances such as cytoplasm, DNA, and fat can be excluded in the extraction process, and high purity collagen purification is possible.

In general, the conventional method of treating collagen with acid and alkali is disadvantageous in that the yield is as low as about 10 to 15%. However, the present invention is based on the finding that the content ratio of pepsin and dermis powder, : When the concentration of pepsin was 1 g: 250 to 350 mg and the concentration of the acidic solution was 1 M, the extraction efficiency of collagen increased 5 times or more as compared with other conditions (see Fig. 1) When the collagen is extracted from the dermis, the extraction efficiency is increased by increasing the surface area upon extraction, and as a result, a high yield of 35% or more can be expected.

When pepsin is used together with an acidic solution, the cross-linked form of the collagen telopeptide helical site can be more easily cleaved, thereby improving the efficiency of collagen extraction.

When the acid concentration is 2M or more, the collagen is decomposed. When the concentration is 1M or less, the extraction efficiency is low.

In addition, it is possible to obtain more collagen under the condition of 1M sodium chloride during dialysis for separation and purification of collagen. It has an intrinsic salt concentration which is precipitated per protein. Collagen derived from a cell-free allogeneic dermis is more easily precipitated near 1M concentration of sodium chloride.

As a result, the efficiency and yield of the extraction process can be maximized through the extraction method of the present invention, and high-purity and high-concentration collagen can be produced with the purified collagen.

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

The present invention relates to a method for producing high-purity human collagen having high biocompatibility and a high yield and a high concentration of collagen to provide a high-purity collagen, which is effective for relieving pain associated with inflammation and treating osteoarthritis Collagen and a method for producing the collagen are provided.

Example  1-1: Acellular cell  Production of homogeneous dermis powder

The donated skin tissue was firstly washed with sterile distilled water and the fat layer was separated from the skin tissue. The neutral protease, Distase, was treated at a concentration of 1.0 units / ml, stirred at 37 ° C for 60 minutes to 120 minutes in a stirred incubator, and then washed three times with sterile distilled water to separate the dermal layer and the epidermal layer to remove the skin layer Respectively.

 The cells of the dermal layer were removed by treatment with a Triton X-100 solution at a concentration of 1% (v / v) for 100 minutes at 30 ° C. The cells were washed three times or more with sterilized distilled water to remove the used materials from the process. Allogeneic dermis was frozen at a temperature below -40 ℃ for more than 2 hours, then put into a freeze dryer and dried for more than 12 hours to remove water.

Allogeneic dermis was cut with a surgical knife to a size of about 1 cm x 1 cm, and then pulverized for 3 minutes at 5000 rpm, which is an optimum condition for preventing denaturation of collagen and elastin by using a micro-pulverizer that does not break the tertiary structure of the same kind of dermis Cell-free allogeneic dermis was prepared by centrifugation through a sieve of 250 μm diameter in a sterile condition.

Example  1-2: Extraction of collagen from human body

From the acellular dermis powder prepared in Example 1-1, collagen extraction was performed under six conditions according to the concentration of acetic acid solution and the addition of pepsin. 1 g of cell-free allogeneic dermis powder was added to 50 ml of acetic acid solution of 0.5, 1 and 2M concentration containing 300 mg of pepsin and stirred at 200 rpm for 24 hours at 4 ° C.

The extracted collagen was centrifuged at 16,000xg for 30 minutes at 4 ° C, and the supernatant was separated and refrigerated.

The solution obtained from the above procedure was filled in a 50 kDa cellulose ultrafiltration membrane, and a solution of 1 M sodium chloride dissolved in 1 M acetic acid was dialyzed at 4 캜 for 24 hours as an external dialysis solution.

At this time, collagen was precipitated by exchanging the external dialysis fluid every 12 hours.

Thereafter, the dialysate external solution was replaced with 1M acetic acid for neutralization, dialyzed for 24 hours, and collagen was precipitated by changing the external dialysis fluid every 12 hours. After the dialysis was completed, the collagen solution was transferred to a tube, frozen at -80 ° C for 2 hours or more, and then dried under reduced pressure so that the water residual rate was 10% or less.

Example  1-3: Setting the extraction time of collagen from human body

1 g of cell-free allogeneic dermis powder was added to 50 ml of 1 M acetic acid solution containing 300 mg of pepsin and stirred at 200 rpm for 24 hours at 4 ° C. The extracted collagen was centrifuged at 16,000xg for 30 minutes at 4 ° C, and the supernatant was separated and refrigerated. After that, fresh solution was added to the remaining tissue and extraction was repeated. This process was continued for up to 5 days (120 hours) to set the extraction time to maximize collagen yield.

The solution obtained from the above procedure was filled in a 50 kDa cellulose ultrafiltration membrane, and a solution of 1 M sodium chloride dissolved in 1 M acetic acid was dialyzed at 4 캜 for 24 hours as an external dialysis solution. At this time, collagen was precipitated by exchanging the external dialysis fluid every 12 hours. Thereafter, the dialysate external solution was replaced with 1M acetic acid for neutralization, dialyzed for 24 hours, and collagen was precipitated by changing the external dialysis fluid every 12 hours. After the dialysis was completed, the collagen solution was transferred to a tube, frozen at -80 ° C for 2 hours or more, and then dried under reduced pressure so that the water residual rate was 10% or less. The longer the extraction time, the greater the amount of extracted collagen (see Figures 3 and 4).

Example  1-4: Infusible  High-purity collagen production

The lyophilized collagen was filled into the syringe and the saline solution was injected into another syringe so that the collagen concentration was 3% (w / v) or 6% (w / v). On the other hand, a solution obtained by dissolving lyophilized collagen in saline at a concentration of 2% (w / v) was mixed with 2% (w / v) hyaluronic acid, and then the crosslinking agent BDDE (1,4-butanediol diglycidyl ether) v / v) and injected into a cylindrical mold. The mold filled with the mixed solution is crosslinked at 37 ° C for 5 hours. Thereafter, after the lyophilization process, a support form made of human collagen was finally obtained.

Example  2-1: Identification and type analysis of extracted collagen

Western blotting was performed to determine the type of collagen extracted. The collagen extracted and purified in Example 1 was subjected to electrophoresis and then moved to 100 V for 1 hour at 4 ° C. The membrane was then reacted with collagen antibody. As a result of western blotting, it was confirmed that the separated and purified collagen was type 1 collagen. In addition, it was confirmed that the longer the extraction time, the higher the yield of extracted collagen, and it was confirmed that the collagen did not denature even when the time was prolonged.

Example  2-2: Analysis of extracted collagen

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed to confirm the purity of the purified collagen in Example 1. Protein 5 × sample buffer was added to the collagen, and the mixture was heated at 100 ° C. for 5 minutes. The gel was then subjected to electrophoresis on a 6% SDS-PAGE gel at 60 V for 30 minutes and 100 V for 90 minutes, followed by staining with Coomassie brilliant blue R250. As shown in FIG. 6, the human collagen produced using the collagen extraction method of the present invention was found to contain an? Chain having a peptide molecular weight of 100 kDa to 130 kDa, and other bands were not visible and contained almost no impurities It was confirmed that it is a high purity human type I collagen.

Example  2-3: Purity analysis of human collagen

The colorimetric method using hydroxyproline was performed to determine the collagen extracted for 5 days in 1 M acetic acid containing pepsin, which is the condition for obtaining the highest yield (yield,% w / w). The content of collagen was 13.5% of hydroxyproline in collagen, so hydroxyproline was quantified and multiplied by conversion factor. 10 mg of the extracted collagen was placed in a test tube in a lyophilized form, and 10 mL of 4N HCl solution was added thereto, followed by hydrolysis in a high pressure autoclave at 110 ° C. for 20 hours. The supernatant of the sample solution was neutralized by the same amount of 4N sodium hydroxide solution (4N sodium hydroxide). To 50 μl of the neutralized test solution, 450 μl of a chloramine-T solution was added, and the mixture was stirred for 25 minutes. Then, 500 μl of enrich's aldehyde reagent was added and reacted at 65 ° C for 40 minutes using a water bath. The absorbance of the solution was measured at 550 nm using a spectrophotometer. The hydroxyproline content was determined by preparing a standard curve using hydroxyproline purchased from Sigma (Fig. 9) and adding the absorbance of the sample to the standard curve formula to determine the concentration of hydroxyproline. The extracted collagen sample had a content of collagen of 97% or more, indicating that the extracted collagen had a high purity with almost no impurities (FIG. 10).

Comparative Example 1  (Acetic acid concentration condition at the time of extraction)

As shown in FIG. 1, when the extract of collagen was added together with pepsin as well as acid, the yield was high and the highest amount of collagen was obtained at 1M acetic acid concentration. At an acetic acid concentration of 2M, there was almost no amount of collagen finally obtained.

Comparative Example 2  (Sodium chloride concentration during dialysis)

In the salt precipitation step, the collagen was dialyzed at different concentrations of sodium chloride.

Pepsin was added to 1 M acetic acid, and the collagen was extracted for 24 hours. The concentration of sodium chloride was further divided by 1M and 2.5M, followed by dialysis and subsequent steps. As shown in Fig. 2, more collagen was obtained under the condition of 1M sodium chloride. This means that the collagen derived from the cell-free allogeneic dermis has a property of being more aggregated at a concentration of 1M sodium chloride.

Claims (10)

Mixing the dermis powder derived from human body with a mixed solution of pepsin and an acidic solution at 200 to 300 rpm to dissolve the dermis powder;
An extraction step of centrifuging the stirred solution to obtain a supernatant from which collagen has been extracted;
Dialyzing the supernatant to precipitate collagen;
Freeze-drying a solution in which collagen has precipitated, followed by freeze-drying under reduced pressure,
The acidic solution has a pH of 4 to 6.5, an acidic solution concentration of 0.5 to 1 M, a content ratio of pepsin and dermis powder is dermis powder: pepsin = 1 g: 250 to 350 mg,
The dermal powder was treated with a Triton X-100 solution at a concentration of 0.5-1.5% (v / v) at 25-35 ° C for 95-105 minutes in skin tissues from which the fat layer and the epidermal layer were removed from the skin tissue, And removing the cells, and lyophilizing the dermis layer from which the cells have been removed.
delete The method for producing collagen according to claim 1, wherein the acidic solution is citric acid, acetic acid, citric acid, or a combination thereof.
delete The method of claim 1, wherein the particle size powder of the dermis powder is 250 탆 or less.
delete The method of claim 1, wherein the collagen preparation comprises: precipitating the extracted collagen; Neutralizing the precipitated collagen; Characterized by further comprising lyophilizing the neutralized collagen.
delete The method of claim 1, wherein the stirring is performed for 24 to 120 hours, and the stirring temperature is 4 to 10 ° C.
Mixing the dermis powder derived from human body with a mixed solution of pepsin and an acidic solution at 200 to 300 rpm to dissolve the dermis powder;
Centrifuging the agitated solution at a temperature of 15,000 to 17,000 x g for 30 minutes at 3 to 6 DEG C to obtain a collagen-extracted supernatant;
The supernatant was filled into a 50 kDa cellulose ultrafiltration membrane and dialyzed against 1 M acetic acid and 1 M sodium chloride solution at 4 째 C for 24 hours in an external dialysis solution. The external dialysis solution was replaced with 1 M acetic acid and dialyzed for 24 hours to precipitate collagen ;
Freeze-drying the collagen-precipitated solution at -70 to -80 ° C for 2 hours or more, followed by lyophilization under reduced pressure so that the water residual rate becomes 10% or less;
Filling the syringe with reduced pressure lyophilized collagen, injecting saline into other syringes so that the concentration of the collagen is 3% (w / v) to 6% (w / v), and then mixing;
A solution prepared by mixing lyophilized collagen dissolved in saline with 2% (w / v) hyaluronic acid and then treating with 4% (v / v) crosslinking agent BDDE (1,4-butanediol diglycidyl ether) ;
Injecting the mixed solution into a cylindrical mold and allowing the crosslinking reaction to proceed at 37 ° C for 5 hours; And
And lyophilizing the mixed solution after completion of the crosslinking reaction
The acidic solution has a pH of 4 to 6.5, an acidic solution concentration of 0.5 to 1 M, a content ratio of pepsin and dermis powder is dermis powder: pepsin = 1 g: 250 to 350 mg,
The dermal powder was treated with a Triton X-100 solution at a concentration of 0.5-1.5% (v / v) at 25-35 ° C for 95-105 minutes in skin tissues from which the fat layer and the epidermal layer were removed from the skin tissue, And removing the cells, followed by freeze-drying the dermal layer. A method for producing an allograft collagen support.

Images