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WO2008131639A1 - Substrat de cornée produit par suppression de cellules et procédé de préparation de celui-ci - Google Patents

Substrat de cornée produit par suppression de cellules et procédé de préparation de celui-ci Download PDF

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Publication number
WO2008131639A1
WO2008131639A1 PCT/CN2008/000661 CN2008000661W WO2008131639A1 WO 2008131639 A1 WO2008131639 A1 WO 2008131639A1 CN 2008000661 W CN2008000661 W CN 2008000661W WO 2008131639 A1 WO2008131639 A1 WO 2008131639A1
Authority
WO
WIPO (PCT)
Prior art keywords
corneal stroma
acellular
cornea
cell
corneal
Prior art date
Application number
PCT/CN2008/000661
Other languages
English (en)
Chinese (zh)
Inventor
Zhichong Wang
Original Assignee
Zhongshan Ophthalmic Center, Sun Yat-Sen University
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 Zhongshan Ophthalmic Center, Sun Yat-Sen University filed Critical Zhongshan Ophthalmic Center, Sun Yat-Sen University
Publication of WO2008131639A1 publication Critical patent/WO2008131639A1/fr

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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
    • A61L27/38—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 containing added animal cells
    • 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
    • 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
    • A61L27/3641—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 site of application in the body
    • 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
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the invention belongs to the field of medical instruments, and in particular relates to a decellularized corneal stroma and a preparation method thereof. Background technique
  • Corneal transplantation is one of the most important methods for treating corneal diseases, defects, turbidity and diopter abnormalities caused by various causes such as infection, trauma and congenital abnormalities. Many patients cannot get timely and effective treatment due to lack of donor sources. Artificial corneas made of synthetic materials have poor biocompatibility, and are difficult to fuse with the periocular tissues after transplantation, which is prone to complications such as loss, water leakage, and anterior and posterior membranes.
  • Pterygium is a common ocular surface disease with the characteristics of easy recurrence after surgery. Simple pterygium excision combined with limbal transplantation can reduce the recurrence rate of the disease. However, the current donor material is insufficient.
  • the ocular surface includes the cornea, conjunctival epithelium and tear film.
  • the treatment of ocular surface diseases caused by different causes includes the effective maintenance and reconstruction of these three aspects of health.
  • the corneal refractive power accounts for 2/3 of the total refractive power of the eye. Surgical changes in the refractive state of the eye can be used to correct different types of refractive errors.
  • Tissue engineered scaffold materials serve to provide nutrients to cells, provide a site for cell proliferation, and determine the size of the tissue being repaired.
  • Natural polymer materials such as collagen, hyaluronic acid, chitin and its derivatives, and polysaccharides have poor mechanical properties, too hydrophobic, and too fast degradation, which is not conducive to the diffusion of cellular nutrients into the material.
  • Synthetic polymer materials (such as polylactic acid, polyglycolic acid) are also used as carriers for tissue engineering corneas, which have poor transparency and stability, and high aseptic inflammatory response rates, which are not suitable for clinical trials.
  • amniotic membrane provides a continuous basement membrane containing collagen, laminin, fibronectin and various integrin proteins, these components are beneficial for the differentiation and migration of epithelial cells, but the material itself is too thin and is not suitable for the repair of deep lesions of the corneal stroma.
  • the technical problem to be solved by the present invention is to provide a decellularized corneal stroma for repairing ocular surface tissue defects, reconstructing and shaping ocular surface tissues, treating ocular surface diseases, and using as a tissue engineered biological cornea.
  • the carrier is intended to provide a sufficient source of material for clinical ocular surface surgery.
  • An acellular corneal stroma consisting of hundreds of mutually parallel layers of collagen fibers, with voids formed between the layers of collagen fibers.
  • the decellularized corneal stroma the basic components of which include collagen fibers, corneal stroma (including mucin and glycoprotein, etc.) and cell membrane and cytosolic components remaining after decellularization.
  • the thickness of the acellular corneal matrix is preferably 0. 01 ⁇ 5. 0mm.
  • the decellularized corneal stroma is preferably lmn in diameter! ⁇ 40mm round.
  • the acellular corneal stroma can also carry drugs and/or cytokines.
  • the method for preparing the above-mentioned acellular corneal stroma is prepared by using a heterologous (animal) or allogeneic corneal stroma as a raw material, and subjected to decellularization to obtain the decellularized corneal stroma.
  • Cell depletion can be performed by cell separation (such as filtration, centrifugation, sedimentation, etc.), or by cell disruption (such as high-pressure homogenization of mechanical crushing, bead milling, ultrasonication, etc., chemical and biochemical permeation of acid and alkali, Chemical reagent treatment, enzymatic dissolution, physical penetration, osmotic pressure shock, freeze-thaw method, etc.).
  • cell separation such as filtration, centrifugation, sedimentation, etc.
  • cell disruption such as high-pressure homogenization of mechanical crushing, bead milling, ultrasonication, etc., chemical and biochemical permeation of acid and alkali, Chemical reagent treatment, enzymatic dissolution, physical penetration, osmotic pressure shock, freeze-thaw method, etc.
  • the decellularization treatment is a chemical permeation method, and the specific steps are as follows: treating the heterogeneous corneal stroma at a concentration of 0.05 to 5% of an anionic surfactant SDS solution at 4 to 50 ° C for 4 to 24 hours.
  • This method does not constitute the destruction of the three-dimensional structure of the cornea and its matrix components while completely removing the corneal tissue cells, so that the most important characteristics of the cornea, transparency and toughness, are fully retained.
  • the decellularized treatment can also be another chemical permeation method, the steps are as follows: treating the heterogeneous corneal stroma at a concentration of 0. 01 ⁇ 5% of the detergent TritonX-100 solution at 4 to 50 ° C for 24 to 48 hours After digestion with trypsin for 2-12 hours.
  • the decellularized corneal stroma can also be treated by removing xenogeneic antigens, and the method of removing xenoantigens can be performed by primary bioseparation techniques (such as membrane separation, extraction, precipitation fractionation, and foam separation), or other biological separation techniques (such as adsorption, chromatography, electrophoresis and electrochromatography, etc.).
  • primary bioseparation techniques such as membrane separation, extraction, precipitation fractionation, and foam separation
  • other biological separation techniques such as adsorption, chromatography, electrophoresis and electrochromatography, etc.
  • the acellular corneal stroma of the present invention may be prepared by using a heterologous (animal) or allogeneic partial cornea, or all corneas, and even a part of the sclera and conjunctiva connected thereto. It can be a fresh tissue or a preserved tissue. Under certain conditions (such as: enzymatic treatment, membrane damper treatment), completely remove the cellular components in the material, maximize the removal of pathogenic microorganisms in the tissue, reduce or eliminate the antigenicity of the tissue, but retain its normal The three-dimensional structure of the matrix fibers.
  • a tissue engineered biokeratome can be constructed by loading the corresponding cells on the surface of the acellular corneal stroma prepared in accordance with the present invention. Since the decellularized corneal stroma has certain hardness and toughness, it has processability and can form a three-dimensional structure; it can be processed into various shapes such as a circular shape, a crescent shape, and the like. It can have a certain curvature or a flat shape. It can also be made into a thickness consistent with the thickness of the ocular surface defect of the recipient to be repaired, and processed to an equal thickness. The acellular corneal stroma does not have diopter, and if processed to unequal thickness, it can have a corresponding diopter.
  • the acellular corneal stroma of the present invention can be long-term or gradually degraded after transplantation, and is highly transparent, the nerve with the receptor can grow, the recipient cells can grow on the surface for a long time, the immunogenicity is low, and the tissue Good compatibility; it can also carry related drugs and/or cytokines when making or processing rehydration, inhibiting the growth of new blood vessels and/or treating primary diseases; It can grow into the matrix, and the epithelial cells of the receptor can adhere to the surface of the substrate; the decellularized corneal stroma also has similar thermal stability to the ocular surface tissue of the recipient.
  • the preparation method of the acellular corneal stroma of the invention is simple and easy to operate, and the prepared acellular corneal stroma has the following advantages:
  • the decellularized keratoconjunctival matrix with conjunctiva can be used as a fibrous collagen scaffold to provide an epithelial, healthy matrix and basement membrane that can be used to reconstruct the ocular surface.
  • the acellular corneal stroma of the present invention is tested by in vivo and in vitro toxicity tests, stimulation sensitization test and immunology, and conforms to biological evaluation criteria, and proves that the material is safe and reliable to the receptor, and does not constitute a local receptor and The biological hazard of the whole body.
  • Example 1 A decellularized corneal stroma and a preparation method thereof according to the present invention are further illustrated by the following examples.
  • Example 1 A decellularized corneal stroma and a preparation method thereof according to the present invention are further illustrated by the following examples.
  • Example 1 A decellularized corneal stroma and a preparation method thereof according to the present invention are further illustrated by the following examples.
  • Example 1 A decellularized corneal stroma and a preparation method thereof according to the present invention are further illustrated by the following examples.
  • An acellular corneal stroma consisting of hundreds (100-250 or so) layers of collagen fibers parallel to each other with a gap formed between the layers of collagen fibers.
  • the collagen fiber slab layers are parallel to each other, have the same size, and are equally spaced.
  • the space between the collagen fiber slab layers also contains corneal stroma (including mucin and glycoprotein) and cell membrane and cytosolic components remaining after decellularization.
  • the thickness of the acellular corneal stroma is 0. 1-0. 5mra or so (the specific shape and specifications depend on the clinical case).
  • a method for preparing acellular corneal stroma, taking a corneal stroma of a heterologous animal as a raw material, and thinning by chemical infiltration Cell processing is available, the steps are as follows:
  • Preparation of decellularized keratin by chemical permeation method The heterogeneous corneal stroma was treated with an anionic surfactant SDS solution at a concentration of 0.1% for 7 hours, and the paraffin sections were taken for histopathological observation. The ingredients can be removed.
  • the acellular cellulite prepared above was used in the following test:
  • the acellular cell membrane is used for lamellar keratoplasty
  • the corresponding size of the acellular corneal stromal graft is fixed to the implanted bed with suture or bioglue, including the eye, to be surfaced. After epithelialization, it is treated by lamellar keratoplasty. The transparency and integrity of the cornea can be restored to varying degrees after surgery.
  • the decellularized corneal stroma can carry different drugs or cytokines through different carriers (such as liposomes, emulsions, nanospheres, etc.), such as VEGF against neovascularization, ICAM-1 promoting epithelial adhesion, and antibiotics. Etc., to improve complications such as neovascularization, slow epithelial growth and secondary infection after lamellar angular displacement.
  • the acellular cell membrane is used for pterygium surgery
  • the wound is cleaned, and the acellular corneal stroma (peripheral margin) with the sclera of the limbal zone is fixed with the suture or bio-adhesive.
  • the conjunctiva is pulled open, covering the scleral portion of the acellular corneal stroma, and the pressure bandaging eye is applied.
  • the acellular cell keratin is used to repair scleral defects
  • An acellular corneal stroma comprising hundreds of mutually parallel layers of collagen fibers with voids formed between the layers of collagen fibers.
  • the layers of collagen fibers are parallel, uniform in size, and equally spaced.
  • the basic components include collagen fibers, corneal stroma (including mucin and glycoprotein), and membrane and cytosolic components remaining after decellularization. 5 ⁇
  • the acellular corneal matrix having a diameter of about 1 to 10mm, a thickness of about 0.3 mm. (The specific diameter and thickness can be shaped according to the refractive state and pathological condition of the affected eye)
  • the preparation method of the acellular corneal stroma adopts the allogeneic corneal stroma as the raw material, and the cornea includes the incidental angle
  • the sclera and conjunctiva on the membrane can be obtained by chemical permeation decellularization.
  • the steps are as follows: Chemical infiltration to prepare decellularized corneal: 1% detergent TritonX-100 solution at 37 ° C
  • the heterogeneous corneal stroma was treated for 24 hours, and paraffin sections were made for histopathological observation. The results showed that the cellular components in the stroma could be removed.
  • the acellular cellulite prepared above was used in the following test:
  • the acellular cell membrane is used for keratomileusis
  • the inlay groove is formed on the surface of the cornea, and the thin peripheral edge of the decellularized corneal matrix sheet with suitable refractive power is inserted into the groove, fixed by suture or bio glue, and the eye is pressurized and bandaged.
  • corneal lens surgery routine treatment after surgery. Postoperative patient diopter can be improved to varying degrees.
  • the acellular keratitis is used for interlamellar implantation of the corneal stroma
  • the decellularized corneal stroma sheet having a suitable diopter is selected according to the diopter of the patient.
  • a lamellar corneal or corneal stroma interstitial capsular bag is implanted, and the selected acellular corneal stroma sheet is implanted into the corneal layer, and after fixation, the dressing is applied under pressure. According to the corneal lens surgery, routine treatment.
  • the decellularized keratocytes are used to construct a tissue engineered biokeratome:
  • epithelial cells are implanted on the anterior surface of the acellular corneal stroma, and endothelial cells are implanted on the posterior surface to form a three-dimensional structure of the cornea.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un substrat de cornée produit par une suppression de cellules, qui est constitué essentiellement de centaines de fibres de collagène parallèles entre lesquelles des espaces intermédiaires sont formés par suppression de cellules. Ce substrat sert à réparer un traumatisme des tissus superficiels de l'oeil, à reconstruire et à façonner le tissu superficiel de l'œil, à traiter une maladie du tissu oculaire superficiel et à construire le support de la cornée par ingénierie tissulaire biologique. Ledit substrat peut être maintenu pendant une longue durée ou se décomposer graduellement, présente une grande transparence, permet le développement nerveux de récepteurs et la prolifération de cellules de récepteurs sur sa surface, est peu immunogène et possède une bonne compatibilité tissulaire après une greffe.
PCT/CN2008/000661 2007-04-25 2008-04-02 Substrat de cornée produit par suppression de cellules et procédé de préparation de celui-ci WO2008131639A1 (fr)

Applications Claiming Priority (2)

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CN200710027698.6 2007-04-25
CN200710027698A CN101066471B (zh) 2007-04-25 2007-04-25 一种脱细胞角膜基质及其制备方法

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CN101066471B (zh) * 2007-04-25 2010-05-19 中山大学中山眼科中心 一种脱细胞角膜基质及其制备方法
CN101433478B (zh) * 2007-11-14 2012-05-23 上海交通大学医学院附属第九人民医院 一种全层生物角膜及其构建方法和用途
CN101274106B (zh) * 2008-03-24 2011-11-09 中山大学中山眼科中心 一种制备脱细胞基质的方法
KR101029887B1 (ko) * 2009-03-04 2011-04-18 서울대학교산학협력단 탈세포화를 위한 돼지 각막의 가공방법
US9901601B2 (en) * 2011-09-02 2018-02-27 Adeka Corporation Method for preparing decellularized tissue product, and graft provided with decellularized tissue product
CN102599991A (zh) * 2012-03-20 2012-07-25 中山大学中山眼科中心 美容性角膜镜片的应用
CN102793593B (zh) * 2012-09-03 2015-06-10 于好勇 用于后巩膜加固术的异种巩膜片及其制备方法
CN103127549A (zh) * 2013-02-01 2013-06-05 中山大学中山眼科中心 一种脱细胞睑板及其制备方法
CN103892938A (zh) * 2014-04-01 2014-07-02 薛春燕 一种角膜板层片及其制备方法
US20160184084A1 (en) * 2014-12-29 2016-06-30 Ofer Daphna Keratoprosthesis
CN105288736B (zh) * 2015-11-24 2016-11-16 北京清源伟业生物组织工程科技有限公司 脱细胞真皮基质角膜及眼表组织修复材料的制备方法
CN107233143A (zh) * 2017-05-31 2017-10-10 广州新诚生物科技有限公司 去细胞角膜基质透镜及其制备方法
CN108852615A (zh) * 2018-05-09 2018-11-23 山东省眼科医院 一种用于角膜基质植入的植片及角膜基质囊袋

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