KR20140104252A

KR20140104252A - Composition for in vivo transplantation comprising micro-cartilage and acellular dermal matrix and method for producing the same

Info

Publication number: KR20140104252A
Application number: KR1020130018211A
Authority: KR
Inventors: 김형구; 이주희; 김기민; 이환철
Original assignee: 주식회사 엘앤씨바이오
Priority date: 2013-02-20
Filing date: 2013-02-20
Publication date: 2014-08-28

Abstract

TECHNICAL FIELD The present invention relates to a composition for living body transplantation containing micro-cartilage and acellular dermis tissue, and more particularly, to a composition for transplantation comprising a cartilage having a fine polyhedral structure with excellent engrafting ratio at the time of transplantation, And a method for producing the same. The composition for implanting a living body containing the cartilage and acellular dermis tissue of the microhedral structure according to the present invention can solve the problem of growth and proliferation of cells due to air bubbles generated when using the micro-cartilage alone, For the purpose of recovery or molding.

Description

TECHNICAL FIELD The present invention relates to a composition for a living body transplantation containing micro-cartilage and acellular dermis tissue and a method for producing the same.

TECHNICAL FIELD The present invention relates to a composition for living body transplantation containing micro-cartilage and acellular dermis tissue, and more particularly, to a composition for transplantation comprising a cartilage having a fine polyhedral structure with excellent engrafting ratio at the time of transplantation, And a method for producing the same.

Transplantation of cartilage has been widely used in the treatment of injured cartilage or in the field of plastic surgery. Especially, as the nose formation is activated day by day, Among them, foreign-made implants, such as Gore-Tex or silicone, are easy to handle, save operative time, and have the advantage of eliminating donor morbidity. Patent literature 1 (artificial cartilage replacement) and patent document 2 (support for articular cartilage regeneration and manufacturing method thereof) are disclosed as prior arts related to the field of using heterogeneous materials. However, long-term follow-up has reported side effects such as foreign body sensation, infection, pain, escape of the graft and mobility.

Therefore, in recent years, autologous or homologous cartilage transplantation has been widely used for the purpose of lowering the infection rate from external infection and the like, rather than using the above-described foreign body forming grafts. Here, various types of implants have been used including autologous cartilage. For example, in the case of nose formation using autogenous cartilage, the cartilage required for the formation can be obtained from the nose or the ear and the ribs, Is called septal cartilage and is used to raise the nasal tip. The ear cartilage is used to raise the nasal dorsum. The costal cartilage attached to the ribs can be used for nose and nose and is used for re-operation of patients already using septate cartilage or ear cartilage. Allogenous cartilage using cadaver's castal cartilage has the advantages of autologous cartilage such as no foreign body sensation and no rejection reaction, none. As a conventional technique using such a cartilage, there is a bio-implantable material derived from the cartilage tissue of a mammal of Patent Document 3. [

In using these cartilage, recently, micro-cartilage, which has been broken down to a fine size, is used. In this case, air bubbles are formed between the micro-cartilages, which has been an obstacle to the growth and proliferation of the cells. There is an inconvenience.

Patent Document 1: Korean Patent Publication No. 10-2011-0012807 Patent Document 2: Korean Patent Publication No. 10-2011-0097662 Patent Document 3: Korean Patent Laid-Open Publication No. 10-2012-0116332

Accordingly, an object of the present invention is to provide a composition for implantation containing a disruption of cartilage and acellular dermis tissue of a microhedral structure.

It is still another object of the present invention to provide a method for producing a composition for implantation of a living body, which contains the disruption of cartilage and acellular dermis tissue of the fine polyhedral structure.

In order to accomplish the above object, the present invention provides a composition for implantation of a living body containing a microsaturated cartilage and a cell-free dermis tissue lump.

In the present invention, the micro-cartilage may be a cartilage having a hexahedron structure with a side length of 1 mm or less.

In the present invention, the cell-free dermal tissue disruption and the micro-cartilage may be mixed in the same ratio.

In the present invention, the cell-free dermal tissue disruption and micro-cartilage may be mixed with physiological saline, tertiary distilled water, or blood.

The present invention also relates to a method of producing a microcrystalline cartilage comprising: (a) finely cutting cartilage to produce fine cartilage; And (b) disrupting the acellular dermis tissue, and mixing the disruption with the micro-cartilage of the step (a).

In the present invention, the acellular dermis tissue may be,

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

(ii) treating the skin from which the skin layer has been removed with a surfactant or an ultrasonic wave to remove cells of the dermal layer,

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In the present invention, the skin layer may be separated from the dermal layer using a protease.

In the present invention, the lysate of the acellular dermis tissue produced in the step (b) may be stored in a lyophilized state.

The composition for living body implantation containing a cartilage and acellular dermis tissue of a microhedral structure according to the present invention functions as a scaffolder in vivo by filling a gap between micro-cartilage with a disruption of an acellular dermis tissue, And it can act as a physiological space in which the cells of the recipient can move and grow. As angiogenesis occurs, capillary blood vessels can be formed and the tissue can become a graft with time, and air bubbles Thereby preventing the growth and proliferation of the cells.

The composition for living body implantation according to the present invention can be utilized for the purpose of recovery or molding of damaged cartilage.

FIG. 1 is a photograph of a process of processing chest cartilage into fine cartilage.
Fig. 2 is a micrograph of the processed cartilage.
Fig. 3 is a micrograph of the cartilage in a hydrated state.
FIG. 4 is a photograph of a particle-type acellular allogeneic dermis in a hydrated state.
Figure 5 is a photograph of a mixture of hydrated micro-cartilage and granular acellular allogeneic dermis.
Fig. 6 is a photograph of a mixer capable of mixing fine cartilage and allogeneic dermis.

Unless otherwise defined, 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 well known and commonly used in the art.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for implantation of a living body including a cell-free dermis tissue crush and fine cartilage.

In the past, mainly, fine cartilage was cut and used for restoration or molding of a damaged cartilage area. These autografts and allografts are used by carving and trimming to suit the purpose of the procedure. In the case of nasal reconstruction, these cartilages are used for the role of a support, but in some procedures, especially for increasing the volume of the nose, fine cartilage is often more suitable than a large size cartilage slit or cartilage slit. Micro-cartilage refers to cartilage that has been cut to a size of about 1 mm on each side. Normally, it is stored in sterilized physiological saline. In the procedure, saline is removed and inserted into the procedure site. In this process, unnecessary air bubbles may be formed between the micro-cartilages. These air bubbles interfere with the filling between the cartilage and the tissue and create an empty space to inhibit the growth of the micro-cartilage, In severe cases, necrosis or infection may occur. In order to prevent this, the micro-cartilage is mixed with the blood drawn from the patient. In this process, the blood cells are destroyed, and congestion such as congestion and edema may occur.

In order to solve this problem, the present inventors have developed a composition for transplantation of a living body in which microcell cartilage is mixed with a cell-free dermis tissue crush.

In the present invention, the micro-cartilage is obtained by separating the cartilage, which is a part of the living tissue, and securing the cartilage secured through a pre-treatment process such as the removal of the periosteum by using a surgical scalp or the like, .

In the present invention, the acellular dermis tissue is a dermis layer that removes cells that can cause immunodeficiency through chemical treatment of the separated dermis, and is composed of collagen and elastin as main components. The "cell-free dermal tissue disruption" in the present invention is a disruption of this acellular allogeneic dermis to give a microstructure of about 500 μm.

In the present invention, the cell-free dermal tissue disruption and the micro-cartilage are mixed and mixed at the same ratio.

The present invention also relates to a method for producing the composition for living body implantation,

(a) finely cutting cartilage to produce fine cartilage; And (b) disrupting the acellular dermis tissue, and mixing the disruption with the micro-cartilage of step (a).

Here, the micro-cartilage is a cartilage having a hexahedron structure with a side of 1 mm or less. To obtain the micro-cartilage, a surgical knife, a forceps, a surgical nipper, scissors, or the like can be used.

In the step (b), the cell-free dermal tissue is disrupted and the thus-obtained disruption is mixed with the micro-cartilage of the step (a). At this time, the physiological saline can be used to mix the micro-cartilage and the cell-free dermis tissue lysate. The mixing ratio of the physiological saline and the cell-free dermis tissue lysate is 1: 1 to 2: 1, for example, 1.5: 1.

In the mixing process as described above, the micro-cartilage and the dermis tissue can be mixed with physiological saline in the syringe, and the mixture can be mixed through the piston movement.

For example, Figs. 1 and 2 are photographs showing the micro-cartilage of the chest cartilage processed into fine cartilage and processed, and Fig. 3 is a photograph of the cartilage hydrated. Here, a mixture obtained by crushing hydrated particle-type microcrystalline allogenic dermis (FIG. 4), and mixing the hydrated micro-cartilage with particle-type acellular allodermic dermis is as shown in FIG. As shown in FIG. 6, there is a method in which a micro-cartilage in a hydrated state is present on one side of the syringe and a micro-cartilage is formed on the other side in a hydrated particle type Cell-free dendritic dermis can be packaged and mixed with each other as needed.

In the present invention, the process of making micro-cartilage is as follows:

The cartilage, which is a part of the living tissue, is separated from the donated body, and the cartilage obtained through a pre-treatment process such as the removal of the periosteum is processed into a hexahedral structure with a variation of 1 mm or less using a surgical scalpel Make cartilage.

Specifically, as a process of processing costal cartilage into micro-cartilage, the tissue of the costal bone tissue is opened and the thawed tissue is cut to an appropriate size for processing and processing

(Figs. 1 and 2). Place the ribs in a sterilized container containing sterile distilled water and shake it off using a roll forceps. This cleaning is carried out three times. Carefully remove soft tissue and cartilage. Cut out unnecessary parts. Wet the tissue surface with sterile distilled water to prevent tissue drying during tissue conditioning and standardization. Wash three times with sterile distilled water. Cut with a surgical knife so that the size is less than 1 mm. The micro-cartilage is cut into physiological saline so that it is not dried. Radiation-sterilized microcosts finished with packaging. At this time, the gamma-dose required for sterilization is 15 to 25 kGy.

In the present invention, the acellular dermis tissue may be a homogeneous dermis tissue,

The process of preparing such an acellular dermis tissue is as follows.

First, the method of transporting and storing the material tissue for obtaining the acellular dermis tissue is as follows:

There is a risk of tissue damage due to hypoxia, degradation by autolytic enzymes, and damage of extracellular epilepsy by proteases when the donated body tissue is transported separately from the body. In addition, physical damage may occur due to the osmotic pressure of the carrier solution. In addition, there is always a risk of contamination by microorganisms such as bacteria and fungi. Therefore, the solution used for transporting tissues should be supplemented with substances that can prevent degradation by hypoxia, degradation by autolytic enzymes, degradation by proteins and degrading enzymes

And antibiotics and antimicrobial agents that can prevent microbial contamination should be added. Appropriate buffer solutions should be included to prevent tissue damage by osmotic pressure. The osmotic pressure of the tissue transport solution should have an osmotic pressure of about 260 to 320 mOsm / kg, which is the plasma osmotic pressure. In the commercial medium, which is widely used for animal cell cultures, the osmotic pressure is about 260-320 mOsm / kg, which is similar to the osmotic pressure of plasma. Therefore, a commercial medium is used as a base solution and various components are added to it.

Antibiotics such as penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, vancomycin, etc., alone or in combination, are added to prevent contamination of bacteria and fungi, and amphotericin-b, nystatin, The same antimicrobial agent is added alone or in combination. An enzyme inhibitor should be added to prevent tissue damage by various enzymes.

Enzyme inhibitors include N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis '-Tetraacetic acid (EGTA) and the like, protease inhibitors such as lupeptin, apoproteinin and the like.

In addition, tissue must be transported in a manner that minimizes physical damage.

Most enzyme reactions are highly influenced by temperature and are most active around the human body temperature of 37 ° C, thus transporting tissues at a low temperature of about 4 ° C.

Generally, ice cubes are used to transport ice cubes. If the transport solution carries tissue to a frost-free temperature, ice crystals can damage the tissue and should be avoided.

The process of obtaining the acellular dermis tissue can be roughly divided into two steps. In the first step, the skin layer is removed from the prepared tissue, and the epidermal layer and the dermal layer are separated.

Generally, various proteolytic enzymes are used to separate the dermal layer and the epidermal layer. When the enzyme is used, if the concentration is too low or the treatment time is too short, the separation will not be performed well. If the concentration is too high or the treatment time is too long, the cell or tissue will be damaged. Therefore, it should be treated according to appropriate concentration and time. Enzymes used to separate the dermis and epidermis include the neutral proteases disaspase, tramolysin, and trypsin. The dermal layer and the epidermal layer can be separated by treatment with 1.0 units / ml of disiaase at 37 ° C for 60 to 120 minutes. Alternatively, treatment with tamolysin at a concentration of 200 占 퐂 / ml for 30 minutes at 37 占 폚 can separate the dermal layer and the epidermal layer. The use of tamoxifen reduces the risk of basement membrane damage compared with the use of distearate. Another method is to separate the two layers of the tissue by changing the ionic strength of the solution. This method also depends on the conditions such as ionic strength, treatment time, and treatment temperature. Treatment with 1 mol or more of sodium chloride solution at 37 ° C for 14 to 32 hours can separate the dermal layer and the epidermal layer. Bacteria and fungi can not grow in a solution of 1 moles or more chloride sodium chloride, so the risk of microbial contamination can be reduced. Or 20 mM of ethylenediaminetetraacetic acid (EDTA) at 37 DEG C for 14 to 32 hours to separate the dermal layer and the epidermal layer. EDTA can reduce tissue damage by proteolytic enzymes because EDTA acts as a protease inhibitor. Therefore, treatment with 1 to 5 mM of EDTA in 1 molar sodium chloride solution can minimize microbial contamination and tissue damage caused by enzymes, and can separate the dermal layer and the epidermal layer.

In the second step, the skin layer is removed as described above, and then the cells of the dermal layer are removed.

The immune response is mainly caused by membrane proteins present in the cell membrane. Therefore, removal of the cells can minimize the immune response. We use a method to selectively remove cells without damage to the tissue using the difference in physical and chemical properties between cells and extracellular epilepsy. The main component of the cell membrane is phospholipid, and various surfactants can be used to remove cells without damaging the tissue.

For this purpose, ionic surfactants such as sodium dodecyl sulfate (SDS), or ionic surfactants such as Triton X-100, Tween 20, Tween 40, Twin 60, Twin 80, Nonidetip- (NP-40), and the like are used.

When the dermal layer is treated at room temperature for 30 to 120 minutes with an SDS solution at a concentration of 0.2 to 1% at room temperature, cells can be removed without damaging the tissue. Or treated with a solution of Tween 20 at a concentration of 0.1 to 2.0% for 30 to 180 minutes at room temperature or treated with a solution of Triton X-100 or Nonidt P-40 at a concentration of 0.2 to 2% at 22 to 37 ° C for 30 to 180 minutes Cells can be removed without tissue damage.

In addition to the above chemical methods, the cells can be removed by a physical method. Ultrasonic waves of 10 to 100 kHz for 5 to 60 minutes can be used to remove the cells. Alternatively, the combination of a surfactant and ultrasonic waves can also remove cells without damaging the tissue. In addition, using a solvent (TNBP) and a surfactant, cell removal and virus removal can be performed at the same time.

In the present invention, as an additional step, an E-beam (electron beam) of 25 kGy dose can be injected to perform the final sterilization for unexpected contamination.

The thus-obtained acellular dermis tissue is disrupted using a micro-mill or the like to have a size of 500 mu m in diameter.

The thus-prepared composition for living body implantation can be used as a therapeutic agent for cartilage damage. That is, it can be used as a filler for implantation in plastic surgery in plastic surgery, and as a filler for implantation in facial surgery, it can be used for reconstruction of breast by breast cancer treatment in ocular depression, facial depression and plastic surgeon.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Preparation of composition for living body implantation according to the present invention

(1) Preparation of fine cartilage

Obtain ribs and open the package and cut the thawed tissue to a size appropriate for processing and processing. Place the ribs in a sterile container containing sterile distilled water, shake it off with a roll forceps, and perform this wash three times. Carefully remove soft tissue and cartilage. Cut out unnecessary parts according to commercialization specifications. Wet the tissue surface with sterile distilled water to prevent tissue drying during tissue conditioning and standardization. Wash three times with sterile distilled water. Cut with a surgical knife so that the size is less than 1 mm. The micro-cartilage is cut into physiological saline so that it is not dried. Radiation-sterilized microcosts finished with packaging. At this time, the gamma-dose required for sterilization is 15 to 25 kGy.

(2) Production of disruption of acellular dermis tissue

The skin tissue (collected from the donated tissue from a tissue bank for the purpose of treatment of a patient for non-profit purposes) was treated with a concentration of 1.0 units / ml of the neutral protease, Dispase, and incubated at 37 < 0 & After stirring for 3 minutes, the epidermis was removed by separating the dermal layer and the epidermal layer by washing three times with sterilized distilled water. The tissue from which the epidermis layer was removed was treated with a 1% Triton X-100 solution at 30 ° C for 100 minutes to remove the cells of the dermal layer.

After washing with sterilized distilled water more than 3 times, the treated materials used in the process were removed and free cell type allogeneic dermis was frozen at a temperature of -40ºC or lower for 2 hours or more, then placed in a freeze dryer and dried for 12 hours or longer to remove moisture Respectively. The homogenous dermis was cut by a surgical knife to a size of about 1 cm in width and about 1 cm in size, and then 100 g of allogeneic dermis was placed in a microfibrillator that did not break the tertiary structure of the allogeneic dermis and the denaturation of collagen and elastin The homogenized dermis of the cell-free allogeneic dermis was passed through a sieve having a diameter of 500 uM which was most advantageous in the living body in the aseptic state, .

(3) Mixing of micro-cartilage and dermis tissue lumps

The freeze-dried dermis tissue lysate was hydrated with a physiological saline: dermis ratio of 1.5: 1 and mixed with the hydrated dermis tissue and the same amount of micro-cartilage.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

A cell-free dermal tissue lumen and micro-cartilage.

[3] The composition according to claim 1, wherein the micro-cartilage is a cartilage having a hexagonal structure with a side length of 1 mm or less.

The composition according to claim 1, wherein the acellular dermis tissue crush and the micro-cartilage are mixed in the same ratio.

[4] The composition of claim 3, wherein the acellular dermis tissue and the micro-cartilage are mixed using physiological saline, tertiary distilled water, or blood.

(a) finely cutting cartilage to produce fine cartilage; And
(b) disrupting the acellular dermis tissue, mixing the disruption with the micro-cartilage of step (a)
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6. The method of claim 5,
(i) removing the epidermal layer from the skin; And
(ii) treating the skin from which the skin layer has been removed to remove cells of the dermal layer,
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The method according to claim 6,
Wherein the cells of the dermal layer are removed through a surfactant or ultrasonic treatment.

8. The method according to claim 6 or 7, wherein the skin layer is separated from the dermal layer using proteolytic enzymes.

[Claim 6] The method according to claim 5, wherein the lysate of the acellular dermis tissue prepared in step (b) is stored in a lyophilized state.

6. The method of claim 5, wherein the acellular dermis tissue crush and the micro-cartilage are mixed in the same ratio.