KR102040592B1 - Method of manufacturing implantation materials - Google Patents

Abstract

The present invention relates to a bag for packaging a living implant, and more particularly, to a bag for packaging a living implant consisting of acellular dermal tissue that can be inserted into the human body in a convenient manner.
The living graft packaging bag according to the present invention, by using the cell-free dermal tissue from which the basement membrane layer is removed when packaging the living implant, the living cells enter the tissue well compared to the conventional dermal membrane has an excellent engraftment rate effect It has the advantage of maintaining the structure because the living implants do not leak out because of the pocket shape.

Description

METHOD OF MANUFACTURING IMPLANTATION MATERIALS

The present invention relates to a method for producing a living implant, and more particularly, to a method for producing a living implant using acellular dermal tissue that can be inserted into the human body in a convenient manner.

For the purpose of treating damaged or damaged tissues, the transplantation of cartilage tissues is increasing rapidly. As a patent related to a conventional transplant, there is a living transplant derived from cartilage tissue of mammal of Patent Document 1.

BACKGROUND ART Conventionally, cartilage transplantation has been considered in the orthopedic field for repairing tissues damaged by arthritis and the like, but recently, the use of cartilage tissues for cosmetic and cosmetic purposes is rapidly increasing. In the shaping using cartilage tissues, especially the rhinoplasty is increasing as the rhinoplasty is activated day by day, and recently, the cartilage is finely crushed and used for transplantation by wrapping it with periosteum or dermis. Although there are attempts to be made, the living grafts are wrapped in a crepe shape, and the living grafts leak out, which is inconvenient even during the procedure, and still shows poor results in the engraftment rate after the procedure, even when inserted into the nose. In some cases, they can't maintain their structure and can't get the effect of the procedure.

Patent Document 1: Republic of Korea Patent Publication 10-2012-0116332

In order to solve the above problems, the present inventors have prepared a living graft packaging bag made of acellular dermal tissue insertable into the human body, and completed the present invention. Accordingly, it is an object of the present invention to provide a method for producing a living implant.

In order to achieve the above object, the present invention provides a living material packaging bag made of acellular dermal tissue insertable into the human body.

In the present invention, the cell-free dermal tissue may be characterized in that the cell-free dermal tissue from which the basement membrane layer has been removed.

In the present invention, the living implant may be characterized in that the cartilage or skin tissue, or a mixture thereof, or the dermal tissue of the fine cartilage or skin.

In the present invention, the living graft material is a mixture of crushed dermal tissue from which the fine cartilage and the basement membrane layer have been removed, and preferably, the cell-free dermal tissue crushed matter and the fine cartilage may be mixed in the same ratio. .

The invention also comprises the steps of (a) removing the basement membrane layer from acellular dermal tissue; And (b) incising one end surface of the cell-free dermal tissue from which the basement membrane layer has been removed to form a bag. It provides a method for manufacturing the living body packaging packaging material comprising a.

In the present invention, the step of removing the base film layer may be characterized by removing the base film layer by using a meat grinder or by treating with hydrogen peroxide.

In the present invention, it may be characterized in that it further comprises the step of hydrating the produced bag.

The living graft packaging bag according to the present invention, by using the cell-free dermal tissue from which the basement membrane layer is removed when packaging the living implants, the living cells enter the tissues well compared to the conventional dermal membranes, and has an excellent engraftment rate. It has the advantage of maintaining the three-dimensional structure well after implantation because the implants do not leak out because of the pocket form.

1 is a photograph showing the cell-free allogeneic dermis from which the basal membrane layer was removed.
Figure 2 is a photograph showing the process of making a package for packaging a living implant prepared using acellular allogeneic dermis.
Figure 3 is a comparison of the degree of fibroblast influx in the dermis depending on the presence of the basement membrane layer.

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 well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

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

Until now, as a method for wrapping microcartilage, it has been used to separate the periosteum (fascia) from the head and percutaneous skin or to wrap the microcartilage like a furoshiki using human acellular dermis, but the microcartilage leaks out. Another method is to use a needle and thread to sew like a pocket and put microcartilage into it, but this is inconvenient for the operator. To reduce this inconvenience and side effects, we cut one side of the lyophilized acellular dermis, make a pocket, and then hydrate the dermis. After sufficient hydration, the dermis is filled with hydrated microdermis, microcartilage, or a mixture of microdermis and microcartilage, and the open side is sewn into a surgical thread to form a bag.

The present invention relates to a living material packaging pouch consisting of acellular dermal tissue insertable into the human body.

In the present invention, such a living implant is cartilage or skin tissue, or a mixture thereof, more specifically, the living implant is fine cartilage alone, or dermal tissue of the skin, specifically, the dermal tissue from which the fine cartilage and basement membrane layers are removed. It may be a mixture of crushed, for example, the cell-free dermal tissue crushed and fine cartilage may be mixed in the same ratio.

When using microcartilage, it can be used by mixing with physiological saline in the ratio of 1.5-2: 1. In the above mixing process, the fine cartilage and dermal tissue debris may be mixed with the saline solution in a syringe, and mixed through a piston movement.

In the present invention, the fine cartilage, the cartilage secured by separating the cartilage which is a part of the living tissue, and through a pre-treatment process such as the removal of the periosteum, using a surgical scalpel, etc. Refers to cartilage processed. Surgical knife may be used to obtain such fine cartilage, and in addition to the cutting process, forceps or medical forceps may be used to fix the cartilage on the opposite side.

In the present invention, the cell-free dermal tissue refers to the dermal layer from which cells that can cause immunorejection of the separated dermis through chemical treatment are composed of collagen and elastin. When it is used as part of a living implant, the cell-free allogeneic dermis is crushed into a microstructure of about 500 μm, which is the “cell-free dermal tissue debris” in the present invention.

In the present invention, the process for producing microcartilage used as a living implant is as follows:

The cartilage, which is a part of the biological tissue, is separated from the donated body, and the cartilage secured through a pretreatment process such as periosteum removal is processed into a hexahedral structure with each side of 1 mm or less using a scalpel or the like. Produce cartilage. Specifically, The process of processing the costal cartilage into microcartilage, the packaging of the costal cartilage tissue is opened and the thawed tissue is cut into a size suitable for processing and processing. Put the costal cartilage into a sterile container containing sterile distilled water and shake it off using a roll forceps. This wash is performed three times. Carefully remove the soft tissue and cartilage. Cut out unnecessary parts. Moisturize the surface of the tissue with sterile distilled water to prevent it from drying out during tissue grooming and standardization. Wash three times with sterile distilled water. Use a surgical knife to cut it down to 1 mm in size. Soak the cut fine cartilage in physiological saline to prevent it from drying and seal it. Packaged fine cartilage finished product is radiation sterilized. At this time, the gamma-dose required for sterilization is 15 to 25 kGy.

In the present invention, the cell-free dermal tissue may be allogeneic dermal tissue, the process of preparing such cell-free dermal tissue is as follows:

When the tissue of donated bodies is separated from the body and transported, there is a risk of tissue damage due to hypoxia, degradation by autolytic enzymes, and damage to extracellular epilepsy by proteolytic enzymes. In addition, depending on the osmotic pressure of the carrier solution may be physically damaged. 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 added with substances that can prevent degradation by hypoxia, degradation by autolytic enzymes, and degradation by proteins and enzymes.

Antibiotic and antimicrobial agent should be added to prevent microbial contamination. Appropriate buffers 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 ~ 320 mOsm / kg of the plasma osmotic pressure. In the case of commercial medium, which is widely used for culturing animal cells, the osmotic pressure is about 260-320mOsm / kg, which is similar to the osmotic pressure of plasma. Therefore, commercial medium is used as a basic solution, and various functional ingredients are added thereto.

Antibacterial agents such as penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamicin, vancomycin, etc. may be added alone or in combination to prevent contamination of bacteria or fungi, such as amphotericin-B, nystatin, polymyxin and the like. The same antimicrobial agent is added alone or in combination. In addition, enzyme inhibitors should be added to prevent tissue damage caused by various enzymes.

Enzyme inhibitors include n-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethyl) -N, N, N ', N Chelating agents such as' -tetraacetic acid (EGTA), protease inhibitors such as lupetin, apoprotinin and the like.

In addition, tissues should be transported in a way that minimizes physical damage.

Most enzymatic reactions are affected by temperature and become the most active around human body temperature of 37 ℃, thus transporting the tissue at low temperature of 4 ℃.

Generally, the ice box is filled with ice. Transporting the tissue at a temperature that is too low for the carrier solution to ice may cause damage to the tissue and should be avoided.

Obtaining acellular dermal tissue can be largely divided into two steps, the first step is to remove the epidermal layer from the tissue prepared as described above, separating the epidermal layer and the dermal layer.

In general, various proteolytic enzymes are used to separate the dermal and epidermal layers. In the case of using an enzyme, if the concentration is low or the processing time is too short, the separation is not good, and if the concentration is high or the processing time is too long, damage to cells or tissues occurs. Therefore, the treatment should be done according to the appropriate concentration and time. Enzymes used to separate the dermal and epidermal layers include neutralases such as dispase, termolysin and trypsin. The epidermal and epidermal layers can be separated by treatment with 1.0 units / mL dispase at 37 ° C for 60 to 120 minutes. Alternatively, the dermal and epidermal layers can be separated by treating termolysin at a concentration of 200 µg / ml for 30 minutes at 37 ° C. Termolysin lowers the risk of damaging the basement membrane than dispase. Another method is to separate the two layers of tissue by varying the ionic strength of the solution, which also depends on conditions such as ionic strength, treatment time, and treatment temperature. Treatment with at least 1 mole of sodium chloride solution at 37 ° C. for 14 to 32 hours can separate the dermal and epidermal layers. More than one mole of sodium chloride solution can reduce the risk of microbial contamination because bacteria and fungi cannot grow. Alternatively, the dermal and epidermal layers may also be separated by treatment with 20 mM ethylenediaminetetraacetic acid (EDTA) at 37 ° C. for 14 to 32 hours. EDTA can reduce tissue damage caused by proteases because EDTA acts as a protease inhibitor. Therefore, when 1-5 mM EDTA is added to 1 mol of sodium chloride solution, the dermal and epidermal layers can be separated while minimizing microbial contamination or enzyme damage.

The second step is to remove the epidermal layer as above and then to remove the cells of the dermal layer.

Immune responses are mainly caused by membrane proteins present in the cell membrane. Therefore, removing the cells can minimize the immune response. By using the difference in physical and chemical properties between cells and extracellular epilepsy, only cells are selectively removed without damage to tissues. The main component of the cell membrane is phospholipids, and various surfactants can be used to remove cells without damaging tissue.

For this purpose, ionic surfactants such as sodium dodecyl sulfate (SDS), or Triton X-100, Tween20, Tween 40, Tween60, Tween80, Nonidet P-10 (NP-10), Nonidet P-40 Nonionic surfactants such as (NP-40) and the like.

 The dermal layer is treated with SDS solution at a concentration of 0.2-1% at room temperature for 30-120 minutes at room temperature to remove cells without damaging the tissue. Alternatively, treatment with Tween 20 solution at a concentration of 0.1 to 2.0% at room temperature for 30 to 180 minutes, or treatment with a Triton X-100 or nonidetpi-40 solution at a concentration of 0.2 to 2% at 22 to 37 ° C for 30 to 180 minutes is also possible. The cells can be removed without damaging the tissue.

In addition to the above chemical method, the cells may be removed by physical methods, and the cells may be removed by treating the dermis with 10 to 100 Hz ultrasonic waves for 5 to 60 minutes. Alternatively, a combination of surfactants and ultrasound can also be used to remove cells without damaging the tissue. In addition, a solvent (TNBP) and a surfactant can be used to simultaneously perform cell removal and virus removal.

The invention also comprises the steps of (a) removing the basement membrane layer from acellular dermal tissue; And (b) incising one end surface of the cell-free dermal tissue from which the basement membrane layer has been removed to form a bag. It relates to a method for manufacturing the living body implant packaging bag comprising a.

Briefly describing the manufacturing process, as shown in Figure 2, (A) is a lyophilized acellular dermal tissue lying down and (B) is a standing state. (C) is the process of making a bag using a surgical knife on the erected surface of the lyophilized acellular dermis, and (D) is the completed bag. Figures (E) and (F) show the complete hydration of the lyophilized dermis bag with these sheaths in place, where the living implant is inserted (F) and the dermal sac filled with the implant (G). .

In the present invention, the step of removing the base film layer may be to use, or process the hydrogen peroxide solution to remove the base film layer a six season.

More specifically, the step of removing the base film layer may include the following process.

Separation of the basement membrane layer from the dermal tissue includes physical methods and chemical methods using chemicals that are harmless to the human body. As a physical method, there is a method of thinly cutting the upper surface of the dermis layer from which the epidermal layer is removed to a thickness of 0.01 to 0.5 mm (preferably 0.05 to 0.2 mm) using a meat grinder. In this case, the meat grinder blade can use carbon steel material to minimize heat generation and prevent degeneration of dermal tissue. Chemical treatment method uses a roller with a microneedle attached to make a small hole in the top surface of the basement membrane layer, and then treated with 0.3% hydrogen peroxide solution for about 1 hour to 3 hours to separate the basement membrane layer into the dermis layer. By loosening the structure, the basement membrane can be removed without destroying the dermal tissue.

In addition, dermal tissue from which the basement membrane layer is removed may be stored by freeze drying.

Specifically, the base film layer is removed, and then stored in a freeze solution treatment and freeze drying.

The freezing solution consists of a buffer solution that maintains the ionic strength or osmotic pressure of the solution, a cryoprotectant that prevents physical and chemical damage of the dermal layer tissue when frozen, and a dry protectant that prevents structural changes of the dermal layer tissue when dried. Cryoprotectants increase the glass transition temperature to increase the stability of frozen tissue. As the glass transition temperature increases, the drying rate can be increased by increasing the specific gravity of the glass or square ice, which is less stable than hexagonal ice. In addition, glassy ice and square ice have smaller ice sizes and less tissue.

Damage. Therefore, cryoprotectant must contain cryoprotectant. Currently used cryoprotectants include dimethyl sulfoxide (DMSO), dextran, sugar, propylene glycol, glycerol, mannitol, sorbitol, fructose, trehalose, raffinose, 2.3-butanediol, ethyl starch (HES), polyethylene glycol, Polyvinylpyrrolidone (PVP), proline, hetastarch, serum albumin and the like. A combination of these components and various base components is used to prepare a freezing solution. Among them, dextran, glycerol, hetastarch, mannitol, and ethyl hydroxide starch are used as serum substitutes, and polyethylene glycol is also used as a stabilizer for protein injection. As such, a freezing solution is prepared based on substances known to have little harm to the human body. The dermal layer is immersed in the thus prepared freezing solution, and the freezing solution can penetrate well using an appropriate method. The dermal layer into which the freezing solution has penetrated is stored in an cryogenic freezer at -70 ° C. or lower (preferably -40 ° C. to -70 ° C.). After freezing for 12 to 48 hours, the freeze drying time is preferably carried out for 24 to 50 hours after freezing drying.

In the present invention, it may be characterized in that it further comprises the step of hydrating the produced bag. Without this hydration, allogeneic dermis is fairly hard and feels like a hard nonwoven fabric. In this state, even if the pocket is made with a knife, it is difficult to insert the implant. Therefore, it is sufficiently hydrated to secure a space for the implant, and it is also easy to suture after the implant is placed.

In the present invention, in the case of a pocket in which the cell-free dermal tissue is obtained by the above-described process, the basement membrane layer is removed, as shown in FIG. 3, the dermis pocket (a) and the dermis pocket without the basement membrane as shown in FIG. 3. Comparing the inflow of the fibrous cells of (b), there is an advantage that the engraftment rate is increased because the inflow of the fibroblasts is not disturbed when the basement membrane is removed.

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

Example  1: according to the invention Cell free  Manufacture of dermal pouch

Skin tissues (collected from donated bodies for treatment of non-profit patients from tissue banks) were treated with a neutral protease dispase of 1.0 units / ml and 60-120 minutes at a temperature of 37 ° C. in a stirred incubator. After stirring for 3 minutes, washed three times with sterile distilled water to separate the dermal layer and the epidermal layer to remove the epidermal layer.

The tissue from which the epidermal layer was removed was treated with Triton X-100 solution at 1% concentration for 100 minutes at 30 ° C. to remove the cells of the dermal layer.

The basement layer was removed by thinly cutting the upper surface of the epidermal layer from which the epidermal layer was removed to a thickness of 0.05 to 0.2 mm using a meat grinder of a carbon steel blade to minimize degeneration of the dermal tissue.

10% dextran solution was used as the freezing solution, and the dermal tissue from which the basement membrane layer was removed was soaked in the freezing solution for 12 hours at -4 ° C to allow the freezing solution to penetrate well, and then the cryogenic freezer at -70 ° C or lower. 12 hours in lyophilizer and 48 hours lyophilized (Fig. 1).

As described above, one side of the lyophilized acellular dermis was cut to form a bag, and the dermis was hydrated. After sufficient hydration, the hydrated microdermis, microcartilage, or a mixture of microdermis and microcartilage were filled in front of this dermal sac, and the opened one side was sewn into a surgical chamber (FIG. 2).

Comparative Example 1 Pocket of Cell-free Dermal Tissue without Basal Membrane Layer Removed

Except for the base film layer removal step described in 1 was produced in the same process.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

Making one end face of the acellular dermal tissue insertable into the human body to produce a packaging bag having the open face;
Filling the packaging bag with crushed material formed by crushing cartilage or skin tissue, or a mixture thereof, in the packaging bag; And
Suturing the open cross-section of the packaging pouch. The method of claim 1,
The method,
Prior to the step of manufacturing the packaging bag, further comprising the step of lyophilizing the cell-free dermal tissue, a living implant manufacturing method. The method of claim 2,
The method,
Prior to lyophilizing the cell free dermal tissue, further comprising removing a basement membrane layer from the cell free dermal tissue. The method according to any one of claims 1 to 3,
Between the step of manufacturing the packaging bag and the step of filling the shredding, further comprising the step of hydrating the packaging bag, a living implant manufacturing method. The method according to any one of claims 1 to 3,
The lysate is a microstructure of less than 500 micrometers, biograft manufacturing method. delete

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