KR20230068010A - Method of making super-micronized acellular dermal matrix derived from animal skin - Google Patents

Abstract

The present invention relates to a method for producing ultrafine acellular dermis. In the present invention, the yield of ultra-fine acellular dermis can be increased through first grinding and second grinding steps for ultra-fine acellular dermis, and through a sieve separation step using negative pressure, the time required to obtain the ultra-fine acellular dermis of a uniform size can be shortened. In addition, even after the acellular dermis becomes ultra-fine, the properties and shape of type 1 collagen, a main component of the acellular dermis, can be maintained.

Description

Method of making super-micronized acellular dermal matrix derived from animal skin}

The present invention relates to a method for producing ultra-fine acellular dermis.

The skin tissue of animals, including humans, consists of three parts: an epidermal layer, the outermost layer of the skin, a dermal layer below it, and a subcutaneous layer. The epidermal layer containing keratin recognizes changes in the external environment, protects the body from external contamination or infection, and prevents excessive evaporation of body moisture. Blood vessels distributed in the dermal layer supply oxygen and nutrients to epidermal and dermal cells, maintain skin elasticity, and protect the body. In addition, the fat distributed in the subcutaneous tissue stores the energy necessary for the body and acts as a buffer against external shocks. As described above, skin tissue maintains the body through various roles such as sensory function, protective function, thermoregulation function, and energy storage function (Non-Patent Document 1).

Acellulardermal Matrix, in which the epidermis layer and subcutaneous tissue are removed from skin tissue and the cells of the dermis are removed, is a dermal layer matrix obtained through acellularization technology from human or animal skin, and is an extracellular matrix composed of pure collagen and elastin ( It means a skin substitute in the form of an Extracellular Matrix). It is known that these biologically derived skin substitutes have a relatively low immune rejection response, unlike organs for transplantation. In addition, human-derived skin substitutes are generally reported to be superior to animal-derived skin substitutes in terms of biological performance and safety. However, since human-derived skin substitutes need to receive skin tissue from a posthumous donor, it may be limited in terms of raw material supply and demand compared to animal-derived skin substitutes.

The form of acellular dermis, which is a bioderived skin substitute, is generally very similar to skin, and is transplanted into a patient's body or damaged skin in the form of a flat sheet (Non-Patent Document 2). However, in order to use the acellular dermis as a tissue repair material for plastic surgery or as a wound dressing material such as bedsores and foot ulcers, it must be sufficiently passable through a small-diameter passage such as an injection needle.

Accordingly, the present invention provides a method for manufacturing ultra-fine acellular dermis that maintains the properties and structure of type 1 collagen, which is the main component of acellular dermis, through a novel ultra-miniaturization process and can be injected into the human body.

1. MCGRATH, J. A.; EADY, R. A. J.; POPE, F. M. Anatomy and organization of human skin. Rook's textbook of dermatology, 2004, 1: 3.2-3.80. 2. SPEAR, S. L., et al. Acellular dermis-assisted breast reconstruction. Aesthetic plastic surgery, 2008, 32.3: 418-425.

An object of the present invention is to provide a method for producing ultra-fine acellular dermis.

Specifically, according to the present invention, an ultra-fine acellular dermis of a uniform size can be obtained without changing the properties and structure of type 1 collagen through the ultra-miniaturization process through the steps of primary grinding, secondary grinding and sieve separation, An object of the present invention is to provide a method for producing ultra-fine acellular dermis that can be injected through a fine-diameter injection needle.

The present invention comprises a first grinding step of grinding the acellular dermis at 8,000 to 6,000 rpm using a rotating rotor;

A secondary crushing step of freezing the primary pulverized acellular dermis, putting the frozen acellular dermis and metal rod into a crushing container, and then freeze-grinding them through impact; and

Provided is a method for producing ultra-fine acellular dermis comprising a sieve separation step of sieving the secondary crushed acellular dermis under negative pressure conditions.

In addition, the present invention is an ultra-fine acellular dermis prepared by the above-described manufacturing method; and

An injectable acellular dermal composition comprising a solution is provided.

The present invention provides a method for preparing an injectable ultra-fine acellular dermis.

In the present invention, the yield of ultra-fine acellular dermis is increased compared to the existing process through the primary grinding and secondary grinding steps to ultra-miniaturize acellular dermis, and ultra-fine acellular dermis of uniform size through sieve separation using negative pressure. It is possible to shorten the time required to obtain. In addition, even after ultra-miniaturization, the properties and shape of type 1 collagen, which is the main component of acellular dermis, can be maintained.

The ultra-fine acellular dermis according to the present invention can be mixed with a solution and provided as a composition that can be injected into the human body through a fine-diameter injection needle.

1 is a flow chart showing a method for producing ultra-fine acellular dermis.
2 is a graph showing the results of particle size analysis of ultra-fine acellular dermis according to the presence or absence of a sieve separation step.
Figure 3 is a photograph taken with a scanning electron microscope (SEM) of the ultra-fine acellular dermis.
4 is a photograph showing type 1 collagen, which is the main component of ultrafine acellular dermis, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
5 is a photograph showing type 1 collagen, which is the main component of ultrafine acellular dermis, by Western blot.
6 is a graph showing the injection power of a composition comprising ultrafine acellular dermis and a solution.

The present invention includes a first grinding step of grinding the acellular dermis at 800 to 6,000 rpm using a rotating rotor;

A secondary crushing step of freezing the primary pulverized acellular dermis, putting the frozen acellular dermis and metal rod into a crushing container, and then freeze-grinding them through impact; and

It relates to a method for producing ultra-fine acellular dermis comprising a sieve separation step of sieving the secondly ground acellular dermis under negative pressure conditions.

In the present invention, the yield of ultra-fine acellular dermis can be increased compared to the existing process through the primary and secondary crushing steps to ultra-miniaturize the acellular dermis, and the sieve separation step using negative pressure can increase the yield of ultra-fine acellular dermis of uniform size. The time for obtaining cell dermis can be shortened. The ultrafine acellular dermis obtained by the production method of the present invention can maintain the shape and properties of type 1 collagen, which is the main component of the acellular dermis. In addition, the prepared ultra-fine acellular dermis can be mixed with a solution and injected into the human body through a fine-diameter injection needle.

In the present invention, the numerical value is limited to more than, less than, or more than or less than, but indicates more or less unless otherwise specified.

Hereinafter, the method for producing ultra-fine acellular dermis according to the present invention will be described in more detail.

In the present invention, the ultra-fine acellular dermis refers to acellular dermal particles having a uniform particle size distribution prepared through the manufacturing method of the present invention, that is, primary grinding, secondary grinding and sieve separation. The average particle size of the ultrafine acellular dermis may be 120 μm or less, 110 μm or less, or 100 μm or less.

In the present invention, the average particle size may be calculated as the average value of D10, D50, and D90 using the wet mode of the particle size analyzer (LS13320XR, BECKMAN COULTER).

In the present invention, acellular dermis (ADM) refers to a dermal layer matrix obtained through acellularization technology, and the dermis may be obtained from allogeneic or heterogeneous skin. The same species refers to humans, and the heterogeneous species may refer to animals other than humans, that is, mammals such as pigs, cows, and horses.

In one embodiment, commercially available acellular dermal products for acellular dermis may be used, or cells may be removed from skin tissue for use.

In one embodiment, acellular dermis is removed by removing lipid components from dermal tissue; and

It can be prepared through the step of removing cells from the dermal tissue from which the lipid component has been removed.

In the step of removing lipid components from the dermal tissue, the dermal tissue may be delipidated.

In one embodiment, delipidation can be performed using a delipidation solution. The degreasing solution may include a polar solvent, a non-polar solvent, or a mixture thereof. Water, alcohol or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol. In addition, hexane, heptane, octane, or a mixed solution thereof may be used as the non-polar solvent. Specifically, a mixed solution of isopropyl alcohol (IPA) and hexane may be used as the degreasing solution. At this time, the mixing ratio (wt%) of isopropyl alcohol and hexane may be 20:80 to 80:20.

In the step of removing cells from the dermal tissue from which the lipid components are removed, the dermal tissue may be decellularized. Decellularization refers to removing other cellular components, such as nuclei, cell membranes, and nucleic acids, except for the extracellular matrix, from dermal tissue.

In one embodiment, decellularization can be performed using a decellularization solution. A surfactant and/or a basic solution may be used as the decellularization solution. Specifically, as a surfactant, an ionic surfactant such as sodium dodecyl sulfate (SDS), or Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidet P-10 (NP-10), Noni Nonionic surfactants such as detpi-40 (NP-40) and the like can be used. In addition, at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may be used as the basic solution.

In one embodiment, the acellular dermis may be composed of an extracellular matrix composed of collagen and elastin.

In the present invention, the first grinding step is a step of grinding the acellular dermis at 800 to 6,000 rpm using a rotating rotor.

In one embodiment, the step may be performed using a rotary grinder. As the rotary grinder, IKA's MF-10 product can be used.

In one embodiment, the acellular dermis can be cut to a size applicable to a rotating rotor and then used for primary crushing. At this time, the size of the acellular dermis may be 0.5 to 2 mm in width and length, respectively.

In one embodiment, the primary grinding may be performed at 1,000 to 5,000 rpm, 2,500 to 5,500 rpm, 2,500 to 4,500 rpm, or 3,000 to 4,000 rpm. In addition, the primary grinding time may be 10 to 50 minutes, 10 to 30 minutes, or 20 to 25 minutes. As the rpm increases, the particle size of the acellular dermis decreases, but since a significant particle size reduction effect is not obtained above 6,000 rpm, it is preferable to adjust the rpm within the above range. That is, the aforementioned rpm and time ranges are preferable for process efficiency.

In one embodiment, the average particle size of the primary ground acellular dermis may be 600 to 900 μm, 600 to 850 μm, or 600 to 750 μm. It is possible to efficiently manufacture acellular dermis suitable for human body injection in the above particle size range, and it is applicable to a grinding device for secondary grinding.

In the present invention, the secondary crushing is a step of secondary crushing the acellular dermis primarily crushed in the above step.

The secondary crushing may be performed by freezing the primary crushed acellular dermis, putting the frozen acellular dermis and metal rod into a crushing container, and then freeze-pulverizing through impact.

In one embodiment, freeze-drying of acellular dermis may be performed through a method common in the art.

In one embodiment, the secondary grinding may be performed using a freeze mill. As the freeze mill, SPEX Sample Prep's 6875D product can be used.

In one embodiment, the material of the metal rod may be stainless steel or titanium. In addition, the size of the metal rod may vary depending on the size of the grinding container, and may be, for example, 5 to 8 cm in width and 1 to 3 cm in length.

In one embodiment, a process of putting the frozen acellular dermis and metal rod into a crushing container and then freezing for 10 to 60 minutes may be further performed.

In one embodiment, the secondary grinding may be performed for 1 to 60 cycles, 1 to 45 cycles, 10 to 40 cycles, or 16 to 30 cycles. In addition, the secondary grinding may be performed for 20 to 100 minutes, 20 to 80 minutes, or 20 to 60 minutes. During the cycle and time range, the sample, that is, the acellular dermis, can be finely ground while the metal rod in the grinding vessel moves rapidly. In excess of 60 cycles, since a significant particle size reduction effect is not obtained, it is recommended to adjust the cycle within the above range.

In one embodiment, the average particle size of the secondly ground acellular dermis may be 350 μm or less, 300 μm or less, 250 μm or less, or 100 to 250 μm. In the above particle size range, acellular dermis suitable for human body injection can be efficiently prepared.

In the present invention, the sieving step is a step of sieving the acellular dermis secondaryly pulverized in the above-mentioned secondary pulverizing step under negative pressure conditions.

In one embodiment, the screen size of the sieve may be 100 to 300 μm, 150 to 250 μm or 200 μm.

In one embodiment, the sieve separation step may be performed by placing the acellular dermis on a sieve, sealing the upper end of the sieve, and suctioning the lower end with negative pressure. This negative pressure may be a pressure of 500 to 5000 mmAq or 1000 to 3000 mmAq.

In one embodiment, the sieving time may be 1 to 20 minutes, or 1 to 10 minutes.

Through the sieving step, it is possible to prepare ultra-fine acellular dermis having a uniform particle size distribution.

The average particle size of the ultrafine acellular dermis prepared by the manufacturing method according to the present invention may be 120 μm or less, 110 μm or less, or 100 μm or less. The lower limit of the average particle size may be 30 μm or more.

Further, the yield of ultrafine acellular dermis may be 60% or more, 70% or more, or 80% or more. That is, through the manufacturing method according to the present invention, ultrafine acellular dermis can be obtained with a short process time and high yield compared to conventional methods.

In addition, the present invention relates to an ultra-fine acellular dermis prepared by the method for producing ultra-fine acellular dermis described above.

The ultra-fine acellular dermis according to the present invention is composed of an extracellular matrix composed of collagen and elastin, and can maintain the properties and structure of type 1 collagen, the main component of acellular dermis, even after undergoing a grinding process. there is.

In one embodiment, the ultrafine acellular dermis has an average particle size of 120 μm or less, 110 μm or less, or 100 μm or less, and through this, it can be applied for injection into the human body through injection.

In addition, the present invention is the above-mentioned ultra-fine acellular dermis; and

It relates to an injectable acellular dermal composition comprising a solution.

In one embodiment, sterile physiological saline or sterile distilled water may be used as the solution.

In the acellular dermal composition, the content of the ultrafine acellular dermis may be 5 to 70 parts by weight or 10 to 50 parts by weight based on the total weight of the composition (100 parts by weight).

In one embodiment, the acellular dermal composition is used for injection, and the injection pressure may be 40 N or less. The injection force may be measured according to the method of Experimental Example 4 of the present invention.

In one embodiment, the acellular dermal composition can be used as a tissue repair material for plastic surgery or a wound covering material such as bedsores and foot ulcers.

The present invention will be described in more detail through the following examples. However, those skilled in the art will understand that the scope of the present invention is not limited to the following examples, and that various changes, modifications, or applications are possible within a range that does not deviate from the technical details derived by the matters described in the appended claims. will be.

Example

Example 1. Ultraminiaturization of acellular dermis

(1) Manufacture of acellular dermis

The animal's dermal tissue was washed with sterile distilled water. Fascia and fat were removed from the washed animal's dermal tissue using a scrapper or the like. The dermal tissue was treated with 0.1M to 15M sodium chloride for 6 to 12 hours to remove the epidermis. The dermal tissue from which the epidermis was removed was washed with sterile distilled water. After washing, it was treated for 1 to 6 hours using 10% to 50% isopropyl alcohol and 10% to 50% hexane to remove fat. Then, cells were removed by treatment with 0.1% to 10% SDS solution. The decellularized dermal tissue was frozen for 2 to 6 hours at a temperature of -70°C or lower. The frozen dermal tissue was dried for 12 to 24 hours to prepare acellular dermis.

(2) primary crushing and secondary crushing of acellular dermis

The micronization of the acellular dermis proceeded in the order of primary crushing, secondary crushing, and sieve (Fig. 1).

The primary crushing was performed by crushing the acellular dermis having a horizontal and vertical size of 10 mm x 10 mm using a rotor (MF-10, IKA Co.) rotating at 1000 rpm to 5000 rpm.

Secondary grinding was performed using a 6875D (SPEX Sample Prep) machine. Specifically, the acellular dermis and metal rods frozen at -70 ° C for 12 to 24 hours are placed in a crushing container, sealed, mounted inside a container filled with liquid nitrogen, frozen for 10 minutes to 1 hour, and then the acellular dermis is The contained grinding container was moved repeatedly for 1 to 60 cycles.

(3) Sieve separation of pulverized acellular dermis

The acellular dermis pulverized through the primary and secondary crushing was placed on a separator having a size of 200 μm, the upper end of the separator was sealed, and the lower end was suctioned at a pressure of 1000 to 3000 mmAq to obtain an ultra-fine acellular dermis.

Experimental Example 1. Verification of primary grinding and secondary grinding effects of acellular dermis

In order to confirm the particle size of the acellular dermis and the time required for grinding according to the rpm difference in the primary grinding, the primary grinding was performed at 1000 rpm or more to less than 2000 rpm, 2000 rpm or more to less than 3000 rpm, 3000 rpm or more to less than 4000 rpm, After proceeding at 4000 rpm or more to less than 5000 rpm, the particle size and required time of the sample were checked.

In addition, in order to confirm the particle size of the acellular dermis and the time required for grinding according to the difference in cycle in the second grinding, the second grinding was performed from 1 cycle to 15 cycle, 16 cycle to 30 cycle, and 31 cycle to 45 cycle, and then the sample The particle size and required time were confirmed.

At this time, the particle size was measured using the wet mode of the particle size analyzer (LS13320XR, BECKMAN COULTER) as an average particle size.

Table 1 below shows the particle size and total grinding time of acellular dermis according to the first and second grinding steps.

As shown in Table 1, the particle size decreases as the rpm increases during the primary grinding, but the particle size of the sample (sample 3) and the sample (sample 4) performed at 3000 rpm to 4000 rpm and 4000 rpm to 5000 rpm It can be confirmed that it is within the margin of error. In addition, the required time was about 1 to 7 minutes shorter than the samples performed at 3000 rpm to 4000 rpm than the samples performed at 4000 rpm to 5000 rpm. Through this, it can be confirmed that a significant particle size reduction effect is not obtained at 4000 rpm or more, and it is preferable to perform the primary grinding at less than 4000 rpm in terms of process efficiency.

In addition, at the time of the secondary grinding, the particle size decreases as the cycle increases, but the sample in which the secondary grinding was performed with 16 to 30 cycles (Sample 6) and the sample with 31 to 45 cycles (Sample 7) showed an error. You can check what is within range. In addition, the required time was about 41 to 47 minutes shorter than the samples performed with 16 cycle to 30 cycle and those with 31 cycle to 45 cycle. Through this, it can be confirmed that a significant particle size reduction effect is not obtained in excess of 30 cycles.

In particular, the particle size of samples 8 to 10 in which the first grinding was performed at 3000 rpm to 4000 rpm and then the second grinding was performed was 93 μm to 247 μm smaller than the sample in which only the second grinding was performed, and about 300 μm or less As a result, it can be confirmed that acellular dermis having a fine particle size can be obtained.

The sample (sample 9) with 16 to 30 cycles of secondary grinding along with the primary grinding and the sample with 31 to 45 cycles (sample 10) had particle sizes within the error range, and the time required for sample 9 was It is about 28 minutes to 50 minutes shorter than 10, and it can be confirmed that it is preferable in terms of process efficiency to proceed with the secondary grinding at 30 cycles or less.

Experimental Example 2. Verification of sieve separation effect of ultrafine acellular dermis

In order to confirm the sieving effect of the ultra-fine acellular dermis, the first grinding was performed at 3000 rpm to 4000 rpm, and the second grinding was performed at 1 to 15 cycles, 16 to 30 cycles, and 31 to 45 cycles. The particle size, yield, and required time were confirmed according to the presence or absence of sieving.

Table 2 below shows the particle size, yield, and total required time of ultrafine acellular dermis according to the first grinding, second grinding, and sieve separation steps.

As shown in Table 2, it can be seen that the particle size of the sample subjected to sieving was about 154 to 279 μm smaller than that of the sample not subjected to sieving. In addition, when comparing the particle size, yield, and time required for samples subjected to sieving, the particle size decreased as the cycle of secondary grinding increased after the primary grinding, but the secondary grinding was performed at 16 to 30 cycles. It can be seen that the sample (Sample 16) and the sample (Sample 17) with 31 to 45 cycles are within the error range. In addition, it can be confirmed that the required time for sample 16 is about 31 to 53 minutes shorter than that for sample 17. Through this, it can be confirmed that it is preferable in terms of process efficiency to perform the secondary grinding at 30 cycles or less.

In addition, the acquisition time according to the presence or absence of negative pressure in the sieving step was confirmed. Specifically, the average yield of 82% of sample 16 in Table 2 was set as the maximum yield of acellular dermis, and the time required for obtaining was confirmed.

Table 3 below is a table showing the sifting time required for obtaining according to the presence or absence of negative pressure in the sieving step.

As shown in Table 3, the time required for the sieving step with negative pressure was 5 minutes, while the time obtained for the sieving step without negative pressure was 90 minutes.

In addition, the coefficient of variation according to the presence or absence of sieving of the samples subjected to the first and second grinding was confirmed.

The coefficient of variation is a value obtained by dividing the standard deviation by the arithmetic mean, and was calculated from values measured using LS13320XR (BECKMAN COULTER) for samples 9 and 16.

2 is a graph showing the results of particle size analysis of ultra-fine acellular dermis with and without a sieve separation step.

As shown in FIG. 2, the coefficient of variation of the sample without sieving (sample 9) was 141%, and the coefficient of variation of the sample (sample 16) that was sieved was 96%, which was the sample subjected to sieving. It can be seen that the coefficient of variation of

Through this, it can be confirmed that the particle size of the sample subjected to sieving is more uniform than that of the sample not subjected to sieving.

Experimental Example 3. Verification of components of ultra-fine acellular dermis

In order to verify the components of the ultra-fine acellular dermis, SEM was taken using the uniformly sized ultra-fine acellular dermis (sample 16) and the acellular dermis that was not subjected to the ultra-miniaturization process (i.e. grinding process and sieve separation process), SDS-PAGE and Western blot were performed.

Figure 3 is a photograph taken with a scanning electron microscope (SEM) of the ultra-fine acellular dermis.

As shown in Figure 3, it can be confirmed that the shape and properties of the collagen fibers are maintained even after the ultra-miniaturization process.

4 is a photograph showing type 1 collagen, which is the main component of ultrafine acellular dermis, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). And, Figure 5 is a photograph showing type 1 collagen, which is the main component of ultrafine acellular dermis, by Western blot.

As shown in FIGS. 4 and 5, as a result of SDS-PAGE and Western blot, it can be confirmed that type 1 collagen is maintained even after the micronization process.

Example 2. Mixture of ultrafine acellular dermis and solution manufacturing

The ultrafine acellular dermis corresponding to Sample 3, Sample 6, Sample 9, Sample 11, Sample 13, and Sample 16 were contained in 5% to 50% respectively in a photophoresis bottle, and a solution (sterile physiological saline or sterile distilled water) was added to each bottle. 50% to 95% of the mixture was mounted on a three-dimensionally rotating rotor and mixed at 10 to 150 rpm for 30 minutes to 2 hours to prepare a mixed solution.

Experimental Example 4. Measuring the injection force of the mixed solution

The injection force of the mixture of the ultrafine acellular dermis and the solution was confirmed.

Depending on the presence or absence of sieving, acellular dermis with only 1st grinding (sample 3, sample 11), acellular dermis with only 2nd grinding (sample 6, sample 13), and 1st and 2nd grinding were sequentially performed. Each of the acellular dermis (sample 9 and sample 16) was mixed with the solution, and a 30G injection needle was placed in a syringe. Then, after mounting the syringe on a UTM (universal testing machine), the injection force was checked by operating at a speed of 12 mm/min.

6 is a graph showing the injection force for a mixture of ultrafine acellular dermis and a solution.

Specifically, FIG. 6a shows sample 3, FIG. 6b shows sample 11, FIG. 6c shows sample 6, FIG. 6d shows sample 13, FIG. 6e shows sample 9, and FIG. 6f shows the injection force of the mixed solution using acellular dermis.

As shown in FIG. 6, it can be seen that only the mixture of the ultra-fine acellular dermis that has been subjected to primary crushing, secondary crushing, and sieving together has an injection force of 40 N or less.

Claims (13)

A first grinding step of grinding the acellular dermis at 800 to 6,000 rpm using a rotating rotor;
A secondary crushing step of freezing the primary pulverized acellular dermis, putting the frozen acellular dermis and metal rod into a crushing container, and then freeze-grinding them through impact; and
A method for producing an ultra-fine acellular dermis comprising a sieve separation step of sieving the secondary pulverized acellular dermis under negative pressure conditions.
According to claim 1,
Dermis is a method for producing an ultra-fine acellular dermis that is allogeneic or heterogeneous dermis.
According to claim 1,
A method for producing an ultra-fine acellular dermis, wherein the acellular dermis is prepared by removing cells from skin tissue.
According to claim 1,
The first crushing step is a method for producing ultra-fine acellular dermis that is performed for 10 to 50 minutes.
According to claim 1,
A method for producing an ultra-fine acellular dermis having an average particle size of 600 to 900 μm of the primary crushed acellular dermis.
According to claim 1,
Secondary grinding is a method for producing an ultra-fine acellular dermis that is performed using a freeze mill.
According to claim 1,
Method for producing ultra-fine acellular dermis in which the material of the metal rod in the secondary grinding step is stainless steel or titanium.
According to claim 1,
The secondary grinding is performed for 1 to 60 cycles,
Method for producing an ultra-fine acellular dermis in which the secondary grinding time is 20 to 100 minutes.
According to claim 1,
A method for producing an ultra-fine acellular dermis in which the size of the sieve in the sieve separation step is 100 to 300 μm.
According to claim 1,
The sieving step is performed at a pressure of 500 to 5000 mmAq,
A method for producing an ultra-fine acellular dermis in which the sieve separation time is 1 to 20 minutes.
According to claim 1,
A method for producing an ultrafine acellular dermis having an average particle size of 120 μm or less.
ultra-fine acellular dermis prepared by the manufacturing method according to claim 1; and
An injectable acellular dermal composition comprising a solution.
According to claim 12,
An acellular dermal composition having an injection force of 40 N or less.

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