WO2020258453A1 - 天然组织来源的脱细胞骨膜基质凝胶材料的制备方法 - Google Patents

天然组织来源的脱细胞骨膜基质凝胶材料的制备方法 Download PDF

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WO2020258453A1
WO2020258453A1 PCT/CN2019/099595 CN2019099595W WO2020258453A1 WO 2020258453 A1 WO2020258453 A1 WO 2020258453A1 CN 2019099595 W CN2019099595 W CN 2019099595W WO 2020258453 A1 WO2020258453 A1 WO 2020258453A1
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periosteal
hours
derived
gel material
periosteum
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French (fr)
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林贤丰
邱朋程
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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    • 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
    • A61L27/3604—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
    • A61L27/3641—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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • 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
    • A61L2430/40—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 method for preparing an acellular periosteal matrix gel material derived from natural tissues.
  • Bone defect is a common clinical disease, which often causes bone disconnection, delayed healing or nonunion, and local dysfunction, causing pain to patients, restricting their mobility, and seriously affecting their quality of life.
  • traditional synthetic materials can provide sufficient mechanical support, they have poor biological activity, insufficient osteoinductivity, and are not easily degraded, and even cause foreign body reactions.
  • the treatment of bone defects and osteonecrosis is mainly based on autologous bone transplantation and allogeneic bone transplantation, but autologous bone transplantation will cause secondary damage, and allogeneic bone transplantation has potential immunity and disease transmission risks, so There is an urgent need to develop new bone tissue engineering materials with good bone healing, regeneration and repair effects.
  • Extracellular matrix is a macromolecule synthesized by cells and secreted to the outside of the cell.
  • the main components include collagen fibers, glycoproteins, polysaccharides, growth factors, etc.
  • ECM constitutes a complex network structure, supports and connects the tissue structure, regulates the growth of tissue cells and other physiological activities.
  • Hydrogel is a kind of scaffold material system that has attracted more attention in tissue engineering. It has the advantages of injectable, small damage, and arbitrary shaping. However, common hydrogel products are composed of a single or a few specific components, and it is difficult to perfectly simulate the components of biological tissues. If they are loaded with other growth factors or active molecules, there are risks of side effects and tumorigenesis.
  • the patent application number 201410145791.7 discloses a decellularized matrix gel and a new preparation method thereof. After decellularization and digestion, incubation at a lower temperature and constant temperature reduces the damage to the tissue.
  • the patent application number 201811452768.7 discloses a method for preparing acellular matrix gel. In addition to adopting a mild decellularization method, the acellular matrix gel is digested and processed under pressure to obtain a better repair. effect.
  • a periosteal ECM gel repair material with high retention of biological activity and unified standards is obtained.
  • cross-linking growth factors through EDC has a very good effect on achieving excellent regeneration of new bone tissue and vascularization. It can be used to repair bone defects and bone nonunion caused by various clinical causes.
  • the purpose of the present invention is to provide an acellular periosteal matrix gel material with high retention of biologically active substances and a preparation method thereof.
  • the present invention provides the following technical solution: After decellularizing the periosteal tissue derived from natural tissues, freeze-drying, grinding, and gel processing to obtain a highly active periosteal ECM gel material. And through the modified glue technology, the introduction of biologically active factors (VEGF, FGF), grafted to the gel collagen fiber network, to achieve a combination of specific functions.
  • VEGF biologically active factors
  • the natural tissue-derived periosteal tissue includes mammalian bone tissue, the periosteum of the limbs long bone periosteum and the skull periosteum; the decellularization technology includes protease inhibitors, KCl, KI, acetone, tributyl phosphate, Triton X-100,
  • the decellularization program consisting of sodium dodecyl sulfate (SDS) and other chemical reagents and physical freeze-thaw methods includes the following steps:
  • the material obtained in g) is irradiated with gamma rays for 10-48 hours to kill possible bacteria and viruses;
  • the frequency of the grinder is 60HZ
  • the set time is 1 minute
  • freeze with liquid nitrogen grind into powder
  • pass through a 40-100 mesh screen and collect the freeze-dried tissue powder
  • the obtained periosteal ECM gel material uses a cross-linking agent to increase the cross-linking degree of the gel fiber network, and the cross-linking agent is EDC and NHS.
  • the radiation dose in h) is 5-40 kGy.
  • the concentration of the glacial acetic acid is 0.3-0.7M.
  • an 80 mesh screen is selected to sift through the tissue particles.
  • the amount of pepsin added is 2-20% of the total solution.
  • EDC is a crosslinking agent with a volume concentration of 0.1-0.4%, and the ratio of EDC:NHS can be 2:1, 3:1, 3:2, 4:3, 5:3.
  • the bioactive factor is introduced and grafted onto the gel collagen fiber network, and the bioactive factor is VEGF or FGF.
  • the natural tissue-derived periosteal ECM gel of the present invention can highly retain a variety of bioactive molecules in the matrix, and the gel particle size is standardized, and through EDC cross-linking a variety of bioactive factors, it can achieve excellent regeneration of new bone tissue and blood vessels. It has a very good promotion effect.
  • the present invention has significant progress in:
  • tissue particles are digested after grinding, and the particle size is not standardized, which is not conducive to the standardization and repeatability of the digestion process.
  • This patent is conducive to stable production of hydrogel with controllable quality through improved digestion control technology.
  • the technology of the present invention improves the retention efficiency of active molecules such as collagen, GAGs and various growth factors in the natural matrix to play the role of affecting cell activity, proliferation, migration, morphology and differentiation, thereby promoting the in situ functional tissue Regeneration, with good biological activity.
  • the periosteal ECM gel of the present invention cross-links various growth factors, such as VEGF, FGF, etc., through EDC, thereby increasing the repair function of the periosteal ECM gel material.
  • EDC cross-linking agents Compared with the current side effects of aldehyde cross-linking agents that cause abnormal calcification, EDC cross-linking agents have better biological safety and can be completely eliminated after catalytic reaction.
  • Periosteum is an important tissue component of bone, its tissue specificity is more conducive to bone regeneration and repair, and it has strong osteoinductivity. Therefore, the development of quality-controllable periosteal ECM gel repair materials is of great significance for bone defect repair.
  • FIG. 1 Schematic diagram of natural tissue-derived periosteal ECM powder, gel solution and gel material.
  • FIG. 3 Scanning electron microscope observation of type I collagen gel 8mg/ml, periosteum ECM gel 4mg/ml and 8mg/ml.
  • Periosteum ECM gel has a highly similar nanofiber network with type I collagen gel under microscopic view. This structure has a good role in promoting tissue repair.
  • FIG. 4 Evaluation of the in vitro biocompatibility of natural tissue-derived periosteal ECM gel materials.
  • mBMSCs Murine bone marrow mesenchymal stem cells
  • periosteum ECM gel The number of viable cells of mBMSCs in the periosteum ECM gel culture process increases with the number of days.
  • CCK8 method also proved that mBMSCs cultured on the periosteum ECM gel had good activity.
  • FIG. 5 Evaluation of angiogenic activity of natural tissue-derived periosteal ECM gel material.
  • Type I collagen and periosteal ECM gel material In the in vitro endothelial cell tube-like formation experiment, the periosteal ECM gel material exhibited stronger angiogenic activity.
  • FIG. 7 Evaluation of periosteal ECM gel materials derived from natural tissues on the osteogenic activity of primary skull cells.
  • Fig. 8 Experimental results of skull formation in vivo with periosteal ECM gel material derived from natural tissues. Periosteum ECM gel material promotes the formation of more new bone in the skull defect.
  • Figure 9 Results of subcutaneous embedding of natural tissue-derived periosteal ECM gel material.
  • Subcutaneous implanted gel (8mg/ml type I collagen hydrogel, 4mg/ml periosteal hydrogel, 8mg/ml periosteal hydrogel) general images of 3, 7, and 21 days, showing a stable degradation trend.
  • the periosteum ECM gel showed a low immune response and subsided rapidly, promoting angiogenesis and migrating into the stent
  • step 2 Rinse the material obtained in step 1 with sterile water 3 times for 20 minutes each time to remove residual muscle, fascia and fat and other tissues, and irradiate it with 24kGy dose of gamma rays for 24h to kill possible bacteria and viruses ;
  • step 3 Treat the material obtained in step 2 with a volume ratio of tributyl phosphate and deionized water of 2% for 24 hours;
  • step 4 Soak the material obtained in step 4 in a 1% SDS solution, shake at a constant temperature of 23°C and shake at 200 rpm for 2 hours, and rinse with ddH 2 O for 12 hours to remove residual SDS solution;
  • step 7 Soak the material obtained in step 6 in PBS buffer containing KI for 10 hours, the concentration of KI is 2M, and wash with ddH 2 O for 12 hours to remove residual PBS buffer.
  • step 7 The material obtained in step 7 is irradiated with 24kGy dose of gamma rays for 24 hours to kill possible bacteria and viruses;
  • step 9 After pre-cooling the sterilized decellularized periosteal material obtained in step 8 in the refrigerator at -80°C, put it in a freeze dryer for 24 hours, keeping the vacuum degree at 1.5mtorr, the temperature at -50°C, and the vacuum pumping rate of 195L/min , 60Hz;
  • step 9 Transfer the freeze-dried material obtained in step 9 to a grinder with a frequency of 60HZ and a set time of 1 minute. After freezing with liquid nitrogen, grind into powder, pass through a 100-mesh screen, and collect the freeze-dried tissue powder.
  • the diameter is about 150 ⁇ m;
  • step 10 Soak the powder obtained in step 10 in a 0.5M glacial acetic acid solution to prepare a 10% pepsin-containing periosteal ECM powder suspension, and digest at room temperature for 48 hours;
  • step 11 Centrifuge the suspension obtained in step 11 at 500 ⁇ g for 10 min. After removing impurities, adjust the pH to about 7.4 with NaOH solution to obtain the periosteal ECM gel material and store it at 4°C. As shown in Figure 1.
  • decellularization treatment steps are the same as the steps 2-7 in the above embodiment 1; wherein the volume ratio of tributyl phosphate to deionized water in step 3 is 1%; the treatment time is 12 hours;
  • step 7 Irradiate the material obtained in step 7 with 40kGy dose of gamma rays for 10 hours to kill possible bacteria and viruses;
  • step 8 After pre-cooling the sterilized decellularized periosteal material obtained in step 8 in the refrigerator at -80°C, put it in a freeze dryer for 24 hours, keeping the vacuum degree at 1.5mtorr, the temperature at -50°C, and the vacuum pumping rate of 195L/min , 60Hz;
  • step 9 Transfer the freeze-dried material obtained in step 9 to a grinder with a grinder frequency of 60HZ and a set time of 1 minute, freeze with liquid nitrogen, grind into powder, pass through an 80 mesh screen, and collect the freeze-dried tissue powder;
  • step 10 Soak the powder obtained in step 10 in a 0.3M glacial acetic acid solution to prepare an ECM powder suspension containing 2% pepsin, and digest it at room temperature for 24 hours;
  • step 11 Centrifuge the suspension obtained in step 11 at 500 ⁇ g for 10 minutes, adjust the pH to about 7.4 with NaOH solution after removing impurities, to obtain the periosteal ECM gel material, and store it at 4°C;
  • decellularization treatment steps are the same as steps 2-7 in the above embodiment 1; wherein the volume ratio of tributyl phosphate to deionized water in step 3 is 5%; the treatment time is 16 hours;
  • step 7 Irradiate the material obtained in step 7 with 5kGy dose of gamma rays for 48 hours to kill possible bacteria and viruses;
  • step 9 Transfer the freeze-dried material obtained in step 9 to a grinder with a grinder frequency of 60HZ and a set time of 1 minute, freeze with liquid nitrogen, grind into powder, pass through a 40-mesh screen, and collect the freeze-dried tissue powder;
  • step 10 Soak the powder obtained in step 10 in a 0.7M glacial acetic acid solution to prepare an ECM powder suspension containing 15% pepsin, and digest for 72 hours at room temperature;
  • VEGF vascular endothelial growth factor
  • FGF human fibroblast growth factor
  • HE staining shows that the natural tissue-derived acellular femoral periosteum ECM gel has a complete structure, the nucleus is completely removed, and there is no residual cell debris; DAPI staining further illustrates the nucleus The ingredients are negative and the antigenicity is completely removed.
  • the periosteal ECM gel material and type I collagen gel obtained in step 11 are solidified at 37°C, cut into 5 ⁇ 5mm size gel particles, and then placed in 0.1M phosphate buffer solution (PBS) at pH 7.0. Fix overnight at 4°C in 2.5% w/v glutaraldehyde.
  • PBS phosphate buffer solution
  • step 3 For the cells in step 3, add 100 ⁇ l of CCK-8 and medium mixture containing 10% CCK-8 reaction solution, incubate for 2h at 37°C, and measure the optical density (OD) value at 450nm wavelength. In the presence of the hydrogel, it was treated in the same way, and 3 sets of experiments were repeated. The result is shown in Figure 4.
  • Example 8 The vascularization ability of natural tissue-derived femoral periosteum ECM gel
  • Type I collagen hydrogel 8mg/ml, periosteum ECM gel 4mg/ml, periosteum ECM gel 8mg/ml, non-hydrogel incubation and other groups were placed in a 24-well plate for curing at 37°C.
  • osteogenic induction medium (OIC, 100nM dexamethasone, 5 ⁇ M ascorbic acid, 1mM b-glycerophosphate) and incubate together, and change the medium every other day.
  • Example 11 The repair effect of natural tissue-derived femoral periosteum ECM gel on skull defect in rats
  • control group was not filled, and 8mg/ml type I collagen hydrogel and 8mg/ml periosteal hydrogel were used to fill the periosteal defect. Then, the periosteum and scalp were sutured with interrupted 4-0 nylon sutures.
  • Example 12 In vivo osteogenic repair effect of natural tissue-derived femoral periosteum ECM gel
  • the natural tissue-derived periosteal ECM gel materials obtained in Examples 2-4 were subjected to histological evaluation, surface property observation, biocompatibility analysis, and in-vivo restoration experiment evaluation. The results are similar to those in Examples 5-12.
  • the results of the femoral periosteal ECM gel materials are similar, which indicates that the preparation of natural tissue-derived periosteal ECM gel materials with similar effects can be achieved through the adjustment of the reagent concentration and treatment time determined after the above optimization, which has good repair and regeneration effects. It can be used as a filling material for clinical treatment of bone defects.
  • the present invention provides a method for preparing natural tissue-derived periosteal ECM gel material, which can induce biomineralization, has osteogenic activity, is beneficial to bone repair in vivo, and can also promote endothelial cell differentiation. Promote angiogenesis in the body. Specific functions can be combined by crosslinking agent modification and growth factor addition. It is a promising bone defect graft material.

Abstract

一种天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，将哺乳动物来源的脱细胞骨膜基质通过冷冻干燥，最大化保留生物活性分子；并将其研磨成粉、控制粉末粒径，增加溶液接触面积从而加速消化，得到具有良好成骨活性潜力的标准统一化的脱细胞骨膜基质凝胶。此外，通过EDC交联生长因子，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，骨不愈合等多种骨再生障碍问题。

Description

天然组织来源的脱细胞骨膜基质凝胶材料的制备方法 技术领域
本发明属于骨组织修复及其再生技术领域，具体涉及天然组织来源的脱细胞骨膜基质凝胶材料的制备方法。
背景技术
骨缺损是临床常见疾病，往往会造成骨不连接、延迟愈合或不愈合及局部功能障碍，对患者造成痛苦，导致其行动受限，生活质量受到严重影响。传统人工合成材料虽然能提供足够的力学支撑，但其生物活性差，骨诱导性不足，不易降解，甚至引起异物反应。目前，治疗骨缺损和骨坏死等的方法以自体骨移植和同种异体骨移植为主，但自体骨移植会产生二次损伤，而同种异体骨移植因存在潜在免疫和疫病传播风险，因此亟需研发新的具有良好骨愈合再生修复效果的骨组织工程材料。
细胞外基质(Extracellular matrix,ECM)是由细胞合成并分泌到胞外的大分子，主要成分包括胶原纤维、糖蛋白、多聚糖、生长因子等。ECM构成复杂的网架结构，支持并连接组织结构，调节组织细胞的生长等系列生理活动。水凝胶是组织工程中一类较受关注的支架材料体系，具有可注射，损伤小，任意塑形等优点。而常见的水凝胶产品有单一或少数特定成分构成，难以完美模拟生物组织成分，若负载另外的生长因子或活性分子，有产生副效应和致瘤等风险。申请号为201410145791.7的专利公开了一种脱细胞基质凝胶及其制备新方法，脱细胞、消化后通过较低温恒温孵育减少了对组织的损伤。申请号为201811452768.7的专利公开了一种脱细胞基质凝胶制备的方法，除了采用温和的脱细胞法，还在脱细胞基质凝胶消化后，在加压条件下进行处理，获得了较好修复效果。
但既往脱细胞基质凝胶材料的制备通过剪碎方式无法获得足够的比表面积，不利于消化液的充分接触，生物活性物质保留低。此外，目前并未有骨膜ECM凝胶标准化制备的产品。骨膜是骨的重要组成部分，骨膜ECM的组织特异性更有利于骨的再生修复，是其他异位组织作为修复材料所无法取代的。因此，基于上述背景，本专利提出一种通过冷冻干燥的方案实现对骨膜ECM活性分子的最大保留；并且将ECM研磨成粉增加溶液接触面积；通过筛网过滤控制粉末粒径，标化消化步骤。从而得到具有生物活性高保留、标准统一化的骨膜ECM凝胶修复材料。此外，通过EDC交联生长因子，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，骨不愈合等多种骨再生障碍问题。
发明内容
有鉴于此，本发明的目的在于提供一种生物活性物质高保留的脱细胞骨膜基质凝胶材料及其制备方法。
为了实现上述发明目的，本发明提供以下技术方案：对天然组织来源的骨膜组织经脱细胞处理后，通过冷冻干燥、磨粉、凝胶处理，得到高活性的骨膜ECM凝胶材料。并通过修饰胶的技术，引入生物活性因子(VEGF、FGF)，将其接枝于凝胶胶原纤维网络，实现特定功能的组合。
所述的天然组织来源的骨膜组织包括哺乳动物骨组织骨膜为四肢长骨骨膜以及颅骨骨膜；所述的脱细胞技术包括蛋白酶抑制剂、KCl、KI、丙酮、磷酸三丁酯、Triton X-100、十二烷基硫酸钠(SDS)等化学试剂和物理冻融法组成的脱细胞方案，具体包括以下步骤：
a)将初步去除骨膜周边肌肉、筋膜和脂肪作为原材料；
b)将骨膜组织材料用无菌水漂洗3次，每次20分钟，去除残余的肌肉、筋膜和脂肪，用24kGy剂量的γ射线照射24h，杀灭存在的细菌和病毒；
c)将b)得到的材料经磷酸三丁酯与去离子水处理12-24小时；磷酸三丁酯与去离子水的体积比为1％-5％；
d)将c)得到的材料浸泡于浓度为1％TritonX-100溶液中，恒温23℃摇床200rpm震荡12小时，用无菌水漂洗3次，每次20分钟，去除残留TritonX-100溶液；
e)将d)得到的材料浸泡于摩尔浓度为1％SDS溶液中，恒温23℃摇床200rpm震荡2小时，用ddH 2O流动冲洗12小时，去除残留SDS溶液；
f)将e)得到的材料浸泡于含KCl的PBS缓冲液中8小时，KCl的浓度为2M；
g)将f)得到的材料浸泡于含KI的PBS缓冲液中10小时，KI的浓度为2M，ddH 2O流动冲洗12小时去除残留PBS缓冲液。
h)将g)得到的材料辐照灭菌；
将g)得到的材料经γ射线照射10-48小时，杀灭可能存在的细菌和病毒；
i)将h)得到的材料冷冻干燥；
将灭菌后的材料先放入-80℃冰箱预冷后，放入冷冻干燥机干燥24小时，保持真空度为1.5mtorr，温度-50℃。
j)将i)得到的材料磨粉
将冻干的材料转移到研磨器中，研磨机频率为60HZ，设定时间1分钟，经液氮冷冻，研磨成粉末，过40-100目筛网，收集冻干组织粉末；
k)将j)得到的材料凝胶；
将将j)得到的粉末浸泡于冰醋酸溶液中，加入胃蛋白酶，常温消化24-72小时，待完全消化，溶液500×g离心10min，去除杂质后用NaOH溶液调节pH至7.4左右，即得骨膜ECM凝胶材料，4℃保存。
将所得骨膜ECM凝胶材料使用交联剂增加凝胶纤维网络的交联度，交联剂为EDC和NHS。
作为优选，h)中的辐照剂量为5-40kGy。
作为优选，所述的冰醋酸浓度为0.3-0.7M。
作为优选，选择80目筛网筛过组织颗粒。
作为优选，胃蛋白酶加入量为占总溶液的2-20％。
作为优选，EDC为交联剂，体积浓度为0.1-0.4％，EDC：NHS比例可为2:1，3:1，3:2，4:3，5:3。
作为优选，引入生物活性因子接枝于凝胶胶原纤维网络，生物活性因子为VEGF或FGF。
本发明的有益效果：
本发明的天然组织来源的骨膜ECM凝胶能够高保留基质中多种生物活性分子，且凝胶颗粒尺寸标准化，并且通过EDC交联多种生物活性因子，能够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。
相比现有技术处理的凝胶或凝胶种类，本发明显著的进步在于：
1)以哺乳动物骨膜为原材料，既往通过剪碎方式无法获得足够的比表面积，不利于消化液的充分接触；本专利通过冻干磨粉处理，增加了消化效率。
2)既往通过研磨后组织颗粒即进行消化，未标控颗粒尺寸，不利于消化流程的标准化和重复性。本专利通过改良的消化控制技术，有利于稳定产生品质可控的水凝胶。
3)本发明技术提高了对天然基质中的胶原、GAGs和多种生长因子等活性分子的保留效率，以发挥影响细胞活性、增殖、迁移、形态和分化等作用，进而促进功能组织的原位再生，具有良好的生物学活性。
4)本发明的骨膜ECM凝胶通过EDC交联多种生长因子，如VEGF、FGF等，增加了骨膜ECM凝胶材料的修复功能。相比目前醛类交联剂引起异常钙化的副作用，EDC交联剂更好的生物安全性，且催化反应后可被完全清除。
5)骨膜作为骨的重要组织成分，其组织特异性更有利于骨的再生修复，骨诱导性强。因此，开发品质可控的骨膜ECM凝胶修复材料，对于骨缺损修复的意义重大。
附图说明
图1天然组织来源的骨膜ECM粉末、凝胶溶液及凝胶材料示意图。
图2天然组织来源的骨膜ECM的HE染色和DAPI染色，证明本材料DNA残留量极低。
图3 Ⅰ型胶原凝胶8mg/ml、骨膜ECM凝胶4mg/ml和8mg/ml扫描电镜观察。骨膜ECM凝胶微观下同I型胶原凝胶有着高度相似的纳米纤维网络，这种结构对于组织修复有着很好的促进作用。
图4天然组织来源的骨膜ECM凝胶材料体外生物相容性的评估。(a)鼠骨髓间充质干细胞(mBMSCs)在骨膜ECM凝胶上培养1、3、7天的细胞活性良好，说明骨膜ECM凝胶安全性及良好的促生长性。(b)mBMSCs在骨膜ECM凝胶培养过程中活细胞的数量随着天数增加。(c)CCK8法同样证明骨膜ECM凝胶上培养的mBMSCs活性良好。
图5天然组织来源的骨膜ECM凝胶材料血管生成活性的评价。I型胶原和骨膜ECM凝胶材料在体外内皮细胞管样的形成实验中，骨膜ECM凝胶材料展现出更强的促血管生成活性。
图6天然组织来源的骨膜ECM凝胶材料在体外自发矿化的评价。(a，b，c)骨膜ECM凝胶材料浸泡在m-SBF(模拟人体液)中的micro-CT扫描图，骨膜ECM凝胶比I型胶原有着更高的矿物含量骨膜ECM。(d)骨膜ECM凝胶材料在Von Kossa染色下有更丰富的矿化结节、扫描电镜下有更大直径的矿化纤维，透射电镜单晶衍射下更显著的矿化衍射环。说明骨膜凝胶支架提供更丰富的活性位点，显示出更高的钙磷沉积水平，有利于自发矿化。
图7天然组织来源的骨膜ECM凝胶材料对原代颅骨细胞成骨活性的评价。(a)成骨细胞在8mg/ml Ⅰ型胶原和PEM水凝胶上生长14天时，Runx 2、ALP、OPN、COL 1α的相对表达。(b)成骨细胞在8mg/ml Ⅰ型胶原和PEM水凝胶上生长14和28天时，Runx 2和OCN在细胞内的表达和位置。表明骨膜ECM凝胶支架上培养的成骨细胞表达更高水平的成骨标志物。
图8天然组织来源的骨膜ECM凝胶材料体内颅骨形成实验结果。骨膜ECM凝胶材料促进颅骨缺损部位更多的新骨形成。
图9天然组织来源的骨膜ECM凝胶材料皮下包埋结果。(a)皮下植入凝胶(8mg/ml Ⅰ型胶原水凝胶，4mg/ml骨膜水凝胶，8mg/ml骨膜水凝胶)3、7、21d的大体图像，呈现稳定降解趋势。(b)皮下植入水凝胶对全身器官毒性损伤的组织学评价，未显现出明显的脏器毒害反应。(c)第3、7、21天皮下植入水凝胶的组织学图像，骨膜ECM凝胶表现出较低的免疫反应并迅速消退，促进血管生成和迁入支架内部
具体实施方式
下面结合实施例对本发明提供的一种天然组织来源的脱细胞骨膜ECM凝胶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 天然组织来源的股骨骨膜ECM凝胶的制备
1、用刀片剔除成年猪股骨上的肌肉、筋膜和脂肪等组织，切取股骨骨膜作为原材料；
2、将步骤1所得的材料用无菌水漂洗3次，每次20分钟，去除残余的肌肉、筋膜和脂肪等组织，用24kGy剂量的γ射线照射24h，杀灭可能存在的细菌和病毒；
3、将步骤2所得的材料经磷酸三丁酯与去离子水的体积比为2％处理24小时；
4、将步骤3所得的材料浸泡于浓度1％TritonX-100溶液中，恒温23℃摇床200rpm震荡12小时，用无菌水漂洗3次，每次20分钟，去除残留TritonX-100溶液；
5、将步骤4所得的材料浸泡于浓度1％SDS溶液中，恒温23℃摇床200rpm震荡2小时，用ddH 2O流动冲洗12小时，去除残留SDS溶液；
6、将步骤5所得的材料浸泡于含KCl的PBS缓冲液中8小时，KCl的浓度为2M；
7、将步骤6所得的材料浸泡于含KI的PBS缓冲液中10小时，KI的浓度为2M，ddH 2O流动冲洗12小时去除残留PBS缓冲液。
8、将步骤7所得的材料用24kGy剂量的γ射线照射24小时，杀灭可能存在的细菌和病毒；
9、将步骤8所得的灭菌脱细胞骨膜材料在-80℃冰箱预冷后，放入冷冻干燥机干燥24小时，保持真空度为1.5mtorr，温度-50℃，真空泵抽气速率195L/min，60Hz；
将步骤9所得的冻干材料转移到研磨器中，研磨机频率为60HZ，设定时间1分钟，经液氮冷冻，研磨成粉末，过100目筛网，收集冻干组织粉末，所获颗粒直径约150μm；
10、将步骤10所得的粉末浸泡于0.5M冰醋酸溶液中，制成含有10％胃蛋白酶的骨膜ECM粉末悬液，常温消化48小时；
11、将步骤11所得的悬液500×g离心10min，去除杂质后用NaOH溶液调节pH至7.4左右，即得骨膜ECM凝胶材料，4℃保存。如图1所示。
实施例2 天然组织来源的肩胛骨骨膜ECM凝胶的制备
1、用刀片剔除成年猪肩胛骨上的肌肉、筋膜和脂肪等组织，切取肩胛骨骨膜作为原材料；
2、脱细胞处理步骤同上述实施例1中步骤2-7；其中步骤3中的磷酸三丁酯与去离子水的体积比为1％；处理时间为12小时；
3、将步骤7所得的材料用40kGy剂量的γ射线照射10小时，杀灭可能存在的细菌和病毒；
4、将步骤8所得的灭菌脱细胞骨膜材料在-80℃冰箱预冷后，放入冷冻干燥机干燥24小时，保持真空度为1.5mtorr，温度-50℃，真空泵抽气速率195L/min，60Hz；
5、将步骤9所得的冻干材料转移到研磨器中，研磨机频率为60HZ，设定时间1分钟，经液氮冷冻，研磨成粉末，过80目筛网，收集冻干组织粉末；
6、将步骤10所得的粉末浸泡于0.3M冰醋酸溶液中，制成含有2％胃蛋白酶的ECM粉末悬液，常温消化24小时；
7、将步骤11所得的悬液500×g离心10min，去除杂质后用NaOH溶液调节pH至7.4左右，即得骨膜ECM凝胶材料，4℃保存；
实施例3 天然组织来源的股骨骨膜ECM凝胶的制备
1、用刀片剔除成年猪股骨上的肌肉、筋膜和脂肪等组织，切取股骨骨膜作为原材料；
8、脱细胞处理步骤同上述实施例1中步骤2-7；其中步骤3中的磷酸三丁酯与去离子水的体积比为5％；处理时间为16小时；
2、将步骤7所得的材料用5kGy剂量的γ射线照射48小时，杀灭可能存在的细菌和病毒；
3、将步骤8所得的灭菌脱细胞骨膜材料在-80℃冰箱预冷后，放入冷冻干燥机干燥24小时，保持真空度为1.5mtorr，温度-50℃，真空泵抽气速率195L/min，60Hz；
4、将步骤9所得的冻干材料转移到研磨器中，研磨机频率为60HZ，设定时间1分钟，经液氮冷冻，研磨成粉末，过40目筛网，收集冻干组织粉末；
5、将步骤10所得的粉末浸泡于0.7M冰醋酸溶液中，制成含有15％胃蛋白酶的ECM粉末悬液，常温消化72小时；
6、将步骤11所得的悬液500×g离心10min，去除杂质后用NaOH溶液调节pH至7.4左右，即得骨膜ECM凝胶材料，4℃保存；
实施例4 天然组织来源的股骨骨膜ECM凝胶交联生长因子的制备
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将步骤11所得的骨膜ECM凝胶材料加入含0.4％EDC和0.24％NHS(N-亚胺)的2-吗啡乙醇磺酸缓冲液(0.05M,pH5.6)，于37℃中孵育4h。
3、随后浸没于1×PBS，100rpm匀速震荡6h。
4、随后将材料浸没于4M NaCl，100rpm匀速震荡6h。
5、随后将材料浸没于ddH2O中，以100rpm匀速震荡6h，洗涤干净后备用。
6、加入生长因子溶液，5ng/mL VEGF(血管内皮生长因子)或5ng/mL FGF(人成纤维生长因子)，37℃孵育24h，获得接枝生长因子的凝胶胶原纤维网络，实现特定功能的组合。
7、即得骨膜ECM凝胶交联生长因子(如VEGF,FGF)材料，4℃保存；
实施例5 天然组织来源的股骨骨膜ECM凝胶的组织学评价
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将步骤11所得的材料进行组织学评价，结果如图2，HE染色显示天然组织来源的脱细胞股骨骨膜ECM凝胶结构完整，细胞核成分完全去除，无细胞碎片残留；DAPI染色进一步说明细胞核成分呈阴性，抗原性得到完全去除。
实施例6 天然组织来源的股骨骨膜ECM凝胶的表面性质
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将步骤11得到的骨膜ECM凝胶材料、Ⅰ型胶原凝胶在37℃固化，切成5×5mm大小的凝胶颗粒，然后在pH7.0的0.1M磷酸盐缓冲溶液(PBS)，2.5％w/v戊二醛中4℃固定过夜。
3、第二天，将样品置于pH7.0的0.1M PBS中清洗3次，每次15min，用1％四氧化锇溶液固定2h，用pH7.0的0.1M PBS中清洗3次，每次15min。
4、在一系列梯度浓度的乙醇溶液(30，50，70，80，90，95和100％w/v，20min)中脱水。
5、将样品转移到乙醇和乙酸异芳酯(1：1v/v)的混合物中30min，然后在纯乙酸异芳酯浸泡过夜，用液态CO 2脱水。随后，喷上金钯，并使用扫描电镜(TM-1000，Hitachi，日本东京)对处理后的样品进行了观察。如图3所示，说明了脱细胞骨膜ECM凝胶与广泛应用于复合材料制备的I型胶原相比，微观纤维排布高度相似，相同浓度下，孔隙大小无显著差异。可以允许细胞在其内生长和传播。
实施例7 天然组织来源的股骨骨膜ECM凝胶的生物相容性分析
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、骨髓间充质干细胞(MSCs)消化、重悬至2×10 4个/mL细胞密度，将步骤11所得的材料加入至96孔板中，每孔加入100μl股骨骨膜ECM凝胶。
3、在凝胶中加入100μl细胞悬液，然后培养1、3、7天。
4、对步骤3中细胞，用活死细胞染色试剂盒(BioVision,Inc)中1mM活染料和2.5mg/ml碘化丙啶(PI)进行染色，状态差或死的细胞会染红，活的细胞会变绿。荧光显微镜(激发/发射，488/518nm)检测染色活细胞；(激发/发射，488/615)检测染色死亡细胞。随机选择三个级别来计数存活细胞的数量。
5、对步骤3中细胞，加入含10％CCK-8反应液的CCK-8和培养基混合液100μl，37℃下孵育2h，测定450nm波长处的光密度(OD)值，对照组在没有水凝胶存在的情况下以同样的方式处理，重复3组实验。结果如图4。
实施例8 天然组织来源的股骨骨膜ECM凝胶的成血管能力
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将人脐静脉内皮细胞消化重悬至1×10 5个/mL细胞密度，在96孔板中加入分别含有100ng/ml VEGF，8mg/ml I型胶原和8mg/ml脱细胞骨膜ECM凝胶的基质凝胶，每孔100μl，37℃凝胶化。
3、在水凝胶中加入100μl细胞悬液，培养6h后，用4％多聚甲醛固定。
4、通过计数内皮细胞网络分支间的交叉数来定量管状结构。
5、对每个条件下的5个高功率场进行了检测，并在三种独立培养物中重复实验。结果如图5所示。
实施例9 天然组织来源的股骨骨膜ECM凝胶的体外自发矿化评价
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将步骤11所得的材料37℃固化。
3、将固化骨膜ECM凝胶浸泡在含聚天冬氨酸(p-Asp)100μg/ml的模拟人体液m-SBF(1.67×10 -3m CaCl2,9.5×10 -3m Na2HPO4,150×10 -3m NaCl)溶液中，恒温4℃，震荡28天，每48小时更换溶液。
4、所有矿化胶原凝胶在达到指定时间后，用去离子水冲洗。
5、采用micro-CT扫描、扫描电镜、透射电镜和von Kossa染色法对样品进行矿化评价。结果如图6所示。
实施例10 天然组织来源的股骨骨膜ECM凝胶的成骨活性
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将颅骨的原代成骨细胞以1×10 5/孔的密度接种于24孔板内。
3、Ⅰ型胶原水凝胶8mg/ml，骨膜ECM凝胶4mg/ml，骨膜ECM凝胶8mg/ml，无水凝胶孵育等组分别置于24孔板内37℃固化。
4、加入成骨诱导培养基(OIC,100nM地塞米松,5μM抗坏血酸,1mM b-甘油磷酸脂)共同孵育，每隔一天换一次液。
5、在14和28天，用PBS轻轻清洗细胞两次，用4％多聚甲醛固定15分钟，然后用0.5％v/v Triton x-100处理10分钟，用10％山羊血清封闭，然后4℃条件下敷：抗Runx 2抗体、抗OCN抗体过夜。
6、用1ml PBS洗涤细胞，洗涤3次。
7、37℃条件下，孵育适应的二抗1h。避光Rhodamine Phalloidin染色1h。
8、用1ml PBS洗涤细胞，洗涤3次。
9、DAPI染色10min后，1ml PBS洗涤细胞，洗涤3次。
10、通过免疫荧光显微镜(Nikon Corporation，Minato，日本东京)观察。结果如图7。
实施例11 天然组织来源的股骨骨膜ECM凝胶的大鼠颅骨缺损修复效果
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、取18只6-8周龄的SD大鼠(250±50g)随机分为3组(对照组、Ⅰ型胶原水凝胶组、骨膜水凝胶组)。
3、腹腔注射戊巴比妥钠(50mg/kg)麻醉动物。消毒后行颅骨缺损造模，双侧全厚度临界大小颅骨缺损直径为4mm(颅骨厚度≈0.5mm，体积≈6.28mm 3)。
4、对照组不填充，将8mg/ml I型胶原水凝胶、8mg/ml骨膜水凝胶填充骨膜缺损部位。然后，用间断的4-0尼龙缝线缝合骨膜和头皮。
5、移植后4、8周，用CO 2窒息处死动物，将头骨固定在4％多聚甲醛内。
6、根据扫描方式(电压为80KV，电流为500mA，各向同性分辨率为14.97mm)，采用micro-ct(西门子Ineon，德国埃斯伯恩)对材料的修复效果进行了分析。采用IneonResearchWorkine2.2(西门子)对新骨形成进行了分析。结果如图8。
实施例12 天然组织来源的股骨骨膜ECM凝胶的在体成骨修复效果
1、脱细胞及凝胶化处理步骤同上述实施例1中步骤1-11；
2、将1ml Ⅰ型胶原、股骨骨膜ECM凝胶分别经26mm针皮下注射至SD大鼠背部。
3、分别于第3、7、21、28天采集皮肤组织标本，将获得的组织固定在4％(w/v)多聚甲醛(PFA)中24小时。
4、PBS洗涤5min×3次后，用50、75、85、95、100％乙醇连续浓度脱水，石蜡包埋。
5、然后将石蜡切片切成7μm厚，通过苏木精和伊红(HE)染色对获得组织进行分析。
6、取大鼠心脏、肝、脾、肺、肾进行内脏毒性评估。结果如图9。
对实施例2-4所得的天然组织来源的骨膜ECM凝胶材料分别进行组织学评价、表面性质观察及生物相容性分析以及材料的在体修复实验评价，结果与实施例5-12中猪股骨骨膜ECM凝胶材料结果类似，这表明可通过上述优化后确定的试剂浓度和处理时间的调整，实现效果类似的天然组织来源的骨膜ECM凝胶材料的制备，其具有良好的修复再生作用，可以作为临床上治疗骨缺损填充材料。
由上述实施例可知，本发明提供的天然组织来源的骨膜ECM凝胶材料制备方法，该材料可以诱发生物矿化，具有成骨诱导活性，有利于体内成骨修复，还可以促进内皮细胞分化，促进体内血管生成。经交联剂修饰和生长因子添加可组合特定功能。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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  1. 天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：
    a)将初步去除骨膜周边肌肉、筋膜和脂肪作为原材料；
    b)将骨膜组织材料用无菌水漂洗3次，每次20分钟，去除残余的肌肉、筋膜和脂肪，用24kGy剂量的γ射线照射24h，杀灭存在的细菌和病毒；
    c)将b)得到的材料经磷酸三丁酯与去离子水处理12-24小时；磷酸三丁酯与去离子水的体积比为1％-5％；
    d)将c)得到的材料浸泡于浓度为1％TritonX-100溶液中，恒温23℃摇床200rpm震荡12小时，用无菌水漂洗3次，每次20分钟，去除残留TritonX-100溶液；
    e)将d)得到的材料浸泡于摩尔浓度为1％SDS溶液中，恒温23℃摇床200rpm震荡2小时，用ddH 2O流动冲洗12小时，去除残留SDS溶液；
    f)将e)得到的材料浸泡于含KCl的PBS缓冲液中8小时，KCl的浓度为2M；
    g)将f)得到的材料浸泡于含KI的PBS缓冲液中10小时，KI的浓度为2M，ddH 2O流动冲洗12小时去除残留PBS缓冲液；
    h)将g)得到的材料辐照灭菌；
    将g)得到的材料经γ射线照射10-48小时，杀灭可能存在的细菌和病毒；
    i)将h)得到的材料冷冻干燥；
    将灭菌后的材料先放入-80℃冰箱预冷后，放入冷冻干燥机干燥24小时，保持真空度为1.5mtorr，温度-50℃；
    j)将i)得到的材料磨粉
    将冻干的材料转移到研磨器中，研磨机频率为60HZ，设定时间1分钟，经液氮冷冻，研磨成粉末，过40-100目筛网，收集冻干组织粉末；
    k)将j)得到的材料凝胶；
    将将j)得到的粉末浸泡于冰醋酸溶液中，加入胃蛋白酶，常温消化24-72小时，待完全消化，溶液500×g离心10min，去除杂质后用NaOH溶液调节pH至7.4左右，即得骨膜ECM凝胶材料，4℃保存。
  2. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：将所得骨膜ECM凝胶材料使用交联剂增加凝胶纤维网络的交联度，交联剂为EDC和NHS。
  3. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：h)中的辐照剂量为5-40kGy。
  4. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：所述的冰醋酸浓度为0.3-0.7M。
  5. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：选择80目筛网筛过组织颗粒。
  6. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：胃蛋白酶加入量为占总溶液的2-20％。
  7. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：EDC为交联剂，体积浓度为0.1-0.4％，EDC：NHS比例可为2:1，3:1，3:2，4:3，5:3。
  8. 根据权利要求1所述的天然组织来源的脱细胞骨膜基质凝胶材料的制备方法，其特征在于：引入生物活性因子接枝于凝胶胶原纤维网络，生物活性因子为VEGF或FGF。
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