WO2020156388A1 - 干细胞发生器制备骨缺损修复材料的新用途 - Google Patents

干细胞发生器制备骨缺损修复材料的新用途 Download PDF

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WO2020156388A1
WO2020156388A1 PCT/CN2020/073592 CN2020073592W WO2020156388A1 WO 2020156388 A1 WO2020156388 A1 WO 2020156388A1 CN 2020073592 W CN2020073592 W CN 2020073592W WO 2020156388 A1 WO2020156388 A1 WO 2020156388A1
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bone
cells
stem cell
cell generator
stem cells
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PCT/CN2020/073592
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English (en)
French (fr)
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刘昌胜
戴凯
王靖
邓顺书
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华东理工大学
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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
    • A61L27/38—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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • A61L27/50—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
    • 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
    • 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
    • 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
    • A61L2430/38—Materials or treatment for tissue regeneration for reconstruction of the spine, vertebrae or intervertebral discs

Definitions

  • the invention belongs to the cross-field of materials, life and medicine, and relates to a novel application method for bone-like organs formed by stem cell generators in vivo.
  • the bone-like organs produced by stem cell generators in vivo can treat spontaneous or traumatic bone defects or bone defects. deformity.
  • bone determines the human's athletic ability.
  • bones are also important endocrine organs involved in regulating many physiological processes. Damage to the bones will seriously affect the quality of life of the individual.
  • a variety of artificial bone products have been developed, most of the artificial bone products used in clinical applications are still insufficient in activity, which is difficult to meet the clinical treatment of large-scale bone defects caused by diseases or trauma. Even more serious is that with the advent of an aging society, the incidence of bone injury continues to rise.
  • autologous bone graft as the gold standard can achieve good therapeutic effects, but the area and amount of bone taken from autologous bone are limited, and autologous bone removal will cause persistent pain in the donor site.
  • autologous bone removal will cause persistent pain in the donor site.
  • the treatment effect of bone defect is not good.
  • other spontaneous diseases such as varying lengths of limbs, maxillofacial bone loss, and femoral head necrosis, also require bone transplantation.
  • the purpose of the present invention is to provide a new method for treating bone defects caused by various reasons by using bone-like organs produced by the constructed stem cell generator.
  • a stem cell generator is provided.
  • the stem cell generator is implanted into an animal or a human body from a biological material with osteogenic inducing ability or a biological material loaded with active substances and/or cells to produce a stem cell generator.
  • the active substance is bone morphogenetic protein-2, or bone morphogenetic protein-7, other growth factors/polypeptides, growth factor/polypeptide combinations, or combinations thereof that have the ability to induce bone regeneration.
  • mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types of cells with osteogenic differentiation capabilities; help mesenchymal stem cells grow Bone differentiated cells, such as vascular endothelial cells.
  • the biological material used is collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester, poly Carbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate, silicon One of calcium acid, bioglass, decalcified bone matrix or a copolymerization/blend combination thereof.
  • the biomaterial with osteoinductive ability is autologous bone or allogeneic bone.
  • the organoids contain pluripotent stem cells and bone marrow cells.
  • the pluripotent stem cells are hematopoietic stem/progenitor cells (HSC/HPC), mesenchymal stem cells (MSC) or other types of pluripotent stem cells.
  • HSC/HPC hematopoietic stem/progenitor cells
  • MSC mesenchymal stem cells
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1.
  • the number of cells used is 1 ⁇ 10 5 -5 ⁇ 10 8 cells per 100-150 mm 3 of biological material.
  • the in vivo stem cell generator is a bone-like organ that uses biological materials loaded with active substances and/or cells or biological materials with osteoinductive ability to develop in the body.
  • the stem cell generator can grow and develop in the body to form tissues with bone-like organs, and have microscopic bone structure and vascularization characteristics similar to normal bone.
  • the research results of the present invention show that the bone-like organ produced by the in vivo stem cell generator can repair bone defects of critical size, and is expected to be applied to the clinical treatment of severe bone defects, nonunion, and elderly patients with weak regeneration ability.
  • the second aspect of the present invention provides the method for constructing the stem cell generator described in the first aspect, including the following steps:
  • the biological material is a biological material loaded with active substances and/or cells, or a biological material with osteoinductive ability.
  • the active substance is bone morphogenetic protein-2 (Bone Morphogenetic Protein-2, BMP-2), bone morphogenetic protein-7 (Bone Morphogenetic Protein-7, BMP-7), osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis, such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • BMP-2 bone morphogenetic Protein-2
  • BMP-7 bone morphogenetic protein-7
  • osteogenesis Osteogenic peptides or other growth factors that have the ability to induce bone regeneration and angiogenesis such as VEGF, PDG, polypeptides, or growth factor/polypeptide combinations.
  • the bone morphogenetic protein-2 is recombinant bone morphogenetic protein-2.
  • the bone morphogenetic protein-7 is recombinant bone morphogenetic protein-7.
  • the biological material is selected from: collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester , Polycarbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate , Calcium silicate, bioglass, decalcified bone matrix, etc. or their copolymerization/blending combination.
  • the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1.
  • the cells are mesenchymal stem cells
  • the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types have osteogenic differentiation Competent cells; cells that assist mesenchymal stem cells in osteogenic differentiation, such as vascular endothelial cells.
  • the number of cells used is 1 ⁇ 10 5 -5 ⁇ 10 8 cells per 100-150 mm 3 of biological material.
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the organoids have structures and functions similar to in situ bones, including complete bone tissues, bone marrow-like tissues and various functional stem cells.
  • the organoid contains stem cells, and the stem cells are hematopoietic stem/progenitor cells, mesenchymal stem cells, endothelial progenitor cells or other types of pluripotent stem cells.
  • the third aspect of the present invention provides a method for preparing a bone graft/filler, the method comprising the following steps:
  • the bone graft/filler is obtained by producing organoids after development in the body, wherein
  • the biological material is a biological material loaded with bone morphogenetic protein-2, or bone morphogenetic protein-7, or other growth factors/polypeptides or growth factor/polypeptide combinations capable of inducing bone regeneration.
  • the biological material used is collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, polyhydroxy fatty acid ester, poly Carbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate, silicon One of calcium acid, bioglass, decalcified bone matrix or a copolymerization/blend combination thereof.
  • the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1.
  • the animal or human body refers to the muscle pocket, muscle space, intramuscular, subcutaneous, or dorsal muscle of the abdominal cavity of the animal or human.
  • the fourth aspect of the present invention provides the use of the stem cell generator described in the first aspect for preparing bone repair materials or as bone repair materials.
  • the bone repair material is used to treat spontaneous or traumatic bone defects or bone deformities.
  • the bone repair material is used for the following occasions or disease treatment:
  • the fifth aspect of the present invention provides a bone defect repair method, which uses bone-like organs produced by a stem cell generator to replace autologous bone and/or other biological materials for bone defect repair.
  • a method for repairing a critical bone defect is provided, using bone-like organs produced by an in vivo stem cell generator to replace autologous bone and/or other biological materials for bone defect repair.
  • the bone-like organs used for bone repair are derived from biomaterials loaded with growth factors and/or cells, or biomaterials with osteoinductive ability implanted in animal/human muscle pockets or subcutaneous parts, etc. It constitutes a stem cell generator and forms a bone-like organ developed over a period of time, in which the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1, and the number of cells used is 1 ⁇ 10 5 -5 ⁇ 10 8 cells.
  • the growth factor used is bone morphogenetic protein-2 (Bone Morphogenetic Protein-2, BMP-2), or bone morphogenetic protein-7 (Bone Morphogenetic Protein-7, BMP-7) or other Growth factor/polypeptide or growth factor/polypeptide combination capable of inducing bone regeneration.
  • the cells used are adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or other cells with osteogenic differentiation ability or a combination thereof.
  • the biological material used is collagen, gelatin, chitosan, alginic acid, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolide, polylactide, Polyhydroxyalkanoate, polycarbonate, polycaprolactone, polyethylene glycol, polyfumaric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, phosphoric acid One of magnesium, pyrophosphate, calcium silicate, bioglass, decalcified bone matrix, etc. or a copolymerization/blending combination thereof.
  • the resulting bone-like organ has a structure and function similar to autologous bone.
  • the bone-like organ used for bone repair is a new tissue induced in the body by a stem cell generator.
  • the bone defects are various types of bone defects or bone deformities caused by spontaneous or trauma.
  • the bone defect repair method can be used for the following occasions or disease treatment:
  • the disease treatment includes the following diseases or conditions:
  • the present invention proposes to use a stem cell generator in the body to construct an autologously developed bone-like organ in an ectopic state for bone defect treatment.
  • the stem cell generator can provide a bone-like organ that is large, functional, reproducible, and non-immunogenic.
  • Osteogenic active proteins represented by bone morphogenetic protein (Bone Morphogenetic Protein, BMP) have the effect of inducing ectopic bone formation, and the bone-like organs induced by biomaterials have the structure and function similar to autologous bone.
  • the bone-like organs constructed by this method are rich in blood vessel tissue and bone marrow tissue. Pathological sections also showed that the resulting bone-like organs were similar in structure to autologous cortical and cancellous bone.
  • large-volume bone-like organs can be constructed in both young and old mice, and critical-size skull defect repair experiments show that the constructed bone-like organ can quickly repair critical-size skull defects, and has a good therapeutic effect.
  • This method has the potential to replace traditional autologous bone grafts, as an innovative treatment technique applied to the treatment of bone defects.
  • Figure 1 shows the overall experimental flow chart of the embodiment.
  • Figure 2 shows a macroscopic view of bone-like organs produced by stem cell generators formed in young and old mice 3 weeks after the material was implanted.
  • Figure 3 shows the H&E stained sections of bone-like organs produced by stem cell generators formed in young and old mice 3 weeks after the material was implanted.
  • Figure 4 shows TRAP stained sections of bone-like organs produced by stem cell generators formed in young mice and old mice 3 weeks after the material was implanted.
  • Figure 5 shows CD31 immunofluorescence slices of bone-like organs produced by stem cell generators formed in young and old mice 3 weeks after the material was implanted.
  • Figure 6 shows a typical flow cytometry diagram of bone-like organs produced by stem cell generators formed in young and old mice 3 weeks after the material was implanted.
  • Figure 7 shows the flow cytometry statistics of the bone-like organs produced by the stem cell generators formed in young and old mice 3 weeks after the material was implanted.
  • Figure 8 shows the experimental process diagram of bone-like organs produced by a 3W stem cell generator in the repair of autologous skull defects in young mice.
  • Figure 9 shows the 2W, 4W, and 6W ⁇ CT images of the bone-like organ produced by the 3W stem cell generator used in the repair of autologous skull defects in young mice.
  • Fig. 10 shows the 2W, 4W, and 6W repair percentage statistics of the bone-like organ produced by the 3W stem cell generator used in the repair of autologous skull defects in young mice.
  • Figure 11 shows the BV/TV statistics of 2W, 4W, and 6W after the bone-like organ produced by the 3W stem cell generator was used in the repair of autologous skull defect in young mice.
  • Figure 12 shows the BMD statistics of 2W, 4W, and 6W after the bone-like organ produced by the 3W stem cell generator is used in the repair of autologous skull defects in young mice.
  • Figure 13 shows the 2W, 4W, and 6W H&E stained sections of the bone-like organ produced by the 3W stem cell generator used in the repair of autologous skull defects in young mice.
  • Figure 14 shows the 2W, 4W, and 6W TRAP stained sections of the bone-like organ produced by the 3W stem cell generator used in the repair of autologous skull defects in young mice.
  • Figure 15 shows the experimental process diagram of bone-like organs produced by a 3W stem cell generator in the repair of autologous skull defects in old mice.
  • Figure 16 shows a 6W ⁇ CT image of bone-like organs produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • Figure 17 shows the 6W repair percentage statistics of bone-like organs produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • Figure 18 shows a 6W BV/TV statistical graph of bone-like organs produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • Figure 19 shows a 6W BMD statistical graph of bone-like organs produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • Figure 20 shows a 6W H&E stained section image of a bone-like organ produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • Figure 21 shows a 6W TRAP stained section image of a bone-like organ produced by a 3W stem cell generator used in the repair of autologous skull defects in old mice.
  • biomaterials loaded with active substances or biomaterials that are themselves active can form stem cell generators in the body and develop into bone-like organs.
  • This bone-like organ not only has cell components and tissue structure similar to autologous bone, but also has the function of bone tissue, and can be used as an effective substitute for bone graft/filler represented by autologous bone to treat bone defects.
  • the in vivo experimental study of the present invention shows that the bone-like organ developed by the stem cell generator formed after the material is loaded with BMP-2 has similar structure and function to autologous bone, and can replace autologous bone for bone repair.
  • the pathological section showed that the bone marrow structure and bone structure of this bone-like organ and autologous bone were similar.
  • Immunofluorescence staining and flow cytometry showed that bone-like organs are rich in blood vessels.
  • the constructed stem cell generator can quickly repair critical skull defects in young or old mice. This method provides a new way to obtain bone-like organs developed from autologous body.
  • the resulting bone-like organs can effectively repair bone defects, and hopefully become a new source of clinical autologous bone transplantation to cope with the increasing incidence of bone defects in an aging society. Treatment of disease.
  • the stem cell generator produced by the method of the present invention develops a bone-like organ with a structure and function similar to autologous bone, and can replace autologous bone for repair or filling of various bone defects/losses.
  • the present invention can construct a stem cell generator by implanting active materials subcutaneously or in a muscle pocket, and the obtained stem cell generator can be used as a bone-like organ after trimming or other suitable operations, and applied to the treatment of bone defects/losses and other orthopedic diseases.
  • the stem cell generator of the present invention can be developed into a bone-like organ for the treatment of various spontaneous or traumatic bone defects/losses and other orthopedic diseases.
  • rhBMP-2 human bone morphogenetic protein-2
  • Example 2 Young mice develop bone-like organs in vivo.
  • Example 1 Using the active material described in Example 1, implanted under the skin of the back of an 8-week-old C57BL/6 male mouse to form a stem cell generator. After 3 weeks of rearing, the bone-like organs developed by the stem cell generator were taken out, and part of it was used to take macro photos , H&E slices and flow cytometry, the other part is used for autologous skull defect transplantation treatment.
  • Example 3 The bone-like organs developed in old mice.
  • Example 1 Using the active material described in Example 1, implanted 52-week-old C57BL/6 male mice under the skin on the back to form a stem cell generator. After 3 weeks of feeding, the bone-like organs developed by the stem cell generator were taken out, and some were used to take macro photos , H&E slices and flow cytometry, the other part is used for autologous skull defect transplantation treatment.
  • Figure 1 shows the flow chart of the entire autologous skull defect transplantation treatment. It can be seen from the flow chart that the stem cell generators implanted in young/old mice develop after 3 weeks to form bone-like organs, and some are used for further characterization , The other part is used to treat autologous skull defects.
  • the macro photograph of Figure 2 shows the stem cell generators formed in young/old mice in Example 2 and Example 3.
  • the developed bone-like organs are dark red, indicating that they are rich in blood cells and vascular networks, and their tissue morphology Also similar to autologous bone.
  • TRAP Temporal Resistant Acid Phosphatase
  • the CD31 immunofluorescence staining shown in Figure 5 proves that the bone-like organ developed by the stem cell generator has a rich vascular network.
  • This bone-like organ is a highly vascularized bionic autologous bone, which can be used as an effective treatment for ischemic bone defects. Bone graft.
  • the flow cytometry detection of Figure 6 and Figure 7 shows that the proportion of CD31 + cells in the bone-like organs constructed subcutaneously in mice of different ages is the same as that of the corresponding in situ bone marrow of mice, that is, as the mice age, CD31 + cells contained ratio decreased, but the proportion of CD31 + cells in aged mice was significantly lower than the proportion of CD31 + cells in young mice, suggesting that the bone marrow vascular density in situ in aged mice is less than in young mice. This phenomenon was not found in bone-like organs, suggesting that the bone-like organs constructed in old mice have the characteristics of young bones.
  • the purpose of this example is to evaluate the therapeutic effect of the bone-like organ produced by the stem cell generator made in the same young mouse on the 5 mm diameter defect of the young mouse's skull.
  • the active material used is the rhBMP-2 containing scaffold described in Example 1;
  • the bone-like organs are produced by the development of stem cell generators after implantation in animals in Example 2.
  • Preparation of bone-like organs implant the scaffold containing rhBMP-2 described in Example 1 subcutaneously to produce bone-like organs after three weeks of development, remove the bone-like organs, and trim the resulting 5mm diameter with a 5mm inner diameter punch Cylindrical bone-like organs.
  • Fig. 8 is an experimental process diagram of bone-like organs developed by stem cell generators for treating autologous skull defects in young mice. It can be seen from the figure that after the constructed stem cell generator is trimmed, the bone-like organ developed in the body can well cover the defect and achieve the purpose of rapid repair.
  • Fig. 9 is a 2W, 4W, and 6W ⁇ CT scan of a bone-like organ developed by a stem cell generator for the treatment of autologous skull defects in young mice. It can be seen from the figure that the bone-like organs produced by the development of stem cell generators can quickly repair bone defects.
  • the quantitative data in Figure 10 further shows that the bone-like organs developed by the stem cell generator can achieve nearly 100% repair coverage of the bone defect.
  • Figures 11 and 12 show that the BV/TV (bone volume/total volume) and BMD (bone mineralization density) of the bone-like organ repair site produced by the development of the stem cell generator are significantly higher than those of the blank control group, showing the development of the stem cell generator The bone-like organs have a better repair effect.
  • BV/TV bone volume/total volume
  • BMD bone mineralization density
  • This example illustrates that the bone-like organ developed by the stem cell generator constructed from the active material described in Example 1 has a structure and function similar to that of autologous bone, and can repair autologous skull defects well, and is hopefully applied to various Repair of bone-like defects.
  • the purpose of this example is to evaluate the effect of a stem cell generator made in the same old mouse on the treatment effect of the bone-like organ produced by the development of the 5mm diameter defect of the skull of the old mouse.
  • the active material used is the rhBMP-2 containing scaffold described in Example 1;
  • the bone-like organs are produced by the development of stem cell generators when implanted into animals in the third embodiment.
  • Preparation of bone-like organs implant the scaffold containing rhBMP-2 described in Example 1 subcutaneously to produce bone-like organs after three weeks of development, remove the bone-like organs, and trim the resulting 5mm diameter with a 5mm inner diameter punch Cylindrical bone-like organs.
  • Fig. 15 is an experimental process diagram of a bone-like organ produced by a stem cell generator for the treatment of autologous skull defects in old mice. It can be seen from the figure that the bone-like organ produced by the constructed stem cell generator can well cover the defect part and fill the bone defect part after trimming, and strive to achieve the purpose of rapid repair.
  • Fig. 16 is a 6W ⁇ CT scan of a bone-like organ produced by a stem cell generator for the treatment of autologous skull defects in young mice. The figure shows that the bone-like organ produced by the stem cell generator can quickly repair the bone defect.
  • the quantitative data in Figure 17 further shows that the bone-like organs produced by the stem cell generator can achieve nearly 100% repair coverage of the bone defect.
  • Figures 18 and 19 show that the bone-like organ produced by the stem cell generator through development, the BV/TV (bone volume/total volume) and BMD (bone mineralization density) of the repair site are significantly higher than the blank control group, showing the stem cell generator
  • the bone-like organs produced by development have a better repair effect.
  • the stem cell generator constructed from the active material described in this example can be developed to have a structure and function similar to autologous bone, and can be used as a bone-like organ; for elderly patients who are difficult to repair critical bone defects It can also perform effective bone repair. This method is expected to be applied to the repair of bone defects in elderly patients with poor autologous bone condition.

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Abstract

干细胞发生器制备骨缺损修复材料的新用途，干细胞发生器由本身具有成骨诱导能力的生物材料或者负载活性物质和/或细胞的生物材料植入动物或人体内经发育后产生类器官而形成，活性物质为骨形态发生蛋白-2、或骨形态发生蛋白-7、其他具有诱导骨再生能力的生长因子/多肽、生长因子/多肽组合、或它们的组合。细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充值干细胞成骨分化的细胞，如血管内皮细胞等。该干细胞发生器用于制备骨修复材料，用于治疗自发或创伤所致各类骨缺损或骨畸形。

Description

干细胞发生器制备骨缺损修复材料的新用途 技术领域
本发明属于材料、生命和医学的交叉领域，涉及新型的体内干细胞发生器形成的类骨器官的应用方法，使用体内干细胞发生器产生的类骨器官，能够治疗自发或创伤所致骨缺损或骨畸形。
背景技术
骨骼作为人体主要的力学承载体系，决定着人类的运动能力。同时骨骼还作为重要的内分泌器官参与调控许多生理过程。骨骼受到损伤，将严重影响个体的生活质量。尽管已研发出多种人工骨产品，但临床应用的人工骨产品大多活性仍不足，难以满足疾病或外伤所致的大段骨缺损的临床治疗。更为严峻的是，随着老龄化社会的到来，骨损伤的发生率持续上升。对于这类骨缺损的治疗，自体骨移植作为金标准能够取得良好的治疗效果，但自体骨取骨区域及取骨量有限，并且自体取骨会导致供区持续性疼痛，对于二次骨折或骨缺损的治疗效果不佳。此外，其他一些自发性的疾病，如四肢长短不一，颌面部骨缺失、股骨头坏死等也都需要进行骨移植治疗。
为了应对自体骨移植的不利因素，各种有机、无机生物材料被开发用于骨缺损治疗。但绝大部分生物材料普遍不具有或仅具有很低的生物活性，对于大段骨缺损或缺血性骨坏死的治疗效果不佳，特别是在针对老年病人的治疗中尤为明显。而同种异体骨作为另一种具有较好治疗效果的自体骨替代物又存在着病原体污染、免疫原性的可能性。
发明内容
本发明的目的是提供一种利用构建的干细胞发生器产生的类骨器官治疗各类原因导致的骨缺损的新方法。
本发明的第一方面，提供一种干细胞发生器，所述干细胞发生器由本身具有成骨诱导能力的生物材料或者负载活性物质和/或细胞的生物材料植入动物或人体内经发育后产生类器官而形成，所述活性物质为骨形态发生蛋白-2、或骨形态发生蛋白-7、其他具有诱导骨再生能力的生长因子/多肽、生长因子/多肽组合、或它们的组合,所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
在另一优选例中，所用的生物材料为胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质中的一种或其共聚/共混组合。
所述本身具有成骨诱导能力的生物材料为自体骨或异体骨。
在另一优选例中，所述类器官中包含多能干细胞、骨髓细胞。
在另一优选例中，所述多能干细胞为造血干/祖细胞(HSC/HPC)、间充质干细胞(MSC)或其他种类多能干细胞。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、 皮下、或腹腔背侧肌肉。
在另一优选例中，所述活性物质与生物材料的质量比范围为0.0001-1:1。
在另一优选例中，所用细胞的接种数量为每100-150mm 3生物材料接种1×10 5-5×10 8个细胞。
体内干细胞发生器是采用负载活性物质和/或细胞的生物材料或具有成骨诱导能力的生物材料，能在体内发育形成的类骨器官。该干细胞发生器能在体内生长发育形成具有类骨器官的组织，具有与正常骨相似的微观骨结构及血管化特征。本发明研究结果表明，使用体内干细胞发生器产生的类骨器官，能够修复临界尺寸的骨缺损，有望应用于严重骨缺损、骨不连、再生能力弱的老龄患者等的临床治疗。
本发明的第二方面，提供第一方面所述的干细胞发生器的构建方法，包括以下步骤：
(1)将生物材料植入动物或人体内；
(2)经体内发育后产生类器官从而形成所述干细胞发生器，其中，
所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
在另一优选例中，所述活性物质为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)、骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)、成骨多肽(Osteogenic peptides)或其他具有诱导骨再生、血管生成能力的生长因子如VEGF，PDG、多肽或生长因子/多肽组合。
在另一优选例中，所述骨形态发生蛋白-2为重组骨形态发生蛋白-2。
在另一优选例中，所述骨形态发生蛋白-7为重组骨形态发生蛋白-7。
在另一优选例中，所述生物材料选自：胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
在另一优选例中，所述活性物质与生物材料的质量比范围为0.0001-1:1。
在另一优选例中，所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
在另一优选例中，所用细胞的接种数量为每100-150mm 3生物材料接种1×10 5-5×10 8个细胞。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
本发明中，所述的类器官具有与原位骨相似的结构与功能，包括完整的骨组织、骨髓样组织及各种功能干细胞。
在另一优选例中，所述类器官中包含干细胞，所述干细胞为造血干/祖细胞、间充质干细胞、内皮祖细胞或其他种类多能干细胞。
本发明的第三方面，提供一种制备骨移植物/填充物的方法，所述方法包括以下步骤：
(1)将生物材料植入动物或人体内；
(2)经体内发育后产生类器官从而获得所述骨移植物/填充物，其中，
所述生物材料为负载骨形态发生蛋白-2，或骨形态发生蛋白-7，或其他具有诱导骨再生能力的生长因子/多肽或生长因子/多肽组合的生物材料。
在另一优选例中，所用的生物材料为胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质中的一种或其共聚/共混组合。
在另一优选例中，所述活性物质与生物材料的质量比范围为0.0001-1:1。
在另一优选例中，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
本发明的第四方面，提供第一方面所述的干细胞发生器的用途，用于制备骨修复材料或用作骨修复材料。
在另一优选例中，所述骨修复材料用于治疗自发或创伤所致骨缺损或骨畸形。
在另一优选例中，所述骨修复材料用于以下场合或病症治疗：
(1)用于创伤导致的骨损伤、骨不连、骨延迟愈合的植骨治疗；
(2)用于骨肿瘤、骨质疏松、骨畸形等疾病产生的骨缺损和脊柱融合治疗；
(3)用于再生能力弱的老龄患者骨缺损的治疗；
(4)其他需要骨移植的疾病治疗。
本发明的第五方面，提供一种骨缺损修复方法，采用干细胞发生器产生的类骨器官替代自体骨和/或其他生物材料用于骨缺损修复。
在另一优选例中，提供一种临界骨缺损修复方法，采用体内干细胞发生器产生的类骨器官替代自体骨和/或其他生物材料用于骨缺损修复。
在另一优选例中，用于骨修复的类骨器官来自于由负载生长因子和/或细胞的生物材料、或具有成骨诱导能力的生物材料植入动物/人肌袋或皮下等部位，构成干细胞发生器，经一段时间发育形成的类骨器官，其中活性物质与生物材料的质量比范围为0.0001-1:1，所用细胞的接种数量为1×10 5-5×10 8个。
在另一优选例中，所用的生长因子为骨形态发生蛋白-2(Bone Morphogenetic Protein-2，BMP-2)，或骨形态发生蛋白-7(Bone Morphogenetic Protein-7，BMP-7)或其他具有诱导骨再生能力的生长因子/多肽或生长因子/多肽组合。
在另一优选例中，所用的细胞为脂肪来源间充质干细胞、骨髓来源间充质干细胞或其他具有成骨分化能力的细胞或其组合。
在另一优选例中，所用的生物材料为具有良好生物相容性的胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质等中的一种或其共聚/共混组合。
在另一优选例中，所产生的类骨器官具有与自体骨相似的结构与功能。
在另一优选例中，用于骨修复的类骨器官为干细胞发生器在体内诱导产生的新组 织。
在另一优选例中，所述骨缺损为自发或创伤所致各类骨缺损或骨畸形。
在另一优选例中，所述骨缺损修复方法，可用于以下场合或病症治疗：
(1)用于创伤导致的骨损伤、骨不连、骨延迟愈合的植骨治疗；
(2)用于骨肿瘤、骨质疏松、骨畸形等疾病产生的骨缺损和脊柱融合治疗；
(3)用于再生能力弱的老龄患者骨缺损的治疗；
(4)其他需要骨移植的疾病治疗。
在另一优选例中，所述的疾病治疗包括以下病症或状况：
(1)创伤或疾病导致的骨缺损/丢失；
(2)早期缺血性股骨头坏死的保髋治疗；
(3)骨质疏松，脊柱压缩性骨折等的填充；
(4)其他需要骨移植/填充的疾病治疗。
本发明提出利用体内干细胞发生器在异位构建由自体发育而成的类骨器官，用于骨缺损治疗。干细胞发生器能够提供大体积、有功能、可重复产生、且无免疫原性的类骨器官。
以骨形态发生蛋白(Bone Morphogenetic Protein，BMP)为代表的成骨活性蛋白具有异位诱导成骨的作用，其在生物材料协助下诱导产生的类骨器官具有类似自体骨的结构与功能。利用该方法构建的类骨器官中含有丰富的血管组织、骨髓组织。病理切片也表明产生的类骨器官与自体的皮质骨与松质骨结构相似。本发明中，在年轻与老年小鼠体内都能够构建大体积的类骨器官，且临界尺寸颅骨缺损修复实验表明，构建的类骨器官能够快速修复临界尺寸颅骨缺损，具有良好的治疗效果。这一方法有希望替代传统的自体骨移植，作为一种创新的治疗手法应用于骨缺损的治疗。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一赘述。
附图说明
图1显示了实施例的总体实验流程图。
图2显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的宏观图。
图3显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的H&E染色切片图。
图4显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的TRAP染色切片图。
图5显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的CD31免疫荧光切片图。
图6显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的流式细胞术典型图。
图7显示了材料植入3周后在年轻小鼠和老年小鼠体内形成的干细胞发生器所产生的类骨器官的流式细胞术统计图。
图8显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复的实验过程图。
图9显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的μCT图。
图10显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的修复百分比统计图。
图11显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的BV/TV统计图。
图12显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的BMD统计图。
图13显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的H&E染色切片图。
图14显示了体内发育3W的干细胞发生器所产生的类骨器官用于年轻小鼠自体颅骨缺损修复治疗后2W、4W、6W的TRAP染色切片图。
图15显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复的实验过程图。
图16显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的μCT图。
图17显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的修复百分比统计图。
图18显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的BV/TV统计图。
图19显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的BMD统计图。
图20显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的H&E染色切片图。
图21显示了体内发育3W的干细胞发生器所产生的类骨器官用于老年小鼠自体颅骨缺损修复治疗后6W的TRAP染色切片图。
具体实施方式
本申请的发明人经过广泛而深入的研究，发现由负载活性物质的生物材料或本身具有活性的生物材料，在体内能够形成干细胞发生器，并发育形成类骨器官。该类骨器官不仅具有与自体骨相似的细胞成分和组织结构，并且具有骨组织的功能，能够作为以自体骨为代表的骨移植物/填充物的有效替代物而应用于骨缺损治疗。
本发明的体内实验研究显示，材料负载BMP-2后植入体内形成的干细胞发生器发育产生的类骨器官，与自体骨具有相似的结构与功能，可替代自体骨用于骨修复。病理切片显示，该类骨器官与自体骨的骨髓结构与骨结构相似。免疫荧光染色及流式细胞术检测表明类骨器官中含有丰富的血管。利用构建的干细胞发生器能够快速修复年轻或老年小鼠临界颅骨缺损。该方法提供一种新的自体发育而成的类骨器官的获取方式，产生的类骨器官能够有效修复骨缺损，有希望成为临床自体骨移植的新来源以应对老年化社会愈加高发的骨缺损疾病的治疗。
本发明所述方法所产生的干细胞发生器，发育产生具有类似自体骨的结构与功能的类骨器官，能够取代自体骨用于各类骨缺损/丢失的修复或填充。
本发明可通过皮下或肌袋等部位植入活性材料构建干细胞发生器，获得的干细胞发生器可经过修剪或其他合适的操作后作为类骨器官，应用于骨缺损/丢失等骨科疾病的治疗。
综上所述，基于本发明的发现，预期可将本发明的干细胞发生器发育形成类骨器官，用于各类自发或创伤导致的骨缺损/丢失等骨科疾病的治疗。
具体而言，可应用于以下方面：
1.各类自发或创伤导致的骨缺损/丢失；
2.早期缺血性股骨头坏死的保髋治疗；
3.骨质疏松、脊柱压缩性骨折的填充治疗；
4.其他相关骨科疾病的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例一植入材料的制备。
将30μg真核或原核表达系统合成的重组人骨形态发生蛋白-2(rhBMP-2)加入明胶海绵(5mm直径×5mm厚，10mg重)，经冻干后形成含有生长因子的活性材料。
实施例二年轻小鼠体内发育形成类骨器官。
使用实施例一所述的活性材料，植入8周龄C57BL/6雄性小鼠背部皮下，形成干细胞发生器，饲养3周后取出干细胞发生器发育形成的类骨器官，一部分用于拍摄宏观照片、H&E切片以及流式细胞术检测，另一部分用于自体颅骨缺损移植治疗。
实施例三老年小鼠体内发育形成类骨器官。
使用实施例一所述的活性材料，植入52周龄C57BL/6雄性小鼠背部皮下，形成干细胞发生器，饲养3周后取出干细胞发生器发育形成的类骨器官，一部分用于拍摄宏观照片、H&E切片以及流式细胞术检测，另一部分用于自体颅骨缺损移植治疗。
图1显示了整个自体颅骨缺损移植治疗的流程图，由流程图可知，植入年轻/老年小鼠体内形成的干细胞发生器经过3周发育后，形成类骨器官，一部分被用于进一步的表征，另一部分被用于治疗自体颅骨缺损。
图2的宏观照片显示实施例二与实施例三中年轻/老年小鼠体内形成的干细胞发生器，经发育形成的类骨器官显深红色，提示其中含有丰富的血细胞及血管网络，其组织形态亦与自体骨相似。
图3所示H&E染色切片及图4所示TRAP(抗酒石酸酸性磷酸酶)染色切片共同表明：干细胞发生器发育形成的类骨器官具有与自体骨类似的微结构与功能。
图5所示的CD31免疫荧光染色证明干细胞发生器发育形成的类骨器官具有丰富的 血管网络，该类骨器官是一种高度血管化的仿生自体骨，可作为缺血性骨缺损治疗的有效骨移植物。
图6、图7的流式细胞术检测表明：不同鼠龄小鼠皮下构建的类骨器官中的CD31 +细胞比例变化趋势与对应鼠龄原位骨髓相同，即随着小鼠的衰老，其中含有的CD31 +细胞比例呈下降趋势，但老年小鼠的CD31 +细胞比例显著低于年轻小鼠的CD31 +细胞比例，提示老年小鼠原位骨髓中的血管密度低于年轻小鼠。而这一现象在类骨器官中未发现，提示在老年小鼠体内构建的类骨器官具有年轻骨的特征。
实施例四
体内构建干细胞发生器产生的类骨器官用于年轻小鼠自体颅骨缺损治疗：
该实施例的目的是评价同一年轻小鼠体内制造的干细胞发生器产生的类骨器官对该年轻小鼠颅骨5 mm直径缺损的治疗效果。
所用的活性材料为实施例一所述的含rhBMP-2的支架；
类骨器官为实施例二植入动物体内经干细胞发生器发育所产生。
方法：
采用SPF级C57BL/6小鼠，雄性，8周龄，随机分组。实验分组如下：
组别 空白组 类骨器官
数量 6 6
类骨器官制备：皮下植入实施例一所述的含rhBMP-2的支架，经三周发育后产生类骨器官，取下类骨器官，并使用5mm内径打孔器修剪得到的5mm直径的圆柱形类骨器官。
自体类骨器官移植：将小鼠麻醉后，使用手术刀将小鼠头部皮肤切开，暴露颅骨，使用5mm外径的环形锯制造小鼠5mm颅骨缺损，同时将上一步制得的自体类骨器官移植到颅骨缺损处。缝合皮肤后将小鼠置于恒温台保温直至小鼠苏醒。按照既定时间点取样检测。空白组小鼠仅制造5mm颅骨缺损，之后便缝合伤口。
图8为干细胞发生器发育产生的类骨器官，用于治疗年轻小鼠自体颅骨缺损的实验过程图。由图可知，构建的干细胞发生器经过修剪后，在体内发育产生的类骨器官能够很好的将缺损部位覆盖，达到快速修复的目的。
图9为干细胞发生器发育产生的类骨器官，用于治疗年轻小鼠自体颅骨缺损后2W、4W、6W的μCT扫描图。由图可见干细胞发生器发育产生的类骨器官能够快速修复骨缺损部位。
图10定量数据进一步表明干细胞发生器发育产生的类骨器官对骨缺损部位能够达到接近100％的修复覆盖度。
图11、12表明干细胞发生器发育产生的类骨器官修复部位的BV/TV(骨体积/总体积)及BMD(骨矿化密度)皆显著高于空白对照组，显示了干细胞发生器发育产生的类骨器官具有更好的修复效果。
图13所示H&E染色切片图、图14所示TRAP染色切片图共同表明干细胞发生器发育产生的类骨器官，移植之后能够在缺损部位存活并与缺损边缘有效整合，达到良好的修复效果。
该实施例说明，实施例一所述活性材料所构建的干细胞发生器发育产生的类骨器官，具有与自体骨类似的结构与功能，能够对自体颅骨缺损进行良好的修复，有希望应用于各类骨缺损的修复。
实施例五
体内构建干细胞发生器发育产生的类骨器官，用于老年小鼠自体颅骨缺损治疗：
该实施例的目的是评价同一老年小鼠体内制造的干细胞发生器，经发育产生的类骨器官对该老年小鼠颅骨5mm直径缺损的治疗效果。
所用的活性材料为实施例一所述的含rhBMP-2的支架；
类骨器官为实施例三植入动物体内经干细胞发生器发育所产生。
方法：
采用SPF级C57BL/6小鼠，雄性，52周龄，随机分组。实验分组如下：
组别 空白组 类骨器官
数量 6 6
类骨器官制备：皮下植入实施例一所述的含rhBMP-2的支架，经三周发育后产生类骨器官，取下类骨器官，并使用5mm内径打孔器修剪得到的5mm直径的圆柱形类骨器官。
自体类骨器官移植：将小鼠麻醉后，使用手术刀将小鼠头部皮肤切开，暴露颅骨，使用5mm外径的环形锯制造小鼠5mm颅骨缺损，同时将上一步制得的自体类骨器官移植到颅骨缺损处。缝合皮肤后将小鼠置于恒温台保温直至小鼠苏醒。按照既定时间点取样检测。空白组小鼠仅制造5mm颅骨缺损，之后便缝合伤口。
图15为干细胞发生器经发育产生的类骨器官，用于治疗老年小鼠自体颅骨缺损的实验过程图。由图可知，构建的干细胞发生器经发育产生的类骨器官，经过修剪后，能够很好地将缺损部位覆盖，较好地填充骨缺损部位，力图达到快速修复的目的。
图16为干细胞发生器经发育产生的类骨器官，用于治疗年轻小鼠自体颅骨缺损后6W的μCT扫描图。由图可见干细胞发生器经发育产生的类骨器官，能够快速修复骨缺损部位。
图17定量数据进一步表明，干细胞发生器经发育产生的类骨器官对骨缺损部位能够达到接近100％的修复覆盖度。
图18、19表明干细胞发生器经发育产生的类骨器官，修复部位的BV/TV(骨体积/总体积)及BMD(骨矿化密度)皆显著高于空白对照组，显示了干细胞发生器经发育产生的类骨器官具有更好的修复效果。
图20所示H&E染色切片图、图21所示TRAP染色切片图共同表明，干细胞发生器经发育产生的类骨器官移植之后，能够在缺损部位存活并与缺损边缘有效整合，达到良好的修复效果。
该实施例说明，本实施例所述活性材料所构建的干细胞发生器，经发育能产生具有与自体骨类似的结构与功能，可用作类骨器官；对于难以进行临界骨缺损修复的老年患者亦能够进行有效的骨修复。该方法有望应用于各类自体骨状态不佳的老年患者的骨缺损修复。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种干细胞发生器，其特征在于，所述干细胞发生器由本身具有成骨诱导能力的生物材料或者负载活性物质和/或细胞的生物材料植入动物或人体内经发育后产生类器官而形成，所述活性物质为骨形态发生蛋白-2、或骨形态发生蛋白-7、其他具有诱导骨再生能力的生长因子/多肽、生长因子/多肽组合、或它们的组合,所述细胞为间充质干细胞，所述间充质干细胞为骨髓来源间充质干细胞、脂肪来源间充质干细胞或其他来源间充质干细胞；其他类型具有成骨分化能力的细胞；辅助间充质干细胞成骨分化的细胞，如血管内皮细胞等。
  2. 如权利要求1所述的干细胞发生器，其特征在于，所用的生物材料为胶原、明胶、壳聚糖、海藻酸、透明质酸、细菌纤维素、聚乳酸、聚乙交酯、聚丙交酯、聚羟基脂肪酸酯、聚碳酸酯、聚己内酯、聚乙二醇、聚富马酸、羟基磷灰石、硫酸钙、磷酸三钙、磷酸四钙、磷酸八钙、偏磷酸钙、磷酸镁、焦磷酸盐、硅酸钙、生物玻璃、脱钙骨基质中的一种或其共聚/共混组合。
  3. 如权利要求1所述的干细胞发生器，其特征在于，所述类器官中包含多能干细胞、骨髓细胞。
  4. 如权利要求1所述的干细胞发生器，其特征在于，所述多能干细胞为造血干/祖细胞(HSC/HPC)、间充质干细胞(MSC)或其他种类多能干细胞。
  5. 如权利要求1所述的干细胞发生器，其特征在于，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
  6. 一种制备骨移植物/填充物的方法，其特征在于，所述方法包括以下步骤：
    (1)将生物材料植入动物或人体内；
    (2)经体内发育后产生类器官从而获得所述骨移植物/填充物，其中，
    所述生物材料为负载活性物质和/或细胞的生物材料、或本身具有成骨诱导能力的生物材料。
  7. 如权利要求6所述的方法，其特征在于，所述动物或人体内是指动物或人的肌袋、肌肉间隙、肌肉内、皮下、或腹腔背侧肌肉。
  8. 如权利要求1所述的干细胞发生器的用途，其特征在于，用于制备骨修复材料。
  9. 如权利要求1所述的干细胞发生器的用途，其特征在于，所述骨修复材料用于治疗自发或创伤所致骨缺损或骨畸形。
  10. 如权利要求1所述的干细胞发生器的用途，其特征在于，所述骨修复材料用于以下场合或病症治疗：
    (1)用于创伤导致的骨损伤、骨不连、骨延迟愈合的植骨治疗；
    (2)用于骨肿瘤、骨质疏松、骨畸形等疾病产生的骨缺损和脊柱融合治疗；
    (3)用于再生能力弱的老龄患者骨缺损的治疗；或
    (4)其他需要骨移植的疾病治疗。
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