WO2003080143A1 - Materiau de base de reconstruction de tissu ou d'organe contenant une matrice tissulaire sans cellule et un facteur de croissance cellulaire - Google Patents

Materiau de base de reconstruction de tissu ou d'organe contenant une matrice tissulaire sans cellule et un facteur de croissance cellulaire Download PDF

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Publication number
WO2003080143A1
WO2003080143A1 PCT/JP2003/003581 JP0303581W WO03080143A1 WO 2003080143 A1 WO2003080143 A1 WO 2003080143A1 JP 0303581 W JP0303581 W JP 0303581W WO 03080143 A1 WO03080143 A1 WO 03080143A1
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WO
WIPO (PCT)
Prior art keywords
tissue
matrix
bladder
cell growth
growth factor
Prior art date
Application number
PCT/JP2003/003581
Other languages
English (en)
Japanese (ja)
Inventor
Yasuhiko Tabata
Osamu Ogawa
Original Assignee
Kaken Pharmaceutical Co., Ltd.
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 Kaken Pharmaceutical Co., Ltd. filed Critical Kaken Pharmaceutical Co., Ltd.
Priority to AU2003221081A priority Critical patent/AU2003221081A1/en
Priority to JP2003577967A priority patent/JP4431399B2/ja
Publication of WO2003080143A1 publication Critical patent/WO2003080143A1/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
    • A61L27/3683—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

Definitions

  • the present invention relates to a substrate for reconstructing a biological tissue or organ, that is, a substrate for regeneration that induces invasion of cells from surrounding tissues and regenerates and repairs the biological tissue or organ.
  • a substrate for reconstructing a tissue or organ comprising a decellularized tissue matrix and a cell growth factor.
  • Biological tissue engineering is an attempt to regenerate and repair damaged or defective parts of living tissues or organs.
  • scaffold materials for proliferation and differentiation of various cells have been researched and developed.
  • synthetic scaffolds have been created, but there are many issues that need to be improved. Therefore, the production of a scaffold material from a biological material has been considered and attempted.
  • attempts to use various biological tissues or organs perform decellularization to suppress immune rejection of tissues or organs, collect only extracellular matrix, and use it as a scaffold There is.
  • These biological scaffolds have better mechanical properties and body fluid storage properties than synthetic scaffolds.
  • the scaffold shrinks.
  • One way to solve this is to insert cells.
  • isolation and seeding of cells are complicated, and another method is desired.
  • a cell growth factor as a method to suppress the contraction of the scaffold by promoting the invasion of the cell into the scaffold material instead of the cell in the body.
  • This is the point of the present invention.
  • various cells and cell growth factors can be combined and put into a decellularized tissue matrix and used.
  • bladder dysfunction due to a congenital disease or bladder enlargement surgery is necessary when the bladder is removed due to cancer.
  • Patch repair is usually performed on the gastrointestinal tract, but there are many problems such as reabsorption of urine from the intestinal tract, rupture and carcinogenesis, and the regeneration of the bladder itself is ideal.
  • the present invention has solved these problems, that is, it can be used not only for bladder but also for reconstruction of various other living tissues or organs, is excellent in biocompatibility, does not cause graft shrinkage, and is easy to operate. It is an object of the present invention to provide a simple and economically inexpensive base material for reconstructing a tissue or organ that can bring out tissue repair ability.
  • the present invention provides a living tissue or a tissue having a function of inducing regeneration of a living tissue or organ and regenerating and repairing the living tissue or organ, comprising a decellularized tissue matrix and a cell growth factor. It is a substrate for organ reconstruction.
  • the decellularized tissue matrix in the present invention refers to a matrix in which most of the cellular components of a living tissue or organ have been removed and a porous extracellular matrix remains, and decellularized from any living tissue or organ. Includes all products that have been processed.
  • the tissue or organ from which the decellularized tissue matrix can be obtained is not particularly limited as long as it is a living tissue or organ.
  • Examples include vas deferens, fallopian tubes, ureters and lymph vessels.
  • Preferred are the bladder, the small intestine and the esophagus, and particularly preferred is the bladder.
  • Cell growth factors in the present invention include cell growth factors (cell growth factors) such as bFGF (basic fibroblast growth factor), aFGF PDGF TGF- ⁇ 1 VEGF HGF HB_EGF CTG FI GF-I and IGF-II. It includes what is called or interleukins, cytokins, bioactive peptides and chemokines, but is preferably bFGF HGF CTGF and PDGF, and particularly preferably bFGF. These growth factors can be used alone or in combination of two or more.
  • bFGF that can be used in the present invention is an organ such as the pituitary gland, brain, retina, corpus luteum, adrenal gland, etc.
  • Extracted products those produced by genetic engineering techniques such as recombinant DNA technology, and modified products thereof that can act as fibroblast growth factors.
  • modified form of bFGF include those obtained by adding, substituting or deleting an amino acid in the amino acid sequence of bFGF obtained by the above-described extraction or genetic engineering technique.
  • the bFGF that can be used in the present invention preferably includes, for example, those described in WO 87/01728 and WO 89704832, particularly those described in the former.
  • the amount of cell growth factor in the tissue or organ reconstruction substrate of the present invention may be determined by the following: decellularized tissue matrix, type of cell growth factor, type of tissue or organ to be reconstructed, lesion site, extent of lesion, patient condition, etc.
  • decellularized tissue matrix 1,000 to 100,000 g, preferably 5,000 to 50,000 g, more preferably 2,000 to 30,000 / g g.
  • the tissue or organ to be reconstructed in the present invention is not particularly limited as long as it is a living tissue or organ, and for example, esophagus, bladder, urethra, small intestine, liver, lung, skeletal muscle, smooth muscle, cardiac muscle, kidney, bone, Cartilage, skin, hair, brain, nerve, muscle, blood vessels, kidney, retina, cornea, diaphragm, pericardium, serosa, amniotic membrane, tendon, ligament, large intestine, duodenum, trachea, intubation, fallopian tubes, ureters and Examples include lymphatic vessels and the like.
  • Preferred are the bladder, small intestine, and urethra, and particularly preferred is the bladder.
  • the tissue or organ for obtaining the decellularized tissue matrix and the tissue or organ to be reconstructed may be the same or different, but both tissues or organs are the same. In some cases, it is preferable.
  • FIG. 1 is a diagram (A) of the decellularized tissue matrix from rat bladder and the HE staining result (B).
  • Figure 2 shows the preparation of decellularized tissue matrix from rat bladder and the inclusion of cell growth factor in rat bladder before decellularization (1) and decellularized tissue matrix from rat bladder ( 2) Decellularized tissue mat after lyophilization Lix (3) and a decellularized tissue matrix (4) after impregnation with an aqueous cell growth factor solution.
  • FIG. 3 is a graph showing the release of cell growth factor from a decellularized tissue matrix containing cell growth factor in an aqueous solution (PBS).
  • FIG. 4 is a graph showing degradation of a decellularized tissue matrix and attenuation of a cell growth factor contained in the decellularized tissue matrix in a subcutaneous mouse.
  • Figure 5 is a graph (top) and a macroscopic image showing the granulation-forming ability of the decellularized tissue matrix containing cell growth factors embedded in the living body for a certain period of time, 7 days after subcutaneous implantation in mice. (Bottom).
  • Figure 6 shows HE staining results of bladder tissue on grafts of rats in groups A, B, C and D 4 weeks after surgery ((A), (B), (C) and ( D)).
  • FIG. 7 shows the bladder ((A), (B), (C) and (D)) of the rats in groups A, B, C and D 4 weeks after the operation.
  • the region surrounded by the arrow is the graft site.
  • FIG. 8 is a graph showing the graft area of rats in groups A, B, C, and D four weeks after surgery.
  • Figure 9 shows HE staining results of bladder tissue on grafts of rats in groups A, B, C and D 12 weeks after surgery ((A), (B), (C) and (D )).
  • the substrate for tissue or organ reconstruction of the present invention can be produced by including a cell growth factor in a decellularized tissue matrix.
  • the decellularized tissue matrix can be obtained by subjecting the cells or tissues to the above-described tissues or organs by a conventional method, for example, treatment with a surfactant such as Triton X-100 or other known treatments, for example, repeated freeze-thawing, osmotic pressure change, etc. Can be prepared according to the destruction method.
  • the decellularized tissue matrix can be lyophilized and stored for later use.
  • the method for incorporating the cell growth factor into the decellularized tissue matrix may be any method that allows the cell growth factor to be uniformly distributed in the decellularized tissue matrix.
  • the method is not particularly limited, and examples thereof include a method of impregnating a cell growth factor solution into a decellularized tissue matrix, a method of freeze-drying a decellularized tissue matrix, and impregnating or adding a cell growth factor solution thereto.
  • the tissue or organ reconstruction base material of the present invention may be used, for example, when there is a tissue dysfunction due to a congenital disease, when the tissue or organ is excised due to cancer, or when the wound healing power is poor due to complications such as diabetes. Or, when wound healing power is poor due to infection of surrounding tissues, etc., it can be used effectively for reconstruction of the tissues or organs.
  • the reconstruction of a tissue or an organ using the reconstruction substrate of the present invention can be performed, for example, by using the reconstruction substrate as a graph and patch-repairing the tissue or organ to be reconstructed.
  • Example 1 Preparation of decellularized tissue matrix from rat bladder and its morphological evaluation
  • the bladder of a rat weighing 250 to 300 g was excised and the surrounding tissues were excised and decellularized by the following method.
  • HE staining Specimens were prepared and the morphology was recorded with a light microscope. Another specimen was fixed with 2.5% aqueous dartaldehyde solution, dehydrated, replaced with t-butyl alcohol, freeze-dried, coated with platinum vapor deposition, and observed and recorded with a scanning electron microscope.
  • Example 2 Cell growth factor binding method by impregnation of lyophilized decellularized tissue matrix in aqueous solution and evaluation of binding state in aqueous solution
  • the matrix prepared by the above method was frozen in distilled water at 180 ° C. for 12 hours, and then lyophilized for 48 hours using a vacuum pump. This was impregnated with an aqueous bFGF solution and allowed to stand at 37 ° C for 1 hour to prepare a decellularized tissue matrix containing cell growth factors. Separately, the matrix before lyophilization was impregnated with an aqueous bFGF solution.
  • FGF basic fibroblast growth factors
  • Example 3 Evaluation of correlation between in vivo attenuation of cell growth factor and matrix degradation (method)
  • bFGF in vivo release experiments A matrix containing radiolabeled cell growth factor, prepared in a similar manner as described above, was implanted subcutaneously in the back of 6-week-old female d dY mice. Thereafter, this was sacrificed over time and the remaining radioactivity of the matrix and the surrounding tissue was measured to examine the remaining bFGF.
  • a 5 mg decellularized tissue matrix from rat bladder is impregnated with an aqueous solution containing 5 g of bFGF or PBS lOl in the same manner as above, and one group is directly implanted under the mouse subcutaneously in the same manner as above.
  • the remaining two groups were sealed in a diffusion chamber and implanted subcutaneously in a mouse in the same manner as described above. After this was removed 7 or 14 days later, the matrix alone was reimplanted subcutaneously in another mouse. Seven days after matrix implantation (or re-implantation) in each group, the mice were sacrificed and subcutaneous tissues 2 cm square around the matrix were collected to evaluate the tissue weight and the like.
  • Example 5 Bladder Patch Repair Surgery Using Decellularized Tissue Matrix from Rat Bladder Containing FGF, Use b Examination of relationship between FGF concentration and bladder reconstructed image on matrix graft, patch size.
  • the rat bladder was decellularized, and the portion corresponding to the bladder apex 2Z3 was removed therefrom to prepare a decellularized tissue matrix containing various concentrations of bFGF.
  • Another 10-week-old female Wistar rat was anesthetized with xylazine-ketamine, a midline incision was made in the lower abdomen to expose the bladder, the apical 2/3 of the bladder was excised, and the decellularized tissue matrix was used. This defect was patch repaired as a graft.
  • the suture between the bladder and the graft was continuously sutured using 8-0 absorbent thread, and the four corners were marked with 7-0 nylon thread. After the restoration was completed, a tube was inserted through the urethra and physiological saline was injected to confirm that there was no leak. At the same time, the graft size at the time of filling was recorded as the major and minor diameters of the marking thread.
  • the rats are sacrificed and the bladder is emptied.After injecting 10% formalin or Krebs solution equivalent to the bladder capacity, the urethral opening is clamped, and the distance between the marking threads is reduced. It was measured. The graft area was defined as major axis X minor axis Z2. Tissues were fixed in formalin, photographed, and dehydrated, paraffin-embedded, and HE-stained in all groups at 4 weeks and 3 animals at 12 weeks in each group.
  • the substrate for tissue or organ reconstruction of the present invention can be used in tissue or organ reconstruction such as patch repair for bladder reconstruction, is excellent in biocompatibility, does not bring about graft repair, is a simple and economical method. Can bring out the tissue repair ability.
  • the substrate for tissue or organ reconstruction according to the present invention suppresses contraction of a graft, which cannot be obtained with a conventional biologically-derived scaffold, by gradually releasing cell growth factors and promoting cell invasion from the tissue to the substrate. Has an effect.
  • the method of the present invention is more convenient than the conventional method of inserting cells into a biologically-derived scaffold.

Abstract

La présente invention concerne un matériau de base de reconstruction de tissu ou d'organe qui est hautement biocompatible et qui présente une contraction de greffe régulée. Ce matériau de base de reconstruction de tissu ou d'organe contient une matrice tissulaire sans cellule et un facteur de croissance cellulaire.
PCT/JP2003/003581 2002-03-25 2003-03-25 Materiau de base de reconstruction de tissu ou d'organe contenant une matrice tissulaire sans cellule et un facteur de croissance cellulaire WO2003080143A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003221081A AU2003221081A1 (en) 2002-03-25 2003-03-25 Base material for reconstructing tissue or organ containing cell-free tissue matrix and cell growth factor
JP2003577967A JP4431399B2 (ja) 2002-03-25 2003-03-25 脱細胞化組織マトリックス及び細胞成長因子を含む組織又は臓器再建用基材

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002/84138 2002-03-25
JP2002084138 2002-03-25

Publications (1)

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WO2003080143A1 true WO2003080143A1 (fr) 2003-10-02

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AU (1) AU2003221081A1 (fr)
WO (1) WO2003080143A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007521114A (ja) * 2003-12-10 2007-08-02 ハンク・シー・ケイ・ウー 軟組織特徴の再構築のための方法および組成物
JP2011528586A (ja) * 2008-07-22 2011-11-24 グランドホープ バイオテック カンパニー リミテッド 鼻筋用生体インプラント及びその製造方法
JP2018198867A (ja) * 2017-05-29 2018-12-20 学校法人成蹊学園 生体適合性高分子材料のマーキング方法及びマーキングされた生体適合性高分子材料

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996024661A1 (fr) * 1995-02-10 1996-08-15 Purdue Research Foundation Sous-muqueuse utilisee comme substrat de croissance pour des cellules
WO1999065470A1 (fr) * 1998-06-19 1999-12-23 Lifecell Corporation Matrice tissulaires acellulaires particulaires

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996024661A1 (fr) * 1995-02-10 1996-08-15 Purdue Research Foundation Sous-muqueuse utilisee comme substrat de croissance pour des cellules
WO1999065470A1 (fr) * 1998-06-19 1999-12-23 Lifecell Corporation Matrice tissulaires acellulaires particulaires

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007521114A (ja) * 2003-12-10 2007-08-02 ハンク・シー・ケイ・ウー 軟組織特徴の再構築のための方法および組成物
JP2011528586A (ja) * 2008-07-22 2011-11-24 グランドホープ バイオテック カンパニー リミテッド 鼻筋用生体インプラント及びその製造方法
JP2018198867A (ja) * 2017-05-29 2018-12-20 学校法人成蹊学園 生体適合性高分子材料のマーキング方法及びマーキングされた生体適合性高分子材料
JP7084566B2 (ja) 2017-05-29 2022-06-15 創生ライフサイエンス株式会社 生体適合性高分子材料のマーキング方法

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JP4431399B2 (ja) 2010-03-10
AU2003221081A1 (en) 2003-10-08
JPWO2003080143A1 (ja) 2005-07-21

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