WO2022088634A1 - 一种注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂及其制备方法和应用 - Google Patents
一种注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂及其制备方法和应用 Download PDFInfo
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- WO2022088634A1 WO2022088634A1 PCT/CN2021/090778 CN2021090778W WO2022088634A1 WO 2022088634 A1 WO2022088634 A1 WO 2022088634A1 CN 2021090778 W CN2021090778 W CN 2021090778W WO 2022088634 A1 WO2022088634 A1 WO 2022088634A1
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- Prior art keywords
- magnesium
- calcium phosphate
- injection
- carbon nanotube
- plga
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- 229910052749 magnesium Inorganic materials 0.000 title claims abstract description 71
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 title claims abstract description 66
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical class [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 title claims abstract description 39
- 239000002131 composite material Substances 0.000 title claims abstract description 36
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 36
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 3
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- JUNWLZAGQLJVLR-UHFFFAOYSA-J calcium diphosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])(=O)OP([O-])([O-])=O JUNWLZAGQLJVLR-UHFFFAOYSA-J 0.000 claims description 3
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 claims description 3
- 229940043256 calcium pyrophosphate Drugs 0.000 claims description 3
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 229910052711 selenium Inorganic materials 0.000 claims description 3
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- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 claims description 3
- YIGWVOWKHUSYER-UHFFFAOYSA-F tetracalcium;hydrogen phosphate;diphosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].OP([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O YIGWVOWKHUSYER-UHFFFAOYSA-F 0.000 claims description 3
- GBNXLQPMFAUCOI-UHFFFAOYSA-H tetracalcium;oxygen(2-);diphosphate Chemical compound [O-2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GBNXLQPMFAUCOI-UHFFFAOYSA-H 0.000 claims description 3
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- 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 orthopaedic degradable biological materials, and relates to an injectable magnesium-based carbon nanotube composite microsphere activated calcium phosphate biological bone adhesive with excellent osteogenic activity and a preparation method and application thereof.
- OVCF osteoporotic vertebral compression fractures
- PGP percutaneous kyphoplasty
- PVP percutaneous vertebroplasty
- CPC calcium phosphate cement cement
- Degradable magnesium (Mg)-based metal material has good biosafety and suitable mechanical strength. It is easily corroded and rapidly degraded in a physical environment, and is eventually replaced by surrounding normal bone tissue. It is a very promising new type of orthopaedic implant. input material. Mg 2+ released from degradation is an important component of human bone tissue and an essential cation for maintaining human life activities. In vitro and in vivo studies have confirmed that Mg 2+ has good osteogenic inductive activity and can effectively improve the effect of bone defect repair (Zhang, Y., et al.. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med, 2016. 22(10):1160-1169).
- the electrode potential of Mg electrode is negative than the equilibrium electrode potential of hydrogen evolution reaction, resulting in Mg hydrogen evolution reaction. It occurs rapidly, releasing a large amount of H 2 in a short period of time, resulting in an overly alkaline local microenvironment that adversely affects cells. Therefore, how to reduce the concentrated release of H2 in a short time has become a difficulty in the in vivo application of Mg-based metals.
- Carbon nanotubes as a form of carbon nanomaterials, have been widely used in the biomedical field due to their unique structure and rich surface modifiability. At the same time, due to its high chemical reactivity, high tensile strength, biodegradability and other characteristics brought about by its nanoscale effect, it also has a large number of applications in the direction of bone tissue engineering scaffolds.
- Mikael combined polylactic-co-glycolic acid (PLGA) and multi-walled carbon nanotubes (MWCNT) to prepare porous bone scaffolds, which can effectively promote the proliferation and mineralization of osteoblasts (Mikael, PE, et al.. Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo evaluation.
- MWCNTs have a very large surface area and can absorb a large amount of hydrogen through physical adsorption.
- the MWCNT interlayer spacing (0.337nm) is larger than the dynamic diameter of H2 molecules (0.289nm), and a large number of H2 molecules can enter between its layers; and the MWCNT pipes have capillary siphon effect, which makes the H2 molecules condense in their hollow pipes,
- the MWCNTs also have a high hydrogen storage density at room temperature and pressure. Therefore, it is possible to take advantage of the physical adsorption of hydrogen by MWCNTs to control the adverse effects of Mg-based metals being easily corroded and rapidly releasing a large amount of hydrogen in vivo.
- a composite bone cement for preventing and treating bone infection and its preparation method uses Mg-based metal particles to blend with CPC bone cement for filling bone defects; The surface of the metal particles is modified.
- the mixed CPC powder reacts with the curing agent configured with the aqueous solution, a large amount of hydrogen will be generated, resulting in the formation of uniform air gaps around the Mg-based metal particles embedded in the CPC material, which cannot be formed with the CPC matrix. Tightly bonded interface, which seriously degrades the mechanical properties of composites.
- Another example is "a surface-degradable injectable bone filling material and its preparation method" mentioned in Chinese invention patent CN107412850A, using PMMA base material to add metal or alloy particles to repair bone defects; but the PMMA base material cannot be degraded in vivo, Clinical use is extremely limited.
- the purpose of the present invention is to provide an injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate biological bone adhesive and its preparation method and application, so as to solve the problems in the above-mentioned prior art.
- the mentioned shortcomings of calcium phosphate cement (CPC) are low mechanical strength, excessive degradation time, and insufficient osteoinductive activity.
- the injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate biological bone adhesive disclosed in the present invention is prepared from solid powder and liquid curing agent according to the dosage ratio of 10g:(5 ⁇ 8)mL;
- the solid powder in terms of mass percentage, includes 1% to 25% of the activation component and 75% to 99% of the substrate component;
- the activation component is magnesium-based polylactic acid-glycolic acid copolymer microspheres Mg-MWCNT-PLGA formed by combining magnesium-based metal particles and multi-walled carbon nanotubes;
- the base material group is calcium phosphate bone cement solid powder;
- the liquid solidifying agent is citric acid or phosphate buffer.
- the magnesium-based polylactic acid-glycolic acid copolymer microspheres are uniform in shape and size, and have a diameter of 50-200 ⁇ m;
- the mass percentage of magnesium-based metal particles in the magnesium-based polylactic acid-glycolic acid copolymer microspheres Mg-MWCNT-PLGA is 0.1%-10%, and the mass percentage of multi-wall carbon nanotubes is 0.1%-0.5%.
- the surface of the magnesium-based metal particles is modified by phosphorylation to be spherical with a diameter of 10-80 ⁇ m; the multi-walled carbon nanotubes are carboxylated, with a diameter of 10-200 nm and a length of 0.5-80 ⁇ m. 10 ⁇ m, the mass percentage of carboxyl groups is 0.5% ⁇ 5.0%.
- the magnesium-based metal particles are pure Mg or Mg alloy, and the Mg alloy contains one or more of zinc, manganese, copper, selenium, strontium and zirconium.
- the calcium-phosphorus ratio in the calcium phosphate bone cement solid powder is 1.5-1.7; and the calcium phosphate bone cement solid powder is composed of tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate, octacalcium phosphate, hydroxyapatite, One or more of fluoroapatite and calcium pyrophosphate.
- the pH of the citric acid or phosphate buffer is neutral.
- the invention also discloses a preparation method of the above-mentioned injection-type magnesium-based carbon nanotube composite microspheres activated calcium phosphate biological bone adhesive, comprising the following steps:
- step 3 Mix the magnesium-based polylactic acid-glycolic acid copolymer microspheres Mg-MWCNT-PLGA prepared in step 2) and the calcium phosphate bone cement solid powder uniformly at room temperature. After 5 min of hydration treatment, the injection-type magnesium-based carbon nanotube composite microspheres activated calcium phosphate biological bone adhesive was prepared.
- the preparation method of the magnesium-based metal particles is as follows: the magnesium-based metal particles are soaked in a potassium dihydrogen phosphate solution at room temperature and then taken out, rinsed with deionized water, and the surface of the magnesium-based metal particles is prepared with phosphorylation modification. Magnesium-based metal particles.
- the preparation method of magnesium-based polylactic acid-glycolic acid copolymer microspheres Mg-MWCNT-PLGA is as follows: dissolving PLGA in dichloromethane, and adding the magnesium-based metal obtained in step 1) according to the dosage ratio granules, add multi-walled carbon nanotubes at the same time, stir well to obtain a mixed solution; then dissolve type I collagen in deionized water, mix well, add to the above mixed solution, ultrasonically vibrate at 4 °C, and then add PVA solution, room temperature After fully stirring, centrifuging, removing the precipitate, washing and drying at low temperature to obtain magnesium-based polylactic acid-glycolic acid copolymer microspheres Mg-MWCNT-PLGA.
- the invention also discloses the application of the above-mentioned injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate biological bone adhesive as a bone implant material.
- the injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate biological bone adhesive can be used for biological applications or clinical applications.
- the present invention has the following beneficial effects:
- the injection-type magnesium-based carbon nanotube composite microspheres disclosed in the invention activate the calcium phosphate biological bone adhesive, and the magnesium-based polylactic acid-glycolic acid copolymer (Mg-MWCNT) is formed by combining magnesium-based metal particles and multi-walled carbon nanotubes.
- Mg-MWCNT magnesium-based polylactic acid-glycolic acid copolymer
- -PLGA magnesium-based polylactic acid-glycolic acid copolymer
- CPC solid powder as the substrate component
- citric acid or phosphate buffer as the curing agent.
- the Mg-MWCNT-PLGA microspheres are used as "aggregates" to improve the mechanical properties of CPC and provide early strong support for fracture repair.
- the microspheres can rapidly degrade in a short period of time, trigger blood and cell migration, accelerate the disintegration of the CPC substrate, form interconnected microporous structures, and promote the expansion of blood vessels and surrounding bone tissue; the released Mg 2+ has good osteogenic induction. Active, accelerates the process of bone tissue regeneration.
- MWCNT absorbed a large amount of H 2 released by Mg-based metal particles through physical adsorption, maintained the stability of the pH value of the local microenvironment in the early stage of fracture repair, and ensured the continuous proliferation of osteoblasts.
- the biological bone adhesive of the present invention has good osteoinductive activity and strong mechanical properties, and can solve the problems of low mechanical strength, long degradation time and osteoinductive calcium phosphate cement (CPC) mentioned in the prior art. Due to the deficiency of insufficient activity, it has broad clinical application value.
- CPC osteoinductive calcium phosphate cement
- Figure 1 is a schematic diagram of each component of the biological bone adhesive of the present invention.
- Fig. 2 is a light mirror picture of the Mg-MWCNT-PLGA composite microsphere in the present invention.
- FIG. 3 is a SEM picture of the Mg-MWCNT-PLGA composite microspheres in the present invention.
- FIG. 4 is the SEM picture of the sample after injection and solidification of the biological bone adhesive of the present invention.
- Fig. 5 is a SEM picture of the co-culture of the sample and osteoblasts after the injection and solidification of the biological bone adhesive of the present invention.
- Figure 6 is a picture of immunofluorescence staining of the co-culture of the sample and osteoblasts after the injection and solidification of the biological bone adhesive of the present invention.
- Fig. 7 is a picture of the hard tissue slices obtained after 12 weeks of implantation of the biological bone adhesive in the rat femoral condyle defect and VG staining.
- CPC substrate 1: CPC substrate; 2: Mg-MWCNT-PLGA composite microspheres; 3: Mg-based metal particles; 4: MWCNT.
- FIG. 1 it is a schematic diagram of the components of the injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate bio-bone adhesive disclosed in the present invention, which is composed of solid powder and liquid curing agent, and the solid component is a magnesium-based metal Magnesium-based polylactic acid-co-glycolic acid (Mg-MWCNT-PLGA) microspheres (marked 2 in Figure 1) formed by combining particles (marked 3 in Figure 1) with multi-walled carbon nanotubes (marked 4 in Figure 1) as Activation component (1-25 wt %), CPC solid powder mixed with various phosphates as substrate component (75-99 wt %) (marked 1 in Figure 1);
- the liquid curing agent is citric acid or phosphate buffered saline.
- the Mg-MWCNT-PLGA microspheres have a fiber diameter of 50-200 ⁇ m, uniform shape and size, and contain surface-modified Mg-based metal particles and carboxylated multi-walled CNTs.
- the above-mentioned Mg-based metal particles after surface phosphorylation modification, are spherical in shape and 10-80 ⁇ m in diameter; the Mg-based metal particles may be pure Mg or Mg alloys, and may contain zinc, manganese, copper, selenium, strontium , one or more of zirconium and other metals.
- the above-mentioned carboxylated multi-walled CNTs after carboxylation treatment, have a diameter of 10-200 nm, a length of 0.5-10 ⁇ m, and a carboxyl group content of 0.50-5.0 wt%.
- the above-mentioned CPC solid phase powder is composed of several calcium phosphate salts mixed in proportion (the ratio of calcium to phosphorus is 1.5 ⁇ 1.7), which can be tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate, octacalcium phosphate, hydroxyapatite, A mixture of one or more of fluoroapatite, calcium pyrophosphate, and the like.
- the above-mentioned liquid curing agent mainly contains components such as citric acid or phosphate buffer, and the pH value is adjusted to neutrality.
- the preparation method of the injection-type magnesium-based carbon nanotube composite microsphere-activated calcium phosphate biological bone adhesive disclosed in the present invention comprises the following steps:
- Mg-based alloy particles were added to 20% potassium dihydrogen phosphate solution, soaked for 12 hours at room temperature, taken out, rinsed with deionized water for 10 s, and detected by X-ray diffraction (XRD), which showed that potassium magnesium phosphate (KMgPO4) was obtained.
- XRD X-ray diffraction
- the collagen type I was dissolved in 4 ml of deionized water and mixed well, added to the above solution, and ultrasonically oscillated for 20s (1000W) at 4°C; the obtained colostrum was added to 1.0% PVA solution (containing 1% Tween 600) , stirred at 200 rpm for 4 hours at room temperature; after centrifugation, the precipitate was removed, washed with deion
- step 3 Mix the Mg-MWCNT-PLGA microspheres and CPC solid powder obtained in step 2) uniformly at room temperature (Mg-MWCNT-PLGA microspheres: 1-25 wt %), and add citric acid or phosphate buffer solution.
- the curing agent is fully stirred for 1min (the ratio of solid powder and curing agent is 10g:6ml); after mixing uniformly according to the above ratio and hydration for 5min, a multifunctional bone adhesive with good osteogenic activity can be obtained. Injection into the fracture site.
- the Mg-MWCNT-PLGA microspheres (Mg: 0.1-10 wt %, CNT: 0.1-0.5 wt %) prepared according to the above method ( Figure 2, Figure 3) and the CPC solid powder were mixed uniformly at room temperature (microspheres : 1-25 wt %), add a solidifying agent containing citric acid or phosphate buffer, fully stir at room temperature for 1min (solid powder and solidifying agent ratio 10g: 6ml); mix uniformly according to the above ratio and hydrate for 5min , injected into a polyethylene mold through a 20mL syringe and allowed to stand for 10-20min and then solidified to prepare a cylindrical bone adhesive sample with a diameter of 3mm and a height of 3mm (as shown in Figure 4), which was sterilized by 60 Co irradiation and packaged for later use ;
- Osteogenic induction activity Put the above sterilized sample into a 6-well culture plate, suck the rat osteoblast suspension slowly with a 20ul pipette and inoculate it on the surface of the sample (10 ⁇ 10 5 cells/block), add fresh The osteoinduction medium was then placed in an incubator to continue culturing.
- the cell proliferation was detected by the CCK-8 method; the adhesion and distribution of the cells on the surface of the sample were observed by SEM (as shown in Figure 5); at the same time, the osteoblasts were immunofluorescently stained, and the nuclei were marked with DAPI as blue fluorescence.
- the osteogenic marker protein-osteocalcin was marked with green fluorescence (as shown in Figure 6); the results showed that osteoblasts adhered well to the surface of the sample, secreted vigorously, and exhibited good proliferation activity; It shows that the bone adhesive can effectively support the adhesion and proliferation of osteoblasts, and has excellent osteogenic induction activity and good biocompatibility.
- Osteogenic repair ability A cancellous bone defect model with a diameter of 3 mm and a depth of 3 mm was prepared in the femoral condyle of the rat, and different repair materials were implanted respectively.
- the biological bone adhesive was used as the experimental group (group A)
- the pure CPC without the above-mentioned composite microspheres was implanted as the negative control group (group B)
- the blank control group was not implanted with any material to retain local bone defects (C Group); at 4, 12, and 24 weeks after operation, the samples were taken for Micro-CT scanning and VG staining of hard tissue sections (bone tissue was orange, new bone tissue and osteoid were blue-purple).
- the results showed that the bone defect of the experimental group was repaired well, and most of the implant materials had been degraded, and the new bone trabecular structure could be seen, which had no obvious boundary with the surrounding normal bone tissue; most of the implant materials in the bone defect of the negative control group None of them were degraded, and the boundary with the surrounding normal bone tissue was obvious; in the blank control group, the bone defect was locally filled with a large number of fibrous tissues, and there was no obvious sign of osteogenic repair.
- the components for preparing the above-mentioned bone adhesive are packaged after being sterilized by 60 Co irradiation, and the above-mentioned Mg-MWCNT-PLGA composite microspheres (Mg: 0.1-10 wt %, CNT: 0.1-0.5 wt %) and CPC solid powder were mixed uniformly at room temperature (microspheres: 1-25 wt %), and a solidifying agent containing citric acid or phosphate buffer was added, and the mixture was fully stirred at room temperature for 1 min (the ratio of solid powder to solidifying agent was 10 g : 6ml), mix uniformly according to the above ratio and after hydration for 5min, inject it into a special push rod through a 20mL syringe for use, and the operation time of the bone adhesive can be controlled within 10-20min.
- OVCF patients need to perform minimally invasive surgical treatment including percutaneous kyphoplasty (PKP)/percutaneous vertebroplasty (PVP) to restore the height of the fractured vertebral body, Relieve the symptoms of low back pain; the patient is placed in a prone position, the chest and iliac area are protected by sponge pads, the pedicles on both sides of the injured vertebra are positioned under the C-shaped arm, and local infiltration anesthesia is performed after routine skin disinfection. An injection channel was established for the pedicle, and the balloon was used to open it according to the compression of the patient's vertebral body.
- PRP percutaneous kyphoplasty
- PVP percutaneous vertebroplasty
- the bone adhesive was slowly injected through the unilateral/bilateral pedicle channel, and the bone adhesive in the injured vertebra was monitored and observed at intervals under fluoroscopy.
- the injection dose is determined according to the situation, and most of the single vertebral bodies are controlled at 1-10mL.
- the components for preparing the above-mentioned bone adhesive are packaged after being sterilized by 60 Co irradiation, and the above-mentioned Mg-MWCNT-PLGA composite microspheres (Mg: 0.1-10 wt %, CNT: 0.1-0.5 wt %) and CPC solid powder at room temperature (fiber: 10-20 wt %), add a curing agent containing citric acid or phosphate buffer, stir well at room temperature for 1min (solid powder to curing agent ratio 10g: 6ml), mix evenly according to the above ratio and after hydration for 5min, put it into a 20mL syringe and connect it with a special push rod for use.
- the operation time of the bone adhesive can be controlled within 10-20min.
- the present invention prepares Mg-CNT-PLGA composite microspheres through a self-assembly method, which is mixed with CPC powder and injected into the body.
- the composite microspheres can enhance the compressive and anti-collapse properties of the CPC substrate, play a good load-bearing and bonding role in the fracture site, provide a stable mechanical microenvironment for fracture repair, and promote new bone tissue. regeneration.
- the PLGA microspheres located on the surface of the CPC substrate first degrade, exposing the surface of Mg-based metal particles, which react with body fluids and release Mg 2+ and a large amount of H 2 . Due to the existence of a large number of MWCNTs in the microsphere matrix, the excess H 2 can be transferred into the wall gaps and hollow pipes of MWCNTs through molecular motion, and temporarily stored therein. The cytostatic effect caused by unfavorable factors such as excessive local concentration and alkalization of body fluids caused by the concentrated release of H2 can be avoided. As a result, the corrosion resistance of Mg-based metal particles is improved and the concentrated release of H2 is reduced, ensuring the biological stability of the binder in the initial stage of injection.
- Mg-CNT-PLGA composite microspheres can play a similar role as "aggregate”, effectively improve the mechanical strength of CPC adhesive, make it closer to the load-bearing requirements of human cancellous bone, and withstand fractures in the early stage of fracture repair The local main pressure and shear force prevent fretting of the fractured end and provide a stable mechanical environment necessary for fracture repair.
- Mg-based metal particles degrade and release Mg 2+ , which has multiple biological activities, which can effectively promote the transformation of local BMSCs to osteoblasts and the proliferation of osteoblasts, accelerate the process of fracture repair and bone reconstruction, and endow the bone adhesive with excellent performance. osteogenic inductive activity.
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Abstract
本发明公开了一种注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂及其制备方法和应用，属于骨科可降解生物材料领域，以镁基金属颗粒与多壁碳纳米管结合形成的镁基聚乳酸-羟基乙酸共聚物（Mg-MWCNT-PLGA）微球作为活化组分，以CPC固体粉末作为基材组分，以柠檬酸或磷酸盐缓冲液作为固化剂。本发明的生物骨粘合剂具备良好的骨诱导活性和较强的力学性能，能够解决现有技术中所提到的磷酸钙骨水泥（CPC）力学强度较低、降解时间过长、骨诱导活性不足的缺陷，因而具备广阔的临床应用价值
Description
本发明属于骨科可降解生物材料领域，涉及一种具有优良成骨活性的可注射镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂及其制备方法和应用。
随着人均寿命的延长，骨质疏松性椎体压缩骨折（OVCF）的发生率日渐增高，经皮椎体后凸成形术（PKP）/ 经皮椎体成形术（PVP）等微创手术成为了其主要治疗手段。磷酸钙骨水泥（phosphate
cement，CPC）也已被应用于OVCF的微创手术治疗，其在体内具有较好的生物相容性，但由于其力学强度较低、体内降解时间过长、不具备成骨活性，限制了其广泛应用。
可降解镁（Mg）基金属材料，具有良好的生物安全性和适宜的力学强度，在体能环境中极易发生腐蚀并快速降解、最终为周围正常骨组织替代，是非常有希望的新型骨科植入材料。降解释放的Mg
2+，是人体骨组织的重要组分，也是维持人体生命活动的必须阳离子。体内外研究已证实Mg
2+具有良好的成骨诱导活性，能够有效提升骨缺损修复效果（Zhang, Y., et al.. Implant-derived magnesium induces local neuronal
production of CGRP to improve bone-fracture healing in rats. Nat Med, 2016.
22(10):1160-1169）。通常Mg较Mg
2+间的标准电极电位为负（Mg
2+/Mg=
-2.372 V），在生物体内液体环境下，Mg电极的电极电位负于析氢反应的平衡电极电位，造成Mg析氢反应快速发生，在短时间内释放大量H
2，导致局部微环境过于碱化、对细胞产生不利影响。因而，如何减少H
2短时间集中释放成为Mg基金属体内应用的难点。
碳纳米管（CNT）作为一种碳纳米材料存在形式，因其独特的结构和丰富的表面可修饰性，在生物医学领域已广泛使用。同时由于其纳米尺度效应所带来的高化学反应性、高拉伸强度、生物降解性等特征，在骨组织工程支架方向也有大量应用。Mikael将聚乳酸-羟基乙酸共聚物（PLGA）和多壁纳米碳管（MWCNT）结合制备了多孔骨支架，能够有效促进成骨细胞的增殖和矿化（Mikael, P.E., et al.. Functionalized carbon nanotube reinforced
scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo
evaluation. Biomed Mater, 2014. 9(3): 035001）。有研究显示，MWCNT具有非常大的表面积，可以通过物理吸附作用吸收大量的氢气。MWCNT层间距（0.337nm）大于H
2分子的动力学直径（0.289nm），大量H
2分子可以进入其层面之间；而且MWCNT管道具有毛细虹吸作用，使H
2分子凝结在其中空管道中，从而使MWCNT在常温、常压下也具有了较高的储氢密度。因而有可能利用MWCNT物理吸附氢气这一特性，调控Mg基金属在体内极易腐蚀、快速释放大量氢气的不利作用。
国内外学者尝试通过多种方法优化CPC骨水泥各项理化性能，但都存在某些不足之处，有待进一步改进。例如中国发明专利CN104511051B中提到的“一种预防和治疗骨感染的复合骨水泥及其制备方法”，采用Mg基金属颗粒与CPC骨水泥共混，用于骨缺损充填；但未对Mg基金属颗粒进行表面改性，当其混合CPC粉末与含水溶液配置的固化剂反应时，会产生大量氢气，导致镶嵌于CPC材料中的Mg基金属颗粒周围形成均匀的气体间隙，无法与CPC基质形成紧密结合的连接界面，从而严重降低复合材料的力学性能。还例如中国发明专利CN107412850A中提到的“一种表面降解的可注射骨填充材料及其制备方法”，采用PMMA基材添加金属或合金颗粒，以修复骨缺损；但PMMA基材无法体内降解，临床使用受到极大限制。
因而，开发既具有优良力学强度、又具有多重生物活性，并能够调控各项理化性能的新型注射型可降解骨修复材料就显得尤为迫切了。
为了克服上述现有技术的缺点，本发明的目的在于提供一种注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂及其制备方法和应用，以解决上述现有技术中所提到的磷酸钙骨水泥（CPC）力学强度较低、降解时间过长、骨诱导活性不足的缺陷。
为了达到上述目的，本发明采用以下技术方案予以实现：
本发明公开的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，由固态粉末和液态固化剂按照10g：（5~8）mL的用量比制成；
其中，所固态粉末以质量百分比计，包括1%~25%的活化组分和75%~99%的基材组分；
所述活化组分为镁基金属颗粒与多壁碳纳米管结合形成的镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA；所述基材组为磷酸钙骨水泥固体粉末；
所述液态固化剂为柠檬酸或磷酸盐缓冲液。
优选地，所述镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA的形态均匀、大小均一，直径为50~200 µm；
镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA中镁基金属颗粒的质量百分比为0.1%~10%，多壁碳纳米管的的质量百分比为0.1%~0.5%。
优选地，所述的镁基金属颗粒的表面经过磷酸化改性，呈球形，直径为10~80 µm；所述多壁碳纳米管经过羧基化处理，直径为10~200 nm，长度0.5~10 µm，羧基质量百分比为0.5%~5.0%。
优选地，所述的镁基金属颗粒为纯Mg或Mg合金，Mg合金中含有锌、锰、铜、硒、锶和锆中的一种或多种。
优选地，所述磷酸钙骨水泥固体粉末中钙磷比为1.5~1.7；且该磷酸钙骨水泥固体粉末由磷酸三钙、磷酸四钙、磷酸氢钙、磷酸八钙、羟基磷灰石、氟磷灰石和焦磷酸钙中的一种或多种制成。
优选地，所述柠檬酸或磷酸盐缓冲液的pH值为中性。
本发明还公开了上述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的制备方法，包括以下步骤：
1）制备镁基金属颗粒；
2）制备镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA；
3）将步骤2）制得的镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA和磷酸钙骨水泥固体粉末在室温下混合均匀，按用量比加入液态固化剂充分搅拌1min，再经水合处理5min后，制得注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂。
优选地，步骤1）中，镁基金属颗粒的制备方法为：将镁基金属颗粒用磷酸二氢钾溶液在室温下浸泡反应后取出，用去离子水冲洗，制得表面经过磷酸化改性镁基金属颗粒。
优选地，步骤2）中，镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA的制备方法为：将PLGA溶解于二氯甲烷，按用量比加入步骤1）制得的镁基金属颗粒，同时加入多壁碳纳米管，充分搅拌均匀，得到混合溶液；然后将I型胶原溶解于去离子水中混合均匀后加入上述混合溶液中，4℃下超声震荡处理，再加入PVA溶液，室温下充分搅拌后离心后去沉淀物，清洗、低温干燥，获得镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA。
本发明还公开了上述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂作为骨植入材料的应用。
优选地，注射型镁基纳米碳管复合微球活化磷酸钙生物骨粘合剂可以为生物应用，也可以是临床应用。
与现有技术相比，本发明具有以下有益效果：
本发明公开的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，以镁基金属颗粒与多壁碳纳米管结合形成的镁基聚乳酸-羟基乙酸共聚物（Mg-MWCNT-PLGA）微球作为活化组分，以CPC固体粉末作为基材组分，以柠檬酸或磷酸盐缓冲液作为固化剂。本发明的生物骨粘合剂中Mg-MWCNT-PLGA微球作为“骨料”提高了CPC力学性能，为骨折修复提供了早期的有力支撑。该微球短期内能够快速降解、引发血液和细胞迁入，加速CPC基材崩解、形成相互连通的微孔结构，促进血管及周围骨组织张入；释放的Mg
2+具有良好成骨诱导活性，加速骨组织再生过程。同时MWCNT通过物理吸附作用，吸收了大量Mg基金属颗粒释放的H
2，维持了骨折修复早期局部微环境pH值稳定，保证了成骨细胞的持续增殖。本发明的生物骨粘合剂具备良好的骨诱导活性和较强的力学性能，能够解决现有技术中所提到的磷酸钙骨水泥（CPC）力学强度较低、降解时间过长、骨诱导活性不足的缺陷，因而具备广阔的临床应用价值。
图1 为本发明生物骨粘合剂各组分示意图。
图2 为本发明中Mg-MWCNT-PLGA复合微球光镜图片。
图3 为本发明中Mg-MWCNT-PLGA复合微球SEM图片。
图4 为本发明生物骨粘合剂注射凝固后试样SEM图片。
图5 为本发明生物骨粘合剂注射凝固后试样与成骨细胞共培养SEM图片。
图6 为本发明生物骨粘合剂注射凝固后试样与成骨细胞共培养免疫荧光染色图片。
图7为本发明生物骨粘合剂植入大鼠股骨髁部缺损12周后取材，将硬组织切片进行VG染色图片。
图中标记如下：
1：CPC基材；2：Mg-MWCNT-PLGA复合微球；3：镁基金属颗粒；4：MWCNT。
为了使本技术领域的人员更好地理解本发明方案，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的实施例，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做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述：
参见图1，为本发明公开的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的组分示意图，由固态粉末和液态固化剂组成，所述固态成分是以镁基金属颗粒（图1中标记3）与多壁碳纳米管（图1中标记4）结合形成的镁基聚乳酸-羟基乙酸共聚物（Mg-MWCNT-PLGA）微球（图1中标记2）作为活化组分（1-25wt %），由多种磷酸盐混合而成的CPC固体粉末作为基材组分（75-99 wt %）（图1中标记1）；所述液体固化剂为柠檬酸或磷酸盐缓冲液。
其中，Mg-MWCNT-PLGA微球的光镜照片如图2所示，SEM照片如图3所示。Mg-MWCNT-PLGA微球的纤维直径50-200 µm，形态均匀、大小均一，内含经过表面改性的Mg基金属颗粒及羧基化多壁CNT。
上述Mg基金属颗粒，经过表面磷酸化改性，形态为球形，直径10-80µm；所述Mg基金属颗粒可以是纯Mg，也可以是Mg合金，可以含有锌、锰、铜、硒、锶、锆等金属中的一种或多种。
上述羧基化多壁CNT，经羧基化处理，直径10-200nm，长度0.5-10µm，羧基含量0.50-5.0 wt%。
上述CPC固相粉末是由数种磷酸钙盐按比例混合而成（钙磷比为1.5~1.7），可以是磷酸三钙、磷酸四钙、磷酸氢钙、磷酸八钙、羟基磷灰石、氟磷灰石、焦磷酸钙等中的一种或多种的混合物。
上述液态固化剂主要包含柠檬酸或磷酸盐缓冲液等组分，调节pH值至中性。
本发明公开的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的制备方法，包括以下步骤：
1）制备镁基金属颗粒
将Mg基合金颗粒2g，加入20%的磷酸二氢钾溶液，室温下浸泡反应12h后取出，去离子水冲洗10s，经X线衍射（XRD）物相检测，显示得到磷酸镁钾（KMgPO4）包覆的Mg基金属颗粒。
2）制备镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA
将PLGA（LA/GA = 50:50, Mw = 3-5万）溶解于二氯甲烷中制备成制备成3-12% (w/v)的溶液，按照0.1-10 wt %的比例加入Mg基金属颗粒，同时按照0.1-0.5 wt %的比例加入羧基化多壁CNT，加入100 mL烧杯中，置于磁力搅拌器中央，内置磁力搅拌子以1000-3000rpm持续搅拌0.1-1h；将0.6g的I型胶原溶解于4ml去离子水中混合均匀，将其加入上述溶液中，4℃下超声震荡20s（1000W）；将获得的初乳加入1.0 %的PVA溶液（内含1%的Tween 600），室温下200rpm搅拌4小时；离心后去沉淀物，去离子水清洗3-5次，随后移入入低温冷冻干燥机中24h，获得冻干的Mg-MWCNT-PLGA微球；通过扫描电镜（SEM）观察其直径50-200 µm，微球形态均匀、大小均一。
3）将步骤2）获得的Mg-MWCNT-PLGA微球和CPC固体粉末在室温下混合均匀（Mg-MWCNT-PLGA微球: 1-25 wt %），加入含有柠檬酸或磷酸盐缓冲液的固化剂充分搅拌1min（固体粉末与固化剂比例10g：6ml）；按上述比例混合均匀并且经水合作用5min后，即获得具有良好成骨活性的多功能骨粘合剂，可通过专用推杆注射入骨折局部。
本发明的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的性能验证：
将按照上述方法制得的Mg-MWCNT-PLGA微球（Mg: 0.1-10 wt %，CNT: 0.1-0.5 wt %）（图2，图3）和CPC固体粉末在室温下混合均匀（微球: 1-25 wt %），加入含有柠檬酸或磷酸盐缓冲液的固化剂，在室温下充分搅拌1min（固体粉末与固化剂比例10g：6ml）；按上述比例混合均匀并且经水合作用5min，通过20mL注射器注入聚乙烯模具静置10-20min后凝固，制备出直径3mm、高度3mm的圆柱状骨粘合剂试样（如图4所示），经
60 Co辐照灭菌后封装备用；
成骨诱导活性：将上述灭菌试样置入6孔培养板，用20ul移液器吸取大鼠成骨细胞悬液缓慢接种至试样表面（10×10
5个/块），添加新鲜成骨诱导培养基后置于培养箱中继续培养。利用CCK-8方法检测细胞增殖；使用SEM观察细胞在试样表面的粘附及分布态势（如图5所示）；同时进行成骨细胞免疫荧光染色，细胞核以DAPI标记为蓝色荧光，细胞内成骨标志性蛋白-骨钙素（OCN）标记为绿色荧光（如图6所示）；结果显示：成骨细胞在试样表面粘附良好、分泌旺盛，并呈现出良好的增殖活性；表明该骨粘合剂能够有效支持成骨细胞的粘附与增殖，具有优秀的成骨诱导活性和良好的生物相容性。
成骨修复能力：在大鼠股骨髁部制备直径3mm、深度3mm的松质骨缺损模型，分别植入不同修复材料，实验分组如下：以植入添加本发明的Mg-MWCNT-PLGA微球的生物骨粘合剂作为实验组（A组），以植入未添加上述复合微球的纯CPC作为阴性对照组（B组），以不植入任何材料保留局部骨缺损作为空白对照组（C组）；分别在术后4、12、24周取材行Micro-CT扫描及硬组织切片VG染色（骨组织呈橙色，新生骨组织及类骨质呈蓝紫色）观察。术后12周结果显示，实验组骨粘合剂部分降解，缺损周围正常骨组织逐步张入材料，骨修复进程良好（图7），阴性对照组骨缺损局部植入材料无显著降解，周围骨组织无明显张入；空白对照组骨缺损局部无组织无明显骨组织填充，缺损明显。术后24周结果显示实验组骨缺损局部修复良好，大部分植入材料均已降解，可见新生骨小梁结构，与周围正常骨组织无明显界限；阴性对照组骨缺损局部大部分植入材料均未降解，与周围正常骨组织界限明显；空白对照组骨缺损局部充斥大量类纤维组织，无明显成骨修复迹象。
本发明制得的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的具体临床应用案例如下：
应用案例1
将制备上述骨粘合剂的各组分经
60 Co辐照灭菌后封装，在手术室环境中将上述Mg-MWCNT-PLGA复合微球（Mg: 0.1-10 wt %，CNT: 0.1-0.5 wt %）和CPC固体粉末在室温下混合均匀（微球: 1-25 wt %），加入含有柠檬酸或磷酸盐缓冲液的固化剂，在室温下充分搅拌1min（固体粉末与固化剂比例10g：6ml），按上述比例混合均匀并且经水合作用5min后，通过20mL注射器注入专用推杆备用，骨粘合剂可推注操作时间控制在10-20min。
高龄OVCF患者经详细术前检查及系统评估后，需要实施包括经皮椎体后凸成形术（PKP）/ 经皮椎体成形术（PVP）等微创手术治疗，以恢复骨折椎体高度、缓解腰背部疼痛症状；患者取俯卧位，胸部及髂部置海绵垫保护，C型臂下定位伤椎两侧椎弓根，常规皮肤消毒后局部浸润麻醉，切开皮肤后以开路尖锥通过椎弓根建立注射通道，根据患者椎体压缩情况决定是否使用球囊撑开，随后通过单侧/双侧椎弓根通道缓慢注入骨粘合剂，在透视下间隔监测观察伤椎内骨粘合剂弥散分布情况，视情况决定注入剂量，单个椎体多数控制在1-10mL。
应用案例1
将制备上述骨粘合剂的各组分经
60 Co辐照灭菌后封装，在手术室环境中将上述Mg-MWCNT-PLGA复合微球（Mg: 0.1-10 wt %，CNT: 0.1-0.5 wt %）和CPC固体粉末在室温下混合均匀（纤维: 10-20 wt %），加入含有柠檬酸或磷酸盐缓冲液的固化剂，在室温下充分搅拌1min（固体粉末与固化剂比例10g：6ml），按上述比例混合均匀并且经水合作用5min后，装入20mL注射器并连接专用推杆备用，骨粘合剂可推注操作时间控制在10-20min。
由于外伤、感染、肿瘤等原因导致的严重骨缺损患者，经详细术前检查及系统评估后，需要使用骨粘合剂填充空腔以修复大段骨缺损；严密清理骨缺损局部残留组织，采用髓内钉固定或重建钢板固定骨折断端，用连接有推杆的20mL注射器将骨粘合剂缓慢注入骨缺损局部，注入同时用手将注入的粘合剂再次塑性以配合骨缺损局部形态。
综上所述，本发明通过自组装方法，制备Mg-CNT-PLGA复合微球，与CPC粉末共混后注入机体。注入骨折局部早期，复合微球能够增强CPC基材的抗压和抗溃散性能，在骨折局部起到良好的承重和粘结作用，为骨折修复提供了稳定的力学微环境，促进了新生骨组织的再生。
注入骨折局部初期，位于CPC基材表层的PLGA微球首先降解，暴露出Mg基金属颗粒表面，其与体液反应后释放出Mg
2+和大量的H
2。由于微球基质内存在有大量MWCNT，过量的H
2能够过分子运动转移进入MWCNT的管壁间隙及中空管道中，暂时贮存其中。避免了H
2集中释放引起的局部浓度过大、体液碱化等不利因素引发的细胞抑制作用。从而提高了Mg基金属颗粒的耐腐蚀性并减少了H
2集中释放，保证了注入初期粘合剂的生物稳定性。
注入骨折局部后期，随着CPC表层的Mg-CNT-PLGA微球逐步降解，形成了相应的孔隙结构，随着成骨细胞的逐步迁入，周围血管结构也逐渐张入，进一步促进CPC基材的崩解并暴露出更多内部的PLGA微球，引发新一轮的降解。从而形成了相互连接的微观孔隙结构，加快了CPC基材的崩解与重塑，并最终在血管化进程中被周围正常骨组织所替代，实现自体骨组织的完全性修复。
本发明利用自组装技术制备的Mg-CNT-PLGA复合微球活化骨粘合剂具备如下优势特征：
（1）Mg-CNT-PLGA复合微球，能够起到类似“骨料”的作用，有效提高CPC粘合剂的力学强度，使其更加接近人体松质骨承重要求，在骨折修复初期承受骨折局部主要的压力和剪切力，防止骨折断端产生微动，提供骨折修复所必须的稳定力学环境。
（2）Mg-CNT-PLGA复合微球中PLGA体内降解速度较快，其内含的Mg基金属颗粒也先于CPC基质溶解，粘合剂注入骨折局部早期，随着粘合剂表层微球逐步降解，在基材中形成了与微球直径相适应的孔隙结构，随着血液渗入及成骨细胞迁入，粘合剂基材逐步降解，同时位于基材核心部位的微球也随之降解，逐渐形成类似于骨小梁的、相互贯通的微孔结构，促进了粘合剂材料的成血管化及周围正常骨组织张入，有效匹配人体骨骼再生过程。
（3）Mg基金属颗粒降解释放Mg
2+，具有多重生物活性，能够有效促进局部BMSCs向成骨细胞转化和成骨细胞增殖，加速骨折修复进程及骨骼改建过程，赋予了骨粘合剂优秀的成骨诱导活性。
（4）PLGA微球表层降解后，暴露出Mg基金属颗粒表面，其与体液反应后释放出Mg
2+和H
2，大量H
2通过分子运动转移进入微球基质中的MWCNT管壁间隙及中空管道中，暂时储存其中。MWCNT通过物理吸附作用，在Mg基金属颗粒降解早期吸收了大量H
2，后期随着MWCNT自身的逐步降解和吸收，其缓慢释放出所吸附的H
2，保证了H
2的有序、缓慢释放，从而避免了骨折修复早期Mg基金属颗粒腐蚀过程中大量H
2的集中释放，维持了局部微环境pH值稳定，保证了成骨细胞的持续增殖和有效迁移。
以上内容仅为说明本发明的技术思想，不能以此限定本发明的保护范围，凡是按照本发明提出的技术思想，在技术方案基础上所做的任何改动，均落入本发明权利要求书的保护范围之内。
Claims (10)
- 一种注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，由固态粉末和液态固化剂按照10g：（5~8）mL的用量比制成；其中，所固态粉末以质量百分比计，包括1%~25%的活化组分和75%~99%的基材组分；所述活化组分为镁基金属颗粒与多壁碳纳米管结合形成的镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA；所述基材组为磷酸钙骨水泥固体粉末；所述液态固化剂为柠檬酸或磷酸盐缓冲液。
- 根据权利要求1所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，所述镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA的形态均匀、大小均一，直径为50~200 µm；镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA中镁基金属颗粒的质量百分比为0.1%~10%，多壁碳纳米管的的质量百分比为0.1%~0.5%。
- 根据权利要求1或2所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，所述的镁基金属颗粒的表面经过磷酸化改性，呈球形，直径为10~80 µm；所述多壁碳纳米管经过羧基化处理，直径为10~200 nm，长度0.5~10 µm，羧基质量百分比为0.5%~5.0%。
- 根据权利要求1或2所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，所述的镁基金属颗粒为纯Mg或Mg合金，Mg合金中含有锌、锰、铜、硒、锶和锆中的一种或多种。
- 根据权利要求1所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，所述磷酸钙骨水泥固体粉末中钙磷比为1.5~1.7；且该磷酸钙骨水泥固体粉末由磷酸三钙、磷酸四钙、磷酸氢钙、磷酸八钙、羟基磷灰石、氟磷灰石和焦磷酸钙中的一种或多种制成。
- 根据权利要求1所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂，其特征在于，所述柠檬酸或磷酸盐缓冲液的pH值为中性。
- 权利要求1~6中任意一下所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂的制备方法，其特征在于，包括以下步骤：1）制备镁基金属颗粒；2）制备镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA；3）将步骤2）制得的镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA和磷酸钙骨水泥固体粉末在室温下混合均匀，按用量比加入液态固化剂充分搅拌1min，再经水合处理5min后，制得注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂。
- 根据权利要求7所述的制备方法，其特征在于，步骤1）中，镁基金属颗粒的制备方法为：将镁基金属颗粒用磷酸二氢钾溶液在室温下浸泡反应后取出，用去离子水冲洗，制得表面经过磷酸化改性镁基金属颗粒。
- 根据权利要求7所述的制备方法，其特征在于，步骤2）中，镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA的制备方法为：将PLGA溶解于二氯甲烷，按用量比加入步骤1）制得的镁基金属颗粒，同时加入多壁碳纳米管，充分搅拌均匀，得到混合溶液；然后将I型胶原溶解于去离子水中混合均匀后加入上述混合溶液中，4℃下超声震荡处理，再加入PVA溶液，室温下充分搅拌后离心后去沉淀物，清洗、低温干燥，获得镁基聚乳酸-羟基乙酸共聚物微球Mg-MWCNT-PLGA。
- 权利要求1~6中任意一项所述的注射型镁基碳纳米管复合微球活化磷酸钙生物骨粘合剂作为骨植入材料的应用。
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