WO2013137664A1

WO2013137664A1 - Cell-free dermal tissue implant

Info

Publication number: WO2013137664A1
Application number: PCT/KR2013/002063
Authority: WO
Inventors: 김준용; 김병무; 황용섭; 이환철; 서수정; 이주희; 김형구
Original assignee: 주식회사 엘앤씨바이오
Priority date: 2012-03-15
Filing date: 2013-03-14
Publication date: 2013-09-19

Abstract

The present invention relates to a cell-free dermal tissue implant. More particularly, the formation of a multiple penetration structure and the removal of a basement membrane and/or an edge slope treatment are carried out on a cell-free dermal tissue, thus providing an environment advantageous to angiogenesis and tissue self-renewal and increasing tissue expandability and flexibility and thus shortening time taken for implantation and allowing stable implantation, and thus minimizing side effects after implantation. Furthermore, introduction of fibroblast is increased and angiogenesis is promoted, thus improving engraftment reaction with a host tissue to allow for stable implantation and thus shortening periods taken for recovery after implantation. Furthermore, not only can subcutaneous injection be easily performed during implantation but also the skin in the implanted part does not project after being implanted, thus maintaining a natural look in outer appearance.

Description

Acellular dermal graft

The present invention relates to cell-free dermal tissue implants and methods for their preparation.

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 his / her skin tissue or organ tissue, 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 manufacturing and implanting a cell-free dermal layer from skin tissue extracted from a donor [Domestic Patent Publication No. 2001-0092985, Domestic Patent Registration No. 791502] is a method for solving the above problems. Used.

Basically, in the situation where the implant is inserted into the human host tissue, the disruption of blood flow and fluid circulation between the host tissues caused by the implant, the compact structure of the extracellular hepatic substance in the implant, the presence of the basement membrane layer, and the relative presence of the implant Thick thickness prevents blood circulation around the implant, blocks body fluid circulation, decreases the formation and infiltration of neovascularization, limits the proliferation of fibroblasts and endogenous collagen tissues, increases foreign body sensation and inflammation and infection due to insufficient transplantation. Problems such as the generation of capsule of scar tissue from forming aroun the implant and the retention of body fluids are likely to occur.

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.

In particular, the cell-free dermal tissue implants currently on the market are mostly implanted in the form of a square, the skin protrudes by the rectangular corner of the graft on the surface of the skin after transplantation has a problem that is not natural in appearance. In addition, the separation of the front and back through the basement membrane layer (base membrane layer: smoothness, subdermal surface: finely rough surface) is not easy to check with the naked eye, it is not easy to find a method when the transplantation is necessary. In addition, it is necessary to minimize the skin incision of the graft site for the rapid recovery of the patient after transplantation, there is a difficulty in inserting the implant in the case of a rectangular graft.

Accordingly, the present inventors have formed a multi-penetration in the existing acellular dermal tissue for transplantation to increase the stretching force and flexibility of the tissue to make the transplantation stable.

In addition, after the transplantation, the inflow rate of the fibroblasts in the existing transplantation site is significantly reduced compared to the other side, and the basement membrane layer is removed, which slows the engraftment rate. Could be kept at the same speed as

Therefore, the present invention can provide a favorable environment for neovascularization and autologous proliferation by forming multiple penetrations in the acellular dermal tissue, preventing dead space, and increasing the stretch and flexibility of the tissue to increase the transplant time. The purpose of the present invention is to provide a cell-free dermal tissue transplant that can minimize the occurrence of side effects after transplantation because the transplantation is fast and stable.

In addition, the present invention removes the basement membrane layer of the acellular dermal tissue implants to increase the influx of fibroblasts after transplantation, promote the neovascularization action to increase the engraftment rate to the skin to stabilize the transplantation and minimize the recovery date after transplantation can do.

In addition, the present invention not only increases the convenience of subcutaneous insertion at the time of implantation by slope cutting the corners of the cell-free dermal tissue, but also maintains the natural appearance by not protruding the skin of the transplantation site after transplantation. The purpose is to provide a cell-free dermal tissue implant.

As a means for solving the above problems, the present invention

In acellular dermal grafts,

Provided are a cell-derived dermal grafts with multiple penetrations and a method of manufacturing the same.

As another means for solving the above problems, the present invention

In acellular dermal grafts,

Provided are a cell-free dermal tissue implants with edges sloped and methods for preparing the same.

As another means for solving the above problems, the present invention

In acellular dermal grafts,

Provided are a cell-free dermal tissue implants in which the basement membrane layer has been removed, and a method of manufacturing the same.

The cell-free dermal tissue implant according to the present invention is a very suitable condition for implantation in the human body, that is, rapid production and sufficient proliferation of renal vessels, increased proliferation and influx of fibroblasts, inhibition of pore formation, prevention of fluid retention, host tissue And stable fixation of the graft, increased graft area due to increased graft flexibility, reduced postoperative pain, and skin necrosis, inflammatory reactions, and infections due to decreased blood flow to the skin and subcutaneous tissue in contact with the graft. It can give you all the protection.

In particular, when the implant is inserted under the subcutaneous tissue, the surgery may not only increase the convenience of the surgery, but also maintain the natural appearance of the implant because the skin of the upper part of the implant does not protrude. It is also possible to distinguish the front and back of the implant.

Figure 1 compares the cell-free dermal tissue implants and conventional cell-free dermal tissue implants [Alloderm, LifeCell Corporation, Branchburg, NJ] according to an embodiment of the present invention (multi-slit).

Figure 2 compares the cell-free dermal tissue implants and conventional cell-free dermal tissue implants [Alloderm, LifeCell Corporation, Branchburg, NJ] according to one embodiment of the present invention (corner slope treatment).

Figure 3 compares the cell-free dermal tissue implants and conventional cell-free dermal tissue implants [Alloderm, LifeCell Corporation, Branchburg, NJ] according to one embodiment of the present invention (base layer removal).

FIG. 4 shows a plan view of an implant in which multiple slits and multiple punctures are formed.

5 is a plan view of an implant having a multiple slit form according to one embodiment of the present invention.

6 is a perspective view of an implant having alternately stacked horizontal axis multi-penetration shapes.

FIG. 7 shows only the multi-penetrating structure in the implant of FIG. 6.

8 is a photograph of an acellular dermal graft with multiple slits formed.

9 is a cross-sectional view of the cell-free dermal tissue implant with a portion of the basement membrane layer removed in various forms [hatched portion: basement membrane layer removal site; White part: suture part with host tissue, part of basement membrane layer].

Figure 10 shows a plan view (left), a perspective view (middle) and a cross-sectional view (right) of a corner-treated, acellular dermal graft.

FIG. 11 is a photograph of a cell-derived dermal graft with one top corner slanted (red dotted line: inclined area).

12 is a perspective view of a cell-free dermal tissue implant in accordance with an embodiment of the present invention.

Figure 13 is a photograph of Hematoxylin & Eosin stained 3 days after the fibroblast culture of acellular dermal tissue [upper: cell-free dermal graft of Example 2, lower: conventional cell-free dermal tissue transplant, black arrow: Hematoxylin Fibroblasts stained by.

14 is a photograph after transplantation of the existing acellular dermal graft [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 dermal tissue, Black arrow: engrafted fibroblasts].

Figure 15 is a photograph after transplantation of the cell-free dermal tissue implant of Example 5 (thick black solid line: basement membrane layer, black dotted line: transplanted acellular dermal tissue, black arrow: engrafted fibroblasts).

16 is a photograph confirming angiogenesis of transplanted acellular dermal tissue [left; Cell-free dermal tissue implant of Example 5, right; Existing acellular dermal grafts, assay arrows: angiogenesis].

17 is a photograph confirming skin engraftment rate of transplanted acellular dermal tissue [left; Cell-free dermal tissue implant of Example 5, right; Existing acellular dermal grafts].

18 is a photograph showing that the implant of Example 2 effectively penetrated the cells (fibroblast) and activated fibroblasts (cell proliferation, collagen production) through double staining of DAPI and Fibronetine [(a ) DAPI (Cell DNA), (b) Fibronectin, (c) DAPI Fibronectin, (d) DAPI Fibronectin Brightfield.

The present invention relates to a cell-free dermal tissue implant having the following characteristics 1), 2) and / or 3):

1) forming multiple penetrations

2) Slope treatment of corners

3) Basement layer removal

The cell-free dermal tissue implant is preferably skin, ligament or cartilage, but is not limited thereto.

First, the implant of the present invention may have multiple penetrating structures.

The multi-penetrating structure refers to a multi-penetration structure of a vertical axis and / or a horizontal layer multi-penetration structure stacked alternately.

The vertical axial penetrating structure includes the form of multiple slits and / or multiple punctures in acellular dermal grafts.

As an embodiment of the present invention, the multi-slit treated implant is shown in Figures 1, 4 and 5, but is not limited thereto.

In the present invention, the shape of the multiple slits is preferably a vertical line, a horizontal line, a Y-shape, a V-shape, a Z-shape, or a mixture thereof (see FIG. 4), but is not limited thereto. Specifically, the spacing between the slits on the plan view may be treated with 2 to 12 mm in the left and right directions, and 3-5 mm in the vertical direction. In the case of slits, the length of the slits in the longitudinal direction is 3-10 mm, in the case of the slits in the cross-section direction, 4 to 6 mm in size, and 3 to 5 mm from the edge of the implant. Post transplantation is such that the implant is not disconnected when sutured with host tissue [FIG. 5].

In the case of the implant formed by the vertical axis penetration of the multiple slits, the stretching force in the front, rear, left and right directions of the implant is increased, so that the implant can be stretched to a larger area by implanting the tissue in the same width. Increased flexibility results in a natural extension of the host tissue in conditions of swelling and stretching, and the graft site into which the implant is inserted reduces pain and discomfort during expansion.

Particularly, the microscopic spaces of the slit provide a favorable environment for the proliferation of fibroblasts, the infiltration and proliferation of new blood vessels, and the rapid recovery of blood circulation in the transplantation process. It can prevent the occurrence of side effects such as skin necrosis, inflammation, infection, stagnation of fluid, and the formation of membranes.

In addition, the form of the multi-puncture is a form of a circle, a triangle, a square, etc., if the form is formed at a predetermined interval can be all. Multiple punctures may also be processed between the multiple slits. The diameter of the multi-puncture is 0.2 ~ 2 mm, which is preferable for facilitating the penetration of fibroblasts and neovascularization, so that the engraftment is easy after transplantation. The spacing between the punctures is 0.5 to 5 mm for maintaining the maximum physical properties of the implant. This is preferred.

In addition, in the case of a multi-puncture structure, a space in which the autologous tissue is rapidly proliferated through the multi-penetrating structure is formed from the host tissue surrounding the implant with the advantage obtained in the slit form. Vascular penetration and proliferation are facilitated. In addition, the endogenous autologous tissue that proliferates through the columnar space serves to support and fix the graft itself in the host tissue. Prevents deformation. In addition, the resulting autologous tissue is mixed with the implant to reduce the occurrence of foreign body sensation by the transplant. In addition, volume expansion by the initial implants after surgery may affect the host tissues around the implants.In general, subcutaneous tissues and skin placed on top of the implants during implantation by inserting the implants under the subcutaneous tissues It is easy to cause pressure necrosis due to low blood flow due to the volume expansion of the implant into which the implant is inserted. However, in the present invention, a direct contact from the host tissue beneath the implant is performed through a plurality of columnar spaces. In addition to this, the rapid creation of new blood vessels is induced to increase the blood flow to the subcutaneous tissue and skin to reduce the occurrence of necrosis due to blood flow of the subcutaneous tissue and skin. In addition, the disconnection between the host tissues due to the implantation of the pores between the graft and the host tissues by inhibiting the retention of body fluids by restoring the fluid circulation and connection between the tissues through the porous structure and the multiple slits, such as space and curbing can be suppressed, and inflammation and infection can be reduced.

For example, penile enlargement surgery in urology can induce rapid autologous infiltration and proliferation from both the grafts of Dartos fascia and Buck's fascia on the penetrating site, acting as a scaffold to move the graft due to physical stimuli such as sexual intercourse. It prevents the phenomenon or the phenomenon of deformation.

In addition, the alternately stacked horizontal axis multi-penetration form means a through structure in which one layer penetrates several left and right lines and the other layer is penetrated several times in a front and rear line alternately. As shown in FIG. 6, another layer in which left and right structures are arranged in a line is stacked on the layers arranged in a line, and the layers are alternately stacked. Such a shape has flexibility of the implant in each direction in the transverse and longitudinal directions, and thus, when the implant is fixed to the host tissue, the stretching force is improved, so that the surgery is easy, the foreign body feeling after the surgery is reduced, and the pain is reduced. In addition, it provides a more favorable transplant environment to facilitate the proliferation and infiltration of neovascularization in host tissues after transplantation and to help the growth of fibroblasts and endogenous collagen tissues. Higher, leading to a successful transplant. In addition, the implant hydration process required for the preparation of the transplant graft can be made faster and evenly throughout the implant. In addition, foreign body feelings that patients complain after transplantation are generally made because the implant itself does not obtain a biocompatible transplant reaction with the host tissue, but the structure optimized to maximize the transplant environment through the above-described horizontal axis multi-penetration form It can make a significant drop in foreign body after transplantation. At this time, the penetration diameter (size) of the penetration portion is preferably 0.2 to 0.5 mm. The spacing between penetrations is preferably 0.3 to 3 mm.

In addition, it is more preferable that the implant according to the present invention is treated with a corner portion.

The expression “the corner portion is sloped” means that the edge of the upper surface of the rectangular implant (the line segments forming the boundary of each side in the polyhedron) is gently processed to form an inclined surface. In addition, the surface can be sloped at one or more (one to four) corners of the upper surface of the implant. The base length (c in FIG. 10) of the inclined surface after the slope treatment is preferably 5 to 10 mm, and the height (d in FIG. 10) of the unsloped implant after the slope treatment is preferably 1 to 2 mm. In particular, the height of the inclined surface after the slope treatment (b of FIG. 10) is 60% or more (preferably 65 to 85%) of the implant height before the slope treatment (a of FIG. 10), and the appearance is natural after transplantation. In addition, it is desirable to minimize the contact between the suture and the implant when suturing the host tissue and reduce the inflammatory response.

As shown in Fig. 2, in the case of conventional cell-free dermal tissue implants [Alloderm, LifeCell Corporation, Branchburg, NJ], the edges of the implants are transplanted without being slanted, so that the skin of the implanted site is transplanted. On the other hand, the implant of the present invention is protruded to have a surgically unnatural problem, while the implant of the present invention is inclined to a corner portion of a cell-shaped dermal tissue for transplantation to reduce the appearance of protrusion of the skin at the implantation site after transplantation. The advantage is that it can maintain the naturalness. In addition, when the suture of the implant is fixed to the host tissue by suture, the host tissue is fixed through the suture to the part of the end of the sloped slope rather than suture of the total thickness of the implant. Minimizing contact can reduce inflammation.

In addition, the implant according to the present invention is more preferably removed from the basement membrane layer.

Human skin tissue consists of the outermost epidermal layer, dermis layer and subcutaneous tissue. In general, the epidermal layer is removed during the formation of the implant using human dermal tissue, and the basement membrane layer, which serves as a combination of the epidermal layer and the dermal layer, is the upper part of the dermal layer. Will remain in the implant. Although the basement membrane layer has a histologically dense structure, it also serves to structurally support the dermal graft, but the dense structure provides disadvantageous conditions for the infiltration of neovascularization and the generation and migration of fibroblasts from the host tissue. Done. Therefore, in the case where the basement membrane layer is not removed from the implant, engraftment with the host tissue is somewhat insufficient, and thus, the transplantation is not performed, so that a space is formed, a film is formed, or fluid is stored.

As shown in FIG. 3, 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 particular, in order to fix the implant in the host tissue, it is more desirable to partially remove the base membrane layer of the implant and leave the base membrane layer in the suture portion with the host tissue. By leaving the basement membrane layer of the suture as described above, when the implant is fixed to the host tissue by using the rigidity of the basement membrane layer, even if tension is generated, it is well fixed to prevent movement or deformation of the implant. In addition, the rest of the implant, except for the seal portion of the implant, removes the basement membrane layer, and when combined with host tissue, the inflow and proliferation of fibroblasts from the host tissue to the implant is quick and easy, and the formation and infiltration of neovascularization is easy. As a result of rapid coupling between the host tissue and the implants, it is possible to prevent side effects after surgery and to prevent the occurrence of mesopores and the retention of body fluids, resulting in rapid transplantation and recovery.

9 shows various types of implants partially removed leaving the basement membrane layer at the site to be sutured.

The present invention also provides

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

It relates to a method for producing a cell-free dermal tissue implant comprising the step of forming multiple penetrations.

Specifically, the present invention

Removing the epidermal layer;

Removing the cells of the dermal layer;

Forming multiple penetrations; And

Cryopreservation stage

It may include.

The present invention also provides

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

It relates to a method for producing a cell-free dermal tissue implant comprising the step of removing the basement membrane layer.

Specifically, the present invention

Removing the epidermal layer;

Removing the cells of the dermal layer;

Removing the base film layer; And

Cryopreservation stage

It may include.

The present invention also provides

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

It relates to a method for producing a cell-free dermal tissue implant comprising the step of slope processing the corner portion.

Specifically, the present invention

Removing the epidermal layer;

Removing the cells of the dermal layer;

Cryopreservation step; And

Steps to Slope Edges

It may include.

More specifically, the 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;

Forming a multi-penetration structure;

Cryopreservation step; And

Steps to Slope Edges

It may include.

Referring to the method for producing a cell-free dermal tissue implant according to the present invention in more detail.

First, a step of preparing a tissue to be manufactured with the implant may be further performed.

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, the solution used for transporting tissues should be added with substances that can prevent degradation by hypoxia, degradation by autolytic enzymes, and degradation by proteolytic enzymes, and antibiotics and antibacterial agents to prevent microbial contamination. do. Appropriate buffers should be included to prevent tissue damage from osmotic pressure. The osmotic pressure of the tissue transport solution should have an osmotic pressure of about 260-320 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, 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, nistatin, polymyxin, 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.

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

Generally, 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 next step is to remove the epidermal layer as above and then 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 the tissues.

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 next step can additionally remove cells from the dermis and then remove the basement membrane. When the basement membrane is removed, the transplantation can be made more stably by increasing the engraftment reaction with the host tissue by increasing the influx of fibroblasts and promoting the neovascularization.

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 0.3% hydrogen peroxide solution is treated for 1 to 3 hours to separate the basement membrane layer into the dermis layer. The basement membrane can be removed without. By removing the basement membrane layer, the influx of fibroblasts, etc., in the existing transplantation site is greatly increased compared to the existing dermal tissue implants, and the engraftment reaction with the host tissues can be improved by promoting neovascularization. At this point, it is more preferable that the basement membrane layer of the site to be sealed with the host tissue after implantation is not removed.

The next step may additionally form multiple penetration structures.

The multi-penetration specifically includes a vertical-axis multi-penetration structure and / or an alternately stacked horizontal-axis multiple penetration structure. The vertical axis through structure penetrates from the top surface to the bottom surface of the implant, but the through shape is preferably a multiple slits and / or a multiple puncture.

As described above, the vertical axis multi-penetration and the horizontal axis multi-penetration structure may be formed in various forms.

A method of forming the vertical axis through and the horizontal axis through structures will be described in detail as follows.

The multi-slit form can be carried out manually by using a blade or by using a device capable of processing multiple slits (for example, a multi-cutting blade, a skin mesher, etc.) In the case of multiple puncture, the vertical layer can be penetrated in one direction after fixing the corner portion of the dermis layer using multiple needle layers. In addition, in the case of the horizontal axis penetrating form, through the penetrating in one direction of one layer while maintaining the horizontal angle, the multi penetrating can be performed by adding penetrating from the side surface of the other layer. At this time, if the physical properties of the tissue is not maintained by violating the penetrating direction of the lower layer by maintaining the horizontal angle of the side, the properties of the product may be distorted after freeze-drying, so be careful.

The next step is freeze solution treatment and freeze drying to preserve.

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 glass or square ice that is less stable than hexagonal ice. In addition, glassy ice and square ice have a smaller ice size, which causes less 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 injections. 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 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 performed for 24 to 50 hours by moving to a freeze dryer.

The next step, after cryopreservation, may be to treat the corners of the dermal tissue.

Specifically, the inclined surface is formed by gently processing the corners of the upper surface of the implant of the rectangular shape. At this time, one or more corners of the upper surface of the implant may be treated. The specific process of the slope treatment is as described above. In addition, the surface that is not inclined should be the site where the base film layer is located. The surface treatment process cuts the side surface while the acellular dermal graft is fixed to the jig using an ultrasonic cutter which does not generate heat of the dermal tissue. In addition, the corner portion may be cut in various patterns such as a wave pattern, a sawtooth pattern, a flat pattern when processing.

Cell-derived dermal grafts thus prepared can be used as therapeutic agents for skin damage, such as repairing, shaping, expanding, filling, and protecting tissues.

That is, the cell-free dermal implant according to the present invention is a skin restoration agent due to burns, traffic accidents, ulcers, etc., full-layer skin reconstruction, septal reconstruction, reconstruction of cerebrospinal epidural defect window, depression correction agent, scar correction agent, half face It can be used in dermatology, plastic surgery, gynecology, surgery, neurosurgery, urology, and otolaryngology, such as atrophy correction.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

실시예 1: 모서리가 사면 처리된 무세포 진피조직 이식체 제조Example 1 Preparation of Cell-free Dermal Tissue Transplant Treated by Edges

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.

10% dextran solution was used as the freezing solution of the dermal layer from which the epidermal layer was removed, and the dermal tissue from which the basement membrane layer was removed was immersed in the freezing solution at -4 ° C. for 12 hours to allow the freezing solution to penetrate well. It was stored for 12 hours in a cryogenic freezer below 70 ℃ and lyophilized in a freeze dryer for 48 hours.

Then, the corner portion of the implant (dermal tissue) having a height of 5 mm was sloped as follows. Using an ultrasonic cutter that does not generate heat of the dermal tissue, the slope of the base of the inclined surface was 5 to 10 mm, and the slope of the unsloped portion after the slope was 2 mm in height (Fig. 2, Fig. 10 and Fig. 2). 11].

실시예 2: 다중 슬릿 처리된 무세포 진피조직 이식체 제조Example 2: Preparation of Multi-slit Treatment Cell-Free Dermal Tissue Transplants

In order to make the epidermal layer from which the epidermal layer was removed, the slits were vertically or horizontally formed using a blade (see FIGS. 4 and 8).

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. It was stored for 12 hours and lyophilized for 48 hours in a freeze dryer.

After that, the process of slope treatment of the corner portion of the 5 mm implant (dermal tissue) can be additionally carried out as follows. Using an ultrasonic cutter that does not generate heat in the dermis, slope the base of the slope to 5 to 10 mm and the slope of the unsloped part to be 2 mm after the slope.

실시예 3: 다중 펑쳐 처리된 무세포 진피조직 이식체 제조Example 3 Preparation of Multiple Puncture-Free Cell Dermal Tissues

In order to make the epidermal layer from which the epidermal layer has been removed into a multi-puncture form, a multiple puncture was performed by fixing the corner portion of the dermis layer and penetrating in one direction using a multiple needle layer.

실시예 4: 수평축 다중 관통 처리된 무세포 진피조직 이식체 제조Example 4 Preparation of Cellular Dermal Transplants Treated with Horizontal Multi-Through Penetration

In order to make the epidermal tissue from which the epidermal layer was removed was formed in a horizontally stacked multi-shape form, the penetrating was carried out in one direction while maintaining the horizontal angle to form a multi-penetration by adding a penetrating right side of the side surface [FIG. 6. And FIG. 7].

실시예 5: 기저막층이 제거된 무세포 진피조직 이식체 제조Example 5 Preparation of Cell-Free Dermal Tissue Transplant with Basement Membrane Removed

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.

실시예 6: 무세포 진피조직 이식체의 제조Example 6: Preparation of Cell-Free Dermal Tissue Implants

Skin tissue (collected from a donated body for treatment of a non-profit patient from a tissue bank) was treated with a neutral protease dispase 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 base film 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 heat generation to prevent degeneration of the dermal tissue. At this time, the basement membrane layer was removed except for the site to be sealed with the host tissue.

In order to make the dermal layer partially removed from the basement membrane layer into horizontally stacked multiple penetration structures, one layer is subjected to multiple penetrations in left and right rows, and the other layer in front and rear rows, while maintaining a horizontal angle. Multiple penetrations were added to form alternately stacked penetrations.

In addition, in order to make a multi-slit form, the work of making a slit vertically penetrating using a blade was manually performed. Further, in order to make a multi-puncture form, the dermal layer was penetrated in one direction after fixing the corner portion using a multiple needle layer [see FIG. 12].

10% dextran solution was used as a freezing solution, and the dermal tissue was soaked in the freezing solution for 12 hours at -4 ° C to allow the freezing solution to penetrate well, and then stored in an ultra-low temperature freezer at -70 ° C or lower for 12 hours. Lyophilizer was lyophilized for 48 hours.

Then, the corner portion of the implant (dermal tissue) having a height of 5 mm was sloped as follows. Using an ultrasonic cutter that does not generate heat in the dermis, the base length (c) of the inclined surface after slope treatment is 5 to 10 mm, and the height (d) of the graft remaining without slope treatment after slope treatment is 2 mm Treated (see FIG. 12).

실험예 1: 자가조직 증식 확인Experimental Example 1: Confirmation of autologous tissue proliferation

Cut the multi-slit acellular dermal tissue implant of Example 2 into 1 X 1 cm 2 soaked in DMEM medium (Dulbeco's Modified Eagle's Medium) for 20 minutes, and then prepared fibroblasts 1 X 10 ⁶ / 30μL After inoculating the implants, the cells were allowed to adhere to the 37 ° C. incubator for 5 hours, and the culture medium was carefully submerged so that the cells were inoculated with the cells, and then submerged for 3 days.

After culturing the cell-free dermal grafts of Example 2 for 3 days in culture with 10% formalin, Hematoxylin & Eosin staining was observed to see if the overall three-dimensional culture was well performed.

Existing acellular dermal grafts [Alloderm, LifeCell Corporation, Branchburg, NJ] were compared and tested by the same method.

As a result, as shown in FIG. 13, fibroblasts penetrated around the cell-free dermal tissue implant of Example 2 and confirmed engraftment (black arrow in the top view of FIG. 13). On the other hand, in the case of the existing acellular dermal grafts, it can be confirmed that the infiltrated fibroblasts are less than the acellular dermal grafts of Example 2 (black arrow in the lower figure of FIG. 13).

실험예 2: 섬유아세포 유입 확인Experimental Example 2: Confirmation of Fibroblast Inflow

180 g of experimental animal white paper (Sprague Dawley rats) were infused with rumoons to induce anesthesia. After anesthesia and the like was cut using a Clipper, 1 × 1 cm of tissue was implanted on both sides. At this time, the existing acellular dermal graft was transplanted to the right side, the cell-free dermal tissue graft of Example 5 to the left. 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 fibroblast influx, neovascularization, and skin engraftment.

As shown in Figures 14 and 15, hematoxylin, a marker capable of identifying fibroblasts, was confirmed that the fibroblasts were gathered on the basement membrane layer above the basement membrane layer without being introduced into the basement membrane layer in the existing acellular dermal tissue implant (FIG. 14). Of black arrows). On the contrary, in the implant of Example 5, the basal membrane layer was removed and fibroblasts were not collected thereon, and the fibroblasts were evenly engrafted on the implant (black arrow in FIG. 15).

실험예 3: 신혈관 생성 확인Experimental Example 3: Confirmation of neovascular formation

Hematoxylin & Eosin staining of the transplanted cell-free dermal tissue was performed in the same manner as in Experimental Example 2 to confirm the neovascular formation of the implant.

As shown in Figure 16, it can be confirmed that the implantation of Example 5 increased the formation of blood vessels (black arrow) compared to the conventional acellular dermal tissue implant (black arrow).

실험예 4: 피부 생착률 확인Experimental Example 4: 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 2 to confirm the skin engraftment rate of the implant.

As shown in Figure 17, in the existing acellular dermal tissue it can be confirmed that the fibroblasts do not penetrate above the basement membrane layer by the basement membrane layer and deposited outside the top of the implant. In contrast, in the cell-free dermal tissue of Example 5, in which the basement membrane layer was removed, fibroblasts penetrated into the implant and were grown. This confirms that a greater number of fibroblasts are present in acellular dermal grafts with the basement membrane layer removed.

실험예 5: 세포 침투 및 섬유아세포 활성화Experimental Example 5: Cell Infiltration and Fibroblast Activation

The transplantation in which the multiple slit structure of Example 2 was formed was confirmed the cell infiltration and fibroblast activation process using double staining of DAPI and Fibronetine.

DAPI image is used as a cell marker (DNA stain of live cell) during fluorescence staining, fibronectin is a marker (fibroblast) that is involved in cell adhesion, growth and migration (fibroblast) is activated.

The implant with the multi-slit structure of Example 2 was immersed in phosphate buffer for at least 30 minutes to fully hydrate, and then the buffer was removed and washed three times with fresh phosphate buffer. The hydrated implant was immersed in the cell culture for at least 30 minutes and sufficiently filled with the cell culture (DMEM medium containing 10% FBS and 1% Pen / Strep), and then placed in a 6-well plate for cell culture. After adding and stabilizing at least 2 ml of fresh cell culture, 1 × 10 ⁵ fibroblasts were placed on the implant. The cell culture plate was placed for 48 hours in a cell culture cell with an environment in which cells can grow (37 ° C. constant temperature supplied with 5% CO ₂ ). After 48 hours, the implants were carefully removed from the plate using forceps, then gently washed three times in phosphate buffer and fixed in 4% paraformaldehyde for 24 hours. The immobilized implant was again infiltrated with 30% sucrose for at least 24 hours and then OTC blocks were made. The sample block made a 6 m thick slide section using a micro-tome. The fragment was blocked in phosphate buffer containing 5% bovine serum albumin (BSA) and treated with a primary antibody that recognizes fibronectine at low temperature for 12 hours. This slide was washed three times with phosphate buffer and treated with a rhodamine B labeled secondary antibody that recognized the primary antibody. Again washing three times with phosphate buffer, DAPI solution was added in the first wash to wash the remaining secondary antibody and at the same time the DNA was stained with DAPI. A drop of the mount solution was dropped onto the finished slide, the cover slide was covered, and the cover slide edge was fixed with nail polish. This completed slide was observed with a fluorescence microscope.

As a result, it was confirmed that cells (fibroblast) effectively penetrated (doubled) and activated fibroblasts (cell proliferation, collagen production) through double staining of DAPI and Fibronetine [FIG. 18].

The present invention is not limited to the above-described specific preferred embodiments, and various modifications can be made by any person having ordinary skill in the art without departing from the gist of the present invention claimed in the claims. Of course, such changes will fall within the scope of the claims.

Claims

In acellular dermal grafts,

Cell-free dermal tissue implant, characterized in that multiple penetration is formed.
In acellular dermal grafts,

Cell-free dermal tissue implant, characterized in that the edge portion is a slope (slope) treatment.
In acellular dermal grafts,

Cell-free dermal tissue implant, characterized in that the basement membrane layer has been removed.
The method according to any one of claims 1 to 3,

The cell-free dermal tissue implant is a cell-free dermal tissue implant, characterized in that the skin, ligaments or cartilage.
The method of claim 1,

The cell-free dermal tissue implant, characterized in that the edge portion is sloped (slope).
The method of claim 1,

The multiple penetrations are through a vertical axis; An acellular dermal graft, characterized in that one layer penetrates in a left and right row and the other layer is a horizontal axis multi-perforated structure alternately stacked by penetrating in a front and rear line.
The method of claim 1,

Cell-free dermal tissue implant, characterized in that the basement membrane layer is further removed.
The method of claim 2,

Cell-free dermal tissue implant, characterized in that one or more of the top edge of the implant is sloped.
The method of claim 2,

The height of the inclined surface after the slope treatment of the corner portion is a cell-free dermal graft, characterized in that more than 60% of the height of the implant before the slope treatment.
It has a vertical axis multiple penetration structure and alternately stacked horizontal axis multiple penetration structure,

The basement membrane layer is removed,

The edges are sloped

Cell-free dermal tissue implant, characterized in that.
In the method for producing a cell-free dermal tissue implant,

A method for producing a cell-free dermal tissue implant, comprising the step of forming multiple penetrations.
In the method for producing a cell-free dermal tissue implant,

A method for producing a cell-free dermal tissue implant, comprising the step of removing the basement membrane layer.
In the method for producing a cell-free dermal tissue implant,

Method for producing a cell-free dermal tissue implant, comprising the step of treating the corner portion.
The method of claim 11,

Method for producing the cell-free dermal tissue implant

Removing the epidermal layer;

Removing the cells of the dermal layer;

Forming multiple penetrations; And

Cryopreservation stage

Method for producing a cell-free dermal tissue implant comprising a.
The method of claim 12,

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 for producing a cell-free dermal tissue implant comprising a.
The method of claim 13,

Method for producing the cell-free dermal tissue implant

Removing the epidermal layer;

Removing the cells of the dermal layer;

Cryopreservation steps and

Steps to Slope Edges

Method for producing a cell-free dermal tissue implant comprising a.
The method of claim 14,

And removing the basement membrane layer after the step of removing the cells of the dermal layer.
The method according to claim 12, 15 or 17,

The basement membrane layer is a method of producing a cell-free dermal tissue implant, characterized in that by using a meat grinder, or by removing the hydrogen peroxide treatment.
The method of claim 14,

After the cryopreservation step, the method for producing a cell-free dermal tissue, characterized in that it further comprises the step of treating the corner portion.
The method according to claim 13, 16 or 19,

Method for producing a cell-free dermal tissue implant, characterized in that the treatment of the corner portion by ultrasonic treatment.
The method according to claim 14, 15 or 16,

The epidermal layer is a method for producing a cell-free dermal tissue implant, characterized in that the separation using the proteolytic enzyme and the dermal layer removed.
The method according to claim 14, 15 or 16,

Cells of the dermal layer is a method for producing a cell-free dermal tissue implant, characterized in that the removal by surfactant or ultrasonic treatment.
The method according to claim 14, 15 or 16,

The cryopreservation method of producing a cell-free dermal tissue implant, characterized in that the dermal layer is immersed in a cryopreservative containing cryopreservative and stored below -70 ℃.
The method of claim 11,

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;

Forming a multiple penetration structure;

Cryopreservation step; And

Steps to Slope Edges

Method for producing a cell-free dermal tissue implant comprising a.
Cell-free dermal tissue implant prepared by the method of claim 11.
Cell-free dermal tissue implant prepared by the method of claim 12.
Cell-free dermal tissue implant prepared by the method of claim 13.
A therapeutic agent for skin damage comprising the cell-free dermal tissue implant according to claim 1, 2, 3, 25, 26, or 27.