WO2020147181A1 - 一种梯度矿化骨细胞外基质材料的制备方法 - Google Patents

一种梯度矿化骨细胞外基质材料的制备方法 Download PDF

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WO2020147181A1
WO2020147181A1 PCT/CN2019/077754 CN2019077754W WO2020147181A1 WO 2020147181 A1 WO2020147181 A1 WO 2020147181A1 CN 2019077754 W CN2019077754 W CN 2019077754W WO 2020147181 A1 WO2020147181 A1 WO 2020147181A1
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bone
deionized water
hours
extracellular matrix
gradient
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PCT/CN2019/077754
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English (en)
French (fr)
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林贤丰
柳世杰
王艺芸
芦雅智
范顺武
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浙江大学医学院附属邵逸夫医院
浙江狄赛生物科技有限公司
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Priority to EP19910842.4A priority Critical patent/EP3785740B1/en
Priority to JP2020543063A priority patent/JP7118161B2/ja
Priority to US17/040,999 priority patent/US11684696B2/en
Publication of WO2020147181A1 publication Critical patent/WO2020147181A1/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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • 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
    • A61L27/50—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
    • A61L2300/40—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
    • 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 technical field of bone tissue repair and regeneration, and specifically relates to a material of gradient mineralized bone extracellular matrix derived from natural tissue and a preparation method thereof.
  • Bone regeneration and repair is a long process, especially the more serious bone defects are difficult to repair by themselves.
  • bone graft materials have been considered as a promising alternative treatment.
  • natural bone tissue especially natural cancellous bone extracellular matrix (Cancellous bone matrix, CBM)
  • CBM cancellous bone extracellular matrix
  • CBM cancellous bone extracellular matrix
  • it also contains a lot of minerals (including Ca 2+ , PO4 -3 , Mg 2+ plasma).
  • mineral ions Ca 2+ , PO4 -3 , Mg 2+, etc.
  • the mineral content also affects the three-dimensional structure, porosity, micro-biomechanical properties of the material, and the above-mentioned material characterization has a significant regulatory effect on bone repair and regeneration [6].
  • material hardness and elastic modulus can significantly promote the behavioral induction and bone repair ability of osteoblasts [7].
  • Biocollagen mineralization is also considered by more and more researchers to significantly promote the process of osteogenesis and bone tissue regeneration [8].
  • this patent proposes a material that can better simulate the regeneration microenvironment required in the natural bone repair process, and better promote regeneration and repair of damaged bone tissue at the "right time and in the right way".
  • a bone ECM material with specific and precise gradient mineralization is prepared.
  • this material also has rich biologically active components , Good biomechanical properties, three-dimensional microstructure, and a certain degree of mineral enrichment (Ca 2+ , PO4 -3, etc.), have a very good promotion effect on achieving excellent regeneration of new bone tissue and vascularization [11- 13].
  • the present invention can also provide a new accurate gradient mineralization material system based on natural bone matrix for the research of biomineralization materials.
  • the present invention provides a material of gradient mineralized bone extracellular matrix (DCBM) derived from natural tissues (Decellularized Cancellous Bone Matrix, DCBM) and a preparation method thereof.
  • DCBM gradient mineralized bone extracellular matrix
  • the method for preparing a gradient mineralized bone extracellular matrix material of the present invention specifically includes: performing immunogenic removal treatment on bone tissue derived from natural tissues, that is, decellularization, and subjecting the obtained decellularized bone to gradient demineralization treatment; Obtain a gradient mineralized bone extracellular matrix material.
  • Step 1 Take any bone tissue from the whole body of the mammal, take out the bone with a drill, and cut into a cylindrical bone block with a scalpel;
  • Step 2 After sampling, rinse with sterile saline for 2 hours, then sterilize by irradiation; the amount of radiation of the irradiation is 5-40w
  • Step 3 Rinse with deionized water containing protease inhibitors to remove blood, adipose tissue and other impurities; for the deionized water containing protease inhibitors, the concentration of protease inhibitor is 10-50 KIU/ml; wash with deionized water The number of times is 2-6 times, and each washing time is 3-20 minutes;
  • Step 4 Put the bone pieces into the embedding box, and put the embedding box into the acetone-containing deionized water solution and shake for 1-4 hours; for the acetone-containing deionized water solution, the volume ratio of acetone to deionized water is preferable 10%-20%;
  • Step 5 Put the deionized water solution containing tributyl phosphate in the embedding box and shake for 1-4 hours; the volume ratio of the tributyl phosphate to deionized water is 1 %-5%
  • Step 6 Put the embedding cassette in a deionized water solution containing protease inhibitors, shake on a shaker for 24-48 hours at 4°C, then freeze-thaw with liquid nitrogen for 2-6 cycles, each cycle is -80°C ⁇ 37°C, the shaking speed is preferably 50 ⁇ 300rpm;
  • Step 7 Put the embedding cassette in the buffer containing Triton X-100, and shake on a constant temperature shaker for 24 hours; the concentration of Triton X-100 is 0.5 to 5%;
  • Step 8 In the buffer solution containing SDS, shake on a constant temperature shaker for 36 hours; the concentration of the SDS is 0.5-10%;
  • Step 9 In a PBS buffer solution containing potassium chloride at a concentration of 0.1-1M, shake with a shaker at 100rpm for 2-12 hours at a temperature of 4°C;
  • Step 10 In PBS buffer containing potassium iodide, shake with a shaker at 100 rpm for 2-12 hours at a temperature of 4°C; the concentration of potassium iodide in deionized water is 1-1.5M
  • Step 11 In a buffer solution containing EDTA2Na and NaOH at 4°C-10°C, perform gradient demineralization treatments for 4, 8, 12, and 24 hours respectively; to obtain 100%, 90%, 60%, and 0% Bone ECM material with degree of mineralization;
  • Step 12 Perform irradiation sterilization again on the obtained material
  • steps 4 to eleven after each step is completed, rinse with deionized water for 6 hours between steps.
  • the concentration of the protease inhibitor is 20-40 KIU in the deionized water containing the protease inhibitor.
  • the volume ratio of acetone to deionized water is preferably 13%-18%.
  • the volume ratio of tributyl phosphate to deionized water is preferably 2% to 5%.
  • the shaker speed is 30-180rpm.
  • the concentration of Triton X-100 is 0.5-3%.
  • the concentration of the SDS is 0.5 to 5%.
  • the concentration of the potassium chloride in deionized water is 0.3-1M.
  • the concentration of the potassium iodide in deionized water is 1-1.4M.
  • the invention also provides a bone ECM scaffold material with specific gradient mineralization.
  • the degree of mineralization of the gradient mineralized bone ECM material is 90% and 60%.
  • the source of the material is pig scapula.
  • the present invention prepares natural tissue-derived gradient mineralized bone extracellular matrix material with better regeneration and repair effects through low-temperature, accurate and rapid super-gradient demineralization treatment, which has low immunogenicity and is also rich in biologically active components.
  • Good biomechanical properties, three-dimensional microstructure, and a certain degree of mineral enrichment (Ca 2+ , PO4 -3, etc.), have a good effect on achieving excellent regeneration of new bone tissue and vascularization. It can be used to repair bone defects and bone non-union problems caused by various clinical causes.
  • it can also provide a new accurate gradient mineralization material system based on natural bone matrix for the research of biomineralization materials.
  • the present invention Compared with the existing non-demineralized or completely demineralized natural bone matrix products, the present invention (90% and 60% mineralized bone ECM materials) has significant progress in:
  • a certain degree of mineral enrichment (Ca 2+ , PO4 -3, etc.) not only preserves the biomechanical properties and three-dimensional microstructure of natural bone ECM scaffolds, but more importantly, it is important for osteogenesis and blood vessels.
  • Collagen mineralization plays a positive role in the formation and early stage of fracture (hematoma organizing stage), which increases the colonization and adhesion of cells and promotes the induction of cell differentiation.
  • Natural tissue-derived gradient mineralization (90% and 60% salinity) bone extracellular matrix materials show greater performance in promoting mesenchymal stem cell differentiation and bone formation than undemineralized or completely demineralized bone matrix materials potential.
  • AB The porosity of the bone ECM material decreases after the degree of mineralization decreases, while the stiffness of the deformation resistance index decreases;
  • C The change of the front force difference of the bone ECM material with different mineralization degrees (scale bar 1 ⁇ m);
  • D The micromechanics of the bone ECM material Property change graph, as the degree of mineralization decreases, the Young's modulus of the material surface decreases, the amount of deformation increases, and the adhesion force decreases.
  • FIG. 7 Shown in Figure 7.
  • FIG 8. Shown in Figure 8.
  • Bone ECM materials were implanted into the bone defect model for 2 and 4 weeks. MicroCT scans and reconstructed three-dimensional composite images. Among them, the bone repair ability of 90% and 60% mineralization materials is better than 100% and 0%;
  • Bone ECM materials were implanted into the bone defect model for 2 and 4 weeks.
  • the H&E staining of the bone defect and the quantitative map of the repaired area indicated that different mineralization degrees of bone ECM materials can promote bone defect repair, while 90% and 60% of the The repairing ability of materials with a degree of chemical conversion is better than that of 100% and 0% materials.
  • the invention provides a gradient mineralized bone extracellular matrix material derived from natural tissues and a preparation method thereof.
  • Atomic force microscopy also showed that 100%, 90%, 60%, and 0% mineralization of bone ECM material surface collagen is more exposed ( Figure 3C-D).
  • Example 4 Transplantation of bone marrow mesenchymal stem cells into 90% and 60% mineralized bone ECM materials
  • the bone mesenchymal stem cells are cultured for 1-5 days with the extract of bone ECM material with a specific degree of mineralization (the extract is derived from the bone ECM material with a degree of mineralization of 90% and 60%).
  • the results show that the cells grow well , Which shows that the material is safe and non-toxic (Figure 5A).
  • Bone marrow mesenchymal stem cells were transplanted into bone ECM materials with 100%, 90%, 60%, and 0% mineralization for 3 days. Observation showed that the cells adhered to the scaffold under a confocal microscope (Figure 4A). In the scanning electron microscope 1000 times the field of view, it is observed that the bone ECM materials with 90% and 60% mineralization levels have more bone marrow mesenchymal stem cells than the bone ECM materials with less than 100% mineralization levels, which shows that the patented material can be effective Promote cell adhesion and colonization and cell proliferation. The number of cells in the bone ECM material with a degree of mineralization of 0% is relatively small, as shown in Figure 4B.
  • the MAPK signaling pathway plays a role in the Ca 2+ -mediated process of bone differentiation. It was found that compared with the bone ECM material group with 100% and 0% mineralization, the bone ECM with 90% and 60% mineralization The expression of MEK-1 in the cells was up-regulated (Figure 6B). In summary, 90% and 60% mineralized bone ECM materials, especially 90% mineralized bone ECM materials, show better promotion than 100% and 0% mineralized bone ECM materials. Osteogenic differentiation of bone marrow mesenchymal stem cells.
  • Example 5 In vivo experiments of 90% and 60% mineralized bone ECM materials significantly promote the repair of early bone defects
  • Example 6 Bone ECM materials with a degree of mineralization of 90% and 60% significantly promote the formation of blood vessels in the bone defect in vivo experiment
  • the bone ECM material group with 90% and 60% mineralization has the thickness of neovascularization of 4.86 ⁇ 0.15 ⁇ m and 5.07 ⁇ 0.20 ⁇ m, respectively.
  • mature new blood vessels were mainly formed, among which the amount of bone ECM materials with 100%, 90%, 60%, and 0% mineralization were 3.5 ⁇ 0.6/500 ⁇ m 2 and 5.8 ⁇ 1.0/500 ⁇ m 2 respectively.
  • the blood vessel area is 41.26 ⁇ 5.69 ⁇ m 2 , 69.92 ⁇ 11.26 ⁇ m 2 , 60.76 ⁇ 8.66 ⁇ m 2 , and 24.87 ⁇ 8.18 ⁇ m 2 ; thickness is compared with transplantation At 2 weeks, there was a corresponding increase in each group, among which 90% of the mineralized bone ECM material (12.18 ⁇ 0.54 ⁇ m) and 60% of the mineralized bone ECM material (12.18 ⁇ 0.32 ⁇ m) were still higher than 100% The mineralized bone ECM material group (11.83 ⁇ 0.49 ⁇ m) and the 0% mineralized bone ECM material group (9.68 ⁇ 1.83 ⁇ m).
  • Example 4 The rest of the subsequent operations are performed with reference to the method of Example 1 to obtain a gradient mineralized bone extracellular matrix material.
  • Example 8 Gradient mineralized bone extracellular matrix material and its preparation method research and evaluation
  • Example 4 The rest of the subsequent operations are performed with reference to the method of Example 1 to obtain a gradient mineralized bone extracellular matrix material.
  • the gradient mineralized bone extracellular matrix materials obtained in Examples 7-9 were subjected to histological evaluation, calcium and phosphorus content detection, surface collagen morphology and content detection, and mechanical detection. The results were similar to those of the gradient mineralized bone extracellular matrix materials in Example 1. The results of the matrix materials are similar, which indicates that the preparation of gradient mineralized bone extracellular matrix materials with similar effects can be achieved by adjusting the reagents and treatment time determined after the above optimization. Histological evaluation, cell culture experiment, and in-vivo repair experiment evaluation of the material were carried out. The tests showed that the 90% and 60% gradient mineralized bone extracellular matrix materials obtained in Examples 7-9 have good repair and regeneration. It can be used as a safe, reliable, effective and rapid biomaterial to promote the repair and regeneration of muscle defects and lesions clinically.
  • the present invention provides a natural tissue-derived gradient mineralized bone extracellular matrix material and its preparation method, which can better promote cell adhesion, induce the differentiation and proliferation of colonized cells, and promote blood vessel formation.
  • the formation of new bone trabeculae achieves a better repair effect and a smaller inflammation reaction. It is a very promising bone defect graft material.

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Abstract

一种梯度矿化骨细胞外基质材料及其制备方法，对天然组织来源的骨组织进行免疫原性去除处理，即脱细胞化，将获得的脱细胞骨进行梯度脱矿处理；获得梯度矿化骨细胞外基质材料。不仅很好的保留了天然骨ECM支架的生物力学特性和三维微观结构，更对成骨、血管生成以及骨折初期胶原矿化起到了积极作用，增加了细胞的定植粘附，促进了细胞的分化诱导。天然组织来源的梯度矿化骨细胞外基质材料在促进间充质干细胞分化和成骨方面比未脱矿或完全脱矿骨基质材料表现出更大的潜力。

Description

[根据细则26改正20.05.2019]　一种梯度矿化骨细胞外基质材料的制备方法 技术领域
本发明属于骨组织修复及其再生技术领域，具体涉及一种天然组织来源的梯度矿化骨细胞外基质的材料及其制备方法。
背景技术
骨再生修复是一个漫长的过程，尤其是较严重的骨缺损大多难以自行修复。目前骨移植材料已被认为是一种有广泛运用前景的替代治疗。其中天然骨组织，尤其是天然松质骨细胞外基质(Cancellous bone matrix，CBM)，含有丰富的胶原、生长因子等促进细胞生长和骨组织修复的物质。不仅如此，其还含有大量的矿物质(包含Ca 2+、PO4 -3、Mg 2+等离子)。而近年来，越来越多的研究表明矿物离子(Ca 2+、PO4 -3、Mg 2+等)对新生骨组织修复、成骨相关血管生成、胶原矿化等具有极其重要的调控促进作用[1-5]。另外，矿物质含量也同时影响着材料的三维立体结构、孔隙率、微观生物力学特性等，而上述材料表征对骨修复再生具有明显的调控作用[6]。例如，研究证明材料硬度及弹性模量对成骨细胞的行为诱导及骨修复能力有明显促进作用[7]。而生物胶原矿化也被越来越多的学者认为可以明显的促进成骨作用和骨组织再生过程[8]。
虽然近年来，基于天然骨基质来源的生物材料逐渐被制备出来，并且初步运用临床。但由于早期骨修复理论机制研究的不足，造成目前普遍骨基质材料都有一定的缺陷。例如，有人报道通过完全去除天然骨基质的细胞成分制备了松质骨ECM(Extracellular matrix)支架材料，以达到降低 免疫原性而促进骨修复的目的[9]。但由于骨修复的过程需要经历血肿机化期、骨痂形成期、骨痂塑形期等阶段，而生物材料的应用往往是在血肿机化期前后，这就造成了成熟致密、已完全矿化的骨ECM材料无法很好的与新生骨进行融合促进再生。也有文献报道，采用完全脱钙骨基质材料用于骨组织的修复再生，传统处理工艺中采用强酸和长期EDTA-2Na浸泡的脱钙[10]，往往会对天然骨ECM支架材料造成不可逆行破坏，并且导致生物力学性能下降、矿物质和生长活性因子的丢失以及三维微观结构的改变，严重影响了支架的生物再生修复活性。
因此，基于上述背景，本专利提出一种能够更好模拟天然骨修复过程中所需的再生微环境材料，对损伤骨组织在“正确的时间和正确的方式”给予更好促进再生修复。采用天然组织来源骨材料，利用去除免疫原性技术和精确低温快速超声软化方式，制备出特定精确梯度矿化的骨ECM材料，该材料除了具备低免疫原性，还具有丰富的生物活性组分、良好的生物力学特性、三维微观结构以及特定程度的矿物富集(Ca 2+、PO4 -3等)，对实现新生骨组织的优异再生、血管化长入具有很好的促进效果[11-13]。并且，本发明还能对生物矿化材料研究提供一种全新的基于天然骨基质的精确梯度矿化材料体系。
发明内容
本发明针对现有技术的不足，提供了一种天然组织来源的梯度矿化骨细胞外基质的材料(Decellularized cancellous bone matrix,DCBM)及其制备方法。
本发明一种梯度矿化骨细胞外基质材料的制备方法，具体为：对天然组织来源的骨组织进行免疫原性去除处理，即脱细胞化，将获得的脱细胞骨进行梯度脱矿处理；获得梯度矿化骨细胞外基质材料。
具体包括以下步骤：
步骤一：取哺乳动物全身任意骨组织，用钻孔器取出骨，用手术刀切成圆柱状骨块；
步骤二：取材完毕后用无菌生理盐水冲洗2小时，后经辐照灭菌；所述辐照的辐射量为5～40w
步骤三：用含蛋白酶抑制剂的去离子水漂洗，去除血液、脂肪组织和其他杂质；所述的含蛋白酶抑制剂的去离子水中，蛋白酶抑制剂浓度为10-50KIU/ml；去离子水洗涤次数为2-6次，每次洗涤时间为3-20分钟；
步骤四：将骨块分装入包埋盒中，将包埋盒放入含丙酮的去离子水溶液震荡1-4小时；所述含丙酮的去离子水溶液，丙酮与去离子水的体积比优选为10％-20％；
步骤五：将包埋盒中放入含磷酸三丁酯的去离子水溶液震荡1-4小时；所述含磷酸三丁酯的去离子水溶液，磷酸三丁酯与去离子水的体积比为1％-5％
步骤六：将包埋盒中放入在含蛋白酶抑制剂的去离子水溶液中，在4℃条件下，摇床震荡24-48小时然后用液氮冻融2～6个循环，每个循环为-80℃～37℃，摇床速度优选为50～300rpm；
步骤七：将包埋盒放入含Triton X-100的缓冲液中，恒温摇床振荡24h；所述的Triton X-100的浓度为0.5～5％；
步骤八：在含SDS的缓冲液中，恒温摇床振荡36h；所述SDS的浓度为0.5-10％；
步骤九：在含氯化钾浓度为0.1-1M的PBS缓冲液中，且在4℃温度下 用摇床100rpm震荡2-12小时；
步骤十：在含碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡2-12小时；所述的碘化钾在去离子水中的浓度为1-1.5M
步骤十一：在含EDTA2Na和NaOH的缓冲液中，4℃-10℃的条件下，进行梯度脱矿处理分别4，8，12,24h；得到100％、90％、60％、0％的矿化程度的骨ECM材料；
步骤十二：将得到材料再次进行辐照灭菌；
在步骤四～步骤十一中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时。
作为优选，所述的含蛋白酶抑制剂的去离子水中，蛋白酶抑制剂浓度为20-40KIU。
作为优选，所述含丙酮的去离子水溶液，丙酮与去离子水的体积比优选为13％-18％。
作为优选，所述含磷酸三丁酯的去离子水溶液，磷酸三丁酯与去离子水的体积比优选为2％-5％。
作为优选，摇床速度为30-180rpm。
作为优选，所述的Triton X-100的浓度为0.5～3％。
作为优选，所述SDS的浓度为0.5～5％。
作为优选，所述的氯化钾在去离子水中的浓度0.3-1M。
作为优选，所述的碘化钾在去离子水中的浓度为1-1.4M。
本发明还提供了一种特定梯度矿化的骨ECM支架材料。
优选的，梯度矿化骨ECM材料矿化度为90％和60％。
优选的，材料来源是猪肩胛骨。
本发明的有益效果：
本发明通过低温、精确快速超生梯度脱矿处理，制备出再生修复效果更佳的天然组织来源的梯度矿化骨细胞外基质的材料，具备低免疫原性，还具有丰富的生物活性组分、良好的生物力学特性、三维微观结构以及特定程度的矿物富集(Ca 2+、PO4 -3等)，对实现新生骨组织的优异再生、血管化长入具有很好的促进效果。可用于修复临床上各类病因造成的骨缺损，骨不愈合等骨再生障碍问题。并且，还能对生物矿化材料研究提供一种全新的基于天然骨基质的精确梯度矿化材料体系。
相比现有不脱矿或完全脱矿天然骨基质产品，本发明(90％和60％矿化度骨ECM材料)显著的进步在于：
1)特定低温、精确部分脱矿处理扩大了骨基质材料的孔隙率及其表面胶原的暴露程度，有效释放生长因子，很好的提高了材料对细胞粘附度，调控细胞再生相关基因和蛋白上调。
2)另一方面，特定程度的矿物富集(Ca 2+、PO4 -3等)不仅很好的保留了天然骨ECM支架的生物力学特性和三维微观结构，更重要的是对成骨、血管生成以及骨折初期(血肿机化期)胶原矿化起到了积极作用，增加了细胞的定植粘附，促进了细胞的分化诱导。
3)天然组织来源的梯度矿化(90％和60％矿化度)骨细胞外基质材料在促进间充质干细胞分化和成骨方面比未脱矿或完全脱矿骨基质材料表现出更大的潜力。
附图说明
图1所示。A-B.新型梯度矿化骨细胞外基质材料的脱细胞及矿化大体外观图；C.材料脱细胞后DNA含量明显减低，几乎不含细胞成分和免疫原 性物质；D.经梯度脱矿后获得100％、90％、60％、0％矿化程度的材料；E.EDS分析见材料矿化程度降低后钙磷含量明显下降；F.Masson染色见材料矿化程度降低后矿化不成熟胶原纤暴露增多，且经过脱细胞后各组细胞均完全去除，无明显免疫原性物质。
图2所示。A.扫描电镜500倍视野下见骨ECM材料矿化程度降低后表面更光整，钙结节分布减少，5000倍视野下见矿化程度降低后胶原纤维暴露增多；B.免疫组织化学染色图显示矿化程度降低后材料中BMP-2暴露逐渐增多。
图3所示。A-B.骨ECM材料矿化程度降低后孔隙率升高，而抵抗形变能力指标刚度下降；C.不同矿化程度骨ECM材料的锋力差变化图(比例尺1μm)；D.骨ECM材料微观力学性质变化图，随着矿化程度降低，材料表面杨氏模量降低，形变量增高，黏附力降低。
图4所示。骨ECM材料种植细胞后细胞黏附效果。A-B.DAPI染色及电镜5000倍视野下见90％、60％矿化程度骨ECM材料的细胞黏附能力，细胞伸展良好，而100％，0％骨材料细胞黏附能力下降，细胞伸展欠佳。
图5所示。A.用不同浓度的骨ECM材料浸提液培养骨间充质干细胞1-5天，提示90％、60％矿化程度骨ECM材料对间充质干细胞增殖活性无明显影响；B.qPCR检测不同矿化程度骨ECM材料复植间充质干细胞后，细胞中Col-1α、ALP、BMP-2基因相对表达情况，其中90％、60％矿化程度材料的成骨诱导蛋白表达量更高。
图6所示。A-B.免疫荧光染色检测复植于不同矿化程度骨ECM材料2和4周后，间充质干细胞中BMP-2、MEK-1基因相对表达水平，其中90％、 60％矿化程度材料中BMP-2及MEK-1表达较多，提示本材料促BMP-2表达可能与适宜浓度钙离子刺激相关。
图7所示。A.骨ECM材料植入兔股骨骨缺损模型2和4周后，骨缺损部位的Masson三色染色图，提示90％、60％矿化程度的材料促骨小梁和血管生长的能力更优；B.免疫组织化学染色显示在植入后2周缺损部位中Ⅱ型胶原表达情况，提示骨ECM材料促骨修复可能涉及软骨内成骨过程。
图8所示。A.骨ECM材料植入骨缺损模型2周后，骨缺损部位内血管生成情况，可见90％、60％矿化程度材料促血管生成能力更优；B.骨ECM材料植入骨缺损模型2周后，骨缺损部位内VEGFA蛋白表达水平，可见90％、60％矿化程度材料促VEGFA蛋白表达能力更佳；C.骨ECM材料植入骨缺损部位2和4周后新生血管数量、面积、厚度量化图，可见90％、60％矿化程度骨促血管生长及成熟能力较强。
图9所示。A-B.骨ECM材料植入骨缺损模型2和4周后，MicroCT扫描和重建的三维复合图像，其中90％、60％矿化程度材料骨修复能力优于100％及0％；C.骨ECM材料植入骨缺损模型2和4周后新生骨小梁数量及厚度，其中90％、60％矿化程度材料促骨小梁长入能力更优。
图10所示。A-B.骨ECM材料植入骨缺损模型2和4周后，骨缺损部位的H&E染色及修复面积量化图，提示不同矿化程度骨ECM材料均可促骨缺损修复，而90％、60％矿化程度材料修复能力优于100％、0％材料。
具体实施方法
本发明提供了一种天然组织来源的梯度矿化骨细胞外基质的材料及其制备方法。
下面结合实施例对本发明提供的一种天然组织来源的梯度矿化骨细胞外基质的材料及其制备方法进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1猪肩胛骨特定部分脱矿松质骨ECM材料制备
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含20KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作10min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含15％丙酮的去离子水溶液，10度恒温震荡2小时；
5)将包埋盒放入5ml含2％的磷酸三丁酯的去离子水溶液，10度恒温震荡4小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床50rpm震荡24小时然后用液氮冻融3个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡6小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡6小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
实施例2猪肩胛骨90％和60％矿化程度的骨ECM材料的检测
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以40w辐射量进行辐照灭菌；
3)用含10KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作20min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含15％丙酮的去离子水溶液，10度恒温震荡4小时；
5)将包埋盒放入5ml含2％的磷酸三丁酯的去离子水溶液，10度恒温震荡4小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床100rpm震荡24小时然后用液氮冻融3个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡6小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡6小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
14)检测脱细胞处理后的材料的DNA含量极少(图1C)；
15)检测100％、90％、60％、0％矿化程度的骨ECM材料的矿化含量(以钙离子含量为例)，具体90％矿化程度的骨ECM材料(4h脱矿处理)组为4.58±0.01mmol/mg，60％矿化程度的骨ECM材料(8h脱矿处理)组为3.26±0.38mmol/mg，0％矿化程度的骨ECM材料(12h脱矿处理)后几乎不含钙离子(100％矿化程度的骨ECM材料(即脱矿前材料)钙离子含量为4.99±0.22mmol/mg)(图1D)；
16)检测100％、90％、60％、0％矿化程度的骨ECM材料的孔隙率，随着脱矿时间，孔隙率增大(图3A)；
17)检测100％、90％、60％、0％矿化程度的骨ECM材料的刚度，刚度是一个抗压力、抗形变的指标，随着矿化程度的降低对应矿化度材料的刚度依次下降，90％、60％、0％矿化程度的骨ECM材料(脱矿4h、8h、12h)分别为5.71±0.46N/mm,3.68±0.18N/mm,and 2.53±1.62N/mm(100％矿化程度的骨ECM材料(即脱矿前材料)刚度为21.55±1.62N/mm)(图 3B)。
实施例3猪肩胛骨90％和60％矿化程度的骨ECM材料的表征
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以5w辐射量进行辐照灭菌；
3)用含50KIU/ml蛋白酶抑制剂的去离子水漂洗2次，每次操作10min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含10％丙酮的去离子水溶液，10度恒温震荡1小时；
5)将包埋盒放入5ml含5％的磷酸三丁酯的去离子水溶液，10度恒温震荡3小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床50rpm震荡36小时然后用液氮冻融2个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡6小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡6小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
14)扫描电镜(SEM)观察，得到100％、90％、60％、0％矿化程度的骨ECM材料的超微结构特征，与其他组相比，100％和90％矿化程度的骨ECM材料(即脱矿0和4h)表面更加粗糙，孔隙更小。此外，胶原纤维的结构和排列在四组间也有差异。在100％矿化程度的骨ECM材料(即脱矿前材料)组中，大部分胶原纤维被表面覆盖，而在90％和60％矿化程度的骨ECM材料(脱矿4h和8h)表面，胶原纤维暴露较多，排列规律良好，从而为细胞产生更多的粘附驻留位点。然而，由于0％矿化程度的骨ECM材料(12h脱矿)，胶原原纤维的结构更加紊乱，致密性更差，不利于细胞的驻留(图2A)。
15)原子力显微镜(AFM)同样显示100％、90％、60％、0％矿化程度的骨ECM材料表面胶原暴露更多(图3C-D)。
16)免疫组织化学染色显示90％和60％矿化程度的骨ECM材料表面BMP-2表达量增高(图2B)。
17)利用EDS法测定了C、P、Ca(碳、磷、钙)在100％、90％、60％、0％矿化程度的骨ECM材料特定区域的比例(图1E、F)。以C为参照物，根据选择性电极法测得的Ca浓度，Ca密度随着材料矿化度的降低而更加分散。此外，磷含量的变化与钙的变化是一致的，随着矿化度的降低，分散度增大。AFM对100％、90％、60％、0％矿化程度的骨ECM材料的超微观力学性能进行了评价。随着天然骨ECM来源矿化度的不同，我们发现90％和60％矿化程度的骨ECM材料拥有更多的原纤维暴露在表面，从而为细胞粘附提供了许多 RGD配体。
实施例4：骨髓间充质干细胞移植于90％和60％矿化程度的骨ECM材料
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含20KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作10min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含15％丙酮的去离子水溶液，10度恒温震荡2小时；
5)将包埋盒放入5ml含2％的磷酸三丁酯的去离子水溶液，10度恒温震荡4小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床震荡24小时然后用液氮冻融3个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡6小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡6小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
14)用特定矿化程度的骨ECM材料的浸提液(浸提液来源于90％和60％矿化程度的骨ECM材料)培养骨间充质干细胞1-5天，结果显示细胞生长良好，说明材料的安全无毒性高(图5A)。
15)骨髓间充质干细胞移植于100％、90％、60％、0％矿化程度的骨ECM材料3天后观察，共聚焦显微镜视野下细胞粘附在支架上(图4A)。在扫描电镜1000倍视野下观察到90％和60％矿化程度的骨ECM材料上，相较未100％矿化程度的骨ECM材料有更多骨髓间充质干细胞，显示本专利材料能够有效促进细胞粘附定植以及细胞增殖。而0％矿化程度的骨ECM材料的细胞数量也相对较少，如图4B所示。
16)随后，我们比较了100％、90％、60％、0％矿化程度的骨ECM材料上定植的骨髓间充质干细胞内成骨基因的相对表达情况(图5B)。ALP是骨髓间充质干细胞早期最重要的成骨指标之一，结果显示90％矿化程度的骨ECM材料中培养1周的细胞内ALP相较未脱矿组上调17倍；Col-1α1也有同样趋势。在培养第2周和第4周时，90％和60％矿化程度的骨ECM材料细胞，尤其是90％矿化程度的骨ECM材料材料，其内细胞的BMP-2表达水平升高(图6A)。此外，MAPK信号通路在Ca 2+介导的促成骨分化过程中起作用，结果发现相较于100％和0％矿化程度的骨ECM材料组，90％和60％矿化程度的骨ECM材料内细胞MEK-1表达上调(图6B)。综上所述，90％和60％矿化程度的骨ECM材料，尤其是90％矿化程度的骨ECM材料相比100％和0％矿化程度的骨ECM 材料，显示出更好的促骨髓间充质干细胞成骨分化的效果。
实施例5：90％和60％矿化程度的骨ECM材料在体实验明显促进早期骨缺损的修复
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含20KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作10min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含20％丙酮的去离子水溶液，10度恒温震荡4小时；
5)将包埋盒放入5ml含1％的磷酸三丁酯的去离子水溶液，10度恒温震荡1小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床300rpm震荡48小时然后用液氮冻融6个循环(-80℃/37℃)；
7)将包埋盒放入5ml 0.5％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡2小时；
10)在含1.2M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡12小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨 块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
14)建立兔股骨上髁双侧缺损模型，植入未100％、90％、60％、0％矿化程度的骨ECM材料，并对其治疗效果进行了评价。
15)Micro-CT分析(图9A-B)显示，经移植术4周后，90％和60％矿化程度的骨ECM材料组，其骨缺损部位几乎被新生骨小梁填充，而未脱矿和完全脱矿材料组填充效果较小。100％、90％、60％、0％矿化程度的骨ECM材料分别为1.23±0.14/mm,2.16±0.03/mm,1.57±0.21/mm,0.94±0.22/mm；骨小梁厚度分别为0.16±0.03μm、0.24±0.04μm、0.18±0.01μm、0.14±0.02μm。90％和60％矿化程度的骨ECM材料对机体自身新生骨小梁生长的促进作用优于其他组(图9C)。
16)H&E染色后(图10A-B)未见明显炎症或炎症细胞。显示出材料的安全性。
17)移植术4周后，90％和60％矿化程度的骨ECM材料已有部分降解，新生骨长入。新生骨组织中胶原纤维矿化多为未成熟状态矿材料表面胶原(图7A、B)。
实施例6：90％和60％矿化程度的骨ECM材料明显促进骨缺损部分血管形成在体实验
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含20KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作3min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml含15％丙酮的去离子水溶液，10度恒温震荡2小时；
5)将包埋盒放入5ml含5％的磷酸三丁酯的去离子水溶液，10度恒温震荡4小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床震荡24小时然后用液氮冻融3个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含10％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.1M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡12小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡2小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 35g)。将骨块取出，250kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)在步骤(4)-(11)中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时；
13)将骨块取出，得到100％、90％、60％、0％矿化程度的骨ECM材料(图1B)，将材料再次进行25w辐照灭菌；
14)在促血管形成方面，我们观察到新生血管在骨小梁中间分布，移植术2周后，大多形成未成熟新生血管，其中100％、90％、60％、0％矿化程度的骨ECM材料的新生血管数量分别为5.5±1.3/500μm 2,8.0±1.6/500μm 2,8.0±1.8/500μm 2,and 5.0±1.6/500μm 2；血管面积分别为23.92±7.25μm 2,38.95±8.12μm 2,45.54±8.70μm 2,and 18.86±9.43μm 2。其中90％和60％矿化程度的骨ECM材料材料组，其新生血管厚度分别为4.86±0.15μm,5.07±0.20μm。高于100％矿化程度的骨ECM材料(4.29±0.38μm)和0％矿化程度的骨ECM材料组(4.41±0.26μm)。而移植术4周后，主要形成成熟的新生血管，其中100％、90％、60％、0％矿化程度的骨ECM材料的数量分别为3.5±0.6/500μm 2,5.8±1.0/500μm 2,5.0±0.8/500μm 2,and 2.8±1.5/500μm 2；血管面积分别为41.26±5.69μm 2,69.92±11.26μm 2,60.76±8.66μm 2,and 24.87±8.18μm 2；厚度相较移植术2周时，各组都有相应上升，其中90％矿化程度的骨ECM材料(12.18±0.54μm)及60％矿化程度的骨ECM材料(12.18±0.32μm)材料组仍然高于100％矿化程度的骨ECM材料组(11.83±0.49μm)和0％矿化程度的骨ECM材料组(9.68±1.83μm)。90％和60％矿化程度的骨ECM材料组显示新生血管、微血管变性、大血管稳定生长相较其他两组加快，并且血管内皮生长因子A(VEGFA)在缺损组织中的分布上调，有更好的修复效果(图8A-E)。
实施例7梯度矿化骨细胞外基质材料及其制备方法研究及评价
1)取新鲜牛肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行 辐照灭菌；
3)用含50KIU/ml蛋白酶抑制剂的去离子水漂洗2次，每次操作5min，去除血液、脂肪组织和其他杂质；
4)其余后续操作参考实施例1的方法进行，得到梯度矿化骨细胞外基质材料。
实施例8梯度矿化骨细胞外基质材料及其制备方法研究及评价
1)取新鲜猪肋骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含10KIU/ml蛋白酶抑制剂的去离子水漂洗5次，每次操作5min，去除血液、脂肪组织和其他杂质；
4)其余后续操作参考实施例1的方法进行，得到梯度矿化骨细胞外基质材料。
实施例9梯度矿化骨细胞外基质材料及其制备方法研究及评价
1)取新鲜猪肩胛骨，用无菌生理盐水清洗4次，用6mm钻孔器取出松质骨，用手术刀切成高度约2mm的圆柱状骨块；
2)取材完毕后用无菌生理盐水冲洗2h，送辐照中心，以25w辐射量进行辐照灭菌；
3)用含20KIU/ml蛋白酶抑制剂的去离子水漂洗3次，每次操作10min，去除血液、脂肪组织和其他杂质；
4)预备高温灭菌的含500ml去离子水的1L玻璃瓶及20个包埋盒。在无菌操作台上，戴无菌手套，将3个灭菌后的骨块分装入包埋盒中，放入10ml 含10％丙酮的去离子水溶液，10度恒温震荡1小时；
5)将包埋盒放入5ml含2％的磷酸三丁酯的去离子水溶液，10度恒温震荡4小时；
6)在含蛋白酶抑制剂的去离子水溶液中，4℃摇床震荡24小时然后用液氮冻融3个循环(-80℃/37℃)；
7)将包埋盒放入5ml 2％Triton X-100，放入10度恒温摇床100rpm振荡24h；
8)将包埋盒放入含5％SDS的去离子水，10度恒温摇床100rpm振荡36h；
9)在含0.5M氯化钾的PBS缓冲液中，4℃摇床100rpm震荡6小时；
10)在含1M碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡6小时，得到脱细胞骨ECM材料(图1A)；
11)配制脱钙液(去离子水1750ml+EDTA-2Na 450g+NaOH 10g)。将骨块取出，350kHz，4℃条件下在脱钙机中分别脱钙4，8，12，24h；
12)其余后续操作参考实施例1的方法进行，得到梯度矿化骨细胞外基质材料。
对实施例7-9所得的梯度矿化骨细胞外基质材料分别进行组织学评价、钙磷含量检测，材料表面胶原形态和含量检测，力学检测，结果与实施例1中梯度矿化骨细胞外基质材料结果类似，这表明可通过上述优化后确定的试剂和处理时间的调整，实现效果类似的梯度矿化骨细胞外基质材料的制备。并且对该材料进行组织学评价、细胞培养实验、以及材料的在体修复实验评价，检测均说明实施例7-9所得的90％，60％梯度矿化骨细胞外基质材料具有良好的修复再生作用，可以作为临床上促进肌肉缺损和病变修复再生的，安全可靠、有效、快速的生物材料。
由上述实施例可知，本发明提供的一种天然组织来源的梯度矿化骨细胞外基质的材料及其制备方法，能够更好地促进细胞粘附、诱导定植细胞的分化增殖、促进血管形成、新生骨小梁形成，实现了更优的修复效果和更小的炎症反应。是一种很有潜力的骨缺损移植材料。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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Claims (10)

  1. 一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：对天然组织来源的骨组织进行免疫原性去除处理，即脱细胞化，将获得的脱细胞骨进行梯度脱矿处理；获得梯度矿化骨细胞外基质材料。
  2. 根据权利要求1所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：具体包括以下步骤：
    步骤一：取哺乳动物全身任意骨组织，用钻孔器取出骨，用手术刀切成圆柱状骨块；
    步骤二：取材完毕后用无菌生理盐水冲洗2小时，后经辐照灭菌；所述辐照的辐射量为5～40w
    步骤三：用含蛋白酶抑制剂的去离子水漂洗，去除血液、脂肪组织和其他杂质；所述的含蛋白酶抑制剂的去离子水中，蛋白酶抑制剂浓度为10-50KIU/ml；去离子水洗涤次数为2-6次，每次洗涤时间为3-20分钟；
    步骤四：将骨块分装入包埋盒中，将包埋盒放入含丙酮的去离子水溶液震荡1-4小时；所述含丙酮的去离子水溶液，丙酮与去离子水的体积比优选为10％-20％；
    步骤五：将包埋盒中放入含磷酸三丁酯的去离子水溶液震荡1-4小时；所述含磷酸三丁酯的去离子水溶液，磷酸三丁酯与去离子水的体积比为1％-5％
    步骤六：将包埋盒中放入在含蛋白酶抑制剂的去离子水溶液中，在4℃条件下，摇床震荡24-48小时然后用液氮冻融2～6个循环，每个循环为-80℃～37℃，摇床速度为50～300rpm；
    步骤七：将包埋盒放入含Triton X-100的缓冲液中，恒温摇床振荡24h；所述的Triton X-100的浓度为0.5～5％；
    步骤八：在含SDS的缓冲液中，恒温摇床振荡36h；所述SDS的浓度 为0.5-10％；
    步骤九：在含氯化钾浓度为0.1-1M的PBS缓冲液中，且在4℃温度下用摇床100rpm震荡2-12小时；
    步骤十：在含碘化钾的PBS缓冲液，在4℃温度下用摇床100rpm震荡2-12小时；所述的碘化钾在去离子水中的浓度为1-1.5M
    步骤十一：在含EDTA2Na和NaOH的缓冲液中，4℃-10℃的条件下，进行梯度脱矿处理分别4，8，12,24h；得到100％、90％、60％、0％的矿化程度的骨ECM材料；
    步骤十二：将得到材料再次进行辐照灭菌；
    在步骤四～步骤十一中，每个步骤完成后，步骤之间均使用去离子水冲洗6小时。
  3. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述的含蛋白酶抑制剂的去离子水中，蛋白酶抑制剂浓度为20-40KIU。
  4. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述含丙酮的去离子水溶液，丙酮与去离子水的体积比优选为13％-18％。
  5. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述含磷酸三丁酯的去离子水溶液，磷酸三丁酯与去离子水的体积比优选为2％-5％。
  6. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：摇床速度为30-180rpm。
  7. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述的Triton X-100的浓度为0.5～3％。
  8. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述SDS的浓度为0.5～5％。
  9. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述的氯化钾在去离子水中的浓度0.3-1M。
  10. 根据权利要求2所述的一种梯度矿化骨细胞外基质材料的制备方法，其特征在于：所述的碘化钾在去离子水中的浓度为1-1.4M。
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CN110227182B (zh) 2020-12-15
US11684696B2 (en) 2023-06-27
EP3785740A1 (en) 2021-03-03
JP7118161B2 (ja) 2022-08-15
US20210121605A1 (en) 2021-04-29
EP3785740A4 (en) 2021-08-18
CN110227182A (zh) 2019-09-13
JP2021523749A (ja) 2021-09-09

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