KR20130105225A - Acellular dermal matrix removed basement membrane - Google Patents

Abstract

PURPOSE: An acellular dermal tissue implant in which basement membranes are removed is provided to increase the inflow of fibroblasts and enhance an engraftment rate to skin by accelerating angiogenesis, thereby stably doing a transplant and shortening recovery dates after the transplant. CONSTITUTION: In an acellular dermal tissue implant, basement membranes are removed. The acellular dermal tissue implant is one selected from a bone, a ligament, a tendon and a skin. The acellular dermal tissue implant increases the inflow of fibroblasts. The acellular dermal tissue implant accelerates angiogenesis. The acellular dermal tissue implant enhances an engraftment rate. [Reference numerals] (AA,BB) Cross sectional mimetic diagram; (CC) Other company; (DD) Our company MegaDerm; (EE) Basilar membrane (B.M); (FF) Fibroblast

Description

ACELLULAR DERMAL MATRIX REMOVED BASEMENT MEMBRANE

The present invention relates to a cell-free dermal tissue graft from which the basal membrane layer has been removed and a method for producing the same.

The skin tissue of the human body is divided into three parts, consisting of the outermost epidermal layer of the skin, the dermis layer below it, and the subcutaneous tissue. The epidermal layer is composed of epithelial cells differentiated into several layers from the basement membrane that allows the epidermal layer and the dermal layer to bind tightly, as well as melanocytes and immune cells.The dermal layer below the epidermal layer is mainly fibroblasts and It consists of several extracellular matrix secreted by cells.

Skin tissue or internal organ tissues can be damaged by burns, trauma, ulcers, etc., in which case transplantation is performed from the skin tissue or internal organ tissues for the purpose of healing or reconstructing the damaged tissues. Use the method. In such a case, the transplant recipient may have a surgical burden of additionally removing skin or organ tissues, which may be dangerous if the recipient is in poor health. In addition, xenograft or synthetic biomaterials can be used for transplantation. In this case, immunorejection reactions may occur, resulting in inflammatory reactions during long-term transplantation and reoperation. In order to solve the above problems, a method of preparing and implanting a cell-free dermal layer for transplantation from skin tissue extracted from a donor [Patent Document 1, Patent Document 2] is used as a method for solving the above difficulties.

In the cell-free dermal tissue, the basement membrane layer is located at the top of the dermal layer, and the epidermal layer and the dermis layer are firmly bonded and remain attached to the dermal layer even after the epidermal layer is removed. In the case of the basement membrane layer, since the tissue is dense, the inflow rate of the fibroblasts of the existing transplantation site is significantly reduced compared to other surfaces, resulting in slow engraftment.

Korean Patent Publication No. 2001-0092985, Domestic Patent Registration No. 791502

Thus, the present inventors removed the basement membrane layer, which is significantly reduced compared to the other side of the existing graft fibroblasts after transplantation, so as to slow the engraftment rate, the inflow of fibroblasts at the bottom, side of the base layer without the basement membrane layer It could be maintained at the same rate as the influx of blasts.

Therefore, the present invention removes the basal membrane layer of acellular dermal grafts, increases the influx of fibroblasts after transplantation, promotes neovascularization, increases the engraftment rate to the skin, and stabilizes the transplantation and minimizes recovery days after transplantation. can do.

As means for solving the above problems,

In acellular dermal grafts,

Provided is a cell-free dermal tissue implant with a mesial layer removed.

Further, as another means for solving the above-mentioned problems,

In the method for producing a cell-free dermal tissue implant,

Provided is a method for preparing a cell-free dermal tissue implant in which the basement membrane layer is removed, comprising the step of removing the basement membrane layer.

The present invention removes the basal membrane layer of acellular dermal tissue implants to increase the influx of fibroblasts after transplantation and promote the neovascularization function, thereby increasing the engraftment rate to the skin, thereby making the transplant stable and shortening the recovery date after transplantation. have.

1 is a comparison of the cell-free dermal tissue implant according to the present invention and the existing acellular dermal tissue implant [Alloderm, LifeCell Corporation, Branchburg, NJ].
Fig. 2 is a photograph after transplantation of the acellular dermal tissue from which the basal membrane layer is removed [coarse solid line: fibroblasts accumulated by the basement membrane layer and the basement membrane layer without penetrating into the dermal graft, and the dotted line: transplanted acellular dermis Tissue, black arrows: engrafted fibroblasts].
Figure 3 is a photograph after transplantation of acellular dermal tissue without basal membrane layer removed (thick black solid line: basement membrane layer, black dotted line: transplanted acellular dermal tissue, black arrow: engrafted fibroblasts).
4 is a photograph confirming angiogenesis of transplanted acellular dermal tissue [left; Acellular dermal tissue with basal membrane layer removed, right; Acellular dermal tissue without basal membrane layer removed, assay arrow: angiogenesis].
5 is a photograph confirming the skin engraftment rate of the transplanted acellular dermal tissue [left; Acellular dermal tissue with basal membrane layer removed, right; Cell free dermal tissue without basal membrane layer removed].

The present invention relates to a cell-free dermal graft with a basement membrane layer removed.

The acellular dermal graft may be bone, ligament, tendon or skin, but is not limited thereto.

As shown in FIG. 1, in the case of conventional acellular dermal grafts [Alloderm, LifeCell Corporation, Branchburg, NJ], the basement membrane layer is located at the upper part of the dermal layer so that the inflow of fibroblasts from the existing graft site after transplantation is performed. Interfering with the skin, the rate of engraftment was significantly reduced than those without the basement layer at the side and bottom of the dermis. In order to solve the problem of the existing implants, the implant of the present invention removes the basement membrane layer so that the influx of fibroblasts, etc., in the existing implantation site is significantly increased compared to the existing dermal tissue implants, and promotes neovascularization to the skin. The engraftment rate of can be improved.

In addition,

In the method for producing a cell-free dermal tissue implant,

It includes a method of producing a cell-free dermal tissue graft from which the vesicle layer is removed, including the step of removing the basement membrane layer.

Specifically, removing the epidermal layer;

Removing the cells of the dermal layer;

Removing the base film layer; And

Cryopreservation Step

. ≪ / RTI >

The acellular dermal graft may be bone, ligament, tendon or skin, but is not limited thereto.

Hereinafter, a method of manufacturing the cell-free dermal tissue implant in which the basement membrane layer is removed will be described in detail.

First, the step of preparing tissue for use in the implant can be added.

When the tissues of donated bodies are separated from the body and transported, there is a risk of tissue damage caused by hypoxia, degradation by autolytic enzymes, and damage of 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, in the solution used for transporting tissues, substances that can prevent degradation by hypoxia, degradation by autolytic enzymes, and degradation by proteins and enzymes should be added, and antibiotics and antibacterial agents that can prevent microbial contamination are added. shall. 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 to 320 mOsm / kg, which is the plasma osmotic pressure. In the case of commercial medium, which is widely used for animal cell culture, 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.

Antibacterial agents such as penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, vancomycin, etc. may be added alone or in combination to prevent bacterial or fungal contamination. 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, 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.

The first step is to remove the epidermal layer from the tissue prepared as above, separating the dermal and epidermal layers.

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 dermal and epidermal layers can be separated by treatment with 1.0 units / ml of 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.

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, removing 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, a solvent (TNBP) and a surfactant can be used to simultaneously perform cell removal and virus removal.

The third step removes the cells from the dermal layer and then removes the basement membrane layer.

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 cutting the upper surface of the dermis layer from which the epidermal layer has been 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. In the chemical treatment method, a small hole is formed in the upper surface of the basement membrane layer using a microneedle-attached roller, 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.

Finally, after removing the base membrane layer, the solution is freeze-dried and freeze-dried to be preserved.

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. The cryoprotectant enhances the stability of frozen tissues by increasing the glass transition temperature. 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. Glassy and square ice also cause less damage to the tissue due to the smaller size of the ice. Therefore, the freezing solution must contain freezing protection. Currently, commonly used cryoprotectants include dimethylsulfoxide (DMSO), dextran, sugar, propylene glycol, glycerol, mannitol, sorbitol, fructose, trehalose, raffinose, 2.3-butanediol, hydroxyethyl starch (HES), polyethylene glycol, Polyvinylpyrrolidone (PVP), proline, hetastarch, and serum albumin. By combining these components with various base components, a freeze solution is prepared and used. 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 injections. As such, the frozen solution is made mainly of materials known to be harmless to human body. The dermal layer is immersed in the prepared freezing solution, and the freezing solution can penetrate well by using an appropriate method. The dermal layer into which the freezing solution has penetrated is stored in cryogenic freezers of -70 ° C or below (preferably -40 ° C to -70 ° C). After freezing for 12 hours to 48 hours, the freeze drying time is preferably carried out for 24 to 50 hours by moving to a freeze dryer.

The cell-free dermal tissue graft from which the basal membrane layer thus prepared was removed is used as a treatment for skin damage.

That is, the cell-free dermal graft from which the basement membrane layer has been removed is a skin restoration agent due to burns, traffic accidents, ulcers, etc. It can be used in dermatology, plastic surgery, gynecology, surgery, neurosurgery, urology, and otolaryngology, such as semi-facial atrophy correction.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention and Comparative Examples which are not based on the present invention, but the scope of the present invention is not limited by the following Examples.

Example  One: Basement layer  Removed Acellular cell  Dermal tissue Graft  Produce

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.

The basement membrane layer was cut to a thickness of 0.05 to 0.2 mm using a carbon steel knife blade to prevent denaturation of the dermis by minimizing heat generation on the upper surface of the dermis layer from which the epidermal layer was removed.

A 10% dextran solution was used as a frozen solution, and the dermis tissue in which the basement membrane layer was removed was immersed in the frozen solution at -4 DEG C for 12 hours to allow the frozen solution to penetrate well. Then, For 12 hours and lyophilized in a freeze dryer for 48 hours.

Comparative Example  One: Basement layer  Not removed Acellular cell  Dermal tissue Graft  Produce

A cell-free dermal tissue implant was prepared in the same manner as in Example 1 except that the step of removing the basement membrane layer was omitted.

Experimental Example  1: Confirmation of Fibroblast Inflow

180 g of experimental animal white paper (Sprague Dawley rat) was induced using anesthesia. After anesthesia, the hair was cut using a Clipper, and 1 × 1 cm of tissue was transplanted to both sides. At this time, the cell-free dermal tissue (Comparative Example 1) in which the basement membrane layer was not removed was transplanted to the right side, and the cell-free dermal tissue (Example 1) from which the basement membrane layer was removed was transplanted to the left side. The implantation method was incision about 5 mm using a blade, inserted subcutaneously and sutured. After suture treatment with betadine, the anesthesia was transferred to the kennel.

Four weeks after transplantation, cell-free dermal tissue transplanted with skin tissue was collected for histology. The method was perfused through the heart with 100 ml of saline followed by 500 ml of 10% formalin solution prepared with phosphate buffer. The first 200 ml of fixative solution was perfused for 5 minutes and the remaining 300 ml for 25 minutes. The tissues were extracted and fixed with fixative solution for 3 hours and placed in phosphate buffered saline (PBS) containing 30% sucrose for 4 days at 4 ° C. Stored. The next day, the skin tissues were rapidly frozen, and the tissues were cut to a size of 30 um. Hematoxylin & Eosin staining was performed to confirm the influx of fibroblasts, neovascularization, and skin engraftment.

2 and 3, when hematoxylin, which is a marker for identifying fibroblasts, is not removed from the basement membrane layer (Comparative Example 1), fibroblasts do not flow downward into the basement membrane layer but are gathered above the basement membrane layer. It can be confirmed that the presence (black arrow of Figure 2). In contrast, when the basement membrane layer was removed (Example 1), fibroblasts were not collected on the upper side, and fibroblasts were evenly engrafted on the implant (black arrow in FIG. 3).

Experimental Example  2: Neovascular  Confirm creation

Hematoxylin & Eosin staining of the transplanted cell-free dermal tissue was performed in the same manner as in Experimental Example 1 to confirm the neovascular formation of the implant.

As shown in FIG. 4, it can be confirmed that the acellular dermal tissue from which the basal membrane layer has been removed is increased compared to the acellular dermal tissue from which the basal membrane layer has not been removed (black arrow).

Experimental Example  3: Confirmation of skin engraftment rate

Hematoxylin & Eosin staining of the transplanted cell-free dermal tissue was carried out in the same manner as in Experimental Example 1 to determine the skin engraftment rate of the implant.

As shown in FIG. 5, in the case of acellular dermal tissue from which the basement membrane layer was not removed, fibroblasts did not penetrate above the basement membrane layer by the basement membrane layer and could be confirmed to be deposited outside the top of the implant. On the other hand, in the acellular dermal tissue from which the basal membrane layer was removed, fibroblasts penetrated into the graft and were found to grow. This confirms that a greater number of fibroblasts are in acellular dermal grafts with the basement membrane layer removed.

Claims (14)

In acellular dermal grafts,
Cell-free dermal tissue implant, characterized in that the basement membrane layer has been removed.
The method of claim 1,
The cell-free dermal tissue implant is a cell-free dermal tissue implant, characterized in that the bone, ligament, tendon or skin.
The method of claim 1,
Cell-free dermal tissue implants characterized by increasing the influx of fibroblasts.
The method of claim 1,
Cell-free dermal tissue implants characterized by promoting neovascular production.
The method of claim 1,
Cell-free dermal tissue transplant, characterized in that to increase the engraftment rate.
In the method for producing a cell-free dermal tissue implant,
A method of making a cell-free dermal tissue implant with a removed basement membrane layer, comprising the step of removing the basement membrane layer.
The method according to claim 6,
The cell-free dermal tissue implant is a method of producing a cell-free dermal tissue implant, the basement membrane layer is removed, characterized in that the bone, ligament, tendon or skin.
The method according to claim 6,
The basement membrane layer is a method of producing a cell-free dermal tissue implant, the basement membrane layer is removed, characterized in that using a meat grinder, or removed by treating with hydrogen peroxide.
The method according to claim 6,
Method for producing the cell-free dermal tissue implant
Removing the epidermal layer;
Removing the cells of the dermal layer;
Removing the base film layer; And
Cryopreservation stage
Method of producing a cell-free dermal tissue implant, characterized in that the basement membrane layer has been removed.
The method of claim 9,
The epidermal layer is separated from the dermal layer using a proteolytic enzyme to remove the basal membrane layer, characterized in that the preparation of acellular dermal tissue implants removed.
The method of claim 9,
Cells of the dermal layer is a method of producing a cell-free dermal tissue implant, the basement membrane layer is removed, characterized in that the removal by surfactant or ultrasonic treatment.
The method of claim 9,
The cryopreservation is a method of producing a cell-free dermal tissue implant, the basement membrane layer has been removed, characterized in that the dermal layer is immersed in a freezing solution containing a cryopreservative and stored below -70 ℃.
Cell-free dermal tissue implant prepared by the method of claim 6.
A skin damage treatment agent comprising the cell-free dermal tissue implant according to claim 1.

Images