WO2017097105A1 - 一种可注射的骨修复用复合材料及其制备方法 - Google Patents

一种可注射的骨修复用复合材料及其制备方法 Download PDF

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WO2017097105A1
WO2017097105A1 PCT/CN2016/106559 CN2016106559W WO2017097105A1 WO 2017097105 A1 WO2017097105 A1 WO 2017097105A1 CN 2016106559 W CN2016106559 W CN 2016106559W WO 2017097105 A1 WO2017097105 A1 WO 2017097105A1
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bone
biological tissue
solution
tissue
bioceramic
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French (fr)
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俞春华
廖化
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俞春华
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Priority to US15/762,023 priority Critical patent/US11471561B2/en
Priority to JP2018536330A priority patent/JP6622416B2/ja
Priority to EP16872297.3A priority patent/EP3338819B1/en
Publication of WO2017097105A1 publication Critical patent/WO2017097105A1/zh

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    • 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
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A—HUMAN NECESSITIES
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    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • 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
    • 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
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    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • 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
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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 belongs to the field of material science, and particularly relates to an injectable composite material for bone repair and a preparation method thereof, and the composite material can be widely applied in the field of orthopedics and plastic surgery.
  • the tissue organ matrix is a three-dimensional framework composed of various complex structural proteins and functional proteins, and contains many other complexes that are active.
  • the main components include collagen fibers, glycoproteins, mucins, etc.
  • Other components include sugars such as aminodextran (hyaluronic acid, chondroitin sulfate), some lipids and growth factors, which can provide an excellent substrate for cell adhesion, and During the degradation process, a large amount of active polypeptide is released, which can promote cell division and differentiation.
  • bioactive tissue matrix As an ideal biomaterial, bioactive tissue matrix has the characteristics required for almost all tissue engineering and regenerative medicine materials, and has been widely used in the repair and regeneration of various tissues and organs.
  • biologically active matrices extracted from various tissues such as the small intestine, skin, liver, spleen, bladder, etc., due to their different macroscopic microstructures, biomechanical properties, in vivo degradation rate, And cell-matrix interactions, used for repair and regeneration of different tissues.
  • all bio-matrix products on the market exist in the form of biofilms, which are roughly classified into dry and water-based. Their applications focus on skin ulcers, peritoneal reconstruction, soft tissue regeneration, wound healing and meningeal wound repair.
  • Calcium phosphate-based bioceramics which are similar to the inorganic components of human bone tissue, have excellent biocompatibility, biodegradability, and bone conduction properties, and have long been used for the preparation and modification of bone repair materials.
  • the currently common calcium phosphate bioceramics include hydroxyapatite, beta phase tricalcium phosphate, calcium hydrogen phosphate and their composite phase ceramics. Numerous studies have shown that calcium phosphate bioceramics can adsorb/enrich the proteins required for bone formation and promote the proliferation and differentiation of osteoblasts, thereby inducing the formation of new bone.
  • the object of the present invention is to provide an injectable composite material for bone repair and a preparation method thereof.
  • the new composite material is mainly composed of these two materials, and the natural biological matrix is biologically active and can be effectively adjusted.
  • the defect induces new bone formation.
  • the materials involved in the present invention also circumvent the shortcomings of a large number of existing bone repairs affecting their clinical efficacy, thus providing a complete bone defect repair solution.
  • An injectable composite for bone repair comprising the following parts by weight of raw materials: 1-7 parts of biological tissue matrix material, 1-9 parts of bioceramic and 2-8 parts of physiological saline or other isotonic aqueous solution.
  • the biological tissue matrix material is an extracellular matrix prepared from mammalian soft tissue and which is mainly composed of collagen fibers and retains its natural crosslinked structure.
  • the soft tissue of the mammal is a soft tissue of a pig, a soft tissue of a cow or a soft tissue of a human body; the soft tissue includes skin, dermis, blood vessels, a diaphragm, a tendon, a ligament, a large intestine, a small intestine, and a nerve tissue.
  • the biological tissue matrix material is microfibrous
  • the diameter of the micro-fibrous biological tissue matrix material is 1-1500 micrometers
  • the ratio of the diameter and the length of the micro-fibrous biological tissue matrix material is It is 0.05-0.95.
  • the bioceramic is replaced with a bioglass, a mineral containing cerium, zinc, magnesium, or silicon or a salt containing cerium, zinc, magnesium, or silicon as a material for the reinforcing phase.
  • the bioceramic is hydroxyapatite [Ca 5 (PO 4 ) 3 OH], ⁇ -tricalcium phosphate, ⁇ -tricalcium phosphate [ ⁇ -Ca 3 (PO 4 ) 2 or ⁇ -Ca 3 ( PO 4 ) 2 ], calcium hydrogen phosphate [CaHPO 4 ], calcium hydrogen phosphate dihydrate [CaHPO 4 ⁇ 2H 2 O], calcium dihydrogen phosphate [Ca(H 2 PO 4 ) 2 ], tetracalcium phosphate [Ca 4 ( PO 4 ) 2 O], octacalcium phosphate [Ca 8 H 2 (PO 4 ) 6 ⁇ 5H 2 O], calcium sulfate [CaSO 4 ] or calcium carbonate [CaCO 3 ].
  • the bioceramic is distributed in the form of particles within the biological tissue matrix material, and the bioceramic particles form a three-dimensional network structure within the biological tissue matrix material.
  • the bioceramic particles have a particle size of from 1 to 500 microns.
  • the preparation method of the injectable bone repair composite material comprises the following steps:
  • tissue raw materials are sterilized by using 0.1% ammonia aqueous solution, and the tissue raw materials are immersed in the solution and shaken slowly for 6-36 hours; thoroughly washed with sterile deionized water, and then rinsed with sterile physiological saline;
  • Tissue breakage crush the sterilized tissue raw materials with a grinder and mix well;
  • the remaining deoxyribonucleic acid is removed by using the deoxyribonuclease solution; and the ⁇ -galactoside is removed by using the ⁇ -galactoside removal solution; using hydrogen peroxide, acetic acid and peracetic acid Mixed solution for virus inactivation;
  • Tissue substrate cleaning The product of step 1.5) is washed three times or more with physiological saline having a mass concentration of 0.9%, and the residue produced by the treatment of step 1.4) is removed;
  • Terminal sterilization sterilization by Co60 gamma ray, X-ray or electron beam, the obtained biological tissue matrix microfibers are sealed in a closed container, and the biological tissue matrix material is stored in a pH neutral buffer.
  • the buffer includes physiological saline and a phosphate buffer;
  • Bioceramics According to the specific application, after mechanical pulverization, high-speed ball milling, and sieving step, bioceramic particles with a particle size of 0.1-500 ⁇ m are obtained, which are then sterilized by high temperature and high pressure to eliminate bacteria and kill viruses;
  • step 1.7 suspending the biological tissue matrix microfiber obtained in step 1.7) in 1-5 times the weight of physiological saline, and shaking well to form a uniform biological tissue matrix microfiber suspension;
  • Terminal sterilization The obtained fluid mixture is sealed in a closed container and sterilized by gamma rays, X-rays or electron beams.
  • the step 1.1) selected tissue material is derived from soft tissue of a mammal including skin, small intestine, large intestine, septum, bladder, stomach and tendon.
  • the step 1.4) the decellularization solution is 1-10% sodium deoxycholate, 2-15 mM ethylenediaminetetraacetic acid, and 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid per liter.
  • the decellularized solution has a pH of 6.8-7.2; the deoxyribonuclease solution contains 0.5-5 mg of deoxyribonuclease per liter, 10-50 mM 4-hydroxyethylpiperazineethanesulfonic acid, and 1-20 mM calcium chloride.
  • the ⁇ -galactoside removal solution contains 0.2-10 mg of ⁇ -galactosidase per liter, 2-40 mM 4-hydroxyethylpiperazineethanesulfonic acid, the ⁇ -half emulsion
  • the glycoside removal solution has a pH of 5.0 to 7.5;
  • the virus inactivated solution is a mixed solution of a concentration of 0.10% hydrogen peroxide, 0.50% acetic acid, and 0.50% peroxyacetic acid.
  • the solution for dispersing the bioceramic particles and the biological tissue matrix material replaces the physiological saline in the step 7) with blood, bone marrow or high-concentration platelet plasma.
  • a method for transporting stem cells to a bone defect the bone marrow mesenchymal stem cells, the adipose stem cells or the stem cells extracted from the blood are formed into a cell suspension, and then mixed with the bone repair composite material obtained by the present invention as a A way of transporting stem cells to a bone defect.
  • the beneficial effects of the present invention are:
  • the present invention relates to an injectable biocomposite material based on biological tissue material and bioceramic and a preparation method thereof.
  • the composite material is organically combined with a biological tissue material and a bioceramic to form an injectable medical material having a three-dimensional scaffold function.
  • the biological tissue material matrix according to the present invention is a microfiber of a natural crosslinked structure, which does not require additional physical or chemical crosslinking, is excellent in biocompatibility, and can be slowly and completely degraded in the body.
  • Biological tissue materials and bioceramics can be combined in a variety of ways.
  • the bioceramic as a reinforcing phase combined with the biological tissue material can provide a good template for bone tissue regeneration in vivo, and can have Effectively induce bone growth.
  • the injectable material prepared by the invention can be used to seamlessly fill bone defects of any size, and a biological agent including bone marrow is added in the process to further enhance its biological activity. Therefore, the material can be widely used for bone loss due to trauma, tumor resection, osteonecrosis and infection.
  • Figure 1 is a differential scanning calorimetry map of biological tissue microfibers
  • Figure 2 is an injectable display of a biological tissue matrix/calcium phosphate composite
  • Figure 3 is a scanning electron micrograph of a biological tissue matrix/calcium hydrogen phosphate composite:
  • Figure 4 shows the implantation of the skull tissue defect repair of the biological tissue matrix/calcium phosphate composite:
  • Figure 5 shows the implantation and repair of the mouse skull by repairing the composite tissue/dicalcium phosphate composite with fresh bone marrow:
  • Figure 6 is a histological observation of the repair of mouse skull after repair of bio-tissue matrix/dicalcium phosphate composite with fresh bone marrow:
  • Example 1 Preparation and Characterization of Biological Tissue Matrix/Calcium Hydrogen Phosphate Composite
  • an injectable composite material is obtained, as shown in Figure 2, which can be applied to the patient's wound with a standard syringe;
  • the composite material is preserved in the form of a paste, and its microstructure is shown in Fig. 3.
  • Scanning electron micrographs show that the calcium hydrogen phosphate microparticles are distributed very uniformly around the acellular matrix microfibers without significant agglomeration;
  • High magnification photographs show that the acellular matrix is composed of a large number of collagen fibers, and the collagen completely retains its natural triple helix structure and is bundled in the composite material.
  • Example 2 Bone defect repair experiment of biological tissue matrix/calcium phosphate composite injectable material
  • the bone defect repair ability of the material was evaluated in a skull defect model of athymic nude mice. After anesthesia in mice (6 cases), the head fur was shaved, a 1 cm opening was opened at the top of the head, and the skin was removed. A dental defect was used to make a 3.5 mm diameter prototype defect on the skull.
  • the material prepared by the present invention was used. After filling the bone defect, the wound was sutured; after six weeks, the human sacrificed the mouse, and the skull was found by the naked eye. The material and the surrounding bone tissue were well combined.
  • X-ray observation showed that a large amount of bone tissue was formed at the defect; bone defect
  • the cross section of the cross section was observed by histological section. It was found that the bone defect was filled with cells, and it was confirmed by bone calcium staining that a large amount of bone calcium was deposited in the defect, and the bone defect was largely repaired, as shown in Fig. 4.
  • large-area bone defect repairs were performed only through biological materials without the addition of any bioactive factors or stem cells.
  • Example 3 Repair of bone defect with biological tissue matrix/calcium phosphate composite injectable material as fresh bone marrow carrier
  • the injectable biological tissue matrix/dicalcium phosphate composite prepared in Example 1 was mixed with freshly extracted mouse whole bone marrow to prepare a paste material; using the mouse model used in Example 2, the mouse whole bone marrow was mixed.
  • the composite composite was filled with bone defects and the wound was sutured.
  • X-ray observation revealed that the entire prototype bone defect had been filled with newly formed bone tissue, and no tissue gap was found at the edge of the bone defect.
  • Micro CT scans show the same result, the entire bone defect has been filled with newly formed bone tissue and a seamless connection between the new bone and the autologous bone.
  • Quantitative analysis of bone formation indicated that the material prepared in Example 1 could form much more bone than the Healos product and its bone density was closer to that of autologous bone tissue (Fig. 5).

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Abstract

一种可注射的骨修复用复合材料，包括生物组织材料和生物陶瓷形成的具有三维支架功能的医用材料。生物组织材料基质为天然交联结构的微纤维，无需额外的物理或化学交联，具备优良的生物相容性，能够在体内缓慢完全降解。复合材料中的生物陶瓷作为增强相，能够与生物组织材料结合，为体内骨组织再生提供模板以有效诱导骨生长。可注射的骨修复用复合材料能够填充任何尺寸的骨缺损，并可以添加包括骨髓在内的生物制剂以增强生物活性。

Description

一种可注射的骨修复用复合材料及其制备方法 技术领域
本发明属于材料科学领域，具体涉及一种可注射的骨修复用复合材料及其制备方法，该复合材料可广泛应用于骨科及整形科领域。
背景技术
受损骨组织修复与重建在世界范围内是整形外科医生需要面对的重要任务，而修复受损的骨组织就需要用到骨移植材料。在世界范围内修复受损骨组织使用的黄金标准为人的自体骨组织，即取自病人自身的健康骨组织。自体骨具有修复骨组织需要的全部特性，比如优异的骨诱导性，骨传导性，成骨性以及安全性。但是，使用自体骨在临床上具有不可避免的缺陷。由于使用自体骨需要从病人身体的供体部位获得骨组织，这额外的手术便增加了手术的时间，费用，以及病人恢复的时间。同时，供体部位的病变及供体部位提供骨组织的有限性都是目前面临的突出问题。
组织器官基质是由各种复杂的结构蛋白质和功能蛋白质构成的三维立体框架，并含有许多有活性的其他复合物。主要成分包括胶原纤维、糖蛋白、黏蛋白等，其他成分有氨基葡聚糖(透明质酸、硫酸软骨素)等糖类、一些脂质和生长因子，可以为细胞黏附提供优异的基底，并且在其降解过程中，会释放大量活性多肽，能够促进细胞分裂及分化。
生物活性组织基质作为一种理想的生物材料，具备几乎一切组织工程及再生医学材料所需的特质，因而已被广泛用于各类组织和器官的修复和再生。目前，从各类组织如小肠，皮肤，肝脏，脾脏，膀胱等提取的生物活性基质，由于其各自不同的宏观微观结构，生物力学性能，体内降解速率，以 及细胞-基质相互作用，被用于不同组织的修复与再生。基本上市场上所有的生物基质产品都是以生物膜的方式存在，大致分为干燥和含水两类。他们的应用主要集中在皮肤溃疡，腹腔膜重建，软组织再生，伤口愈合和脑膜创伤修复。
磷酸钙基生物陶瓷，由于和人体骨组织的无机成分相似，具备优异的生物相容性，生物降解性，和骨传导性能，被长期用于骨修复材料的制备和改性。目前常见的磷酸钙生物陶瓷包括羟基磷灰石，β相磷酸三钙，磷酸氢钙及它们的复合物相陶瓷。大量研究表明磷酸钙生物陶瓷能够吸附/富集骨形成所需的蛋白及，促进成骨细胞增殖和分化，从而诱导新骨的生成。近期研究表明，磷酸钙生物陶瓷降解过程中所释放的钙离子和磷酸根离子，能够积极地影响骨环境中的成骨及破骨细胞，在骨修复及再生过程中扮演重要的角色，因此调控生物陶瓷降解速率也成为一种调控其生物活性的新的途径，具有重大的潜力。
目前市面上几乎所有涉及生物制剂的骨修复材料都使用化学提纯的I型胶原蛋白。这一类胶原蛋白的缺点在于提取过程复杂，蛋白在提取过程中变性，无法发挥其最佳生物学性能；此外，其成分相对单一，无法满足骨修复过程中的复杂的信号通路需求；使用过程中需要使用毒性较大的化学交联剂，易在植入过程中引起免疫及炎症反应。
发明内容
本发明的目的在于提供一种可注射的骨修复用复合材料及其制备方法，这种新的复合材料是以这两种材料为主要成分的，既吸取天然生物基质具有生物活性且能有效调节细胞行为等多方面的优点，又兼备生物陶瓷等无机材料在骨修复过程中的优异成骨能力，具有高度骨诱导性及成骨性的活性生物支架，因此能够通过多重生物学机理快速在骨缺损处诱导新骨生成。同时，本发明所涉及的材料还规避了大量现存骨修复存在的影响其临床疗效的缺点，因此提供了一种完备的骨缺损修复解决方案。
本发明的目的是通过以下技术方案来实现：
一种可注射的骨修复用复合材料，包括以下重量份数的原料：1-7份的生物组织基质材料、1-9份的生物陶瓷和2-8份的生理盐水或其他等渗水溶液。
优选地，所述生物组织基质材料为由哺乳动物软组织制备的、以胶原纤维为主的、保持其天然交联结构的细胞外基质。
优选地，所述哺乳动物的软组织为猪的软组织、牛的软组织或人体的软组织；所述软组织包括皮肤、真皮、血管、隔膜、肌腱、韧带、大肠、小肠和神经组织。
优选地，所述生物组织基质材料呈微纤维状，微纤维状的所述生物组织基质材料的直径为1-1500微米，微纤维状的所述生物组织基质材料的直径和长度的比值分布区间为0.05-0.95。
优选地，用生物玻璃、含锶、锌、镁、或者硅的矿物质或含锶、锌、镁、或者硅的盐代替所述生物陶瓷作为增强相所用材料。
优选地，所述生物陶瓷为羟基磷灰石[Ca5(PO4)3OH]、α-磷酸三钙、β-磷酸三钙[α-Ca3(PO4)2或β-Ca3(PO4)2]、磷酸氢钙[CaHPO4]、二水合磷酸氢钙[CaHPO4·2H2O]、磷酸二氢钙[Ca(H2PO4)2]、磷酸四钙[Ca4(PO4)2O]、磷酸八钙[Ca8H2(PO4)6·5H2O]、硫酸钙[CaSO4]或碳酸钙[CaCO3]。
优选地，所述生物陶瓷以颗粒形式分布于所述生物组织基质材料内，生物陶瓷颗粒在所述生物组织基质材料内形成三维的网状结构。
优选地，所述生物陶瓷颗粒的粒径为1-500微米。
所述的可注射的骨修复用复合材料的制备方法，包括以下步骤：
1)微纤维状的生物组织基质材料的制备，包括以下步骤：
1.1)收集组织原材料，清洗血污后，切割成宽为0.5-2厘米、长为4-8厘米的长条状，-20℃保存；
1.2)消毒灭菌：组织原材料使用质量浓度为0.1％氨水溶液灭菌，将组织原材料浸泡于溶液中缓慢摇动6-36小时；并用无菌去离子水充分清洗，然后用无菌生理盐水漂洗；
1.3)组织破碎：将消毒过的组织原材料用研磨器粉碎，打匀；
1.4)采用脱细胞液脱去细胞后，采用脱氧核糖核酸酶溶液脱去剩余脱氧核糖核酸；并采用α-半乳糖苷去除液脱去α-半乳糖苷；采用双氧水，乙酸和过氧乙酸的混合溶液进行病毒灭活；
1.5)组织基质清洗：用质量浓度为0.9％的生理盐水对步骤1.5)的产物清洗三遍以上，去除步骤1.4)处理产生的残余物；
1.6)清洗：将步骤1.5)的产物用无菌去离子水清洗三次以上；
1.7)终端灭菌：采用Co60伽玛射线、X射线或者电子束进行灭菌，将得到的生物组织基质微纤维密封于密闭容器内保存，生物组织基质材料保存于pH值中性的缓冲液内，所述缓冲液包括生理盐水和磷酸缓冲液；
2)生物陶瓷微颗粒的制备，包括以下步骤：
2.1)生物陶瓷根据具体应用，经机械粉碎，高速球磨，以及过筛步骤后，得到粒径在0.1-500微米范围的生物陶瓷颗粒，再经高温高压消毒，除菌及杀灭病毒；
2.2)将生物陶瓷微颗粒与无菌生理盐水以1:1到1：5的比例混合，并经充分搅拌及震荡，形成均匀的悬浊液；
3)将步骤1.7)得到的生物组织基质微纤维悬浊在1-5倍重量的生理盐水中，充分摇匀，形成均匀的生物组织基质微纤维悬浊液；
4)本发明所述的原料配比，将生物陶瓷颗粒与生物组织基质微纤维混合；
5)将混合物置于涡旋混合器上充分混合一个小时，直至生物陶瓷颗粒完全吸附于生物组织基质微纤维上；
6)用离心法以200～10000rpm转速去除多余水分，并使混合物以固态方式沉积于底部；
7)加入1-5倍混合物重量的生理盐水，置于涡旋混合器上形成均匀的流体状混合物，其中水分含量的质量百分比为20-80％；
8)终端灭菌：将得到的流体状混合物密封于密闭容器内保存，用伽玛射线、X射线或者电子束进行灭菌。
优选地，所述步骤1.1)所选用的组织原材料来源于哺乳动物的软组织，其包括皮肤、小肠、大肠、隔膜、膀胱、胃和肌腱。
优选地，所述步骤1.4)所述脱细胞溶液为每升含1-10％脱氧胆酸钠、2-15mM乙二胺四乙酸以及10-50mM 4-羟乙基哌嗪乙磺酸，所述脱细胞溶液pH值为6.8-7.2；所述脱氧核糖核酸酶溶液每升含0.5-5毫克脱氧核糖核酸酶、10-50mM 4-羟乙基哌嗪乙磺酸、1-20mM氯化钙以及1-20mM氯化镁；所述α-半乳糖苷去除液为每升含0.2-10毫克α-半乳糖苷酶、2-40mM 4-羟乙基哌嗪乙磺酸，所述α-半乳糖苷去除液pH值为5.0-7.5；所述用病毒灭活的溶液为浓度为0.10％双氧水、0.50％乙酸和0.50％过氧乙酸的混合溶液。
进一步地，用于分散生物陶瓷颗粒和生物组织基质材料的溶液用血液、骨髓或高浓度血小板血浆替代所述步骤7)中的生理盐水。
进一步地，一种向骨缺损处输送干细胞的方法，将骨髓间充质干细胞、脂肪干细胞或者提取自血液的干细胞形成细胞悬液，再与本发明所获得的骨修复用复合材料混合，作为一种向骨缺损处输送干细胞的方式。
本发明的有益效果为：本发明涉及一种基于生物组织材料和生物陶瓷的可注射用的生物复合材料及其制备方法。该复合材料以生物组织材料和生物陶瓷有机结合，形成一种具有三维支架功能的可注射用医学材料。本发明所涉及的生物组织材料基质为天然交联结构的微纤维，无需额外涉及物理或化学交联，生物相容性优良，并且可以在体内被缓慢完全降解。生物组织材料和生物陶瓷可以用多种方式结合。本发明的复合材料中，作为增强相的生物陶瓷与生物组织材料结合后，可以为体内骨组织再生提供良好的模板，能有 效地诱导骨生长。本发明制备的可注射材料，可以用以无缝填充任何尺寸的骨缺损，并在过程中添加包括骨髓在内的生物制剂，进一步增强其生物活性。因此，该材料可广泛用于由于创伤，肿瘤切除，骨坏死及感染等造成的骨缺失。
附图说明
图1为生物组织微纤维的差示扫描量热图谱；
图2为生物组织基质/磷酸氢钙复合材料的可注射性展示；
图3为生物组织基质/磷酸氢钙复合材料的扫描电镜照片：
A.复合材料典型形貌；
B&C.磷酸氢钙微颗粒的分布；
D.微纤维内的胶原蛋白三螺旋结构；
图4为生物组织基质/磷酸氢钙复合材料的头盖骨缺损修复的植入实验：
A.修复后的小鼠头盖骨形貌；
B.修复后小鼠头盖骨的X射线观察；
C.骨缺损处组织染色；
图5为生物组织基质/磷酸氢钙复合材料与新鲜骨髓复合后修复小鼠头盖骨的植入修复实验：
A.修复后小鼠头盖骨的X射线观察；
B.修复后小鼠头盖骨的微型CT扫描图谱；
C.微型CT扫描的定量分析；
图6为生物组织基质/磷酸氢钙复合材料与新鲜骨髓复合后修复小鼠头盖骨修复后的组织学观察：
A.Von-Kassa染色(骨钙沉积)；
B.MASSON三色染色法；
C.苏木精—伊红染色法。
具体实施方式
下面通过实施例对本发明作进一步的详细说明，旨在用于说明本发明而 非限定本发明。
实施例1：生物组织基质/磷酸氢钙复合材料的制备及表征
1)取经过Co60伽马射线灭菌的猪皮脱细胞去抗原后的微纤维状生物组织基质材料，以10毫克/毫升的浓度悬浮于0.9％生理盐水中；
2)用无菌去离子水清洗三次以上，所制备的脱细胞基质微纤维参数如图1所示，其平均长宽比为0.05-0.95，其粒径分布为40-1000微米；
3)取磷酸氢钙微颗粒，加入无菌去离子水，制备成10毫克/毫升的均匀悬浊液
4)将磷酸氢钙微颗粒迅速通过粒径40微米的滤膜，除去其中过大的团聚颗粒；
5)将悬浊液在1200rpm离心2分钟，去除上清液；
6)在1g磷酸氢钙微颗粒内加入0.5毫升无菌去离子水，反复漩涡震荡及搅拌，制备成糊状物；
7)以脱细胞微纤维基质与磷酸氢钙微颗粒的质量比为4：6的比例，将磷酸氢钙微颗粒加入脱细胞微纤维基质内；
8)置于涡旋混合器上充分混合一个小时，使二者混合均匀；
9)在1200rpm下离心2分钟，去除上清液；
10)在4重量份脱细胞基质微纤维及6重量份磷酸氢钙微颗粒内，加入2重量份的无菌生理盐水；
11)充分搅拌后，获得可注射的复合材料，如图2所示，可以用标准注射器将该材料应用于患者伤处；
12)复合材料以糊状物形态保存，其微观结构如图3所示，扫描电镜照片表明磷酸氢钙微颗粒非常均匀地分布于脱细胞基质微纤维的周围，没有发生明显团聚；其外，高放大倍数照片显示，脱细胞基质由大量胶原蛋白纤维构成，并且胶原蛋白完整保持了其天然的三螺旋结构，并且成束状分布于复合材料内。
实施例2：生物组织基质/磷酸氢钙复合可注射材料的骨缺损修复实验
用此发明所制备的可注射的生物组织基质/磷酸氢钙复合材料，以无胸腺裸鼠的头骨缺损模型来评估该材料的骨缺损修复能力。小鼠(6例)麻醉后，剃去头部皮毛，在头顶开一个1厘米的口子，拨开皮肤后，用牙科钻头在头盖骨上打一个直径3.5毫米的原型缺损；用本发明制备的材料将骨缺损填充满后，将伤口缝合；六周后，人道牺牲小鼠，肉眼观察修复后头盖骨发现，材料和周围骨组织复合良好，X射线观测表明在缺损处有大量骨组织形成；骨缺损的横切面用组织学切片观察，发现骨缺损处被细胞填满，并通过骨钙染色表明，有大量骨钙在缺损处沉积，骨缺损在很大程度上得到修复，见图4，本实施例是第一次实现仅通过生物材料，而不添加任何生物活性因子或干细胞的大面积骨缺损修复。
实施例3：生物组织基质/磷酸氢钙复合可注射材料作为新鲜骨髓载体的骨缺损修复实验
将实例1制备的可注射的生物组织基质/磷酸氢钙复合材料与新鲜提取的小鼠全骨髓混合，调制成糊状材料；使用实例2中所使用小鼠模型，将混合小鼠全骨髓的混合复合材料填充骨缺损，然后缝合伤口；六周后，X射线观测发现，整个原型骨缺损已被新形成的骨组织填满，没有在骨缺损边缘发现任何组织间隙。微型CT扫描图谱显示同样结果，整个骨缺损已被新形成的骨组织填充，并在新骨和自体骨之间形成无缝连接。骨形成定量分析表明，实施例1制备的材料可以形成远远多于Healos产品的骨量，并且其骨密度更接近于自体骨组织(图5)。组织学观察表明，整个骨缺损已被钙化的骨组织所连接，大量骨小梁已经形成；Masson三色染色说明大量胶原蛋白沉积于骨缺损处，并发现有成骨细胞在新骨表明，参与新骨形成。苏木精—伊红染色法的结果与前面两种染色得到的结论一致，并且没有在骨缺损周围发现有纤维组织形成，新骨和自体骨以之间为骨连接，使得本实例所展示的修复方法为一种可行的临床策略。
上述仅对本发明中的几种具体实施例加以说明，但并不能限定为本发明的保护范围，凡是依据本发明中的设计精神所作出的等效变化或修饰，均应认为落入本发明的保护范围。

Claims (10)

  1. 一种可注射的骨修复用复合材料，其特征在于，包括以下重量份数的原料：1-7份的生物组织基质材料、1-9份的生物陶瓷和2-8份的生理盐水或其他等渗水溶液。
  2. 根据权利要求1所述的可注射的骨修复用复合材料，其特征在于：所述生物组织基质材料为由哺乳动物软组织制备的、以胶原纤维为主的、保持其天然交联结构的细胞外基质。
  3. 根据权利要求1所述的可注射的骨修复用复合材料，其特征在于：所述哺乳动物的软组织为猪的软组织、牛的软组织或人体的软组织；所述软组织包括皮肤、真皮、血管、隔膜、肌腱、韧带、大肠、小肠和神经组织。
  4. 根据权利要求1所述的可注射的骨修复用复合材料，其特征在于：所述生物组织基质材料呈微纤维状，微纤维状的所述生物组织基质材料的直径为1-1500微米，微纤维状的所述生物组织基质材料的直径和长度的比值分布区间为0.05-0.95。
  5. 根据权利要求1所述的可注射的骨修复用复合材料，其特征在于：用生物玻璃、含锶、锌、镁、或者硅的矿物质或含锶、锌、镁、或者硅的盐代替所述生物陶瓷作为增强相所用材料；所述生物陶瓷为羟基磷灰石、α-磷酸三钙、β-磷酸三钙、磷酸氢钙、二水合磷酸氢钙、磷酸二氢钙、磷酸四钙、磷酸八钙、硫酸钙或碳酸钙。
  6. 根据权利要求5所述的可注射的骨修复用复合材料，其特征在于：所述生物陶瓷以颗粒形式分布于所述生物组织基质材料内，生物陶瓷颗粒在所述生物组织基质材料内形成三维的网状结构，其中陶瓷颗粒的粒径为1-500微米。
  7. 权利要求1-6任一项所述的可注射的骨修复用复合材料的制备方法，其特征在于，包括以下步骤：
    1)微纤维状的生物组织基质材料的制备，包括以下步骤：
    1.1)收集组织原材料，清洗血污后，切割成宽为0.5-2厘米、长为4-8厘米的长条状，-20℃保存；
    1.2)消毒灭菌：组织原材料使用质量浓度为0.1％氨水溶液灭菌，将组织原材料浸泡于溶液中缓慢摇动6-36小时；并用无菌去离子水充分清洗，然后用无菌生理盐水漂洗；
    1.3)组织破碎：将消毒过的组织原材料用研磨器粉碎，打匀；
    1.4)采用脱细胞液脱去细胞后，采用脱氧核糖核酸酶溶液脱去剩余脱氧核糖核酸；并采用α-半乳糖苷去除液脱去α-半乳糖苷；采用双氧水，乙酸和过氧乙酸的混合溶液进行病毒灭活；
    1.5)组织基质清洗：用质量浓度为0.9％的生理盐水对步骤1.5)的产物清洗三遍以上，去除步骤1.4)处理产生的残余物；
    1.6)清洗：将步骤1.5)的产物用无菌去离子水清洗三次以上；
    1.7)终端灭菌：采用Co60伽玛射线、X射线或者电子束进行灭菌，将得到的生物组织基质微纤维密封于密闭容器内保存，生物组织基质材料保存于pH值中性的缓冲液内，所述缓冲液包括生理盐水和磷酸缓冲液；
    2)生物陶瓷微颗粒的制备，包括以下步骤：
    2.1)生物陶瓷根据具体应用，经机械粉碎，高速球磨，以及过筛步骤后，得到粒径在0.1-500微米范围的生物陶瓷颗粒，再经高温高压消毒，除菌及杀灭病毒；
    2.2)将生物陶瓷微颗粒与无菌生理盐水以1:1到1：5的比例混合，并经充分搅拌及震荡，形成均匀的悬浊液；
    3)将步骤1.7)得到的生物组织基质微纤维悬浊在1-5倍重量的生理盐水中，充分摇匀，形成均匀的生物组织基质微纤维悬浊液；
    4)按权利要求1所述的原料配比，将生物陶瓷颗粒与生物组织基质微纤维混合；
    5)将混合物置于涡旋混合器上充分混合一个小时，直至生物陶瓷颗粒完全吸附于生物组织基质微纤维上；
    6)用离心法以200～10000rpm转速去除多余水分，并使混合物以固态方式沉积于底部；
    7)加入1-5倍混合物重量的生理盐水，置于涡旋混合器上形成均匀的流体状混合物，其中水分含量的质量百分比为20-80％；
    8)终端灭菌：将得到的流体状混合物密封于密闭容器内保存，用伽玛射线、X射线或者电子束进行灭菌。
  8. 根据权利要求7所述的可注射的骨修复用复合材料的制备方法，其特征在于：所述步骤1.4)所述脱细胞溶液为每升含1-10％脱氧胆酸钠、2-15mM乙二胺四乙酸以及10-50mM 4-羟乙基哌嗪乙磺酸，所述脱细胞溶液pH值为6.8-7.2；所述脱氧核糖核酸酶溶液每升含0.5-5毫克脱氧核糖核酸酶、10-50mM 4-羟乙基哌嗪乙磺酸、1-20mM氯化钙以及1-20mM氯化镁；所述α-半乳糖苷去除液为每升含0.2-10毫克α-半乳糖苷酶、2-40mM 4-羟乙基哌嗪乙磺酸，所述α-半乳糖苷去除液pH值为5.0-7.5；所述用病毒灭活的溶液为浓度为0.10％双氧水、0.50％乙酸和0.50％过氧乙酸的混合溶液。
  9. 根据权利要求7所述的可注射的骨修复用复合材料的制备方法，其特征在于：用于分散生物陶瓷颗粒和生物组织基质材料的溶液用血液、骨髓或高浓度血小板血浆替代所述步骤7)中的生理盐水。
  10. 一种向骨缺损处输送干细胞的方法，其特征在于：将骨髓间充质干细胞、脂肪干细胞或者提取自血液的干细胞形成细胞悬液，再与权利要求7-9任一项所获得的骨修复用复合材料混合，作为一种向骨缺损处输送干细胞的方式。
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