WO2024055997A1 - 一种具有高效促成骨作用的压电多孔支架及其制备方法 - Google Patents

一种具有高效促成骨作用的压电多孔支架及其制备方法 Download PDF

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WO2024055997A1
WO2024055997A1 PCT/CN2023/118601 CN2023118601W WO2024055997A1 WO 2024055997 A1 WO2024055997 A1 WO 2024055997A1 CN 2023118601 W CN2023118601 W CN 2023118601W WO 2024055997 A1 WO2024055997 A1 WO 2024055997A1
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piezoelectric
porous
scaffold
component
porous scaffold
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English (en)
French (fr)
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刘昌胜
陈芳萍
毛丽杰
沈泽昊
韩啸天
管顾嵩
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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
    • 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
    • A61L27/02—Inorganic materials
    • A61L27/10—Ceramics or glasses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, 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
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained 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
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

  • the invention belongs to the technical field of biomedical materials and relates to a piezoelectric porous scaffold with efficient bone-promoting effect, adjustable piezoelectric properties and excellent osteogenic activity. More specifically, it relates to a piezoelectric porous bone repair scaffold that can carry bioactive factors.
  • Human bone tissue itself has a piezoelectric effect, which generates surface potential under mechanical stress.
  • This piezoelectric response microenvironment can regulate cell osteogenic differentiation and stimulate cells to remodel bone structure.
  • clinical application technologies developed using the electrical properties of bone such as electrical stimulation osteogenesis technology and electromagnetic field stimulation osteogenesis technology
  • electrical stimulation devices are complex and difficult to implant, and can easily lead to infection and secondary diseases.
  • the superposition of electric and magnetic fields also risks causing normal tissue lesions. Therefore, the development of bone replacement materials with piezoelectric properties similar to human bone is a hot research topic in the field of biological tissue engineering.
  • Bone tissue regeneration is a complex biological process involving multiple cytokines. Therefore, immobilizing bioactive factors in tissue engineering scaffolds is an effective means to improve their bioactivity.
  • bioactive factors in tissue engineering scaffolds.
  • the three-dimensional connected pore structure can not only ensure mechanical stimulation Effective delivery, while its high specific surface area provides sufficient space for the immobilization of bioactive factors.
  • the piezoelectric porous scaffold can exert the synergistic effect of the scaffold's piezoelectric effect, porous structure, and factor biological activity to promote efficient and rapid repair of bone defects.
  • the object of the present invention is to provide a piezoelectric porous scaffold for rapid repair and treatment of bone defects.
  • a first aspect of the present invention provides a piezoelectric porous stent, which is obtained by mixing biological components and piezoelectric components through a binder, sintering and polarization treatment, wherein,
  • the biological components include hydroxyapatite (HA), tricalcium phosphate ( ⁇ -TCP), bioglass (MBG), calcium polyphosphate, tetracalcium phosphate, octacalcium phosphate, biphasic calcium phosphate, and calcium polyphosphate.
  • HA hydroxyapatite
  • ⁇ -TCP tricalcium phosphate
  • MSG bioglass
  • calcium polyphosphate calcium polyphosphate
  • tetracalcium phosphate tetracalcium phosphate
  • octacalcium phosphate biphasic calcium phosphate
  • biphasic calcium phosphate and calcium polyphosphate.
  • calcium polyphosphate One or a mixture of two or more;
  • the piezoelectric component is one of calcium titanate (CaTiO 3 ), potassium sodium niobate (K 0.5 Na 0.5 NbO 3 ), barium titanate (BaTiO 3 ) or a mixture of two or more thereof;
  • the binder is one or a mixture of two or more of sodium alginate, polyvinyl alcohol, and cellulose.
  • the binder is used in the form of an aqueous solution. In another preferred embodiment, the binder is used in the form of an aqueous solution with a mass concentration of 5%-20%, 8%-15% or 10%.
  • the biological component is ⁇ -TCP
  • the piezoelectric component is CaTiO 3
  • the binder used is an aqueous solution with a mass concentration of 5%-20%, 8%-15% or 10% polyvinyl alcohol.
  • the mass fraction of the biological component ⁇ -TCP is 5%-50%, 8%-35% or 10%-20%.
  • the mass fraction of the piezoelectric component BaTiO 3 is 95%-50%, 92%-65% or 90%-80%.
  • the binder is 5%-20%, 8%-15% or 10% polyvinyl alcohol solution, accounting for 5%-50%, 8% of the total mass of the biological component and the piezoelectric component. -35% or 10%-20%.
  • the mass of the biological component accounts for 5% to 95% of the total mass of the biological component and the piezoelectric component
  • the piezoelectric component accounts for 5% to 95% of the total mass of the biological component and the piezoelectric component. 95% to 5% of the total mass
  • the mass of the binder accounts for 5% to 30% of the total mass of the biological component and the piezoelectric component.
  • the mass of the biological component accounts for 10% to 80% of the total mass of the biological component and the piezoelectric component
  • the piezoelectric component accounts for 10% to 80% of the total mass of the biological component and the piezoelectric component. 90% to 20% of the total quality.
  • the mass of the biological component accounts for 20% to 50%, 5% to 50%, 8% to 35% or 10% to 20% of the total mass of the biological component and the piezoelectric component.
  • the piezoelectric component accounts for 80% to 50%, 95% to 50%, 92% to 65% or 90% to 80% of the total mass of the biological component and the piezoelectric component.
  • the mass of the binder accounts for 5% to 5% of the total mass of the biological component and the piezoelectric component. 10%, 5%-50% or 8%-35%.
  • the piezoelectric constant d 33 of the obtained piezoelectric porous scaffold is 8 pC/N to 13 pC/N. It is equivalent to d 33 (7.5pC/N ⁇ 10pC/N) of natural human bone.
  • a second aspect of the present invention provides an active piezoelectric porous scaffold, including the piezoelectric porous scaffold described in the first aspect and a bioactive factor loaded on the piezoelectric porous scaffold, wherein the bioactive factor is VEGF, One or a mixture of more than two of BMP-2, TGF- ⁇ 1, and FGF-9.
  • the biologically active factor is BMP-2.
  • the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.1-0.5:1.
  • the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.2-0.3:1.
  • the mass ratio of the bioactive factor to the piezoelectric porous scaffold is 0.26:1.
  • the load is physical adsorption or coupling agent coupling, preferably freeze-drying physical adsorption.
  • the bioactive factor is physically adsorbed on the surface of the piezoelectric porous scaffold described in the first aspect to impart bioactivity to it.
  • Rat bone marrow stromal stem cells were cultured on the surface of the active piezoelectric porous scaffold. After culturing for a specific period of time, the number of cells and the mass density of bone morphogenetic protein in the above scaffold and the ordinary scaffold were detected to determine that the surface of the piezoelectric porous scaffold with high efficiency in promoting bone was obtained.
  • the synergistic effect of osteogenesis regulatory factors and porous piezoelectric scaffolds in promoting bone repair is used to achieve rapid bone repair and treatment of bone defects.
  • the present invention has better biocompatibility than ordinary piezoelectric porous scaffolds and facilitates further medical applications.
  • the electrostatic interaction between the surface electrical signal of the piezoelectric porous scaffold that efficiently promotes bone effects and the bioactive factors can firmly adsorb the factors on the surface of the material and achieve long-term sustained release of the factors, which not only improves the efficiency of the active factors, but also It avoids potential side effects caused by excessive use of active factors; at the same time, it combines the rapid activation of cell response by electrical signals with the induction of osteogenesis by active factors to achieve rapid and efficient repair and functional reconstruction of defective parts.
  • a third aspect of the present invention provides a method for preparing the piezoelectric porous stent described in the first aspect, the preparation method comprising the following steps:
  • the present invention adopts the polyurethane sponge template method to provide a new preparation idea for the porous stent.
  • the mixed slurry and the polyurethane sponge are fully mixed in advance, and then sintered together to remove the polyurethane sponge to obtain the piezoelectric porous scaffold.
  • this method has low preparation cost, simple operation and short material forming time.
  • due to the diverse selectivity of the mixed slurry various physical properties of the obtained scaffold can be improved by changing the composition and proportion of the mixed slurry.
  • piezoelectric ceramic powder, inorganic bioactive components and binders are combined according to a certain ratio, and are evenly mixed with the pretreated porous template. After drying, shaping and high-temperature sintering, a piezoelectric porous scaffold with efficient bone-promoting effect was prepared.
  • the preparation method of this stent is simple and convenient, and it has a three-dimensional connected hole structure, which ensures the effective transmission of mechanical stress inside the stent.
  • the prepared piezoelectric porous scaffold can efficiently carry and adsorb bioactive protein factors to achieve effective sustained release of proteins.
  • BMSCs bone marrow stromal stem cells
  • the mass of the biological component accounts for 5% to 95% of the total mass of the biological component and the piezoelectric component
  • the piezoelectric component accounts for 5% to 95% of the total mass of the biological component and the piezoelectric component. 95% to 5% of the total quality.
  • the mass of the biological component accounts for 20% to 80% of the total mass of the biological component and the piezoelectric component
  • the piezoelectric component accounts for 20% to 80% of the total mass of the biological component and the piezoelectric component. 80% to 20% of the total quality.
  • the mass of the biological component accounts for 20% to 50%, 5% to 50%, 8% to 35% or 10% to 20% of the total mass of the biological component and the piezoelectric component.
  • the piezoelectric component accounts for 80% to 50%, 95% to 50%, 92% to 65% or 90% to 80% of the total mass of the biological component and the piezoelectric component.
  • the mass of the adhesive accounts for 5% to 30% of the total mass of the biological component and the piezoelectric component.
  • the mass of the binder accounts for 5% to 10%, 5% to 50%, or 8% to 35% of the total mass of the biological component and the piezoelectric component.
  • the porous template is polymethyl methacrylate (PMMA) colloid template, polyurethane (PU) sponge template, polycarbonate (PC) membrane template or cellulose template.
  • PMMA polymethyl methacrylate
  • PU polyurethane
  • PC polycarbonate
  • the sintering temperature is 800-2000°C, preferably 1100-1500°C or 1400°C.
  • the sintering time is 1-5 hours, preferably 2-4 hours.
  • the polarization voltage during the polarization process is 2-15kV/mm, preferably 5-10kV/mm.
  • the present invention obtains a porous scaffold material with a certain porosity and pore size distribution through a new preparation method.
  • the obtained piezoelectric scaffold can change its shape at will, providing a suitable environment for the next step of physical adsorption.
  • a fourth aspect of the present invention provides the use of the piezoelectric porous scaffold described in the first aspect or the active piezoelectric porous scaffold described in the second aspect for preparing materials for repairing and treating bone defects.
  • the preparation method used in the present invention is the polyurethane sponge method.
  • this method has simple preparation steps, low cost, short time consumption, and is convenient for large-scale production.
  • product performance the product obtained by the present invention
  • the piezoelectric performance is better than that of traditional preparation products. This method can solve the problems of difficult preparation and high cost of the original preparation method, and provides the possibility of broad clinical application.
  • the piezoelectric porous scaffold prepared by the present invention has a three-dimensional connected pore structure, which ensures the effective transmission of mechanical signals inside the scaffold; at the same time, the high specific surface area of the scaffold provides sufficient space for the immobilization of biologically active factors, and is conducive to new life The growth of bone tissue and the transportation of substances; in addition, the unique electrical signal on the surface of the piezoelectric scaffold can achieve strong adsorption and effective slow release of bioactive factors, improve the utilization rate of factors, and avoid the risks caused by sudden release and excessive use of factors. Potential side effects.
  • the present invention combines the piezoelectric effect with bioactive factors.
  • the electrical signal can quickly activate the early behavioral response and osteogenic differentiation of cells, and works together with the high-efficiency osteoinductive activity of the bioactive factors to synergistically promote the growth of cells in the bone defect site on the scaffold. Rapid recruitment, adhesion, and osteogenic differentiation of surfaces.
  • the present invention achieves a faster and more significant bone repair effect by combining the piezoelectric effect with bioactive factors through the obtained high-efficiency bone-promoting piezoelectric porous scaffold.
  • the present invention combines the piezoelectric effect with bioactive factors through the obtained high-efficiency bone-promoting piezoelectric porous scaffold, quickly starts the early adhesion, spreading and migration of BMSCs, enhances the expression of BMPRs, and cooperates with rhBMP-2 to quickly repair Bone defects, it is expected to significantly reduce the dosage of factors and avoid the potential risks caused by their high-dose use. .
  • Figure 1 is a flow chart for the preparation of piezoelectric porous scaffolds.
  • Figure 2 shows the digital photos and scanning electron microscope images of the piezoelectric porous scaffold.
  • Figure 3 shows the BMSCs cytotoxicity evaluation results of the piezoelectric porous scaffold (A) and the scanning electron microscope image of the adhesion and spreading of BMSCs on the scaffold surface (B).
  • FIG. 4 shows the in vitro ALP activity expression results.
  • FIG. 5 shows the osteogenesis-related gene expression results.
  • the present invention solves the problems of difficulty in preparing traditional bone repair scaffolds, high cost and mediocre effects. It adopts a new polyurethane sponge method to prepare porous piezoelectric scaffolds, and at the same time, it greatly improves the efficiency of traditional bone repair by loading bioactive factors. its bone repair effect.
  • the two complement each other, and the porous scaffold provides an attachment matrix for bioactive factors, thereby indirectly improving the loading capacity of the active factors and the ability to promote bone; accordingly, compared with ordinary porous active scaffolds, the scaffold of the present invention is a piezoelectric scaffold.
  • its surface electrical signal can firmly load the active factor on the surface of the stent through electrostatic interaction, which not only achieves effective sustained release of the active factor and improves its utilization efficiency, but also reduces the usage of the active factor and avoids overdose to a certain extent.
  • Potential side effects caused by use; in addition, the electrical signal on the surface of the piezoelectric scaffold can effectively activate early behavioral responses such as cell adhesion, spreading, and migration, which is conducive to subsequent active factors to fully exert their osteoinductive effects. Both (piezoelectric effect + biological Active factors) work together to quickly achieve high-quality repair and functional reconstruction of defective parts.
  • the piezoelectric porous stent of the present invention is a porous stent material with simple preparation steps, short time consumption and excellent piezoelectric properties.
  • the biological component used is tricalcium phosphate ( ⁇ -TCP)
  • the piezoelectric component used is calcium titanate (BaTiO 3 )
  • the binder used It is polyvinyl alcohol (PVA) solution.
  • the bioactive factor loaded in this experiment is bone morphogenetic protein-2 (BMP-2).
  • BMP-2 bone morphogenetic protein-2
  • the present invention uses a freeze-drying physical adsorption method to load BMP-2 onto the surface of a piezoelectric porous scaffold to achieve an efficient bone-promoting effect.
  • Table 1 shows the naming and component definitions of each group of materials.
  • the piezoelectric coefficient d 33 data of the P-BTCP sample obtained by 6kV polarization is shown in Table 2. Although the lower density of the P-BTCP sample due to its porous structure will affect its piezoelectric properties, its average piezoelectric coefficient d 33 still reaches 10pC/N, which is the same as the piezoelectric coefficient d 33 of natural bone (7.5pC/N ⁇ 10pC/N), which can significantly improve the electrical activity of the implanted site, meet the requirements for the piezoelectric response of the material during the bone repair process, and better accelerate the process of bone regeneration.
  • Table 2 shows the piezoelectric coefficient d 33 of the P-BTCP sample with a polarization strength of 6kV.
  • the density and electric domain structure of the piezoelectric porous scaffold will affect its piezoelectric properties, its average piezoelectric coefficient d 33 still reaches 10pC/N, which is similar to the d 33 of natural bone (7.5pC/N ⁇ 10pC/N ), it can significantly improve the electrical activity of the implanted site, meet the requirements for the piezoelectric response of the material during bone repair, and better accelerate the process of bone regeneration.
  • CCK-8 (Beyotime, Shanghai). Sterilize O-BTCP and P-BTCP for 30 minutes at 121°C and 0.12kPa, place them in 48-well plates respectively, seed BMSCs on the surface of the scaffold at a density of 1 ⁇ 10 4 cells/well, and incubate them in a cell culture incubator. were cultured for 1, 3, and 5 days respectively.
  • Detection steps Discard the old culture medium, fix the cells with glutaraldehyde for 15 minutes at room temperature, and rinse gently with PBS buffer three times. Add 40 ⁇ L CCK-8 to each well (protect from light), incubate at 37°C for 2 hours, and measure the absorbance at 450 nm with a continuous spectrum microplate reader (use the absorbance at 650 nm as the background value). The results are shown in Figure 3A shown; gradient ethanol was added to each well for dehydration, and the surface of the scaffold was covered with isoamyl acetate. After drying in a 37°C oven overnight, the adhesion and spreading morphology of the cells was observed with a scanning electron microscope. The results are shown in B in Figure 3 .
  • BSA bovine serum albumin
  • Table 3 shows the amount of bioactive protein adsorbed on the surface of the piezoelectric porous scaffold.
  • the scaffold sample with a three-dimensional connected pore structure provides sufficient space for protein loading due to its high specific surface area; as the concentration of the protein solution continues to increase, the protein adsorption capacity of the porous scaffold first increases rapidly, Then it tends to stabilize, that is, saturated adsorption is reached.
  • the protein saturated adsorption amount on the surface of P-BTCP scaffold is about twice that of O-BTCP scaffold, indicating that polarization operation can significantly enhance the protein adsorption capacity of porous ceramics, improve the bioactivity of the implanted site, and better promote bone repair and bone repair. regeneration.
  • the O-BTCP and P-BTCP scaffolds were soaked in 8 mg/mL BSA protein solution for 2 hours respectively. After being taken out, they were gently washed twice with PBS to remove unadsorbed protein. After freeze-drying, a BSA-immobilized porous scaffold was obtained. Add 1mL PBS to the BSA-loaded stent, place it in a constant temperature shaking box at 37°C, and shake at 20 rpm. Collect 20 ⁇ L of supernatant at regular intervals and supplement with an equal amount of fresh PBS. After the sustained release, the BSA sustained-release concentration at each time point was measured using the BCA method, and the cumulative sustained-release amount percentage was calculated.
  • the O-BTCP sample released a large amount of protein within the first day, and the protein release amount in the first 7 days was about 50%; while the protein release amount of the P-BTCP sample in the first 7 days was only 20%; this shows that the surface charge of P-BTCP is related to The electrostatic interaction between protein molecules can achieve long-term sustained release of proteins.
  • the utilization of bioactive factors can be greatly improved and negative effects caused by sudden release or excessive use of bioactive factors can be avoided.
  • the main ways of loading biologically active factors include physical adsorption or coupling with coupling agents (such as silane coupling agents, etc.). Since biologically active factors are easily deactivated when the temperature is high and no longer have biological activity, sometimes The high thermal weight loss rate of the coupling agent will lead to lower loading content of bioactive factors. Therefore, in order to increase the total amount of bioactive factors adsorbed and reduce the impact on protein activity, the present invention preferably adopts the freeze-drying physical adsorption method. .
  • VEGF vascular endothelial growth factor
  • BMP-2 bone morphogenetic protein 2
  • TGF- ⁇ 1 transforming growth factor ⁇ 1
  • FGF-9 human fibroblast growth factor 9
  • Table 4 shows scaffold samples loaded with different bioactive factors.
  • O-BTCP Place the high-temperature and high-pressure sterilized O-BTCP, P-BTCP, O-BTCP/BMP-2 and P-BTCP/BMP-2 scaffolds in a 24-well plate, and connect BMSCs at a density of 1 ⁇ 10 5 cells/well. Seed on the surface of the scaffold and culture in a cell culture incubator for 24 hours.
  • Detection steps Change the medium to osteogenic induction medium, and culture it in a cell culture incubator for 7 days (change the medium every two days); gently rinse 3 times with PBS buffer, and add 500 ⁇ L NP-40 to each well to lyse cells.
  • the solution was cultured at 37°C for 90 minutes; the total protein amount was measured using the BCA standard curve method; 50 ⁇ L of lysis solution was taken from each well and placed in a 96-well plate, and 100 ⁇ L of ALP working solution was added to each well (the concentration of PNPP-Na was 1mg/mL), incubate for 2 hours at 37°C, and measure the absorbance at 405nm with a continuous spectrum microplate reader. The results are shown in Figure 4.
  • the ALP activity of P-BTCP is significantly higher than that of O-BTCP, indicating that the surface electroactivity of P-BTCP samples can induce the osteogenic differentiation process of BMSCs by activating early behavioral responses of cells; O-BTCP/BMP- loaded with growth factors 2.
  • the ALP activity of the P-BTCP/BMP-2 scaffold was significantly higher than that of the O-BTCP and P-BTCP porous scaffolds not loaded with growth factors, indicating that the high-activity expression of growth factors can also improve the maturity and formation of osteogenic differentiation of BMSCs. The ability of bone to mineralize.
  • O-BTCP Place the high-temperature and high-pressure sterilized O-BTCP, P-BTCP, O-BTCP/BMP-2 and P-BTCP/BMP-2 scaffolds in a 24-well plate, and connect BMSCs at a density of 1 ⁇ 10 5 cells/well. Seed on the surface of the scaffold and culture in a cell culture incubator for 3 days.
  • Detection steps Add cell lysis solution to each well, extract mRNA, configure reverse transcription reagent, and use Prime Script TM RT reagent Kit reverse-transcribes mRNA into cDNA; uses the reverse-transcribed cDNA as a template, adds SYBR reagent and upstream and downstream primers, and uses an RT-qPCR instrument for amplification; finally, the expression of the target receptor and gene is measured to obtain The results are shown in Figure 5.
  • BMP receptors on the membrane surface compared with O-BTCP, P-BTCP and O-BTCP/BMP-2 both enhanced the expression of downstream osteogenesis-related genes, indicating that the electroactive and bioactive factors BMP-2 can pass through respectively
  • the ability to stimulate osteogenesis-related signaling pathways improves the osteogenic differentiation of BMSCs; among them, the expression of osteogenesis-related genes in BMSCs on the surface of the P-BTCP/BMP-2 scaffold is the most significant, proving that electrical signals on the scaffold surface can cooperate with the bioactive factor BMP-2 Promote the rapid recruitment, adsorption and osteogenic differentiation of cells in bone defect areas on the surface of the scaffold, demonstrating a faster and more significant bone repair effect.

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Abstract

本发明公开了一种具有高效促成骨作用的压电多孔支架及其制备方法，所述压电多孔支架由生物组分与压电组分通过粘结剂混合、烧结后经极化处理后得到。本发明的压电多孔支架可高效固载和吸附生物活性蛋白因子，实现蛋白的有效缓释，同时具备压电性能和成骨活性。在支架压电效应、多孔结构和蛋白的生物活性等多重作用下，相互协同快速启动骨髓基质干细胞早期响应，进而促进细胞粘附、增殖和成骨分化，对于大段骨缺损、骨不连等骨缺损的高效、快速修复具有重要意义。

Description

一种具有高效促成骨作用的压电多孔支架及其制备方法 技术领域
本发明属于生物医用材料技术领域，涉及一种具有高效促成骨作用的压电多孔支架，具有可调的压电性能和优异的成骨活性。更具体地，涉及一种可以携带生物活性因子的压电多孔骨修复支架。
背景技术
随着生物医学技术的不断发展，人们开始着手解决因疾病、事故、老龄化而导致的骨组织创伤和缺损问题。与常规骨折不同，大段骨损伤(骨缺损长度大于骨直径1.5～2倍)在当今的生物医学领域依旧十分棘手，因为人体骨组织对于超过临界尺寸的骨缺损不具备自我修复的能力。面对这类疾病，大量研究以及医学实践表明，植入多孔支架来促使骨组织在支架内生长，从而实现骨缺损快速和高质量重建是目前较为常用且高效的解决方案。
人体骨组织本身具有压电效应，在机械应力下产生表面电势，该压电响应微环境可以调控细胞成骨分化、刺激细胞重塑骨结构。目前，利用骨电学特性发展的临床应用技术(如电刺激成骨技术、电磁场刺激成骨技术)在治疗骨折、骨缺损、骨关节炎和骨坏死等骨科疾病中取得良好治疗效果。然而，电刺激装置复杂，植入困难，易造成感染引发二次疾病。另外，电场和磁场的叠加还存在引发正常组织病变的风险。因此，开发具有类似人骨压电特性的骨替代材料是生物组织工程领域研究的热点。
研究发现，钛酸钡等无铅压电陶瓷材料的极性不对称结构赋予其优异的铁电和压电性，在外部应力作用下材料内电轴发生偏转，产生压电电荷，从而模拟人体骨组织在运动受力时产生的压电微环境，有效促进骨细胞再生。通过复合非压电生物活性组分，旨在得到兼具压电性和生物相容性的复合材料，并在一定程度上解决压电陶瓷的生物不可降解问题，以更加快速有效的促进骨整合、成骨和骨化。
骨组织再生是一个涉及多种细胞因子的复杂生物学过程。因此，在组织工程支架中固载生物活性因子是提高其生物活性的有效手段。然而，目前少有研究将材料的压电效应与生物活性因子相结合，研究电信号与生物活性因子对细胞生物相容性和成骨活性的共同作用。
因此，迫切需要研究一种压电多孔支架，三维连通孔结构不仅可以保证力学刺激 的有效传递，同时其高比表面积为生物活性因子的固载提供了充足空间。压电多孔支架可发挥支架压电效应、多孔结构以及因子生物活性的协同作用，以促进骨缺损的高效快速修复。
发明内容
本发明的目的在于提供一种压电多孔支架，用于快速修复和治疗骨缺损。
本发明的第一方面，提供一种压电多孔支架，所述压电多孔支架由生物组分与压电组分通过粘结剂混合、烧结后经极化处理后得到，其中，
所述生物组分为羟基磷灰石(HA)、磷酸三钙(β-TCP)、生物玻璃(MBG)、聚磷酸钙、磷酸四钙、磷酸八钙、双相磷酸钙、聚磷酸钙中的一种或两种以上的混合物；
所述压电组分为钛酸钙(CaTiO3)、铌酸钾钠(K0.5Na0.5NbO3)、钛酸钡(BaTiO3)中的一种或两种以上的混合物；
所述粘结剂为海藻酸钠、聚乙烯醇、纤维素中的一种或两种以上的混合物。
在另一优选例中，所述粘结剂以水溶液形式使用。在另一优选例中，所述粘结剂以质量浓度为5％-20％、8％-15％或10％的水溶液的形式使用。
在另一优选例中，生物组分为β-TCP，压电组分为CaTiO3，所用粘结剂为质量浓度5％-20％、8％-15％或10％聚乙烯醇的水溶液。在另一优选例中，所用生物组分β-TCP质量分数占比为5％-50％、8％-35％或10％-20％。在另一优选例中，压电组分BaTiO3质量分数占比为95％-50％、92％-65％或90％-80％。在另一优选例中，粘结剂为5％-20％、8％-15％或10％聚乙烯醇溶液，占生物组分与压电组分总质量的5％-50％、8％-35％或10％-20％。
在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的5％～95％，所述压电组分占所述生物组分与压电组分总质量的95％～5％；和/或
所述粘结剂的质量占所述生物组分与压电组分总质量的5％～30％。
在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的10％～80％，所述压电组分占所述生物组分与压电组分总质量的90％～20％。
在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的20％～50％、5％-50％、8％-35％或10％-20％，所述压电组分占所述生物组分与压电组分总质量的80％～50％、95％-50％、92％-65％或90％-80％。
在另一优选例中，所述粘结剂的质量占所述生物组分与压电组分总质量的5％～ 10％、5％-50％或8％-35％。
在另一优选例中，所得压电多孔支架的压电常数d33为8pC/N～13pC/N。与人自然骨的d33(7.5pC/N～10pC/N)相当。
本发明的第二方面，提供一种活性压电多孔支架，包括第一方面所述的压电多孔支架以及负载在压电多孔支架上的生物活性因子，其中，所述生物活性因子为VEGF、BMP-2、TGF-β1、FGF-9中的一种或者两者以上的混合物。
在另一优选例中，所述生物活性因子为BMP-2。
在另一优选例中，所述生物活性因子与所述压电多孔支架的质量比为0.1-0.5:1。
在另一优选例中，所述生物活性因子与所述压电多孔支架的质量比为0.2-0.3:1。
在另一优选例中，所述生物活性因子与所述压电多孔支架的质量比为0.26:1。
在另一优选例中，所述负载为物理吸附或偶联剂偶联，较佳为冷冻干燥物理吸附。
在另一优选例中，在第一方面所述的压电多孔支架表面物理吸附生物活性因子，赋予其生物活性。
在活性压电多孔支架表面培养大鼠骨髓基质干细胞(BMSCs)，培养特定时间后，检测上述支架与普通支架的细胞数量与骨形成蛋白的质量密度，确定获得高效促成骨压电多孔支架表面。
利用成骨调控因子与多孔压电支架在促进骨修复的协同作用，达到快速骨修复以及治疗骨缺陷的目的。本发明通过负载生物活性因子，与普通压电多孔支架相比，生物相容性更加出色，便于进一步医疗方面的应用。
高效促成骨作用的压电多孔支架表面电信号与生物活性因子间的静电相互作用可以将因子牢牢吸附在材料表面，并实现因子的长效缓释，不仅提高了活性因子的作用效率，同时避免了活性因子过量使用带来的潜在副作用；同时将电信号快速启动细胞响应与活性因子诱导成骨相结合，实现缺损部位的快速高效修复与功能重建。
本发明的第三方面，提供第一方面所述的压电多孔支架的制备方法，所述制备方法包括以下步骤：
(i)将压电组分、生物组分和粘结剂混合均匀，得到复合浆料；
(ii)将所述复合浆料与多孔模板混合均匀，得到复合支架；
(iii)将所述复合支架高温烧结，得到多孔支架；
(iv)将所述多孔支架进行高压极化处理，得到压电多孔支架。
本发明采用聚氨酯海绵模板法，为多孔型支架提供了一种新的制备思路。本方法通过将混合浆料与聚氨酯海绵提前充分混合，之后共同烧结，去除聚氨酯海绵，得到所述压电多孔支架。与传统的制备方法相比，此方法制备成本低廉，操作简单，材料成型时间短。同时，由于混合浆料的多样选择性，所得支架的各项物理性能可以通过改变混合浆料的成分与比例得到改进。
本发明以压电陶瓷粉末、无机生物活性组分和粘结剂按照一定的配比组合，并与预处理好的多孔模板混合均匀。经干燥、成型和高温烧结，制备了具有高效促成骨作用的压电多孔支架。该支架制备方式简单便捷，具有三维连通孔结构，保证了机械应力在支架内部的有效传递。同时，制备的压电多孔支架可以高效搭载和吸附生物活性蛋白因子，实现蛋白的有效缓释。在支架压电性能、多孔结构和生物活性蛋白的三重作用下，可协同快速启动骨髓基质干细胞(BMSCs)早期响应，大大促进BMSCs粘附、增殖和成骨分化。该压电多孔支架兼具优异的压电性能和成骨活性，对于大段骨缺损、骨不连等骨缺损的高效、快速修复具有重要意义。
在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的5％～95％，所述压电组分占所述生物组分与压电组分总质量的95％～5％。在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的20％～80％，所述压电组分占所述生物组分与压电组分总质量的80％～20％。
在另一优选例中，所述生物组分的质量占所述生物组分与压电组分总质量的20％～50％、5％-50％、8％-35％或10％-20％，所述压电组分占所述生物组分与压电组分总质量的80％～50％、95％-50％、92％-65％或90％-80％。
在另一优选例中，所述粘结剂的质量占所述生物组分与压电组分总质量的5％～30％。在另一优选例中，所述粘结剂的质量占所述生物组分与压电组分总质量的5％～10％、5％-50％或8％-35％。
在另一优选例中，所述所述多孔模板为聚甲基丙烯酸甲酯(PMMA)胶体模板、聚氨酯(PU)海绵模板、聚碳酸酯(PC)膜模板或纤维素模板。
在另一优选例中，烧结温度为800-2000℃，较佳为1100-1500℃或1400℃。
在另一优选例中，烧结时间为1-5小时，较佳为2-4小时。
在另一优选例中，极化过程中极化电压为2-15kV/mm，较佳为5-10kV/mm。
本发明通过新型制备方法，得到一定孔隙率和孔径分布的多孔支架材料，所得压电支架可任意改变形状，为下一步的物理吸附提供适宜的环境。
本发明的第四方面，提供第一方面所述的压电多孔支架或第二方面所述的活性压电多孔支架的用途，用于制备修复和治疗骨缺损的材料。
本发明的有益效果在于：
(1)本发明采用的制备方法为聚氨酯海绵法，从制备方案而言，本方法制备步骤简单，成本较低，耗时短，便于大规模生产使用；从产品性能而言，本发明所得产品压电性能优于传统制备成品。此方法可以解决原有制备方法存在的制备困难，成本较高等问题，为其广阔的临床应用提供了可能性。
(2)本发明制备的压电多孔支架具有三维连通孔结构，保证了力学信号在支架内部的有效传递；同时支架的高比表面积为生物活性因子的固载提供了充足空间，并有利于新生骨组织的长入和物质的运输；此外，压电支架表面特有的电信号可实现生物活性因子的强力吸附和有效缓释，提高了因子使用率，避免了因子突释和过量使用带来的潜在副作用。
(3)本发明将压电效应与生物活性因子相结合，电信号能够快速激活细胞早期行为响应和成骨分化，与生物活性因子的高效骨诱导活性共同作用，协同促进骨缺损部位细胞在支架表面的快速募集、粘附和成骨分化。与传统骨修复支架相比，本发明通过所得高效促成骨压电多孔支架将压电效应与生物活性因子相结合，起到了更加快速、显著的骨修复效果。
结合压电效应和生物因子两种促成骨因素，将生物活性因子负载在多孔压电支架上，得到一种具有压电性能和生物活性的骨修复压电多孔支架。与传统骨修复支架相比，本发明通过所得高效促成骨压电多孔支架将压电效应与生物活性因子结合起来，快速启动BMSCs早期黏附、铺展和迁移，增强BMPRs表达，协同rhBMP-2快速修复骨缺损，有望大幅降低因子的使用量，避免其大剂量使用带来的潜在风险。。
应当理解，在本发明专利中，本发明的上述技术特征和下文中(如实施例)中具体描述的各技术特征之间可以互相结合，从而构成新的或者优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或者相似目的的代替性取代，限于篇幅，在此不再一一赘述。
附图说明
图1为压电多孔支架的制备流程图。
图2为压电多孔支架的数码照片和扫描电镜图片。
图3为压电多孔支架的BMSCs细胞毒性评价结果(A)与支架表面BMSCs粘附与铺展的扫描电镜图(B)。
图4示出体外ALP活性表达结果。
图5示出成骨相关基因表达结果。
具体实施方式
本发明通过多次长期实验研究，针对传统骨修复所用支架制备困难，成本高昂且作用效果一般等问题，采用全新的聚氨酯海绵法制备多孔压电支架，同时又通过负载生物活性因子，极大地提高了其骨修复效果。两者相辅相成，多孔支架为生物活性因子提供了附着基体，从而间接提高了活性因子的固载量和促成骨能力；相应地，与普通多孔活性支架相比，由于本发明的支架为压电支架，其表面电信号可以通过静电相互作用将活性因子牢牢固载在支架表面，不仅可以实现活性因子的有效缓释，提高其利用效率，同时降低了活性因子使用量，在一定程度上避免了过量使用带来的潜在副作用；另外压电支架表面电信号能够有效激活细胞的粘附、铺展和迁移等早期行为响应，有利于后续活性因子充分发挥其骨诱导效应，二者(压电效应+生物活性因子)协同作用，可快速实现缺损部位的高质量修复与功能重建。
压电多孔支架
本发明所述压电多孔支架，是一种制备步骤简单，耗时短，同时压电性能优异的多孔支架材料。在一优选例中，该多孔支架材料在制备过程中，所用的生物组分为磷酸三钙(β-TCP)，所用的压电组分为钛酸钙(BaTiO3)，所用的粘结剂为聚乙烯醇(PVA)溶液。
生物活性因子
本实验所负载生物活性因子为骨形成发生蛋白-2(BMP-2)。本发明通过冷冻干燥物理吸附法，将BMP-2负载到压电多孔支架表面，实现了高效促成骨效应。
接下来将结合具体实施例，进一步阐述本发明。应当理解，下列所述实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例未注明的实验方法，通常按照常规方法。
为了更好地阐述本发明，现对实施例中出现的组别及组分定义作出解释，如表1所示。
表1为各组材料命名及组分定义
实施例1
β-TCP的制备
分别配置500mL的Ca(NO3)2溶液和(NH4)2HPO4溶液。在37℃的水浴条件下，将0.6mol/L的Ca(NO3)2溶液逐滴滴加至0.4mol/L的(NH4)2HPO4溶液中，充分搅拌。滴加过程中用氨水调节其pH值，严格控制在7.0-7.5之间，滴加完成之后，pH值应为7.2，继续反应5小时，真空抽滤并洗涤后得到前驱体。最后在高温电阻炉中800℃烧结2小时得到β-TCP，过400目分样筛备用。
实施例2
2.1多孔支架的制备
将聚氨酯海绵模板裁剪成所需尺寸的圆柱体后，浸泡在10％NaOH溶液中清洗1小时，用清水洗净后，烘干备用。按照β-TCP：BaTiO3：10％聚乙烯醇溶液＝1：4：5的比例配置混合浆料，并将混合好的浆料浸渍到聚氨酯海绵中，反复挤压至浆料充分挂在海绵中。置于60℃烘箱中干燥72小时后，在高温电阻炉中1400℃烧结180分钟，随炉冷却后得到O-BTCP。制备流程图如图1所示。
2.2 O-BTCP的极化
在室温条件下，将O-BTCP置于高压极化装置的负极板上，调节极化针与支架表面的间距为1mm，打开高压电源并缓慢升高电压至6kV，极化20分钟后，得到P-BTCP。
实施例3
3.1 O-BTCP的制备
将聚氨酯海绵模板裁剪成所需尺寸的圆柱体后，浸泡在10％NaOH溶液中清洗1小时，用清水洗净后，烘干备用。按照β-TCP：BaTiO3：10％聚乙烯醇溶液＝1：4：5的比例配置混合浆料，并将聚氨酯海绵模板浸渍其中，反复挤压使浆料充分挂在聚氨酯海绵模板上。置于60℃烘箱中干燥72小时后，在高温电阻炉中1200℃烧结120分钟，随炉冷却后得到O-BTCP。制备流程图如图1所示。
3.2 O-BTCP的极化
在室温条件下，将O-BTCP置于高压极化装置的负极板上，调节极化针与支架表面的间距为1mm，打开高压电源并缓慢升高电压至3kV，极化20分钟后，得到P-BTCP。
6kV极化得到的P-BTCP样品的压电系数d33数据如表2所示。虽然P-BTCP样品因多孔结构而较低的致密度会影响其压电性能，但是其平均压电系数d33依然达到了10pC/N，与天然骨的压电系数d33(7.5pC/N～10pC/N)相当，能够显著提高植入部位的电活性，满足骨修复过程中对材料压电响应的要求，更好地加快骨再生的进程。
表2为极化强度为6kV的P-BTCP样品的压电系数d33
实施例4
P-BTCP的形貌及性能表征
对P-BTCP的表面进行喷金处理45秒后，用数码相机和扫描电子显微镜观察其结构形貌和电畴结构，得到的结果如图2所示。可见P-BTCP很好地复制了模板的连通孔结构，孔径大小在100-500μm之间，能够较好地实现营养物质或代谢废物的运输、骨组织的粘附与生长，局部放大图显示支架中BaTiO3晶体形成了电畴结构(黑色虚线框)，对P-BTCP的压电性能具有积极贡献。
虽然压电多孔支架的致密度和电畴结构会影响其压电性能，但是它的平均压电系数d33依然达到了10pC/N，与天然骨的d33(7.5pC/N～10pC/N)相当，能够显著提高植入部位的电活性，满足骨修复过程中对材料压电响应的要求，更好地加快骨再生的进程。
实施例5
BTCP支架的细胞毒性测试
采用CCK-8(Beyotime,shanghai)。将O-BTCP与P-BTCP在121℃、0.12kPa的条件下灭菌30分钟，分别置于48孔板中，按照1×104细胞/孔的密度接种BMSCs于支架表面，在细胞培养箱中分别培养1、3、5天。
检测步骤：弃置旧培养基，在室温条件下用戊二醛固定细胞15分钟，用PBS缓冲液轻柔冲洗3次。每孔加入40μL CCK-8(避光操作)，在37℃的条件下孵育2小时，用连续光谱酶标仪测定450nm处的吸光度(以650nm处的吸光度为背景值)，结果如图3A所示；每孔加入梯度乙醇进行脱水处理，用乙酸异戊酯覆盖支架表面，在37℃烘箱中干燥过夜后，用扫描电子显微镜观察细胞的粘附与铺展形态，结果如图3中B所示。
通过图3中A可知，O-BTCP、P-BTCP表面的细胞增殖情况与空白对照组没有显著性差异，说明其具有良好的细胞相容性，既有利于细胞在材料表面的粘附、增殖与分化，也有利于激活成骨相关基因的表达。通过图3中B可知，细胞在连通孔的表面(特别是P-BTCP)大量粘附且形态良好，伪足充分延伸并与材料紧密结合，进一步证明了P-BTCP作为植入材料应用于骨组织修复再生领域的潜力与优势。
实施例6
极化操作对多孔支架蛋白饱和吸附量的影响
分别配置浓度为0.5mg/mL、1mg/mL、2mg/mL、4mg/mL、8mg/mL、16mg/mL的牛血清白蛋白(BSA)溶液，将高温高压灭菌后的O-BTCP和P-BTCP支架置于24孔板中，每孔加入1mL不同浓度的蛋白溶液，在37℃的条件下孵育2小时；用PBS缓冲液轻柔冲洗支架后，加入蛋白裂解液，使牢固吸附的蛋白全部掉落；用BCA标准曲线法测定总蛋白量、用做差法计算出计算出O-BTCP与P-BTCP支架的蛋白吸附量，得到的结果如表3所示。
表3为压电多孔支架表面的生物活性蛋白吸附量
通过表3可知，具有三维连通孔结构的支架样品因其高比表面积而为蛋白负载提供了充足空间；随着蛋白溶液浓度的不断提高，多孔支架的蛋白吸附量先快速增加， 然后趋于稳定，即达到了饱和吸附。P-BTCP支架表面的蛋白饱和吸附量约为O-BTCP支架的两倍，说明极化操作可以显著增强多孔陶瓷的蛋白吸附能力，提高植入部位的生物活性，更好地促进骨修复与骨再生。
分别将O-BTCP和P-BTCP支架浸泡于8mg/mL的BSA蛋白溶液中2h，取出后用PBS轻柔冲洗两遍以去除未吸附的蛋白，冷冻干燥后得到固载BSA的多孔支架。向固载BSA的支架中加入1mL PBS，置于37℃恒温震荡箱中，20rpm震荡。每隔一段时间收集20μL上清液并补充等量的新鲜PBS。缓释结束后，用BCA法测定各个时间点的BSA缓释浓度，并计算累积缓释量百分比。
O-BTCP样品在第1天内便释放大量蛋白，前7天的蛋白释放量约为50％；而P-BTCP样品前7天的蛋白释放量仅为20％；这说明P-BTCP表面电荷与蛋白分子间的静电相互作用可以实现蛋白的长效缓释，当其负载生物活性因子后，可以大大提高生物活性因子的利用率，避免因生物活性因子突释或过量使用而产生的负面影响。
实施例7
7.1生物活性因子的负载方式
本发明中，负载生物活性因子的方式主要有物理吸附或偶联剂偶联(如硅烷偶联剂等)，由于生物活性因子在温度较高时极易失活而不再具有生物活性，偶联剂的高热失重率会导致生物活性因子的负载含量较低等原因，所以为了在提高生物活性因子吸附总量的同时减小对蛋白活性的影响，本发明优选采用的是冷冻干燥物理吸附法。
7.2负载生物活性因子
将高温高压灭菌后的O-BTCP和P-BTCP分别浸泡于0.25mg/mL的血管内皮生长因子(VEGF)、骨形态发生蛋白2(BMP-2)、转化生长因子β1(TGF-β1)和人成纤维细胞生长因子9(FGF-9)溶液中，每组样品200μL，充分覆盖支架。37℃恒温震荡箱24h使样品充分吸收生物活性因子。取出支架并进行无菌冷冻干燥，得到负载生物活性因子的支架样品，如表4所示。
表4为负载不同生物活性因子的支架样品

实施例8
BMSCs碱性磷酸酶(ALP)活性检测
将O-BTCP和P-BTCP支架高温高压灭菌后，每组加入200μL的BMP-2(0.25mg/mL)蛋白溶液。37℃恒温震荡箱24h后，取出支架并无菌冷冻干燥得到负载BMP-2的O-BTCP/BMP-2和P-BTCP/BMP-2支架。
将高温高压灭菌后的O-BTCP、P-BTCP、O-BTCP/BMP-2和P-BTCP/BMP-2支架置于24孔板中，按照1×105细胞/孔的密度接BMSCs种于支架表面，在细胞培养箱中培养24小时。
检测步骤：更换培养基为成骨诱导培养基，在细胞培养箱中培养7天(每隔两天换1次液)；用PBS缓冲液轻柔冲洗3次，每孔加入500μL NP-40细胞裂解液，在37℃的条件下培养90分钟；用BCA标准曲线法测定总蛋白量；每孔吸取50μL裂解液，置于96孔板中，每孔加入100μL的ALP工作液(PNPP-Na浓度为1mg/mL)，在37℃的条件下孵育2小时，用连续光谱酶标仪测定405nm处的吸光度，得到的结果如图4所示。
P-BTCP的ALP活性明显高于O-BTCP，说明P-BTCP样品的表面电活性可以通过激活细胞早期的行为响应，从而诱导BMSCs的成骨分化进程；负载生长因子的O-BTCP/BMP-2、P-BTCP/BMP-2支架的ALP活性明显高于未负载生长因子的O-BTCP、P-BTCP多孔支架，说明生长因子的高活性表达同样能够提高BMSCs成骨分化的成熟度和成骨矿化的能力。
实施例9
生长因子受体和成骨相关基因的表达分析
将高温高压灭菌后的O-BTCP、P-BTCP、O-BTCP/BMP-2和P-BTCP/BMP-2支架置于24孔板中，按照1×105细胞/孔的密度接BMSCs种于支架表面，在细胞培养箱中培养3天。
检测步骤：每孔加入细胞裂解液，提取mRNA，配置逆转录试剂，使用Prime ScriptTM  RT reagent Kit试剂盒将mRNA逆转录为cDNA；将逆转录得到的cDNA作为模板，加入SYBR试剂和上下游引物，用RT-qPCR仪器进行扩增；最后测定目标受体和基因的表达情况，得到的结果如图5所示。
与O-BTCP相比，P-BTCP支架上的BMP受体表达明显增强，但是负载BMP-2生物活性因子后BMP受体并没有显著变化，说明P-BTCP支架表面的电信号可以显著促进BMSCs膜表面BMP受体的表达；与O-BTCP相比，P-BTCP、O-BTCP/BMP-2均使下游成骨相关基因的表达增强，说明电活性和生物活性因子BMP-2可以分别通过刺激成骨相关信号通路提高BMSCs成骨分化的能力；其中，P-BTCP/BMP-2支架表面BMSCs成骨相关基因的表达最为显著，证明了支架表面电信号与生物活性因子BMP-2能够协同促进骨缺损部位细胞在支架表面的快速募集、吸附和成骨分化，展现出更加快速、显著的骨修复效果。
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。此外应理解，在阅读了本发明的上述讲授内容后，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. 一种压电多孔支架，其特征在于，所述压电多孔支架由生物组分与压电组分通过粘结剂混合、烧结后经极化处理后得到，其中，
    所述生物组分为羟基磷灰石(HA)、磷酸三钙(β-TCP)、生物玻璃(MBG)、聚磷酸钙、磷酸四钙、磷酸八钙、双相磷酸钙、聚磷酸钙中的一种或两种以上的混合物；
    所述压电组分为钛酸钙(CaTiO3)、铌酸钾钠(K0.5Na0.5NbO3)、钛酸钡(BaTiO3)中的一种或两种以上的混合物；
    所述粘结剂为海藻酸钠、聚乙烯醇、纤维素中的一种或两种以上的混合物。
  2. 如权利要求1所述的压电多孔支架，其特征在于，所述生物组分的质量占所述生物组分与压电组分总质量的5％～95％，所述压电组分占所述生物组分与压电组分总质量的95％～5％；和/或
    所述粘结剂的质量占所述生物组分与压电组分总质量的5％～30％。
  3. 如权利要求1所述的压电多孔支架，其特征在于，所得压电多孔支架的压电常数d33为8pC/N～13pC/N。
  4. 一种活性压电多孔支架，其特征在于，所述活性压电多孔支架包括权利要求1所述的压电多孔支架以及负载在压电多孔支架上的生物活性因子，其中，所述生物活性因子为VEGF、BMP-2、TGF-β1、FGF-9中的一种或者两者以上的混合物。
  5. 如权利要求4所述的活性压电多孔支架，其特征在于，所述生物活性因子与所述压电多孔支架的质量比为0.1-0.5:1。
  6. 如权利要求1所述的压电多孔支架的制备方法，其特征在于，所述制备方法包括以下步骤：
    (i)将压电组分、生物组分和粘结剂混合均匀，得到复合浆料；
    (ii)将所述复合浆料与多孔模板混合均匀，得到复合支架；
    (iii)将所述复合支架高温烧结，得到多孔支架；
    (iv)将所述多孔支架进行高压极化处理，得到压电多孔支架。
  7. 如权利要求6所述的制备方法，其特征在于，所述所述多孔模板为聚甲基丙烯酸甲酯(PMMA)胶体模板、聚氨酯(PU)海绵模板、聚碳酸酯(PC)膜模板或纤维素模板。
  8. 如权利要求6所述的制备方法，其特征在于，烧结温度为800-2000℃，较佳为1100-1500℃或1400℃。
  9. 如权利要求6所述的制备方法，其特征在于，极化过程中极化电压为2-15kV/mm， 较佳为5-10kV/mm。
  10. 一种权利要求1所述的压电多孔支架或权利要求5所述的活性压电多孔支架的用途，其特征在于，用于制备修复和治疗骨缺损的材料。
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