WO2022080876A1 - Breast reconstruction support using dermal tissue-derived extracellular matrix and fabrication method therefor - Google Patents

Breast reconstruction support using dermal tissue-derived extracellular matrix and fabrication method therefor Download PDF

Info

Publication number
WO2022080876A1
WO2022080876A1 PCT/KR2021/014170 KR2021014170W WO2022080876A1 WO 2022080876 A1 WO2022080876 A1 WO 2022080876A1 KR 2021014170 W KR2021014170 W KR 2021014170W WO 2022080876 A1 WO2022080876 A1 WO 2022080876A1
Authority
WO
WIPO (PCT)
Prior art keywords
extracellular matrix
dermal tissue
breast reconstruction
gamma
tissue
Prior art date
Application number
PCT/KR2021/014170
Other languages
French (fr)
Korean (ko)
Inventor
이기원
김형구
이환철
Original Assignee
주식회사 엘앤씨바이오
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 엘앤씨바이오 filed Critical 주식회사 엘앤씨바이오
Publication of WO2022080876A1 publication Critical patent/WO2022080876A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • 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
    • A61F2/12—Mammary prostheses and implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/04—Materials or treatment for tissue regeneration for mammary reconstruction

Definitions

  • the present invention relates to a transplant material for breast reconstruction using a dermal tissue-derived extracellular matrix and a method for manufacturing the same.
  • Breast reconstruction is performed to restore the shape of the breast not only when there is no or poor breasts congenitally, but also when there is acquired breast loss, that is, when the breast is resected for breast cancer treatment or when the breast is lost due to burns or trauma. It is an operation performed
  • breast reconstruction performed after breast cancer surgery involves continuous radiation therapy after reconstruction. After irradiating radiation, a thick film is formed in the area around the surgery where breast reconstruction is performed due to the inserted material. This is the most frequent problem in breast reconstruction surgery due to capsular contracture in which the muscles around the surgical site gradually harden. Therefore, in breast reconstruction surgery performed after breast cancer surgery, the choice of surgical material is important in order to suppress the spherical contracture that occurs during radiation therapy after surgery.
  • Autologous tissue transplantation is a method of reconstructing the breast using one's own tissue. Because it uses autologous tissue, there is less feeling of foreign body and it is soft to the touch. On the other hand, there are disadvantages in that the operation time is long, the size that can be reconstructed is limited, and scars may remain in the area where the tissue has been removed.
  • Breast implant insertion is a method of restoring the removed breasts using implants.
  • a tissue expander is inserted into the surgical site to gradually increase the dermal tissue to secure space for the implant, and then insert the implant to create a voluminous breast.
  • this breast implant insertion is simple, takes a relatively short time, and leaves no additional scars.
  • a feeling of foreign body may be felt after insertion, and a thick film may be formed around the implanted implant, resulting in spherical contracture in which the surrounding muscles gradually harden.
  • the engraftment rate is lower than that of autologous tissue.
  • a method of removing foreign body feeling, inhibiting spheroid contracture and increasing engraftment rate is used by wrapping the breast implant with an extracellular matrix derived from allogeneic and heterogeneous dermal tissue after using the breast implant.
  • the dermal tissue-derived extracellular matrix is a dermal layer matrix obtained through acellularization technology from human or animal skin, and refers to a skin substitute in the form of collagen, elastin and fibronectin. These bio-derived skin substitutes are known to have a relatively low immune rejection response, unlike transplant organs.
  • human-derived skin substitutes are superior to animal-derived skin substitutes in terms of biological performance and safety in the case of transplantation of human-derived skin substitutes and animal-derived skin substitutes.
  • human-derived skin substitutes must be donated by a donor after death, there may be restrictions in terms of supply and demand of raw materials for animal-derived skin substitutes.
  • spheroid contracture is suppressed, volume is maintained, and shape change is minimized even with continuous radiation treatment performed after breast cancer resection surgery by irradiating gamma rays after manufacturing the delocalized and decellularized dermal tissue-derived extracellular matrix.
  • the present invention is to sterilize and crosslink the dermal tissue-derived extracellular matrix support with gamma rays, so that the volume is maintained even after continuous irradiation with radiation after implantation in the body, and to provide a graft material and a method for manufacturing the same The purpose.
  • the present invention comprises the steps of a) removing a lipid component from a dermal tissue;
  • step c) the dose of gamma rays is 16 to 50 kGy to provide a method for manufacturing a transplant material for breast reconstruction.
  • the present invention provides a transplant material for breast reconstruction manufactured by the above-described manufacturing method.
  • the implant for breast reconstruction manufactured by the manufacturing method according to the present invention may be used for breast reconstruction.
  • the implantation material for breast reconstruction according to the present invention suppresses spherical contracture even with continuous radiation treatment after breast cancer resection surgery, and can maintain volume and minimize shape change.
  • 1 is a photograph taken with a scanning electron microscope of collagen fibers in an extracellular matrix support according to a change in gamma-irradiation dose.
  • FIG. 2 is a photograph taken with a three-dimensional camera of the extracellular matrix support according to the change in gamma-irradiation dose.
  • Figure 3 is a photograph (a) and graph (b) confirming the volume by removing the scaffold at 4 weeks and 8 weeks, respectively, after irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after the insertion of the extracellular matrix support into the mouse.
  • Figure 4 is a photograph of H&E staining for histological analysis after inserting the extracellular matrix support into the mouse and irradiating 0 Gy, 10 Gy, and 20 Gy of radiation, respectively, at 4 weeks and 8 weeks (a) and cell influx is a quantified graph (b).
  • Figure 5 is a photograph of ⁇ -SMA immunohistochemical staining to confirm tissue fibrosis by irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after inserting the extracellular matrix into the mouse, and then removing the support at 4 and 8 weeks, respectively. (a) and a graph (b) quantifying the area of cells stained with ⁇ -SMA.
  • CTGF connective tissue growth factor
  • FIG. 8 is a photograph (a) and CD3 stained with CD3 to confirm the inflammatory response by removing the scaffold at 4 and 8 weeks, respectively, after irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after the insertion of the extracellular matrix support into the mouse. It is a graph (b) in which the stained cells were quantified.
  • the present invention comprises the steps of: a) removing the lipid component from the dermal tissue (hereinafter, delipidation step);
  • decellularization step removing cells from the dermal tissue from which the lipid component has been removed
  • gamma-irradiation step comprising the step of gamma-irradiating the dermal tissue from which the cells have been removed (hereinafter, gamma-irradiation step),
  • the gamma-ray dose in step c) relates to a method for manufacturing a breast reconstruction implant of 16 to 50 kGy.
  • the optimal manufacturing conditions for the production of implants for breast reconstruction were confirmed by checking whether or not crosslinking, sterilization, and shape maintenance of the extracellular matrix support according to the gamma radiation dose.
  • the extracellular matrix scaffold prepared by the present invention is continuously irradiated with radiation after implantation in the body. It was confirmed that the shape change was minimized and inflammation was suppressed by maintaining the volume and suppressing the spherical contracture.
  • graft material for breast reconstruction refers to a product manufactured by treating dermal tissue according to the manufacturing method according to the present invention.
  • the implant material for breast reconstruction is manufactured from dermal tissue and includes an extracellular matrix, so it can be expressed as an "extracellular matrix support”.
  • the dermal tissue may be an allogeneic or heterogeneous dermal tissue.
  • the homogeneous refers to a human, and the heterogeneous refers to animals other than humans, ie, mammals such as pigs, cattle, and horses.
  • a transplant material for breast reconstruction can be manufactured according to the manufacturing method of the present invention using a dermal tissue derived from allogeneic or heterogeneous.
  • the step of pre-treating the dermal tissue (hereinafter, the pre-treatment step) may be additionally performed.
  • the pretreatment step includes washing the dermal tissue; Physically removing fascia tissue, adipose tissue and other foreign substances from the dermal tissue; and removing the epidermis and scab from the dermal tissue; may include one or more of
  • washing may be performed using sterile distilled water.
  • fascia tissue, adipose tissue, and other foreign substances may be physically removed using a scraper or scissors.
  • the epidermis and sicum may be removed using sodium chloride (NaCl) and hydrogen peroxide (H 2 O 2 ).
  • NaCl sodium chloride
  • H 2 O 2 hydrogen peroxide
  • 0.1M to 10M sodium chloride and 1% to 10% hydrogen peroxide may be used, and the treatment time may be 10 to 48 hours or 20 to 30 hours.
  • step a) is a delipidation step of removing the lipid component from the dermal tissue, and the dermal tissue may be delipidated.
  • the delipidation refers to the removal of lipid components from the dermal tissue.
  • defatification may be performed using a delipidation solution.
  • the delipidation solution may include a polar solvent, a non-polar solvent, or a mixed solvent thereof.
  • Water, alcohol, or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol.
  • a mixed solution of isopropyl alcohol (IPA) and hexane may be used as the delipidation solution.
  • the mixing ratio (wt%) of isopropyl alcohol and hexane may be 20:80 to 80:20.
  • the treatment time of the delipidation solution may be 1 to 8 hours.
  • step b) is a decellularization step of removing cells from the dermal tissue from which the lipid component has been removed by step a), and the dermal tissue may be decellularized.
  • Decellularization refers to the removal of other cellular components other than the extracellular matrix from the dermal tissue, for example, the nucleus, cell membrane, nucleic acid, and the like.
  • the dermal tissue that has undergone delocalization and decellularization can be expressed as acellular dermis.
  • decellularization can be performed using a decellularization solution.
  • a surfactant and/or a basic solution may be used as the decellularization solution.
  • an ionic surfactant such as sodium dodecyl sulfate (SDS), or Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidet P-10 (NP-10), Noni A nonionic surfactant such as Det P-40 (NP-40) may be used.
  • SDS sodium dodecyl sulfate
  • Triton X-100 Triton X-100
  • Tween 20 Tween 40
  • Tween 60 Tween 80
  • Nonidet P-10 Nonidet P-10
  • Noni A nonionic surfactant such as Det P-40 (NP-40)
  • one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may
  • an SDS solution having a concentration of 0.1% to 10% may be used. It is easy to remove the cells in the above concentration range.
  • the decellularization step may be performed for 30 minutes to 10 hours. Removal of cells in this time range is easy.
  • step b after performing step b), the step of washing the acellular dermis may be further included.
  • washing may be performed using sterile distilled shoes.
  • step c) is a step of gamma-irradiating the dermal tissue from which the cells were removed in step b).
  • the dermal tissue from which cells have been removed that is, the acellular dermis can be cross-linked and sterilized.
  • the cell-free dermis maintains its volume even after continuous irradiation with radiation after implantation in the body and suppresses spherical contracture to minimize shape change, and also to suppress inflammation.
  • the term “sterilization” refers to making a sterile state by killing or removing infectious bacteria, parasites, and pests present in the acellular dermis.
  • sterilizing the cell-free dermis without increasing the temperature by irradiating gamma rays with strong penetrating power without using heat or chemicals it is possible to completely kill deadly infectious bacteria, parasites and pests.
  • crosslinking refers to the addition of covalent bonds between polypeptides, which are the basic structure of a protein.
  • the three-dimensional structure of the protein may be maintained or the hardness may be modified by the crosslinking.
  • the binding of amino acids, which is a constituent of a protein can be increased, and a sterilization effect can be obtained along with a cross-linking effect, stability is secured and no residue is left.
  • the presence or absence of such crosslinking can be determined by evaluating morphologically based on the arrangement and density of collagen fibers through scanning electron microscopy.
  • the acellular dermis before gamma irradiation has a sparsely arranged, low density of collagen fibers and an unstable collagen fiber structure.
  • the collagen fibers are arranged in parallel, and the density between the collagen fibers is increased to show a stable collagen fiber structure.
  • the amount of gamma radiation is excessive, the shape of the acellular dermis cannot be maintained, so it is important to properly control the gamma radiation dose.
  • the dose of gamma rays may be 16 to 50 kGy, 21 to 29 kGy, or 23 to 27 kGy.
  • crosslinking and sterilization of acellular dermis can be achieved without change in shape. If the dose is less than 16 kGy, cross-linking and sterilization of the acellular dermis is not performed, and if it exceeds 50 kGy, cross-linking and sterilization are achieved, but the dermis cannot maintain its shape and tissue may be destroyed. unsuitable for use
  • gamma rays may be irradiated to the acellular dermis for 18 to 60 hours or 27 to 33 hours at a dose rate of 0.5 to 1.0 kGy/hr or 0.7 to 0.9 kGy/hr.
  • the volume change rate of the acellular dermis according to gamma irradiation may be 30 vol% or less, 1 to 30 vol%, 1 to 28 vol%, 1 to 20 vol% or less, or 1 to 15 vol% or less.
  • the volume of the cell-free dermis can be performed according to the experimental method of "whether or not to maintain shape" of Experimental Example 1 of the present invention.
  • the present invention relates to a transplant material for breast reconstruction manufactured by the above-described manufacturing method.
  • the implant material for breast reconstruction of the present invention may be used as a transplant material for breast reconstruction surgery.
  • the implant for breast reconstruction may be used as an implant for breast reconstruction after a surgical operation on the breast to maintain and/or increase the space of the breast removed during the operation.
  • the surgical procedure may be a mastectomy, a lumpectomy, or a revision breast augmentation procedure.
  • the implant material for breast reconstruction may be used together with the breast implant, and may be implanted in the body in a form that surrounds a part or the whole of the breast implant.
  • the shape of the implant for breast reconstruction of the present invention is not particularly limited, and may have, for example, a sheet shape.
  • the implant material for breast reconstruction according to the present invention can minimize the immune response in the body through the decellularization process.
  • spherical contracture is suppressed even after continuous radiation therapy performed after breast cancer resection surgery, and volume can be maintained and shape change can be minimized.
  • the volume change rate after 8 weeks after irradiation with gamma rays at a dose of 20 Gy to the implant for breast reconstruction according to the present invention may be 1 to 15 vol%.
  • the volume of the implant for breast reconstruction can be performed according to the experimental method of (1) "Verification of in-body volume maintenance capacity" of Experimental Example 2 of the present invention.
  • Human dermal tissue (collected from a cadaver donated by a tissue bank for non-profit patient care) was washed with sterile distilled water. Fascia and fat were removed from the washed dermal tissue using scissors or the like. In order to remove the epidermis and sebum from the fascia and the dermal tissue from which the fat was removed, it was treated with 0.1M to 10M sodium chloride and 1% to 10% hydrogen peroxide for 24 hours. In order to remove fat from the epidermis and tissue from which the scabs were removed, it was treated with 40% to 60% isopropyl alcohol and 40% to 60% hexane for 2 hours. Cells were removed by treating the fat-removed tissue with 0.1% to 10% SDS solution.
  • the washed extracellular matrix support was cross-linked and sterilized by gamma-irradiation of cobalt-60 gamma rays at room temperature at a dose rate of 0.84 kGy/hr for 6 to 60 hours at a dose of 5 to 50 kGy.
  • An extracellular matrix support was prepared in the same manner as in Example 1, except that it was sterilized with 70% ethanol instead of gamma irradiation.
  • the presence or absence of crosslinking was evaluated by visually checking the arrangement and density of collagen fibers from the photograph taken after photographing the support with an electron scanning microscope. Specifically, when the collagen fibers in the support are arranged in parallel and densely arranged between the fibers, it was judged to be crosslinked, and when the collagen fibers were irregularly and sparsely arranged, it was judged that the collagen fibers were not crosslinked.
  • the presence or absence of sterilization was evaluated by visual inspection for the presence or absence of proliferation of microorganisms in the medium using the extract of the dermal tissue-derived extracellular matrix support.
  • the presence or absence of shape maintenance was evaluated by measuring the volume of the dermal tissue-derived extracellular matrix support using a three-dimensional camera. Specifically, it was evaluated that the shape could not be maintained when the volume of the extracellular matrix support was deformed by 20 vol% or more after irradiation with gamma rays.
  • FIG. 1 shows a photograph taken with a scanning electron microscope of collagen fibers in order to confirm the presence or absence of crosslinking of the extracellular matrix support according to the gamma-ray dose.
  • the collagen fibers in the support are sparsely arranged, and when the gamma-ray dose is 20 kGy or more, it can be confirmed that the collagen fibers are arranged in parallel and densely arranged. That is, it can be confirmed that the crosslinking was made at a gamma-ray dose of 20 kGy or more.
  • Figure 2 shows a picture taken with a three-dimensional camera in order to check whether the shape of the extracellular matrix support according to the gamma-ray dose is maintained.
  • the volume change rates of the scaffold measured at each dose with respect to the volume of the scaffold were 5.5 vol% (10 kGy), 12.4 vol% (20 kGy), and 27.7 vol% (30 kGy), respectively.
  • Table 1 shows the results of confirming the presence or absence of crosslinking, sterilization, and shape maintenance of the human dermal tissue-derived extracellular matrix support according to the change in gamma-irradiation dose.
  • the optimal gamma irradiation dose for crosslinking, sterilization, and shape maintenance of the human dermal tissue-derived extracellular matrix support is 20 to 40 kGy, specifically, 25 kGy.
  • the human dermal tissue-derived extracellular matrix scaffold sterilized with 70% ethanol was used as an existing method instead of gamma irradiation of Comparative Example 1, and irradiated with the optimal gamma radiation dose (25 kGy) in Example 1
  • the extracellular matrix scaffold was used as an experimental group (the present invention).
  • FIG 3 shows a photograph (b) and a graph (b) confirming the volume of the excised extracellular matrix support.
  • H&E hematoxylin and eosin
  • FIG. 4 shows a photograph (a) of H&E staining of the excised extracellular matrix support and a graph (b) of quantifying cell influx.
  • tissue fibrosis was confirmed by immunohistochemical staining and real-time PCR for the extracellular matrix scaffolds extracted in (1).
  • ⁇ -SMA smooth muscle actin
  • CTGF connective tissue growth factor
  • FIG. 5 shows a photograph (a) of ⁇ -SMA immunohistochemical staining of the excised extracellular matrix support and a graph (b) of quantifying the cell area stained with ⁇ -SMA.
  • FIG. 6 shows a graph of quantifying the mRNA level of CTGF by real-time PCR to confirm the gene expression of connective tissue growth factor (CTGF), which is a major mediator involved in tissue fibrosis.
  • CTGF connective tissue growth factor
  • the extracellular matrix scaffold (experimental group) prepared by the method according to the present invention exhibits tissue fibrosis similar to that of the scaffold prepared by the existing method.
  • MT Masson's Trichrome staining was performed to confirm the spherical contracture in the excised extracellular matrix scaffolds, and the thickness of the dermis covering the scaffolds was quantified.
  • FIG. 7 shows a photograph (a) of MT staining of the excised extracellular matrix support and a graph (b) quantifying the thickness of the dermis covering the support.
  • the thickness of the dermis covering the scaffold does not increase in the extracellular matrix scaffold of the experimental group at the 4th and 8th weeks after irradiation with 20 Gy of radiation compared to the conventional method.
  • the extracellular matrix support prepared by the manufacturing method according to the present invention suppresses the occurrence of spheroid contracture.
  • CD3 immunohistochemical staining was performed to confirm the inflammatory response in the extracellular matrix scaffolds, and CD3 stained cells were quantified.
  • FIG. 8 shows a photograph (a) in which the excised extracellular matrix was stained with CD3 and a graph (b) in which the cells stained with CD3 were quantified.
  • the implant for breast reconstruction manufactured by the manufacturing method according to the present invention may be used for breast reconstruction.
  • the implantation material for breast reconstruction according to the present invention suppresses spherical contracture even with continuous radiation treatment after breast cancer resection surgery, and can maintain volume and minimize shape change.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The present invention relates to a graft for breast reconstruction by using a dermal tissue-derived extracellular matrix and a fabrication method therefor. In the present invention, a delipidated and decellularized, dermal tissue-derived extracellular matrix is constructed and then irradiated with gamma rays, thereby fabricating a graft for breast reconstruction which can suppress capsular contracture, maintain volume, and minimize a morphological change in spite of continual radiotherapy after mastectomy.

Description

진피 조직 유래 세포외기질을 이용한 유방재건용 지지체 및 그 제조 방법Support for breast reconstruction using dermal tissue-derived extracellular matrix and method for manufacturing the same
본 발명은 진피 조직 유래 세포외기질을 이용한 유방재건용 이식재 및 그 제조 방법에 관한 것이다.The present invention relates to a transplant material for breast reconstruction using a dermal tissue-derived extracellular matrix and a method for manufacturing the same.
유방재건술은 선천적으로 유방이 없거나 빈약한 경우뿐 아니라 후천적으로 유방이 소실된 경우, 즉 유방암 치료를 위해 유방을 절제한 경우나 화상이나 외상에 의해 유방이 소실된 경우에, 유방의 모양 복원을 위해 시행하는 수술이다. Breast reconstruction is performed to restore the shape of the breast not only when there is no or poor breasts congenitally, but also when there is acquired breast loss, that is, when the breast is resected for breast cancer treatment or when the breast is lost due to burns or trauma. It is an operation performed
유방암 수술 후 시행하는 유방재건술은 일반적인 유방재건술과 다르게 재건 후 지속적인 방사선 치료를 병행한다. 방사선을 조사한 후, 유방재건술이 이루어진 수술 주변 부위에는 삽입한 재료로 인해 두꺼운 피막이 형성된다. 이는 수술 부위 주변 근육이 점진적으로 딱딱해지는 구형구축(capsular contracture)으로 유방재건술에서 가장 빈번하게 발생하는 문제점이다. 따라서, 유방암 수술 후 시행하는 유방재건술에서 시술 후 방사선 치료 시 발생하는 구형구축을 억제하기 위해서는 수술 재료의 선택이 중요하다.Unlike general breast reconstruction, breast reconstruction performed after breast cancer surgery involves continuous radiation therapy after reconstruction. After irradiating radiation, a thick film is formed in the area around the surgery where breast reconstruction is performed due to the inserted material. This is the most frequent problem in breast reconstruction surgery due to capsular contracture in which the muscles around the surgical site gradually harden. Therefore, in breast reconstruction surgery performed after breast cancer surgery, the choice of surgical material is important in order to suppress the spherical contracture that occurs during radiation therapy after surgery.
현재, 유방재건술은 수술 재료에 따라 크게 자가조직이식술과 유방보형물 삽입술로 나뉜다. 자가조직이식술은 본인의 조직을 활용해 가슴을 재건하는 방법으로 뱃살, 옆구리, 등 및 엉덩이의 조직을 제거해 유방 쪽으로 이식한다. 자가 조직을 활용하기 때문에 이물감이 덜하며 촉감도 부드럽다. 반면, 수술 시간이 길고 재건할 수 있는 크기가 제한되며 조직이 제거된 부위에 흉터가 남을 수 있다는 단점이 있다.Currently, breast reconstruction surgery is largely divided into autologous tissue transplantation and breast implant insertion depending on the surgical material. Autologous tissue transplantation is a method of reconstructing the breast using one's own tissue. Because it uses autologous tissue, there is less feeling of foreign body and it is soft to the touch. On the other hand, there are disadvantages in that the operation time is long, the size that can be reconstructed is limited, and scars may remain in the area where the tissue has been removed.
유방보형물 삽입술은 보형물을 사용해 절제된 가슴을 복원시키는 방법이다. 수술 부위에 조직확장기를 삽입해 진피 조직을 서서히 늘려 보형물이 들어갈 공간을 확보한 다음, 보형물을 삽입해 볼륨감 있는 가슴을 만든다. 이러한 유방보형물 삽입술은 자가조직이식술에 비해, 수술이 간단하고 시간이 상대적으로 짧으며 추가적인 흉터가 남지 않는다. 그러나, 삽입 후 이물감을 가질 수 있으며, 삽입된 보형물 주위에 두꺼운 피막이 형성되어 점진적으로 주변 근육이 딱딱해지는 구형구축이 발생할 수 있다. 또한, 자가조직에 비해 생착률이 떨어지는 단점이 있다. 특히, 구형구축은 방사선 치료 시 발생할 확률이 높아지는 것으로 보고되어 있다.Breast implant insertion is a method of restoring the removed breasts using implants. A tissue expander is inserted into the surgical site to gradually increase the dermal tissue to secure space for the implant, and then insert the implant to create a voluminous breast. Compared to autologous tissue transplantation, this breast implant insertion is simple, takes a relatively short time, and leaves no additional scars. However, a feeling of foreign body may be felt after insertion, and a thick film may be formed around the implanted implant, resulting in spherical contracture in which the surrounding muscles gradually harden. In addition, there is a disadvantage that the engraftment rate is lower than that of autologous tissue. In particular, it has been reported that the probability of occurrence of spheroid contracture increases during radiation therapy.
이와 같은 단점을 극복하기 위해, 유방보형물을 사용한 후, 상기 유방보형물을 동종 및 이종 진피 조직 유래 세포외기질 등으로 감싸줌으로써 이물감 제거, 구형구축 억제 및 생착률을 증가시키는 방법을 사용한다. 진피 조직 유래 세포외기질은 사람 내지 동물피부로부터 무세포화 기술을 통해 얻어지는 진피층 기질로서, 콜라겐, 엘라스틴 및 피브로넥틴 등으로 구성되어 있는 형태의 피부대체재를 의미한다. 이러한 생체유래 피부대체재는 이식용 장기와는 달리 면역거부반응이 상대적으로 낮은 것으로 알려져 있다. 사람유래 피부대체재와 동물유래 피부대체재의 이식 시 면역거부반응은 일반적으로 사람유래 피부대체재가 생물학적 성능 및 안전성에 대해서 동물유래 피부대체재 보다 우수한 것으로 보고되어 있다. 다만, 사람유래 피부대체재는 사후 기증자로부터 피부조직을 기증받아야 하므로, 동물유래 피부대체재의 원료수급 면에서는 제한이 있을 수 있다.In order to overcome this disadvantage, a method of removing foreign body feeling, inhibiting spheroid contracture and increasing engraftment rate is used by wrapping the breast implant with an extracellular matrix derived from allogeneic and heterogeneous dermal tissue after using the breast implant. The dermal tissue-derived extracellular matrix is a dermal layer matrix obtained through acellularization technology from human or animal skin, and refers to a skin substitute in the form of collagen, elastin and fibronectin. These bio-derived skin substitutes are known to have a relatively low immune rejection response, unlike transplant organs. In general, it is reported that human-derived skin substitutes are superior to animal-derived skin substitutes in terms of biological performance and safety in the case of transplantation of human-derived skin substitutes and animal-derived skin substitutes. However, since human-derived skin substitutes must be donated by a donor after death, there may be restrictions in terms of supply and demand of raw materials for animal-derived skin substitutes.
반면, 유방재건 목적으로는 대부분 동종 진피 조직 유래 세포외기질을 사용하며, 미국의 알로덤(AlloDerm®, 라이프셀), 국내의 슈어덤(SureDermTM, 한스바이오메드), 시지크라이오덤(CGCryoDermTM, 시지바이오) 등과 같은 제품이 있다. 동종 진피 조직 유래 세포외기질에 함유된 콜라겐, 엘라스틴 및 피브로넥틴은 세포의 부착 및 증식을 유도하여 생착을 도와줄 수 있다. 그러나, 유방암 수술 후 시행하는 유방재건술은 일반적인 유방재건술과는 달리, 수술 및 재건 후 환자들의 치료와 재발방지 목적으로 방사선 치료를 병행하게 되는데, 이러한 방사선의 지속적인 조사는 기존 동종 진피 조직 유래 세포외기질 지지체에서 구형구축 및 부피 감소로 인한 형태 유지력 저하를 유발할 수 있다. On the other hand, for the purpose of breast reconstruction, most allogeneic dermal tissue-derived extracellular matrix is used. ), etc. Collagen, elastin and fibronectin contained in the allogeneic dermal tissue-derived extracellular matrix can aid in engraftment by inducing cell adhesion and proliferation. However, in breast reconstruction after breast cancer surgery, unlike general breast reconstruction, radiation therapy is used in parallel with the treatment of patients after surgery and reconstruction and for the purpose of preventing recurrence. It may cause a decrease in shape retention capacity due to spherical contracture and volume reduction in the support.
따라서, 본 발명에서는 탈지방화 및 탈세포화된 진피 조직 유래 세포외기질을 제작한 후, 감마선을 조사하여 유방암 절제 수술 후 행해지는 지속적인 방사선 치료에도 구형구축이 억제되며 부피를 유지하고 형태변화를 최소화하는 유방재건용 이식재 제조방법을 제시하고자 한다.Therefore, in the present invention, spheroid contracture is suppressed, volume is maintained, and shape change is minimized even with continuous radiation treatment performed after breast cancer resection surgery by irradiating gamma rays after manufacturing the delocalized and decellularized dermal tissue-derived extracellular matrix. We would like to present a method for manufacturing a transplant material for breast reconstruction.
[선행기술문헌][Prior art literature]
[비특허문헌][Non-patent literature]
1. Plast Reconstr Surg. 2012 Nov;130(5 Suppl 2):159S-172S. doi: 10.1097/PRS.0b013e3182634e62.1. Plast Reconstr Surg. 2012 Nov;130(5 Suppl 2):159S-172S. doi: 10.1097/PRS.0b013e3182634e62.
본 발명은 탈지방화 및 탈세포화된 진피 조직 유래 세포외기질 지지체를 제작한 후, 감마선을 조사한 유방재건용 이식재 및 그 제조 방법을 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a graft material for breast reconstruction, which is irradiated with gamma rays, and a method for manufacturing the same after preparing a delipidated and decellularized dermal tissue-derived extracellular matrix scaffold.
또한, 본 발명은 진피 조직 유래 세포외기질 지지체에 감마선으로 멸균 및 가교를 진행하여, 체내 이식 후 방사선의 지속적인 조사에도 부피가 유지되고 구형구축을 억제시키는 특성을 갖는 이식재 및 그 제조 방법 제공하는 것을 목적으로 한다. In addition, the present invention is to sterilize and crosslink the dermal tissue-derived extracellular matrix support with gamma rays, so that the volume is maintained even after continuous irradiation with radiation after implantation in the body, and to provide a graft material and a method for manufacturing the same The purpose.
본 발명은 a) 진피 조직에서 지질 성분을 제거하는 단계;The present invention comprises the steps of a) removing a lipid component from a dermal tissue;
b) 상기 지질 성분이 제거된 진피 조직에서 세포를 제거하는 단계; 및 b) removing cells from the dermal tissue from which the lipid component has been removed; and
c) 상기 세포가 제거된 진피 조직을 감마선 조사하는 단계를 포함하며, c) comprising gamma-irradiating the dermal tissue from which the cells have been removed,
단계 c)에서 감마선 선량은 16 내지 50 kGy인 유방재건용 이식재의 제조 방법을 제공한다. In step c), the dose of gamma rays is 16 to 50 kGy to provide a method for manufacturing a transplant material for breast reconstruction.
또한, 본 발명은 전술한 제조 방법에 의해 제조된 유방재건용 이식재를 제공한다. In addition, the present invention provides a transplant material for breast reconstruction manufactured by the above-described manufacturing method.
본 발명에 따른 제조 방법에 의해 제조된 유방재건용 이식재는 유방재건 용도로 사용될 수 있다. The implant for breast reconstruction manufactured by the manufacturing method according to the present invention may be used for breast reconstruction.
본 발명에 따른 유방재건용 이식재는 유방암 절제 수술 후 행해지는 지속적인 방사선 치료에도 구형구축이 억제되며 부피를 유지하고 형태변화를 최소화할 수 있다.The implantation material for breast reconstruction according to the present invention suppresses spherical contracture even with continuous radiation treatment after breast cancer resection surgery, and can maintain volume and minimize shape change.
도 1은 감마선 조사 선량 변화에 따른 세포외기질 지지체 내 콜라겐 섬유를 전자주사현미경으로 촬영한 사진이다.1 is a photograph taken with a scanning electron microscope of collagen fibers in an extracellular matrix support according to a change in gamma-irradiation dose.
도 2는 감마선 조사 선량 변화에 따른 세포외기질 지지체를 3차원 카메라로 촬영한 사진이다.2 is a photograph taken with a three-dimensional camera of the extracellular matrix support according to the change in gamma-irradiation dose.
도 3은 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 부피를 확인한 사진(a) 및 그래프(b)이다. Figure 3 is a photograph (a) and graph (b) confirming the volume by removing the scaffold at 4 weeks and 8 weeks, respectively, after irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after the insertion of the extracellular matrix support into the mouse.
도 4는 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 조직학적 분석을 위해 H&E 염색한 사진(a)과 세포 유입을 정량한 그래프(b)이다.Figure 4 is a photograph of H&E staining for histological analysis after inserting the extracellular matrix support into the mouse and irradiating 0 Gy, 10 Gy, and 20 Gy of radiation, respectively, at 4 weeks and 8 weeks (a) and cell influx is a quantified graph (b).
도 5는 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 조직 섬유화를 확인하기 위해 α-SMA 면역조직화학 염색한 사진(a)과 α-SMA 염색된 세포 면적을 정량한 그래프(b)이다.Figure 5 is a photograph of α-SMA immunohistochemical staining to confirm tissue fibrosis by irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after inserting the extracellular matrix into the mouse, and then removing the support at 4 and 8 weeks, respectively. (a) and a graph (b) quantifying the area of cells stained with α-SMA.
도 6은 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 조직 섬유화에 관여하는 주요 매개물질인 결합조직성장인자(CTGF)의 유전자 발현을 확인하기 위해 real-time PCR에 의한 CTGF의 mRNA 레벨을 정량한 그래프이다.6 shows connective tissue growth factor (CTGF), which is a major mediator involved in tissue fibrosis, after the insertion of the extracellular matrix scaffold into the mouse and after irradiation with 0 Gy, 10 Gy, and 20 Gy of radiation, the scaffolds were removed at 4 and 8 weeks, respectively. ) is a graph quantifying the mRNA level of CTGF by real-time PCR to confirm the gene expression.
도 7은 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 구형구축을 확인하기 위해 MT 염색한 사진(a)과 지지체를 덮고 있는 진피의 두께를 정량한 그래프(b)이다.7 is a photograph showing MT staining to confirm spherical contracture by removing the scaffold at 4 and 8 weeks, respectively, after irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after the insertion of the extracellular matrix support into the mouse (a) and the scaffold It is a graph (b) quantifying the thickness of the dermis covering the
도 8은 마우스에 세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 염증반응을 확인하기 위해 CD3 염색한 사진(a)과 CD3 염색된 세포를 정량한 그래프(b)이다.8 is a photograph (a) and CD3 stained with CD3 to confirm the inflammatory response by removing the scaffold at 4 and 8 weeks, respectively, after irradiating 0 Gy, 10 Gy, and 20 Gy of radiation after the insertion of the extracellular matrix support into the mouse. It is a graph (b) in which the stained cells were quantified.
본 발명은 a) 진피 조직에서 지질 성분을 제거하는 단계(이하, 탈지질화 단계);The present invention comprises the steps of: a) removing the lipid component from the dermal tissue (hereinafter, delipidation step);
b) 상기 지질 성분이 제거된 진피 조직에서 세포를 제거하는 단계(이하, 탈세포화 단계); 및 b) removing cells from the dermal tissue from which the lipid component has been removed (hereinafter, decellularization step); and
c) 상기 세포가 제거된 진피 조직을 감마선 조사하는 단계(이하, 감마선 조사 단계)를 포함하며, c) comprising the step of gamma-irradiating the dermal tissue from which the cells have been removed (hereinafter, gamma-irradiation step),
단계 c)에서 감마선 선량은 16 내지 50 kGy인 유방재건용 이식재의 제조 방법에 관한 것이다. The gamma-ray dose in step c) relates to a method for manufacturing a breast reconstruction implant of 16 to 50 kGy.
본 발명의 실시예에서는 감마선 선량에 따른 세포외기질 지지체의 가교, 멸균 및 형태 유지 유무를 확인하여 유방재건용 이식재의 제조를 위한 최적의 제조 조건을 확인하였다. 또한, 제조된 세포외기질 지지체의 체내 부피유지 능력, 자가조직화, 조직 섬유화, 구형구축 억제효과 및 엄증 억제효과를 검증하여, 본 발명에 의해 제조된 세포외기질 지지체가 체내 이식 후 방사선의 지속적인 조사에도 부피를 유지하고 구형구축을 억제하여 형태변화를 최소화하고 염증을 억제함을 확인하였다. In an embodiment of the present invention, the optimal manufacturing conditions for the production of implants for breast reconstruction were confirmed by checking whether or not crosslinking, sterilization, and shape maintenance of the extracellular matrix support according to the gamma radiation dose. In addition, by verifying the body volume maintenance ability, self-organization, tissue fibrosis, spheroid contracture inhibitory effect, and severe inhibitory effect of the prepared extracellular matrix scaffold, the extracellular matrix scaffold prepared by the present invention is continuously irradiated with radiation after implantation in the body. It was confirmed that the shape change was minimized and inflammation was suppressed by maintaining the volume and suppressing the spherical contracture.
이하, 본 발명에 따른 유방재건용 이식재의 제조 방법을 보다 상세하게 설명한다. Hereinafter, a method of manufacturing the implant for breast reconstruction according to the present invention will be described in more detail.
본 발명에서 "유방재건용 이식재"는 본 발명에 따른 제조 방법에 따라 진피 조직을 처리하여 제조된 생성물을 의미한다. 본 발명에서 유방재건용 이식재는 진피 조직으로부터 제조되며 세포외기질을 포함하므로 "세포외기질 지지체"라 표현할 수 있다.In the present invention, "graft material for breast reconstruction" refers to a product manufactured by treating dermal tissue according to the manufacturing method according to the present invention. In the present invention, the implant material for breast reconstruction is manufactured from dermal tissue and includes an extracellular matrix, so it can be expressed as an "extracellular matrix support".
본 발명에서 진피 조직은 동종 또는 이종의 진피 조직일 수 있다. 상기 동종은 인간을 의미하며, 이종은 인간 이외의 동물, 즉, 돼지, 소, 말 등의 포유류를 의미할 수 있다.In the present invention, the dermal tissue may be an allogeneic or heterogeneous dermal tissue. The homogeneous refers to a human, and the heterogeneous refers to animals other than humans, ie, mammals such as pigs, cattle, and horses.
즉, 본 발명에서는 동종 또는 이종 유래의 진피 조직을 사용하여 본 발명의 제조 방법에 따라 유방재건용 이식재를 제조할 수 있다.That is, in the present invention, a transplant material for breast reconstruction can be manufactured according to the manufacturing method of the present invention using a dermal tissue derived from allogeneic or heterogeneous.
본 발명에서는 단계 a)를 수행하기 전에 진피 조직을 전처리하는 단계(이하, 전처리 단계)를 추가로 수행할 수 있다. In the present invention, before performing step a), the step of pre-treating the dermal tissue (hereinafter, the pre-treatment step) may be additionally performed.
상기 전처리 단계는 진피 조직을 세척하는 단계; 진피 조직에서 근막 조직, 지방 조직 및 기타 이물질을 물리적으로 제거하는 단계; 및 진피 조직에서 표피 및 시반을 제거하는 단계; 중 하나 이상을 포함할 수 있다. The pretreatment step includes washing the dermal tissue; Physically removing fascia tissue, adipose tissue and other foreign substances from the dermal tissue; and removing the epidermis and scab from the dermal tissue; may include one or more of
일 구체예에서, 세척은 멸균 증류수를 이용하여 세척할 수 있다. In one embodiment, washing may be performed using sterile distilled water.
일 구체예에서, 스크래퍼(scrapper) 또는 가위를 사용하여 근막 조직, 지방 조직 및 기타 이물질을 물리적으로 제거할 수 있다. In one embodiment, fascia tissue, adipose tissue, and other foreign substances may be physically removed using a scraper or scissors.
일 구체예에서, 표피 및 시반은 염화나트륨(NaCl) 및 과산화수소(H2O2)를 사용하여 제거할 수 있다. 이때, 0.1M 내지 10M의 염화나트륨 및 1% 내지 10% 과산화수소를 사용할 수 있으며, 처리 시간은 10 내지 48 시간 또는 20 내지 30 시간일 수 있다. In one embodiment, the epidermis and sicum may be removed using sodium chloride (NaCl) and hydrogen peroxide (H 2 O 2 ). In this case, 0.1M to 10M sodium chloride and 1% to 10% hydrogen peroxide may be used, and the treatment time may be 10 to 48 hours or 20 to 30 hours.
본 발명에서 단계 a)는 진피 조직에서 지질 성분을 제거하는 탈지방화 단계로, 진피 조직을 탈지방화(delipidation)할 수 있다. 상기 탈지방화는 진피 조직으로부터 지질 성분을 제거하는 것을 의미한다.In the present invention, step a) is a delipidation step of removing the lipid component from the dermal tissue, and the dermal tissue may be delipidated. The delipidation refers to the removal of lipid components from the dermal tissue.
일 구체예에서, 탈지방화는 탈지질 용액을 사용하여 수행할 수 있다. 상기 탈지질 용액은 극성 용매, 비극성 용매 또는 이들의 혼합 용매를 포함할 수 있다. 상기 극성 용매로는 물, 알코올 또는 이들의 혼합 용액을 사용할 수 있으며, 알코올로는 메탄올, 에탄올 또는 이소프로필 알코올을 사용할 수 있다. 또한, 비극성 용매로는 헥산, 헵탄, 옥탄, 또는 이들의 혼합 용액을 사용할 수 있다. 구체적으로, 본 발명에서는 탈지질 용액으로 이소프로필 알코올(IPA) 및 헥산(Hexane)의 혼합 용액을 사용할 수 있다. 이때, 이소프로필 알코올 및 헥산의 혼합 비율(중량%)은 20:80 내지 80:20일 수 있다. In one embodiment, defatification may be performed using a delipidation solution. The delipidation solution may include a polar solvent, a non-polar solvent, or a mixed solvent thereof. Water, alcohol, or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol. In addition, as the non-polar solvent, hexane, heptane, octane, or a mixed solution thereof may be used. Specifically, in the present invention, a mixed solution of isopropyl alcohol (IPA) and hexane may be used as the delipidation solution. At this time, the mixing ratio (wt%) of isopropyl alcohol and hexane may be 20:80 to 80:20.
상기 탈지질 용액의 처리 시간은 1 내지 8 시간일 수 있다. The treatment time of the delipidation solution may be 1 to 8 hours.
본 발명에서 단계 b)는 단계 a)에 의해 지질 성분이 제거된 진피 조직에서 세포를 제거하는 탈세포화 단계로, 진피 조직을 탈세포화(decellularization)할 수 있다. 탈세포화는 진피 조직으로부터 세포외기질을 제외한 다른 세포 성분, 예를 들면 핵, 세포막, 핵산 등을 제거하는 것을 의미한다. 본 발명에서는 탈지방화 및 탈세포화를 거친 진피 조직을 무세포 진피로 표현할 수 있다.In the present invention, step b) is a decellularization step of removing cells from the dermal tissue from which the lipid component has been removed by step a), and the dermal tissue may be decellularized. Decellularization refers to the removal of other cellular components other than the extracellular matrix from the dermal tissue, for example, the nucleus, cell membrane, nucleic acid, and the like. In the present invention, the dermal tissue that has undergone delocalization and decellularization can be expressed as acellular dermis.
일 구체예에서, 탈세포화는 탈세포 용액을 사용하여 수행할 수 있다. 상기 탈세포 용액으로 계면활성제 및/또는 염기성 용액을 사용할 수 있다. 구체적으로, 계면활성제로 소듐도데실 설페이트(SDS)와 같은 이온성 계면활성제, 또는 트리톤 엑스-100, 트윈 20, 트윈 40, 트윈 60, 트윈 80, 노니데트 피-10(NP-10), 노니데트 피-40(NP-40) 등과 같은 비이온성 계면활성제 등을 사용할 수 있다. 또한, 염기성 용액으로 수산화나트륨, 수산화칼륨, 수산화암모늄, 칼슘카보네이트, 수산화마그네슘, 수산화칼슘 및 암모니아로 이루어진 그룹으로부터 선택된 하나 이상을 사용할 수 있다. 본 발명에서는 탈세포 용액으로 SDS를 사용할 수 있다. In one embodiment, decellularization can be performed using a decellularization solution. A surfactant and/or a basic solution may be used as the decellularization solution. Specifically, as the surfactant, an ionic surfactant such as sodium dodecyl sulfate (SDS), or Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidet P-10 (NP-10), Noni A nonionic surfactant such as Det P-40 (NP-40) may be used. In addition, one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may be used as the basic solution. In the present invention, SDS can be used as the decellularization solution.
일 구체예에서, 탈세포 용액으로 SDS를 사용할 경우, 0.1% 내지 10% 농도의 SDS 용액을 사용할 수 있다. 상기 농도 범위에서 세포의 제거가 용이하다. In one embodiment, when SDS is used as the decellularization solution, an SDS solution having a concentration of 0.1% to 10% may be used. It is easy to remove the cells in the above concentration range.
또한, 일 구체예에서, 탈세포화 단계는 30 분 내지 10 시간 동안 수행될 수 있다. 상기 시간 범위에서 세포의 제거가 용이하다.Also, in one embodiment, the decellularization step may be performed for 30 minutes to 10 hours. Removal of cells in this time range is easy.
본 발명에서는 단계 b)를 수행한 후, 무세포 진피를 세척하는 단계를 추가로 포함할 수 있다. 상기 단계에서는 멸균 증류슈를 이용하여 세척을 수행할 수 있다. In the present invention, after performing step b), the step of washing the acellular dermis may be further included. In the above step, washing may be performed using sterile distilled shoes.
본 발명에서 단계 c)는 상기 단계 b)에서 세포가 제거된 진피 조직을 감마선 조사하는 단계이다. 상기 단계를 통해 세포가 제거된 진피 조직, 즉, 무세포 진피를 가교 및 멸균할 수 있다. 또한, 가교 및 멸균에 의해 무세포 진피는 체내 이식 후 방사선의 지속적인 조사에도 부피를 유지하고 구형구축을 억제하여 형태 변화를 최소화할 수 있으며, 또한, 염증을 억제할 수 있다.In the present invention, step c) is a step of gamma-irradiating the dermal tissue from which the cells were removed in step b). Through the above step, the dermal tissue from which cells have been removed, that is, the acellular dermis can be cross-linked and sterilized. In addition, by crosslinking and sterilization, the cell-free dermis maintains its volume even after continuous irradiation with radiation after implantation in the body and suppresses spherical contracture to minimize shape change, and also to suppress inflammation.
일 구체예에서, 용어 "멸균"은 무세포 진피 내에 존재하는 전염성 세균들이나 기생충 및 해충 등을 사멸 또는 제거시켜 무균 상태로 만드는 것을 의미한다. 본 발명에서는 열이나 화학약품을 사용하지 않고, 강력한 투과력을 지닌 감마선을 조사하여 온도상승 없이 무세포 진피를 멸균함으로써, 치명적인 전염성 세균들이나 기생충 및 해충들을 완전히 사멸시킬 수 있다. In one embodiment, the term “sterilization” refers to making a sterile state by killing or removing infectious bacteria, parasites, and pests present in the acellular dermis. In the present invention, by sterilizing the cell-free dermis without increasing the temperature by irradiating gamma rays with strong penetrating power without using heat or chemicals, it is possible to completely kill deadly infectious bacteria, parasites and pests.
일 구체예에서, 용어 "가교"는 단백질의 기본구조인 폴리펩티드 사이에 공유결합을 첨가하는 것을 의미한다. 상기 가교에 의해 단백질의 3차원 구조를 유지시키거나 경도를 변형시킬 수 있다. 본 발명에서는 감마선(gamma ray)을 사용한 물리적 가교를 이용함으로써, 단백질의 구성물질인 아미노산의 결합을 증가시킬 수 있고, 가교 효과와 더불어 멸균 효과를 가질 수 있으며, 안정성이 확보되고 잔존물이 남지 않는다는 장점을 가질 수 있다. 이러한 가교의 유무는 전자주사현미경 사진을 통하여 형태학적으로 콜라겐 섬유의 배열과 치밀도를 바탕으로 평가하여 결정할 수 있다. 감마선 조사 전의 무세포 진피는 성기게 배열되고 낮은 콜라겐 섬유의 치밀도를 가지며 불안정한 콜라겐 섬유 구조를 가진다. 여기에 감마선을 조사하면 콜라겐 섬유가 평행하게 배열되며 상기 콜라겐 섬유간의 치밀도가 높아져 안정적인 콜라겐 섬유 구조를 나타내게 된다. 다만, 감마선 조사량이 과도할 경우 무세포 진피의 형태가 유지되지 못하므로 감마선 선량을 알맞게 조절하는 것이 중요하다.In one embodiment, the term "crosslinking" refers to the addition of covalent bonds between polypeptides, which are the basic structure of a protein. The three-dimensional structure of the protein may be maintained or the hardness may be modified by the crosslinking. In the present invention, by using physical cross-linking using gamma ray, the binding of amino acids, which is a constituent of a protein, can be increased, and a sterilization effect can be obtained along with a cross-linking effect, stability is secured and no residue is left. can have The presence or absence of such crosslinking can be determined by evaluating morphologically based on the arrangement and density of collagen fibers through scanning electron microscopy. The acellular dermis before gamma irradiation has a sparsely arranged, low density of collagen fibers and an unstable collagen fiber structure. When gamma rays are irradiated here, the collagen fibers are arranged in parallel, and the density between the collagen fibers is increased to show a stable collagen fiber structure. However, if the amount of gamma radiation is excessive, the shape of the acellular dermis cannot be maintained, so it is important to properly control the gamma radiation dose.
일 구체예에서, 감마선의 선량은 16 내지 50 kGy, 21 내지 29 kGy, 또는 23 내지 27 kGy일 수 있다. 상기 선량 범위에서 형태의 변화 없이 무세포 진피의 가교 및 멸균이 이루어질 수 있다. 상기 선량이 16 kGy 미만이면, 무세포 진피의 가교 및 멸균이 수행되지 않으며, 50 kGy를 초과하면 가교 및 멸균이 되나 진피는 그 형태를 유지하지 못하여 조직이 파괴될 수 있으며 유방재건용 이식재로의 사용에 부적합하다. In one embodiment, the dose of gamma rays may be 16 to 50 kGy, 21 to 29 kGy, or 23 to 27 kGy. In the above dose range, crosslinking and sterilization of acellular dermis can be achieved without change in shape. If the dose is less than 16 kGy, cross-linking and sterilization of the acellular dermis is not performed, and if it exceeds 50 kGy, cross-linking and sterilization are achieved, but the dermis cannot maintain its shape and tissue may be destroyed. unsuitable for use
일 구체예에서, 감마선은 0.5 내지 1.0 kGy/hr 또는 0.7 내지 0.9 kGy/hr의 선량률로 18 내지 60 시간 또는 27 내지 33 시간 동안 무세포 진피에 조사될 수 있다. In one embodiment, gamma rays may be irradiated to the acellular dermis for 18 to 60 hours or 27 to 33 hours at a dose rate of 0.5 to 1.0 kGy/hr or 0.7 to 0.9 kGy/hr.
일 구체예에서, 감마선 조사에 따른 무세포 진피의 체적 변화율은 30 vol% 이하, 1 내지 30 vol%, 1 내지 28 vol%, 1 내지 20 vol% 이하, 또는 1 내지 15 vol% 이하일 수 있다. 이때, 무세포 진피의 체적은 본 발명의 실험예 1의 "형태유지 유무"의 실험 방법에 따라 수행할 수 있다. In one embodiment, the volume change rate of the acellular dermis according to gamma irradiation may be 30 vol% or less, 1 to 30 vol%, 1 to 28 vol%, 1 to 20 vol% or less, or 1 to 15 vol% or less. At this time, the volume of the cell-free dermis can be performed according to the experimental method of "whether or not to maintain shape" of Experimental Example 1 of the present invention.
또한, 본 발명은 전술한 제조 방법에 의해 제조된 유방재건용 이식재에 관한 것이다. In addition, the present invention relates to a transplant material for breast reconstruction manufactured by the above-described manufacturing method.
본 발명의 유방재건용 이식재는 유방재건 수술을 위한 이식재로 사용될 수 있다. The implant material for breast reconstruction of the present invention may be used as a transplant material for breast reconstruction surgery.
일 구체예에서, 유방재건용 이식재는 유방 상에 외과 시술 후, 시술 동안 제거된 유방의 공간을 유지 및/또는 증대시키기 위한 유방재건술의 이식재로서 사용될 수 있다. 이때, 외과 시술은 유방절제술(mastectomy), 유방보존술(lumpectomy) 또는 교정 유방 확대 시술(revision breast augmentation procedure) 등일 수 있다.In one embodiment, the implant for breast reconstruction may be used as an implant for breast reconstruction after a surgical operation on the breast to maintain and/or increase the space of the breast removed during the operation. In this case, the surgical procedure may be a mastectomy, a lumpectomy, or a revision breast augmentation procedure.
일 구체예에서, 유방재건용 이식재는 유방보형물과 함께 사용될 수 있으며, 상기 유방보형물의 일부 또는 전체를 감싸는 형태로 체내에 이식될 수 있다. In one embodiment, the implant material for breast reconstruction may be used together with the breast implant, and may be implanted in the body in a form that surrounds a part or the whole of the breast implant.
일 구체예에서, 본 발명의 유방재건용 이식재의 형태는 특별히 제한되지 않으며, 예를 들어, 시트 형태를 가질 수 있다. In one embodiment, the shape of the implant for breast reconstruction of the present invention is not particularly limited, and may have, for example, a sheet shape.
본 발명에 따른 유방재건용 이식재는 탈세포화 과정을 통해 체내 면역반응을 최소화할 수 있다. 또한, 유방재건 용도에 적용되어, 유방암 절제 수술 후 행해지는 지속적인 방사선 치료에도 구형구축이 억제되며 부피를 유지하고 형태변화를 최소화할 수 있다. The implant material for breast reconstruction according to the present invention can minimize the immune response in the body through the decellularization process. In addition, when applied to breast reconstruction, spherical contracture is suppressed even after continuous radiation therapy performed after breast cancer resection surgery, and volume can be maintained and shape change can be minimized.
일 구체예에서, 본 발명에 따른 유방재건용 이식재에 20 Gy의 선량으로 감마선 조사 후 8 주 후의 체적 변화율은 1 내지 15 vol%일 수 있다. 이때, 유방재건용 이식재의 체적은 본 발명의 실험예 2의 (1) "체내 부피유지 능력 검증"의 실험 방법에 따라 수행할 수 있다. In one embodiment, the volume change rate after 8 weeks after irradiation with gamma rays at a dose of 20 Gy to the implant for breast reconstruction according to the present invention may be 1 to 15 vol%. At this time, the volume of the implant for breast reconstruction can be performed according to the experimental method of (1) "Verification of in-body volume maintenance capacity" of Experimental Example 2 of the present invention.
하기 실시예를 통하여 본 발명을 보다 구체적으로 설명하기로 한다. 그러나, 본 발명의 범주는 하기 실시예에 한정되는 것이 아니며 첨부된 특허청구범위에 기재된 사항에 의해 도출되는 기술적 사항을 벗어나지 않는 범위 내에서 다양한 변형, 수정 또는 응용이 가능하다는 것을 당업자는 이해할 수 있을 것이다. The present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and it will be understood by those skilled in the art that various changes, modifications or applications are possible within the scope without departing from the technical matters derived from the matters described in the appended claims. will be.
실시예Example
실시예 1. 인체 진피 조직 유래 세포외기질을 이용한 세포외기질 지지체 제조Example 1. Preparation of extracellular matrix scaffold using human dermal tissue-derived extracellular matrix
인체 진피 조직(조직은행으로부터 비영리 목적의 환자 치료를 위해 기증받은 시신으로부터 채취)을 멸균 증류수로 세척하였다. 세척된 진피 조직을 가위 등을 이용하여 근막 및 지방을 제거하였다. 근막과 지방이 제거된 진피 조직에서 표피 및 시반을 제거하기 위해 0.1M 내지 10M 염화나트륨과 1% 내지 10% 과산화수소로 24시간 동안 처리하였다. 표피 및 시반이 제거된 조직에서 지방을 제거하기 위해 40% 내지 60% 아이소프로필 알코올과 40% 내지 60% 헥산을 이용하여 2시간 동안 처리하였다. 지방이 제거된 조직에 0.1% 내지 10% SDS 용액을 처리하여 세포를 제거하였다. 지방 및 세포 제거가 완료된 세포외기질을 세척하기 위해 멸균 증류수로 2시간 동안 세척하였다. 세척 완료된 세포외기질 지지체는 코발트-60의 감마선을 선원으로 실온에서 0.84 kGy/hr의 선량률로 6 내지 60 시간 동안 5 내지 50 kGy의 선량으로 감마선 조사하여 가교 및 멸균하였다.Human dermal tissue (collected from a cadaver donated by a tissue bank for non-profit patient care) was washed with sterile distilled water. Fascia and fat were removed from the washed dermal tissue using scissors or the like. In order to remove the epidermis and sebum from the fascia and the dermal tissue from which the fat was removed, it was treated with 0.1M to 10M sodium chloride and 1% to 10% hydrogen peroxide for 24 hours. In order to remove fat from the epidermis and tissue from which the scabs were removed, it was treated with 40% to 60% isopropyl alcohol and 40% to 60% hexane for 2 hours. Cells were removed by treating the fat-removed tissue with 0.1% to 10% SDS solution. It was washed with sterile distilled water for 2 hours to wash the extracellular matrix from which fat and cells were removed. The washed extracellular matrix support was cross-linked and sterilized by gamma-irradiation of cobalt-60 gamma rays at room temperature at a dose rate of 0.84 kGy/hr for 6 to 60 hours at a dose of 5 to 50 kGy.
비교예 1.Comparative Example 1.
감마선 조사 대신 70% 에탄올로 멸균한 것을 제외하고는 실시예 1과 동일한 방법으로 세포외기질 지지체를 제조하였다. An extracellular matrix support was prepared in the same manner as in Example 1, except that it was sterilized with 70% ethanol instead of gamma irradiation.
실험예 1. 감마선 선량에 따른 지지체의 물성 측정 Experimental Example 1. Measurement of physical properties of support according to gamma-ray dose
감마선 선량에 따른 지지체의 가교 유무, 멸균 유무 및 형태 유지 유무를 평가하였다. The presence or absence of crosslinking, sterilization, and shape maintenance of the support according to the gamma radiation dose were evaluated.
가교 유무는 전자주사현미경으로 지지체를 촬영한 후 촬영한 사진으로부터 콜라겐 섬유의 배열과 치밀도를 육안으로 확인함으로써 평가하였다. 구체적으로, 지지체 내의 콜라겐 섬유가 평행하게 배열되고 섬유간 치밀하게 배열된 경우 가교된 것으로 판단하였으며, 상기 콜라겐 섬유가 불규칙적이고 또한 성기게 배열되면 가교가 되지 않은 것으로 판단하였다.The presence or absence of crosslinking was evaluated by visually checking the arrangement and density of collagen fibers from the photograph taken after photographing the support with an electron scanning microscope. Specifically, when the collagen fibers in the support are arranged in parallel and densely arranged between the fibers, it was judged to be crosslinked, and when the collagen fibers were irregularly and sparsely arranged, it was judged that the collagen fibers were not crosslinked.
멸균 유무는 대한민국약전 무균시험법을 바탕으로, 진피 조직 유래 세포외기질 지지체의 용출물을 사용하여 배지에서의 미생물의 증식 유무를 육안검사를 통하여 평가하였다. Based on the Korean Pharmacopoeia sterility test method, the presence or absence of sterilization was evaluated by visual inspection for the presence or absence of proliferation of microorganisms in the medium using the extract of the dermal tissue-derived extracellular matrix support.
또한, 형태유지 유무는 3차원 카메라를 사용하여 진피 조직 유래 세포외기질 지지체의 체적을 측정하여 평가하였다. 구체적으로, 감마선 조사 후 상기 세포외기질 지지체의 체적에 20 vol% 이상의 변형이 이루어진 경우 형태가 유지되지 못한 것으로 평가하였다.In addition, the presence or absence of shape maintenance was evaluated by measuring the volume of the dermal tissue-derived extracellular matrix support using a three-dimensional camera. Specifically, it was evaluated that the shape could not be maintained when the volume of the extracellular matrix support was deformed by 20 vol% or more after irradiation with gamma rays.
본 발명에서 도 1은 감마선 선량에 따른 세포외기질 지지체의 가교 유무를 확인하기 위해 콜라겐 섬유를 전자주사현미경으로 촬영한 사진을 나타낸다.In the present invention, FIG. 1 shows a photograph taken with a scanning electron microscope of collagen fibers in order to confirm the presence or absence of crosslinking of the extracellular matrix support according to the gamma-ray dose.
상기 도 1에 나타난 바와 같이, 감마선 선량이 10 kGy이면, 지지체 내 콜라겐 섬유가 성기게 배열되며, 감마선 선량이 20 kGy 이상인 경우 상기 콜라겐 섬유가 평행하게 배열되고 치밀하게 배열됨을 확인할 수 있다. 즉, 20 kGy 이상의 감마선 선량에서 가교가 이루어진 것을 확인할 수 있다. As shown in FIG. 1, when the gamma-ray dose is 10 kGy, the collagen fibers in the support are sparsely arranged, and when the gamma-ray dose is 20 kGy or more, it can be confirmed that the collagen fibers are arranged in parallel and densely arranged. That is, it can be confirmed that the crosslinking was made at a gamma-ray dose of 20 kGy or more.
도 2는 감마선 선량에 따른 세포외기질 지지체의 형태유지 유무를 확인하기 위해 3차원 카메라로 촬영한 사진을 나타낸다.Figure 2 shows a picture taken with a three-dimensional camera in order to check whether the shape of the extracellular matrix support according to the gamma-ray dose is maintained.
상기 도 2에 나타난 바와 같이, 감마선 선량이 30 kGy 미만인 경우 뚜렷한 체적 변화는 관찰되지 않았으나, 감마선 선량이 30 kGy인 경우 세포외기질 지지체의 체적이 감소한 것을 확인할 수 있다. 구체적으로, 감마선 선량이 0 kGy일때 지지체의 체적에 대한 각 선량에서 측정한 지지체의 체적 변화율은 각각 5.5 vol%(10 kGy), 12.4 vol%(20 kGy) 및 27.7 vol%(30 kGy)였다.As shown in FIG. 2 , when the gamma-ray dose was less than 30 kGy, a clear volume change was not observed, but when the gamma-ray dose was 30 kGy, it was confirmed that the volume of the extracellular matrix was decreased. Specifically, when the gamma-ray dose was 0 kGy, the volume change rates of the scaffold measured at each dose with respect to the volume of the scaffold were 5.5 vol% (10 kGy), 12.4 vol% (20 kGy), and 27.7 vol% (30 kGy), respectively.
또한, 표 1은 감마선 조사 선량 변화에 따른 인체 진피 조직 유래 세포외기질 지지체의 가교, 멸균 및 형태유지 유무를 확인한 결과를 나타낸다.In addition, Table 1 shows the results of confirming the presence or absence of crosslinking, sterilization, and shape maintenance of the human dermal tissue-derived extracellular matrix support according to the change in gamma-irradiation dose.
Figure PCTKR2021014170-appb-img-000001
Figure PCTKR2021014170-appb-img-000001
상기 표 1에 나타난 바와 같이, 인체 진피 조직 유래 세포외기질 지지체의 가교, 멸균 및 형태유지를 위한 최적의 감마선 조사 선량은 20 내지 40 kGy, 구체적으로, 25 kGy임을 확인할 수 있다.As shown in Table 1, it can be confirmed that the optimal gamma irradiation dose for crosslinking, sterilization, and shape maintenance of the human dermal tissue-derived extracellular matrix support is 20 to 40 kGy, specifically, 25 kGy.
실험예 2. 인체 진피 조직 유래 세포외기질 지지체의 Experimental Example 2. Human dermal tissue-derived extracellular matrix scaffold in vivoin vivo 성능 검증 Performance Verification
지지체의 성능 검증을 위한 동물 실험은 비교예 1의 감마선 조사 대신 70% 에탄올로 멸균된 인체 진피 조직 유래 세포외기질 지지체를 기존 방법으로 하였고, 실시예 1에서 최적의 감마선 선량(25 kGy)으로 조사된 세포외기질 지지체를 실험군(본 발명)으로 사용하였다.In the animal experiment for verifying the performance of the scaffold, the human dermal tissue-derived extracellular matrix scaffold sterilized with 70% ethanol was used as an existing method instead of gamma irradiation of Comparative Example 1, and irradiated with the optimal gamma radiation dose (25 kGy) in Example 1 The extracellular matrix scaffold was used as an experimental group (the present invention).
ICR 마우스의 배부 피하에 가로 0.5 cm, 세로 0.5 cm, 높이 0.2 cm의 세포외기질 지지체를 이식한 후, 방사선을 0 Gy(control), 10 Gy, 20 Gy의 선량으로 조사한 후, 4 주와 8 주에 각 실험동물을 희생하여 결과를 분석하였다. 이때, 10 Gy는 7 분, 20 Gy는 15 분 동안 감마선을 조사하였다. After implanting an extracellular matrix scaffold with a width of 0.5 cm, a length of 0.5 cm, and a height of 0.2 cm under the dorsal skin of ICR mice, after irradiating at doses of 0 Gy (control), 10 Gy, and 20 Gy, 4 weeks and 8 weeks The results were analyzed by sacrificing each experimental animal during the week. At this time, 10 Gy was irradiated with gamma rays for 7 minutes and 20 Gy for 15 minutes.
(1) 체내 부피유지 능력 검증 (1) Verification of body volume maintenance ability
세포외기질 지지체 삽입 후 방사선을 0 Gy, 10 Gy, 20 Gy 조사한 후, 4 주와 8 주에 각각 지지체를 적출하여 사진을 촬영하였고, 3D 카메라를 통해 부피를 측정하였다. After insertion of the extracellular matrix scaffold, 0 Gy, 10 Gy, and 20 Gy of radiation was irradiated, and the scaffolds were removed at 4 and 8 weeks, respectively, and photographs were taken, and the volume was measured using a 3D camera.
도 3은 적출된 세포외기질 지지체의 부피를 확인한 사진(b) 및 그래프(b)를 나타낸다.3 shows a photograph (b) and a graph (b) confirming the volume of the excised extracellular matrix support.
상기 도 3에 나타난 바와 같이, 감마선을 10 Gy와 20 Gy 조사한 후 8 주차에서 부피를 측정한 결과, 실험군의 세포외기질 지지체가 기존 방법보다 체내 부피 유지력이 우수함을 확인할 수 있다. 또한, 3D 카메라를 통한 부피 정량 그래프 결과, 실험군이 기존 방법에 비해 유의미하게 부피가 유지됨을 확인할 수 있다. As shown in FIG. 3 , as a result of measuring the volume at week 8 after irradiating 10 Gy and 20 Gy of gamma rays, it can be confirmed that the extracellular matrix support of the experimental group has better body volume retention than the conventional method. In addition, as a result of the volume quantification graph through the 3D camera, it can be confirmed that the experimental group significantly maintains the volume compared to the existing method.
(2) 자가조직화 검증 (2) Self-organization verification
(1)에서 적출한 세포외기질 지지체들에 대해 조직 염색으로 자가조직화 여부를 확인하였다. 구체적으로, H&E(hematoxylin and eosin) 염색하여 조직분석을 진행했고, 세포 유입을 정량하였다. For the extracellular matrix scaffolds extracted in (1), autologous tissue was confirmed by tissue staining. Specifically, hematoxylin and eosin (H&E) staining was performed for tissue analysis, and cell influx was quantified.
도 4는 적출된 세포외기질 지지체를 H&E 염색한 사진(a)과 세포 유입을 정량한 그래프(b)를 나타낸다.4 shows a photograph (a) of H&E staining of the excised extracellular matrix support and a graph (b) of quantifying cell influx.
상기 도 4에 나타난 바와 같이, 실험군의 지지체와 기존 방법 모두 유사하게 자가조직화 되는 것을 확인할 수 있다.As shown in FIG. 4 , it can be confirmed that both the support of the experimental group and the existing method are similarly self-organized.
(3) 조직 섬유화 검증 (3) Tissue fibrosis verification
(1)에서 적출한 세포외기질 지지체들에 대해 면역조직화학 염색과 real-time PCR로 조직 섬유화 여부를 확인하였다.The presence of tissue fibrosis was confirmed by immunohistochemical staining and real-time PCR for the extracellular matrix scaffolds extracted in (1).
구체적으로, 적출된 세포외기질 지지체들 내 조직 섬유화(fibrosis)를 확인하기 위해, 조직 섬유화에 중요한 역할을 하는 근섬유아세포(myofibroblast) 표시인자인 α-SMA(smooth muscle actin) 면역조직화학 염색을 진행하였고, α-SMA 염색된 세포의 면적을 정량하였다. Specifically, to confirm tissue fibrosis in the excised extracellular matrix scaffolds, immunohistochemical staining of α-SMA (smooth muscle actin), a marker for myofibroblast that plays an important role in tissue fibrosis, was performed. and the area of α-SMA stained cells was quantified.
또한, 적출된 시료들 내 조직 섬유화에 관여하는 주요 매개물질인 결합조직성장인자(connective tissue growth factor, CTGF)의 유전자 발현을 확인하기 위해 real-time PCR로 CTGF의 mRNA 레벨을 정량하였다.In addition, the mRNA level of CTGF was quantified by real-time PCR to confirm the gene expression of connective tissue growth factor (CTGF), which is a major mediator involved in tissue fibrosis in the extracted samples.
도 5는 적출된 세포외기질 지지체를 α-SMA 면역조직화학 염색한 사진(a)과 α-SMA 염색된 세포 면적을 정량한 그래프(b)를 나타낸다.5 shows a photograph (a) of α-SMA immunohistochemical staining of the excised extracellular matrix support and a graph (b) of quantifying the cell area stained with α-SMA.
상기 도 5에 나타난 바와 같이, 실험군의 세포외기질 지지체는 기존 방법과 유의미한 차이를 보이지 않는 것을 확인할 수 있다. As shown in FIG. 5, it can be confirmed that the extracellular matrix support of the experimental group does not show a significant difference from the existing method.
또한, 도 6은 조직 섬유화에 관여하는 주요 매개물질인 결합조직성장인자(CTGF)의 유전자 발현을 확인하기 위해 real-time PCR에 의한 CTGF의 mRNA 레벨을 정량한 그래프를 나타낸다.In addition, FIG. 6 shows a graph of quantifying the mRNA level of CTGF by real-time PCR to confirm the gene expression of connective tissue growth factor (CTGF), which is a major mediator involved in tissue fibrosis.
상기 도 6에 나타난 바와 같이, 실험군의 세포외기질 지지체는 기존 방법과 유의미한 차이를 보이지 않는 것을 확인할 수 있다. As shown in FIG. 6 , it can be confirmed that the extracellular matrix support of the experimental group does not show a significant difference from the existing method.
상기 결과를 통해, 본 발명에 따른 방법에 의해 제조된 세포외기질 지지체(실험군)는 기존 방법에 의해 제조된 지지체와 유사한 조직 섬유화를 나타냄을 확인할 수 있다.Through the above results, it can be confirmed that the extracellular matrix scaffold (experimental group) prepared by the method according to the present invention exhibits tissue fibrosis similar to that of the scaffold prepared by the existing method.
(4) 구형구축 억제효과 검증 (4) Verification of spherical contracture inhibitory effect
(1)에서 적출한 세포외기질 지지체들에 대해 구형구축 억제효과를 검증하였다. The effect of inhibiting spheroid contracture was verified for the extracellular matrix scaffolds extracted in (1).
구체적으로, 적출된 세포외기질 지지체들 내 구형구축을 확인하기 위해 MT(Masson's Trichrome) 염색을 진행하였고, 지지체를 덮고 있는 진피의 두께를 정량하였다. Specifically, MT (Masson's Trichrome) staining was performed to confirm the spherical contracture in the excised extracellular matrix scaffolds, and the thickness of the dermis covering the scaffolds was quantified.
도 7은 적출된 세포외기질 지지체를 MT 염색한 사진(a)과 지지체를 덮고 있는 진피의 두께를 정량한 그래프(b)를 나타낸다. 7 shows a photograph (a) of MT staining of the excised extracellular matrix support and a graph (b) quantifying the thickness of the dermis covering the support.
도 7에 나타난 바와 같이, 방사선을 20Gy 조사한 후 4 주차와 8 주차에서 실험군의 세포외기질 지지체는 기존 방법보다 지지체를 덮고 있는 진피의 두께가 증가하지 않는 것을 확인할 수 있다. 이를 통해, 높은 선량으로 방사선을 조사하여도, 본 발명에 따른 제조 방법에 의해 제조된 세포외기질 지지체가 구형구축의 발생을 억제하는 것을 확인할 수 있다. As shown in FIG. 7 , it can be confirmed that the thickness of the dermis covering the scaffold does not increase in the extracellular matrix scaffold of the experimental group at the 4th and 8th weeks after irradiation with 20 Gy of radiation compared to the conventional method. Through this, even when irradiated with a high dose, it can be confirmed that the extracellular matrix support prepared by the manufacturing method according to the present invention suppresses the occurrence of spheroid contracture.
또한, 진피의 두께를 정량한 그래프 결과, 기존 방법과 실험군 간에 유의미하게 진피의 두께 차이가 나는 것을 확인할 수 있다. In addition, as a result of quantifying the thickness of the dermis, it can be confirmed that there is a significant difference in the thickness of the dermis between the existing method and the experimental group.
(5) 염증 억제효과 검증 (5) Verification of anti-inflammatory effect
(1)에서 세포외기질 지지체들에 대해 염증 억제 효과를 검증하였다. In (1), the anti-inflammatory effect was verified on the extracellular matrix scaffolds.
구체적으로, 세포외기질 지지체들 내 염증반응을 확인하기 위해 CD3 면역조직화학 염색을 진행했고, CD3 염색된 세포를 정량하였다.Specifically, CD3 immunohistochemical staining was performed to confirm the inflammatory response in the extracellular matrix scaffolds, and CD3 stained cells were quantified.
도 8은 적출된 세포외기질 지지체를 CD3 염색한 사진(a)과 CD3 염색된 세포를 정량한 그래프(b)를 나타낸다. 8 shows a photograph (a) in which the excised extracellular matrix was stained with CD3 and a graph (b) in which the cells stained with CD3 were quantified.
상기 도 8에 나타난 바와 같이, 실험군의 세포외기질 지지체는 기존 방법보다 염증반응을 덜 일으키는 것을 확인할 수 있다. As shown in FIG. 8, it can be confirmed that the extracellular matrix support in the experimental group causes less inflammatory response than the conventional method.
본 발명에 따른 제조 방법에 의해 제조된 유방재건용 이식재는 유방재건 용도로 사용될 수 있다. The implant for breast reconstruction manufactured by the manufacturing method according to the present invention may be used for breast reconstruction.
본 발명에 따른 유방재건용 이식재는 유방암 절제 수술 후 행해지는 지속적인 방사선 치료에도 구형구축이 억제되며 부피를 유지하고 형태변화를 최소화할 수 있다.The implantation material for breast reconstruction according to the present invention suppresses spherical contracture even with continuous radiation treatment after breast cancer resection surgery, and can maintain volume and minimize shape change.

Claims (9)

  1. a) 진피 조직에서 지질 성분을 제거하는 단계;a) removing the lipid component from the dermal tissue;
    b) 상기 지질 성분이 제거된 진피 조직에서 세포를 제거하는 단계; 및 b) removing cells from the dermal tissue from which the lipid component has been removed; and
    c) 상기 세포가 제거된 진피 조직을 감마선 조사하는 단계를 포함하며, c) comprising gamma-irradiating the dermal tissue from which the cells have been removed,
    상기 단계 c)에서 감마선 선량은 16 내지 50 kGy인 유방재건용 이식재의 제조 방법. In step c), the gamma-ray dose is 16 to 50 kGy of a method of manufacturing a transplant material for breast reconstruction.
  2. 제 1 항에 있어서, The method of claim 1,
    진피 조직은 동종 또는 이종의 진피 조직인 유방재건용 이식재의 제조 방법.Dermal tissue is a method of manufacturing a transplant material for breast reconstruction, which is an allogeneic or heterogeneous dermal tissue.
  3. 제 1 항에 있어서, The method of claim 1,
    단계 a)는 탈지질 용액을 사용하여 수행하며, Step a) is carried out using a delipidation solution,
    상기 탈지질 용액은 극성 용매, 비극성 용매, 또는 이들의 혼합 용매를 포함하는 것인 유방재건용 이식재의 제조 방법.The delipidation solution is a method of manufacturing a breast reconstruction implant comprising a polar solvent, a non-polar solvent, or a mixed solvent thereof.
  4. 제 1 항에 있어서, The method of claim 1,
    단계 b)는 탈세포 용액을 사용하여 수행하며, Step b) is performed using a decellularization solution,
    탈세포 용액은 계면활성제 및 염기성 용액으로 이루어진 그룹으로부터 선택된 하나 이상을 포함하는 것인 유방재건용 이식재의 제조 방법.The cell decellularization solution is a method for producing a breast reconstruction implant comprising at least one selected from the group consisting of a surfactant and a basic solution.
  5. 제 1 항에 있어서, The method of claim 1,
    단계 c)에서 감마선 선량은 21 내지 29 kGy인 유방재건용 이식재의 제조 방법.In step c), the gamma-ray dose is 21 to 29 kGy of a method of manufacturing a transplant material for breast reconstruction.
  6. 제 1 항에 있어서, The method of claim 1,
    단계 c)에서 감마선 조사 시간은 18 내지 60 시간인 유방재건용 이식재의 제조 방법. In step c), the gamma-irradiation time is 18 to 60 hours, a method of manufacturing a transplant material for breast reconstruction.
  7. 제 1 항에 있어서, The method of claim 1,
    단계 c)에서 감마선 조사 후 세포가 제거된 진피 조직의 체적 변화율은 30 vol% 이하인 것인 유방재건용 이식재의 제조 방법.In step c), the volume change rate of the dermal tissue from which cells are removed after irradiation with gamma rays is 30 vol% or less.
  8. 제 1 항에 따른 제조 방법에 의해 제조되는 유방재건용 이식재. A transplant material for breast reconstruction manufactured by the manufacturing method according to claim 1 .
  9. 제 8 항에 있어서, 9. The method of claim 8,
    20 Gy의 선량으로 감마선 조사 후 8 주 후의 유방재건용 이식재의 체적 변화율은 1 내지 15 vol%인 유방재건용 이식재.The volume change rate of the transplant material for breast reconstruction 8 weeks after irradiation with gamma rays at a dose of 20 Gy is 1 to 15 vol%.
PCT/KR2021/014170 2020-10-15 2021-10-14 Breast reconstruction support using dermal tissue-derived extracellular matrix and fabrication method therefor WO2022080876A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2020-0133382 2020-10-15
KR20200133382 2020-10-15

Publications (1)

Publication Number Publication Date
WO2022080876A1 true WO2022080876A1 (en) 2022-04-21

Family

ID=81208496

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/014170 WO2022080876A1 (en) 2020-10-15 2021-10-14 Breast reconstruction support using dermal tissue-derived extracellular matrix and fabrication method therefor

Country Status (2)

Country Link
KR (1) KR20220050065A (en)
WO (1) WO2022080876A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070244568A1 (en) * 2003-12-26 2007-10-18 Cardio Incorporated Decellularized Tissue and Method of Preparing the Same
US20120264190A1 (en) * 2009-12-21 2012-10-18 The Regents Of The University Of California Decellularized and Delipidized Extracellular Matrix and Methods of Use
KR20130041560A (en) * 2011-10-17 2013-04-25 이희영 Bio-fat material eliminated immunity
KR20140104252A (en) * 2013-02-20 2014-08-28 주식회사 엘앤씨바이오 Composition for in vivo transplantation comprising micro-cartilage and acellular dermal matrix and method for producing the same
US20160256606A1 (en) * 2012-09-26 2016-09-08 Lifecell Corporation Processed Adipose Tissue
US20180214609A1 (en) * 2015-07-30 2018-08-02 Ucl Business Plc Methods and devices for the production of decellularised tissue scaffolds

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070244568A1 (en) * 2003-12-26 2007-10-18 Cardio Incorporated Decellularized Tissue and Method of Preparing the Same
US20120264190A1 (en) * 2009-12-21 2012-10-18 The Regents Of The University Of California Decellularized and Delipidized Extracellular Matrix and Methods of Use
KR20130041560A (en) * 2011-10-17 2013-04-25 이희영 Bio-fat material eliminated immunity
US20160256606A1 (en) * 2012-09-26 2016-09-08 Lifecell Corporation Processed Adipose Tissue
KR20140104252A (en) * 2013-02-20 2014-08-28 주식회사 엘앤씨바이오 Composition for in vivo transplantation comprising micro-cartilage and acellular dermal matrix and method for producing the same
US20180214609A1 (en) * 2015-07-30 2018-08-02 Ucl Business Plc Methods and devices for the production of decellularised tissue scaffolds

Also Published As

Publication number Publication date
KR20220050065A (en) 2022-04-22

Similar Documents

Publication Publication Date Title
KR102116620B1 (en) Preparation and use of cell-free cartilage graft
KR100791502B1 (en) Production methods of virus inactivated and cell-free body implant
US11179505B2 (en) Methods for stabilizing collagen-containing tissue products against enzymatic degradation
WO2017155328A1 (en) Method for producing extracellular matrix membrane derived from biocompatible porcine cartilage capable of regulating in vivo decomposition rate and physical properties, and composition for preventing adhesion containing extracellular matrix derived from porcine cartilage as active ingredient
Tran et al. Preparation and characterization of acellular porcinepericardium for cardiovascular surgery
Bush et al. Process development and manufacturing of human and animal acellular dermal matrices
Kumar et al. Use of the bubaline acellular diaphragm matrix for umbilical hernioplasty in pigs
WO2021012677A1 (en) Bionic pre-vascular material and preparation method and use therefor
Łabuś et al. Tissue engineering in skin substitute
AU2015397501B2 (en) Method for manufacturing collagen film using ultraviolet light, collagen film manufactured by using same, and biomaterial prepared using collagen film
WO2022080876A1 (en) Breast reconstruction support using dermal tissue-derived extracellular matrix and fabrication method therefor
CN106390201A (en) Absorbable medical biological membrane and preparation method thereof
CN107412867B (en) Preparation method of heterogenous acellular dermal matrix
EP3188596B1 (en) Human dermis, preparation and use thereof
CN114796615B (en) Cartilage acellular matrix and preparation method thereof
US20110236949A1 (en) Methods for Processing Biological Tissues
US20210015973A1 (en) Human nipple areolar complex extracellular matrix scaffold and methods relating thereto
WO2022138992A1 (en) Acellular nerve graft material and method for producing same
WO2023003430A1 (en) Method for increasing thickness of acellular dermal layer graft
TWI791290B (en) Use of collagen particles in hair follicles formation or angiogenesis
WO2022086209A1 (en) Adhesive graft for engraftment enhancement
WO2023136538A1 (en) Acellular dermal matrix for implantation in body, using vegetable complex natural surfactant, and preparation method therefor
KR20230166079A (en) Use of collagen particles to promote hair follicle formation or angiogenesis
Rosadas et al. Decellularized dermal matrices: unleashing the potential in tissue engineering and regenerative medicine
Raghuvanshi et al. Tissue Scaffolds Derived from Goat Rumen and Clinical Applications

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21880523

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21880523

Country of ref document: EP

Kind code of ref document: A1