Low-extrusion composition comprising acellular dermal matrix and method for preparing same

Abstract

The present invention relates to a low-pressure extrusion composition includes an acellular dermal matrix having improved extrusion force properties compared to existing ones by applying maltitol, and a preparation method therefor. The low-extrusion composition including an acellular dermal matrix comprises: 100 parts by weight of the acellular dermal matrix; 100-3000 parts by weight of water; and 1-1000 parts by weight of maltitol, and the preparation method includes the steps of: (a) preparing a de-epidermized, defatted, and decellularized human-derived acellular dermal matrix; (b) adding and infiltrating a maltitol solution to the acellular dermal matrix; (c) freeze-drying and pulverizing the acellular dermal matrix that has completely undergone the infiltration; and (d) hydrating and forming a paste by adding water to the pulverized acellular dermal matrix.

Description

Low-extrusion composition comprising acellular dermal matrix and method for producing the same

[0001] The present invention relates to a low-extrusion composition comprising an acellular dermal matrix (ADM) and a method for producing the same.

[0002] Acellular dermal matrices are injectable biological tissue substitutes. They promote the regeneration of existing tissue and repair damaged areas, enabling them to be used not only for wound healing but also for soft tissue reinforcement. These acellular dermal matrices are applied in various forms for wound treatment, including injectables, dressings, adhesives, surgical and medical devices, artificial skin, bandages, foams, films, anti-adhesives, and grafts.

[0003] Meanwhile, when acellular dermal matrix is ​​used in an injectable form, the high extrusion force makes it difficult for the practitioner to control, increasing patient pain and potentially leading to momentary shock. Attempts have been made to reduce extrusion force by adding lubricants such as hyaluronic acid. However, the lubricant's properties limit its use, hindering its widespread application.

[0004] Therefore, there is an urgent need to develop a component that can replace the existing lubricants while reducing extrusion force.

[0005] The purpose of the present invention is to solve the above-described problem, and to provide a low-extrusion composition including an acellular dermal matrix having reduced extrusion force characteristics compared to conventional compositions by applying maltitol, and a method for producing the same.

[0006] In order to achieve the above purpose, a low-extrusion composition comprising an acellular dermal matrix according to one embodiment of the present invention comprises 100 parts by weight of an acellular dermal matrix, 100 to 3000 parts by weight of water, and 1 to 1000 parts by weight of maltitol.

[0007] A method for producing a low-extrusion composition comprising an acellular dermal matrix according to another embodiment of the present invention comprises the steps of (a) producing a human-derived acellular dermal matrix that has been de-epidermized, defatted, and decellularized, (b) adding a maltitol solution to the acellular dermal matrix and infiltrating it, (c) freeze-drying the infiltrated acellular dermal matrix and then pulverizing it, and (d) adding water to the pulverized acellular dermal matrix to hydrate and form a paste.

[0008] According to one embodiment of the present invention, a low-extrusion composition including an acellular dermal matrix can improve convenience and safety of the procedure by lowering the average extrusion force by applying 1 to 1000 parts by weight of maltitol.

[0009] FIG. 1 and FIG. 2 are process diagrams illustrating a method for manufacturing a low-extrusion composition including an acellular dermal matrix according to another embodiment of the present invention.

[0010] Figure 3 is a graph showing the results according to Experimental Example 1.

[0011] Figures 4 to 6 are graphs showing the results according to Experimental Example 2.

[0012] Figure 7 is a photograph showing a mixture of ADM and purified water, and Figure 8 is a graph showing the results according to Experimental Example 3.

[0013] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0014] In addition, the terms used in this specification are terms used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0015] Throughout this specification, '%' used to indicate the concentration of a particular substance is %(w / w) for solid / solid, %(w / v) for solid / liquid, and %(v / v) for liquid / liquid, unless otherwise stated.

[0016]

[0017] Hereinafter, a low-extrusion composition including an acellular dermal matrix according to one embodiment of the present invention is described in detail.

[0018] The low-extrusion composition comprising the acellular dermal matrix of the present embodiment comprises an acellular dermal matrix, water, and maltitol. In the present embodiment, the extrusion force, which was relatively high at 100 N or more, was reduced to 20 N or less by applying maltitol, which acts as a lubricant, thereby improving the convenience and safety of the procedure. In other words, although individual differences may occur, the present invention was able to achieve an extrusion force of 20 N or less, which is generally a level at which injection without clogging is possible.

[0019] Acellular dermal matrix (ADM) is a widely used ingredient in plastic and orthopedic surgery for the reconstruction, regeneration, and strengthening of skin, tendons, and ligaments due to its superior biocompatibility, high cell adhesion, and low immune response compared to conventional animal-derived products. This acellular dermal matrix can be included in amounts of 100 parts by weight.

[0020] Water is an ingredient added for hydration, and may be included in an amount of 100 to 3,000 parts by weight per 100 parts by weight of the acellular dermal matrix. If the amount of water is less than 100 parts by weight, it may be difficult to hydrate the acellular dermal matrix and maltitol, and if it exceeds 3,000 parts by weight, it may be difficult to implement the desired formulation due to excessive use.

[0021] Maltitol is a component that acts as a lubricant and prevents a decrease in extrusion force within a syringe and browning of tissue. It can be included in an amount of 1 to 1000 parts by weight per 100 parts by weight of acellular dermal matrix. If maltitol is less than 1 part by weight, its effect on the decrease in extrusion force may be minimal, and if it exceeds 1000 parts by weight, the effect on the decrease in extrusion force due to excessive addition may not be that great.

[0022] In this embodiment, one or more additives selected from a storage solution, an isotonic solution, a buffer solution, a surfactant, glycerol, and propylene glycol may be further included, and since the additive is preferably managed to be 50 ppm or less as a decellularizing reagent, it may be included in an amount of 0.00001 to 0.00125 parts by weight per 100 parts by weight of the acellular dermal matrix.

[0023] It goes without saying that the low-extrusion composition comprising the acellular dermal matrix in this embodiment can be used as an injectable, a covering, an adhesive, a surgical and medical device, an artificial skin, a bandage, a foam, a film, an anti-adsorption agent or an implantable material.

[0024] Meanwhile, when a low-extrusion composition including the acellular dermal matrix of the present invention is injected into the body, it acts as a scaffold, thereby activating cells and growth factors in the body to help not only regenerate but also recover and strengthen soft tissues, and therefore can be used for the purpose of recovering and regenerating damaged or missing muscles, biomembranes, ligaments, and tendons during internal and external surgical procedures and operations. Here, soft tissue refers to muscles, ligaments, fat, blood vessels, nerves, tendons, fibrous tissues, synovial tissues, etc., and among the connective tissues of the body, it collectively refers to the rest excluding hard tissues, i.e., cartilage and bone, and blood and hematopoietic tissue.

[0025]

[0026] Hereinafter, a method for manufacturing a low-extrusion composition including an acellular dermal matrix according to another embodiment of the present invention will be described in detail with reference to the drawings.

[0027] FIG. 1 and FIG. 2 are process diagrams illustrating a method for manufacturing a low-extrusion composition including an acellular dermal matrix according to another embodiment of the present invention.

[0028] Referring to FIGS. 1 and 2, first, a de-epidermized, de-fatted, and decellularized human-derived acellular dermal matrix is ​​manufactured (S10).

[0029] An acellular dermal matrix can be manufactured by the steps of: preparing human-derived skin tissue without performing separate de-epidermization and defatting processes (S11); treating the skin tissue with a hypotonic solution containing a surfactant (S12); and washing the treated skin tissue with an isotonic solution (S13).

[0030] For example, the surfactant may be an ionic surfactant, preferably sodium dodecyl sulfate (SDS). The storage solution may include at least one selected from Tris-HCl, EDTA (ethylenediaminetetraacetic acid), and NaOH (sodium hydroxide), preferably 0.1 to 1.5 M EDTA, and the osmolality may be 270 mOsm / L or less. The isotonic solution may include at least one selected from Tris-HCl, EDTA (ethylenediaminetetraacetic acid), NaOH (sodium hydroxide), and NaCl (sodium chloride), and the osmolality may be 270 to 300 mOsm / L.

[0031] In the present invention, osmolarity refers to the expression of osmotic concentration per volume of solution (mOsm / L), and the osmolar concentration of plasma and other body fluids is 270 to 300 mOsm / L.

[0032] Between the above steps S12 and S13 and after the above step S13, a step of washing the skin tissue with a buffer solution may be further included, wherein the buffer solution is PBS (phosphate buffer saline), distilled water, physiological saline, HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), Bicine (N,N-Bis(2-hydroxyethyl) glycine), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), PIPES (piperazine- N,N'-bis(2-ethanesulfonic acid), sodium cacodylate, potassium cacodylate, MES (2-(N-morpholino)ethanesulfonic acid), It may be at least one selected from MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI1640, IMDM (Iscove's Modified Dulbecco's Media), Keratinocyte-SFM (without BPE (bovine pituitary extract)), Keratinocyte-SFM (with BPE), KnockOut DMEM, AmnioMAX-II Complete Medium, and AmnioMAX-C100 Complete Medium.

[0033]

[0034] Next, a maltitol solution is added to the acellular dermal matrix and allowed to penetrate (S20).

[0035] In the above step S10, 20 to 40 parts by weight of a maltitol solution is added to 100 parts by weight of the acellular dermal matrix, and the solution can be infiltrated in a low-temperature reactor at 3 to 5°C for 10 to 30 hours. If the maltitol solution is added in an amount less than 20 parts by weight, the content of maltitol that penetrates into the acellular dermal matrix may be low, and the effect of reducing extrusion force may be minimal. If the content exceeds 40 parts by weight, the content of maltitol that penetrates into the acellular dermal matrix may be high, but the effect of reducing extrusion force may be minimal. If the infiltration time is less than 10 hours, the effect of improving the maltitol penetration rate into the acellular dermal matrix may be minimal. If it exceeds 30 hours, the effect due to the excess time may not be very large.

[0036] In this embodiment, the maltitol solution may be a mixed solution of glycerol, propylene glycol, and a buffer solution containing maltitol at a concentration of 20 to 40%, preferably 25 to 35%. The above-mentioned concentration of maltitol may be taken into consideration for the content of maltitol that penetrates into the acellular dermal matrix. The mixed solution may be a mixture of glycerol, propylene glycol, and a buffer solution at a weight ratio of 1: 0.1 to 2: 7 to 9. Since the buffer solution has been described in detail above, a detailed description thereof will be omitted.

[0037]

[0038] Then, the acellular dermal matrix that has been completely penetrated is freeze-dried and then pulverized (S30).

[0039] The fibers can be crushed into a fiber form using one or more crushers selected from a cutting mill, a food processor, a mano crusher, and a freeze crusher so that the ratio of the short axis to the long axis is 1:1 to 2,000, and the ratio is 70% or more. In the present embodiment, the long axis length can be in the range of 10 to 2,000 μm. If the ratio of the short axis to the long axis or the long axis length is outside the above-mentioned ratio of the short axis to the long axis or the long axis length, it may be difficult to implement the viscosity and flowability required in the present embodiment when pasted as described below.

[0040]

[0041] Finally, water is added to the crushed acellular dermal matrix to hydrate and form a paste (S40).

[0042] In the above step S30, 100 to 2,500 parts by weight of water can be added to 100 parts by weight of the pulverized acellular dermal matrix to hydrate and form a paste. If the water content is less than 100 parts by weight, it may be difficult to hydrate the acellular dermal matrix permeated with maltitol, and if it exceeds 2,500 parts by weight, it may be difficult to implement the desired formulation due to excessive use.

[0043]

[0044] After the above step S40, a step (S50) of sterilizing the pasted acellular dermal matrix by irradiating it with radiation (E-beam) at a dose ranging from 11 to 30 kGy may be further included.

[0045] In the above step S40, the paste-formed acellular dermal matrix is ​​filled into a syringe and then sterilized by irradiating with radiation (E-beam) at a dose ranging from 11 to 30 kGy. However, if the dose exceeds 30 kGy, the relatively high dose may cause tissue degeneration and destruction of the structure in human tissue. In the present embodiment, by irradiating with radiation at the dose range mentioned above, only bacteria can be killed while minimizing destruction and degeneration of human tissue.

[0046]

[0047] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0048]

[0049] Manufacturing Example 1. Manufacturing of acellular dermal matrix

[0050] Skin tissues were purchased from EURO skin bank, Allosource, and CTS, and tissues with a thickness of 1 mm or more were selected. The adipose tissue attached to the skin tissues was removed using forceps, and the tissues were washed three times with sterile water. Then, the tissues were immersed in a hypotonic solution (Tris-HCl, EDTA, NaOH, and SDS) and treated for 6 hours. Then, the skin tissues were washed with PBS at 4°C to remove the remaining fat, epidermis, cells, and hypotonic solution, and left overnight. After washing with an isotonic solution (Tris-HCl, EDTA, NaCl, and NaOH) for 6 hours, a deepidermal, defatted, and decellularized human-derived acellular dermal matrix was prepared.

[0051]

[0052] Manufacturing Examples 2 to 7. Preparation of maltitol solution

[0053] A mixed solution containing glycerol (Sigma, USA), glycol (Sigma, USA), and phosphate buffer solution (Gibco, USA) in a weight ratio of 1:1:8 was prepared so that the maltitol concentration was within the range shown in Table 1 below.

[0054] Maltitol concentration (%) Manufacturing example 20 Manufacturing example 37.15 Manufacturing example 414.3 Manufacturing example 517.9 Manufacturing example 621.45 Manufacturing example 725

[0055]

[0056] Examples 1 to 16. Preparation of low-extrusion compositions

[0057] In Manufacturing Example 1, a maltitol solution was added to the acellular dermal matrix and infiltrated in a low-temperature reactor at 4°C. Then, it was placed in a Tyvek (Korea Advanced Materials, Korea), freeze-dried for 24 hours in a vacuum freeze-dryer at 5 torr, and then pulverized using a cutting mill (Pulverisette19, FRITSCH, Germany). Then, 3 cc of distilled water was added to 1 g of the pulverized acellular dermal matrix, mixed with a stirrer at room temperature, placed in a syringe, double-packed in a PET tray and Tyvek, irradiated with 11 to 30 kGy of radiation (E-beam), and sterilized to prepare a low-extrusion composition (hereinafter referred to as “ADM composition or ADM”).

[0058] ClassificationMaltitol contentMaltitol penetration timeSterilizationExample 1 Manufacturing Example 212OExample 2 Manufacturing Example 312OExample 3 Manufacturing Example 412OExample 4 Manufacturing Example 612OExample 5 Manufacturing Example 224XExample 6 Manufacturing Example 324XExample 7 Manufacturing Example 424XExample 8 Manufacturing Example 524XExample 9 Manufacturing Example 624XExample 10 Manufacturing Example 724XExample 11 Manufacturing Example 224XExample 12 Manufacturing Example 324XExample 13 Manufacturing Example 424XExample 14 Manufacturing Example 524XExample 15 Manufacturing Example 624XExample Manufacturing Example 16 724O

[0059]

[0060] Examples 17 to 22. Preparation of low-extrusion compositions

[0061] A low-extrusion composition was prepared by mixing ADM and purified water according to Example 13 in the weight ratio shown in Table 3 below (hereinafter referred to as 'ADM composition or ADM').

[0062] Purified water content per 1 part by weight of ADM (parts by weight)Example 1750Example 1820Example 1910Example 203Example 212Example 221

[0063]

[0064] Comparative Example 1: DMSO treatment group

[0065] Comparative Example 2: ADM 0% (only media)

[0066]

[0067] Experimental Example 1: Quantitative Analysis of Maltitol

[0068] The content of maltitol in the ADM compositions according to Examples 1 to 16 was measured and expressed as the maltitol content (%) in Table 4. Sample photographs and detected maltitol contents of the ADM compositions according to Examples 1 to 4 are shown in Fig. 3.

[0069] Referring to FIG. 3 and Table 4, it was confirmed that maltitol was detected in the ADM composition manufactured according to the present invention under both 12-hour and 24-hour maltitol penetration conditions.

[0070] Referring to FIGS. 3 and 4, it was confirmed that as the concentration of maltitol in the ADM composition increased, the content of maltitol remaining in the tissue powder increased, which affected the extrusion force. It was confirmed that the extrusion force decreased from Example 1 to Example 4, and it was found that the average and maximum extrusion forces decreased depending on the content of maltitol.

[0071]

[0072] Experimental Example 2: Evaluation of extrusion force according to the presence / absence and content of maltitol

[0073] In order to confirm the extrusion force characteristics according to the presence / absence and content of maltitol, the ADM compositions according to Examples 1 to 16 were placed in a 3cc syringe (Needle: 18G), and the reagent was extruded from the syringe at a speed of 50 mm / min and a constant force using a UTM (Universal Testing Machine), and the results are shown in Table 4 and Figures 4 to 6. At this time, the average value was the average value of the extrusion force that appeared in the total time section during which the reagent was continuously extruded, and the maximum value was the maximum value of the extrusion force that appeared in the total time section during which the reagent was continuously extruded.

[0074] Partial pressure output average / maximum value (N) Maltitol content (%) Partial pressure output average / maximum value (N) Maltitol content (%) Example 155.2 / 157.3 Not detected Example 92.01 / 3.85 17.16 Example 24.57 / 11.4 46.23 Example 101.85 / 3.3 419.10 Example 32.86 / 6.3 09.79 Example 115 0.2 / 139.5 Not detected Example 42.85 / 6.2 310.68 Example 123.84 / 12.5 04.74 Example 52.83 / 4.18 Not detected Example 132.02 / 7.9 47.49 Example 62.20 / 3.904.74Example 141.80 / 4.9513.74Example 71.44 / 3.667.49Example 151.46 / 3.4817.16Example 81.94 / 3.9013.74Example 161.16 / 3.3119.10

[0075] Referring to Table 4 and Figures 4 to 6, it was confirmed that as the concentration of maltitol in the ADM composition increased, the content of maltitol remaining in the tissue powder increased, which affected the extrusion force to decrease.

[0076] In addition, it was confirmed that the low-extrusion composition according to the embodiment of the present invention exhibited an extrusion force of 20 N or less in both cases where the maltitol solution was infiltrated for 12 hours (overnight) and 24 hours.

[0077] In addition, referring to Examples 5 to 10 illustrated in FIG. 5, it was confirmed that no significant difference occurred before sterilization depending on the presence or absence of maltitol, but that a significant difference occurred after sterilization for commercialization depending on the presence or absence of maltitol, as in Examples 11 to 16 illustrated in FIG. 6. In addition, referring to Examples 5 and 11 corresponding to the case where maltitol was not included, it was confirmed that the extrusion force increased rapidly during the sterilization process, but referring to Examples 6 to 10 and Examples 12 to 16 corresponding to the case where maltitol was included, it was confirmed that the extrusion force was maintained at 20 N or less even after sterilization.

[0078] That is, the ADM compositions of Examples 1 and 11, which were sterilized and did not contain maltitol, were confirmed to have an extrusion force exceeding 20 N in both the average and maximum values, whereas the ADM compositions of Examples 12 to 16, which were sterilized and contained maltitol, were all confirmed to have an extrusion force of less than 20 N. That is, it was confirmed that the extrusion force was significantly low when the concentration of the maltitol solution of the present invention was in the range of more than 0% and less than 25%.

[0079] In addition, the average extrusion force of the ADM composition generally increases during the sterilization process, but it was confirmed that the average extrusion force of Examples 14 to 16 was rather reduced.

[0080]

[0081] Experimental Example 3: Wound Healing Evaluation

[0082] In order to confirm the wound healing effect according to the content of ADM manufactured according to the embodiment of the present invention, the migratory ability of human dermal fibroblasts (HDF) was evaluated through a scratch assay using Examples 17 to 22.

[0083] After creating a scratch, cell migration to the scratch site was observed over a period of 20 hours, and the results are shown in Figs. 7 and 8.

[0084] Referring to Figures 7 and 8, it was confirmed that cells died and did not grow in the DMSO treatment group, but that the ADM treatment group had a wound healing effect. That is, it was confirmed that the wound healing effect was present in all ranges of ADM and purified water ratios manufactured according to the embodiment of the present invention, ranging from 1:1 to 50. Among these, excellent effects were confirmed in the range of 1:2 to 50, and very excellent effects were confirmed in the range of 1:3 to 1:20.

[0085]

[0086] Although exemplary embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0087] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. The contents of all publications cited herein as references are incorporated herein by reference.

Claims

1. 100 parts by weight of acellular dermal matrix; 100 to 3000 parts by weight of water; and A low-extrusion composition comprising an acellular dermal matrix, comprising 1 to 1000 parts by weight of maltitol.

2. In paragraph 1, A low-extrusion composition comprising an acellular dermal matrix having an extrusion force of 20 N or less. 3.(a) A step of manufacturing a de-epidermized, de-adipized and decellularized human-derived acellular dermal matrix; (b) a step of adding a maltitol solution to the acellular dermal matrix and allowing it to penetrate; (c) a step of freeze-drying and then crushing the acellular dermal matrix after the above penetration is completed; and (d) a method for producing a low-extrusion composition comprising an acellular dermal matrix, comprising the step of adding water to the pulverized acellular dermal matrix to hydrate and form a paste; 4. In paragraph 3, A method for producing a low-extrusion composition including an acellular dermal matrix, wherein in the step (b) above, the maltitol solution is added in an amount of 20 to 40 parts by weight to 100 parts by weight of an acellular dermal matrix, and penetration is performed for 10 to 30 hours.

5. In paragraph 3, A method for producing a low-extrusion composition including an acellular dermal matrix, wherein in the step (b), the maltitol solution is a mixed solution of glycerol, propylene glycol, and a buffer solution, wherein maltitol is contained at a concentration of 20 to 40%.

6. In paragraph 3, A method for producing a low-extrusion composition including an acellular dermal matrix, wherein in the step (c) above, the crushing is performed so that the ratio of the short axis to the long axis is 1:1 to 2,000, and the ratio is 70% or more.

7. In paragraph 3, A method for producing a low-extrusion composition including an acellular dermal matrix, wherein in the step (d) above, water is added in an amount of 100 to 2,500 parts by weight to 100 parts by weight of the pulverized acellular dermal matrix.

8. In paragraph 3, A method for producing a low-extrusion composition including an acellular dermal matrix, further comprising the step of sterilizing the paste-formed acellular dermal matrix by irradiating it with a dose ranging from 11 to 30 kGy after the step (d).

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