WO2023179544A1 - 一种负载间充质干细胞外基质的3d打印骨缺损修复支架及其制备方法 - Google Patents

一种负载间充质干细胞外基质的3d打印骨缺损修复支架及其制备方法 Download PDF

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WO2023179544A1
WO2023179544A1 PCT/CN2023/082528 CN2023082528W WO2023179544A1 WO 2023179544 A1 WO2023179544 A1 WO 2023179544A1 CN 2023082528 W CN2023082528 W CN 2023082528W WO 2023179544 A1 WO2023179544 A1 WO 2023179544A1
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scaffold
stem cell
printed
mesenchymal stem
bone defect
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PCT/CN2023/082528
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English (en)
French (fr)
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涂欣冉
江千舟
郭吕华
谭国忠
陈荣丰
张阳
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广州医科大学附属口腔医院（广州医科大学羊城医院）
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Publication of WO2023179544A1 publication Critical patent/WO2023179544A1/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
    • A61L27/14—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
    • A61L27/02—Inorganic materials
    • A61L27/10—Ceramics or glasses
    • 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
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P10/00—Technologies related to metal processing
    • Y02P10/25—Process efficiency

Definitions

  • the invention belongs to the technical field of bone tissue engineering repair and reconstruction, and mainly relates to a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix and a preparation method thereof.
  • bone transplantation methods such as autologous bone transplantation, allogeneic bone transplantation and artificial bone transplantation are usually used.
  • Autologous bone transplantation is the "gold standard" for defect repair, but the source of autologous bone is limited and is often in short supply.
  • Allogeneic bone transplantation carries the risk of infectious diseases, while artificial bone transplantation lacks osteoinductive activity and has poor osteogenic efficiency, making it difficult to form healthy bone.
  • bone defect repair scaffolds are divided into synthetic materials and biological materials according to materials.
  • synthetic materials have control advantages in terms of strength and configuration, but their compatibility and degradability in living organisms need to be improved; while biological materials have good compatibility and degradability, but it is difficult to meet the requirements in strength and shape.
  • the three-dimensional structure of the scaffold is more conducive to cell differentiation and proliferation.
  • Bioactive glass is an important scaffold material for bone tissue engineering. It can effectively promote biomineralization in the body and release silicon and calcium ions to promote stem cell osteogenesis and blood vessel formation.
  • Gelatin/sodium alginate hydrogel is a mixture of natural polymer materials. It has the advantages of good biocompatibility, tissue absorbability, and low immunogenicity. In particular, it is conducive to combining with highly bioactive inorganic powders. 3D printing is performed, but its mechanical strength is relatively poor. 3D printing can effectively construct porous bioglass bone tissue engineering scaffolds, accurately control parameters such as porosity and pore size, and give it better biological activity. Repair scaffolds usually need to have good mechanical properties, biocompatibility, osteoconductivity, bone Inductive.
  • the present invention can provide theoretical guidance and experimental data for the development of new and effective jaw bone defect repair materials, and provide new exploration for the clinical development of bone defect replacement materials.
  • the purpose of the present invention is to provide a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix, which uses gelatin, sodium alginate and 58S bioglass and adjusts the content ratio of the three to make the prepared scaffold high in strength and phase-resistant. It has good capacitance and good in vivo degradation effect, and rBMSC can adhere and proliferate on the scaffold, significantly improving the osteogenesis efficiency of bone tissue.
  • a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extra matrix including a 3D printed scaffold and mesenchymal stem cell extra matrix loaded on the scaffold;
  • the preparation method of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix includes the following steps:
  • the first layer includes multiple parallel lines, and the second layer is vertically connected to the first layer. There are multiple parallel lines on the upper surface of the lines, and the third layer is multiple parallel lines vertically connected to the upper surface of the second layer of lines, and so on, printing 4-8 layers;
  • the semi-finished stent After printing, the semi-finished stent is obtained.
  • the semi-finished stent is first chemically cross-linked with calcium chloride solution for 0.5 hours, and then immersed in glutaraldehyde solution for chemical cross-linking for 6 hours. Finally, it is washed and freeze-dried.
  • the sodium alginate gel has a three-dimensional culture structure suitable for cell nutrient exchange, and can maintain a specific shape due to a large surface area and many pores. Adding gelatin can improve the mechanical strength of sodium alginate gel, simulate the internal environment required for cell growth, and have good biocompatibility.
  • 58S bioglass can stably release Si ions, Ca ions, etc. in the scaffold, stimulate osteoprogenitor cells at the genetic level, and promote the growth of new bone.
  • the content selection of gelatin, sodium alginate and 58S bioglass has a greater impact on the overall performance of the stent.
  • High gelatin content can achieve good biocompatibility; sodium alginate has good consolidation and molding capabilities, and bio Glass promotes bone growth. Therefore, how to adjust the dosage of these three substances to obtain a scaffold with good biocompatibility, cell adhesion, biodegradability and bioactive factor loading capacity requires a lot of experimental work.
  • the 58S bioglass component in it improves the smoothness of the filament during printing, making the printed scaffold structure more regular, and the porosity and spacing more qualified.
  • the structural holes of the 3D printed scaffold of the present invention are straight. Because of the regular structure, there are no obvious obstacles and no strong fluid resistance in fluid mechanics, which is conducive to the penetration of nutrients and cells into the interior of the scaffold and accelerates the repair process. Osteogenic efficiency.
  • Extracellular matrix is a general term for a series of proteins and other components secreted by cells. It plays a key role in cell signal transduction and regulating cell physiological functions.
  • the ECM of rBMSC cells can not only provide necessary protein factors for osteogenic differentiation, but also is more conducive to the formation of bone tissue because of its special "cell sheet" structure.
  • the 3D printed scaffold of the present invention can load the ECM of rBMSC cells, and rBMSC can adhere and proliferate on the scaffold.
  • the 3D printing in S12 uses a needle with an aperture of 0.41mm, and prints at a printing speed of 8mm/s under the conditions of 0.42Mpa air pressure and 30°C.
  • the above-mentioned printing parameter settings can maintain the configuration in the stent molding, and at the same time, the size is more accurate and regular, and there will be no adhesion.
  • the solution is stirred evenly by magnetic stirring and/or mechanical stirring to obtain 3D printing slurry.
  • the 3D printing slurry is injected into the 3D printing barrel, and printing is started after bubble removal and homogenization.
  • the distance between adjacent lines in each layer of the stent is 300-500 ⁇ m, and the stent The number of layers is 6.
  • the vertical stacking of the scaffolds up and down and the adjustment of the distance between the holes expand and enrich the diversity of the pores, which is conducive to the penetration of nutrients and cells into the interior of the scaffold.
  • the 58s biological glass is ground and then sieved, so that the particle diameter of the 58s biological glass powder ranges from 4 to 10 microns, and the chemical composition of the 58s biological glass is 58% SiO 2 -33% CaO -9%P 2 O 5 .
  • the particle size of 58s bioglass powder is selected so that it has a large specific surface area and releases more ions. When the particle size is less than 4 microns, its dispersion uniformity in the solution becomes poor, which is not conducive to its efficacy.
  • the operation of changing the medium in S3 is to use a pipette to suck up the culture medium in the dish, rinse the cells with PBS three times, and add fresh culture medium;
  • the decellularization treatment in S4 is: 10mM ammonia water Soak the scaffold in +0.1% SDS for 30 minutes, rinse with distilled water 3 times, soak in 0.1% DNase solution for 10 minutes, and rinse with distilled water 3 times.
  • the lyophilization described in S4 is stored at -40°C for 12 hours.
  • the concentration of the calcium chloride solution is 5% to 6%, and is prepared by adding calcium chloride powder to distilled water and dissolving it.
  • the concentration of the glutaraldehyde solution is 1.0% to 1.5%, which is obtained by diluting 50% glutaraldehyde solution with distilled water.
  • the concentration of calcium chloride solution and glutaraldehyde solution is selected to make the cross-linking effect of the stent better.
  • the present invention selected the mass/volume concentration of each component of the 3D printing scaffold to be 18% gelatin, 5% sodium alginate, and 5.5% 58S bioglass; without affecting the osteogenesis efficiency,
  • the 58S bioglass component in the printing slurry is reduced, and the smoothness of the filament output during printing is improved, making the printed scaffold structure more regular, and the porosity and spacing more qualified.
  • the structural holes of the 3D printed scaffold are straight. Because of the regular structure, there are no obvious obstacles and there is no strong fluid resistance in hydrodynamics, which is conducive to the penetration of nutrients and cells into the interior of the scaffold and accelerates osteogenesis during the repair process. efficiency.
  • the present invention has discovered through research that when ECM loaded with rBMSC cells on a gelatin/sodium alginate/58S bioglass scaffold is used to repair bone defects, the area and number of branches forming vascular tissue are significantly increased, effectively promoting bone tissue and vascular tissue. formation, significantly improving the efficiency of bone defect repair.
  • the present invention studies and optimizes the preparation process parameters of the extracellular matrix loaded with rBMSC cells on the gelatin/sodium alginate/58S bioglass scaffold, and achieves good loading effects.
  • Figure 1 is a photo of the 3D printed bracket of the present invention (a: front photo, b: side photo).
  • Figure 2 shows the live/dead staining results of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix. It shows that rBMSC can adhere and proliferate on the composite scaffold.
  • Figure 3 shows the SEM observation of the surface micromorphology of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix and the adhesion and extension of rBMSC on the scaffold.
  • Figure 4 shows the expression levels of osteogenesis-related genes in 3D printed bone defect repair scaffolds loaded with mesenchymal stem cell extramatrix (a: RUNX-2; b: BMP2).
  • Figure 5 is a graph showing the osteogenic efficiency of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix in animal experiments (left: scaffold group of the present invention, middle: blank group, right: BIO-OSS bone powder positive control group).
  • the chemical composition of the 58s bioglass used is 58% SiO 2 -33% CaO - 9% P 2 O 5 , with a diameter ranging from 4 to 10 microns.
  • a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extra matrix includes a 3D printed scaffold and mesenchymal stem cell extra matrix loaded on the scaffold.
  • the preparation method of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix includes the following steps:
  • the first layer includes multiple parallel lines
  • the second layer is multiple parallel lines vertically connected to the upper surface of the lines on the first layer.
  • the third floor is vertically connected to the second floor with lines Multiple parallel lines on the upper surface, and so on, print 4 layers;
  • the semi-finished stent is obtained.
  • the semi-finished stent is first chemically cross-linked with calcium chloride solution for 0.5 hours, and then immersed in glutaraldehyde solution for chemical cross-linking for 6 hours; finally, it is washed and freeze-dried; the chlorinated
  • the concentration of the calcium solution is 5%, which is obtained by adding calcium chloride powder to distilled water and dissolving it; the concentration of the glutaraldehyde solution is 1.0%, which is obtained by diluting 50% glutaraldehyde solution with distilled water;
  • the operation of changing the medium specifically includes using a pipette to suck up the culture medium in the dish, rinsing the cells 3 times with PBS, and adding fresh culture medium;
  • a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extra matrix includes a 3D printed scaffold and mesenchymal stem cell extra matrix loaded on the scaffold.
  • the preparation method of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix includes the following steps:
  • the first layer includes multiple parallel lines
  • the second layer is multiple parallel lines vertically connected to the upper surface of the lines on the first layer.
  • the third layer is a number of parallel lines vertically connected to the upper surface of the second layer of lines, and so on, printing 6 layers;
  • the semi-finished stent is obtained.
  • the semi-finished stent is first chemically cross-linked with calcium chloride solution for 0.5 hours, and then immersed in glutaraldehyde solution for chemical cross-linking for 6 hours; finally, it is washed and freeze-dried; the chlorinated
  • the concentration of the calcium solution is 5.5%, which is obtained by dissolving calcium chloride powder in distilled water;
  • the concentration of the glutaraldehyde solution is 1.5%, which is obtained by diluting 50% glutaraldehyde solution with distilled water;
  • the operation of changing the medium specifically includes using a pipette to suck up the culture medium in the dish, rinsing the cells 3 times with PBS, and adding fresh culture medium;
  • a 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extra matrix includes a 3D printed scaffold and mesenchymal stem cell extra matrix loaded on the scaffold.
  • the preparation method of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix includes the following steps:
  • the first layer includes multiple parallel lines
  • the second layer is multiple parallel lines vertically connected to the upper surface of the lines on the first layer.
  • the third layer is a number of parallel lines vertically connected to the upper surface of the second layer of lines, and so on, printing 8 layers;
  • the semi-finished stent is obtained.
  • the semi-finished stent is first chemically cross-linked with calcium chloride solution for 0.5 hours, and then immersed in glutaraldehyde solution for chemical cross-linking for 6 hours; finally, it is washed and freeze-dried; the chlorinated
  • the concentration of the calcium solution is 6%, which is obtained by adding calcium chloride powder to distilled water and dissolving it; the concentration of the glutaraldehyde solution is 1.5%, which is obtained by diluting 50% glutaraldehyde solution with distilled water;
  • the operation of changing the medium specifically includes using a pipette to suck up the culture medium in the dish, rinsing the cells 3 times with PBS, and adding fresh culture medium;
  • PCR experiment rBMSCs were seeded at 10 5 cells/well on the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix, cultured in low-sugar DMEM containing 10% FBS, and the medium was changed every 3 days. , extract total cellular RNA on days 0, 7, and 14 for PCR detection.
  • Penicillin sodium is injected intramuscularly to fight infection. Materials were collected at two time points of 4 weeks and 8 weeks. The rats were euthanized by carbon dioxide asphyxiation. The mandibles including the defect area were removed and fixed in 10% neutral buffered formalin for 24 hours, and then Micro-CT scans were performed. Use NRecon software Skyscan to scan and reconstruct the image file, and select a region of interest (ROI) from the scanned image for analysis.
  • ROI region of interest
  • the structural holes of the prepared 3D printed scaffold are straight. Because of the regular structure, there are no obvious obstacles and there is no strong fluid resistance in hydrodynamics, which is conducive to the penetration of nutrients and cells. Inside the scaffold, it accelerates the osteogenesis efficiency during the repair process. As can be seen from Figures 2 and 3, rBMSCs can adhere and proliferate on the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix. As can be seen from Figure 4, the expression levels of osteogenesis-related genes are high. As can be seen from Figure 5, the osteogenesis efficiency of the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix is not much different from that of BIO-OSS bone powder.
  • the 3D printed bone defect repair scaffold loaded with mesenchymal stem cell extramatrix prepared by the present invention has achieved the purpose of the present invention and has a good loading effect.
  • the obtained composite scaffold can be used for bone defect repair and can effectively promote bone defect repair.
  • the formation of tissue and vascular tissue significantly improves the efficiency of bone defect repair.

Abstract

本发明公开了一种负载间充质干细胞外基质的3D打印骨缺损修复支架，制备步骤如下：S1.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，溶液搅拌均匀，获得3D打印浆料，然后进行3D打印；打印成品先用氯化钙溶液进行化学交联，后浸泡于戊二醛溶液中化学交联；最后清洗，冻干即得3D打印支架；S2.将3D打印支架进行消毒处理；S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖DMEM培养基培养2周；S4.取出支架，进行脱细胞处理，冻干即得。本发明制备的负载间充质干细胞外基质的3D打印骨缺损修复支架应用于骨缺损修复，能够有效促进骨组织及血管组织形成，明显提高骨缺损修复的效率。

Description

一种负载间充质干细胞外基质的3D打印骨缺损修复支架及其制备方法 技术领域
本发明属于骨组织工程修复及重建的技术领域，主要涉及一种负载间充质干细胞外基质的3D打印骨缺损修复支架及其制备方法。
背景技术
随着老龄化、关节退行性病变、车祸等外伤引起的骨组织损伤的增多，骨缺损修复越来越受到重视，临床中通常采用自体骨移植、异体骨移植和人工骨移植等骨移植方法。自体骨移植是进行缺损修复的“金标准”，但自体骨来源有限，往往供不应求，异体骨移植有感染疾病的风险，而人工骨移植缺乏骨诱导活性，成骨效率差，难以形成与健康骨组织相似结构的新生组织。因此，研究具有高生物活性和能够高效促成骨的新型再生骨缺损修复材料成为近年来的难点和热点，且具有巨大的临床需求和市场前景。
目前骨缺损修复支架按材料分为人工合成材料和生物材料。其中，人工合成材料在强度和构型方面具有控制优势，但在生物体内的相容性和降解性有待提高；而生物材料的相容性和降解性好，但强度和形状较难达到要求。另外，三维结构的支架更有利于细胞的分化和增殖。
生物活性玻璃是一种重要的骨组织工程的支架材料，能在体内有效促进生物矿化，释放硅、钙离子促进干细胞成骨、成血管。明胶/海藻酸钠水凝胶是由天然高分子材料混合而成，具有良好的生物相容性、组织可吸收性、低免疫原性等优点，特别是其利于和高生物活性无机粉体结合进行3D打印成型，但其力学强度比较差。3D打印能有效构建多孔生物玻璃骨组织工程支架，精确调控孔隙率、孔径等参数，赋予其较好的生物活性，修复支架通常需要具备良好的力学性质、生物相容性、骨传导性、骨诱导性。
在复合材料支架的基础上，为进一步提高支架的成骨效率，在支架上负载促生长因子或药物成为提高骨组织修复的另一有效手段。其中，生长因子因在生物体内高效的促进细 胞增殖分化及功能性蛋白形成，在人工合成的复合生物支架中的应用引起了研究者们极大的兴趣。在复合支架中添加生长因子例如骨基质蛋白2(one matrix protein-2，BMP-2)，可促进干细胞成骨向分化，但由于其在体内半衰期短，为长期保持有效剂量，需要在支架中大量添加，超过了安全标准剂量1.5mg/ml，因此导致了一系列不良反应，例如炎症，异位骨和肿瘤形成。因此，如何在不引起不良反应的情况下进一步提高骨组织形成效率，成为目前亟待解决的问题。研究表明，rBMSC细胞的ECM不仅能够为成骨分化提供必要的蛋白因子，同时因其存在特殊的“cell sheet”结构，更加利于骨组织的形成。本发明可为开发新型有效的颌骨缺损修复材料提供理论指导及实验数据，为临床研发骨缺损替代材料提供新探索。
发明内容
本发明的目的是提供一种负载间充质干细胞外基质的3D打印骨缺损修复支架，利用明胶、海藻酸钠和58S生物玻璃并调节三者的含量配比，使制备的支架强度高、相容性好、体内降解效果好，并且rBMSC能够在支架上黏附，增殖，明显提高骨组织的成骨效率。
为实现上述目的，本发明提供的技术方案如下：
一种负载间充质干细胞外基质的3D打印骨缺损修复支架，包括3D打印支架和负载在支架上的间充质干细胞外基质；
所述负载间充质干细胞外基质的3D打印骨缺损修复支架的制备方法包括如下步骤：
S1.3D打印支架的制备：
S11.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，其中，溶液中各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；
S12.将溶液搅拌均匀，获得3D打印浆料，然后进行3D打印；采用0.40-0.50mm孔径的针头进行打印，第一层包括多条相互平行的线条，第二层为垂直连接于第一层线条上表面的多条相互平行的线条，第三层为垂直连接于第二层线条上表面的多条相互平行的线条，以此类推，打印4-8层；
S13.打印完成获得支架半成品，支架半成品先用氯化钙溶液进行化学交联0.5小时，后浸泡于戊二醛溶液中进行化学交联6小时；最后清洗，冻干即得；
S2.将3D打印制备好的支架进行消毒处理；
S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖 DMEM培养基进行培养，每3天进行1次换液，在支架上培养rBMSC 2周；
S4.取出支架，脱细胞处理，冻干即得。
作为应用于骨修复的支架材料，其必须具备以下几个条件：1、生物降解不产生有毒物质；2、为新生组织提供良好的力学支持；3、降解速度与组织新生速度相匹配；4、具有孔隙，允许营养物质及代谢产物的弥散；5、匹配支架和正常软骨的抗压特性。在本发明中，海藻酸钠凝胶具有适合细胞营养交换的三维培养结构，并能保持因表面积大、气孔多而形成的特定形态。加入明胶，能够提高海藻酸钠凝胶的机械强度，模拟细胞生长所需要的内环境，生物相容性好。58S生物玻璃能够在支架中稳定释放Si离子、Ca离子等，能够在基因水平上刺激骨祖细胞，促进新生骨的生长。
明胶、海藻酸钠和58S生物玻璃三者的含量选择对支架的整体性能影响较大，明胶的含量高，可以获得良好的生物相容性；海藻酸钠具有良好的固结成型的能力，生物玻璃促进骨的生长。因此，如何调整这三种物质的用量，获得具有良好的生物相容性、细胞粘附性、生物降解性和生物活性因子负载能力的支架，是需要大量试验工作的。发明人经过大量实验研究后选择了各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；在几乎不影响成骨效率的情况下，降低了打印浆料中的58S生物玻璃组分，提高了打印时出丝的流畅度，使得打印的支架结构更加规则，孔隙率和间距更加合格。本发明3D打印支架的结构孔洞是平直的，因为结构规则，没有比较明显的阻碍物，在流体力学上没有较强的流体阻力，有利于营养物质和细胞渗入支架内部，加快修复过程中的成骨效率。
细胞外基质(extracellular matrix，ECM)是由细胞分泌的一系列蛋白及其他成分的总称，在细胞信号转导，调节细胞生理功能等方面起到关键的作用。rBMSC细胞的ECM不仅能够为成骨分化提供必要的蛋白因子，同时因其存在特殊的“cell sheet”结构，更加利于骨组织的形成。本发明3D打印支架可以负载rBMSC细胞的ECM，rBMSC能够在支架上黏附，增殖。
在本发明中，优选地，S12中的3D打印采用0.41mm孔径的针头，在0.42Mpa气压、30℃条件下，按照8mm/s的打印速度进行打印。上述打印参数的设置，在支架成型方面能够保持构型，同时尺寸更准确规整，不会有黏连产生。优选地，S12中，通过磁力搅拌和/或机械搅拌将溶液搅拌均匀，获得3D打印浆料，将3D打印浆料注入3D打印料筒，除泡均化后开始打印。
在本发明中，优选地，S12中，支架中每一层相邻线条之间的距离为300-500μm，支架 的层数为6层。支架上下垂直堆叠的设置以及孔洞距离的调整，扩大丰富了孔道的多样性，有利于营养物质和细胞渗入支架内部。
在本发明中，优选地，所述58s生物玻璃经过研磨后过筛，使58s生物玻璃粉体的颗粒直径范围为4～10微米，58s生物玻璃的化学组成为58％SiO2-33％CaO-9％P2O5。58s生物玻璃粉体粒径的选择，使其比表面积大，释放的离子较多，粒径小于4微米时，其在溶液中的分散均匀性变差，不利于其功效的发挥。
在本发明中，优选地，S3所述换液的操作具体为用移液枪吸干皿内培养液，用pbs漂洗细胞3次，加入新鲜培养液；S4所述脱细胞处理为：10mM氨水+0.1％SDS浸泡支架30min，蒸馏水漂洗3次，0.1％DNA酶溶液浸泡10min，蒸馏水漂洗3次。
在本发明中，优选地，S4所述冻干在-40℃温度下保存12小时。
本发明支架负载间充质干细胞外基质的换液操作、脱细胞处理以及最后的冻干保存，可以采用本领域的常规技术手段。上述参数设置为优化方案，能够使支架负载更多的间充质干细胞外基质，负载率高。
在本发明中，优选地，所述氯化钙溶液的浓度为5％～6％，通过将氯化钙粉体加入蒸馏水中溶解而成。所述戊二醛溶液浓度为1.0％～1.5％，通过用蒸馏水对50％的戊二醛溶液进行稀释获得。氯化钙溶液和戊二醛溶液的浓度选择，使支架的交联效果较好。
与现有技术相比，本发明的有益效果：
1、本发明经过大量实验研究后选择了3D打印支架各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；在几乎不影响成骨效率的情况下，降低了打印浆料中的58S生物玻璃组分，提高了打印时出丝的流畅度，使得打印的支架结构更加规则，孔隙率和间距更加合格。3D打印支架的结构孔洞是平直的，因为结构规则，没有比较明显的阻碍物，在流体力学上没有较强的流体阻力，有利于营养物质和细胞渗入支架内部，加快修复过程中的成骨效率。
2、本发明经过研究发现，在明胶/海藻酸钠/58S生物玻璃支架上负载rBMSC细胞的ECM，应用于骨缺损修复，其形成血管组织面积、分支数量显著提高，有效促进骨组织及血管组织形成，明显提高骨缺损修复的效率。
3、本发明研究和优化了明胶/海藻酸钠/58S生物玻璃支架上负载rBMSC细胞的细胞外基质的制备工艺参数，获得了良好的负载效果。
附图说明
图1为本发明3D打印支架的照片(a：正面照片，b：侧面照片)。
图2为负载间充质干细胞外基质的3D打印骨缺损修复支架的live/dead染色结果图。显示rBMSC能够在复合支架上黏附，增殖。
图3为SEM观察负载间充质干细胞外基质的3D打印骨缺损修复支架的表面微观形貌及rBMSC在支架上的黏附伸展情况。
图4为负载间充质干细胞外基质的3D打印骨缺损修复支架的成骨相关基因表达水平(a：RUNX-2；b：BMP2)。
图5为负载间充质干细胞外基质的3D打印骨缺损修复支架的动物实验成骨效率图(左：本发明支架组，中：空白组，右：BIO-OSS骨粉阳性对照组)。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合说明书附图和具体实施例，对本发明进一步详细说明，但本发明要求的保护范围并不局限于实施例。
下述实施例所采用的原料如无特殊说明，均为市售。
其中，采用的58s生物玻璃的化学组成为58％SiO2-33％CaO-9％P2O5，直径范围为4～10微米。
实施例1:
一种负载间充质干细胞外基质的3D打印骨缺损修复支架，包括3D打印支架和负载在支架上的间充质干细胞外基质。
所述负载间充质干细胞外基质的3D打印骨缺损修复支架的制备方法包括如下步骤：
S1.3D打印支架的制备：
S11.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，其中，溶液中各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；
S12.通过磁力搅拌和/或机械搅拌将溶液搅拌均匀，获得3D打印浆料，将3D打印浆料注入3D打印料筒，除泡均化后开始打印；采用0.41mm孔径的针头，在0.42Mpa气压、30℃条件下，按照8mm/s的打印速度进行打印，第一层包括多条相互平行的线条，第二层为垂直连接于第一层线条上表面的多条相互平行的线条，第三层为垂直连接于第二层线条 上表面的多条相互平行的线条，以此类推，打印4层；
S13.打印完成获得支架半成品，支架半成品先用氯化钙溶液进行化学交联0.5小时，后浸泡于戊二醛溶液中进行化学交联6小时；最后清洗，冻干即得；所述氯化钙溶液的浓度为5％，通过将氯化钙粉体加入蒸馏水中溶解而成；所述戊二醛溶液浓度为1.0％，通过用蒸馏水对50％的戊二醛溶液进行稀释获得；
S2.将3D打印制备好的支架进行消毒处理；
S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖DMEM培养基进行培养，每3天进行1次换液，在支架上培养rBMSC 2周；所述换液的操作具体为用移液枪吸干皿内培养液，用pbs漂洗细胞3次，加入新鲜培养液；
S4.取出支架，脱细胞处理：10mM氨水+0.1％SDS浸泡支架30min，蒸馏水漂洗3次，0.1％DNA酶溶液浸泡10min，蒸馏水漂洗3次；最后在-40℃温度下保存12小时即得。
实施例2:
一种负载间充质干细胞外基质的3D打印骨缺损修复支架，包括3D打印支架和负载在支架上的间充质干细胞外基质。
所述负载间充质干细胞外基质的3D打印骨缺损修复支架的制备方法包括如下步骤：
S1.3D打印支架的制备：
S11.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，其中，溶液中各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；
S12.通过磁力搅拌和/或机械搅拌将溶液搅拌均匀，获得3D打印浆料，将3D打印浆料注入3D打印料筒，除泡均化后开始打印；采用0.41mm孔径的针头，在0.42Mpa气压、30℃条件下，按照8mm/s的打印速度进行打印，第一层包括多条相互平行的线条，第二层为垂直连接于第一层线条上表面的多条相互平行的线条，第三层为垂直连接于第二层线条上表面的多条相互平行的线条，以此类推，打印6层；
S13.打印完成获得支架半成品，支架半成品先用氯化钙溶液进行化学交联0.5小时，后浸泡于戊二醛溶液中进行化学交联6小时；最后清洗，冻干即得；所述氯化钙溶液的浓度为5.5％，通过将氯化钙粉体加入蒸馏水中溶解而成；所述戊二醛溶液浓度为1.5％，通过用蒸馏水对50％的戊二醛溶液进行稀释获得；
S2.将3D打印制备好的支架进行消毒处理；
S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖DMEM培养基进行培养，每3天进行1次换液，在支架上培养rBMSC 2周；所述换液的操作具体为用移液枪吸干皿内培养液，用pbs漂洗细胞3次，加入新鲜培养液；
S4.取出支架，脱细胞处理：10mM氨水+0.1％SDS浸泡支架30min，蒸馏水漂洗3次，0.1％DNA酶溶液浸泡10min，蒸馏水漂洗3次；最后在-40℃温度下保存12小时即得。
实施例3:
一种负载间充质干细胞外基质的3D打印骨缺损修复支架，包括3D打印支架和负载在支架上的间充质干细胞外基质。
所述负载间充质干细胞外基质的3D打印骨缺损修复支架的制备方法包括如下步骤：
S1.3D打印支架的制备：
S11.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，其中，溶液中各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；
S12.通过磁力搅拌和/或机械搅拌将溶液搅拌均匀，获得3D打印浆料，将3D打印浆料注入3D打印料筒，除泡均化后开始打印；采用0.41mm孔径的针头，在0.42Mpa气压、30℃条件下，按照8mm/s的打印速度进行打印，第一层包括多条相互平行的线条，第二层为垂直连接于第一层线条上表面的多条相互平行的线条，第三层为垂直连接于第二层线条上表面的多条相互平行的线条，以此类推，打印8层；
S13.打印完成获得支架半成品，支架半成品先用氯化钙溶液进行化学交联0.5小时，后浸泡于戊二醛溶液中进行化学交联6小时；最后清洗，冻干即得；所述氯化钙溶液的浓度为6％，通过将氯化钙粉体加入蒸馏水中溶解而成；所述戊二醛溶液浓度为1.5％，通过用蒸馏水对50％的戊二醛溶液进行稀释获得；
S2.将3D打印制备好的支架进行消毒处理；
S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖DMEM培养基进行培养，每3天进行1次换液，在支架上培养rBMSC 2周；所述换液的操作具体为用移液枪吸干皿内培养液，用pbs漂洗细胞3次，加入新鲜培养液；
S4.取出支架，脱细胞处理：10mM氨水+0.1％SDS浸泡支架30min，蒸馏水漂洗3次，0.1％DNA酶溶液浸泡10min，蒸馏水漂洗3次；最后在-40℃温度下保存12小时即得。
性能测试
对上述实施例2制备的负载间充质干细胞外基质的3D打印骨缺损修复支架进行性能测试，如下：
1、拍照，负载间充质干细胞外基质的3D打印骨缺损修复支架的结构尺寸如附图1所示。
2、PCR实验：将rBMSC以105个/孔接种于负载间充质干细胞外基质的3D打印骨缺损修复支架上，使用含10％FBS的低糖DMEM进行培养，每3天进行1次换液，在第0，7，14天提取细胞总RNA，进行PCR检测。
3、动物实验：
SPF级雄性SD大鼠64只，体重280-320g，随机分成ECM支架组、BIO-OSS骨胶原组和对照组。大鼠无菌状态下腹腔注射麻醉，在平行下颌骨下缘上作1.0-1.5cm厘米切口，皮下组织分层切开后钝性分离暴露下颌骨，利用直径5mm环骨钻配合生理盐水灌注冷却制作直径为5mm圆形全层骨缺损，分别植入载ECM支架和BIO-OSS骨胶原，空白组不放任何材料，组织内伤口采用5-0缝合线分层缝合，术后连续3天予青霉素钠肌肉注射抗感染。4周和8周两个时间点取材，大鼠采用二氧化碳窒息法行安乐死，摘取缺损区在内的下颌骨固定在10％中性缓冲福尔马林24小时，然后Micro-CT进行扫描。使用NRecon软件Skyscan对图像文件进行扫描重建，并从扫描图像中选择感兴趣的区域(ROI)进行分析。
从图1中可以看出，制备的3D打印支架的结构孔洞是平直的，因为结构规则，没有比较明显的阻碍物，在流体力学上没有较强的流体阻力，有利于营养物质和细胞渗入支架内部，加快修复过程中的成骨效率。从图2、图3可以看出，rBMSC能够在负载间充质干细胞外基质的3D打印骨缺损修复支架上黏附，增殖。从图4可以看出，成骨相关基因表达水平高。从图5可以看出，负载间充质干细胞外基质的3D打印骨缺损修复支架的成骨效率与BIO-OSS骨粉相差不大。
从上述测试结果来看，本发明制备的负载间充质干细胞外基质的3D打印骨缺损修复支架达到了本发明的目的，负载效果良好，获得的复合支架应用于骨缺损修复，能够有效促进骨组织及血管组织形成，明显提高骨缺损修复的效率。
根据上述说明书的揭示和教导，本发明所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对发明的一些 修改和变更也应当落入本发明的权利要求的保护范围内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。

Claims (10)

  1. 一种负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，包括3D打印支架和负载在支架上的间充质干细胞外基质；
    所述负载间充质干细胞外基质的3D打印骨缺损修复支架的制备方法包括如下步骤：
    S1. 3D打印支架的制备：
    S11.将明胶、海藻酸钠和58S生物玻璃溶于水中获得溶液，其中，溶液中各成分的质量/体积的浓度为明胶18％、海藻酸钠5％、58S生物玻璃5.5％；
    S12.将溶液搅拌均匀，获得3D打印浆料，然后进行3D打印；采用0.40-0.50mm孔径的针头进行打印，第一层包括多条相互平行的线条，第二层为垂直连接于第一层线条上表面的多条相互平行的线条，第三层为垂直连接于第二层线条上表面的多条相互平行的线条，以此类推，打印4-8层；
    S13.打印完成获得支架半成品，支架半成品先用氯化钙溶液进行化学交联0.5小时，后浸泡于戊二醛溶液中进行化学交联6小时；最后清洗，冻干即得；
    S2.将3D打印制备好的支架进行消毒处理；
    S3.将rBMSC细胞悬液以至少106个/孔的浓度接种于支架上，使用含10％FBS的低糖DMEM培养基进行培养，每3天进行1次换液，在支架上培养rBMSC 2周；
    S4.取出支架，脱细胞处理，冻干即得。
  2. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，S12中的3D打印采用0.41mm孔径的针头，在0.42Mpa气压、30℃条件下，按照8mm/s的打印速度进行打印。
  3. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，S12中，支架中每一层相邻线条之间的距离为300-500μm，支架的层数为6层。
  4. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，所述58s生物玻璃经过研磨后过筛，使58s生物玻璃粉体的颗粒直径范围为4～10微米，58s生物玻璃的化学组成为58％SiO2-33％CaO-9％P2O5。
  5. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，S3所述换液的操作具体为用移液枪吸干皿内培养液，用pbs漂洗细胞3次，加入新鲜培养液；S4所述脱细胞处理为：10mM氨水+0.1％SDS浸泡支架30min，然后蒸馏水漂洗3次，0.1％DNA酶溶液浸泡10min，蒸馏水漂洗3次。
  6. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征 在于，S4所述冻干操作为在-40℃温度下保存12小时。
  7. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，所述氯化钙溶液的浓度为5％～6％，通过将氯化钙粉体加入蒸馏水中溶解而成。
  8. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，所述戊二醛溶液浓度为1.0％～1.5％，通过用蒸馏水对50％的戊二醛溶液进行稀释获得。
  9. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，S12中，将3D打印浆料注入3D打印料筒，除泡均化后开始打印。
  10. 根据权利要求1所述负载间充质干细胞外基质的3D打印骨缺损修复支架，其特征在于，S12中，通过磁力搅拌和/或机械搅拌将溶液搅拌均匀，获得3D打印浆料。
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