WO2021189192A1 - 一种具有逐步抗菌和促进骨再生功能的骨组织工程支架及其制备方法和应用 - Google Patents

一种具有逐步抗菌和促进骨再生功能的骨组织工程支架及其制备方法和应用 Download PDF

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WO2021189192A1
WO2021189192A1 PCT/CN2020/080673 CN2020080673W WO2021189192A1 WO 2021189192 A1 WO2021189192 A1 WO 2021189192A1 CN 2020080673 W CN2020080673 W CN 2020080673W WO 2021189192 A1 WO2021189192 A1 WO 2021189192A1
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scaffold
black phosphorus
hydroxyapatite
ligand
antibacterial
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French (fr)
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王怀雨
伍煜政
童丽萍
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • 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
    • A61L27/50—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
    • A61L27/58—Materials at least partially resorbable by the body

Definitions

  • the invention belongs to the technical field of biomedical materials, and relates to a bone tissue engineering scaffold with gradual antibacterial and bone regeneration functions, and a preparation method and application thereof.
  • Bone implant is a kind of biomedical material, which can be applied to the bone defect of the organism to achieve the purpose of repairing or replacing the original bone tissue function. It can be divided into inorganic, polymer, metal and composite bone implants by composition classification. body. Because bone implants need to be used inside the human body for a long time and are in direct contact with human blood, body fluids, tissues, etc., bone implants need to have non-toxicity, stable chemical properties, and mechanical properties that match the bone at the implantation site and are excellent. Biocompatibility and other properties. In addition, there is a possibility of bacterial infection at the implant site after orthopedic implant surgery, and bacterial infection will reduce the success rate of implant surgery. If the infection is serious, a second operation is required to remove the infected tissue. Therefore, if bone implants can prevent or treat bacterial infections that may occur after implantation, the efficacy will be greatly improved.
  • the antibacterial performance of bone implants is mainly determined by the physical and chemical properties of the surface, so surface modification can ensure that the antibacterial performance of the material itself is not affected.
  • high-temperature hydrolysis [1] , vapor deposition [2] , chemical grafting [3] , ion implantation [4] and other methods can be used to establish antibacterial surfaces on bone implants.
  • These antibacterial surfaces usually have physicochemical properties that can kill bacteria. nature.
  • the nanosheets can capture bacteria and stretch the cell membrane of the bacteria to put the bacteria in a stress state, resulting in a sharp increase in the level of active oxygen inside the bacteria, which is effective Kill bacteria [1] .
  • a nanotube structure loaded with antibacterial drugs on the surface of the implant and then use a degradable polymer film to coat the surface of the nanotubes, so as to obtain the implant surface that can slowly release antibacterial drugs [8] ;
  • the surface modification of titanium-based materials contains silver nanoparticles, and the slow release of silver ions from silver nanoparticles can impart certain antibacterial properties to the titanium-based materials [9] .
  • the modified material can also have a certain antibacterial effect after being implanted in the human body, but the more prominent problem is the explosive release of the drug or the safety of the ion; and the material can only passively exert the antibacterial effect after being implanted in the human body. It is not possible to actively control antibacterial or promote tissue regeneration according to the actual situation of the wound.
  • the TiO 2 coated with the up-conversion material is absorbing After ultraviolet light, it can react with oxygen and water in the solution to produce reactive oxygen molecules that can destroy the bacterial cell wall and DNA structure, thereby achieving the photocatalytic antibacterial effect [11] ; Wu Shuilin and others constructed a bismuth sulfide nanorod coating on the surface of titanium metal, Then use silver phosphate nanoparticles to modify bismuth sulfide, so that bismuth sulfide produces more active oxygen molecules under near-infrared irradiation to achieve the effect of near-infrared regulation of antibacterial [12] ; in addition, Wu Shuilin and others also regulate the surface polymerization of molybdenum sulfide through near-infrared.
  • gentamicin encapsulated by ethylene glycol achieves antibacterial activity.
  • the photothermal properties of molybdenum sulfide make it heat up rapidly under near-infrared radiation, melting the polyethylene glycol wrapped outside gentamicin to release the drug to achieve near-infrared Regulate the antibacterial effect of drug release [13] .
  • near-infrared responsive materials near-infrared light is used as an external excitation condition, and the activated active oxygen or gentamicin is the final antibacterial effect factor; in addition, whether it is an up-conversion luminescent material (containing rare earth ions) or a sulfide Bismuth, molybdenum sulfide, and even antibacterial silver ions will introduce elements that are not contained in the human body, and their lower biological safety will limit their clinical application [14] .
  • Polydopamine which has good biocompatibility, has also been tried to be used in near-infrared-responsive antibacterial biomaterials [15,16] , but the synthesis mechanism of polydopamine is not yet clear, and the by-products produced during the synthesis process are difficult to separate. The poor dispersion properties of nanoparticles [17] and other factors are obstacles to their clinical application.
  • the present invention provides a new type of degradable bone implant material, the composition of which includes: main body hydroxyapatite (HA) Porous scaffolds, two-dimensional black phosphorus nanomaterials modified with divalent metal ions loaded on the surface of the HA scaffolds, and a degradable polymer coating layer.
  • the present invention uses two-dimensional black phosphorous nanomaterials modified with divalent metal ions as the core component of near-infrared photothermal response, and is loaded on the surface of the HA stent by degradable polymers, and only needs to pass through a near-infrared laser generator outside the body. Irradiation can adjust the temperature of the implant site immediately, realize the different functions of regulating the antibacterial and promoting bone regeneration of the implant site of the bone defect, avoid the use of systemic antibiotics and other drugs, and shorten the healing period of the defect site.
  • HA main body hydroxyapatite
  • One of the objectives of the present invention is to provide a bone tissue engineering scaffold with gradual antibacterial and bone regeneration functions.
  • the technical solution adopted by the present invention is: a bone tissue engineering scaffold with the function of gradual antibacterial and bone regeneration, including a hydroxyapatite porous scaffold and a divalent metal loaded on the surface of the hydroxyapatite porous scaffold Ion-modified two-dimensional black phosphorus nanomaterials and degradable polymer coating.
  • the divalent metal particles include Zn ions and Ca ions;
  • the degradable polymer coating layer includes polylactic acid-glycolic acid copolymer PLGA, polylactic acid PLA, polyglycolic acid, polycaprolactone, and polybutylene succinate.
  • the temperature of the bone tissue engineering scaffold with gradual antibacterial function can be adjusted by adjusting the near-infrared light density (through in vitro adjustment).
  • the intensity of near-infrared light adjusts its heating rate and final holding temperature), and achieves antibacterial and bone-promoting functions at higher temperatures (45-60°C) and lower temperatures (39-42°C).
  • the second objective of the present invention is to provide a method for preparing the aforementioned bone tissue engineering scaffold with the functions of stepwise antibacterial and bone regeneration.
  • the technical solution adopted by the present invention is: a method for preparing the above-mentioned bone tissue engineering scaffold with gradual antibacterial and bone regeneration functions, including the following steps:
  • the hydroxyapatite scaffold is immersed in the blend and then taken out to dry. After the bone implant is taken out of the solution, it is necessary to ensure that the solvent on its surface evaporates slowly to form a uniform and non-porous film.
  • the loading amount of the divalent metal ion modified BP nanomaterial on the surface of the bone implant can be adjusted according to the concentration of the BP nanomaterial contained in the suspension of the polymer and the BP nanomaterial and the total immersion time.
  • the preparation method of the hydroxyapatite scaffold in step 1) is: adding the hydroxyapatite powder to the sodium alginate aqueous solution, stirring uniformly at room temperature, filling the slurry into the cavity of the 3D printer, and adding After pressing and extruding to obtain the hydroxyapatite scaffold embryo body;
  • the hydroxyapatite scaffold body After the hydroxyapatite scaffold body is dried at room temperature, it is transferred to the muffle furnace for sintering and forming;
  • the 3D printed HA stent can be designed according to the specific shape of the patient's defect.
  • the mass fraction of the sodium alginate aqueous solution is 2% to 3%
  • the mass ratio of the hydroxyapatite powder to the sodium alginate aqueous solution is 0.8:1 to 1.2:1
  • the stirring time is 6 to For 12 hours
  • the extrusion pressure is 80-300kPa
  • the extrusion rate is 1-1.5cm/s
  • the sintering temperature is 1100-1300°C, and the sintering time is 6-10 hours.
  • the preparation method of the metal ligand black phosphorus in step 2) is: disperse the black phosphorus in a mixed solvent of N-methylpyrrolidone and water, and then add a divalent metal ion ligand, under the protection of argon After stirring at room temperature and avoiding light, washing and centrifuging with deionized water, ligand-modified black phosphorus nanosheets are obtained, which are labeled as metal ligand black phosphorus.
  • the volume ratio of water to N-methylpyrrolidone in the mixed solvent is 1:9 to 1:1, and the molar ratio of the black phosphorus to the divalent metal ion ligand is 1:1 to 1:30.
  • the time for stirring at room temperature and avoiding light is 3-12 hours, and the number of washing centrifugation is 3-5 times;
  • the divalent metal ion ligand includes salts such as zinc p-toluenesulfonate, zinc chloride, zinc nitrate, zinc sulfate, calcium nitrate, calcium chloride and calcium gluconate.
  • step 3) is specifically: dissolving the degradable polymer in dichloromethane or chloroform to obtain a degradable polymer solution, and dispersing the metal ligand black phosphorus in absolute ethanol to obtain the metal ligand black phosphorus Disperse liquid, then add the metal ligand black phosphorus dispersion to the degradable polymer solution, and obtain a blended solution of the metal ligand black phosphorus and the polymer after ultrasonic dispersion;
  • Step 4) specifically includes: immersing the hydroxyapatite scaffold prepared in step 1) into the blend solution of metal ligand black phosphorus and degradable polymer in step 3), and placing the scaffold in the refrigerator for a period of time after taking it out, and then at room temperature After vacuum drying, a hydroxyapatite bone tissue engineering scaffold of a blend of surface-modified metal ligand black phosphorus and a degradable polymer is obtained.
  • the concentration of the degradable polymer solution is 20-100 mg/mL
  • the concentration of the metal ligand black phosphorus dispersion is 1-10 mg/mL
  • the degradable polymer solution is dispersed with the metal ligand black phosphorus
  • the volume ratio of the liquid is 1-3mL: 0.05-0.3mL
  • the ultrasonic power is 100-500W
  • the ultrasonic time is 10-60 minutes
  • the degradable polymer includes polylactic acid-glycolic acid copolymer, polylactic acid, Fully biodegradable polymers such as polyglycolic acid, polycaprolactone and polybutylene succinate;
  • the immersion time mentioned in step 4) is 0.5 to 3 minutes. After taking out the stent, place it in a refrigerator at 4°C for 1 to 3 days, and then vacuum dry at room temperature for 6 to 18 hours. The air pressure for vacuum drying is 1 ⁇ 10 4 ⁇ 2 ⁇ 10 4 Pa.
  • the third objective of the present invention is to provide an application of the aforementioned bone tissue engineering scaffold with the functions of gradual antibacterial and bone regeneration.
  • the technical solution adopted by the present invention is the application of the above-mentioned bone tissue engineering scaffold with the functions of gradual antibacterial and bone regeneration as a medical material for orthopedics implantation.
  • the present invention uses modified black phosphorus two-dimensional nanosheets as the core element for introducing light and heat.
  • the preparation process of black phosphorus is mature, has good dispersibility and biodegradability, and is degraded into non-biologically toxic phosphate ions in the organism. It is an essential element contained in a large amount in the human body, so it has good biological safety.
  • the black phosphorous nanosheets have good near-infrared responsiveness. Under the irradiation of near-infrared light, the temperature of the black phosphorous will rise rapidly, and the high-temperature environment generated by it can be directly sterilized. The local micro-thermal environment generated by black phosphorous nanosheets under low-power near-infrared irradiation can also promote the formation of new bones.
  • the present invention uses divalent metal ions (Zn 2+ , Ca 2+, etc.) to covalently modify black phosphorus, which not only improves the stability of black phosphorus in the body, but also slowly releases these divalent metal ions as the black phosphorus degrades , Synergistically with heat, it promotes antibacterial and bone regeneration at the defect site.
  • divalent metal ions Zn 2+ , Ca 2+, etc.
  • the present invention adopts 3D printing technology to prepare structured hydroxyapatite stent, immersing the stent in a solution containing divalent metal ion ligand black phosphorus, and then a functionalized coating with photothermal responsiveness can be constructed on the surface of the stent , Has a good antibacterial and bone-promoting effect.
  • the present invention has the following advantages:
  • the bone implant prepared by 3D printing technology has a structure matching human bone, which can induce the formation of new bone.
  • the functional components are dispersed in a fluid polymer solution. Even if the shape of the bone implant is irregular, it can be immersed in the solution to achieve a uniform load.
  • the oxidation speed slows down and the stability in the phosphate buffer is improved, thereby ensuring that the metal ligand black phosphorus has stable and long-lasting photothermal performance in the body.
  • the near-infrared responsive bone implant prepared by the present invention can control the antibacterial effect of the bone implant in vivo by irradiating near-infrared light outside the body.
  • the near-infrared responsive bone implant prepared by the present invention can be slowly degraded in the body and does not need to be taken out after the completion of the bone repair.
  • the near-infrared responsive bone implant prepared by the present invention has the characteristics of gradual degradation in the body, and the final degradation products are carbon dioxide, water, zinc ions, phosphate ions and calcium ions that are common in the human body, which are harmless to the human body. Has high biological safety.
  • FIG. 1 is a macro scanning electron micrograph of the hydroxyapatite scaffold obtained by 3D printing and sintering in Example 1.
  • FIG. 1 is a macro scanning electron micrograph of the hydroxyapatite scaffold obtained by 3D printing and sintering in Example 1.
  • FIG. 2 is an X-ray diffraction spectrum of the hydroxyapatite scaffold in Example 2.
  • FIG. 3a is a scanning electron micrograph of the pure black phosphorus nanosheets without zinc p-toluenesulfonate coordination in Example 3.
  • FIG. 3a is a scanning electron micrograph of the pure black phosphorus nanosheets without zinc p-toluenesulfonate coordination in Example 3.
  • FIG. 3b is a scanning electron micrograph of the zinc ligand black phosphorus coordinated by zinc p-toluenesulfonate in Example 3.
  • FIG. 3b is a scanning electron micrograph of the zinc ligand black phosphorus coordinated by zinc p-toluenesulfonate in Example 3.
  • FIG. 4 is a full spectrum of X-ray photoelectron spectroscopy of black phosphorus and zinc ligand black phosphorus in Example 4.
  • FIG. 4 is a full spectrum of X-ray photoelectron spectroscopy of black phosphorus and zinc ligand black phosphorus in Example 4.
  • Figure 5 is a graph of the Zeta potential of zinc p-toluenesulfonate, black phosphorus and zinc ligand black phosphorus in Example 5.
  • Fig. 6 is an optical photograph of black phosphorus and zinc ligand black phosphorus dispersed in water for 7 days in Example 6.
  • FIG. 7a is a scanning electron microscope image of the surface micro-topography of the hydroxyapatite scaffold in Example 7.
  • FIG. 7b is a scanning electron microscope image of the surface micro-topography of ZnL 2 -BPs@HA-PLGA in Example 7.
  • FIG. 7b is a scanning electron microscope image of the surface micro-topography of ZnL 2 -BPs@HA-PLGA in Example 7.
  • Fig. 8 is a heating curve of the sample in Example 8 under near-infrared irradiation.
  • Figure 9 is a comparison of optical photographs of the sample in Example 9 incubating in a phosphate solution for 10 weeks.
  • Fig. 10 shows the change of light and heat efficiency of ZnL 2 -BPs@HA-PLGA in the phosphate solution for 10 weeks in Example 10.
  • FIG. 11 is a comparison of optical photographs of the implantation site in Example 11.
  • FIG. 11 is a comparison of optical photographs of the implantation site in Example 11.
  • Figure 12 is a schematic diagram of the antibacterial rate of the material in Example 12 against Staphylococcus aureus in vivo.
  • Fig. 13 is a three-dimensional reconstruction diagram of bone tissue around the implant in Example 13.
  • FIG. 14 is a comparison diagram of bone volume fraction around the implant in Example 14.
  • FIG. 14 is a comparison diagram of bone volume fraction around the implant in Example 14.
  • the present invention first prepares a three-dimensional ordered hydroxyapatite scaffold through 3D printing technology, and then uses the coordination of divalent metal ions and black phosphorus to prepare a positively charged and stable metal ligand black phosphorus (through The coordination of black phosphorus and divalent metal ions enhances the stability of black phosphorus), then the black phosphorus modified by the ligand is blended with the degradable polymer, the hydroxyapatite scaffold is immersed in the blend and then taken out, blended After the solvent of the substance evaporates, the surface of the stent is loaded with a metal ligand black phosphorus with near-infrared responsiveness, and a metal ligand black phosphorus modified bone implant that can realize photothermal antibacterial and thermally promoted bone formation is obtained.
  • HA is a layered scaffold composed of regular cylindrical hydroxyapatite.
  • the diameter of a single hydroxyapatite cylinder is about 550 ⁇ m, and the spacing between each cylinder is about 180 ⁇ m.
  • the angle between the layers is 60°, which shows that HA has a regular and orderly three-dimensional structure, which is beneficial to induce bone regeneration.
  • Example 2 The HA sample processed in Example 1 was characterized by an X-ray diffractometer, and the X-ray spectrum shown in Fig. 2 was obtained. It can be seen from the spectrum in Figure 2 that the crystal structure of HA after sintering and cleaning is consistent with the standard spectrum of pure hydroxyapatite powder. The composition of hydroxyapatite.
  • FIG. 3 The black phosphorus and zinc ligand black phosphorus were observed by scanning electron microscope, and the topography picture shown in Fig. 3 was obtained.
  • Figure 3a shows that the black phosphorus presents a nanoplatelet structure with a size between 100-500nm.
  • the zinc ligand black phosphorus exhibits a structure and size similar to that of unmodified black phosphorus, which indicates that the coordination with zinc p-toluenesulfonate will not affect the structure and size of black phosphorus.
  • XPS X-ray photoelectron spectroscopy
  • the Zeta potential test was performed on zinc p-toluenesulfonate (ZnL 2 ), black phosphorus (BPs) and zinc ligand black phosphorus ( ZnL 2 -BPs) in Example 3 using a Zeta potential meter, and the results shown in FIG. 5 were obtained. The results showed that the zeta potential of ZnL 2 was +31.2mV. After the surface of BPs coordinated with ZnL 2 , the zeta potential changed from -25.8mV to +28.2mV.
  • PLGA polylactic acid-glycolic acid copolymer
  • ZnL 2 -BPs zinc ligand black phosphorus
  • Example 3 100 ⁇ L of absolute ethanol
  • the ZnL 2 -BPs dispersion was added to the PLGA solution, and ultrasonically dispersed for 30 minutes.
  • the hydroxyapatite scaffold (HA) in step 1 is immersed in the mixture of ZnL 2 -BPs and PLGA, taken out after 1 minute, placed in a refrigerator at 4°C for 3 days, and then vacuum dried at room temperature for 12 hours.
  • a stent with ZnL 2 -BPs on the surface is obtained, and the processed sample is called ZnL 2 -BPs@HA-PLGA.
  • the HA selected above has a diameter of 7mm and a thickness of 2.5mm.
  • the sample prepared by immersing HA in a PLGA solution is called HA-PLGA; the sample prepared by immersing HA in a mixture of black phosphorus (BPs) and PLGA is called BPs@HA-PLGA.
  • the surface morphology of HA and ZnL 2 -BPs@HA-PLGA was observed with a scanning electron microscope, and the morphology picture shown in Figure 7 was obtained. It can be seen from Figure 7a that HA is sintered from nano-sized spherical hydroxyapatite with a large number of pores on the surface. In Figure 7b, the surface of ZnL 2 -BPs@HA-PLGA is relatively smooth and flat. During preparation, the mixture of ZnL 2 -BPs and PLGA fills the pores on the surface of the HA substrate. After the solvent evaporates, the surface of HA is loaded with ZnL 2- PLGA film of BPs.
  • the HA, HA-PLGA, BPs@HA-PLGA and ZnL 2 -BPs@HA-PLGA in Example 7 were soaked in 300 ⁇ L phosphate buffer respectively, and the sample was irradiated with a near-infrared laser with a laser power of 1.0W/cm -2 , the irradiation distance is 10cm, and the temperature change of the sample during near-infrared irradiation is recorded with a thermal imager. The result is shown in Figure 8.
  • the temperature change of HA and HA-PLGA is not obvious under near-infrared irradiation, indicating that HA and PLGA do not have photothermal effect; while under near-infrared irradiation, both BPs@HA-PLGA and ZnL 2 -BPs@HA-PLGA rapidly heat up to 50 °C, indicating that black phosphorus has a good photothermal effect, and the addition of zinc p-toluenesulfonate will not affect the photothermal performance of black phosphorus.
  • Example 7 First put the HA, HA-PLGA, BPs@HA-PLGA and ZnL 2 -BPs@HA-PLGA in Example 7 into glass bottles containing 4 mL of phosphate buffer, where the liquid in the glass bottle can submerge the sample Then place the glass bottle containing the sample in a shaker at 37°C for 10 weeks and incubate the shaker at a speed of 100 rpm/min. Use a camera to record the macroscopic changes of the sample during the incubation.
  • 10-week-old SD rats were selected as the in vivo experimental objects, and the experiment was divided into HA+Light, ZnL 2 -BPs@HA-PLGA and ZnL 2 -BPs@HA-PLGA+Light 3 groups.
  • the three groups of samples were respectively immersed in normal saline with a concentration of Staphylococcus aureus of 5 ⁇ 10 4 CFU/mL for 1 minute, and then the samples were taken out and implanted in the tibial defect of the rat.
  • the tibia in the HA+Light and ZnL 2 -BPs@HA-PLGA+Light groups were irradiated with 808nm near-infrared laser at 1, 2, and 3 days after implantation .
  • the laser power was 1.5W/cm -2 and the irradiation time was For 10 minutes, the distance between the laser light source and the rat tibia was 15cm, and the irradiation temperature of the ZnL 2 -BPs@HA-PLGA+Light group was 50 ⁇ 0.5°C.
  • Example 11 The samples of each group in Example 11 were taken out from the implantation site, and the antibacterial rate of each group was calculated by the plate counting method. As shown in Figure 12, ZnL 2 -BPs@HA-PLGA+Light reached 98.5%, indicating that the antibacterial behavior of ZnL 2 -BPs@HA-PLGA in vivo can be adjusted by irradiating near infrared in vitro to achieve good The antibacterial effect.
  • the time was 6 minutes, the distance between the laser light source and the rat tibia was 15cm, and the highest temperature at the implantation site in the ZnL 2 -BPs@HA-PLGA(++) group was 40 ⁇ 0.5°C during irradiation.
  • the samples of ZnL 2 -BPs@HA-PLGA+Light that have not undergone weekly near-infrared irradiation are marked as ZnL 2 -BPs@HA-PLGA(+-).
  • the CTAn software was used to analyze the micro-CT three-dimensional images in Example 13, and the bone volume fraction (BV/TV) map as shown in FIG. 14 was obtained.
  • the BV/TV of the HA(++) group was only 31.49%; after the bacterial infection was eliminated, the BV/TV of ZnL 2 -BPs@HA-PLGA(+-) reached 45.26%; in the elimination of bacteria After infection, if the samples were continuously irradiated with near-infrared light, the BV/TV of the ZnL 2 -BPs@HA-PLGA(++) group increased to 57.72%.

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Abstract

一种具有逐步抗菌与促进骨再生功能的骨组织工程支架及其制备方法和应用。其组成包括：羟基磷灰石多孔支架、羟基磷灰石多孔支架表面负载的二价金属离子修饰的二维黑磷纳米材料、以及可降解高分子包裹层。首先通过3D打印技术制备出三维有序的羟基磷灰石支架，然后利用二价金属离子与黑磷的配位作用制备出带正电荷的、稳定性良好的金属配体黑磷，再将配体修饰后的黑磷与可降解高分子共混，羟基磷灰石支架浸入共混物后取出，共混物的溶剂挥发后，支架的表面即负载上具有近红外响应性的金属配体黑磷，得到可实现光热抗菌及热促成骨的复合骨植入体。

Description

一种具有逐步抗菌和促进骨再生功能的骨组织工程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技术领域，涉及一种具有逐步抗菌和促进骨再生功能的骨组织工程支架及其制备方法和应用。
背景技术
骨植入体是生物医用材料的一种，可应用于生物体骨缺损部位以达到修复或替换原有骨组织功能的目的，由组成分类可分为无机、高分子、金属以及复合骨植入体。因为骨植入体需长时间应用于人体内部，并与人体的血液、体液、组织等直接接触，所以骨植入体需要具有无毒性、化学性能稳定、力学性能与植入部位骨匹配和优异的生物相容性等性能。此外，骨科植入手术后植入部位有发生细菌感染的可能，而细菌感染会降低植入手术的成功率，感染严重时还需进行二次手术将被感染的组织清除。因此，若骨植入体能预防或治疗植入后可能发生的细菌感染，疗效则会有大幅提高。
骨植入体的抗菌性能主要由其表面的物化性质所决定，因此对其进行表面改性可以保证在材料本体性能不受影响的前提下提高其抗菌性能。目前已有高温水解 [1]、气相沉积 [2]、化学接枝 [3]、离子注入 [4]等方法可在骨植入体上建立抗菌表面，这些抗菌表面通常具有可杀伤细菌的理化性质。例如，采用水热法在金属镁表面原位生长氢氧化镁纳米片，该纳米片可捕获细菌并通过拉伸细菌的细胞膜使细菌处于应激状态，导致细菌内部活性氧水平急剧上升，从而有效杀伤细菌 [1]。
在骨科植入手术中，手术后患者所面临的第一个问题是术后感染问题，如果在手术后植入部位发生细菌感染，将会影响后期的新骨形成，严重时会引发骨髓炎 [5]，需要进行二次手术将感染组织清除。目前临床采用的方法是通过全身性抗生素的使用以降低感染风险，而全身性抗生素的使用会带来系统问题 [6]。因此有材料学家提出在植入体表面构建抗菌功能层以降低植入后的感染风险，提高骨科植入手术的成功率。绝大多数对植入材料表面抗菌都是基于药物负载或者抗菌离子释放来完成的 [7]。例如在植入体表面构建负载抗菌药物的纳米管结构，然后利用可降解的高分子薄膜对纳米管表面进行包覆，从而获得可缓慢释放抗菌药物的植入物表面 [8]；又例如在钛基材料的表面修饰含银纳米颗粒，从银纳米颗粒缓慢释放出的银离子可赋予钛基材料一定的抗菌性能 [9]。改性后的材料在植入人体后也能有一定的抗菌效果，但较为突出的问题是药物的暴释或离子的安全性问题；并且材料在植入人体后只能被动地发挥抗菌效果，无法根据伤口实际情况主动调控抗菌或促进组织再生功能。
随着对植入材料性能的要求越来越高，主动调控已成为未来技术发展的趋势。将外界调控因子与植入材料结合使用，可根据损伤组织的恢复情况，更精准的实现对植入材料功能的主动调控。近红外光调控因其良好的生物组织穿透性 [10]，临床使用广泛等特点，成为较受关注的外界调控因子。多种通过近红外调控植入材料抗菌性能的研究和专利已相继发表，如宋焱焱等利用上转换发光材料将近红外光转换成可见光与紫外光，上转换材料包覆下的TiO 2在吸收紫外光后可与溶液中的氧和水反应产生可破坏细菌细胞壁及DNA结构的活性氧分子，从而实现光催化抗菌的效果 [11]；吴水林等在钛金属表面构建硫化铋纳米棒涂层，再利用磷酸银纳米粒子修饰硫化铋，使硫化铋在近红外照射下产生更多的活性氧分子，以达到近红外调控抗菌的效果 [12]；此外吴水林等还通过近红外调控硫化钼表面聚乙二醇包裹的庆大霉素释放实现抗菌，硫化钼具有的光热性能使其在近红外照射下快速升温，融化包裹在庆大霉素外的聚乙二醇从而释放药物，达到近红外调控药物释放的抗菌效果 [13]。
在上述的近红外响应材料中，近红外光在其中作为外界激发条件，激发的活性氧或庆大霉素是最终抗菌作用因素；此外，无论是上转换发光材料(含有稀土离子)，还是硫化铋、硫化钼、甚至抗菌银离子，都会引入非人体所含元素，较低的生物安全性会限制他们在临床方面的应用 [14]。生物相容性较好的聚多巴胺也被尝试应用于近红外响应的抗菌生物材料中 [15,16]，但聚多巴胺的合成机理尚未明确、其合成过程中产生的副产物难以分离、聚多巴胺纳米粒子的分散性能不佳 [17]等因素均是其临床应用的阻碍。
为了解决植入物放入体内后需要通过全身性的抗生素用药预防或治疗因细菌感染引起的炎症问题，本发明提供了一种新型可降解骨植入材料，其组成包括：主体羟基磷灰石(HA)多孔支架、HA支架表面负载的二价金属离子修饰的二维黑磷纳米材料、以及可降解高分子包裹层。本发明利用二价金属离子修饰的二维黑磷纳米材料作为近红外光热响应的核心部件，通过可降解高分子负载至HA支架表面，仅需通过在体外的近红外激光发生器远程近红外照射，即时调节植入部位的温度，实现调控骨缺损植入部位的抗菌和促进骨再生的不同功能，避免全身性抗生素等药物的使用，缩短缺损部位的愈合期。
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发明内容
本发明的目的之一是提供一种具有逐步抗菌和促进骨再生功能的骨组织工程支架。
为了达到上述目的，本发明所采用的技术方案为：一种具有逐步抗菌和促进骨再生功能的骨组织工程支架，包括羟基磷灰石多孔支架、羟基磷灰石多孔支架表面负载的二价金属离子修饰的二维黑磷纳米材料、以及可降解高分子包裹层。
进一步地，所述二价金属粒子包括Zn离子、Ca离子；
所述可降解高分子包裹层包括聚乳酸-羟基乙酸共聚物PLGA、聚乳酸PLA、聚羟基乙酸、聚己内酯和聚丁二酸丁二醇酯。
进一步地，通过体外即时调控的近红外激光与具有逐步抗菌功能的骨组织工程支架协调作用，通过对近红外光密度的调节实现对具有逐步抗菌功能的骨组织工程支架温度的调节(通过体外调节近红外光强度调节其升温速度和最终保持温度)，在较高温度(45～60℃)和较低温度(39～42℃)时分别实现抗菌和促成骨的功能。
本发明的目的之二是提供一种上述所述具有逐步抗菌和促进骨再生功能的骨组织工程支架的制备方法。
为了达到上述目的，本发明所采用的技术方案为：一种上述所述具有逐步抗菌和促进骨再生功能的骨组织工程支架的制备方法，包括以下步骤：
1)制备羟基磷灰石支架；
2)二价金属离子与黑磷的配位作用制备金属配体黑磷；
3)将金属配体黑磷与可降解高分子溶液共混；
4)羟基磷灰石支架浸入共混物后取出干燥。骨植入体从溶液中取出后，需保证其表面的溶剂缓慢挥发以形成均匀无气孔的薄膜。二价金属离子修饰BP纳米材料在骨植入体表面的负载量，可根据高分子与BP纳米材料悬液中所含BP纳米材料的浓度及总浸入时间来调节。
进一步地，步骤1)中所述羟基磷灰石支架的制备方法为：将羟基磷灰石粉末加到海藻酸钠水溶液中，室温搅拌均匀后，浆料填入3D打印机的腔室，经加压后挤出得到羟基磷灰石支架胚体；
羟基磷灰石支架胚体在室温下干燥后，转移到马弗炉中烧结成型；
成型后的支架用去离子水清洗3遍后，即得到三维有序的羟基磷灰石支架。3D打印HA支架可根据患者缺损部位的具体形状进行设计。
进一步地，所述海藻酸钠水溶液的质量分数为2％～3％，所述羟基磷灰石粉末与海藻酸钠水溶液的质量比为0.8：1～1.2：1，所述搅拌时间为6～12小时，所述挤出气压为80～300kPa，所述挤出速率为1～1.5cm/s；
所述烧结温度为1100～1300℃，所述烧结时间为6～10小时。
进一步地，步骤2)中所述金属配体黑磷的制备方法为：将黑磷分散于N-甲基吡咯烷酮与水的混合溶剂中，然后加入二价金属离子配体，在氩气保护下，室温避光搅拌，去离子水清洗离心后，得到配体修饰的黑磷纳米片，标记为金属配体黑磷。
进一步地，所述混合溶剂中水与N-甲基吡咯烷酮的体积比为1：9～1：1，所述黑磷与二价金属离子配体的投料摩尔比为1：1～1：30，所述室温避光搅拌的时间为3～12小时，水洗离心次数为3～5次；
所述二价金属离子配体包括对甲苯磺酸锌、氯化锌、硝酸锌、硫酸锌、硝酸钙、氯化钙和葡萄糖酸钙等在内的盐类。
进一步地，步骤3)具体为：将可降解的高分子溶于二氯甲烷或三氯甲烷中得到可降解高分子溶液，将金属配体黑磷分散于无水乙醇中得到金属配体黑磷分散液，然后将金属配体黑磷分散液加入到可降解高分子溶液中，超声分散后得到金属配体黑磷与高分子的共混溶液；
步骤4)具体为：将步骤1)制备的羟基磷灰石支架浸入步骤3)的金属配体黑磷与可降解高分子的共混溶液中，支架取出后放置在冰箱中一段时间，然后室温真空干燥，最后得到表面修饰金属配体黑磷与可降解高分子共混物的羟基磷灰石骨组织工程支架。
进一步地，步骤3)所述可降解高分子溶液的浓度为20～100mg/mL，金属配体黑磷分散液的浓度为1～10mg/mL，可降解高分子溶液与金属配体黑磷分散液的体积比为的体积为1～3mL：0.05～0.3mL，超声功率为100～500W，超声时间为10～60分钟；所述可降解高分子包括聚乳酸-羟基乙酸共聚物、聚乳酸、聚羟基乙酸、聚己内酯和聚丁二酸丁二醇酯等可完全生物降解的高分子；
步骤4)中所述浸入时间为0.5～3分钟，支架取出后放置在4℃冰箱中1～3天，然后室温真空干燥6～18小时，真空干燥的气压为1×10 4～2×10 4Pa。
本发明的目的之三是提供一种上述所述具有逐步抗菌和促进骨再生功能的骨组织工程支架的应用。
为了达到上述目的，本发明所采用的技术方案为：上述所述具有逐步抗菌和促进骨再生功能的骨组织工程支架作为骨科植入医用材料的应用。
本发明采用修饰的黑磷二维纳米片作为引入光热的核心元素，黑磷的制备工艺成熟、分散性良好和可生物降解，在生物体内降解为无生物毒性的磷酸根离子，且磷元素为人体中大量含有的必须元素，因此生物安全性良好。此外，黑磷纳米片具有良好的近红外响应性，在近红外光的照射下，黑磷的温度会快速升高，利用其产生的高温环境可以直接杀菌。黑磷纳米片在较低功率近红外照射下产生的局部微热环境也可促进新骨的形成。
本发明采用二价金属离子(Zn 2+，Ca 2+等)对黑磷进行共价修饰，不仅可以提高黑磷在体内稳定性，随着黑磷的降解，这些二价金属离子可缓慢释放，与热协同作用，促进缺损部位的抗菌和骨再生。
本发明采用3D打印技术制备结构有序的羟基磷灰石支架，将该支架浸入含有二价金属离子配体黑磷的溶液中，即可在其表面构建具有光热响应性的功能化涂层，具有良好的抗菌及促成骨效果。与现有技术相比，本发明具有以下优点：
1)通过3D打印技术制备的骨植入体具有与人体骨相匹配的结构，该结构可诱导新骨的形成。
2)功能成分分散在具有流动性的高分子溶液中，即使骨植入体的形状不规则，也可通过浸入溶液中来实现均匀的负载。
3)骨植入体浸入溶液后取出，溶剂挥发后即可在骨植入体表面修饰功能成分，不影响骨植入体本身的性能。
4)黑磷与二价金属离子配位后，氧化速度减慢，在磷酸盐缓冲液中的稳定性提高，从而保证了金属配体黑磷在体内具有稳定、长效的光热性能。
5)本发明制备的近红外响应性骨植入体，可通过在体外照射近红外光的方法，控制骨植入体在体内的抗菌效果。
6)本发明制备的近红外响应性骨植入体在达到抗菌目的后，还可通过定期的近红外照射，来促进骨植入体附近新骨的形成。
7)本发明制备的近红外响应性骨植入体可在体内缓慢降解，在完成骨修复后无需二次手术取出。
8)本发明制备的近红外响应性骨植入体在体内具有逐渐降解的特性，最终降解产物为人体中常见的二氧化碳、水、锌离子、磷酸根离子和钙离子等，对人体无害，具有较高生物安全性。
附图说明
图1是实施例1中经3D打印和烧结处理得到的羟基磷灰石支架的宏观扫描电镜图。
图2是实施例2中羟基磷灰石支架的X射线衍射谱图。
图3a是实施例3中未经对甲苯磺酸锌配位的纯黑磷纳米片的扫描电镜图。
图3b是实施例3中经对甲苯磺酸锌配位的锌配体黑磷的扫描电镜图。
图4是实施例4中黑磷和锌配体黑磷的X射线光电子能谱全谱谱图。
图5是实施例5中对甲苯磺酸锌、黑磷和锌配体黑磷的Zeta电位图。
图6是实施例6中黑磷和锌配体黑磷分散在水中7天的光学照片。
图7a是实施例7中羟基磷灰石支架的表面微观形貌扫描电镜图。
图7b是实施例7中ZnL 2-BPs@HA-PLGA的表面微观形貌扫描电镜图。
图8是实施例8中样品在近红外照射下的升温曲线。
图9是实施例9中样品在磷酸盐溶液里孵育10周期间的光学照片对比。
图10是实施例10中ZnL 2-BPs@HA-PLGA在磷酸盐溶液里孵育10周期间的光热效率变化。
图11是实施例11中植入部位的光学照片对比。
图12是实施例12中材料在体内对金黄色葡萄球菌的抗菌率示意图。
图13是实施例13中植入体周围骨组织的三维重建图。
图14是实施例14中植入体周围的骨体积分数对比图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明首先通过3D打印技术制备出三维有序的羟基磷灰石支架，然后利用二价金属离子与黑磷的配位作用制备出带正电荷的、稳定性良好的金属配体黑磷(通过黑磷与二价金属离子的配位作用增强黑磷的稳定性)，再将配体修饰后的黑磷与可降解高分子共混，羟基磷灰石支架浸入共混物后取出，共混物的溶剂挥发后，支架的表面即负载上具有近红外响应性的金属配体黑磷，得到可实现光热抗菌及热促成骨的金属配体黑磷改性骨植入体。
实施例1
将10g羟基磷灰石粉末加入到10mL质量分数为3％的海藻酸钠溶液中，室温下搅拌6小时，然后将搅拌后的浆料填入3D打印机的腔室中，在120kPa的气压下，以1cm/s的打印速度挤出浆料。在胚体室温干燥后，将其转移到马弗炉中，在1200℃下烧结8小时，烧结后的羟基磷灰石支架用去离子水超声清洗3次。以该步骤得到的样品称为HA。
通过扫描电子显微镜对HA进行观察，得到图1所示的形貌照片。由图1可知，经烧结后，HA呈规则的圆柱状羟基磷灰石组成的层状支架，其中单根羟基磷灰石圆柱的直径约为550μm，每根圆柱之间的间距约为180μm，层间夹角为60°，可见HA具有规整有序的立体结构，有利于诱导骨再生。
实施例2
采用X射线衍射仪对实施例1中处理得到的HA样品进行表征，得到图2所示的X射线 谱图。由图2中的谱图可知，烧结清洗后HA的晶体结构与纯净的羟基磷灰石粉末的标准谱图一致，这说明了HA中的有机相在烧结过程中已完全挥发，HA支架由单纯的羟基磷灰石组成。
实施例3
将3mg黑磷分散到6mL的N-甲基吡咯烷酮与水的混合溶液中，其中N-甲基吡咯烷酮与水的体积比为9：1，然后加入60mg对甲苯磺酸锌。在氩气保护下避光搅拌12小时后，以12000rpm的转速离心15分钟，倒掉上清液后，用去离子水超声清洗沉淀，12000rpm的转速离心15分钟后收集经对甲苯磺酸锌修饰的黑磷沉淀，标记为ZnL 2-BPs(锌配体黑磷)。
通过扫描电子显微镜对黑磷与锌配体黑磷进行观察，得到图3所示的形貌照片。图3a表明黑磷呈现出纳米片状结构，尺寸在100-500nm之间。在图3b中，锌配体黑磷呈现出与未改性黑磷相似的结构与尺寸，这说明与对甲苯磺酸锌的配位不会影响黑磷的结构与尺寸。
实施例4
对实施案例3中的黑磷(BPs)与锌配体黑磷(ZnL 2-BPs)进行X射线光电子能谱(XPS)宽场扫描，得到图4所示的XPS全谱谱图。其中横坐标表示结合能，纵坐标表示峰强。通过观察图4中各试样的信号峰可知，ZnL 2-BPs除了呈现出与BPs一样的O 1s峰、C 1s峰和P2p峰外，还存在与对甲苯磺酸锌相关的Zn 2p峰、Zn的俄歇电子信号峰和S 2p峰，说明ZnL 2-BPs由对甲苯磺酸锌与黑磷配位形成。
实施例5
采用Zeta电位仪对实施案例3中的对甲苯磺酸锌(ZnL 2)、黑磷(BPs)与锌配体黑磷(ZnL 2-BPs)进行Zeta电位测试，得到图5所示结果。结果显示，ZnL 2的zeta电位为+31.2mV，BPs表面与ZnL 2配位后，zeta电位从-25.8mV变至+28.2mV。
实施例6
将实施案例3中的黑磷(BPs)与锌配体黑磷(ZnL 2-BPs)分别分散在去离子水中，用相机记录0、1、3、5、7天后分散液的颜色变化，结果如图6所示。由于黑磷易被氧化成可溶于水的磷酸根离子，因此BPs分散液的颜色逐渐变透明。然而，ZnL 2-BPs分散液在放置7天后依然呈不透明的棕色，这说明ZnL 2-BPs的稳定性良好，可以降低被氧化的速率。
实施例7
将100mg聚乳酸-羟基乙酸共聚物(PLGA)溶解在2mL三氯甲烷中，同时将1.0mg实施案例3中的锌配体黑磷(ZnL 2-BPs)分散在100μL无水乙醇中，然后把ZnL 2-BPs分散液加入到PLGA溶液中，超声分散30分钟。把实施步骤1中的羟基磷灰石支架(HA)浸入到 ZnL 2-BPs与PLGA的混合液中，1分钟后取出，放置在4℃冰箱中3天，然后室温下真空干燥12小时后，得到表面负载有ZnL 2-BPs的支架，该处理后的样品称为ZnL 2-BPs@HA-PLGA。以上所选用的HA直径为7mm，厚度为2.5mm。相似地，把HA浸入到PLGA溶液中制得的样品称为HA-PLGA；把HA浸入到黑磷(BPs)与PLGA的混合液中制得的样品称为BPs@HA-PLGA。
采用扫描电子显微镜观察HA和ZnL 2-BPs@HA-PLGA的表面形貌，得到图7所示的形貌图片。从图7a可知，HA由纳米级的球形羟基磷灰石烧结而成，表面存在大量孔隙。图7b中ZnL 2-BPs@HA-PLGA的表面相对光滑平整，在制备时，ZnL 2-BPs与PLGA的混合物填充了HA基底表面的孔隙，溶剂挥发后在HA表面形成了负载有ZnL 2-BPs的PLGA薄膜。
实施例8
将实施例7中的HA、HA-PLGA、BPs@HA-PLGA和ZnL 2-BPs@HA-PLGA分别浸泡在300μL磷酸盐缓冲液中，用近红外激光器照射样品，激光功率为1.0W/cm -2，照射距离为10cm，使用热成像仪记录近红外照射时样品的温度变化，结果如图8所示。HA和HA-PLGA在近红外照射下温度变化不明显，说明HA与PLGA均不具有光热效应；而在近红外照射下BPs@HA-PLGA和ZnL 2-BPs@HA-PLGA均快速升温至50℃，说明黑磷具有良好的光热效果，并且对甲苯磺酸锌的加入不会影响黑磷的光热性能。
实施例9
先将实施例7中的HA、HA-PLGA、BPs@HA-PLGA和ZnL 2-BPs@HA-PLGA分别放入装有4mL磷酸盐缓冲液的玻璃瓶中，其中玻璃瓶内液体可淹没样品，然后将装有样品的玻璃瓶放置在37℃的摇床中孵育10周，摇床的转速为100rpm/min，使用相机记录孵育过程中样品的宏观形貌变化。如图9所示，HA和HA-PLGA在孵育过程中宏观形貌未发生明显变化；而BPs@HA-PLGA在孵育的4周内颜色逐渐由深棕色变为白色，这是BPs@HA-PLGA中的黑磷降解造成的。相反，ZnL 2-BPs@HA-PLGA在孵育时的褪色速度明显较慢，到第6周时依然呈浅棕色，这说明ZnL 2-BPs具有更好的稳定性。
实施例10
将实施例9中的ZnL 2-BPs@HA-PLGA从玻璃瓶中取出，然后对其进行近红外激光照射，激光功率为1.0W/cm -2，照射距离为10cm，同时用热成像仪监测样品的温度变化，得到图10所示的温度-时间变化图。从图10可知，ZnL 2-BPs@HA-PLGA具有稳定的光热性能，在孵育的前6周内，1.0W/cm -2近红外照射下样品的升温幅度可达到15℃以上。
实施例11
选用10周大的SD大鼠作为体内实验对象，实验分为HA+Light、ZnL 2-BPs@HA-PLGA和ZnL 2-BPs@HA-PLGA+Light 3组。先将3组样品分别浸泡在金黄色葡萄球菌浓度为5×10 4CFU/mL的生理盐水中1分钟，然后将样品取出植入到大鼠胫骨缺损部位。在植入后的1、2、3天对HA+Light和ZnL 2-BPs@HA-PLGA+Light组的胫骨进行808nm的近红外激光照射，激光功率为1.5W/cm -2，照射时间为10分钟，激光光源与大鼠胫骨的距离为15cm，ZnL 2-BPs@HA-PLGA+Light组的辐照温度为50±0.5℃。
植入后的第14天对部分大鼠进行安乐死，用相机记录各组植入部位的细菌感染情况，得到如图11所示的照片。由图11可知，HA+Light组的植入部位附近存在大量的脓液，这说明HA+Light组在植入后发生了细菌感染；虽然ZnL 2-BPs@HA-PLGA组的感染情况没有HA+Light组严重，但其植入部位附近依然有脓液出现，说明ZnL 2-BPs@HA-PLGA中虽然含有具抗菌效果的锌元素，但并不足以完全杀伤附着在其中的金黄色葡萄球菌；而在ZnL 2-BPs@HA-PLGA+Light组中没有发现脓液，说明近红外光照射ZnL 2-BPs@HA-PLGA时产生的局部高温可协同锌元素有效地杀伤细菌，达到良好的抗菌效果。
实施例12
将实施案例11中各组的样品从植入部位取出，通过平板计数法计算出各组的抗菌率。如图12所示，ZnL 2-BPs@HA-PLGA+Light达到了98.5％，说明可以通过在体外照射近红外的方法，调控ZnL 2-BPs@HA-PLGA在体内的抗菌行为，以达到良好的抗菌效果。
实施例13
在实施案例11中的HA+Light和ZnL 2-BPs@HA-PLGA+Light植入到大鼠胫骨后的前6周，每周都对HA+Light和部分ZnL 2-BPs@HA-PLGA+Light的大鼠的胫骨进行808nm的近红外激光照射，对应的样品分别标记为HA(++)和ZnL 2-BPs@HA-PLGA(++)，激光功率为1.0W/cm -2，照射时间为6分钟，激光光源与大鼠胫骨的距离为15cm，辐照时ZnL 2-BPs@HA-PLGA(++)组植入部位处的最高温度为40±0.5℃。没有经过每周近红外照射的ZnL 2-BPs@HA-PLGA+Light样品标记为ZnL 2-BPs@HA-PLGA(+-)。
在植入后的第10周，将各组大鼠进行安乐死，胫骨取出进行micro-CT扫描，扫描结果经数据重建后得到如图13所示的三维图片。从图13可知，HA(++)组植入物附近的骨组织最少，这是因为前期的细菌感染影响了后期的新骨形成；在前期已经消除细菌感染的两组中，ZnL 2-BPs@HA-PLGA(++)附近的骨组织比ZnL 2-BPs@HA-PLGA(+-)要多，这说明在前期利用近红外达到抗菌效果后，后期还可利用照射近红外的方法提升ZnL 2-BPs@HA-PLGA在体内的成骨效果。
实施例14
使用CTAn软件对实施例13中的micro-CT三维图片进行分析，得到如图14所示的骨体积分数(BV/TV)图。其中，因为细菌感染，HA(++)组的BV/TV只有31.49％；在消除细菌感染后，ZnL 2-BPs@HA-PLGA(+-)的BV/TV达到了45.26％；在消除细菌感染后，若继续对样品照射近红外光，ZnL 2-BPs@HA-PLGA(++)组的BV/TV则提高至57.72％。
最后应说明的是，以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种具有逐步抗菌和促进骨再生功能的骨组织工程支架，其特征在于，包括羟基磷灰石多孔支架、羟基磷灰石多孔支架表面负载的二价金属离子修饰的二维黑磷纳米材料、以及可降解高分子包裹层。
  2. 根据权利要求1所述的具有逐步抗菌和促进骨再生功能的骨组织工程支架，其特征在于，所述二价金属粒子包括Zn离子、Ca离子；
    所述可降解高分子包裹层包括聚乳酸-羟基乙酸共聚物PLGA、聚乳酸PLA、聚羟基乙酸、聚己内酯和聚丁二酸丁二醇酯。
  3. 一种权利要求1或2所述具有逐步抗菌和促进骨再生功能的骨组织工程支架的制备方法，其特征在于，包括以下步骤：
    1)制备羟基磷灰石支架；
    2)二价金属离子与黑磷的配位作用制备金属配体黑磷；
    3)将金属配体黑磷与可降解高分子溶液共混；
    4)羟基磷灰石支架浸入共混物后取出干燥。
  4. 根据权利要求3所述的制备方法，其特征在于，步骤1)中所述羟基磷灰石支架的制备方法为：将羟基磷灰石粉末加到海藻酸钠水溶液中，室温搅拌均匀后，浆料填入3D打印机的腔室，经加压后挤出得到羟基磷灰石支架胚体；
    羟基磷灰石支架胚体在室温下干燥后，转移到马弗炉中烧结成型；
    成型后的支架用去离子水清洗3遍后，即得到三维有序的羟基磷灰石支架。
  5. 根据权利要求4所述的制备方法，其特征在于，所述海藻酸钠水溶液的质量分数为2％～3％，所述羟基磷灰石粉末与海藻酸钠水溶液的质量比为0.8：1～1.2：1，所述搅拌时间为6～12小时，所述挤出气压为80～300kPa，所述挤出速率为1～1.5cm/s；
    所述烧结温度为1100～1300℃，所述烧结时间为6～10小时。
  6. 根据权利要求3所述的制备方法，其特征在于，步骤2)中所述金属配体黑磷的制备方法为：将黑磷分散于N-甲基吡咯烷酮与水的混合溶剂中，然后加入二价金属离子配体，在氩气保护下，室温避光搅拌，去离子水清洗离心后，得到配体修饰的黑磷纳米片，标记为金属配体黑磷。
  7. 根据权利要求6所述的制备方法，其特征在于，所述混合溶剂中水与N-甲基吡咯烷酮的体积比为1：9～1：1，所述黑磷与二价金属离子配体的投料摩尔比为1：1～1：30，所述室温避光搅拌的时间为3～12小时，水洗离心次数为3～5次；
    所述二价金属离子配体包括对甲苯磺酸锌、氯化锌、硝酸锌、硫酸锌、硝酸钙、氯化钙 和葡萄糖酸钙。
  8. 根据权利要求3所述的制备方法，其特征在于，步骤3)具体为：将可降解的高分子溶于二氯甲烷或三氯甲烷中得到可降解高分子溶液，将金属配体黑磷分散于无水乙醇中得到金属配体黑磷分散液，然后将金属配体黑磷分散液加入到可降解高分子溶液中，超声分散后得到金属配体黑磷与高分子的共混溶液；
    步骤4)具体为：将步骤1)制备的羟基磷灰石支架浸入步骤3)的金属配体黑磷与可降解高分子的共混溶液中，支架取出后放置在冰箱中一段时间，然后室温真空干燥，最后得到表面修饰金属配体黑磷与可降解高分子共混物的羟基磷灰石骨组织工程支架。
  9. 根据权利要求8所述的制备方法，其特征在于，步骤3)中所述可降解高分子溶液的浓度为20～100mg/mL，金属配体黑磷分散液的浓度为1～10mg/mL，可降解高分子溶液与金属配体黑磷分散液的体积比为的体积为1～3mL：0.05～0.3mL，超声功率为100～500W，超声时间为10～60分钟；所述可降解高分子包括聚乳酸-羟基乙酸共聚物、聚乳酸、聚羟基乙酸、聚己内酯和聚丁二酸丁二醇酯；
    步骤4)中所述浸入时间为0.5～3分钟，支架取出后放置在4℃冰箱中1～3天，然后室温真空干燥6～18小时，真空干燥的气压为1×10 4～2×10 4Pa。
  10. 权利要求1所述具有逐步抗菌和促进骨再生功能的骨组织工程支架作为骨科植入医用材料的应用。
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CN115679541A (zh) * 2022-11-14 2023-02-03 郑州大学第一附属医院 一种静电纺复合纳米纤维材料及其制备方法和应用
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CN117069502A (zh) * 2023-07-14 2023-11-17 东莞理工学院 一种生物陶瓷材料及其制备方法和应用

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CN114113240A (zh) * 2021-11-22 2022-03-01 中南大学 一种室温氨气传感材料及其制备方法
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CN114015120A (zh) * 2021-12-03 2022-02-08 江苏万纳普新材料科技有限公司 一种基于黑磷烯的塑料改性用抗菌剂及其制备方法
CN114452402A (zh) * 2022-01-26 2022-05-10 中山大学 一种口服黑磷纳米材料的制备及其在胃肠道疾病中的应用
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CN115068686A (zh) * 2022-05-18 2022-09-20 复旦大学附属中山医院 一种天然来源的黑磷-丝蛋白-纤维素生物支架
CN115006591A (zh) * 2022-06-13 2022-09-06 江西理工大学 一种具有抗菌和骨缺损修复的双功能骨支架的制备方法
CN115154670A (zh) * 2022-07-26 2022-10-11 江西理工大学 一种石墨烯相氮化碳-硫化铋/高分子复合气管支架
CN115154670B (zh) * 2022-07-26 2024-02-02 江西理工大学 一种石墨烯相氮化碳-硫化铋/高分子复合气管支架
CN115177784A (zh) * 2022-07-31 2022-10-14 西南大学 具近红外光触发抗菌抗炎功能的钛骨钉
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CN115300482B (zh) * 2022-08-17 2023-11-28 山东大学 一种牛血清蛋白包覆的氯己定负载磷化镍纳米胶囊及其制备方法与抗菌应用
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