KR20220115134A - Wound Dressing Comprising Fiberized Acellular Dermal Matrix and Biocompatible Polymer, and Method for Preparation Thereof - Google Patents

Abstract

The present invention is a sheet-type freeze-dried type of acellular allogeneic dermis pulverized, fibrous, and pulverized to rehydrate with a matrix made of fibrotic acellular dermis and soft tissue, then reprocessed into a sheet type, and then freeze-dried. Provided is a composition that is non-crosslinked and can be crosslinked as a characteristic composition.

Description

Composition comprising a fibrotic acellular dermal matrix and a biocompatible polymer, and a method for preparing the same

The present invention relates to a wound covering material comprising a fibrous acellular dermal matrix and an aqueous solution of a biocompatible polymer, and more particularly, 5 to 20 wt% of a fibrotic acellular dermal matrix, 0.5 to 5 wt% of a biocompatible polymer and It relates to a wound covering material comprising 75 to 94.5% by weight of water and a method for preparing the same.

In addition, the present invention relates to a composition comprising a human-derived acellular dermal matrix and gelatin, and more particularly, the fibrous acellular dermal matrix and the gelatin composition have a crosslinking degree, viscoelasticity, curing degree and/or extrusion force value after sterilization. It relates to a composition characterized in that the degree of change is 30% or less, preferably 20% or less, and most preferably 10% or less compared to the value before sterilization, a method for preparing the same, and a sheet production using the same.

A wound is a state in which the continuity of the skin or other tissues is broken or damaged by external pressure, and the continuity of the tissue is destroyed, and usually refers to a phenomenon in which the skin is opened due to damage to the dermis layer of the skin. Wound surgery basically prevents infection and suppresses the inflammatory response by blocking exposure to the external environment by suturing and dressing the wound area.

The material of the wound covering material can be largely divided into the allogeneic dermis and the heterogeneous dermal material, and the wound covering material can be manufactured by a method of extracting a specific polymer from the material and a method using a dermal matrix. The dermal tissue consists of 80-90% of collagen, elastin, and glycosaminoglycan (GAG).

On the other hand, LifeCell company decellularized and freeze-dried skin tissue collected from cadavers in 1994 to commercialize Acellular Dermal Matrix (ADM) (Alloderm (allograft)) and used it for burn treatment and skin reconstruction. did. Alloderm (allograft) was safer than other products and showed significantly better engraftment rate and healing effect. Similar products have been developed such as Bard Dabol's AlloMax, which is used as a graft material, and Ethicon's FlexHD with increased adhesion.

However, the sheet-type acellular dermal matrix covering material as described above is difficult to use for curved or deeply dug wounds, and it is inefficient because the tissue sheet must be cut or used by attaching several sheets depending on the size and shape of the wound site.

Accordingly, LifeCell applied for a technology for granulating the acellular dermal matrix in 1999 (US6933326B1), and in 2008 applied for a patent on a composition in which the granulated acellular dermal matrix and an acidic solution were mixed (US9382422B2). AlloDerm (Cymetra) product in injectable micronized form was released. In addition, Wright Medical Group developed the Graftjacket product, which is a sheet-type acellular dermal matrix, in an injectable form and released the Graftjacket Xpress product.

In addition, Korea's L&C Bio Inc. applied for a patent on a composition in which hyaluronic acid was cross-linked after granulating acellular dermal matrix in 2014 (KR10-1523878B1).

However, the composition obtained by crosslinking acellular dermal matrix particles and a biocompatible polymer as described above has high structural stability, but also has high degree of crosslinking, viscoelasticity, curing and extrusion force, so it is suitable for use as fillers and implants, but as a wound covering material. is insufficient, and the acellular dermal matrix in micronized form is not fixed to the wound site in liquid form and is not evenly distributed on the wound, such as flowing out of the dressing.

In addition, even if a crosslinking agent is not artificially added to the acellular dermal matrix particles and the biocompatible polymer composition, crosslinking occurs when E-beam is irradiated for sterilization purposes, and the composition increases the degree of hardening as the storage period increases. changes in the physical properties or formulation of The composition with increased degree of curing is not suitable for use as an injection for wound covering pastes, gels, ointments, emulsions, creams and suspensions due to poor applicability.

In order to develop an acellular dermal matrix suitable as a wound covering material, the present inventors studied a paste-form composition by combining the acellular dermal matrix and a biocompatible polymer with excellent hemostatic performance, that is, gelatin. However, it was confirmed that the degree of curing and extrusion force increased when stored for a long period (one year or more) or at a low temperature after radiation sterilization, resulting in poor stability of the composition.

Accordingly, the present inventors have made diligent efforts to develop a composition of a paste formulation that can be used irrespective of the curvature, size and shape of the wound site, is smooth, has good spreadability, and has high stability. As a result, acellular dermal matrix, preferably sterilization of a composition in which a fibrous acellular dermal matrix and a biocompatible polymer, preferably alginate, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid and hyaluronic acid, more preferably gelatin are mixed, preferably By constant heat treatment before and/or after radiation sterilization, it was confirmed that the degree of crosslinking was suppressed, the physical properties of the soft paste were maintained and storage stability was improved, and the present invention was completed.

In addition, the present inventors have made diligent efforts to develop a wound covering sheet that can adjust the width, length and thickness and length suitable for the wound site, and as a result, the acellular dermal matrix and the gelatin composition are pasted and then placed in a mold (mold or frame). The sheet was prepared by application and freeze-drying, and it was confirmed that the sheet was stable even in a long-term rehydration process, had excellent bioadhesiveness, and had excellent therapeutic effect, and completed the present invention.

The present invention is a sheet-type freeze-dried type of acellular allogeneic dermis pulverized, fibrous, and pulverized to rehydrate with a matrix made of fibrotic acellular dermis and soft tissue, then reprocessed into a sheet type, and then freeze-dried. It is to provide a composition that is non-crosslinked and can be crosslinked as a characteristic composition.

The present invention is a sheet-type freeze-dried type of acellular allogeneic dermis pulverized, fibrous, and pulverized to rehydrate with a matrix made of fibrotic acellular dermis and soft tissue, then reprocessed into a sheet type, and then freeze-dried. Provided is a composition that is non-crosslinked and can be crosslinked as a characteristic composition.

For example, the fibrous cell-free particles are characterized by skin-derived acellular dermis and may include all soft tissues (amniotic membrane, fascia, pericardium, etc.).

As an example, the size of the fibrous cell-free particles may include those having a major axis length of 100 to 2,000 μm.

For example, it may include a product prepared through a crosslinking process through crosslinking using radiation, reduced pressure and heat, and chemical crosslinking using a crosslinking agent.

For example, the fibrotic acellular dermis and soft tissue may include human-derived allogeneic and heterogeneous soft tissue.

For example, the sheet-type composition made through hydration and freeze-drying may include a thickness of 0.1-2 mm.

For example, it may include a product containing 4-20% of moisture by lyophilization.

For example, the composition may further include antibacterial agents, excipients and additives used pharmaceutically.

As an example, the composition may include stem cells and growth factors.

For example, the composition may include natural and synthetic polymers.

The sheet according to the present invention is stable even in a long-term rehydration process, has excellent bioadhesiveness, and has excellent therapeutic effect.

The paste for wound covering material according to the present invention can be easily applied to areas or localized areas where it is difficult to apply the conventional sheet-type acellular dermal matrix, and is a ready-to-use product that can be used immediately. It can also be cured quickly without surgery.

In addition, it has excellent stability of physical properties even when stored for a long period or at low temperature, so that when applied to a local area, it is soft and has excellent adhesion. Accordingly, by protecting the wound site from external contaminants and maintaining a moist environment, it can effectively help the wound site treatment.

In addition, the sheet for a wound covering material according to the present invention has excellent bioadhesiveness, and the polymer structure does not dissolve well even during a long rehydration process, so it is excellent in shape maintenance ability during a specialist operation. In addition, since the sheet can adjust the horizontal, vertical and thickness sizes required by the wound site or surgical characteristics, wound treatment can be qualitatively improved.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In the present invention, human-derived cell-free, characterized in that it is sterilized and ready-to-use, can be applied to curved or deep-cut wounds, and maintains soft physical properties even when stored for a long period of time or at low temperatures An attempt was made to prepare a composition comprising the dermis and gelatin.

In the present invention, it is confirmed that the composition changes physical properties such as degree of crosslinking, degree of curing, and extrusion force by radiation sterilization, and accordingly, changes in properties of the composition are minimized by including a certain heat treatment process before and/or after radiation sterilization. and improved stability.

Accordingly, in one aspect, the present invention provides a composition comprising a human-derived acellular dermal matrix and gelatin, wherein the composition is sterilized and ready-to-use, and crosslinking after sterilization, It relates to a composition and a method for preparing the same, characterized in that the change in values of viscoelasticity, degree of curing and/or extrusion force is 30% or less compared to the value before sterilization.

The dermal tissue of the present invention may be bone, ligament, tendon, or skin, and may be a heterogeneous or allogeneic dermal tissue, but is preferably a human dermal tissue.

In the present invention, the raw material strength of the gelatin is 250 Bloom or more, the viscosity is 30 to 45 mps, the pH is 5 to 6.5, and it may be characterized in that the loss on drying is 5 to 15%. In the present invention, in addition to the gelatin, alginate, agarose, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid, hyaluronic acid, polyethylene glycol, chondroital Biocompatible polymers of , dermatan, polysaccharide, mucopolysaccharide, hydrogel, dextran, amylose, protein, glycoprotein and derivatives thereof may be further included.

On the other hand, the constituent content of the composition may be 5 to 20% by weight of acellular dermal matrix, 0.5 to 5% by weight of gelatin and 75 to 94.5% by weight of water, but preferably 8 to 12% by weight of acellular dermal matrix, 1 to gelatin 2% by weight and 86-91% by weight of water.

In an embodiment of the present invention, before and/or after sterilization of the composition, 30 to 60 ° C, preferably 35 to 55 ° C, most preferably 40 to 50 ° C for 1 to 180 minutes, preferably 60 to It was confirmed that the stability of the physical properties was maintained by performing the heat treatment for 120 minutes.

The sterilization process may include a dry heating method, a pasteurization method, a radiation sterilization method, a gas sterilization method, etc., and preferably may include a radiation sterilization method performed by E-beam irradiation. It was confirmed that E-beam irradiation induces cross-linking in the acellular dermis and the gelatin composition to cause changes in viscoelasticity, degree of curing and extrusion force, which are correlated with this. In addition, crosslinking by E-beam irradiation increased as the storage period after manufacturing the finished product increased, and in particular, when stored at a low temperature, crosslinking increased even more. The measurement of changes in physical properties, such as the degree of crosslinking, viscoelasticity, degree of curing, and extrusion force, is not limited to a specific experimental method. In the case of the degree of crosslinking, indirect indicators such as a gel fraction and a swelling ratio may be used.

In another aspect, the present invention is a composition comprising a fibrous acellular dermal matrix and a biocompatible polymer, wherein the fibrous acellular dermal matrix has a ratio of short axis to long axis of 1:30 to 1:2,000. It relates to a composition and a method for preparing the same, characterized in that the proportion thereof is 70%, preferably 80%, most preferably 90% or more.

The acellular dermal matrix of the present invention is in a fibrotic form. In the present invention, in order to prepare a fibrotic acellular dermal matrix, a dermis having a thickness of 1 mm or more was selected and used. After decellularizing the dermal matrix with a thickness of 1 mm or more, it was dried or lyophilized to contain an appropriate moisture content, for example, 5 to 10% of moisture. Thereafter, the acellular dermal matrix was pulverized and fibrous using a pulverizer such as a cutting mill, a food processor, an agate pulverizer, or a freeze pulverizer.

The ratio of the length of the short axis to the long axis of the fibrous acellular dermal matrix of the present invention is 1:30 to 1:2,000, and 70% or more, preferably the ratio of the short axis to the long axis is 1:30 to 1 Fiber particles of :2,000 may account for 80% of the total composition.

The fibrous acellular dermal matrix under the above conditions coexists in a bundle-like form and a thin and long single fiber form like a thread, and when used as a wound covering material, it has excellent preservation in the body, maintains a moist environment, and separate chemical It shows a structure that is stably supported with a polymer without bonding.

In another aspect, the present invention is a sheet for a wound covering material comprising 75 to 85% by weight of acellular dermal matrix (ADM), 15 to 25% by weight of a biocompatible polymer, and 3 to 8% by weight of water, the horizontal length is 4 to It relates to a sheet for a wound covering material, characterized in that it has a length of 20 cm, a length of 4 to 20 cm, and a thickness of 1.5 to 3 mm.

In the present invention, the composition containing the cell-free dermal matrix in the form of a paste and a biocompatible polymer was applied to a specific mold (mold or frame) and lyophilized to prepare a wound covering material.

The freeze-drying may be performed until the moisture content of the sheet becomes 3 to 8%, preferably 4 to 5%.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Method for fibrosis of human-derived acellular dermal matrix

Skin tissues isolated from cadavers were purchased from EURO skin bank, Allosource, and CTS, and tissues with a thickness of 1 mm or more were selected and used to prepare acellular dermal matrix.

The skin tissue was reacted with 1M NaCl solution at 38° C. for 6 to 24 hours, and then the epidermis was removed using forceps. The dermis from which the epidermis was removed was washed with a phosphate buffer solution, and then reacted with 0.5% sodium dodecyl sulfate (SDS) at room temperature for 1 hour to remove cells in the dermis.

Glycerol (Sigma, USA), propylene glycol (Sigma, USA) and phosphate buffer solution (Gibco, USA) are mixed in a weight ratio of 1:1:8, and the final concentration of maltitol, sucrose or sorbitol is adjusted to 30%. Thus, a cryoprotectant was made. The maltitol was confirmed to prevent browning of the dermal tissue, and maltitol was used for this.

The cryoprotectant was infiltrated into the dermis in a low temperature reactor at 4° C. for 12 hours. The infiltrated dermis was placed in a tie bag (Korea Advanced Materials, Korea) and dried for 24 hours in a freeze dryer with a vacuum of 5 torr to make a freeze-dried cell-free dermal matrix.

The freeze-dried acellular dermal matrix was pulverized using a cutting mill (Pulverisette 19, FRITSCH, Germany) to form fibers.

1 is a comparison of the appearance of a fibrous acellular dermal matrix (A) prepared by the above method and a bead-shaped acellular dermal matrix (B).

The fibrous cell-free dermal matrix was in the form of cotton, that is, in the form of entangled fibers, which had a larger volume (surface area) than the bead-type and the structural shape of the pulverized matrix was stable.

The bead type was prepared by selecting an acellular dermal matrix having a thickness of 1 mm or less, and then decellularizing, lyophilizing, pulverizing and preparing in the same manner as above.

Example 2: Measurement of the length of the short axis of the fibrotic acellular dermal matrix, its distribution and the ratio of the length of the short axis and the intestine

The long and short lengths of the fibrotic acellular dermal matrix particles obtained in Example 1 were photographed with a Zoom Stereomicroscope (SMZ745, Nikon, Japan) microscope, and then an Image Analyzer program (i-SolutionTM IMT i-Solution Inc. , Korea) was used.

As shown in FIG. 2, the long axis length and distribution of the fibrotic acellular dermal matrix are about 33% for 500-1,000 μm, about 30% for 1,000-1,500 μm, about 27% for 1,500-2,000 μm, and 2,000-2,500 μm. was about 7% and 2,500-3,000 μm was about 3%. That is, fiber particles with a major axis length of 1,000 to 2,000 μm accounted for about 60% of the total particles, and fiber particles with a length of 500 to 2,000 μm accounted for about 90% of the total particles.

As shown in FIG. 3, the short axis length and distribution of the fibrotic acellular dermal matrix are about 20% for 10-20 μm, about 39% for 20-30 μm, about 28% for 30-40 μm, and 40-50 μm. was about 9% and 50-60 μm was about 4%. That is, it was confirmed that about 70% of the fiber particles had a minor axis length of 20 to 40 μm. That is, fiber particles with a minor axis length of 20 to 40 μm accounted for 70% of the total particles.

As shown in Fig. 4A, the length and shortening ratios of the fibrotic acellular dermal matrix are about 3% for 20-30:1, about 23% for 30-40:1, and about 33% for 40-50:1, 50 ~60:1 measured about 34% and 60-70:1 was about 7%. That is, fiber particles having a length/short axis ratio of 40 to 60:1 accounted for 70% of the total particles, and fiber particles having a length of 40 to 70:1 accounted for about 75%.

On the other hand, as shown in FIG. 4B, about 90% of the particles exhibited a ratio of 1 to 2:1 in the bead-type cell-free dermal matrix. Compared with the fibrous particles, the bead type was confirmed to be a particle shape close to a spherical shape.

4C is a micrograph of a fibrous acellular dermal matrix, and FIG. 4D is a micrograph of a bead-type acellular dermal matrix.

Example 3: Preparation of paste using acellular dermal matrix

After adding water to 1.25 g of pork skin gelatin (Sammi, Korea) to make 100 ml of a mixture, it was dissolved at 60° C. to prepare an aqueous gelatin solution. The strength of the gelatin is 299 bloom, the viscosity is 38.3 mps, the pH is 5.83, and the loss on drying is 11.4%. was measured with A paste was prepared by mixing 89 g of the aqueous gelatin solution and 11 g of the fibrous acellular dermal matrix obtained in Example 1 at 60°C.

E-beam radiation sterilization was carried out after putting the paste in a syringe and double packaging P.E.T tray and tie bag. The crosslinking reaction of the paste by radiation sterilization was suppressed by heat treatment for 2 hours or 1 hour at 45°C incubation before and/or after sterilization. 5 is a photograph showing the formulation of the paste.

Example 4: Viscosity Measurement of Pastes

The paste of 5 samples was prepared by the method of Example 3, and the paste viscosity was measured in the initial state and after storage for 3 months.

The viscosity of the paste was measured using the torque generated due to the viscosity of the fluid by rotating the disk in the fluid according to the rotational viscometer method (the Korean Pharmacopoeia general test method), and a circulator of the rotational viscometer (DV2THB Viscometer, Brookfield, USA) ( circulator) conditions were set to 15 rpm and 25° C., and the viscosity of 1 g of the sample was repeatedly measured.

As a result, as shown in FIG. 6, both the initial state and the paste after storage for 3 months exhibited a viscosity between 20,000 and 25,000 cps. That is, it was confirmed that the viscosity and physical properties of the paste were maintained unchanged for 3 months.

Example 5: Measurement of the extrusion force of the paste

The extrusion force is a measure of the load value generated for the force when work is applied to the specimen in a certain time and force and in a certain direction. The extrusion force of the paste according to the reaction temperature conditions before and after radiation sterilization or whether radiation sterilization was performed in the paste preparation step was compared.

Put the paste without E-beam sterilization and the paste whose reaction temperature was adjusted to 10, 25 and 45 ℃ before and after E-beam sterilization into a 3cc syringe, and inject with a constant force using a UTM (Universal Testing Machine). The sample was extruded from

As a result, the extrusion force of the paste without E-beam sterilization was maintained between about 10 and 15 N (average 12.1 N), and showed no increase or decrease with time. Among the pastes subjected to E-beam sterilization, the size of the extrusion force increased in direct proportion with time in the paste having a temperature response of 10 °C. After 30 seconds of extrusion, the extrusion force reached about 40 N, and the average value was measured to be 26.82 N. The paste with a temperature response at 25 °C showed an extrusion force value of about 15 to 20 N, an average value of 16.06 N, and showed no increase or decrease with time.

The paste having a temperature response at 45 °C showed an extrusion force value of about 6-8 N, an average value of 6.645 N, and the change in extrusion force with time was the most gentle among the experimental groups (FIG. 7). This means that the paste with a temperature reaction at 45 °C does not gel and the particles are evenly distributed. On the other hand, the paste without E-beam treatment and the paste having a temperature response at 10 °C and 25 °C along with E-beam sterilization were recognized for particles due to gelation, and the extrusion force curve was not gentle but sharply bent. became

The average extrusion force of the paste without E-beam sterilization and the paste subjected to E-beam sterilization at 45°C was about 12.1 N and 6.645 N, respectively, and the change in the extrusion force value was about 45%.

When E-beam sterilization was applied to the paste formulation, the extrusion force increased due to cross-linking. However, when 45 ℃ heat was applied during E-beam sterilization, cross-linking was inhibited and the extrusion force was reduced, and the extrusion force value was stable compared to other experimental groups.

Example 6: Determination of gel fraction (degree of crosslinking)

In the paste preparation step of Example 3, the gel fraction (degree of crosslinking) of the cell-free dermal matrix paste according to the reaction temperature before and after radiation sterilization was measured ( FIG. 8 ). In order to analyze the gel fraction, the initial weight of the reacted paste at each temperature was measured, and then immersed in deionized water and treated at room temperature for 48 hours or 72 hours in a constant temperature shaking water bath (60 rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a dry oven at 50 °C.

Gel Fraction (%) = Wd/Wi * 100

Wi: Initial sample weight

Wd: dry weight after extraction

The gel fractions of the paste reacted at 5, 15, 25, 35, 45 and 55 °C were analyzed to be 99, 96, 78, 18, 6 and 3%, respectively. Accordingly, it was confirmed that the higher the reaction temperature of the paste before and after radiation sterilization, the lower the gel fraction, that is, the lower the degree of crosslinking.

In addition, the gel fraction of the paste without E-beam sterilization was about 2%, and it was analyzed that crosslinking was not made. On the other hand, when E-beam sterilization was treated at room temperature, the value of the gel fraction was about 79%.

The gel fractions of the paste without E-beam sterilization and the paste subjected to E-beam sterilization at 45 °C and 55 °C were about 2%, 6.3%, and 3%, respectively, and the change in the gel fraction was about 68% and 33 % was analyzed.

When E-beam sterilization is performed on a paste formulation, the gel fraction is increased by cross-linking, but when heat of 45 ° C or 55 ° C is applied during E-beam sterilization, cross-linking is inhibited and the gel fraction is reduced. .

Example 7: Clinical therapeutic effect of paste

7-1: Clinical treatment effect of wide and deep wounds

2cc of the paste of the present invention was applied to the wound site where bones and tendons were exposed, twice for 2 weeks, and treated with Curabag. After 2 weeks, it was confirmed that the granulation tissue in the area where the paste was engrafted was re-formed to cover the exposure of the bone (FIG. 9).

7-2: Clinical treatment effect of foot wounds

The paste of the present invention was treated with Yugo Tool Barrier and Curabag on pocket wounds caused by foot trauma. As a result, it was confirmed that granulation tissue was formed on the 7th day of application and the depth of the pocket wound became shallow, and then it was confirmed that the foot ulcer was treated without surgery by repeating the treatment (FIG. 10).

7-3: Clinical Treatment Effects of Chronic Wounds

This is a case where the operating window was opened due to infection after chest surgery, and the patient's condition was not good, so reoperation was difficult, and even with the use of Curavac alone, granulation tissue was not formed for 6 months. It was confirmed that this was completely cured (FIG. 11)

7-4: Clinical therapeutic effect of tissue cut wounds

The paste of the present invention and Curabag were concurrently treated on a deeply dented wound of a patient whose toe was amputated due to diabetic complications. As a result, it was confirmed that blood vessels were filled into the paste on the 9th day of treatment and the formation of granulation tissue was promoted, and it was confirmed that it was cured in 44 days (FIG. 12). Since it is known that it takes about 90 days on average to cure the toe amputation wound as described above, it was confirmed that the healing period was reduced by half when the paste of the present invention was applied.

7-5: Clinical treatment effect of intractable wounds

Although flap surgery was performed on the exposed bone in the soft tissue injury, the wound was re-opened due to the tunneling wound, and granulation tissue was not formed, so it developed into an intractable wound. As described above, when the paste of the present invention was applied to a case that was not healed by conventional treatment and was difficult to operate, it was confirmed that blood vessels were filled up and granulation tissue was formed, which was cured by final suture (FIG. 13).

Example 8: Preparation of sheet for wound covering material

The paste prepared in Example 3 was applied to molds having horizontal, vertical and thickness values of 10 cm, 10 cm and 2.5 mm, and then freeze-dried.

FIG. 14 is an external photograph of the sheet manufactured by the above method, and FIG. 15 is a photograph taken after rehydrating the sheet in a saline solution for 1 hour. It was confirmed that the shape of the sheet was maintained without significant change in appearance even after hydration for 1 hour.

Example 9: Confirmation of storage stability according to heat treatment before sterilization

9-1. Viscosity check

The viscosity of the paste was measured using the torque generated by the viscosity of the fluid by rotating the disk in the fluid according to the rotational viscometer method (the general test method of the Korean Pharmacopoeia). ) conditions were set at 15 rpm and 25° C., and the viscosity of 1 g of the sample was repeatedly measured. A comparative test was performed between the sample treated at the optimized temperature (45° C.) and the non-sterilized sample before sterilization.

As a result, as shown in FIG. 16 , it was confirmed that 20,000-3,000cp of viscosity similar to the initial state appeared after radiation sterilization in the heat-treated sample, whereas crosslinking occurred after radiation sterilization in other samples and the viscosity was measured to be three times higher. .

9-2. Check the gel fraction

In the paste preparation step, the gel fraction (crosslinking degree) of the acellular dermal matrix paste with or without heat treatment before radiation sterilization was measured. In order to analyze the gel fraction, the initial weight of the reacted paste at each temperature was measured, and then immersed in deionized water and treated at room temperature for 48 hours or 72 hours in a constant temperature shaking water bath (60 rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a dry oven at 50 °C.

Gel Fraction (%) = Wd/Wi * 100

Wi: Initial sample weight

Wd: dry weight after extraction

As a result, in the heat-treated sample as shown in FIG. 17, the gel fraction after radiation sterilization is maintained at about 6% even after time passes, whereas in non-radiation-sterilized samples, crosslinking occurs after radiation sterilization, and a gel fraction of 70-85% is measured. confirmed that.

Example 10: Confirmation of adhesion according to the shape of the pulverized ADM

In order to compare and measure the adhesiveness of ADM pulverized into a fibrous paste with an ADM pulverized into a bead form, it was measured using TPA (texture profile analysis) (FIG. 18). Measured by pressing the sample with a compression plate at a speed of 50 mm/min.

As a result, as shown in FIG. 19 , it was confirmed that the paste prepared with the ADM pulverized into a fibrous form showed about 26,000 g.s, whereas the paste prepared with the ADM pulverized into the bead form showed a low adhesion of about 3,000 g.s.

Since the use of human skin tissue from a donor (cadaver) for experimental purposes is limited, pig skin close to human skin was used and manufactured according to the method in Examples below.

Example

(1) Pig skin was thoroughly washed with physiological saline.

(2) Pig skin was cut into a size of 5 × 10 cm2, respectively.

(3) Pig skin was put in 1M NaCl (Sigma, USA) solution.

(4) A 38°C reactor (P-039, Coretech) was prepared.

(5) Pig skin soaked in 1M NaCl (Sigma, USA) solution was stirred in a 38°C reactor (P-039, Coretech) and reacted for about 6 to 24 hours.

(6) The epidermis was removed using forceps.

(7) The dermis from which the epidermis was removed was washed with a phosphate buffer solution.

(8) The washed dermis was placed in 0.5% SDS and reacted with stirring at room temperature for 1 hour to remove cells in the dermis.

(9) The dermis from which cells were removed was washed with a phosphate buffer solution.

(10) Glycerol (Sigma, USA), propylene glycol (Sigma, USA) and phosphate buffer solution (GIBCO, USA) were mixed in a weight ratio of 1: 1:8.

(11) Sucrose (Sigma, USA) was added and dissolved in the solution of (10) so that the final concentration was 30% to prepare a cryoprotectant.

(12) 4 ℃ low temperature reactor (P-039, Coretech) was prepared.

(13) The pig skin of (9) was put into a 4°C low-temperature reactor, and then the cryoprotectant of (11) was infiltrated for 12 hours.

(14) Pig skin, which had already penetrated the cryoprotectant, was placed in a Tyvek bag (Tyvek bag, Korea Advanced Materials, Korea).

(15) A freeze dryer (Genesis 25XL, VirTis, USA) was prepared.

(16) The Tyvek bag of (14) was put into a lyophilizer and frozen at a rate of -1°C per minute to -70°C, and then placed in a lyophilizer with a vacuum of 5 torr and dried for 24 hours to obtain a freeze-dried cell-free dermal matrix. .

(17) The freeze-dried acellular dermal matrix was pulverized and fibrous using a cutting mill (Pulverisette 19, FRITSCH, Germany).

(18) Water was added to 1.25 g of gelatin to make 100 ml, and then dissolved at 60° C. to prepare an aqueous gelatin solution.

(19) 11 g of the fibrous acellular dermal matrix of (17) was mixed with 89 g of an aqueous gelatin solution of (18) to prepare a paste.

(20) The paste of (19) was put in a syringe, P.E.T tray and Tyvek double packaging, followed by E-beam radiation sterilization.

Experimental Example 1: Viscosity Measurement

Viscosity measurement was carried out by rotating the disk in the fluid according to the rotational viscometer method and measuring the torque generated due to the viscosity of the fluid. The following method was repeated 6 times, and the results are shown in Table 1.

(1) To measure the viscosity at room temperature (25°C), the rotational viscometer was adjusted to 25°C using a circulator.

(2) Put 1 ml of the sample in the rotational viscometer and rotate the disk to measure the viscosity value due to torque.

Experimental Example 2: Measurement of loss on drying

Loss on drying was measured by the following method, and the results are shown in FIG. 2 .

(1) About 1 g of the sample was put into the weighing bottle, and the initial weighing bottle and the sample were weighed.

(2) After drying at 105° C. for 4 hours, the weighing bottle containing the sample was weighed.

(3) The weight before and after drying was measured using the following formula to measure the loss on drying.

a: Initial weight of the weighing bottle

b: sample weight

c: Weighing bottle and sample weight after drying at 105℃ for 4 hours

Experimental Example 3: Damaged skin regeneration efficacy evaluation

According to Table 3 below, 6-week-old Sprague Dawley rats were divided into groups. The collagen sponge was prepared by freeze-drying bovine-derived collagen (Bioland) and using carbodiamide as a crosslinking agent. It was conducted in the Medi-Campus SPF Clean Animal Room.

In order to prevent the implants from falling off after application and transplantation of Examples and Controls on the wounds in two places of each rat, the first dressing with Tegaderm™, 3M, followed by the second dressing with an elastic bandage and the dressing was changed daily for about 10 days when exudate occurred.

The weight of each rat was measured and the results are shown in FIG. 4 . The weight difference between the normal rats in the positive control group and the experimental group was within 20%, and no weight change was observed due to surgery or dressing.

3-1. Evaluation of wound shrinkage and dermal regeneration

After calculating the area of the wound by the Leopard program, the result is shown in FIG. 5 as the wound contraction rate, and it is shown in Table 4 below along with the dermal regeneration result.

As can be seen from the above results, the paste of Example exhibited a much superior effect compared to the control group.

3-2. Histological observation (H&E staining and MT staining)

After 6 weeks of the experiment, the animals were sacrificed and the tissue containing the wound site was extracted, fixed in 10% neutral formalin solution for 24 hours, and the tissue damaged by burns was taken and embedded in paraffin after dehydration. Using a tissue sectioner, the tissue was sliced to a thickness of 5 μm and attached to a slide to remove paraffin and hydrous. Then, hematoxylin-eosin (H&E) staining and Masson's trichrome (MT) staining were performed. .

For H&E staining, the tissue sections were paraffin-removed with xylene, then hydrated with 100, 90, 80, 70% ethanol and distilled water for 5 minutes, respectively, and washed with distilled water before use. It was stained with Harris hematoxylin for 3 minutes, and the tissue was washed with distilled water for 5 minutes. The washed tissue was stained with eosin for 5 minutes, dehydrated using 70, 80, 90, and 100% ethanol and xylene, and then sealed with Shandon Synthetic Mountant (Thermo scientific, USA). For MT staining, the tissue sections were deparaffinized with xylene and then hydrated with 100, 90, 80, and 70% ethanol and distilled water for 5 minutes, respectively, and washed with distilled water before use. After reacting the tissue in Bouin's (IMEB, USA) solution of 60 for one hour, it was washed with distilled water. After the reaction and washing, the tissues were again treated with Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue stain solution (IMEB, USA) for 5 minutes, respectively, and then washed with distilled water. After dehydration using 70, 80, 90, 100% ethanol and xylene, it was sealed with Shandon Synthetic Mountant (Thermo scientific, USA).

The results of H&E staining and MT staining are shown in FIGS. 6 and 7, respectively. 6, it can be seen that in the case of the Example group, regeneration was not made to the epidermal layer, but in the dermal layer, new blood vessels, fibroblasts, monocytes and some macrophages were observed. In FIG. 7 , it could be seen that the initial regeneration of the dermis occurred in the Example group, and the regeneration of collagen fibers was thicker than that of the other groups.

3-3. CD 31 immunostaining

Since the generation of new blood vessels is important in the treatment process for skin damage, the results of CD 31 immunostaining of tissues 2 and 6 weeks after the experiment were shown in FIGS. 8 and 9, respectively, in order to evaluate the formation of blood vessels.

As can be seen from FIG. 8, in the tissue after 2 weeks of the experiment, no neovascularization was observed in the negative control group and the control group 1, but brown neovascularization was observed in the control groups 2 and 3, and the inflow of many new blood vessels was observed in the Example group. . From FIG. 9, in the tissue after 6 weeks of the experiment, no new blood vessels were observed in the other experimental groups, but new blood vessels were still observed in the Example group, from which it could be seen that the regeneration of the dermis was continued.

3-4. Victoria blue dyed

The formation of elastin, which has an important effect on the elasticity of the skin, plays an important role in the completeness of dermal regeneration. Accordingly, Victoria blue staining was performed to observe the formation of elastin in the dermis 6 weeks after wound treatment, and the results are shown in FIG. 10 .

As can be seen from FIG. 10, blue elastin was observed in the positive control group and the Example group, and weak elastin expression was also observed in Control 3.

3-5. fibronectin immunostaining

Fibronectin is an extracellular matrix (ECM) that has an important effect on the elasticity of the skin and plays an important role in dermal regeneration. Accordingly, immunostaining was performed to observe the formation of fibronectin in the dermis 6 weeks after wound treatment, and the results are shown in FIG. 11 .

As can be seen from FIG. 11 , brown fibronectin was observed in the positive control group and the Example group, and the expression of fibronectin was also observed in Control 2 and Control 3. However, fibronectin was expressed very weakly in the negative control group and control group 1.

3-6. Laminin staining

The basement membrane between the dermis and the epidermis is an important extracellular matrix (ECM) for the regeneration of the epidermal layer. If the regeneration of the basement membrane is weak, skin diseases such as blister formation occur. Accordingly, after 6 weeks of wound treatment, laminin staining was performed to observe the regeneration of the basement membrane, and the results are shown in FIG. 12 .

As can be seen from FIG. 12 , brown laminin was observed in the Example group and Control 3 similar to the positive control, and in Control 2, a lot of laminin was expressed in the dermal layer other than the basement membrane. However, in the negative control group and control group 1, laminin was expressed very weakly.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A composition characterized in that it is made by pulverizing and fibrotic allogeneic dermis of a sheet type freeze-dried type, rehydrating it with a matrix made of fibrotic acellular dermis and soft tissue, then reprocessing it into a sheet type and freeze-drying it As a non-crosslinked and crosslinkable composition.
The method according to claim 1,
The fibrous cell-free particles are characterized by skin-derived acellular dermis, and a composition comprising all soft tissues (amniotic membrane, fascia, pericardium, etc.).
The method according to claim 1,
The composition, characterized in that the long axis length of the fibrous cell-free particle size is 100 to 2,000um.
The method according to claim 1,
A composition characterized in that it is prepared through a crosslinking process through crosslinking using radiation, reduced pressure and heat, and chemical crosslinking using a crosslinking agent.
The method according to claim 1,
The fibrotic acellular dermis and soft tissue is a composition comprising human-derived allogeneic and heterogeneous soft tissue.
The method according to claim 1,
The sheet-type composition made through hydration and freeze-drying has a thickness of 0.1-2 mm.
The method according to claim 1,
A composition comprising 4-20% moisture content by freeze-drying.
The method according to claim 1,
Composition, characterized in that it further comprises an antibacterial agent, excipient and additive used pharmaceutically in the composition.
The method according to claim 1,
A composition comprising stem cells and growth factors in the composition.
The method according to claim 1,
A composition comprising natural and synthetic polymers in the composition.