WO2017031906A1 - Bone repair porous compound stent based on 3d -bioplotter printing technology and preparation method therefor - Google Patents

Bone repair porous compound stent based on 3d -bioplotter printing technology and preparation method therefor Download PDF

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WO2017031906A1
WO2017031906A1 PCT/CN2015/100017 CN2015100017W WO2017031906A1 WO 2017031906 A1 WO2017031906 A1 WO 2017031906A1 CN 2015100017 W CN2015100017 W CN 2015100017W WO 2017031906 A1 WO2017031906 A1 WO 2017031906A1
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bioplotter
bone repair
stent
composite
microspheres
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PCT/CN2015/100017
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French (fr)
Chinese (zh)
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魏坤
胡露
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华南理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic 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
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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

Definitions

  • the invention relates to the field of biomedical engineering and biomedical material technology, in particular to a 3D-Bioplotter Printed bone repair porous composite scaffold and preparation method thereof.
  • Bone is an important organ of the human body, and it is responsible for supporting, exercising, protecting, hematopoiesis, storing minerals and metabolism. Clinically, due to trauma, infection, tumor and congenital dysplasia, large areas of bone defects and osteoporosis, beyond the bone's self-repair ability, require bone repair treatment.
  • Traditional bone defect treatment methods mainly include autologous bone transplantation and allogeneic bone transplantation. Autologous bone transplantation has a very limited source and produces secondary surgery, which brings more pain to patients. Allogeneic bone transplantation has immune rejection and carries virus. And the risk of bacteria, so the application is limited. How to find better bone defect repair materials has become the goal pursued by many scientists around the world.
  • PLGA is one of the first biomaterials approved by the US Food and Drug Administration (FDA) for use in humans. It has good biocompatibility and can be adjusted for LA and GA by degradation rate. The ratio is regulated, and the degradation products are non-toxic and can be eliminated by metabolism in the body.
  • the ideal bone tissue engineering scaffolding material should have the following properties: 1) Good biocompatibility, ie no obvious cytotoxicity, inflammatory reaction and immune rejection, can be safely used in human body; (2 Suitable biodegradable absorption, ie degradation rate corresponding to cell and tissue growth rate; (3) suitable pore size (200-400 ⁇ m), high porosity (90% or more) And three-dimensional porous structure to facilitate the proliferation of a large number of cells, the growth of tissues, the formation of extracellular matrix, the transmission of oxygen and nutrients, the excretion of metabolites, and the growth of blood vessels and nerves; (4) a specific three-dimensional shape to obtain the desired shape of the tissue or organ; (5) high specific surface area and suitable surface physical and chemical properties to facilitate cell adhesion, proliferation and differentiation, and biosignal molecules such as growth factors; Good mechanical shape
  • the preparation methods of bone tissue engineering mainly include phase separation method, gas foaming method, particle leaching method, particle sintering method, template method, electrospinning technology and the like.
  • the above method generally can only prepare a pore diameter smaller than
  • the bracket has the disadvantages of small porosity, difficult to control the geometry of the bracket, and poor connection between the pores.
  • the pore size of the scaffold prepared by the phase separation method is small; the gas foaming method cannot accurately control the porosity and the shape of the stent, and the pore diameter cannot be made; the porogen content of the particle leaching method will seriously affect the mechanical strength of the stent. And the residue of the porogen may cause cytotoxicity; the mechanical properties of the stent prepared by the electrospinning technique are low. Rapid prototyping technology can accurately control the geometry, pore size, porosity and pore distribution of the stent according to the requirements of different patients and the characteristics of the material, and develop a personalized treatment plan.
  • the object of the present invention is to provide a 3D-Bioplotter based on the deficiencies of the prior art.
  • the present invention will be 3D-Bioplotter Printing technology combined with microsphere technology to produce a new composite porous bone tissue engineering scaffold.
  • the stent not only has a simple and rapid preparation process, but also has a controllable shape and a multi-stage pore structure, is beneficial to the transportation of nutrients and oxygen, promotes cell adhesion, proliferation and differentiation, and can induce long-term formation of bone tissue; Good drug release properties.
  • a bone-repairing porous composite scaffold of a printing technique which is composed of a matrix having a three-dimensional macroporous structure and a drug-loaded microsphere; the three-dimensional macroporous matrix is composed of 3D-Bioplotter Instrument preparation; drug-loaded microspheres were prepared by emulsification method; composite stents were prepared by low-temperature sintering method at 37 ⁇ 65 °C.
  • the porous structure of the bone repair porous composite stent is composed of a large hole having a pore diameter of 0.1 to 1.2 mm, Micropores of 0.05 ⁇ 150 ⁇ m and mesopores of 2 ⁇ 50nm, the macropores are the pores in the matrix of the three-dimensional scaffold, and the micropores are microspheres 37 ⁇ 65 °C
  • the inter-microsphere pores formed after low-temperature sintering, the mesopores are holes in hexagonal mesoporous silicon (HMS).
  • the bone repair porous composite stent has a porosity of 60 to 80% and a pore connectivity of 90% or more.
  • the bone repair porous composite stent is filled in the matrix by the microspheres in a specific arrangement to form a regular pore structure composite stent having a specific pattern, and the pore structure composite stent of different patterns can be prepared according to requirements.
  • the matrix material of the bone repair porous composite stent is one or more of the following materials: polylactic acid - A glycolic acid copolymer, polylactic acid, polyglycolic acid, polycaprolactone, and a polyhydroxy fatty acid ester.
  • a method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology comprising the following steps:
  • the PLGA/CS/HMS composite microspheres loaded with drug or growth factor are uniformly filled in the scaffold channel and placed at 37 ⁇ 65 °C. In the oven, heat for 2 ⁇ 4h until the microspheres are firmly bonded in the channel of the scaffold to obtain a porous composite scaffold for bone repair.
  • the CAD The model is a three-dimensional structural model of a specific shape, a specific specification, and a specific size designed according to different repair parts of the human body using computer-aided software.
  • the printing temperature is 130 to 160 ° C, and the platform temperature is 20 to 40 ° C.
  • the needle size is 0.2 ⁇ 0.4mm, the extrusion pressure is 1.0 ⁇ 3.5bar, the extrusion speed is 1.0 ⁇ 5.0mm/s, the aperture is 0.1 ⁇ 1.2mm; the internal structure is the nozzle angle 0 ⁇ 179° staggered, layer thickness is 0.16 ⁇ 0.32mm.
  • the drug or growth factor is any one or more of the following: isoniazid, rifampicin, gentamicin, BMP-1, BMP-2, BMP-7, BMP-14, TGF- ⁇ and TGF- ⁇ .
  • the hexagonal mesoporous silicon is a laboratory-made mesoporous silicon, and the preparation method is as follows: 2 to 5 g of dodecylamine, 20 ⁇ 60ml absolute ethanol, 20 ⁇ 60ml deionized water is added into a 250ml beaker and stirred with a magnetic stirrer; then 5 ⁇ 10g of tetraethyl orthosilicate is added and stirred for 8 ⁇ 12h. After 30 min; washed with deionized water and ethanol, dried in an oven at 640 ° C for 4 h, and then ground through a 160 mesh screen to obtain hexagonal mesoporous silica powder.
  • the calcium silicate powder is a laboratory-made calcium silicate, and the preparation method is as follows: mixing sodium silicate and calcium nitrate by 1:1 mixing for 12 hours, then aging for 30 minutes, washing with deionized water 4 Then, it is freeze-dried in a vacuum state for 24 to 48 hours until it is completely dehydrated and calcined at 800 °C for 2 hours, and then ground through a 160 mesh sieve to obtain a calcium silicate powder.
  • the concentration of the drug or growth factor is 0.2-2 mg/ml, and the concentration of hexagonal mesoporous silicon is 10 ⁇ 40mg/ml, the concentration of calcium silicate powder is 10 ⁇ 60mg/ml; polyvinyl alcohol is 1788 type, 1799 One of the types; the stirring speed of the blended droplets added to the polyvinyl alcohol aqueous solution to separate the composite microspheres is 180-350 r/min; and the composite microspheres have a particle diameter of 100-600 ⁇ m.
  • the ml in the concentration unit mentioned above is based on the volume of the blend obtained in this step.
  • the present invention has the following advantages and effects:
  • the base material is made of 3D-Bioplotter
  • the instrument is prepared, which not only has a simple and rapid preparation process, but also can accurately control the geometric shape, pore size, porosity and pore distribution of the stent according to different patient requirements and material characteristics, and formulate a personalized treatment plan.
  • the composite stent has a multi-stage pore structure from nanometer to millimeter, and the porous structure is composed of a large pore of 0.1 to 1.2 mm. Micron holes of 0.05 ⁇ 100 ⁇ m and 2 ⁇ 50nm
  • the mesoporous composition is beneficial to the proliferation of a large number of cells, the growth of tissues, the formation of extracellular matrix, the transmission of oxygen and nutrients, the excretion of metabolites, and the angiogenesis of blood vessels and nerves, which can induce the formation of bone tissue for a long time.
  • PLGA/CS/HMS PLGA/CS/HMS
  • the addition of composite microspheres not only greatly improves the mechanical properties, biocompatibility and osteoinductive effects of the stent, but also has good drug-loading properties.
  • the microspheres can be filled in the matrix in different arrangements according to requirements, and individualized composite stents of different patterns are formed.
  • FIG. 1 is a schematic view showing a process of preparing a bone repair porous composite stent according to an embodiment of the present invention.
  • FIG. 2 is a scanning electron micrograph (200 ⁇ ) of a composite microsphere according to an embodiment of the present invention.
  • FIG. 3 is a scanning electron micrograph (1000 ⁇ ) of a composite microsphere according to Embodiment 1 of the present invention.
  • FIG. 4 is a three-dimensional microscope picture of a stent base according to an embodiment of the present invention.
  • Fig. 5 is a three-dimensional microscope picture of a composite stent according to an embodiment of the present invention.
  • Figure 6 is a scanning electron micrograph (25x) of the composite stent of the embodiment 1 of the present invention.
  • Figure 7 is a scanning electron micrograph (200x) of the composite stent of the embodiment 1 of the present invention.
  • the hexagonal mesoporous silicon described below is a laboratory-made mesoporous silicon prepared as follows: 5 g of dodecylamine, 60 ml of absolute ethanol, 60 ml. Deionized water was added to a 250 ml beaker and stirred with a magnetic stirrer for 30 min; then 22.3 ml of tetraethyl orthosilicate was slowly added and stirred for 12 h and aged for 30 min. Washed twice with deionized water and ethanol, placed in an oven at 80 ° C for 2 h, then calcined at 640 ° C for 4 h, and then ground through a 160 mesh screen to obtain an average pore diameter of 2.5 nm. Hexagonal mesoporous silicon powder.
  • the calcium silicate powder described below is a laboratory-made calcium silicate, and the preparation method is as follows: the sodium silicate and the calcium nitrate are mixed by a 1:1 mixing and stirring for 12 hours. , then aged for 30 min, washed 4 times with deionized water, lyophilized under vacuum for 48 h to fully dehydrated and calcined at 800 °C for 2 h, then ground 160 The mesh screen is used to prepare calcium silicate powder.
  • PLA PGA monomer ratio of 50:50, molecular weight of 3.1 Million of polylactic acid monoglycolic acid copolymer (PLGA).
  • Cube model STL 10 mm long, 10 mm wide and 2 mm high using Bioplotter RP software Format data is layered, 2gPLGA is added to the stainless steel barrel, 0.3mm needle is used, VisualMachines software is opened, and the printing temperature is set to 150 °C.
  • the platform temperature is 25 °C
  • the extrusion pressure is 1.5bar
  • the extrusion speed is 3mm/s
  • the internal structure is set to the nozzles 0° and 90° alternately
  • the layer thickness is 0.24mm.
  • the hole diameter is 1.2mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD
  • the regular three-dimensional macroporous structure of the PLGA cube support matrix in the model as shown in Figure 4;
  • the composite microspheres are uniformly filled in the channel in the middle of the stent, and the rows around the edge are not filled, and then placed in an oven at 45 °C for 4 hours until the microspheres are firmly bonded in the pores of the stent, and the average pores between the microspheres for 120 ⁇ m, as shown in Fig. 5, Fig. 6 and Fig. 7, the composite scaffold obtained by CT measurement has a porosity of 78.5% and a pore connectivity of 97.6%.
  • Cylindrical model STL with diameter 10 mm and height 2 mm using Bioplotter RP software Format data is layered, 2gPLGA is added to the stainless steel barrel, 0.2mm needle is used, VisualMachines software is opened, and the printing temperature is set to 150 °C.
  • the platform temperature is 25 °C
  • the extrusion pressure is 3.0bar
  • the extrusion speed is 2mm/s
  • the internal structure is set to nozzles 0°, 45°, 90°, 145°.
  • the layer thickness is 0.16mm
  • the hole diameter is 1.0mm
  • the material is heated to the specified temperature and then kept for 30min to start 3D-Bioplotter
  • the three-dimensional structure model is layer-by-layer printed to form a PLGA cylinder support matrix with a regular three-dimensional macroporous structure in the CAD model;
  • the PLGA/CS/HMS composite microspheres carrying gentamicin are uniformly filled in the channels in the middle of the stent, and the filled channels and unfilled channels are pressed. Arrange in 1:1, then place in an oven at 55 °C for 4 h until the microspheres are firmly bonded in the pores of the scaffold. The average pore volume between the microspheres is 96 ⁇ m. The porosity of the composite scaffold obtained by CT measurement is obtained. 73.8%, the pore connectivity was 95.6%.
  • Cube model STL 10 mm long, 10 mm wide and 2 mm high using Bioplotter RP software The format data is layered. Add 2gPCL to the stainless steel barrel, select the 0.4mm needle, open the VisualMachines software, and set the printing temperature to 160 °C.
  • the platform temperature is 25 °C
  • the extrusion pressure is 1.2bar
  • the extrusion speed is 3mm/s
  • the internal structure is set.
  • the nozzles are alternately 0° and 90°
  • the layer thickness is 0.32mm.
  • the hole diameter is 1.2mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD a PCL cube support matrix with a regular three-dimensional macroporous structure in the model;
  • the above blended liquid was slowly added dropwise to an aqueous solution of polyvinyl alcohol, and stirred at a rate of 280 rpm for 12 hours, and then the composite microspheres at the bottom of the vessel were separated and freeze-dried under vacuum for 48 h.
  • the isoniazid-loaded PLGA/CS/HMS composite microspheres were prepared, and the composite microspheres with a particle size of 200-450 ⁇ m were separated by a stainless steel mesh;
  • the isoniazid-loaded PLGA/CS/HMS composite microspheres are uniformly filled in the channel in the middle of the stent, and the filled and unfilled channels are pressed. Arrange in 2:1, then place in an oven at 55 °C for 3 h until the microspheres are firmly bonded in the pores of the scaffold. The average pore volume between the microspheres is 108 ⁇ m. The porosity of the composite scaffold obtained by CT measurement is obtained. 76.4%, the pore connectivity was 96.8%.
  • Cylindrical model STL with diameter 5mm and height 2mm using Bioplotter RP software The format data is layered. Add 2gPCL to the stainless steel barrel, select 0.3mm needle, open VisualMachines software, set the printing temperature to 160 °C. The platform temperature is 25 °C, the extrusion pressure is 1.5bar, the extrusion speed is 3mm/s, and the internal structure is set to the nozzles 0° and 90° alternately, the layer thickness is 0.24mm.
  • the hole diameter is 0.8mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD a PCL cylinder support matrix with a regular three-dimensional macroporous structure in the model;
  • the PLGA/CS/HMS composite microspheres carrying BMP-2 were evenly filled in the channel in the middle of the stent, and then placed at 45 °C. In the oven, the temperature was kept for 4 hours until the microspheres were firmly bonded in the pores of the stent.
  • the average pore volume between the microspheres was about 114 ⁇ m.
  • the porosity of the composite stent obtained by CT measurement was 77.9%, and the pore connectivity was 97.3%.

Abstract

A bone repair porous compound stent based on 3D-Bioplotter printing technology and a preparation method therefor. The stent is formed by compounding a matrix provided with a 3D macroporous structure and a drug carrying microsphere. The preparation method comprises the following steps: printing a stent matrix having a regular 3D macroporous structure by means of 3D-Bioplotter; preparing a drug carrying microsphere compounding hexagonal mesoporous silica (HMS), calcium silicate (CS) powder and PLGA by means of an emulsion solvent volatilization method; finally, fixing the compound microsphere into the matrix by means of low-temperature sintering, so as to obtain the bone repair porous compound stent based on 3D-Bioplotter printing technology.

Description

基于3D-Bioplotter打印技术的骨修复多孔复合支架及其制备方法Bone repair porous composite scaffold based on 3D-Bioplotter printing technology and preparation method thereof
技术领域Technical field
本发明涉及生物医学工程和生物医用材料技术领域，具体涉及一种基于 3D-Bioplotter 打印技术的骨修复多孔复合支架及其制备方法。  The invention relates to the field of biomedical engineering and biomedical material technology, in particular to a 3D-Bioplotter Printed bone repair porous composite scaffold and preparation method thereof.
背景技术Background technique
骨骼是人体的重要器官，担负着支撑、运动、保护、造血、储存矿物质和代谢等功能。临床上由于外伤、感染、肿瘤和先天性发育不良等造成的大面积骨缺损以及骨质疏松，超过了骨的自我修复能力，需要进行骨修复治疗。传统的骨缺损治疗方法主要包括自体骨移植和异体骨移植，自体骨移植由于来源十分有限，且产生二次手术，给患者带来更多痛苦；异体骨移植又存在着免疫排斥反应和携带病毒及细菌的风险，因此应用受到限制。如何找到更好的骨缺损修复材料已经成为全世界众多科学家共同追求的目标。 Bone is an important organ of the human body, and it is responsible for supporting, exercising, protecting, hematopoiesis, storing minerals and metabolism. Clinically, due to trauma, infection, tumor and congenital dysplasia, large areas of bone defects and osteoporosis, beyond the bone's self-repair ability, require bone repair treatment. Traditional bone defect treatment methods mainly include autologous bone transplantation and allogeneic bone transplantation. Autologous bone transplantation has a very limited source and produces secondary surgery, which brings more pain to patients. Allogeneic bone transplantation has immune rejection and carries virus. And the risk of bacteria, so the application is limited. How to find better bone defect repair materials has become the goal pursued by many scientists around the world.
目前，对于骨组织工程材料的研究，主要在无机材料和高分子材料方面，虽然许多研究成果已达到一定的骨修复效果，但总体上还未达到理想骨组织工程支架材料的要求。如无机材料存在降解速度慢、力学性能较差等问题；天然高分子材料则存在不能大规模生产、机械强度不够等问题；人工合成高分子材料的亲水性、机械强度和有机溶剂残留物引起细胞中毒等是该类材料迫切需要解决的问题。 PLGA 是最早经过美国食品药品局 (FDA) 认证可用于人体的生物材料之一，其生物相容性好，降解速率可通过调节 LA 和 GA 的比例来调控，且降解产物均无毒，可通过体内代谢排除，是骨组织工程中良好的支架材料，但也存在一些缺点，如亲水性较差、机械强度不足，降解后产生的酸性物质会降低聚合物周围的 pH 值，对细胞生长不利。因此，若能选择一种具有生物活性，可缓解 pH 值下降，能增强支架机械强度的无机材料与 PLGA 复合，使两种材料之间互相取长补短 , 并以合适的工艺方法制备出理想结构的骨组织工程复合支架，具有极其重要的意义。 At present, the research on bone tissue engineering materials mainly focuses on inorganic materials and polymer materials. Although many research results have achieved certain bone repair effects, the requirements of ideal bone tissue engineering scaffold materials have not yet been reached. For example, inorganic materials have problems such as slow degradation rate and poor mechanical properties; natural polymer materials have problems such as large-scale production and insufficient mechanical strength; hydrophilicity, mechanical strength and residual organic solvents caused by synthetic polymer materials Cell poisoning and the like are urgent problems to be solved for such materials. PLGA is one of the first biomaterials approved by the US Food and Drug Administration (FDA) for use in humans. It has good biocompatibility and can be adjusted for LA and GA by degradation rate. The ratio is regulated, and the degradation products are non-toxic and can be eliminated by metabolism in the body. It is a good scaffold material in bone tissue engineering, but there are also some shortcomings, such as poor hydrophilicity, insufficient mechanical strength, and acidity after degradation. Substance will reduce the surrounding polymer pH is not good for cell growth. Therefore, if you choose a biological material that can alleviate the pH drop and strengthen the mechanical strength of the stent, it can be combined with PLGA to make the two materials complement each other. It is of great significance to prepare a bone tissue engineering composite scaffold with an ideal structure by a suitable process.
组织工程中，不仅要考虑材料的物理化学性质、表面性能等，组织工程支架作为组织再生的框架，其结构特性在组织工程中也起着关键性的作用。一般来讲，理想的骨组织工程支架材料应该具备以下性能：（ 1 ） 良好的生物相容性，即无明显的细胞毒性、炎症反应和免疫排斥反应，能安全用于人体；（ 2 ）合适的可生物降解吸收性，即与细胞、组织生长速率相适应的降解吸收速率；（ 3 ）合适的孔尺寸（ 200-400μm ）、高的孔隙率 (90% 以上 ) 和三维多孔结构，以利于大量细胞的增殖、组织的生长、细胞外基质的形成、氧气和营养的传输、代谢物的***以及血管和神经的内生长；（ 4 ）特定的三维外形以获得所需的组织或器官形状；（ 5 ）高的比表面积和合适的表面理化性能以利于细胞粘附、增殖和分化，以及负载生长因子等生物信号分子；（ 6 ）良好的可塑形性与植入部位组织的力学性能相匹配的机械强度，以在体内生物力学微环境中保持结构稳定性和完整性，并为植入细胞提高合适的微应力环境；（ 7 ）来源不受限制，易消毒、运输方便。 In tissue engineering, not only the physical and chemical properties of materials, surface properties, etc., but also the tissue engineering scaffolds as a framework for tissue regeneration, its structural properties play a key role in tissue engineering. In general, the ideal bone tissue engineering scaffolding material should have the following properties: 1) Good biocompatibility, ie no obvious cytotoxicity, inflammatory reaction and immune rejection, can be safely used in human body; (2 Suitable biodegradable absorption, ie degradation rate corresponding to cell and tissue growth rate; (3) suitable pore size (200-400 μm), high porosity (90% or more) And three-dimensional porous structure to facilitate the proliferation of a large number of cells, the growth of tissues, the formation of extracellular matrix, the transmission of oxygen and nutrients, the excretion of metabolites, and the growth of blood vessels and nerves; (4) a specific three-dimensional shape to obtain the desired shape of the tissue or organ; (5) high specific surface area and suitable surface physical and chemical properties to facilitate cell adhesion, proliferation and differentiation, and biosignal molecules such as growth factors; Good mechanical shape matching the mechanical properties of the implant site tissue to maintain structural stability and integrity in the in vivo biomechanical microenvironment and to enhance the appropriate microstress environment for implanted cells; (7) The source is unrestricted, easy to disinfect and convenient to transport.
骨组织工程的制备方法主要有相分离法、气体发泡法、粒子沥滤法、颗粒烧结法、模板法、静电纺丝技术等。而上述方法一般只能制备孔径小于 The preparation methods of bone tissue engineering mainly include phase separation method, gas foaming method, particle leaching method, particle sintering method, template method, electrospinning technology and the like. The above method generally can only prepare a pore diameter smaller than
200μm 的支架，且有孔隙率小、支架的几何形状不易控制，孔隙之间的连通率不佳等缺点。如相分离法制备的支架孔径偏小；气体发泡法无法精确控制孔隙率、支架形状，且无法做出较大孔径；粒子沥滤法的致孔剂含量又会严重的影响支架的机械强度，且致孔剂的残留会导致细胞毒性；静电纺丝技术制备的支架力学性能较低等。快速成型技术可以根据不同病人的要求和材料的特点，精确控制支架的几何外形、孔径、孔隙率及孔的分布，制定个性化的治疗方案。 200μm The bracket has the disadvantages of small porosity, difficult to control the geometry of the bracket, and poor connection between the pores. For example, the pore size of the scaffold prepared by the phase separation method is small; the gas foaming method cannot accurately control the porosity and the shape of the stent, and the pore diameter cannot be made; the porogen content of the particle leaching method will seriously affect the mechanical strength of the stent. And the residue of the porogen may cause cytotoxicity; the mechanical properties of the stent prepared by the electrospinning technique are low. Rapid prototyping technology can accurately control the geometry, pore size, porosity and pore distribution of the stent according to the requirements of different patients and the characteristics of the material, and develop a personalized treatment plan.
发明内容Summary of the invention
本发明的目的在于针对现有技术的不足，提供一种 基于 3D-Bioplotter 打印技术的骨修复多孔复合支架及其制备方法。 The object of the present invention is to provide a 3D-Bioplotter based on the deficiencies of the prior art. Printed bone repair porous composite scaffold and preparation method thereof.
本发明将 3D-Bioplotter 打印技术与微球技术结合起来，制得一种新型复合多孔骨组织工程支架。该支架不仅制备工艺简便快捷，且具有可控的外形和多级孔结构，有利于营养物质与氧的运输，促进细胞的黏附、增殖和分化，可长效诱导骨组织的形成；同时还具有良好的载药释药性能。 The present invention will be 3D-Bioplotter Printing technology combined with microsphere technology to produce a new composite porous bone tissue engineering scaffold. The stent not only has a simple and rapid preparation process, but also has a controllable shape and a multi-stage pore structure, is beneficial to the transportation of nutrients and oxygen, promotes cell adhesion, proliferation and differentiation, and can induce long-term formation of bone tissue; Good drug release properties.
本发明目的通过如下技术方案实现： The object of the present invention is achieved by the following technical solutions:
一种基于 3D-Bioplotter 打印技术的骨修复多孔复合支架，所述骨修复多孔复合支架由具有三维大孔结构的基体和载药微球复合而成；所述的三维大孔结构基体由 3D-Bioplotter 仪器制备；载药微球由乳化法制备；复合支架由 37~65 ℃ 低温烧结法制备而成。 One based on 3D-Bioplotter A bone-repairing porous composite scaffold of a printing technique, which is composed of a matrix having a three-dimensional macroporous structure and a drug-loaded microsphere; the three-dimensional macroporous matrix is composed of 3D-Bioplotter Instrument preparation; drug-loaded microspheres were prepared by emulsification method; composite stents were prepared by low-temperature sintering method at 37~65 °C.
进一步地，本发明中，所述骨修复多孔复合支架的多孔结构由孔径为 0.1~1.2mm 的大孔、 0.05~150μm 的微米孔和 2~50nm 的介孔组成，大孔为三维支架基体中的孔洞，微米孔为微球 37~65 ℃ 低温烧结后形成的微球间孔隙，介孔为六方介孔硅（ HMS ）中的孔洞。 Further, in the present invention, the porous structure of the bone repair porous composite stent is composed of a large hole having a pore diameter of 0.1 to 1.2 mm, Micropores of 0.05~150μm and mesopores of 2~50nm, the macropores are the pores in the matrix of the three-dimensional scaffold, and the micropores are microspheres 37~65 °C The inter-microsphere pores formed after low-temperature sintering, the mesopores are holes in hexagonal mesoporous silicon (HMS).
进一步地，本发明中，所述骨修复多孔复合支架的孔隙率为 60~80% ，孔隙连通率为 90% 以上。 Further, in the present invention, the bone repair porous composite stent has a porosity of 60 to 80% and a pore connectivity of 90% or more.
进一步地，本发明中，所述骨修复多孔复合支架由微球按照特定的排列方式填充在基体中，形成具有特定图案的规则孔结构复合支架，可根据需求制备不同图案的孔结构复合支架。 Further, in the present invention, the bone repair porous composite stent is filled in the matrix by the microspheres in a specific arrangement to form a regular pore structure composite stent having a specific pattern, and the pore structure composite stent of different patterns can be prepared according to requirements.
进一步地，本发明中，所述骨修复多孔复合支架的基体材料为以下材料中的一种或几种：聚乳酸 - 羟基乙酸共聚物、聚乳酸、聚羟基乙酸、聚己内酯和聚羟基脂肪酸酯。 Further, in the present invention, the matrix material of the bone repair porous composite stent is one or more of the following materials: polylactic acid - A glycolic acid copolymer, polylactic acid, polyglycolic acid, polycaprolactone, and a polyhydroxy fatty acid ester.
本发明的另一目的通过如下技术方案实现： Another object of the present invention is achieved by the following technical solutions:
一种基于 3D-Bioplotter 打印技术的骨修复多孔复合支架的制备方法，包括以下步骤： A method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology, comprising the following steps:
(1) 制备具有规则三维大孔结构的支架基体： (1) Preparation of a stent substrate having a regular three-dimensional macroporous structure:
利用计算机辅助设计软件 CAD 设计出骨缺损部位的三维结构模型，并把该三维模型保存为 STL 格式文件，然后导入 3D-Bioplotter 中，用 Bioplotter RP 软件对 CAD 模型的数据进行分层处理，然后将基体材料加入不锈钢料筒中，在 VisualMachines 软件中设置打印温度、平台温度、针头大小、挤出压力、挤出速度、内部结构和孔径参数，然后启动 3D-Bioplotter 将骨缺损部位的三维结构模型逐层打印成型，形成 CAD 模型中的规则三维大孔结构的支架基体； Design a three-dimensional structural model of the bone defect using CAD, and save the 3D model as STL Format the file, then import it into 3D-Bioplotter, layer the data of the CAD model with Bioplotter RP software, and then add the matrix material to the stainless steel barrel. Set the print temperature, platform temperature, needle size, extrusion pressure, extrusion speed, internal structure and pore size parameters in the VisualMachines software, then start 3D-Bioplotter The three-dimensional structure model of the bone defect portion is printed layer by layer to form a stent base body of a regular three-dimensional macroporous structure in the CAD model;
(2) 制备载药或生长因子的 PLGA/CS/HMS 复合微球： (2) Preparation of PLGA/CS/HMS composite microspheres loaded with drugs or growth factors:
将药物或生长因子与六方介孔硅混合得到药物或生长因子与介孔硅的混合粉体，然后将 PLGA （聚乳酸 - 羟基乙酸共聚物）溶于二氯甲烷中，待 12~24h 完全溶解后，加入上述混合粉体和硅酸钙粉体 (CS) ，用高速分散均质机搅拌均匀，得到载药或生长因子的 PLGA/CS/HMS 共混液；用去离子水配制 10~30mg/ml 的聚乙烯醇水溶液，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，搅拌 8~20h 后将容器底部的复合微球分离出来，真空状态下冷冻干燥 24~48 h 至完全脱水，制得载药或生长因子的 PLGA/CS/HMS 复合微球，并用不锈钢筛网分离出目标粒径的复合微球备用； Mixing drugs or growth factors with hexagonal mesoporous silicon to obtain a mixed powder of drug or growth factor and mesoporous silicon, and then PLGA (polylactic acid - The glycolic acid copolymer) is dissolved in methylene chloride, and after completely dissolved in 12 to 24 hours, the above mixed powder and calcium silicate powder (CS) are added, and uniformly stirred by a high-speed dispersing homogenizer to obtain a drug-loading or growth factor. of PLGA/CS/HMS blend; prepare 10~30mg/ml aqueous solution of polyvinyl alcohol in deionized water, then slowly add the above blend to the aqueous solution of polyvinyl alcohol, stir for 8~20h After that, the composite microspheres at the bottom of the vessel were separated and lyophilized under vacuum for 24 to 48 h to complete dehydration to obtain PLGA/CS/HMS loaded with drugs or growth factors. Composite microspheres, and the composite microspheres of the target particle size are separated by a stainless steel mesh;
(3) 基体材料与微球的复合： (3) Composite of matrix material and microsphere:
将载药或生长因子的 PLGA/CS/HMS 复合微球均匀填充在支架孔道中，置于 37~65 ℃ 烘箱中，保温 2~4h 直至微球牢固的粘结在支架孔道中，得到骨修复多孔复合支架。 The PLGA/CS/HMS composite microspheres loaded with drug or growth factor are uniformly filled in the scaffold channel and placed at 37~65 °C. In the oven, heat for 2~4h until the microspheres are firmly bonded in the channel of the scaffold to obtain a porous composite scaffold for bone repair.
进一步地，本发明步骤 (1) 中，所述的 CAD 模型为根据人体不同修复部位利用计算机辅助软件设计的特定形状、特定规格、特定尺寸的三维结构模型。 Further, in the step (1) of the present invention, the CAD The model is a three-dimensional structural model of a specific shape, a specific specification, and a specific size designed according to different repair parts of the human body using computer-aided software.
进一步地，本发明步骤 (1) 中，所述打印温度为 130~160 ℃ ，平台温度为 20~40 ℃ ，针头大小为 0.2~0.4mm ，挤出压力为 1.0~3.5bar ，挤出速度为 1.0~5.0mm/s ，孔径为 0.1~1.2mm ；内部结构为喷头角度 0~179° 交错排列，分层厚度为 0.16~0.32mm 。 Further, in the step (1) of the present invention, the printing temperature is 130 to 160 ° C, and the platform temperature is 20 to 40 ° C. The needle size is 0.2~0.4mm, the extrusion pressure is 1.0~3.5bar, the extrusion speed is 1.0~5.0mm/s, the aperture is 0.1~1.2mm; the internal structure is the nozzle angle 0~179° staggered, layer thickness is 0.16~0.32mm.
进一步地，本发明步骤 (2) 中，所述药物或生长因子为以下任意一种或几种：异烟肼、利福平、庆大霉素、 BMP-1 、 BMP-2 、 BMP-7 、 BMP-14 、 TGF-α 和 TGF -β 。 Further, in the step (2) of the present invention, the drug or growth factor is any one or more of the following: isoniazid, rifampicin, gentamicin, BMP-1, BMP-2, BMP-7, BMP-14, TGF-α and TGF-β.
进一步地，本发明步骤 (2) 中，所述六方介孔硅为实验室自制介孔硅，制备方法如下：将 2~5g 十二胺、 20~60ml 无水乙醇、 20~60ml 去离子水加入 250ml 的烧杯中，用磁力搅拌器搅拌；然后加入 5~10g 正硅酸乙酯搅拌 8~12h 后陈化 30min ；分别用去离子水和乙醇洗涤，放入烘箱干燥后 640 ℃ 锻烧 4h ，然后研磨过 160 目筛网，制得六方介孔硅粉末。 Further, in the step (2) of the present invention, the hexagonal mesoporous silicon is a laboratory-made mesoporous silicon, and the preparation method is as follows: 2 to 5 g of dodecylamine, 20~60ml absolute ethanol, 20~60ml deionized water is added into a 250ml beaker and stirred with a magnetic stirrer; then 5~10g of tetraethyl orthosilicate is added and stirred for 8~12h. After 30 min; washed with deionized water and ethanol, dried in an oven at 640 ° C for 4 h, and then ground through a 160 mesh screen to obtain hexagonal mesoporous silica powder.
进一步地，本发明步骤 (2) 中，所述硅酸钙粉体为实验室自制硅酸钙，制备方法如下：将硅酸钠和硝酸钙按摩尔比为 1:1 混合搅拌 12h ，然后陈化 30min ，用去离子水洗涤 4 次，真空状态下冷冻干燥 24~48h 至完全脱水后 800 ℃ 煅烧 2h ，然后研磨过 160 目筛网，制得硅酸钙粉体。 Further, the steps of the present invention (2) The calcium silicate powder is a laboratory-made calcium silicate, and the preparation method is as follows: mixing sodium silicate and calcium nitrate by 1:1 mixing for 12 hours, then aging for 30 minutes, washing with deionized water 4 Then, it is freeze-dried in a vacuum state for 24 to 48 hours until it is completely dehydrated and calcined at 800 °C for 2 hours, and then ground through a 160 mesh sieve to obtain a calcium silicate powder.
进一步地，本发明步骤 (2) 中，所述药物或生长因子浓度为 0.2-2mg/ml ，六方介孔硅的浓度为 10~40mg/ml ，硅酸钙粉体的浓度为 10~60mg/ml ；聚乙烯醇为 1788 型、 1799 型中的一种；所述共混液滴加到聚乙烯醇水溶液到分离复合微球时的搅拌速度均为 180~350r/min ；复合微球粒径为 100~600μm ；以上所述浓度单位中的 ml 均以该步骤得到的共混液的体积为计算基准。 Further, in the step (2) of the present invention, the concentration of the drug or growth factor is 0.2-2 mg/ml, and the concentration of hexagonal mesoporous silicon is 10~40mg/ml, the concentration of calcium silicate powder is 10~60mg/ml; polyvinyl alcohol is 1788 type, 1799 One of the types; the stirring speed of the blended droplets added to the polyvinyl alcohol aqueous solution to separate the composite microspheres is 180-350 r/min; and the composite microspheres have a particle diameter of 100-600 μm. The ml in the concentration unit mentioned above is based on the volume of the blend obtained in this step.
与现有技术相比，本发明具有如下优点及效果： Compared with the prior art, the present invention has the following advantages and effects:
（ 1 ）本发明中，基体材料由 3D-Bioplotter 仪器制备而成，不仅制备工艺简便快捷，而且可以根据不同病人的要求和材料的特点，精确控制支架的几何外形、孔径、孔隙率及孔的分布，制定个性化的治疗方案。 (1) In the present invention, the base material is made of 3D-Bioplotter The instrument is prepared, which not only has a simple and rapid preparation process, but also can accurately control the geometric shape, pore size, porosity and pore distribution of the stent according to different patient requirements and material characteristics, and formulate a personalized treatment plan.
（ 2 ）本发明中，所述复合 支架具有从纳米到毫米的多级孔结构，多孔结构由 0.1~1.2mm 的大孔、 0.05~100μm 的微米孔和 2~50nm 的介孔组成，有利于大量细胞的增殖、组织的生长、细胞外基质的形成、氧气和营养的传输、代谢物的***以及血管和神经的内生长，可长效诱导骨组织的形成。 (2) In the present invention, the composite stent has a multi-stage pore structure from nanometer to millimeter, and the porous structure is composed of a large pore of 0.1 to 1.2 mm. Micron holes of 0.05~100μm and 2~50nm The mesoporous composition is beneficial to the proliferation of a large number of cells, the growth of tissues, the formation of extracellular matrix, the transmission of oxygen and nutrients, the excretion of metabolites, and the angiogenesis of blood vessels and nerves, which can induce the formation of bone tissue for a long time.
（ 3 ）本发明中， PLGA/CS/HMS 复合微球的加入，不仅能大幅度地提高支架的力学性能、生物相容性和骨诱导效果，还具有良好的载药释药性能。 (3) In the present invention, PLGA/CS/HMS The addition of composite microspheres not only greatly improves the mechanical properties, biocompatibility and osteoinductive effects of the stent, but also has good drug-loading properties.
（ 4 ）本发明中，微球可以根据需求以不同的排列方式填充在基体中，形成不同图案的个性化复合支架。 (4) In the present invention, the microspheres can be filled in the matrix in different arrangements according to requirements, and individualized composite stents of different patterns are formed.
附图说明DRAWINGS
图 1 为本发明具体实施例制备骨修复多孔复合支架的过程示意图。FIG. 1 is a schematic view showing a process of preparing a bone repair porous composite stent according to an embodiment of the present invention.
图 2 为本发明实施例 1 复合微球的扫描电镜图（ 200× ）。 2 is a scanning electron micrograph (200× ) of a composite microsphere according to an embodiment of the present invention.
图 3 为本发明实施例 1 复合微球的扫描电镜图（ 1000× ）。 3 is a scanning electron micrograph (1000× ) of a composite microsphere according to Embodiment 1 of the present invention.
图 4 为本发明实施例 1 支架基体三维显微镜图片。 4 is a three-dimensional microscope picture of a stent base according to an embodiment of the present invention.
图 5 为本发明实施例 1 复合支架三维显微镜图片。 Fig. 5 is a three-dimensional microscope picture of a composite stent according to an embodiment of the present invention.
图 6 为本发明实施例 1 复合支架的扫描电镜图（ 25× ）。 Figure 6 is a scanning electron micrograph (25x) of the composite stent of the embodiment 1 of the present invention.
图 7 为本发明实施例 1 复合支架的扫描电镜图（ 200× ）。 Figure 7 is a scanning electron micrograph (200x) of the composite stent of the embodiment 1 of the present invention.
具体实施方式detailed description
为进一步理解本发明，下面结合实施例对本发明作进一步的描述，但是本发明要求保护的范围并不局限于实施例。 In order to further understand the present invention, the present invention is further described below in conjunction with the embodiments, but the scope of the invention is not limited to the embodiments.
以下所述六方介孔硅为实验室自制介孔硅，制备方法如下：将 5g 十二胺、 60ml 无水乙醇、 60ml 去离子水加入 250ml 的烧杯中，用磁力搅拌器搅拌 30min ；然后缓慢加入 22.3ml 正硅酸乙酯搅拌 12h 后陈化 30min ；分别用去离子水和乙醇洗涤 2 次，放入烘箱 80 ℃ 干燥 2h 后 640 ℃ 锻烧 4h ，然后研磨过 160 目筛网，制得平均孔径为 2.5nm 的六方介孔硅粉末。 The hexagonal mesoporous silicon described below is a laboratory-made mesoporous silicon prepared as follows: 5 g of dodecylamine, 60 ml of absolute ethanol, 60 ml. Deionized water was added to a 250 ml beaker and stirred with a magnetic stirrer for 30 min; then 22.3 ml of tetraethyl orthosilicate was slowly added and stirred for 12 h and aged for 30 min. Washed twice with deionized water and ethanol, placed in an oven at 80 ° C for 2 h, then calcined at 640 ° C for 4 h, and then ground through a 160 mesh screen to obtain an average pore diameter of 2.5 nm. Hexagonal mesoporous silicon powder.
以下所述硅酸钙粉体为实验室自制硅酸钙，制备方法如下：将硅酸钠和硝酸钙按摩尔比为 1:1 混合搅拌 12h ，然后陈化 30min ，用去离子水洗涤 4 次，真空状态下冷冻干燥 48 h 至完全脱水后 800 ℃ 煅烧 2h ，然后研磨过 160 目筛网，制得硅酸钙粉体。 The calcium silicate powder described below is a laboratory-made calcium silicate, and the preparation method is as follows: the sodium silicate and the calcium nitrate are mixed by a 1:1 mixing and stirring for 12 hours. , then aged for 30 min, washed 4 times with deionized water, lyophilized under vacuum for 48 h to fully dehydrated and calcined at 800 °C for 2 h, then ground 160 The mesh screen is used to prepare calcium silicate powder.
以下说述 PLGA 均采用 PLA:PGA 单体比为 50:50 、分子量为 3.1 万的聚乳酸一羟基乙酸共聚物（ PLGA ）。 The following description of PLGA uses PLA: PGA monomer ratio of 50:50, molecular weight of 3.1 Million of polylactic acid monoglycolic acid copolymer (PLGA).
实施例 1 Example 1
(1) 制备具有规则三维大孔结构的 PLGA 正方体支架基体： (1) Preparation of a PLGA cube support matrix with a regular three-dimensional macroporous structure:
用 Bioplotter RP 软件对长 10mm ，宽 10mm ，高 2mm 的正方体模型 STL 格式数据进行分层处理，将 2gPLGA 加入不锈钢料筒中，选用 0.3mm 的针头，打开 VisualMachines 软件，设置打印温度为 150 ℃ ，平台温度为 25 ℃ ，挤出压力为 1.5bar ，挤出速度为 3mm/s ，设置内部结构为喷头 0° 和 90° 依次交替，分层厚度为 0.24mm ，孔径为 1.2mm ，然后将材料加热到指定温度后保温 30min ，启动 3D-Bioplotter 将三维结构模型逐层打印成型，形成 CAD 模型中的规则三维大孔结构的 PLGA 正方体支架基体，如图 4 ； Cube model STL 10 mm long, 10 mm wide and 2 mm high using Bioplotter RP software Format data is layered, 2gPLGA is added to the stainless steel barrel, 0.3mm needle is used, VisualMachines software is opened, and the printing temperature is set to 150 °C. The platform temperature is 25 °C, the extrusion pressure is 1.5bar, the extrusion speed is 3mm/s, and the internal structure is set to the nozzles 0° and 90° alternately, the layer thickness is 0.24mm. , the hole diameter is 1.2mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD The regular three-dimensional macroporous structure of the PLGA cube support matrix in the model, as shown in Figure 4;
(2) 制备载利福平的 PLGA/CS/HMS 复合微球： (2) Preparation of rifampicin-loaded PLGA/CS/HMS composite microspheres:
将 30mg 利福平与 1g 平均孔径为 2.5nm 的六方介孔硅混合均匀，然后将 1g PLGA 溶于 5ml 二氯甲烷中，待 12h 完全溶解后，加入 0.1g 利福平与六方介孔硅的混合粉末和 0.1g 硅酸钙粉体，用高速分散均质机以 2000rmp 的速率搅拌均匀，得到载利福平的 PLGA/CS/HMS 共混液；用去离子水配制 10mg/ml 的 1788 型聚乙烯醇水溶液 300ml ，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，以 300rmp 的速率搅拌 12h 后，将容器底部的复合微球分离出来，真空状态下冷冻干燥 48h 至完全脱水，制得载利福平的 PLGA/CS/HMS 复合微球，并用不锈钢筛网分离出粒径为 200~450μm 的复合微球（如图 2 与图 3 ）备用； Mix 30mg of rifampicin with 1g of hexagonal mesoporous silicon with an average pore diameter of 2.5nm, then dissolve 1g of PLGA In 5 ml of dichloromethane, after completely dissolved in 12 h, add 0.1 g of mixed powder of rifampicin and hexagonal mesoporous silica and 0.1 g of calcium silicate powder, and use a high-speed dispersing homogenizer to 2000 rmp. Stirring at a uniform rate to obtain a PLGA/CS/HMS blend containing rifampicin; preparing 10 mg/ml 1788 polyvinyl alcohol aqueous solution with deionized water 300 ml Then, the above blended liquid was slowly added dropwise to an aqueous solution of polyvinyl alcohol, and stirred at a rate of 300 rpm for 12 hours, and then the composite microspheres at the bottom of the vessel were separated and freeze-dried under vacuum for 48 hours. To complete dehydration, the PLGA/CS/HMS composite microspheres loaded with rifampicin were prepared, and the composite microspheres with a particle size of 200-450 μm (Fig. 2 and Fig. 3) were separated by a stainless steel sieve;
(3)PLGA 正方体支架基体与微球的复合： (3) Composite of PLGA cube support matrix and microspheres:
将载利福平的 PLGA/CS/HMS 复合微球均匀填充在支架中间的孔道中，四周靠近边缘处那一排均不填充，然后置于 45 ℃ 烘箱中，保温 4h 直至微球牢固的粘结在支架孔道中，微球间平均孔隙为 120μm ，如图 5 、图 6 与图 7 ，经 CT 测量，得到的复合支架孔隙率为 78.5% ，孔隙连通率为 97.6% 。 Will be loaded with rifampicin PLGA/CS/HMS The composite microspheres are uniformly filled in the channel in the middle of the stent, and the rows around the edge are not filled, and then placed in an oven at 45 °C for 4 hours until the microspheres are firmly bonded in the pores of the stent, and the average pores between the microspheres for 120μm, as shown in Fig. 5, Fig. 6 and Fig. 7, the composite scaffold obtained by CT measurement has a porosity of 78.5% and a pore connectivity of 97.6%.
实施例 2 Example 2
(1) 制备具有规则三维大孔结构的 PLGA 圆柱体支架基体： (1) Preparation of a PLGA cylinder support matrix with a regular three-dimensional macroporous structure:
用 Bioplotter RP 软件对直径为 10mm ，高为 2mm 的圆柱体模型 STL 格式数据进行分层处理，将 2gPLGA 加入不锈钢料筒中，选用 0.2mm 的针头，打开 VisualMachines 软件，设置打印温度为 150 ℃ ，平台温度为 25 ℃ ，挤出压力为 3.0bar ，挤出速度为 2mm/s ，设置内部结构为喷头 0° 、 45° 、 90° 、 145° 依次交替，分层厚度为 0.16mm ，孔径为 1.0mm ，然后将材料加热到指定温度后保温 30min ，启动 3D-Bioplotter 将三维结构模型逐层打印成型，形成 CAD 模型中的规则三维大孔结构的 PLGA 圆柱体支架基体； Cylindrical model STL with diameter 10 mm and height 2 mm using Bioplotter RP software Format data is layered, 2gPLGA is added to the stainless steel barrel, 0.2mm needle is used, VisualMachines software is opened, and the printing temperature is set to 150 °C. The platform temperature is 25 °C, the extrusion pressure is 3.0bar, the extrusion speed is 2mm/s, and the internal structure is set to nozzles 0°, 45°, 90°, 145°. Alternately, the layer thickness is 0.16mm, the hole diameter is 1.0mm, then the material is heated to the specified temperature and then kept for 30min to start 3D-Bioplotter The three-dimensional structure model is layer-by-layer printed to form a PLGA cylinder support matrix with a regular three-dimensional macroporous structure in the CAD model;
(2) 制备载庆大霉素的 PLGA/CS/HMS 复合微球： (2) Preparation of PLGA/CS/HMS composite microspheres loaded with gentamicin:
将 30mg 庆大霉素与 1g 平均孔径为 2.5nm 的六方介孔硅混合均匀，然后将 1g PLGA 溶于 5ml 二氯甲烷中，待 12h 完全溶解后，加入 0.2g 庆大霉素与六方介孔硅的混合粉末和 0.2g 硅酸钙粉体，用高速分散均质机以 3000rmp 的速率搅拌均匀，得到载庆大霉素的 PLGA/CS/HMS 共混液；用去离子水配制 12mg/ml 的 1788 型聚乙烯醇水溶液 300ml ，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，以 300rmp 的速率搅拌 12h 后，将容器底部的复合微球分离出来，真空状态下冷冻干燥 48 h 至完全脱水，制得载庆大霉素的 PLGA/CS/HMS 复合微球，并用不锈钢筛网分离出粒径为 200~450μm 的复合微球备用； Mix 30mg of gentamicin with 1g of hexagonal mesoporous silicon with an average pore diameter of 2.5nm, then dissolve 1g of PLGA In 5ml of dichloromethane, after completely dissolved in 12h, add 0.2g of mixed powder of gentamicin and hexagonal mesoporous silicon and 0.2g of calcium silicate powder, and use high-speed dispersion homogenizer to 3000rmp. Stirring at a uniform rate to obtain a PLGA/CS/HMS blend containing gentamicin; preparing 12 mg/ml 1788 polyvinyl alcohol aqueous solution with deionized water 300 ml Then, the above blended liquid was slowly added dropwise to an aqueous solution of polyvinyl alcohol, and stirred at a rate of 300 rpm for 12 hours, and then the composite microspheres at the bottom of the vessel were separated and freeze-dried under vacuum for 48 h. To complete dehydration, PLGA/CS/HMS composite microspheres loaded with gentamicin were prepared, and composite microspheres with a particle size of 200-450 μm were separated by stainless steel mesh;
(3)PLGA 圆柱体支架基体与微球的复合： (3) Composite of PLGA cylinder support matrix and microspheres:
将载庆大霉素的 PLGA/CS/HMS 复合微球均匀填充在支架中间的孔道中，填充的孔道与不填充的孔道按 1:1 排列，然后置于 55 ℃ 烘箱中，保温 4h 直至微球牢固的粘结在支架孔道中，微球间平均孔隙为 96μm ，经 CT 测量，得到的复合支架孔隙率为 73.8% ，孔隙连通率为 95.6% 。 The PLGA/CS/HMS composite microspheres carrying gentamicin are uniformly filled in the channels in the middle of the stent, and the filled channels and unfilled channels are pressed. Arrange in 1:1, then place in an oven at 55 °C for 4 h until the microspheres are firmly bonded in the pores of the scaffold. The average pore volume between the microspheres is 96 μm. The porosity of the composite scaffold obtained by CT measurement is obtained. 73.8%, the pore connectivity was 95.6%.
实施例 3 Example 3
(1) 制备具有规则三维大孔结构的聚己内酯（ PCL ）正方体支架基体： (1) Preparation of a polycaprolactone (PCL) cube support matrix having a regular three-dimensional macroporous structure:
用 Bioplotter RP 软件对长 10mm ，宽 10mm ，高 2mm 的正方体模型 STL 格式数据进行分层处理，将 2gPCL 加入不锈钢料筒中，选用 0.4mm 的针头，打开 VisualMachines 软件，设置打印温度为 160 ℃ ，平台温度为 25 ℃ ，挤出压力为 1.2bar ，挤出速度为 3mm/s ，设置内部结构为喷头 0° 和 90° 依次交替，分层厚度为 0.32mm ，孔径为 1.2mm ，然后将材料加热到指定温度后保温 30min ，启动 3D-Bioplotter 将三维结构模型逐层打印成型，形成 CAD 模型中的规则三维大孔结构的 PCL 正方体支架基体； Cube model STL 10 mm long, 10 mm wide and 2 mm high using Bioplotter RP software The format data is layered. Add 2gPCL to the stainless steel barrel, select the 0.4mm needle, open the VisualMachines software, and set the printing temperature to 160 °C. The platform temperature is 25 °C, the extrusion pressure is 1.2bar, the extrusion speed is 3mm/s, and the internal structure is set. The nozzles are alternately 0° and 90°, and the layer thickness is 0.32mm. , the hole diameter is 1.2mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD a PCL cube support matrix with a regular three-dimensional macroporous structure in the model;
(2) 制备载异烟肼的 PLGA/CS/HMS 复合微球： (2) Preparation of isoniazid-loaded PLGA/CS/HMS composite microspheres:
将 30mg 异烟肼与 1g 平均孔径为 2.5nm 的六方介孔硅混合均匀，然后将 1g PLGA 溶于 5ml 二氯甲烷中，待 12h 完全溶解后，加入 0.1g 异烟肼与六方介孔硅的混合粉末和 0.2g 硅酸钙粉体，用高速分散均质机以 2000rmp 的速率搅拌均匀，得到载异烟肼的 PLGA/CS/HMS 共混液；用去离子水配制 10mg/ml 的 1788 型聚乙烯醇水溶液 300ml ，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，以 280rmp 的速率搅拌 12h 后，将容器底部的复合微球分离出来，真空状态下冷冻干燥 48 h 至完全脱水，制得载异烟肼的 PLGA/CS/HMS 复合微球，并用不锈钢筛网分离出粒径为 200~450μm 的复合微球备用； Mix 30 mg of isoniazid with 1 g of hexagonal mesoporous silicon with an average pore diameter of 2.5 nm, and then dissolve 1 g of PLGA. In 5ml of dichloromethane, after completely dissolved in 12h, add 0.1g of mixed powder of isoniazid and hexagonal mesoporous silicon and 0.2g of calcium silicate powder, and use high-speed dispersion homogenizer to 2000rmp. The rate is evenly stirred to obtain the isoniazid-loaded PLGA/CS/HMS blend; 10 mg/ml of the 1788-type polyvinyl alcohol aqueous solution is prepared with deionized water. Then, the above blended liquid was slowly added dropwise to an aqueous solution of polyvinyl alcohol, and stirred at a rate of 280 rpm for 12 hours, and then the composite microspheres at the bottom of the vessel were separated and freeze-dried under vacuum for 48 h. To complete dehydration, the isoniazid-loaded PLGA/CS/HMS composite microspheres were prepared, and the composite microspheres with a particle size of 200-450 μm were separated by a stainless steel mesh;
(3)PCL 正方体支架基体与微球的复合： (3) Composite of PCL cube support matrix and microspheres:
将载异烟肼的 PLGA/CS/HMS 复合微球均匀填充在支架中间的孔道中，填充的孔道与不填充的孔道按 2:1 排列，然后置于 55 ℃ 烘箱中，保温 3h 直至微球牢固的粘结在支架孔道中，微球间平均孔隙为 108μm ，经 CT 测量，得到的复合支架孔隙率为 76.4% ，孔隙连通率为 96.8% 。 The isoniazid-loaded PLGA/CS/HMS composite microspheres are uniformly filled in the channel in the middle of the stent, and the filled and unfilled channels are pressed. Arrange in 2:1, then place in an oven at 55 °C for 3 h until the microspheres are firmly bonded in the pores of the scaffold. The average pore volume between the microspheres is 108 μm. The porosity of the composite scaffold obtained by CT measurement is obtained. 76.4%, the pore connectivity was 96.8%.
实施例 4 Example 4
(1) 制备具有规则三维大孔结构的聚己内酯（ PCL ）圆柱体支架基体： (1) Preparation of a polycaprolactone (PCL) cylinder support matrix having a regular three-dimensional macroporous structure:
用 Bioplotter RP 软件对直径为 5mm ，高为 2mm 的圆柱体模型 STL 格式数据进行分层处理，将 2gPCL 加入不锈钢料筒中，选用 0.3mm 的针头，打开 VisualMachines 软件，设置打印温度为 160 ℃ ，平台温度为 25 ℃ ，挤出压力为 1.5bar ，挤出速度为 3mm/s ，设置内部结构为喷头 0° 和 90° 依次交替，分层厚度为 0.24mm ，孔径为 0.8mm ，然后将材料加热到指定温度后保温 30min ，启动 3D-Bioplotter 将三维结构模型逐层打印成型，形成 CAD 模型中的规则三维大孔结构的 PCL 圆柱体支架基体； Cylindrical model STL with diameter 5mm and height 2mm using Bioplotter RP software The format data is layered. Add 2gPCL to the stainless steel barrel, select 0.3mm needle, open VisualMachines software, set the printing temperature to 160 °C. The platform temperature is 25 °C, the extrusion pressure is 1.5bar, the extrusion speed is 3mm/s, and the internal structure is set to the nozzles 0° and 90° alternately, the layer thickness is 0.24mm. , the hole diameter is 0.8mm, then the material is heated to the specified temperature and then kept for 30min, start 3D-Bioplotter to print the 3D structure model layer by layer to form CAD a PCL cylinder support matrix with a regular three-dimensional macroporous structure in the model;
(2) 制备载 BMP-2 的 PLGA/CS/HMS 复合微球： (2) Preparation of PLGA/CS/HMS composite microspheres carrying BMP-2:
将 10mg BMP-2 与 1g 平均孔径为 2.5nm 的六方介孔硅混合均匀，然后将 1g PLGA 溶于 5ml 二氯甲烷中，待 12h 完全溶解后，加入 0.1g BMP-2 与六方介孔硅的混合粉末和 0.3g 硅酸钙粉体，用高速分散均质机以 3000rmp 的速率搅拌均匀，得到载 BMP-2 的 PLGA/CS/HMS 共混液；用去离子水配制 10mg/ml 的 1788 型聚乙烯醇水溶液 300ml ，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，以 300rmp 的速率搅拌 12h 后，将容器底部的复合微球分离出来，真空状态下冷冻干燥 48 h 至完全脱水，制得载 BMP-2 的 PLGA/CS/HMS 复合微球，并用不锈钢筛网分离出粒径为 200~450μm 的复合微球备用； Mix 10 mg of BMP-2 with 1 g of hexagonal mesoporous silicon with an average pore diameter of 2.5 nm, and then add 1 g of PLGA. Dissolved in 5ml of dichloromethane, after 12h complete dissolution, add 0.1g of mixed powder of BMP-2 and hexagonal mesoporous silicon and 0.3g of calcium silicate powder, using high-speed dispersion homogenizer Stirring at a rate of 3000rmp to obtain a PLGA/CS/HMS blend containing BMP-2; 10mg/ml 1788 polyvinyl alcohol aqueous solution prepared with deionized water 300ml, then the above blend was slowly added dropwise to the aqueous solution of polyvinyl alcohol, stirred at 300rmp for 12h, the composite microspheres at the bottom of the container were separated, and freeze-dried under vacuum for 48 h. To complete dehydration, the PLGA/CS/HMS composite microspheres carrying BMP-2 were prepared, and the composite microspheres with a particle size of 200-450 μm were separated by a stainless steel mesh;
(3)PCL 圆柱体支架基体与微球的复合： (3) Composite of PCL cylinder support matrix and microspheres:
将载 BMP-2 的 PLGA/CS/HMS 复合微球均匀填充在支架中间的孔道中，然后置于 45 ℃ 烘箱中，保温 4h 直至微球牢固的粘结在支架孔道中，微球间平均孔隙约为 114μm ，经 CT 测量，得到的复合支架孔隙率为 77.9% ，孔隙连通率为 97.3% 。 The PLGA/CS/HMS composite microspheres carrying BMP-2 were evenly filled in the channel in the middle of the stent, and then placed at 45 °C. In the oven, the temperature was kept for 4 hours until the microspheres were firmly bonded in the pores of the stent. The average pore volume between the microspheres was about 114 μm. The porosity of the composite stent obtained by CT measurement was 77.9%, and the pore connectivity was 97.3%.
以上所述实施例仅表达了本发明的几种实施方式，但并不能理解为对本发明范围的限制。对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出其他不同形式的变形和改进，这些都属于本发明的保护范围。因此，本发明的保护范围应以所附权利要求为准。 The above-described embodiments are merely illustrative of several embodiments of the invention, but are not to be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various other forms of modifications and improvements can be made without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.
以下以实施例 1 中的一种基于 3D-Bioplotter 打印技术的骨修复多孔复合支架及其制备方法进行附图说明，实施例 2-4 与实施例 1 基本相似，不一一说明。 图 1 为本发明具体实施例制备骨修复多孔复合支架的过程示意图，附图仅用作示例性说明，不能理解为对本发明的限制。 The following is based on one of the examples 1 based on 3D-Bioplotter The bone repair porous composite stent of the printing technique and the preparation method thereof are described in the drawings, and the embodiments 2-4 are basically similar to the embodiment 1, and are not described one by one. figure 1 The process of preparing a bone repair porous composite stent for a specific embodiment of the present invention is illustrated by way of example only and is not to be construed as limiting the invention.

Claims (10)

  1. 基于3D-Bioplotter打印技术的骨修复多孔复合支架，其特征在于，所述骨修复多孔复合支架由具有三维大孔结构基体和载药微球复合而成；所述的三维大孔结构基体由3D-Bioplotter仪器制备；载药微球由乳化法制备；复合支架由37~65℃低温烧结法制备而成。A bone repair porous composite scaffold based on 3D-Bioplotter printing technology, characterized in that the bone repair porous composite scaffold is composed of a three-dimensional macroporous structure matrix and drug-loaded microspheres; the three-dimensional macroporous structure matrix is composed of 3D - Bioplotter instrument preparation; drug-loaded microspheres are prepared by emulsification method; composite stents are prepared by low-temperature sintering method at 37~65 °C.
  2. 根据权利要求1所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架，其特征在于，所述骨修复多孔复合支架的多孔结构由孔径为0.1~1.2mm的大孔、0.05~150μm的微米孔和2~50nm的介孔组成，大孔为三维支架基体中的孔洞，微米孔为微球37~65℃低温烧结后形成的微球间孔隙，介孔为六方介孔硅即HMS中的孔洞。 The bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 1, wherein the porous structure of the bone repair porous composite stent has a large pore diameter of 0.1 to 1.2 mm, 0.05 to 150 μm. The micropores and the mesopores of 2~50nm, the macropores are the pores in the matrix of the three-dimensional scaffold, the micropores are the microspheres formed by the microspheres at 37~65°C, and the mesopores are hexagonal mesoporous silicon or HMS. The hole in it.
  3. 根据权利要求1所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架，其特征在于，所述骨修复多孔复合支架的孔隙率为60~80%，孔隙连通率为90%以上。The bone repair porous composite stent based on the 3D-Bioplotter printing technology according to claim 1, wherein the bone repair porous composite stent has a porosity of 60 to 80% and a pore connectivity of 90% or more.
  4. 根据权利要求1所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架，其特征在于，所述骨修复多孔复合支架的基体材料为以下材料中的一种或几种：聚乳酸-羟基乙酸共聚物、聚乳酸、聚羟基乙酸、聚己内酯和聚羟基脂肪酸酯。The bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 1, wherein the matrix material of the bone repair porous composite stent is one or more of the following materials: polylactic acid- A glycolic acid copolymer, polylactic acid, polyglycolic acid, polycaprolactone, and a polyhydroxy fatty acid ester.
  5. 制备权利要求1所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的方法，其特征在于，包括以下步骤，A method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 1, comprising the following steps,
    (1)制备具有规则三维大孔结构的支架基体：(1) Preparation of a stent substrate having a regular three-dimensional macroporous structure:
    利用计算机辅助设计软件CAD设计出骨缺损部位的三维结构模型，并把该三维模型保存为STL格式文件，然后导入3D-Bioplotter中，用Bioplotter RP软件对CAD模型的数据进行分层处理，然后将基体材料加入不锈钢料筒中，在VisualMachines软件中设置打印温度、平台温度、针头大小、挤出压力、挤出速度、内部结构和孔径参数，然后启动3D-Bioplotter将骨缺损部位的三维结构模型逐层打印成型，形成CAD模型中的规则三维大孔结构的支架基体；The three-dimensional structure model of the bone defect site was designed by computer aided design software CAD, and the three-dimensional model was saved as an STL format file, and then imported into 3D-Bioplotter with Bioplotter. The RP software layered the data of the CAD model, then added the matrix material to the stainless steel barrel, and set the printing temperature, platform temperature, needle size, extrusion pressure, extrusion speed, internal structure and pore size parameters in VisualMachines software, and then The 3D-Bioplotter is used to print the three-dimensional structure model of the bone defect layer layer by layer to form a stent base of a regular three-dimensional macroporous structure in the CAD model;
    (2)制备载药或生长因子的PLGA/CS/HMS复合微球：(2) Preparation of PLGA/CS/HMS composite microspheres loaded with drugs or growth factors:
    将药物或生长因子与六方介孔硅混合得到药物或生长因子与六方介孔硅的混合粉体，然后将聚乳酸一羟基乙酸共聚物即PLGA溶于二氯甲烷中，待12~24h完全溶解后，加入上述混合粉体和硅酸钙粉体即CS，用高速分散均质机搅拌均匀，得到载药或生长因子的PLGA/CS/HMS共混液；用去离子水配制10~30mg/ml的聚乙烯醇水溶液，然后将上述共混液缓慢滴加到聚乙烯醇水溶液中，搅拌8~20h后将容器底部的复合微球分离出来，真空状态下冷冻干燥24~48 h至完全脱水，制得载药或生长因子的PLGA/CS/HMS复合微球，并用不锈钢筛网分离出目标粒径的复合微球备用；Mixing a drug or a growth factor with hexagonal mesoporous silicon to obtain a mixed powder of a drug or a growth factor and hexagonal mesoporous silicon, and then dissolving the polylactic acid monoglycolic acid copolymer, PLGA, in dichloromethane, and completely dissolving in 12-24 hours. After that, the above mixed powder and calcium silicate powder, CS, are added, and uniformly stirred by a high-speed dispersing homogenizer to obtain a PLGA/CS/HMS blend containing drug or growth factor; 10~30 mg/ml is prepared with deionized water. The aqueous solution of polyvinyl alcohol is then slowly added dropwise to the aqueous solution of polyvinyl alcohol. After stirring for 8 to 20 hours, the composite microspheres at the bottom of the container are separated and freeze-dried under vacuum for 24 to 48. From h to complete dehydration, a PLGA/CS/HMS composite microsphere loaded with a drug or a growth factor is prepared, and a composite microsphere of a target particle size is separated by a stainless steel mesh;
    (3)基体材料与微球的复合：(3) Composite of matrix material and microsphere:
    将载药或生长因子的PLGA/CS/HMS复合微球均匀填充在支架孔道中，置于37~65℃烘箱中，保温2~4h直至微球牢固的粘结在支架孔道中，得到一种基于3D-Bioplotter打印技术的骨修复多孔复合支架。The PLGA/CS/HMS composite microspheres loaded with drug or growth factor were uniformly filled in the pores of the stent and placed in an oven at 37-65 ° C for 2 to 4 hours until the microspheres were firmly bonded in the pores of the stent. Bone repair porous composite scaffold based on 3D-Bioplotter printing technology.
  6. 根据权利要求5所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的制备方法，其特征在于，步骤(1)中，所述打印温度为130~160℃，平台温度为20~40℃，针头大小为0.2~0.4mm，挤出压力为1.0~3.5bar，挤出速度为1.0~5.0mm/s，孔径为0.1~1.2mm；内部结构为喷头角度0~179°交错排列，分层厚度为0.16~0.32mm。The method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 5, wherein in the step (1), the printing temperature is 130-160 ° C, and the platform temperature is 20~ 40 ° C, the needle size is 0.2 ~ 0.4mm, the extrusion pressure is 1.0 ~ 3.5bar, the extrusion speed is 1.0 ~ 5.0mm / s, the aperture is 0.1 ~ 1.2mm; the internal structure is the nozzle angle 0 ~ 179 ° staggered, The layer thickness is 0.16~0.32mm.
  7. 根据权利要求5所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的制备方法，其特征在于，步骤(2)中，所述药物或生长因子为以下任意一种或几种：异烟肼、利福平、庆大霉素、BMP-1、BMP-2、BMP-7、BMP-14、TGF-α和TGF-β。The method for preparing a bone repair porous composite stent based on the 3D-Bioplotter printing technology according to claim 5, wherein in the step (2), the drug or the growth factor is any one or more of the following: Isoniazid, rifampicin, gentamicin, BMP-1, BMP-2, BMP-7, BMP-14, TGF-α and TGF-β.
  8. 根据权利要求5所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的制备方法，其特征在于，步骤(2)中，所述六方介孔硅为实验室自制介孔硅，制备方法如下：将2~5g十二胺、20~60ml无水乙醇、20~60ml去离子水加入250ml的烧杯中，用磁力搅拌器搅拌；然后加入5~10g正硅酸乙酯搅拌8~12h后陈化30min；分别用去离子水和乙醇洗涤，放入烘箱干燥后640℃锻烧4h，然后研磨过160目筛网，制得六方介孔硅粉末。The method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 5, wherein in the step (2), the hexagonal mesoporous silicon is prepared by using mesoporous silicon in a laboratory. The method is as follows: 2~5g dodecylamine, 20~60ml anhydrous ethanol, 20~60ml deionized water is added into a 250ml beaker and stirred by a magnetic stirrer; then 5~10g orthosilicate is added and stirred for 8~12h. After aging for 30 min; respectively, it was washed with deionized water and ethanol, dried in an oven, and calcined at 640 ° C for 4 h, and then ground through a 160 mesh screen to obtain hexagonal mesoporous silicon powder.
  9. 根据权利要求5所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的制备方法，其特征在于，步骤(2)中，所述硅酸钙粉体为实验室自制硅酸钙，制备方法如下：将硅酸钠和硝酸钙按摩尔比为1:1混合搅拌12h，然后陈化30min，用去离子水洗涤4次，真空状态下冷冻干燥24~48 h至完全脱水后800℃煅烧2h，然后研磨过160目筛网，制得硅酸钙粉体。The method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 5, wherein in the step (2), the calcium silicate powder is a laboratory-made calcium silicate. The preparation method is as follows: mixing sodium silicate and calcium nitrate in a molar ratio of 1:1 for 12 hours, then aging for 30 minutes, washing 4 times with deionized water, and freeze-drying under vacuum 24~48 After h to complete dehydration, it was calcined at 800 ° C for 2 h, and then ground through a 160 mesh screen to obtain a calcium silicate powder.
  10. 根据权利要求5所述的一种基于3D-Bioplotter打印技术的骨修复多孔复合支架的制备方法，其特征在于，步骤(2)中，所述药物或生长因子浓度为0.2-2mg/ml，六方介孔硅的浓度为10~40mg/ml，硅酸钙粉体的浓度为10~60mg/ml；聚乙烯醇为1788型、1799型中的一种；所述共混液滴加到聚乙烯醇水溶液到分离复合微球时的搅拌速度均为180~350r/min；复合微球粒径为100~600μm；以上所述浓度单位中的ml均以该步骤得到的共混液的体积为计算基准。The method for preparing a bone repair porous composite stent based on 3D-Bioplotter printing technology according to claim 5, wherein in the step (2), the concentration of the drug or growth factor is 0.2-2 mg/ml, hexagonal The concentration of mesoporous silicon is 10~40mg/ml, the concentration of calcium silicate powder is 10~60mg/ml; the polyvinyl alcohol is one of type 1788 and 1799; the blended droplets are added to polyvinyl alcohol The stirring speed of the aqueous solution to the separation composite microspheres is 180-350 r/min; the composite microspheres have a particle diameter of 100-600 μm; the ml in the above concentration unit is calculated based on the volume of the blend obtained in this step.
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