WO2021169759A1 - 中药调控型复合活性骨支架及其制备方法和应用 - Google Patents

中药调控型复合活性骨支架及其制备方法和应用 Download PDF

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WO2021169759A1
WO2021169759A1 PCT/CN2021/074988 CN2021074988W WO2021169759A1 WO 2021169759 A1 WO2021169759 A1 WO 2021169759A1 CN 2021074988 W CN2021074988 W CN 2021074988W WO 2021169759 A1 WO2021169759 A1 WO 2021169759A1
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scaffold
composite active
active bone
bone scaffold
preparation
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PCT/CN2021/074988
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English (en)
French (fr)
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刘昌胜
陈芳萍
梁青
毛丽杰
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华东理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/42—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
    • A61L27/425—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix of phosphorus containing material, e.g. apatite
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • 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
    • A61L2300/23—Carbohydrates
    • A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention belongs to the field of biomedical materials science, and relates to the loading of active factors and biomolecules and a multi-level drug release system. Specifically, it relates to a traditional Chinese medicine-regulated composite active bone scaffold with both defect repair and treatment effects.
  • Osteoporotic fracture is a disease caused by the breakdown of the dynamic balance between bone formation by osteoblasts and bone resorption by osteoclasts.
  • the scaffold is one of the key elements of osteoporotic defect repair, and its composition and structure affect cell adhesion, proliferation and bone tissue formation.
  • Sichuan Dipsacus is a traditional Chinese medicinal material in Asia. It contains saponins and other important components. It has the effects of invigorating the liver and kidney, continued fractures, and strengthening the muscles and bones. It is mainly used for paralysis of limbs, backaches, bruises, damaged muscles and bones. As well as bleeding and other diseases. Although Chuanduan has achieved many results in the prevention and treatment of osteoporosis, it is mainly administered by oral drinking. At the same time, there are very few reports on the effects of drugs on bone metabolism at the cellular level. See report. From the perspective of molecular biology and cell biology, how to enhance the role of its drugs or active factors in bone repair requires in-depth research.
  • the purpose of the present invention is to provide a traditional Chinese medicine-regulated composite active bone scaffold with excellent mechanical strength, simple operation, rapid prototyping, controllable pore size, degradability, excellent biocompatibility, and both defect repair and treatment effects.
  • a composite active bone scaffold is provided.
  • the composite active bone scaffold is composed of an organic component and an inorganic component, and the scaffold also carries Dipsacopsis VI;
  • the organic component is selected from the group consisting of: polylactic acid-glycolic acid copolymer (PLGA), dextro-polylactic acid (PDLA), levorotatory polylactic acid (PLLA), poly(citrate-1,8 octanediol) ester (POC) );
  • PLGA polylactic acid-glycolic acid copolymer
  • PDLA dextro-polylactic acid
  • PLLA levorotatory polylactic acid
  • POC poly(citrate-1,8 octanediol) ester
  • the inorganic component is selected from: ⁇ -tricalcium phosphate, hydroxyapatite.
  • the organic component is preferably PLGA, and the inorganic component is preferably ⁇ -tricalcium phosphate.
  • the mass ratio of the organic component and the inorganic component is 1:9-5:5, preferably 3:7-5:5.
  • the mass ratio of the organic component to the inorganic component is 4:6.
  • the mass ratio of the organic component to the inorganic component is 5:5.
  • the optimal ratio of the organic component to the inorganic component is 3:7.
  • the mass ratio of Dipsacus saponins VI to the inorganic components is 0.1% to 5%.
  • the mass ratio of the traditional Chinese medicine Dipsaclopsis saponins VI to the inorganic components is 0.2%, 0.5%, 1%, and 5%.
  • the scaffold also carries BMP-2 and/or 6-O-sulfonated chitosan (26CSC).
  • the amount of BMP-2 loaded for example, 1 ⁇ g BMP-2 is instilled into each cylindrical scaffold with a diameter of 10 mm and a height of 4 mm at the cell level.
  • the concentration of the 26CSC PBS solution is 1-10 mg/ml, preferably 2-8 mg/ml, more preferably 5 mg/ml.
  • the pore size of the composite active bone scaffold is 300-500 microns.
  • the compressive stress when the composite active bone scaffold is broken is 20-30Mpa.
  • the compressive stress when the composite active bone scaffold is broken is 25.41Mpa.
  • the second aspect of the present invention provides a method for preparing the composite active bone scaffold described in the first aspect, which includes the following steps:
  • a mixture is obtained by adding inorganic components to the above-mentioned mixed solution;
  • the concentration of the Dipsacus saponins VI solution is 0.1-0.5 mg/ml.
  • the concentration of the organic component solution is 1-5 g/ml.
  • the mass ratio of the organic component and the inorganic component is 1:9-5:5, preferably 3:7-5:5.
  • the mass ratio of the organic component to the inorganic component is 4:6.
  • the mass ratio of the organic component to the inorganic component is 5:5.
  • the mass ratio of Dipsacus saponins VI to the inorganic components is 0.1% to 5%.
  • the mass ratio of the traditional Chinese medicine Dipsaclopsis saponins VI to the inorganic components is 0.2%, 0.5%, 1%, and 5%.
  • the amount of BMP-2 loaded for example, 1 ⁇ g BMP-2 is instilled into each cylindrical scaffold with a diameter of 10 mm and a height of 4 mm at the cell level.
  • the concentration of the 26CSC PBS solution is 1-10 mg/ml, preferably 2-8 mg/ml, more preferably 5 mg/ml.
  • the preparation method includes the following steps:
  • the organic solvent dissolves the degradable polymer component PLGA, the ratio of the organic solvent to the polymer is 0.5ml/g, the dissolution temperature environment is 60°C water bath heating, the time used is 30min, and the magnetic stirrer stirs;
  • the 3D printing adopts a normal temperature printing nozzle.
  • the printing parameters are: air pressure 0.2-0.4Mpa, preferably 0.3Mpa; speed 2.0-2.5mm/s, preferably 2.0mm/s; filling distance 0.7-0.9mm.
  • the broken wire elevation is 0.4-0.8mm, preferably 0.5mm.
  • the preparation method includes the following steps:
  • the organic solvent dissolves the degradable polymer component PLGA, the ratio of the organic solvent to the polymer is 0.5ml/g, the dissolution temperature environment is 60°C water bath heating, the time used is 30min, and the magnetic stirrer stirs;
  • the organic component solution is prepared by dissolving the organic component in 1,4-dioxane, dichloromethane, chloroform or a mixed solvent thereof.
  • the ⁇ -TCP is ground with a ball mill and passed through a 325-mesh sieve with a vibrating sieve.
  • the preparation method of ⁇ -TCP includes the following steps:
  • the suspension of Ca(OH) 2 and the solution of H 3 PO 4 are separately prepared.
  • 0.6 mol/L Ca(OH) 2 suspension was added dropwise to 0.4 mol/L H 3 PO 4 solution, and stirred vigorously. After the addition is completed, the reaction is continued for 5 hours, and the precursor is obtained by centrifugation and lyophilization.
  • ⁇ -TCP was obtained by sintering in a muffle furnace at 900°C for 2 hours.
  • the traditional Chinese medicine-regulated composite active bone scaffold with both defect repair and treatment effects is placed in an oven at 60° C. to evaporate the solvent.
  • the method further includes the steps of physically adsorbing 26CSC, after the adsorption is saturated, instilling BMP-2, and then aseptically drying.
  • the third aspect of the present invention provides the use of the composite active bone scaffold described in the first aspect to prepare repair materials or tissue engineering materials for the treatment of bone defects.
  • the prepared composite scaffold is used to solve the filling and repair of large bone defects caused by trauma, inflammation, infection, tumor and dysplasia, and postmenopausal women due to estrogen deficiency, and due to aging and drugs. Defect repair caused by increased side effects.
  • the composite material of the present invention complements the advantages of organic and inorganic components, and has excellent mechanical properties; the traditional Chinese medicine Chuanhuashang Saponin VI can promote osteogenesis, inhibit osteogenesis, and maintain the balance of osteogenesis and osteoclast activities; BMP-2 It has high osteogenic activity and can participate in regulating the process of bone repair; 26CSC has the function of promoting angiogenesis, thereby promoting osteogenesis.
  • the active factors and biomolecules on the surface of the composite scaffold are first released to promote rapid cell proliferation, and then the drugs inside the scaffold are slowly released over time to provide follow-up strength, thereby improving the utilization of local drug delivery.
  • the drug-loaded scaffold can significantly promote cell proliferation and osteogenic gene expression, promote osteogenic bone mineralization and other differentiation behaviors, thereby increasing its bone repair ability.
  • the preparation method of the organic-inorganic Chinese medicine composite material of the present invention is simple and fast, has a regular and controllable pore structure, provides a good microenvironment for the growth of cell growth vessels, and is conducive to cell adhesion and proliferation.
  • Figure 1 shows the digital and SEM pictures of the porous composite scaffold.
  • FIG 2 shows the rapid degradation curve of NaOH in vitro for the porous composite scaffold (a) and the pH change of degradation in Tris-HCl (b).
  • Figure 3 shows the cytotoxicity evaluation results of the composite scaffold.
  • Figure 4 shows the results of the in vitro ALP activity analysis of Dipsacus saponins VI.
  • Figure 5 is a graph showing the results of in vitro gene expression analysis of Dipsacus saponins VI.
  • Figure 6 shows the results of the compression performance test.
  • the organic component of the composite material is preferably polylactic acid glycolic acid copolymer (PLGA).
  • the inorganic component is preferably ⁇ -tricalcium phosphate ( ⁇ -TCP), and the traditional Chinese medicine-regulated composite active bone scaffold is loaded with traditional Chinese medicine Chuanduan Saponin VI, which innovatively combines traditional Chinese medicine ingredients into the material.
  • the material preparation process is easy to operate and environmentally friendly. Dipsacus saponins VI promotes osteogenesis and inhibits osteoclasts. It not only has a therapeutic effect, but also has a preventive effect.
  • BMP-2 has high osteogenic activity and can participate in regulating the process of bone repair; 26CSC has the function of promoting angiogenesis, thereby promoting osteogenesis.
  • the active factors and biomolecules on the surface of the composite scaffold are first released to promote rapid cell proliferation, and then the drugs inside the scaffold are slowly released over time to provide follow-up strength, thereby improving the utilization of local drug delivery.
  • BMP-2 and 26CSC which play a role in the early stage of bone repair, rapidly stimulate cell proliferation and differentiation, and cooperate with Dipsacanthus saponin VI, which plays a role in promoting bone formation and inhibiting osteoclastosis in the later stage of bone repair, to play a synergistic effect.
  • materials with regular and connected pore diameters, excellent degradability and biocompatibility are prepared to achieve high-efficiency bone defect repair effects.
  • the composite active bone scaffold provided by the present invention is a traditional Chinese medicine-regulated composite active bone scaffold with both defect repair and treatment effects.
  • the organic component of the composite material is preferably polylactic acid glycolic acid copolymer (PLGA); the inorganic component is preferably It is ⁇ -tricalcium phosphate ( ⁇ -TCP).
  • the composite active bone scaffold also contains the traditional Chinese medicine Chuan Dipuan VI, BMP-2 and 26CSC (6-O-sulfonated chitosan).
  • the invention discloses a traditional Chinese medicine control type composite active bone scaffold material with both defect repair and treatment effects.
  • the degradable polymer PLGA and the inorganic material ⁇ -TCP are compounded with the traditional Chinese medicine Chuanhuasaponin VI through a solvent method, prepared by 3D printing rapid prototyping technology or conventional tableting technology, and simultaneously adsorbs bone morphogenetic protein BMP and promotes angiogenesis
  • the drug 26CSC (6-O-sulfonated chitosan) is compounded.
  • the traditional Chinese medicine Chuan Dipuan saponins VI can inhibit bone resorption and promote bone formation. At the same time, it has a good anti-osteoporosis effect.
  • PLGA has good biocompatibility.
  • the degradation products are lactic acid and glycolic acid. They are also by-products of human metabolic pathways and will not have toxic side effects;
  • ⁇ -TCP has excellent osteoconductivity and is widely used as bone repair materials, but It is brittle and difficult to form. Mixing the two together through the solvent method makes the advantages of the two complement each other, and obtains a composite scaffold with excellent performance.
  • BMP-2 has high osteogenic activity, can participate in the regulation of bone repair process, and is an effective way to speed up material bone repair and regeneration.
  • 26CSC as a heparin-like biomolecule, can promote the bone formation of BMP-2 and promote angiogenesis.
  • the composite scaffold material realizes the preparation of the composite scaffold with multi-level regulation and control activity carrying traditional Chinese medicine by physically adsorbing the growth factors and biomolecules. Orderly pore structure, growth factor loading, cell adhesion growth, extracellular matrix deposition, nutrition and oxygen entry, and metabolite discharge are also conducive to the growth of blood vessels and nerves.
  • the composite scaffold can be directly used for filling and repairing large-scale bone defects caused by trauma, inflammation, infection, tumor and dysplasia, and has great clinical significance and social value.
  • the invention discloses a preparation process of a traditional Chinese medicine control type composite active bone scaffold with both defect repair and treatment effects.
  • the specific preparation process includes the compounding of the organic component and the inorganic component, the preparation of the printing paste, and the drying step.
  • ⁇ -TCP powder preparation Ca(OH) 2 suspension and H 3 PO 4 solution were prepared according to the Ca/P ratio of 1.5. At room temperature, the 0.6 mol/L Ca(OH) 2 suspension was added to the 0.4 mol/L H 3 PO 4 solution at 1-3 drops per second, and stirred vigorously. During the dripping process, the pH is adjusted with ammonia water, strictly controlled between 7.0-7.5, after the dripping is completed, the pH should be 7.2, continue the reaction for 5h, centrifuge and freeze-dry to obtain the precursor. ⁇ -TCP was obtained by sintering in a muffle furnace at 800°C for 2 hours. Pass through a 325-mesh sieve for later use.
  • the molding preparation method of the composite material includes the following steps:
  • the organic solvent dissolves the degradable polymer component PLGA, the ratio of the organic solvent to the polymer is 0.5ml/g, the dissolution temperature environment is 60°C water bath heating, the time used is 30min, and the magnetic stirrer stirs;
  • the organic solvent is 1,4-dioxane.
  • the inorganic component ⁇ -TCP is all ground with a ball mill and passed through a 325-mesh sieve with a vibrating machine.
  • the 3D printing adopts a normal temperature printing nozzle and a 0.41 mm needle.
  • the printing parameters also include model and size design, filling path, extrusion pressure, nozzle movement speed, wire ejection distance, wire breakage elevation, etc.
  • the print model file is in the .stl format; the filling path is linear filling; the extrusion pressure is 0.35Mpa; the movement speed of the nozzle is 3mm/s; the wire spacing is 0.85mm, the layer thickness is 0.2mm, and the broken wire is lifted. 0.5mm high.
  • the printed composite stent material in the step b), is placed in an oven at 60° C. to evaporate the solvent.
  • the composite scaffold described in the present invention can be used for bone defect repair and replacement materials, and can also be used as tissue engineering bone.
  • the composite material combines the excellent performance of the organic component and the inorganic component, greatly improves the formability and mechanical strength of the material, makes the advantages of the two complement each other, and obtains a composite scaffold material with excellent performance.
  • the porous material prepared by 3D printing is simple and fast, has a regular and controllable pore structure, provides a good microenvironment for cell growth and blood vessel growth, and is conducive to cell adhesion and proliferation.
  • this molding method is suitable for all similar materials.
  • the suspension of Ca(OH) 2 and the solution of H 3 PO 4 are separately prepared.
  • the 0.6 mol/L Ca(OH) 2 suspension was added to the 0.4 mol/L H 3 PO 4 solution at 1-3 drops per second, and stirred vigorously.
  • the pH is adjusted with ammonia water, strictly controlled between 7.0-7.5, after the dripping is completed, the pH should be 7.2, continue the reaction for 5h, centrifuge and freeze-dry to obtain the precursor.
  • ⁇ -TCP was obtained by sintering in a muffle furnace at 800°C for 2 hours. Pass through a 325-mesh sieve for later use.
  • 1,4-dioxane dissolves the degradable polymer component PLGA, the ratio of 1,4-dioxane to the polymer is 0.5ml/g, and the dissolved temperature environment is 60°C water bath heating, the time required For 30min, stir with a magnetic stirrer;
  • 1,4-dioxane dissolves the degradable polymer component PLGA, the ratio of 1,4-dioxane to the polymer is 0.5ml/g, and the dissolved temperature environment is 60°C water bath heating, the time required For 30 minutes, stir with a magnetic stirrer; the traditional Chinese medicine Chuan Dipuan VI was fully dissolved in DMSO (1 mg/5 ⁇ l) and then added to the above PLGA solution and mixed.
  • 1,4-dioxane dissolves the degradable polymer component PLGA, the ratio of 1,4-dioxane to the polymer is 0.5ml/g, the dissolved temperature environment is 60°C water bath heating, the time used is 30min , Magnetic stirrer stirring;
  • 1,4-dioxane dissolves the degradable polymer component PLGA, the ratio of 1,4-dioxane to the polymer is 0.5ml/g, the dissolved temperature environment is 60°C water bath heating, the time used is 30min , Stir with a magnetic stirrer;
  • the traditional Chinese medicine Chuan Dipuan VI is fully dissolved in DMSO (1mg/5 ⁇ l) and then added to the above PLGA solution and mixed.
  • the amount of BMP-2 depends on the size of the stent and the size of the animal load. At the cell level, 1 ⁇ g was instilled into each cylindrical stent with a diameter of 10 mm and a height of 4 mm. The PBS solution concentration of 26CSC is 5mg/ml.
  • the digital photo and SEM of the porous composite scaffold are shown in Figure 1 for the pore size. It can be seen that the macroscopic pore size and morphology of the scaffold material are regular, and the microscopic surface is rough, with exposed inorganic particles.
  • the pore size is about 300-500 microns.
  • the pH change of the porous composite scaffold in Tris-HCl degradation and the rapid degradation curve of NaOH in vitro are shown in Figure 2. It can be seen that at 7 days, the pH is still around 7.10, and in the in vitro NaOH rapid degradation curve, it can be seen that as the proportion of polymer increases, the degradation rate of the composite material increases, and the final degradation rate and its composite ratio are basically Unanimous.
  • the degradation rate of 3:7 composite material in 5 days reaches 33.22%; the degradation rate of 4:6 composite material reaches 48.06%; the degradation rate of 5:5 reaches 64.70%.
  • Tris-HCl standard buffer as the degradation medium, select the composite scaffold as the test sample, weigh G1, put it into the degradation bottle, soak the scaffold in a ratio of 0.1g/10ml, and incubate in a constant temperature shaking box at 37°C, every three days Change the liquid once, take it out after a certain period of time, and measure the change in pH value
  • a concentration of 0.1M NaOH is used.
  • the quality of the selected samples is approximately the same.
  • Each sample is added with 10ml NaOH solution and placed in a constant temperature shaking box at 37°C. , Take out after 5D, pour out the supernatant, rinse with distilled water, dry overnight in a vacuum drying oven at 40°C, and weigh.
  • CCK-8 (Beyotime, shanghai).
  • the rBMSCs cells were seeded in a 24-well plate at a cell concentration of 2 ⁇ 10 4 cells/well, and cultured for 1, 2, and 3 days respectively.
  • Detection steps Discard the medium, add 400 ⁇ l of new medium ( ⁇ -MEM containing 10% FBS) to each well, then add 40 ⁇ l of CCK-8 to each well (operate in the dark), incubate in a constant temperature shake flask at 37°C for 2 hours, at 450nm Measure the absorbance at 650nm as the background value.
  • Example 3 Taking the TCP synthesized in Example 1 and the TCP/PLGA composite scaffold prepared in Example 3.1 as an example, the cytotoxicity of each group was compared and compared with the blank control group. The results are shown in FIG. 3. It can be seen that the synthesized organic-inorganic composite material has excellent cell compatibility.
  • rBMSCs cells were seeded at a density of 1 ⁇ 10 4 cells/well. Add BMP-2, 26CSC, and Dipsacopsis VI in groups according to the experiment shown in Table 2, where the concentration in the table is the concentration of the factor and the drug in the base. The erythrocyte alkaline phosphatase activity was detected on the 4th, 7th, and 14th day.
  • Detection steps Discard the culture medium, wash with PBS 3 times, add 500 ⁇ l NP-40 cell lysate to each well, incubate in a constant temperature shake flask at 37°C for 90 minutes to completely lyse the cells.
  • the total protein was determined by the BCA standard curve method. Take 50 ⁇ l of lysate from each well, place it in a 96-well plate, add 100 ⁇ l of alkaline phosphatase working solution (PNPP-Na concentration is 1mg/ml) and incubate in a constant temperature shake flask at 37°C for 2 hours. The absorbance at 405 nm was measured. The final result is 405nm OD value/(total protein amount * incubation time), and the unit is 405nm OD value/mg protein/min.
  • the addition of BMP-2 and 26CSC significantly improved the effect of bone formation and mineralization.
  • the ALP value of 10 -5 M was the highest at 7d and 14d; especially On the 14th day, the group containing Dipsaclopsis saponins VI was significantly higher than the group stimulated by BMP-2 and 26CSC.
  • the addition of BMP-2 and 26CSC would highlight the effect of the drug.
  • the rBMSCs cells were seeded in a 24-well plate at a cell concentration of 4 ⁇ 10 4 cells/well, cultured normally for 3 days, and stimulated by replacing the media with drugs and factors for 24 hours.
  • the experimental groups are shown in Table 3.
  • Detection steps In short, the cells are lysed first, after the mRNA is extracted, a reverse transcription reagent is prepared, and the mRNA is transcribed into cDNA using PrmeScriptTM RT reagent kit. Finally, the cDNA obtained by reverse transcription is used as a template, SYBR reagents and upstream and downstream primers are added, and the rt-qPCR instrument is used for amplification and detection according to the program. Determine the expression of BMP-2, COL1, OSX, Smad1/4/5, TGF- ⁇ 1, VEGF, RANKL genes.
  • the determination process is divided into 3 stages: the first stage is 95°C, 3min; the second stage is mainly the amplification stage, a total of 40 cycles occur, and each cycle is 95°C for 10s and the temperature is reduced to 60°C for 20s; In the third stage, the temperature was raised to 95°C again until the end of the program.
  • the combination of osteoclast and osteoclast factor Chuandasaponin VI contributes to the expression of osteogenic genes, while inhibiting osteoclast factors, thereby promoting the formation of new bone.
  • Sichuan Dipsacus saponins VI (ASP VI) and BMP-2 and 26CSC have a synergistic effect, have significant proliferation and mineralization effects, enhance the effect of ASP VI, and have significant osteogenesis effects.
  • the composite scaffold material prepared as in Example 2.1 was explored for its compressive strength.
  • the size of the printed scaffold was 10 ⁇ 10 ⁇ 6mm 3.
  • the compressive performance of the scaffold was measured by a universal material testing machine and compressed at an application speed of 1 mm/min.
  • a is the average data of compression experiments on multiple groups of 10 ⁇ 10 ⁇ 6mm 3 composite stents.
  • the compressive stress when the stent is broken is 25.41Mpa, which is higher than that of the simple ⁇ -TCP stent, and the average compressive modulus is higher. It is 21.74Mpa and the compressive strain is 20.8%. This is mainly due to the bonding effect of PLGA to the inorganic filler.
  • the stress-strain curve of the composite scaffold has no yield platform and is a typical plastic compression curve.
  • the addition of PLGA can improve the brittleness of the traditional ⁇ -TCP scaffold.
  • the simple ⁇ -TCP stent becomes powder after being compressed by force, while the PLGA/ ⁇ -TCP composite stent collapses slightly after compression, but the surface pore structure remains intact without obvious cracking, and the shape is basically maintained. After being implanted in the defect in the body, the stent can withstand greater adjacent tissue stress without being damaged.

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Abstract

一种中药调控型复合活性骨支架及其制备方法和应用，所述复合活性骨支架由有机组分和无机组分复合而成，该支架还载有川续断皂苷VI，所述有机组分选自：PLGA、PLLA、PDLA、POC；所述无机组分选自：β-磷酸三钙、羟基磷灰石。该复合活性骨支架力学强度优异，操作简单，快速成型、孔径可控，可降解，生物相容性优异，抑制破骨，维持成骨和破骨活动的平衡，兼具骨缺损修复和治疗作用。

Description

中药调控型复合活性骨支架及其制备方法和应用 技术领域
本发明属于生物医用材料学领域，涉及活性因子和生物分子的负载以及多级药物释放体系，具体来说，涉及一种兼具缺损修复和治疗作用的中药调控型复合活性骨支架。
背景技术
因创伤、炎症、感染、肿瘤及发育异常等造成的大段骨缺损(骨缺损长度大于骨直径1.5～2倍)严重危害着人类的健康和生活质量，其修复治疗是现代医学力图解决的棘手难题。随着社会发展，人口激增、交通繁忙、生活节奏加快、疾病和自然灾害增多、运动创伤以及战争等等诸多问题都造成人们意外伤害频发，伴随而来的是日益增多的大段骨组织缺损、骨修复等社会问题。大段骨缺损因受疤痕组织多、血液循环差和潜在感染等条件的限制，在临床中常伴以骨不连或短缩畸形等并发症，且病发率高，给个人、家庭和社会带来了沉重的精神负担和巨大的经济压力。
骨质疏松性骨折是由于成骨细胞的骨形成和破骨细胞的骨吸收之间的动态平衡被打破而引起的疾病。其中，支架是骨质疏松性缺损修复的关键要素之一，其组成和结构影响着细胞的黏附、增殖以及骨组织的形成。
川续断为亚洲传统的中药材，有皂苷等重要组成成分，具有补肝肾、续折伤、强筋骨之功效，主要用于肢节麻痹、腰酸背痛、跌打创伤、损筋折骨以及血崩下血等疾病。尽管川续断在预防和治疗骨质疏松症的研究中虽然取得了很多一定的成果，但主要仍以口服饮用的方式给药，同时药物在细胞水平对骨代谢影响的研究极少报道也鲜见报道。从分子生物学和细胞生物学的角度，如何增强其药物或活性因子在骨修复中的作用需要深入研究。
发明内容
本发明的目的在于提供一种力学强度优异，操作简单，快速成型、孔径可控，可降解，生物相容性优异，兼具缺损修复和治疗作用的中药调控型复合活性骨支架。
本发明第一方面，提供一种复合活性骨支架，所述复合活性骨支架由有机组分和无机组分复合而成，所述支架还载有川续断皂苷VI；
其中所述有机组分选自：聚乳酸-羟基乙酸共聚物(PLGA)、右旋聚乳酸(PDLA)、左旋聚乳酸(PLLA)、聚(柠檬酸-1,8辛二醇)酯(POC)；
所述无机组分选自：β-磷酸三钙、羟基磷灰石。
在另一优选例中，有机组分优选PLGA，无机组分优选β-磷酸三钙。
在另一优选例中，PLGA规格为乳酸:乙醇酸＝50:50；分子量3万-5万。
在另一优选例中，有机组分和无机组分的质量比例为1:9-5:5，较佳为3:7-5:5。
在另一优选例中，所述有机组分与无机组分的质量比例为4:6。
在另一优选例中，所述有机组分与无机组分的质量比例为5:5。
在另一优选例中，综合其降解性及其机械强度，所述有机组分与无机组分的最佳比例为3:7。
在另一优选例中，川续断皂苷VI占无机组分的质量比例为0.1％-5％。
在另一优选例中，所述中药川续断皂苷VI占无机组分的质量比例为0.2％、0.5％、1％、5％。
在另一优选例中，所述支架还载有BMP-2和/或6-O-磺化壳聚糖(26CSC)。
在另一优选例中，依据支架大小以及动物大小负载BMP-2的量，例如对于细胞水平每个直径10mm高4mm的圆柱形支架滴注1μg BMP-2。
在另一优选例中，26CSC的PBS溶液浓度为1-10mg/ml，较佳为2-8mg/ml，更佳佳为5mg/ml。
在另一优选例中，所述复合活性骨支架的孔径大小在300-500微米。
在另一优选例中，所述复合活性骨支架破坏时的压缩应力为20-30Mpa。
在另一优选例中，所述复合活性骨支架破坏时的压缩应力为25.41Mpa。
本发明的第二方面，提供第一方面所述的复合活性骨支架的制备方法，包括以下步骤：
(i)混合川续断皂苷VI溶液和有机组分的溶液得到混合溶液；
从向上述混合溶液中加入无机组分得到混合物；
(iii)3D打印上述混合物或对混合物压片得到所述复合活性骨支架。
在另一优选例中，所述川续断皂苷VI溶液的浓度为0.1-0.5mg/ml。
在另一优选例中，所述有机组分的溶液的浓度为1-5g/ml。
在另一优选例中，有机组分和无机组分的质量比例为1:9-5:5，较佳为3:7-5:5。
在另一优选例中，所述有机组分与无机组分的质量比例为4:6。
在另一优选例中，所述有机组分与无机组分的质量比例为5:5。
在另一优选例中，川续断皂苷VI占无机组分的质量比例为0.1％-5％。
在另一优选例中，所述中药川续断皂苷VI占无机组分的质量比例为0.2％、0.5％、1％、5％。
在另一优选例中，依据支架大小以及动物大小负载BMP-2的量，例如对于细胞水平每个直径10mm高4mm的圆柱形支架滴注1μg BMP-2。
在另一优选例中，26CSC的PBS溶液浓度为1-10mg/ml，较佳为2-8mg/ml，更佳佳为5mg/ml。
在另一优选例中，所述制备方法包括以下步骤：
(i)有机溶剂溶解可降解聚合物组分PLGA，所述有机溶剂占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；
(ii)中药川续断皂苷VI充分溶于DMSO(1mg/5μl)后加入(i)中所述溶液混匀。向此溶解完全的溶液中加入β-TCP粉末，充分搅拌均匀制得粘稠合适的打印浆料，进行打印。
在另一优选例中，所述3D打印采用常温打印喷头。所述打印参数为：气压0.2-0.4Mpa，优选为0.3Mpa；速度2.0-2.5mm/s，优选为2.0mm/s；填充距离为0.7-0.9mm。断丝抬高为0.4-0.8mm，较佳为0.5mm。
在另一优选例中，所述制备方法包括以下步骤：
(i)有机溶剂溶解可降解聚合物组分PLGA，所述有机溶剂占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；
(ii)中药川续断皂苷VI充分溶于DMSO(1mg/5μl)后加入(i)中所述溶液混匀。 向此溶解完全的溶液中加入β-TCP粉末，充分搅拌均匀制得面团状均匀物料，搓成细条后截成等长小团，于聚四氟乙烯模具中压片制得。
在另一优选例中，有机组分的溶液是由有机组分溶解于1,4-二氧六环、二氯甲烷、氯仿或其混合溶剂中配制而成。
在另一优选例中，所述β-TCP用球磨机磨粉并且用振筛机过325目筛子。
在另一优选例中，β-TCP制备方法包括以下步骤：
按照Ca/P比为1.5分别配置Ca(OH) 2的悬浮液和H 3PO 4溶液。在室温下，将0.6mol/L的Ca(OH) 2悬浮液逐滴加入0.4mol/L的H 3PO 4溶液中，强烈搅拌。滴加完成之后，继续反应5h，离心冻干得到前驱体。900℃马弗炉中烧结2h得到β-TCP。
在另一优选例中，兼具缺损修复和治疗作用的中药调控型复合活性骨支架置于60℃烘箱中挥干溶剂。
在另一优选例中，所述方法还包括物理吸附26CSC，吸附饱和后，滴注BMP-2冷冻后无菌干燥的步骤。
本发明的第三方面，提供第一方面所述的复合活性骨支架的用途，用于制备治疗骨缺损的修复材料或组织工程材料。
在另一优选例中，制备的复合支架用于解决因创伤、炎症、感染、肿瘤及发育异常等造成的大段骨缺损的填充修复以及绝经后妇女因雌激素缺乏、以及因老龄化和药物副作用的加剧所引发的缺损修复。
本发明中的复合材料，使有机和无机组分优势互补，具有优异的力学性能；中药川续断皂苷VI能够促进成骨，抑制破骨，维持成骨和破骨活动的平衡；BMP-2具有很高的成骨活性，能够参与调节骨修复的进程；26CSC具有促进血管发生的功能，从而促进成骨。此复合支架表面的活性因子和生物分子最先释放起到促进细胞快速增殖的作用，随后支架内部的药物随着时间慢慢释放出来，提供后续力量，从而提高局部给药的利用率。载药支架能够明显促进细胞的增殖以及成骨基因的表达、促进成骨骨矿化等分化行为，从而增大其骨修复能力。
同时本发明中有机无机中药复合材料所述制备方法，简单快捷，具有规则可控的孔道结构，为细胞生长血管的生长提供良好的微环境，有利于细胞的黏附增殖。
在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为多孔复合支架的数码照片和SEM图片。
图2为多孔复合支架体外NaOH快速降解曲线(a)以及在Tris-HCl中降解的pH值变化情况(b)。
图3为复合支架的细胞毒性评价结果。
图4为川续断皂苷VI体外ALP活性分析结果图。
图5为川续断皂苷VI体外基因表达分析结果图。
图6为压缩性能测试结果图。
具体实施方式
本申请的发明人经过广泛而深入地研究，首次研发出一种兼具缺损修复和治疗作用的中药调控型复合活性骨支架，该复合材料有机组分优选为聚乳酸乙醇酸共聚物(PLGA)；无机组分优选为β-磷酸三钙(β-TCP)，中药调控型复合活性骨支架载有中药川续断皂苷VI，创新性地将中药成分复合到材料中。材料制备过程操作简单，环境友好。川续断皂苷VI促进成骨，抑制破骨，不仅具有治疗作用，还具有预防作用。BMP-2具有很高的成骨活性，能够参与调节骨修复的进程；26CSC具有促进血管发生的功能，从而促进成骨。此复合支架表面的活性因子和生物分子最先释放起到促进细胞快速增殖的作用，随后支架内部的药物随着时间慢慢释放出来，提供后续力量，从而提高局部给药的利用率。简言之，在骨修复早期发挥作用的BMP-2和26CSC，快速刺激细胞增殖分化，与骨修复后期发挥促成骨抑破骨作用的川续断皂苷VI配合，起到协同增强作用。在此基础上，制备具有规则连通的孔径以及优异降解性和生物相容性的材料，达到高效骨缺损修复效果。
复合活性骨支架
本发明提供的复合活性骨支架，是一种兼具缺损修复和治疗作用的中药调控型复合活性骨支架，该复合材料有机组分优选为聚乳酸乙醇酸共聚物(PLGA)；无机组分优选为β-磷酸三钙(β-TCP)。复合活性骨支架还包含中药川续断皂苷VI、BMP-2和26CSC(6-O-磺化壳聚糖)。
本发明公开了一种兼具缺损修复和治疗作用的中药调控型复合活性骨支架材料。本发明将可降解聚合物PLGA和无机材料β-TCP通过溶剂法和中药川续断皂苷VI复合，采用3D打印快速成型技术或常规压片技术制备，同时吸附骨形态发生蛋白BMP和促血管发生药物26CSC(6-O-磺化壳聚糖)复合而成。其中，中药川续断皂苷VI能够抑制骨吸收促进骨形成，同时，有较好的抗骨质疏松作用，在后续的骨修复过程中起到续力的作用，对促进骨质疏松等引起的骨缺损的修复具有巨大的临床应用价值。PLGA具有良好的生物相容性，降解产物是乳酸和羟基乙酸，同时也是人代谢途径的副产物，不会有毒副作用；β-TCP具有优异的骨传导性，被广泛用于骨修复材料，但本身脆性大，难以成型。将二者通过溶剂法混合在一起使二者优点互补，获得性能优异的复合支架。此外，BMP-2成骨活性高，能参与调节骨修复进程，是加快材料骨修复和再生的有效途径。同时，26CSC作为一种类肝素生物分子，可促进BMP-2的成骨且促进血管发生。该复合支架材料通过物理吸附此生长因子和生物分子，实现载中药多级调控活性复合支架的制备。有序的孔道结构生长因子的负载、细胞粘附生长、细胞外基质沉积、营养和氧气进入以及代谢产物排出，也利于血管和神经长入。该复合支架可直接用于解决因创伤、炎症、感染、肿瘤及发育异常等造成的大段骨缺损的填充修复，具有重大的临床意义和社会价值。
制备方法
本发明公开了一种兼具缺损修复和治疗作用的中药调控型复合活性骨支架的制备工艺。
具体制备工序包括有机组分与无机组分的复合，打印浆料的制备，以及干燥步骤。
β-TCP粉体制备：按照Ca/P比为1.5分别配置Ca(OH) 2的悬浮液和H 3PO 4溶液。 在室温下，将0.6mol/L的Ca(OH) 2悬浮液按照1-3滴/秒加入0.4mol/L的H 3PO 4溶液中，强烈搅拌。滴加过程中用氨水调节其pH，严格控制在7.0-7.5之间，滴加完成之后，pH应为7.2，继续反应5h，离心冻干得到前驱体。800℃马弗炉中烧结2h得到β-TCP。过325目筛备用。
在另一优选例中，所述复合材料的成型制备方法包括以下步骤：
a)有机溶剂溶解可降解聚合物组分PLGA，所述有机溶剂占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；
b)向上述溶解完全的溶液中加入充分物理混匀的β-TCP和中药续断粉末，充分搅拌均匀制得粘稠合适的打印浆料，进行打印或者压片。
在另一优选例中，所述有机溶剂选择1,4-二氧六环。
在另一优选例中，所述无机组分β-TCP均用球磨机磨粉并且用振筛机过325目筛子。
在另一优选例中，先溶解川续断皂苷VI，然后充分溶解于上述a)中。
在另一优选例中，所述3D打印采用常温打印喷头，0.41mm针头。
在另一优选例中，所述打印参数还包括模型及尺寸设计，填充路径，挤料压力，喷头运动速度，出丝间距，断丝抬高等。所述打印模型文件为.stl格式；所述填充路径为线性填充；挤料压力为0.35Mpa；喷头的运动速度为3mm/s；出丝间距为0.85mm，层厚为0.2mm，断丝抬高0.5mm。
在另一优选例中，所述步骤b)中，打印成型过后的复合支架材料置于60℃烘箱中挥干溶剂。
用途
本发明中所述的复合支架可用于骨缺损修复、替代材料，也可用作组织工程骨。
本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
本发明的优势在于：
(1)该复合材料结合有机组分和无机组分的优异性能，极大提高材料成型性及力学强度，使二者优点互补，获得性能优异的复合支架材料。
(2)中药川续断皂苷VI促进成骨，抑制破骨，维持成骨和破骨活动的平衡。
(3)通过3D打印制备的多孔材料，简单快捷，具有规则可控的孔道结构，为细胞生长血管的生长提供良好的微环境，有利于细胞的黏附增殖。
(4)同时此成型方法适用于所有同类材料。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
除非另外说明，否则百分比和份数是重量百分比和重量份数。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
β-TCP的制备
按照Ca/P比为1.5分别配置Ca(OH) 2的悬浮液和H 3PO 4溶液。在室温下，将0.6mol/L的Ca(OH) 2悬浮液按照1-3滴/秒加入0.4mol/L的H 3PO 4溶液中，强烈搅拌。滴加过程中用氨水调节其pH，严格控制在7.0-7.5之间，滴加完成之后，pH应为7.2，继续反应5h，离心冻干得到前驱体。800℃马弗炉中烧结2h得到β-TCP。过325目筛备用。
实施例2
2.1复合支架的3D打印成型制备
a)1,4-二氧六环溶解可降解聚合物组分PLGA，1,4-二氧六环占聚合物的比例为 0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；
b)向上述溶解完全的溶液中加入充分物理混匀的β-TCP粉末，充分搅拌均匀制得粘稠合适的打印浆料，进行打印。有机组分与无机组分重量比例为3:7-5:5。具体打印参数如表1所示。
2.2载中药川续断皂苷VI的复合支架的3D打印成型制备
a)1,4-二氧六环溶解可降解聚合物组分PLGA，1,4-二氧六环占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；中药川续断皂苷VI充分溶于DMSO(1mg/5μl)后加入上述PLGA溶液混匀。
b)向上述步骤a)获得的溶液中加入充分物理混匀的β-TCP粉末，充分搅拌均匀制得粘稠合适的打印浆料，进行打印。有机组分与无机组分重量比例为3:7-5:5。具体打印参数如表1所示。
表1 PLGA/β-TCP支架的3D打印成型参数
Figure PCTCN2021074988-appb-000001
实施例3
3.1复合支架的压片成型制备
1,4-二氧六环溶解可降解聚合物组分PLGA，1,4-二氧六环占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；
向上述溶解完全的溶液中加入充分物理混匀的β-TCP粉末，充分搅拌均匀制得面团状均匀物料，搓成细条后截成等长小团，于聚四氟乙烯模具中压片制得。有机组 分与无机组分比例为3:7-5:5。
3.2载中药川续断皂苷VI的复合支架的压片成型制备
1,4-二氧六环溶解可降解聚合物组分PLGA，1,4-二氧六环占聚合物的比例为0.5ml/g，溶解的温度环境为60℃水浴加热，所用时间为30min，磁力搅拌器搅拌；；中药川续断皂苷VI充分溶于DMSO(1mg/5μl)后加入上述PLGA溶液混匀。
向上述溶解完全的溶液中加入充分物理混匀的β-TCP粉末，充分搅拌均匀制得面团状均匀物料，搓成细条后截成等长小团，于聚四氟乙烯模具中压片制得。有机组分与无机组分比例为3:7-5:5。
实施例4复合支架的吸附活性因子步骤
如实施例2.2和3.2制备的中药调控型复合活性骨支架制备好之后，进行钴60灭菌，然后于无菌条件浸泡26CSC溶液，物理吸附饱和后，滴注BMP-2冷冻后无菌干燥。
BMP-2的量依据支架大小以及动物大小负载。细胞水平每个直径10mm高4mm的圆柱形支架滴注1μg。26CSC的PBS溶液浓度为5mg/ml。
实施例6复合支架形貌及结构表征
以实施例2.1制备的空白复合支架为例，多孔复合支架的数码照片和SEM如图1所示孔径。可见支架材料的宏观孔径和形貌规整，并且微观表面粗糙，有裸露无机粒子。孔径大小在300-500微米左右。
实施例7复合支架体外降解性能
以实施例3.1制备的空白复合支架为例，多孔复合支架在Tris-HCl中降解的pH值变化情况以及体外NaOH快速降解曲线如图2所示。可以看出7天时，pH依然维持在7.10附近，而在体外NaOH快速降解曲线图中可以看出随着聚合物比例增大，复合材料的降解率增大，最终的降解率与其复合的比例基本一致。3：7的复合材料5天降解率达33.22％；4：6的复合材料降解率达48.06％；5：5的降解率达64.70％。
体外Tris-HCL降解具体实施步骤如下：
采用Tris-HCl标准缓冲液为降解介质，选用复合支架作为测试样品，称重G1,放入降解瓶中，按照0.1g/10ml的比列浸泡支架，37℃恒温震荡箱进行孵育，每三天换一次液，一定时间后取出，测量其pH值变化情况
体外NaOH快速降解具体实施步骤如下：
为了在短时间内获得复合材料的降解性，采用浓度为0.1M的NaOH，选取的样品的质量大致相同，每个样品加入10ml NaOH溶液，放入37℃恒温震荡箱中，分别于1D,2D,5D后取出，倒出上清液，用蒸馏水冲洗，与40℃真空干燥箱中过夜烘干，称重。以G＝(w0-w1)/w0×100％计算降解率。
实施例8复合支架的细胞毒性的评价
采用CCK-8(Beyotime，shanghai)。将rBMSCs细胞接种于24孔板中，细胞浓度为2×10 4细胞/孔，分别培养1、2、3天。检测步骤：弃置培养基，每孔加入400μl新培养基(含10％FBS的α-MEM)，然后每孔加入40μl CCK-8(避光操作)，37℃恒温摇瓶孵育2小时，在450nm处测定吸光度，以650nm处测定吸光度为背景值。
以实施例1所合成的TCP和实施例3.1制备的复合支架TCP/PLGA为例，比较各组细胞毒性情况并于空白对照组相比，其结果如图3所示。可以看出所合成的有机无机复合材料具有优异的细胞相容性。
实施例9体外ALP活性评价
在24个孔板中，以1×10 4细胞/孔的密度接种rBMSCs细胞。根据表2所示的实验分组加入BMP-2、26CSC、川续断皂苷VI，其中表中浓度为培基中因子以及药物的浓度。分别于第4、7、14天检测红细胞碱性磷酸酶活性。
检测步骤：弃去培养基，PBS洗涤3次，每孔加入500μl NP-40细胞裂解液，37℃恒温摇瓶中培养90min，使细胞完全裂解。采用BCA标准曲线法测定总蛋白。从各孔取50μl裂解液，置96孔板中，加入100μl碱性磷酸酶工作液(PNPP-Na浓度为1mg/ml)于37℃恒温摇瓶中孵育2小时。测定了405nm的吸光度。最终结果为405nm OD值/(总蛋白量*培养时间)，单位为405nm OD值/mg蛋白/min。
表2 ALP活性分析实验分组
Figure PCTCN2021074988-appb-000002
如图4，与control组相比，BMP-2和26CSC的加入显著提高成骨矿化效果，不同浓度川续断皂苷VI相比，7d和14d时10 -5M的ALP值最高；尤其在14d时，含有川续断皂苷VI组别明显高于只有BMP-2和26CSC刺激的组别，BMP-2和26CSC的加入会突出药物的作用效果。
实施例10川续断皂苷VI的基因表达分析
将rBMSCs细胞接种于24孔板中，细胞浓度为4×10 4细胞/孔，正常培养3天，更换带有药物和因子的培基刺激24h。实验组别如表3所示。
检测步骤：简言之，首先将细胞裂解，提取mRNA后，配制逆转录试剂，使用PrmeScriptTM RT reagent kit将mRNA转录为cDNA。最后通过逆转录所得到的cDNA作为模板，加入SYBR试剂和上下游的引物，通过rt-qPCR仪按照程序进行扩增检测。测定BMP-2,COL1,OSX,Smad1/4/5,TGF-β1,VEGF,RANKL基因的表达情况。测定过程分为3个阶段：第一阶段是95℃，3min；第二阶段主要为扩增阶段，总共有40个循环发生，并且每个循环为95℃保持10s降温至60℃保持20s；第三阶段再次升温至95℃直至程序结束。
如图5，综合成骨和破骨因子川续断皂苷VI有助于成骨基因的表达，同时抑制破骨因子，从而促进新骨形成。
综合ALP活性检测和PCR表达分析，川续断皂苷VI(ASP VI)和BMP-2以及26CSC具有协同作用，显著的增殖和矿化效果，增强ASP VI的作用效果，具有显著的成骨效果。
表3 ALP活性分析实验分组
Figure PCTCN2021074988-appb-000003
实施例11复合支架的力学性能表征
如实施例2.1制备的复合支架材料，探究其压缩强度，打印支架大小为10×10×6mm 3，通过万能材料试验机测定支架的压缩性能，以1mm/min的施加速度进行压缩。
图6中a为对多组10×10×6mm 3复合支架进行压缩实验的平均数据，可得支架破坏时的压缩应力为25.41Mpa，高于单纯的β-TCP支架，平均压缩模量较高为21.74Mpa，压缩应变20.8％，这主要得益于PLGA对无机填料的粘结作用，复合支架应力应变曲线没有屈服平台，是典型的塑性压缩曲线。
如图6中b-c所示，在相同的应力条件下，PLGA的添加，可以改善传统β-TCP支架的脆性。单纯的β-TCP支架受力压缩后会变成粉末，而PLGA/β-TCP复合支架压缩之后支架高度略有坍塌，但表面孔结构保持完整，并未出现明显开裂，外形基本保持，这说明支架在植入体内缺损处后可以承受较大的相邻组织应力而不被破坏。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种复合活性骨支架，其特征在于，所述复合活性骨支架由有机组分和无机组分复合而成，所述支架还载有川续断皂苷VI；
    其中所述有机组分选自：PLGA、PDLA、PLLA、POC；
    所述无机组分选自：β-磷酸三钙、羟基磷灰石。
  2. 如权利要求1所述的复合活性骨支架，其特征在于，有机组分和无机组分的质量比例为1:9-5:5。
  3. 如权利要求1所述的复合活性骨支架，其特征在于，川续断皂苷VI占无机组分的质量比例为0.1％-5％。
  4. 如权利要求1所述的复合活性骨支架，其特征在于，所述支架还载有BMP-2和/或6-O-磺化壳聚糖。
  5. 如权利要求1所述的复合活性骨支架，其特征在于，所述复合活性骨支架的孔径大小在300-500微米。
  6. 如权利要求1所述的复合活性骨支架的制备方法，其特征在于，所述制备方法包括以下步骤：
    (i)混合川续断皂苷VI溶液和有机组分的溶液得到混合溶液；
    从向上述混合溶液中加入无机组分得到混合物；
    (iii)3D打印上述混合物或对混合物压片得到所述复合活性骨支架。
  7. 如权利要求6所述的制备方法，其特征在于，所述川续断皂苷VI溶液的浓度为0.1-0.5mg/ml。
  8. 如权利要求6所述的制备方法，其特征在于，所述有机组分的溶液的浓度为1-5g/ml。
  9. 如权利要求6所述的制备方法，其特征在于，所述方法还包括物理吸附26CSC，吸附饱和后，滴注BMP-2冷冻后无菌干燥的步骤。
  10. 如权利要求1所述的复合活性骨支架的用途，其特征在于，用于制备治疗骨缺损的修复材料或组织工程材料。
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