WO2013137664A1 - Implant de type tissu dermique acellulaire - Google Patents

Implant de type tissu dermique acellulaire Download PDF

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Publication number
WO2013137664A1
WO2013137664A1 PCT/KR2013/002063 KR2013002063W WO2013137664A1 WO 2013137664 A1 WO2013137664 A1 WO 2013137664A1 KR 2013002063 W KR2013002063 W KR 2013002063W WO 2013137664 A1 WO2013137664 A1 WO 2013137664A1
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Prior art keywords
cell
dermal tissue
layer
tissue implant
free
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Ceased
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PCT/KR2013/002063
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English (en)
Korean (ko)
Inventor
김준용
김병무
황용섭
이환철
서수정
이주희
김형구
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L&C Bio Co Ltd
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L&C Bio Co Ltd
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Priority claimed from KR1020120064024A external-priority patent/KR101362403B1/ko
Priority claimed from KR1020120064022A external-priority patent/KR101362402B1/ko
Priority claimed from KR1020120064026A external-priority patent/KR20130105227A/ko
Application filed by L&C Bio Co Ltd filed Critical L&C Bio Co Ltd
Publication of WO2013137664A1 publication Critical patent/WO2013137664A1/fr
Priority to US14/484,031 priority Critical patent/US9532866B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin

Definitions

  • the present invention relates to cell-free dermal tissue implants and methods for their preparation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention As a means for solving the above problems, the present invention
  • a cell-derived dermal grafts with multiple penetrations Provided are a cell-derived dermal grafts with multiple penetrations and a method of manufacturing the same.
  • a cell-free dermal tissue implants with edges sloped are provided.
  • 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.
  • 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.
  • FIG. 5 is a plan view of an implant having a multiple slit form according to one embodiment of the present invention.
  • FIG. 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.
  • FIG. 8 is a photograph of an acellular dermal graft with multiple slits formed.
  • FIG. 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).
  • 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.
  • FIG. 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].
  • FIG. 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].
  • FIG. 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].
  • the present invention relates to a cell-free dermal tissue implant having the following characteristics 1), 2) and / or 3):
  • 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 multi-slit treated implant is shown in Figures 1, 4 and 5, but is not limited thereto.
  • 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.
  • 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.
  • 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].
  • 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.
  • 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.
  • 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.
  • the endogenous autologous tissue that proliferates through the columnar space serves to support and fix the graft itself in the host tissue. Prevents deformation.
  • the resulting autologous tissue is mixed with the implant to reduce the occurrence of foreign body sensation by the transplant.
  • volume expansion by the initial implants after surgery may affect the host tissues around the implants.
  • 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.
  • a direct contact from the host tissue beneath the implant is performed through a plurality of columnar spaces.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the height (d in FIG. 10) of the unsloped implant after the slope treatment is preferably 1 to 2 mm.
  • 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.
  • 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.
  • the epidermal layer is removed during the formation of the implant using human dermal tissue
  • 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.
  • 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.
  • 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.
  • 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.
  • the implant 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.
  • the basement membrane layer of the suture 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.
  • 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.
  • 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.
  • It relates to a method for producing a cell-free dermal tissue implant comprising the step of forming multiple penetrations.
  • the present invention also provides
  • It relates to a method for producing a cell-free dermal tissue implant comprising the step of removing the basement membrane layer.
  • the present invention also provides
  • 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.
  • 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.
  • 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.
  • NEM n-ethylmaleimide
  • PMSF phenylmethylsulfonyl fluoride
  • EDTA ethylenediaminetetraacetic acid
  • EGTA 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.
  • tissues should be transported in a way that minimizes physical damage.
  • the next step is to remove the epidermal layer from the tissue prepared as above, separating the epidermal and dermal layers.
  • proteolytic enzymes are used to separate the dermal and epidermal layers.
  • concentration 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.
  • the dermal and epidermal layers can be separated by treating termolysin at a concentration of 200 ⁇ g / 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.
  • Immune responses are mainly caused by membrane proteins present in the cell membrane. Therefore, removing the cells can minimize the immune response.
  • the main component of the cell membrane is phospholipids, and various surfactants can be used to remove cells without damaging the tissues.
  • 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.
  • 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.
  • 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.
  • 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.
  • a combination of surfactants and ultrasound can also be used to remove cells without damaging the tissue.
  • TNBP solvent
  • 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.
  • 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.
  • 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.
  • the meat grinder blade can use carbon steel material to minimize heat generation and prevent degeneration of dermal tissue.
  • 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.
  • 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.
  • the vertical axis multi-penetration and the horizontal axis multi-penetration structure may be formed in various forms.
  • 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.)
  • a device capable of processing multiple slits for example, a multi-cutting blade, a skin mesher, etc.
  • the vertical layer can be penetrated in one direction after fixing the corner portion of the dermis layer using multiple needle layers.
  • 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • dextran sugar
  • propylene glycol glycerol
  • mannitol sorbitol
  • fructose trehalose
  • raffinose 2.3-butanediol
  • HES ethyl starch
  • PVP Polyvinylpyrrolidone
  • proline proline
  • hetastarch serum albumin and the like.
  • 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.
  • 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.
  • 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.
  • 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 °C and lyophilized in a freeze dryer for 48 hours.
  • 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].
  • 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 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.
  • 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.
  • a multiple puncture was performed by fixing the corner portion of the dermis layer and penetrating in one direction using a multiple needle layer.
  • 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.
  • 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 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].
  • the process of slope treatment of the corner portion of the 5 mm implant 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Example 2 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 / 30 ⁇ L 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.
  • DMEM medium Dulbeco's Modified Eagle's Medium
  • the infiltrated fibroblasts are less than the acellular dermal grafts of Example 2 (black arrow in the lower figure of FIG. 13).
  • 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).
  • 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.
  • Example 5 increased the formation of blood vessels (black arrow) compared to the conventional acellular dermal tissue implant (black arrow).
  • 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.
  • 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 ).
  • 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.
  • BSA bovine serum albumin
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Transplantation (AREA)
  • Vascular Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Dermatology (AREA)
  • Cardiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Materials For Medical Uses (AREA)
  • Prostheses (AREA)
PCT/KR2013/002063 2012-03-15 2013-03-14 Implant de type tissu dermique acellulaire Ceased WO2013137664A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/484,031 US9532866B2 (en) 2012-03-15 2014-09-11 Acellular dermal graft

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
KR10-2012-0026617 2012-03-15
KR10-2012-0026618 2012-03-15
KR10-2012-0026616 2012-03-15
KR20120026618 2012-03-15
KR20120026616 2012-03-15
KR20120026617 2012-03-15
KR1020120064024A KR101362403B1 (ko) 2012-03-15 2012-06-15 다중 관통이 형성된 무세포 진피조직 이식체
KR1020120064022A KR101362402B1 (ko) 2012-03-15 2012-06-15 기저막층이 제거된 무세포 진피조직 이식체
KR10-2012-0064022 2012-06-15
KR10-2012-0064026 2012-06-15
KR10-2012-0064024 2012-06-15
KR1020120064026A KR20130105227A (ko) 2012-03-15 2012-06-15 모서리 부분이 경사 처리된 무세포 진피조직 이식체

Related Child Applications (1)

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US14/484,031 Continuation US9532866B2 (en) 2012-03-15 2014-09-11 Acellular dermal graft

Publications (1)

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WO2013137664A1 true WO2013137664A1 (fr) 2013-09-19

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PCT/KR2013/002063 Ceased WO2013137664A1 (fr) 2012-03-15 2013-03-14 Implant de type tissu dermique acellulaire

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD856517S1 (en) 2016-06-03 2019-08-13 Musculoskeletal Transplant Foundation Asymmetric tissue graft
USD895812S1 (en) 2018-09-07 2020-09-08 Musculoskeletal Transplant Foundation Soft tissue repair graft
US10813743B2 (en) 2018-09-07 2020-10-27 Musculoskeletal Transplant Foundation Soft tissue repair grafts and processes for preparing and using same
CN112352044A (zh) * 2018-06-29 2021-02-09 莱雅公司 包含真皮-皮下接合的纤维母细胞的细胞和组织模型及其应用
US10945831B2 (en) 2016-06-03 2021-03-16 Musculoskeletal Transplant Foundation Asymmetric tissue graft
WO2022131407A1 (fr) * 2020-12-18 2022-06-23 주식회사 엘앤씨바이오 Peau artificielle à base de derme comprenant une couche de membrane basale et son procédé de fabrication
US12458730B2 (en) 2019-09-25 2025-11-04 Allosource Pre-shaped allograft implant for reconstructive surgical use and methods of manufacture and use, and tools for forming a pre-shaped allograft implant for reconstructive surgical use
USD1101168S1 (en) 2023-08-09 2025-11-04 Allosource Acellular dermal matrix sheet allograft

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KR20010092985A (ko) * 2000-03-28 2001-10-27 황호찬 이식용 무세포 진피층 가공 및 분리방법
US20050021141A1 (en) * 2001-10-26 2005-01-27 Bleyer Mark W. Medical graft device with meshed structure
JP3658385B2 (ja) * 2002-09-20 2005-06-08 佳宏 高見 皮膚の無細胞化方法、該方法による無細胞化真皮マトリックス及びその製造方法並びに該マトリックスを用いた複合培養皮膚
EP1020198B1 (fr) * 1999-01-12 2006-07-12 Jentec, Inc. Pansement résistant au froissement

Patent Citations (4)

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EP1020198B1 (fr) * 1999-01-12 2006-07-12 Jentec, Inc. Pansement résistant au froissement
KR20010092985A (ko) * 2000-03-28 2001-10-27 황호찬 이식용 무세포 진피층 가공 및 분리방법
US20050021141A1 (en) * 2001-10-26 2005-01-27 Bleyer Mark W. Medical graft device with meshed structure
JP3658385B2 (ja) * 2002-09-20 2005-06-08 佳宏 高見 皮膚の無細胞化方法、該方法による無細胞化真皮マトリックス及びその製造方法並びに該マトリックスを用いた複合培養皮膚

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD856517S1 (en) 2016-06-03 2019-08-13 Musculoskeletal Transplant Foundation Asymmetric tissue graft
US10945831B2 (en) 2016-06-03 2021-03-16 Musculoskeletal Transplant Foundation Asymmetric tissue graft
CN112352044A (zh) * 2018-06-29 2021-02-09 莱雅公司 包含真皮-皮下接合的纤维母细胞的细胞和组织模型及其应用
USD895812S1 (en) 2018-09-07 2020-09-08 Musculoskeletal Transplant Foundation Soft tissue repair graft
US10813743B2 (en) 2018-09-07 2020-10-27 Musculoskeletal Transplant Foundation Soft tissue repair grafts and processes for preparing and using same
US11642216B2 (en) 2018-09-07 2023-05-09 Musculoskeletal Transplant Foundation Soft tissue repair grafts and processes for preparing and using same
US12458730B2 (en) 2019-09-25 2025-11-04 Allosource Pre-shaped allograft implant for reconstructive surgical use and methods of manufacture and use, and tools for forming a pre-shaped allograft implant for reconstructive surgical use
WO2022131407A1 (fr) * 2020-12-18 2022-06-23 주식회사 엘앤씨바이오 Peau artificielle à base de derme comprenant une couche de membrane basale et son procédé de fabrication
USD1101168S1 (en) 2023-08-09 2025-11-04 Allosource Acellular dermal matrix sheet allograft

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