WO2018120672A1 - 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 - Google Patents

一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 Download PDF

Info

Publication number
WO2018120672A1
WO2018120672A1 PCT/CN2017/088415 CN2017088415W WO2018120672A1 WO 2018120672 A1 WO2018120672 A1 WO 2018120672A1 CN 2017088415 W CN2017088415 W CN 2017088415W WO 2018120672 A1 WO2018120672 A1 WO 2018120672A1
Authority
WO
WIPO (PCT)
Prior art keywords
tendon
bioactive
scaffold
slice
tissue
Prior art date
Application number
PCT/CN2017/088415
Other languages
English (en)
French (fr)
Inventor
秦廷武
宁良菊
张亚靖
崔静
姚璇
罗静聪
Original Assignee
四川大学华西医院
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 四川大学华西医院 filed Critical 四川大学华西医院
Priority to US16/475,016 priority Critical patent/US20200121828A1/en
Publication of WO2018120672A1 publication Critical patent/WO2018120672A1/zh

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
    • 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
    • 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
    • A61L27/3645—Connective tissue
    • A61L27/3662—Ligaments, tendons
    • 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
    • A61L27/3687—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 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • A61L27/3691—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 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/38—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
    • A61L27/3839—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 characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/386—Ligaments, tendons
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61L27/58—Materials at least partially resorbable by 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention relates to a preparation method of a biologically active stent with tendon tissue regeneration and a use thereof, and belongs to the field of biological materials.
  • tendon injury caused by diseases, trauma, degeneration, etc. has become a health problem that cannot be ignored.
  • tendon injury there are various methods for repairing tendon injury, including autologous tendon transplantation, allogeneic or heterogeneous tendon transplantation, and artificial material replacement.
  • Autologous tendon transplantation can cause pain, infection and injury in the donor area, and the source of tendon can be limited.
  • Allogeneic tendon transplantation may cause disease transmission, different degrees of rejection, and limited sources; heterogeneous tendon transplantation It may also cause disease transmission, but the most important problem is the existence of immune rejection; artificial tendon due to poor mechanical compatibility or degradation rate is difficult to synchronize with tissue regeneration, so that the long-term effect of tendon defect repair remains to be seen, and the price is expensive .
  • the ideal tendon repair material should have the following conditions: (1) good biocompatibility; (2) good mechanical compatibility; (3) potential to induce tendon tissue regeneration; (4) shape and size controllable, processing The preparation method is operable.
  • a large number of literatures have shown that the currently used tendon injury repair materials have their own defects and deficiencies, so the search for more ideal tendon repair materials has never stopped.
  • tissue engineering technology researchers have been encouraged to try to solve the problem of difficulty in repairing tendon injuries by tissue engineering tendons. With the joint efforts of scholars at home and abroad, the research on tissue engineering tendon has made great progress.
  • many technical problems remain unresolved: the source, quantity and quality of seed cells; lack of ideal scaffold materials; how the concentration of exogenous growth factors is controlled by tissue release in the process of tissue engineering tendon through sustained release technology, How to solve the synergistic effect and ordering effect of factors; the mechanism of in vitro mechanical stimulation and the optimal stress stimulation conditions need to be studied.
  • the large-scale application of traditional engineered tendons needs to rely on the construction of engineered tendons and long-term preservation and transportation.
  • tissue engineering tendon products are not ideal, difficult to preserve and transport, and it is difficult to achieve clinical application. Therefore, the development of tissue engineering tendons has encountered technical bottlenecks, and tissue engineering tendons constructed using traditional tissue engineering methods are still difficult to be used for clinically achieving the goal of repair and regeneration of tendon defects.
  • the growth environment is conducive to the transfer of information between cells and scaffolds and between cells and cells; (3) the removal of cell-related substances and xenoantigens during processing, the antigenicity is significantly reduced; (4) the basic mechanical strength of the tendon is retained; 5) The processing and processing methods of the stent are relatively mild, which is beneficial to retain the biologically active substances in the material; (6) The animal tendon tissue is rich in source, no need to take materials from human tissues, reduce the trauma, and avoid the source of autologous or allogeneic tendon tissue. Limit problem.
  • the bio-modification of scaffolds to improve biological activity is usually a direct combination of bioactive factors, but the composite scaffold prepared by the method has a limited variety of bioactive factors, and the appropriate concentration of each factor is difficult to determine, so the biological activity is limited, and the effect is limited. Not quite satisfactory.
  • the present invention provides a novel bioactive scaffold and a preparation method and use thereof.
  • the method for preparing a biologically active stent for inducing regeneration of tendon tissue comprises the following steps:
  • the extracellular matrix can be compounded on the above scaffold.
  • the preparation method of the acellular cell tendon tablet holder in the step (1) is as follows:
  • step 3 Repeated freezing and thawing of the compressed tendon in step 2); the repeated freezing and thawing is performed by placing the compressed tendon in step 2) in liquid nitrogen for 1 to 3 minutes, and placing it at 25 to 37 ° C for 3 to 10 minutes, repeating operation 4 ⁇ 6 times.
  • the nuclease treatment method comprises the steps of: treating the tendon after the step 3), placing the nuclease solution having a DNase concentration of 120-180 IU/ml and an RNase concentration of 80-120 ⁇ g/ml, and treating at room temperature or 37 ° C at a constant temperature. ⁇ 24h.
  • the preparation method of the acellular cell tendon slice stent in the step (1) is as follows:
  • step 2) repeatedly freeze-thaw the tendon; the repeated freezing and thawing is to remove the tendon in step 1) in liquid nitrogen for 1 to 3 minutes, 25 to 37 ° C for 3 to 10 minutes, and repeat the operation for 4 to 6 times;
  • Step 2) freeze-thaw treatment of the tendon along the length of the frozen section, the thickness of the slice is 300 ⁇ 900 ⁇ m;
  • the nuclease treatment method comprises: taking the tendon slice obtained in the step 3), placing the nuclease solution having a DNase concentration of 120-180 IU/ml and an RNase concentration of 80-120 ⁇ g/ml, and treating at room temperature or 37 ° C at a constant temperature. ⁇ 24h.
  • step (2) the method of compounding the extracellular matrix is:
  • a, preparation of decellularized tendon gel take the tendon tissue, decellularization, freeze-dried, pulverized, added to 1mg / ml of pepsin solution, digested at room temperature for 24h, neutralized with a base to neutral, and then added to the final volume 1 /10 PBS solution, placed in a 37 ° C incubator to incubate into a tendon gel;
  • step b Take the decellularized tendon gel prepared in step a, and sputum on the decellularized tendon tablet or slice holder of step (1) so that the tendon gel completely covers the tendon tablet or the slice holder.
  • step a the step of decellularization treatment is: repeated freezing and thawing: 1 to 3 minutes in liquid nitrogen, 3 to 10 minutes at 25 to 37 ° C, repeated operation 4 to 6 times; longitudinal sectioning: freezing along the length of the tendon
  • the slice was sliced to a thickness of 300-900 ⁇ m; the nuclease solution was treated with 120-150 IU/ml DNase and 80-100 ⁇ g/ml RNase, and shaken at 37 ° C for 12 h under a constant temperature shaker;
  • step a neutralization with 0.2 N NaOH.
  • step (2) the method of compounding the extracellular matrix is:
  • step (1) taking tendon cells or stem cells, inoculated on the acellular tendon tablet or the section scaffold of step (1);
  • the cells are grown to a dense cell sheet with a degree of fusion of 100%, and subjected to decellularization treatment.
  • the stem cells are bone marrow stromal stem cells, adipose stem cells, and tendon stem cells.
  • step 2 when the cells reach 90% confluence, 50 ⁇ Vc is added to continue to culture for 6-8 days to a dense cell sheet with a degree of fusion of 100%.
  • step 2 the decellularization treatment was first treated with 0.5% Triton X-10037 °C containing 20 mM ammonia water for 15 min, and then treated with 100 U/mL DNase at 37 ° C for 2 h.
  • the present invention also provides a bioactive scaffold for inducing tendon tissue regeneration prepared by the foregoing method.
  • the present invention also provides the use of the aforementioned bioactive scaffold for inducing tendon tissue regeneration in the preparation of a soft tissue defect repair material.
  • the soft tissue defect repairing material is a tendon tissue or a ligament tissue defect repairing material.
  • the prepared bioactive scaffold substrate is composed of acellular cell tendon sections or compressed tablets, and is a natural tendon-derived material;
  • the prepared bioactive scaffold is subjected to decellularization and de-antigen treatment, thereby effectively reducing the antigenicity of the scaffold;
  • the prepared bioactive scaffold fully utilizes the extracellular matrix environment of the tendon tissue, and provides a good microenvironment and mechanical support for the cell, which facilitates cell adhesion, proliferation and function;
  • the prepared bioactive scaffold has good mechanical properties, has similar mechanical properties to normal tendon tissue, and can well overcome the problem of poor mechanical compatibility of artificial materials for tendon repair;
  • the prepared bioactive scaffold has a degradation rate matched with tendon regeneration, and the degradation product is non-toxic and harmless;
  • the prepared bioactive scaffold is modified by a tendon gel, tendon cell or stem cell extracellular matrix, and contains various biological active factors (transforming growth factor, insulin-like growth factor, vascular endothelial growth factor), structural protein (fibronectin). , glass adhesion protein) and proteoglycan substances (Biglycan, Fibromodulin), etc., is conducive to the induction of tendon tissue regeneration and repair.
  • various biological active factors transforming growth factor, insulin-like growth factor, vascular endothelial growth factor
  • structural protein fibronectin
  • fibronectin structural protein
  • glass adhesion protein glass adhesion protein
  • proteoglycan substances Biglycan, Fibromodulin
  • the prepared bioactive scaffold is easy to be trimmed, which is advantageous for suturing and fixing the tendon defect.
  • the preparation process of the bioactive scaffold of the present invention is simple, and can be used for large-scale production, and the product is easy to store and transport, and the added value of the product is high.
  • the bioactive scaffold for inducing tendon tissue regeneration can effectively repair tendon defect by compound tendon cells, a matrix secreted by stem cells or an extracellular matrix of natural tendon, and the repair effect is good, and the method is superior to direct composite cytokines.
  • the clinical application prospects are good.
  • Figure 1 is a schematic diagram of the preparation process of the bioactive scaffold.
  • FIG. 1 Bioactive scaffolds The tendon tendon gel contains a variety of growth factors.
  • FIG. 3 Bioactive scaffold histology of extracellular matrix modification of tendon stem cells: H&E staining, Masson staining and DAPI staining confirmed that the cell fraction was effectively removed by decellularization, and ECM components were retained on the surface of the tendon section. DTSs are acellular cell tendon sections.
  • Fig. 4 Observation of the surface morphology of the bioactive scaffold: The tendon stem cells were cultured on the surface of the tendon slice to form a cell-scaffold complex. After decellularization, the surface of the scaffold showed a large amount of stem cell ECM deposition. The DTSs in the figure are acellular cell tendon sections.
  • Fig. 5 Detection of bioactive scaffold growth factor in extracellular matrix modified tendon sections of tendon stem cells: ELISA quantitative detection showed that the growth factors (TGF- ⁇ 1, VEGF and IGF-1) in the bioactive scaffolds modified by ECM of tendon stem cells were significantly increased.
  • DTSs are acellular cell tendon sections, and ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • FIG. 6 Cytocompatibility of bioactive scaffolds of stem cell extracellular matrix modified tendon sections: SEM analysis confirmed that the surface of ECM modified bioactive scaffolds of tendon stem cells is suitable for the growth of bone marrow stromal stem cells (BMSCs) with good cytocompatibility.
  • BMSCs bone marrow stromal stem cells
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • FIG. 7 West-blot quantitative detection of extracellular matrix protein content before and after stenting of tendon stem cell ECM modified tendon sections. The results showed that the content of extracellular matrix protein components (Biglycan, Fibromodulin, Fibronectin and Vitronectin) in the bioactive scaffold modified by ECM of tendon stem cells was significantly increased.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells. * indicates a significant difference compared to the DTSs group.
  • FIG. 8 Quantitative detection of the effect of bioactive scaffolds obtained from tendon stem cell ECM modified tendon sections on the proliferation of BMSCs.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells. * indicates a significant difference compared to the DTSs group.
  • FIG. 9 Live cell staining results showed that BMSCs maintained higher cell viability on the ECM modified bioactive scaffold of tendon stem cells.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • FIG. 10 Construction and surgical repair of a rabbit Achilles tendon defect model. SDFT is superficial flexor tendon, DBTs are tendon compression stents, and AT is Achilles tendon.
  • FIG. 11 Ultrasound observations of different groups at different time points after surgery.
  • FIG. 12 MRI observations of different groups at different time points after surgery. B-ultrasound and MRI tests were performed on rabbit Achilles tendon at 4, 8, and 12 weeks after operation. It was found that the three groups of repaired and reconstructed Achilles tendon decreased with time, and the abnormal echo and abnormal signals were reduced. The bioactive stent was obviously visible. It is gradually remodeled to the same signal as the autologous Achilles tendon tissue, and at 12w, the difference between the tendon of the autologous tendon and the bioactive stent can not be distinguished.
  • Figure 13 is a general observation of different groups at different time points after surgery.
  • Figure 14 shows histological (H&E staining) observations of different groups at different time points after surgery. Gross and histological observations showed that autologous tendon and autologous tendon and tendon compression stents were repaired at the 4th hour, and the inflammatory reaction around the tendon compression stent was obvious; the bioactive stent group was followed by the 12th postoperative period. The defect has been reconstructed, the fiber bundle is continuous, and the inflammatory response is significantly reduced. DBTs are tendon compression scaffolds and ECM is a tendon gel.
  • the bioactive scaffold was prepared by modifying the tendon slices of the tendon stem cell extracellular matrix with the longitudinal section of the hind limbs of the canine as the base material.
  • the specific preparation method is as follows:
  • the method of treating the nuclease is: taking the tendon slice obtained in the step 3), placing the nuclease solution having a DNase concentration of 120-180 IU/ml and an RNase concentration of 80-120 ⁇ g/ml, at room temperature. Or heat treatment at 37 ° C for 6 ⁇ 24h. Freeze and disinfect.
  • the culture conditions were 5% CO 2 and 37 ° C.
  • 50 ⁇ M Vc vitamin C
  • the tendon stem cells form a dense cell sheet with a cell fusion degree of 100% on the surface of the Achilles tendon slice.
  • the cultured tendon stem cell-Achilles tendon section complex was then subjected to decellularization treatment, first treated with 0.5% Triton X-100 containing 20 mM ammonia water at 37 ° C for 15 min, and then treated with 100 U/ml DNase at 37 ° C for 2 h.
  • the DNA cells were completely removed by DNA quantification and histological observation.
  • the extracellular matrix of tendon stem cells was left on the Achilles tendon slice scaffold, and the tendon stem cell extracellular matrix modified tendon slice scaffold was obtained.
  • the scaffold was a simple tendon slice. A better bioactive scaffold for the stent can be used to repair tendon defects and rebuild tendon function.
  • the tendon tablet was modified by the extracellular matrix of tendon stem cells.
  • Bioactive scaffolds are prepared, and the specific preparation methods are as follows:
  • Preparation of the tablet holder can be prepared according to the method of claim 1 of Patent No.: ZL201310636964.0, specifically, 1) taking fresh tendon, washing; 2) compressing the tendon in the thickness direction, the compression ratio is 60-90 %, compressed into a thickness of about 1.0 ⁇ 1.2mm tendon compression; 3) step 2) compression of the tendon after repeated freezing and thawing; the repeated freezing and thawing is the step 2) compressed muscle tendon in liquid nitrogen 1 ⁇ 3min, 25 ⁇ 37°C, 3 ⁇ 10min, repeated operation 4 ⁇ 6 times; 4)
  • the tendon treated in step 3) is treated with nuclease and washed, wherein the nuclease treatment method is: taking steps 3)
  • the treated tendon is placed in a nuclease solution having a DNase concentration of 120-180 IU/ml and an RNase concentration of 80-120 ⁇ g/ml, and is thermostated at room temperature or 37 ° C for 6-24 hours; freeze-dried and disinfect
  • the third-generation rat tendon stem cells were cultured with the above-mentioned Achilles tendon tablet for 7 days.
  • the culture conditions were 5% CO 2 and 37 ° C.
  • 50 ⁇ Vc was added and culture was continued for 8 days.
  • the tendon stem cells form a dense cell sheet with a cell fusion degree of 100% on the surface of the Achilles tendon tablet.
  • the cultured tendon stem cell-Achilles tendon tableting complex was subjected to decellularization treatment, first treated with 0.5% Triton X-100 containing 20 mM ammonia water at 37 ° C for 15 min, and then treated with 100 U/ml DNase at 37 ° C for 2 h.
  • the calf and the sputum tablet were used as the base material to prepare the tendon gel and the tablet scaffold composite for repairing the Achilles tendon defect and reconstructing the Achilles tendon function.
  • the specific preparation and use methods are as follows:
  • Preparation of the tablet holder can be prepared according to the method of claim 1 of Patent No.: ZL201310636964.0, specifically, 1) taking fresh tendon, washing; 2) compressing the tendon in the thickness direction, the compression ratio is 60-90 %, compressed into a thickness of about 1.0 ⁇ 1.2mm tendon compression; 3) step 2) compression of the tendon after repeated freezing and thawing; the repeated freezing and thawing is the step 2) compressed muscle tendon in liquid nitrogen 1 ⁇ 3min, 25 ⁇ 37°C, 3 ⁇ 10min, repeated operation 4 ⁇ 6 times; 4)
  • the tendon treated in step 3) is treated with nuclease and washed, wherein the nuclease treatment method is: taking steps 3)
  • the treated tendon is placed in a nuclease solution having a DNase concentration of 120-180 IU/ml and an RNase concentration of 80-120 ⁇ g/ml, and is thermostated at room temperature or 37 ° C for 6-24 hours; freeze-dried and disinfect
  • the rhesus monkey decellularized tendon gel prepared above is combined with the rehydrated tendon tablet to prepare a bioactive scaffold, and the specific method is to knead the prepared tendon gel on the surface of the tendon tablet to make the tendon gel. Completely covering the tendon tablet holder.
  • the results of ELISA showed that the growth factors VEGF, TGF- ⁇ 1 and IGF-1 associated with tendon repair were 1.30 ⁇ 0.53 pg/mg, 1.56 ⁇ 0.24 pg/mg, and 8.07 ⁇ 0.89 pg/mg, respectively.
  • the bioactive scaffold prepared according to the method of Example 1 was tested for its properties as follows:
  • H&E staining, Masson staining, and DAPI staining were performed according to the method described in the literature Ning LJ, et al. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. Journal of Biomedical Materials Research Part A, 2012: 100A: 1448-1456.
  • the above scanning electron microscope (SEM) observation method The samples were observed to be inoculated with BMSCs on the surface of the tendon slice scaffold and the tendon stem cell ECM modified tendon slice.
  • the sample processing method was the same as above.
  • BMSCs were inoculated on the surface of the scaffold, and stained with live cells at 1 and 3 days after 5% CO 2 and 37 ° C: 3 times with sterile PBS; live dead cell stain (1 ml sterile PBS + 1 ⁇ l calcein + 1 ⁇ l propidium iodide was incubated at 37 ° C for 30 min in the dark; washed 3 times with sterile PBS, observed with a fluorescence microscope.
  • the composite material prepared in Example 3 (wherein the thickness of the tendon gel covering the tendon tablet holder was 100 to 300 ⁇ m) was subjected to an in vivo repair experiment as follows: 63 male adult New Zealand white rabbits were randomly selected. The lateral side was a blank group (as a control group), the simple tendon compression group (DBTs), and the tendon gel composite tendon compression group (ECM+DBTs), and 2 cm from the upper part of the Achilles tendon calcaneus to the proximal end. The Achilles tendon tissue was used to make the Achilles tendon defect model (Fig.
  • the Achilles tendon compression stent was used to reconstruct the Achilles tendon defect with the tendon compression stent or the tendon gel composite tendon compression stent.
  • the blank group was simply removed after the Achilles tendon was removed.
  • MRI tests were performed on the 4th, 8th, and 12th postoperatively, the samples were taken again. Further histological analysis (H&E staining) and biomechanical testing.
  • DTSs are acellular cell tendon sections.
  • the tendon stem cells were cultured on the surface of the tendon slice to form a cell-scaffold complex, and after decellularization treatment, a large amount of stem cell ECM deposition was observed on the surface of the stent.
  • the DTSs in the figure are acellular cell tendon sections.
  • the ELISA quantitative results showed that the growth factor (TGF- ⁇ 1, VEGF and IGF-1) levels in the bioactive scaffolds modified by ECM of tendon stem cells were significantly increased.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells. * indicates a significant difference compared to the DTSs group.
  • the DTSs in the figure are decellularized tendon sections, and the ECM-DTSs are biologically active scaffolds modified by the extracellular matrix of tendon stem cells. * indicates a significant difference compared to the DTSs group.
  • DTSs are acellular cell tendon sections
  • ECM-DTSs are biologically active scaffolds modified by extracellular matrix of tendon stem cells.
  • the rabbit Achilles tendon defect model was constructed and surgically repaired.
  • SDFT is superficial flexor tendon
  • DBTs are tendon compression stents
  • AT is Achilles tendon.
  • the bioactive scaffold obtained by the specific method for inducing regeneration of tendon tissue has high cytokine content, good biocompatibility, excellent in vivo repairing effect, and has good application prospect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Dermatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • Botany (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Vascular Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rehabilitation Therapy (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种制备诱导肌腱组织再生的生物活性支架的方法，包括如下步骤：（1）取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；（2）在支架上复合细胞外基质，即可。前述方法制备的脱细胞肌腱生物活性支架及其用途也被提及。该脱细胞肌腱支架生物活性高，体内修复效果良好，免疫原性低，生物相容性好，制备方法简单，成本低廉。

Description

一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 技术领域
本发明涉及一种具有肌腱组织再生的生物活性支架的制备方法及其用途，属于生物材料领域。
背景技术
由于疾病、创伤、退变等原因导致的肌腱损伤已成为不可忽视的健康问题。目前肌腱损伤修复的方法多种多样，包括自体肌腱移植、同种或异种肌腱移植和人工材料替代等。自体肌腱移植会造成供区疼痛、感染、损伤，同时所能供给的肌腱来源有限；同种异体肌腱移植可能引起疾病的传播，出现不同程度的排斥反应，也存在来源有限的问题；异种肌腱移植也可能引起疾病传播，但最主要的问题是存在免疫排斥反应；人工肌腱由于力学相容性较差或降解速率难与组织再生同步等问题，使得肌腱缺损修复的长期效果有待观察，且价格昂贵。理想的肌腱修复材料应具备以下条件：(1)良好的生物相容性；(2)良好的力学相容性；(3)具有诱导肌腱组织再生的潜能；(4)形状大小可控，加工制备方法有可操作性。查阅大量文献结果表明，目前使用的肌腱损伤修复材料均存在各自的缺陷和不足，因此寻求更加理想的肌腱修复材料的脚步从未停止。
随着组织工程技术的发展，激励着研究人员试图通过组织工程肌腱来解决肌腱损伤修复困难这一难题。在国内外学者的共同努力探索下，组织工程肌腱的相关研究已取得巨大进展。然而，到目前为止，很多技术问题依然悬而未决：种子细胞的来源、数量以及质量等问题；缺乏理想的支架材料；外源性生长因子通过缓释技术用于组织工程肌腱时其浓度如何控制，多因子的协同作用及有序作用如何解决；体外力学刺激的作用机制及最佳的应力刺激条件尚需研究。而且传统的工程化肌腱的大规模应用，需要依赖于工程化肌腱的构建和长期保存及运输，其生产制备过程耗时，保存费用昂贵。总之，现有的组织工程肌腱产品的生物功能不甚理想，难于保存和运输，很难实现临床推广应用。因此，组织工程肌腱的发展遇到了技术瓶颈，利用传统的组织工程方法构建的组织工程肌腱尚难以用于临床实现肌腱缺损修复和再生的目标。
前期研究(Preparation and characterization of decellularized tendon slices for tendon tissue engineering.Journal of Biomedical Materials Research Part A，2012:100A:1448–1456)和发明专利(专利号：ZL201310636964.0)，已经证实脱细胞肌腱切片和压片支架是较好的肌腱组织工程的支架材料。其优越性包括： (1)这些支架主要是由平行排列的I型胶原构成，具有天然肌腱的结构和组成成分；(2)具有肌腱特有的三维空间结构，有利于细胞粘附和增殖，能为细胞提供良好的生长环境并有利于细胞与支架及细胞与细胞间的信息传递；(3)加工处理过程中除去了细胞相关物质和异种抗原，抗原性显著降低；(4)保留了肌腱的基本力学强度；(5)支架的加工和处理方法相对温和，有利于保留材料中的生物活性物质；(6)动物的肌腱组织来源丰富，无需从人体组织取材，减少了创伤，避免了自体或异体肌腱组织来源受限的问题。尽管如此，上述肌腱切片或压片支架经过一系列物理加工和酶学处理，生物活性物质部分丢失，在体外诱导干细胞腱向分化或者体内促进肌腱组织再生的能力受到限制。
因此，对上述肌腱切片或压片支架进行生物修饰，以增强其生物活性非常必要。
目前对支架进行生物修饰以提高生物活性的方法通常是直接复合生物活性因子，但是该方法制备得到的复合支架中生物活性因子种类有限，每种因子的合适浓度难以确定，因此生物活性有限，效果不尽如人意。
发明内容
为了解决上述问题，本发明提供了一种新的生物活性支架及其制备方法和用途。
本发明制备诱导肌腱组织再生的生物活性支架的方法，包括如下步骤：
(1)取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；
(2)在上述支架上复合细胞外基质，即可。
步骤(1)所述脱细胞肌腱压片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱沿厚度方向进行压缩，压缩率为60～90％；
3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次。
4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
步骤(1)所述脱细胞肌腱切片支架的制备方法如下：
1)取新鲜肌腱，清洗；
2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；
4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；
其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
步骤(2)中，复合细胞外基质的方法是：
a、制备脱细胞肌腱凝胶：取肌腱组织，脱细胞处理，冻干，粉碎，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用碱中和成中性，然后加入终体积1/10的PBS溶液，置于37℃培养箱中孵育成肌腱凝胶；
b、取步骤a制备的脱细胞肌腱凝胶，裱籿在步骤(1)的脱细胞肌腱压片或切片支架上，使肌腱凝胶完全覆盖肌腱压片或切片支架。
步骤a中，所述脱细胞处理的步骤是：反复冻融：在液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；纵向切片：沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm；核酸酶溶液处理：置于120～150IU/ml DNA酶和80～100μg/ml RNA酶中，37℃恒温摇床震荡12h；
和/或，步骤a中，用0.2N NaOH中和成中性。
步骤(2)中，复合细胞外基质的方法是：
①取肌腱细胞或者干细胞，接种于步骤(1)的脱细胞肌腱压片或切片支架上培养；
②待细胞生长至融合度为100％的致密细胞片层，进行脱细胞处理。
步骤①中，所述干细胞是骨髓基质干细胞、脂肪干细胞、肌腱干细胞。
步骤②中，待细胞达到90％融合时，加入50μΜVc继续培养6-8d至融合度为100％的致密细胞片层。
步骤②中，所述脱细胞处理是先用含20mM氨水的0.5％Triton X-10037℃处理15min，再用100U/mL DNA酶37℃处理2h。
本发明还提供了前述方法制备的诱导肌腱组织再生的生物活性支架。
本发明还提供了前述的诱导肌腱组织再生的生物活性支架在制备治疗软组织缺损修复材料中的用途。
其中，所述软组织缺损修复材料是肌腱组织或韧带组织缺损修复材料。
本发明的优点在于：
(1)制备的生物活性支架基底由脱细胞肌腱切片或压片构成，是天然的肌腱来源材料；
(2)制备的生物活性支架经过脱细胞和去抗原处理，有效地降低了支架的抗原性；
(3)制备的生物活性支架充分利用了肌腱组织的胞外基质环境，能为细胞提供良好的微环境和力学支撑，利于细胞粘附、增殖和发挥功能；
(4)制备的生物活性支架具有良好的力学性能，具有与正常肌腱组织相近的力学特性，能很好的克服肌腱修复的人工材料力学相容性差的问题；
(5)制备的生物活性支架具有与肌腱再生相匹配的降解速率，且降解产物无毒无害；
(6)制备的生物活性支架通过肌腱凝胶、肌腱细胞或干细胞胞外基质修饰，含有多种生物活性因子(转化生长因子、胰岛素样生长因子、血管内皮生长因子)、结构蛋白(纤维连接蛋白、玻璃粘连蛋白)和蛋白多糖物质(Biglycan，Fibromodulin)等，有利于诱导肌腱组织再生与修复。
(7)制备的生物活性支架易于修剪，利于肌腱缺损的缝合与固定。
(8)本发明的生物活性支架的制备工艺路线简洁，可用于大规模生产，产品易于保存和运输，产品附加值高。
综上，本发明诱导肌腱组织再生的生物活性支架，通过复合肌腱细胞、干细胞分泌的基质或者天然肌腱的细胞外基质，可以有效修复肌腱缺损，修复效果良好，优于直接复合细胞因子的方式，临床应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1生物活性支架制备流程示意图。
图2生物活性支架裱籿肌腱凝胶中含有多种生长因子。
图3肌腱干细胞胞外基质修饰的生物活性支架组织学：H&E染色、Masson染色和DAPI染色结果证实经过脱细胞处理有效地去除了细胞组分，同时在肌腱切片支架表面有ECM组分保留。DTSs为脱细胞肌腱切片。
图4生物活性支架表面形态观察：在肌腱切片表面复合肌腱干细胞培养，形成细胞-支架复合物，对其再次脱细胞处理后，可见支架表面有大量的干细胞ECM沉积。图中DTSs为脱细胞肌腱切片。
图5肌腱干细胞胞外基质修饰肌腱切片的生物活性支架生长因子检测：ELISA定量检测结果显示肌腱干细胞ECM修饰后的生物活性支架中生长因子(TGF-β1，VEGF和IGF-1)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图6干细胞胞外基质修饰的肌腱切片生物活性支架的细胞相容性观察：SEM检测证实肌腱干细胞ECM修饰的生物活性支架表面很适合骨髓基质干细胞(BMSCs)生长，具有良好的细胞相容性。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图7 West-blot定量检测肌腱干细胞ECM修饰肌腱切片支架前后胞外基质蛋白含量变化。检测结果显示肌腱干细胞ECM修饰后的生物活性支架中胞外基质蛋白组分(Biglycan，Fibromodulin，Fibronectin和Vitronectin)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
图8
Figure PCTCN2017088415-appb-000001
定量检测肌腱干细胞ECM修饰肌腱切片得到的生物活性支架对BMSCs细胞增殖能力的影响。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
图9活死细胞染色结果发现BMSCs在肌腱干细胞ECM修饰的生物活性支架上可维持更高的细胞活力。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
图10兔跟腱缺损模型构建及手术修复情况。SDFT为浅表性屈肌腱，DBTs为肌腱压片支架，AT为跟腱。
图11术后不同时间点不同组别的超声观察。
图12术后不同时间点不同组别的MRI观察。术后4、8、12w分别对兔子跟腱进行B超及MRI检测实验发现，三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少，生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异。
图13术后不同时间点不同组别的大体观察。
图14术后不同时间点不同组别的组织学(H&E染色)观察。大体及组织学观察发现，可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架组跟腱缺损已重建，纤维束连续，炎性反应明显减轻。DBTs为肌腱压片支架，ECM为肌腱凝胶。
图15术后不同时间生物活性支架修复跟腱的生物力学检测。结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组及肌腱凝胶复合肌腱压片组最大载荷及刚度高(P<0.05)。DBTs为肌腱压片支架，ECM为肌腱凝胶。
具体实施方式
主要材料、试剂与仪器：
新鲜肌腱、骨髓基质干细胞、肌腱干细胞、肌腱细胞；细胞培养基、核酸酶、DNA检测试剂盒；冰冻切片机、CO2培养箱、生物力学测试系统、扫描电子显微镜、荧光显微镜，冷冻干燥机(Christ，德国)、低温球磨仪(Retsch，德国)。
统计学方法：
采用SPSS16.0软件包进行数据统计分析和处理，全部数据用均数±标准差表示，数据满足正态分布，方差齐，组间两两比较采用单因素方差分析(Sceffe法)，检验水准α＝0.05，即P<0.05时差异有统计学意义。
实施例1 诱导肌腱组织再生的生物活性支架
以犬后肢跟腱纵向切片为基底材料，通过肌腱干细胞胞外基质修饰肌腱切片制备生物活性支架，具体制备方法如下：
制备切片支架：1)取新鲜肌腱，清洗；2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。冻干、消毒备用。
如图1所示，将大鼠肌腱干细胞与跟腱切片支架复合培养7d后，培养条件是5％CO2、37℃，待细胞达到90％融合时，加入50μΜVc(维生素C)继续处理8d，至肌腱干细胞在跟腱切片支架表面形成细胞融合度为100％的致密细胞片层。然后将上述培养的肌腱干细胞-跟腱切片复合物进行脱细胞处理，先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/ml DNA酶37℃处理2h。
通过DNA定量和组织学观察证实干细胞已经全部脱除，在跟腱切片支架上留下肌腱干细胞的胞外基质，获得肌腱干细胞胞外基质修饰的肌腱切片支架，该支架是一种比单纯肌腱切片支架更好的生物活性支架，可用于修复肌腱缺损，重建肌腱功能。
实施例2 诱导肌腱组织再生的生物活性支架
以小牛跟腱压片为基底材料，通过肌腱干细胞胞外基质修饰肌腱压片制 备生物活性支架，具体制备方法如下：
制备压片支架：可以按照专利号：ZL201310636964.0的专利的权利要求1的方法制备，具体地，1)取新鲜肌腱，清洗；2)将肌腱沿厚度方向进行压缩，压缩率为60～90％，压缩成厚度约1.0～1.2mm的肌腱压片；3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h；冻干、消毒备用。
如图1所示，取第三代大鼠肌腱干细胞与上述跟腱压片支架复合培养7d，培养条件是5％CO2、37℃，待细胞达到90％融合时，加入50μΜVc继续培养8d，至肌腱干细胞在跟腱压片支架表面形成细胞融合度为100％的致密的细胞片层。然后将上述培养的肌腱干细胞-跟腱压片复合物进行脱细胞处理，先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/ml DNA酶37℃处理2h。
通过DNA定量和组织学观察证实干细胞已经全部脱除，在跟腱压片支架上留下肌腱干细胞的胞外基质，获得肌腱干细胞胞外基质修饰的肌腱压片支架，该支架是一种比单纯肌腱压片支架更好的生物活性支架，可用于修复肌腱缺损，重建肌腱功能。
实施例3 诱导肌腱组织再生的生物活性支架
1、制备方法
以小牛跟腱压片为基底材料，制备肌腱凝胶与压片支架复合物，用于修复大白兔跟腱缺损，重建跟腱功能，具体制备和使用方法如下：
制备压片支架：可以按照专利号：ZL201310636964.0的专利的权利要求1的方法制备，具体地，1)取新鲜肌腱，清洗；2)将肌腱沿厚度方向进行压缩，压缩率为60～90％，压缩成厚度约1.0～1.2mm的肌腱压片；3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h；冻干、消毒备用。
取新鲜恒河猴跟腱先进行脱细胞处理，即反复冻融(液氮中1～3min， 25～37℃放置3～10min，重复操作4～6次)、纵向切片(沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm)和核酸酶溶液(120～150IU/ml DNA酶和80～100μg/ml RNA酶)处理，37℃恒温摇床震荡12h，然后冻干，-80℃冷冻，-20℃球磨，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用0.2N NaOH中和成中性，最后加入终体积1/10的1×PBS，置于37℃培养箱中孵育成肌腱凝胶。
将上述制备的恒河猴脱细胞肌腱凝胶，与复水的肌腱压片复合制备生物活性支架，复合的具体方式是将上述制备的肌腱凝胶裱籿在肌腱压片表面，使肌腱凝胶完全覆盖肌腱压片支架。
取成年大白兔，制备后肢双侧跟腱缺损动物模型，分别用上述单纯肌腱压片支架、生物活性支架和大白兔自体跟腱修复，重建跟腱功能。术后经大体、组织学、影像学、生物力学检测，证实生物活性支架具有诱导跟腱组织再生、重建跟腱功能的效果，详细修复实验及结果见实验例1。
2、性质检测
在肌腱凝胶中加入组织裂解液(FNN0071,Invitrogen；凝胶质量/组织裂解液体积＝1g/10ml)，混匀，冰浴条件下静置1.5h。匀浆液在4℃条件下10,000g离心20min，取上清液待测。ELISA检测方法分别按照VEGF、TGF-β1和IGF-1检测试剂盒说明书进行。
ELISA检测结果显示，肌腱凝胶中含有与肌腱修复相关的生长因子VEGF、TGF-β1和IGF-1分别为1.30±0.53pg/mg、1.56±0.24pg/mg、8.07±0.89pg/mg。
以下用实验例的方式来说明本发明的有益效果：
实验例1 本发明诱导肌腱组织再生的生物活性支架的性质
1、实验方法
按照实施例1的方法制备的生物活性支架，按照下述方法检测其性质：
(1)生物活性支架组织学检测
按照文献Ning LJ,et al.Preparation and characterization of decellularized tendon slices for tendon tissue engineering.Journal of Biomedical Materials Research Part A，2012:100A:1448–1456记载的方法，进行H&E染色、Masson染色和DAPI染色。
(2)表面形态观察
表面形态检测采用扫描电子显微镜(SEM)观察。具体方法是将待测样本(对照组和实验组)用2.5％戊二醛4℃固定2h以上。PBS清洗30min×3次。 酒精梯度脱水：50％、70％、80％、90％、100％各15min。临界点干燥。真空喷金后扫描电镜观察。
(3)生长因子检测
同上述ELISA检测方法。按照待测生长因子检测试剂盒说明书进行。
(4)细胞相容性观察
同上述扫描电镜(SEM)观察方法。观察样本为单纯的肌腱切片支架和肌腱干细胞ECM修饰的肌腱切片支架表面接种BMSCs，样本处理方法同上。
(5)West-blot定量检测肌腱干细胞ECM修饰肌腱切片支架前后胞外基质蛋白含量变化。
Western-Blot定量检测生物活性支架中胞外基质蛋白含量的方法，包括：待测支架中总蛋白的提取，所有蛋白样本的总蛋白含量均采用BCA蛋白浓度测定试剂盒(按说明书方法)；生物活性支架相关蛋白(Biglycan，Fibromodulin，Fibronectin和Vitronectin)的电泳及抗体孵育；采用Gel-Pro Analyzer4软件对蛋白条带进行灰度分析；所有样品的目的蛋白的相对表达情况均由其内参的表达量进行归一化处理，即：目的蛋白相对表达量＝目的蛋白灰度值/GAPDH灰度值。
(6)
Figure PCTCN2017088415-appb-000002
定量检测肌腱干细胞ECM修饰肌腱切片得到的生物活性支架对BMSCs细胞增殖能力的影响
按照文献Ning LJ,et al.Preparation and characterization of decellularized tendon slices for tendon tissue engineering.Journal of Biomedical Materials Research Part A，2012:100A:1448–1456记载的方法检测。
(7)活死细胞染色
将脱细胞肌腱切片和生物活性肌腱切片支架表面加无血清DMEM培养基孵育过夜。将BMSCs接种于支架表面，5％CO2、37℃条件下培养第1d和3d时行活死细胞染色：无菌PBS清洗3次；活死细胞染液(1ml无菌PBS+1μl钙黄绿素+1μl碘化丙啶)37℃避光孵育30min；无菌PBS清洗3次，荧光显微镜观察并采图。
2、体内修复实验
取实施例3制备的复合材料(其中，覆盖在肌腱压片支架上的肌腱凝胶的厚度是100～300μm)，按照如下方法进行体内修复实验：取63只雄性成年新西兰大白兔，随机选取一侧为空白组(作为对照组)、单纯肌腱压片组(DBTs)，及肌腱凝胶复合肌腱压片组(ECM+DBTs)，自跟腱跟骨止点上方1cm处向近端取2cm的跟腱组织制作跟腱缺损模型(图10)，然后利用单纯肌腱压片支架或肌腱凝胶复合肌腱压片支架重建跟腱缺损，空白组则单纯切断跟腱后进行修复。术后第4、8、12w分别进行B超和MRI检测后，再取材 进一步行组织学分析(H&E染色)和生物力学检测。
研究结果：术后4、8、12w分别对兔子跟腱进行B超及MRI检测，实验发现三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少，生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异(图11和图12)。取三组标本行大体观察(图13)及组织学观察和生物力学测试。大体及组织学观察显示，对照组可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架组跟腱缺损已重建，纤维束连续，炎性反应明显减轻(图13和图14)。生物力学测试结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组及生物活性支架组最大载荷及刚度高(P<0.05)(图15)。
3、实验结果
如图3所示，H&E染色、Masson染色和DAPI染色结果证实第二次脱细胞处理有效地去除了细胞组分，同时在肌腱切片支架表面有ECM组分保留。DTSs为脱细胞肌腱切片。
如图4所示，在肌腱切片表面复合肌腱干细胞培养，形成细胞-支架复合物，对其再次脱细胞处理后，可见支架表面有大量的干细胞ECM沉积。图中DTSs为脱细胞肌腱切片。
如图5所示，ELISA定量检测结果显示肌腱干细胞ECM修饰后的生物活性支架中生长因子(TGF-β1，VEGF和IGF-1)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图6所示，SEM检测证实肌腱干细胞ECM修饰的生物活性支架表面很适合BMSCs生长，具有良好的细胞相容性。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图7所示，检测结果显示肌腱干细胞ECM修饰后的生物活性支架中胞外基质蛋白组分(Biglycan，Fibromodulin，Fibronectin和Vitronectin)含量显著提高。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
如图8所示，图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。*表示与DTSs组相比具有显著性差异。
如图9所示，活死细胞染色结果发现BMSCs在肌腱干细胞ECM修饰的生物活性支架上可维持更高的细胞活力。图中DTSs为脱细胞肌腱切片，ECM-DTSs为肌腱干细胞胞外基质修饰的生物活性支架。
如图10所示，兔跟腱缺损模型构建及手术修复情况。SDFT为浅表性屈肌腱，DBTs为肌腱压片支架，AT为跟腱。
如图11所示，术后不同时间点不同组别的超声观察；如图12所示，术后不同时间点不同组别的MRI观察。术后4、8、12w分别对兔子跟腱进行B超及MRI检测实验发现，三组修复和重建的跟腱随着时间的延长，其异常回声及异常信号均减少；生物活性支架从外形明显逐渐经过重塑而与自体跟腱组织信号相同，在第12w时无法分辨自体肌腱与生物活性支架修复的肌腱的差异
如图13所示，术后不同时间点不同组别的大体观察；如图14所示，术后不同时间点不同组别的组织学(H&E染色)观察。大体组织学观察发现，对照组可见自体肌腱及自体肌腱和肌腱压片支架修复处，在第4w时有瘢痕组织形成，肌腱压片支架周围炎性反应明显；术后第12w时生物活性支架修复跟腱缺损已重建，纤维束连续，炎性反应明显减轻。DBTs为肌腱压片支架，ECM为肌腱凝胶。
如图15所示，术后不同时间生物活性支架修复跟腱的生物力学检测。结果显示，随时间延长，实验组最大载荷和刚度均呈逐渐增加趋势；12w时对照组和生物活性支架组最大载荷比较差异无统计学意义(P>0.05)，其余在4w时对照组较单纯肌腱压片组，及生物活性支架组最大载荷及刚度高(P<0.05)。DBTs为肌腱压片支架，ECM为肌腱凝胶。
综上，本发明通过特定方法制备得到的诱导肌腱组织再生的生物活性支架，细胞因子含量高，生物相容性好，体内修复效果优良，具有较好的应用前景。

Claims (12)

  1. 一种制备诱导肌腱组织再生的生物活性支架的方法，其特征在于：包括如下步骤：
    (1)取新鲜肌腱，脱细胞处理，得脱细胞肌腱压片或切片支架；
    (2)在上述支架上复合细胞外基质，即可。
  2. 根据权利要求1所述的方法，其特征在于：步骤(1)所述脱细胞肌腱压片支架的制备方法如下：
    1)取新鲜肌腱，清洗；
    2)将肌腱沿厚度方向进行压缩，压缩率为60～90％；
    3)对步骤2)压缩后的肌腱进行反复冻融；所述反复冻融是将步骤2)压缩后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
    4)将步骤3)处理后的肌腱用核酸酶处理，清洗，即可；
    其中，核酸酶处理的方法为：取步骤3)处理后的肌腱，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
  3. 根据权利要求1所述的方法，其特征在于：步骤(1)所述脱细胞肌腱切片支架的制备方法如下：
    1)取新鲜肌腱，清洗；
    2)将肌腱进行反复冻融；所述反复冻融是将步骤1)清洗后的肌腱置于液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；
    3)对步骤2)冻融处理后的肌腱沿长度方向进行冰冻切片，切片厚度为300～900μm；
    4)将步骤3)获得的肌腱切片用核酸酶处理，清洗，即可；
    其中，核酸酶处理的方法为：取步骤3)得到的肌腱切片，置于DNase浓度为120～180IU/ml和RNase浓度为80～120μg/ml的核酸酶溶液中，室温或37℃恒温处理6～24h。
  4. 根据权利要求1所述的方法，其特征在于：步骤(2)中，复合细胞外基质的方法是：
    a、制备脱细胞肌腱凝胶：取肌腱组织，脱细胞处理，冻干，粉碎，加入到1mg/ml的胃蛋白酶溶液中，室温消化24h，用碱中和成中性，然后加入终体积1/10的PBS溶液，置于37℃培养箱中孵育成肌腱凝胶；
    b、取步骤a制备的脱细胞肌腱凝胶，裱籿在步骤(1)的脱细胞肌腱压片或切片支架上，使肌腱凝胶完全覆盖肌腱压片或切片支架。
  5. 根据权利要求1所述的方法，其特征在于：
    步骤a中，所述脱细胞处理的步骤是：反复冻融：在液氮中1～3min，25～37℃放置3～10min，重复操作4～6次；纵向切片：沿肌腱长度方向进行冰冻切片，切片厚度为300～900μm；核酸酶溶液处理：置于120～150IU/ml DNA酶和80～100μg/ml RNA酶中，37℃恒温摇床震荡12h；
    和/或，步骤a中，用0.2N NaOH中和成中性。
  6. 根据权利要求1所述的方法，其特征在于：步骤(2)中，复合细胞外基质的方法是：
    ①取肌腱细胞或者干细胞，接种于步骤(1)的脱细胞肌腱压片或切片支架上培养；
    ②待细胞生长至汇合度为100％的致密细胞片层，进行脱细胞处理。
  7. 根据权利要求6所述的方法，其特征在于：步骤①中，所述干细胞是骨髓基质干细胞、脂肪干细胞、肌腱干细胞。
  8. 根据权利要求6所述的方法，其特征在于：步骤②中，待细胞达到90％融合时，加入50μΜ Vc继续培养6-8d至融合度为100％的致密细胞片层。
  9. 根据权利要求6所述的方法，其特征在于：步骤②中，所述脱细胞处理是先用含20mM氨水的0.5％Triton X-100 37℃处理15min，再用100U/ml DNA酶37℃处理2h。
  10. 权利要求1～9任意一项所述方法制备的诱导肌腱组织再生的生物活性支架。
  11. 权利要求10所述的诱导肌腱组织再生的生物活性支架在制备治疗软组织缺损修复材料中的用途。
  12. 根据权利要求11所述的用途，其特征在于：所述软组织缺损修复材料是肌腱组织或韧带组织缺损修复材料。
PCT/CN2017/088415 2016-12-28 2017-06-15 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 WO2018120672A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/475,016 US20200121828A1 (en) 2016-12-28 2017-06-15 Bioactive scaffold for inducting tendon regeneration, preparation method therefor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611263819.2 2016-12-28
CN201611263819.2A CN108245708B (zh) 2016-12-28 2016-12-28 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途

Publications (1)

Publication Number Publication Date
WO2018120672A1 true WO2018120672A1 (zh) 2018-07-05

Family

ID=62706856

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/088415 WO2018120672A1 (zh) 2016-12-28 2017-06-15 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途

Country Status (3)

Country Link
US (1) US20200121828A1 (zh)
CN (1) CN108245708B (zh)
WO (1) WO2018120672A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109675113A (zh) * 2018-11-26 2019-04-26 中国人民解放军总医院 一种组织工程肌腱微组织的制备方法
CN110448732B (zh) * 2019-08-27 2022-03-29 中南大学湘雅医院 区域干细胞诱导活性去细胞骨腱界面书页支架
US11376347B1 (en) * 2021-06-21 2022-07-05 The Florida International University Board Of Trustees Materials and methods for accelerating cardiovascular tissue regeneration
CN114533959B (zh) * 2022-04-02 2022-12-09 山东隽秀生物科技股份有限公司 一种肌腱修复材料、制备方法及在制备肌腱修复产品中的应用
CN115607740B (zh) * 2022-12-21 2023-04-21 北京德益达美医疗科技有限公司 一种生长因子复合肌腱及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101147810A (zh) * 2007-11-01 2008-03-26 南昌大学第一附属医院 细胞-生物可降解材料复合物及其制备方法和应用
CN104208750A (zh) * 2013-12-02 2014-12-17 四川大学华西医院 一种脱细胞肌腱材料的制备方法及其用途
CN106075585A (zh) * 2016-07-08 2016-11-09 福建师范大学 一种基于人工肌腱支架材料的组织工程化人工肌腱移植物的制备方法
WO2017031167A1 (en) * 2015-08-17 2017-02-23 The Johns Hopkins University Fiber-hydrogel composite surgical meshes for tissue repair
WO2017031169A1 (en) * 2015-08-17 2017-02-23 The Johns Hopkins University Mesenchymal cell-binding composite material for tissue restoration

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060153815A1 (en) * 2004-12-21 2006-07-13 Agnieszka Seyda Tissue engineering devices for the repair and regeneration of tissue
WO2010123500A1 (en) * 2009-04-22 2010-10-28 University Of Utah Research Foundation Methods of making and using three-dimensional extracellular matrices
US8945536B2 (en) * 2011-01-26 2015-02-03 The Chinese University Of Hong Kong Stem cell sheet for tissue repair
US9101458B2 (en) * 2011-11-10 2015-08-11 Lifecell Corporation Device for tendon and ligament treatment
EP3690028A1 (en) * 2012-09-04 2020-08-05 Anthrogenesis Corporation Methods of tissue generation
CN104971380B (zh) * 2014-04-11 2017-12-15 山东隽秀生物科技股份有限公司 一种脱细胞基质修复凝胶及其制备新方法
CN103966158B (zh) * 2014-05-28 2016-09-21 中国人民解放军第四军医大学 一种牙周组织特异性细胞外基质ecm的制备方法及其应用
CN105169483B (zh) * 2015-10-20 2018-10-12 中山大学 一种脱细胞基质凝胶的制备方法及其脱细胞基质凝胶

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101147810A (zh) * 2007-11-01 2008-03-26 南昌大学第一附属医院 细胞-生物可降解材料复合物及其制备方法和应用
CN104208750A (zh) * 2013-12-02 2014-12-17 四川大学华西医院 一种脱细胞肌腱材料的制备方法及其用途
WO2017031167A1 (en) * 2015-08-17 2017-02-23 The Johns Hopkins University Fiber-hydrogel composite surgical meshes for tissue repair
WO2017031169A1 (en) * 2015-08-17 2017-02-23 The Johns Hopkins University Mesenchymal cell-binding composite material for tissue restoration
CN106075585A (zh) * 2016-07-08 2016-11-09 福建师范大学 一种基于人工肌腱支架材料的组织工程化人工肌腱移植物的制备方法

Also Published As

Publication number Publication date
US20200121828A1 (en) 2020-04-23
CN108245708A (zh) 2018-07-06
CN108245708B (zh) 2021-04-23

Similar Documents

Publication Publication Date Title
US10729813B2 (en) Extracellular matrix-derived gels and related methods
WO2018120672A1 (zh) 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途
KR101056069B1 (ko) 동물조직 분말을 이용한 다공성 3차원 지지체의 제조방법
Hoganson et al. The retention of extracellular matrix proteins and angiogenic and mitogenic cytokines in a decellularized porcine dermis
Brown et al. Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix
CN101563450B (zh) 细胞衍生细胞外基质膜的制备方法
Bondioli et al. Development and evaluation of a decellularized membrane from human dermis
Parmaksiz et al. Decellularization of bovine small intestinal submucosa and its use for the healing of a critical‐sized full‐thickness skin defect, alone and in combination with stem cells, in a small rodent model
Sano et al. Acellular adipose matrix as a natural scaffold for tissue engineering
CN108310467A (zh) 一种组装型细胞衍生细胞外基质膜复合骨修复材料及其制备方法和应用
CN107810014B (zh) 包含间充质干细胞的组合物及其用途
WO2008083223A1 (en) Methods for culturing dermal cells for treatment of skin injuries such as burns
Liu et al. Evaluation of the biocompatibility and mechanical properties of xenogeneic (porcine) extracellular matrix (ECM) scaffold for pelvic reconstruction
CN105682697A (zh) 去除α-半乳糖的方法
Eren et al. Decellularized porcine Achilles tendon induces anti-inflammatory macrophage phenotype in vitro and tendon repair in vivo
CN1836034A (zh) 生产神经元的方法
CN112870445A (zh) 一种软组织修复材料的制备方法及应用
Lim et al. A decellularized scaffold derived from squid cranial cartilage for use in cartilage tissue engineering
Lin et al. Fabrication and evaluation of an optimized xenogenic decellularized costal cartilage graft: preclinical studies of a novel biocompatible prosthesis for rhinoplasty
CN107890586B (zh) 一种同种异体生物乳房补片的制备方法
CN108066750A (zh) 干细胞及其分泌物用于治疗皮肤烧烫伤的新用途
Pajoum et al. In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold
CN110152067B (zh) 组织工程骨支架及其制备方法
US20240091407A1 (en) Cartilage tissue engineering complex and use thereof
Łabuś et al. Own experience from the use of a substitute of an allogeneic acellular dermal matrix revitalized with in vitro cultured skin cells in clinical practice

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: 17888520

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: 17888520

Country of ref document: EP

Kind code of ref document: A1