CN114129776B

CN114129776B - Composite stent material and preparation method and application thereof

Info

Publication number: CN114129776B
Application number: CN202111473702.8A
Authority: CN
Inventors: 吴钊英; 赵蒙恩; 张超
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-11-29
Filing date: 2021-11-29
Publication date: 2022-10-14
Anticipated expiration: 2041-11-29
Also published as: CN114129776A

Abstract

The invention discloses a composite scaffold material and a preparation method and application thereof, wherein the composite scaffold material comprises polyether-ether-ketone and drug-loaded metal-doped bioglass microspheres, the drug-loaded metal-doped bioglass microspheres are dispersed in the polyether-ether-ketone, the drug loaded on the drug-loaded metal-doped bioglass microspheres is a bone metabolism regulator, and the metal comprises at least one of strontium, zinc, lithium and calcium. The composite scaffold material has a large number of through holes, can induce apatite mineralization by soaking in SBF solution, and can promote cell adhesion, proliferation and differentiation by co-culturing with rBMSCs; when the cells are cultured together with RAW264.7, the TRAP activity of the cells can be reduced, and the generation and the maturation of osteoclasts are influenced. The in vivo implantation experiment shows that the composite scaffold material can promote the regeneration of new bones, has excellent bone-related biological properties and has potential development prospect in the repair of osteoporotic bone defects.

Description

Composite stent material and preparation method and application thereof

Technical Field

The invention belongs to the field of materials, and particularly relates to a composite stent material as well as a preparation method and application thereof.

Background

PEEK (polyetheretherketone) has excellent biological properties, mechanical properties, elastic modulus similar to bone, wear resistance and biological stability, and is widely applied to the field of bone defect repair and replacement. However, PEEK is not biologically active and cannot integrate with bone, easily resulting in implant failure. The Bioglass (BG) HAs excellent bioactivity and special mesoporous structural characteristics, and can promote HA deposition, cell adhesion, proliferation, osteogenic differentiation in vitro and new bone regeneration in vivo.

Under the condition of osteoporosis, bone resorption is greater than bone formation, and the activity of osteoclast is greater than that of osteoblast, so that the repair process of bone defect is delayed. Therefore, increasing the functional activity of osteoblasts and inhibiting the functional activity of osteoclasts are key to the repair of bone defects in osteoporotic conditions. Modulators of bone metabolism may inhibit osteoclast activity and promote osteoblast activity, for example: alendronate sodium (ALN) is a potent regulator of bone metabolism, and can inhibit osteoclast activity, promote osteoclast apoptosis, reduce bone resorption, and slow bone mass loss. However, the traditional treatment methods (such as oral administration, intravenous injection and the like) have the defects of too low concentration of target drugs, large systemic toxic and side effects and the like.

Disclosure of Invention

In order to overcome the problems of the prior art, an object of the present invention is to provide a composite stent material.

The second purpose of the invention is to provide a preparation method of the composite scaffold material.

The invention also aims to provide the application of the composite scaffold material in the material for repairing bone defects.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a composite scaffold material, which comprises polyether-ether-ketone and drug-loaded metal-doped bioglass microspheres, wherein the drug-loaded metal-doped bioglass microspheres are dispersed in the polyether-ether-ketone, the drug loaded on the drug-loaded metal-doped bioglass microspheres is a bone metabolism regulator, and the metal comprises at least one of strontium, zinc, lithium and calcium.

Preferably, the metal comprises at least one of strontium and calcium; further preferably, the metals are strontium and calcium.

Preferably, the mass ratio of the drug-loaded metal-doped bioglass microspheres to the polyether-ether-ketone is 1: (1-6); further preferably, the mass ratio of the drug-loaded metal-doped biological glass microspheres to the polyether-ether-ketone is 1: (1.5-4); still further preferably, the mass ratio of the drug-loaded metal-doped biological glass microspheres to the polyether-ether-ketone is 1: (3.5-4); still more preferably, the mass ratio of the drug-loaded metal-doped bioglass microspheres to polyether-ether-ketone is 1:4.

preferably, the composite scaffold material meets at least one of the following conditions:

(1) The composite scaffold material contains interconnected pores;

(2) The composite scaffold material contains macropores with the aperture of 200-400 mu m and micropores with the aperture of 0.1-2 nm;

(3) The porosity of the composite scaffold material is 60-80%.

Preferably, the porosity of the composite scaffold material is 65-75%; further preferably, the porosity of the composite scaffold material is 68-72%; still further preferably, the porosity of the composite scaffold material is 70%.

Preferably, the composite scaffold material contains macropores with the pore diameter of 250-400 mu m; further preferably, the composite scaffold material contains macropores with the pore diameter of 300-400 microns.

Preferably, the composite scaffold material contains micropores with the pore diameter of 0.5-2 nm; further preferably, the composite scaffold material contains micropores with the pore diameter of 1-2 nm.

The drug-loaded metal-doped bioglass microspheres meet at least one of the following conditions:

(1) The particle size of the drug-loaded metal-doped bioglass microspheres is 100-500 nm;

(2) The drug-loading amount of the metal-doped bioglass microspheres is 3-6%;

(3) The content of metal in the drug-loaded metal-doped bioglass microspheres is 10-20%.

Preferably, the bone metabolism regulator is at least one of alendronate sodium, pamidronate sodium and zoledronic acid. Further preferably, the bone metabolism regulator is alendronate sodium. The bone metabolism regulator can be chelated with calcium to form a three-ligand, so that the loading of the bone metabolism regulator is realized, and the slow release effect is realized, such as: alendronate sodium has strong affinity to calcium ions and can be chelated with calcium to form a three-ligand. Therefore, the alendronate sodium-mesoporous strontium glass (A-SrBG) prepared by chelation can achieve the slow release effect of alendronate sodium in vivo, and well solves the problem of continuous administration in the slow process of new bone formation.

Preferably, the particle size of the drug-loaded metal-doped bioglass microspheres is 350-450 nm; further preferably, the particle size of the drug-loaded metal-doped bioglass microspheres is 380-420 nm.

Preferably, the drug loading amount of the drug-loaded metal-doped bioglass microspheres is 4-5%; further preferably, the drug loading amount in the drug-loaded metal-doped bioglass microspheres is 4.7%.

Preferably, the content of the metal in the drug-loaded metal-doped bioglass microspheres is 15-20%; further preferably, the content of metal in the drug-loaded metal-doped bioglass microspheres is 17%.

In a second aspect, the present invention provides a method for preparing the composite scaffold material provided in the first aspect, comprising the following steps:

preparing metal-doped bioglass microspheres by a sol-gel method;

mixing the metal-doped bioglass microspheres with a bone metabolism regulator to prepare drug-loaded metal-doped bioglass microspheres;

mixing the drug-loaded metal-doped bioglass microspheres, polyether-ether-ketone and pore-foaming agent, and then preparing the composite scaffold material by adopting a hot pressing-particle leaching method.

Preferably, the hot press-particle leaching method specifically comprises the steps of:

mixing polyether-ether-ketone and the drug-loaded metal-doped bioglass microspheres, and ball-milling by using a ball mill;

and mixing the ball-milled mixture with a pore-foaming agent, hot-press molding, and removing the pore-foaming agent.

Preferably, the step of removing the porogen is: and soaking the hot-press formed product in deionized water to remove the pore-foaming agent.

Preferably, the pressure of the hot-press molding is 16-24 Mpa; further preferably, the pressure of the hot press molding is 18-22 Mpa; still more preferably, the pressure of the hot press forming is 20Mpa.

Preferably, the hot-press molding temperature is 150-200 ℃; further preferably, the temperature of the hot press molding is 170-190 ℃; preferably, the temperature of the hot press forming is 180 ℃.

Preferably, the time for hot press molding is 24-40 min; further preferably, the time of the hot press molding is 25min to 35min.

Preferably, the preparation steps of the metal-doped bioglass microspheres are as follows:

s1: preparing a mixed solution A of alkali liquor, alcohol solution and water, and mixing the mixed solution A with a silicon source solution for reaction to prepare a mixed solution B;

s2: mixing the mixed solution B with a pore-making agent for reaction, and then mixing with a metal salt for reaction;

s3: and (3) sintering the product obtained in the step (S2) to obtain the metal-doped bioglass microspheres.

Preferably, the alcohol solution is selected from at least one of methanol, ethanol, butanol and propanol.

Preferably, the silicon source solution is an ethyl orthosilicate solution; further preferably, the silicon source solution is an alcohol solution of tetraethoxysilane.

Preferably, the alkali liquor is at least one of ammonia water, sodium hydroxide solution and potassium hydroxide.

Preferably, the mixing reaction in step S1 and step S2 are carried out under stirring.

Preferably, in the step S1, an alcohol solution of tetraethoxysilane is added to the mixed solution a for mixing reaction.

Preferably, in the step S2, the pore-forming agent is added to the mixed solution B to mix and react.

Preferably, the step S2 further comprises a step of centrifugally collecting and purifying the product, wherein the step of centrifugally collecting and purifying is located at the step of mixing and reacting with the metal salt.

Preferably, the metal salt is at least one of strontium salt, calcium salt, zinc salt and lithium salt; further preferably, the metal salt is at least one of strontium salt and calcium salt; still further preferably, the metal salts are strontium salts and calcium salts.

Preferably, the sintering conditions are: firstly, heating to a first temperature of 180-240 ℃ at a first heating rate of 0.5-2 ℃/min, and preserving heat for 1.5-2.5 h; then heating to the second temperature of 600-700 ℃ at a second heating rate of 0.5-2 ℃/min, and keeping the temperature for 1.5-2.5 h.

Preferably, the first heating speed is 0.8-1.5 ℃/min; further preferably, the first temperature rise rate is 0.8 to 1.2 ℃/min; still more preferably, the first temperature increase rate is 1 ℃/min.

Preferably, the second heating speed is 0.8-1.5 ℃/min; further preferably, the second temperature rise rate is 0.8 to 1.2 ℃/min; still more preferably, the second temperature increase rate is 1 ℃/min.

Preferably, the first temperature is 190-220 ℃; further preferably, the first temperature is 190-210 ℃; still further preferably, the first temperature is 200 ℃.

Preferably, the second temperature is 630 to 680 ℃; further preferably, the second temperature is 650 to 670 ℃; still further preferably, the second temperature is 650 ℃.

Preferably, the heat preservation time is 1.8-2.2 h; further preferably, the holding time is 2h.

Preferably, the pore-forming agent is at least one of cetyltrimethylammonium chloride and cetyltrimethylammonium bromide.

Preferably, the pore-foaming agent is sodium chloride with the particle size of 300-500 mu m; further preferably, the pore-foaming agent is sodium chloride with the particle size of 350-450 μm.

In a third aspect, the invention provides a use of the composite scaffold material provided in the first aspect of the invention in a material for repairing a bone defect.

The invention has the beneficial effects that: the composite scaffold material provided by the invention can effectively improve the biological activity of the material, effectively regulate and control the imbalance of bone reconstruction and improve the biological inertia of the polyether-ether-ketone material.

Specifically, the method comprises the following steps: the composite scaffold material has a large number of through holes, can induce apatite mineralization when being soaked in SBF solution, and can promote cell adhesion, proliferation and differentiation when being co-cultured with rBMSCs; when the cells are cultured together with RAW264.7, the TRAP activity of the cells can be reduced, and the generation and the maturation of osteoclasts are influenced. The in vivo implantation experiment shows that the composite scaffold material can promote the regeneration of new bones, has excellent bone-related biological performance and has potential development prospect in the repair of osteoporotic bone defects.

Drawings

Fig. 1 is an SEM image of the composite scaffold materials in examples 1 to 2 and comparative example 1.

FIG. 2 is an FTIR chart of A-SrBG, PEEK-1, ASP20, and ASP40.

FIG. 3 is an XRD pattern of A-SrBG, PEEK-1, ASP20, and ASP40.

FIG. 4 is an SEM image of PEEK-1, ASP20, and ASP40 after soaking in an SBF solution.

FIG. 5 is an EDS elemental analysis chart of ASP20 and ASP40 surface deposits.

FIG. 6 is a graph showing the change in ion concentration after soaking the composite scaffolds in the SBF solutions of examples 1-2 and comparative example 1.

FIG. 7 is a graph of the in vitro release of ALN.

FIG. 8 is a graph showing cell proliferation on the surface of PEEK-1, ASP20 and ASP40 materials.

FIG. 9 is an SEM image of cells after co-culturing PEEK-1, ASP20 and ASP40 materials with the cells.

FIG. 10 is a graph showing the effect of PEEK-1, ASP20 and ASP40 on osteoblasts.

FIG. 11 shows the expression profiles of osteogenesis-related genes after co-culture of PEEK-1, ASP20 and ASP40 with rBMSCs.

FIG. 12 is a TRAP activity profile of cells after 5 days co-culture of PEEK-1, ASP20 and ASP40 with RAW 264.7.

FIG. 13 is a CLSM plot after 5 days co-culture of PEEK-1, ASP20 and ASP40 with RAW 264.7.

FIG. 14 is the expression pattern of the osteoclast-associated gene after co-culture of PEEK-1, ASP20 and ASP40 with RAW 264.7.

FIG. 15 is a graph showing the effect of PEEK-1, ASP20 and ASP40 groups in a co-culture system.

FIG. 16 is an mRNA expression pattern of the osteogenesis-related gene and osteoclastogenesis-related gene of the PEEK-1, ASP20 and ASP40 groups.

FIG. 17 is a graph of Micro-CT images of PEEK-1, ASP20, and ASP40 implanted in vivo at 6 and 12 weeks.

FIG. 18 is a BV/TV image after 6 and 12 weeks of implantation of PEEK-1, ASP20 and ASP40 in vivo.

FIG. 19 is a sectional view of a bone defect repair tissue at 6 weeks and 12 weeks after implantation of PEEK-1, ASP20, and ASP40 in vivo.

Detailed Description

Specific embodiments of the present invention are described in further detail below with reference to the figures and examples, but the practice and protection of the present invention is not limited thereto. It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated by the manufacturer, and are regarded as conventional products commercially available. All data below are expressed as mean ± standard deviation. Statistics of differences between data groups were performed using the One way ANOVA function of Origin 8.0. p <0.05 (. Sup.) and p <0.01 (. Sup.) -are significantly different.

The formulations of the composite scaffold materials of example 1, example 2 and comparative example 1 are shown in table 1 below,

table 1 formulations of composite scaffold materials of example 1, example 2 and comparative example 1

The composite scaffold material of example 1 was prepared as follows:

(1) Preparation of strontium-doped bioglass microspheres (SrBG)

Synthesizing strontium-doped bioglass microspheres (SrBG) by a sol-gel method, which comprises the following steps: cetyl Trimethyl Ammonium Chloride (CTAC) is selected as a pore-making agent. 2g CTAC was dissolved in 10mL of deionized water as solution A; dissolving 3mL of Tetraethoxysilane (TEOS) in 15mL of absolute ethyl alcohol to obtain a solution B;10mL of deionized water, 4.5mL of ammonia water and 15mL of alcohol are mixed to obtain a solution C; the solutions A, B and C are in a rapid stirring state, the stirring state is maintained, the solution B is added into the solution C, and after 15 minutes of reaction, the solution A is added into the solution; after 30 minutes of reaction, 0.25g of strontium nitrate (Sr (NO) was added ₃ ) ₂ ) And 0.28g of calcium nitrate tetrahydrate (Ca (NO) ₃ ) ₂ ·4H ₂ O); after further reaction for 2 hours, a turbid solution was obtained. Centrifuging the turbid solution at 8000rpm; centrifuging to obtain a white powder product; washing with anhydrous ethanol for 2 times and deionized water for 3 times at the same rotation speed to obtain white powder; drying the white powder product in an oven at 100 ℃ overnight; sintering the white powder product by using a muffle furnace under the following temperature conditions: the temperature-rising speed is 1 ℃And min, heating to 200 ℃, keeping the temperature for 2h, heating to 650 ℃ again, wherein the heating rate is 1 ℃/min, and the keeping time is 2h, and finally obtaining the strontium-doped biological glass microsphere (SrBG), wherein the strontium doping amount is 17%.

(2) Preparation of drug-loaded strontium-doped bioglass microspheres (A-SrBG)

And (2) soaking 5g of strontium-doped bioglass microspheres (SrBG) prepared in the step (1) in 100ml of a saturated solution of alendronate sodium, stirring for 24 hours, washing with deionized water, and drying to obtain strontium-doped bioglass microspheres (A-SrBG) carrying drugs, wherein the drug loading is 4.7%.

(3) Preparation of the composite scaffold Material of this example

The preparation method of the composite stent material in the embodiment is a hot pressing-particle leaching method; the method comprises the following specific steps: PEEK powder and a-SrBG powder were mixed in the proportions described in example 1 in table 1, and mixed using a planetary ball mill; after the mixing is finished, the mixed powder and sodium chloride particles (300-500 micrometers) are uniformly mixed according to the mass ratio of 1. Tabletting the mixture in the die by a powder tabletting machine, wherein the pressure during tabletting is 20MPa, the temperature is 180 ℃, the mixture is subjected to compression molding for 30 minutes, then the surface is polished by using sand paper, and after washing for 5 times by ultrasonic oscillation, the mixture is soaked in deionized water for three days to remove the pore-foaming agent sodium chloride particles; deionized water was replaced every 12 hours. The sample obtained by the above procedure was finally placed in an oven (60 ℃ C.) overnight to produce a composite scaffold material in this example, designated ASP20.

Example 2 a composite scaffold material according to example 2 of ASP40 of example 2 was prepared according to the formulation of example 2 of table 1 using the procedure of example 1.

Comparative example 1 the composite scaffold material of comparative example 1, designated PEEK-1, was prepared according to the formulation of comparative example 1 of table 1 using the preparation method of example 1.

And (3) performance testing:

(1) Characterization and in vitro bioactivity test

The composite scaffold materials of examples 1-2 and comparative example 1 were characterized by their composition and microstructure using XRD, SEM and FTIR, wherein the SEM, FTIR, and XRD patterns of the composite scaffold materials of examples 1-2 and comparative example 1 are shown in fig. 1, fig. 2, and fig. 3, respectively. The composite scaffold materials of examples 1 to 2 and comparative example 1 were respectively soaked in Simulated Body Fluid (SBF) to evaluate the biological activity of the composite scaffold materials. The in vitro bioactivity test was carried out in a constant temperature shaker at 37 ℃ for 10 days. After soaking, samples were collected from SBF, rinsed with deionized water, and dried in an electric air blast drying oven at 37 ℃. The surface morphology and composition of the composite scaffold material samples in examples 1-2 and comparative example 1 were characterized by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS), wherein the SEM image is shown in fig. 4 and the EDS image is shown in fig. 5. The specific test results are as follows:

the surface morphology structures of the composite scaffold materials in examples 1 to 2 and comparative example 1 were observed and analyzed by SEM, and the results are shown in fig. 1. Wherein, FIG. 1 (a) is an SEM topography of PEEK-1 in comparative example 1; FIG. 1 (b) is an SEM topography of ASP20 of example 1; FIG. 1 (c) is an SEM topography of the ASP40 of example 2; FIG. 1 (d) is an SEM sectional view of PEEK-1 in comparative example 1; FIG. 1 (e) is an SEM cross-sectional view of the ASP20 in example 1; FIG. 1 (f) is an SEM cross-sectional view of ASP40 in example 2. As can be seen from fig. 1 (a) to 1 (c), the composite scaffold materials (PEEK-1, aspp 20, and ASP 40) in examples 1 to 2 and comparative example 1 each have a macroporous structure with a pore diameter of about 200 to 400 μm, which is consistent with the particle size of the pore-forming agent (NaCl) used in the sample preparation process, and the macropores are interconnected, and the results show that: the addition of the bioglass cannot influence the macroporous structure of the bioglass, and the bioglass, the bioglass and the bioglass all have similar macroporous structures. In vivo, the macroporous structure can provide enough growth space for new bones and blood vessels. As can be seen from FIG. 1 (d), the PEEK-1 of comparative example 1 had a smooth and flat cross-section. It can be seen in fig. 1 (e) and 1 (f) that many a-SrBG particles are uniformly distributed in the ASP20 and the ASP40. Therefore, the introduction of the bioactive glass can improve the adhesion of cells on the PEEK material, so that the bioactivity of the inert PEEK is improved.

Fourier transform Infrared Spectroscopy (FTIR) of A-SrBG, PEEK-1, ASP20, and ASP40 is shown in FIG. 2. PEEK-1 at 1652cm ^-1 Peak at (C = O) is a stretching vibration band of carbonyl (C = O), 1597cm ^-1 Is an in-plane vibration band of R-O-R benzene ring, 1226cm ^-1 Is an asymmetric stretching vibration band of R-O-R. In addition, characteristic peaks of A-SrBG and PEEK were observed in both ASP20 and ASP40.

The X-ray diffraction patterns of A-SrBG, PEEK-1, ASP20 and ASP40 are shown in FIG. 3. As can be seen from FIG. 3, characteristic peaks of PEEK-1 appear at 2 θ =18 ℃, 20 ℃, 22 ℃ and 28 ℃ respectively, indicating that PEEK-1 is a semicrystalline polymer structure. A-SrBG shows a broad peak at 2 theta =26 ℃, which indicates that the A-SrBG is in an amorphous phase. In addition, the crystallinity of the composite scaffold materials of examples 1 to 2 was significantly reduced due to the addition of A-SrBG.

FIG. 4 is an SEM image of the deposition of apatite on the surface of a composite scaffold material of PEEK-1, ASP20 and ASP40 after soaking in an SBF solution for 10 days, wherein FIG. 4 (a) is an SEM image of the surface of the PEEK-1 material; FIG. 4 (b) is an SEM image of the surface of an ASP20 material; FIG. 4 (c) is an SEM image of the surface of an ASP40 material. The surface of the material does not change obviously before and after the PEEK-1 is soaked, and no sediment is generated; after ASP20 is soaked, more particle sediments appear on the porous surface; the ASP40 has the most deposits on the surface, and an apatite deposition layer is formed on the surface of the composite scaffold material.

EDS analysis of ASP20 and ASP40 surface particle deposits is shown in fig. 5, where: FIG. 5 (a) is an EDS map of ASP20 surface deposits; FIG. 5 (b) is an EDS map of ASP40 surface deposits; as can be seen from FIG. 5, the particle deposits on the surfaces of ASP20 and ASP40 contain Ca and P elements, which indicates that the deposits are apatite, and the mineralization capacity of the apatite on the surface of the composite scaffold material is improved along with the increase of the A-SrBG content in PEEK.

FIG. 6 is a graph showing the change in ion concentration after soaking the composite scaffolds in SBF solutions of examples 1-2 and comparative example 1, wherein: FIG. 6 (a) is a graph showing the change in the ion concentration of PEEK-1; FIG. 6 (b) is a graph showing the change in ion concentration of ASP 20; FIG. 6 (c) is a graph showing the change in ion concentration of ASP40. As can be seen from fig. 6, the ion concentrations of ASP20 and ASP40 after soaking in SBF solution exhibit similar rules, which are specifically: during the first day, the concentration of Ca ions is slightly increased, the process corresponds to the dissolving process of A-SrBG distributed on the surface of the composite scaffold material, the bioactive glass is amorphous, so that the bioactive glass can exchange with ions in a solution, and the A-SrBG is quickly dissolved so as to release a large amount of Ca; the ion concentration in the ASP40 solution has a stronger trend of increasing obviously, which indicates that the dissolution process of the ASP40 solution occurs more strongly. This is mainly due to the higher content of a-SrBG in the ASP40 scaffold, the more a-SrBG is dissolved and thus more Ca ions can be dissolved more and faster. After 3 days, the Ca ions begin to decrease, mainly because the Ca ions deposit on the surface to form a core, the osteoid apatite begins to grow, and Ca and P ions are deposited on the surface of the sample along with the growth of the osteoid apatite, so that the Ca and P ions in the solution decrease, and the process continues for about 10 days. While the concentrations of Si and Sr ions steadily increase, which corresponds to the dissolution process of A-SrBG. While no deposit was found on the surface of PEEK-1 in comparative example 1, PEEK-1 itself is an inert substance and has good stability in various environments, so that the ion concentration after soaking in SBF solution is not greatly changed. This result is consistent with SEM and EDS results for composite scaffold materials.

In conclusion, the bioglass material has bioactivity, can induce apatite deposition, can stimulate cell response (adhesion, proliferation and osteogenic differentiation) when being implanted into a body, enhances the combination of the material and bone tissues, reduces the generation of fibrous tissues and is beneficial to bone repair. PEEK is not bioactive and therefore soaking in SBF solutions does not induce apatite formation. After bioactive glass loaded with A-SrBG is added into PEEK, the composite support material is soaked in SBF solution, ca, si, sr and the like generated by degradation of the A-SrBG on the surfaces of the porous surface and the dense layer can interact with ions in the SBF solution, and apatite is induced to be deposited on the surface of the composite support material.

(2) ALN in vitro Release assay

The composite scaffolds of examples 1-2 were immersed in 2mL of PBS (phosphate buffer solution) at different time points (12 hours, 1 day, 2 days)3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 20 days) 200 μ L of PBS was taken out of the PBS solution soaked with the composite scaffold material, and then 200 μ L of fresh PBS solution was supplemented, and the experiment was performed in a shaker at 37 ℃. A working solution of derivatizing reagent was prepared by dissolving 10.0mg of o-phthalaldehyde (OPA) in 2.0mL of 0.05M NaOH, to which was added 50. Mu.L of 2-mercaptoethanol (2 ME), and the solution was topped up to 10.0mL in a volumetric flask using 0.05M NaOH. The removed release medium (composite scaffold material) was mixed with 100. Mu.L OPA/2ME and 1.7mL NaOH for 10min. On FS5 fluorescence spectrometer at 360nm (. Lamda.) _max =470 nm) the emission intensity of the solution is measured at 360-600 nm. The cumulative release amount of ALN was calculated from the calibration curve by plotting the curve between the concentration at 470nm and the emission intensity. The specific test results are as follows:

FIG. 7 is a graph of ALN release in vitro, with time on the abscissa and ALN concentration on the ordinate of FIG. 7; as can be seen from fig. 7: within 1 day, ALN is released rapidly because the surface of the strontium-doped bioactive glass is porous, ALN can be adsorbed in the pore channels of the bioactive glass, and the adsorption is relatively unstable, so that ALN is released relatively rapidly in the early stage. In addition, ALN has a strong affinity for calcium ions and can chelate with calcium to form a tri-ligand. The late ALN release is relatively slow. Therefore, the A-SrBG prepared by chelation can achieve the slow release effect of ALN in vivo and well solve the problem of continuous administration in the slow process of new osteogenesis. Therefore, the composite scaffold materials of examples 1-2 release ALN in situ at the bone defect site, so that the effective concentration of the peri-implant drug can be maintained for a long time, the drug effect is improved, and the bone healing under the condition of osteoporosis is accelerated.

(3) Effect of composite scaffold materials on osteoblasts

Male SD rats of 3 weeks old are sacrificed by cervical dislocation, and soaked in 75% anhydrous ethanol for 5min each time for 3 times of sterilization. Then, the rat was transferred to a clean bench for working, and the skin of the rat lower limb was peeled off with tweezers and scissors, and the lower limb was separated from the hip joint. The hind paw was excised from the ankle joint, soaked in 5% double antibody (mixed solution of penicillin and streptomycin) in PBS buffer solution, and thenThe leg bones of the mice were obtained by stripping the muscle and tendon tissues as completely as possible. The leg bones were transferred to a PBS buffer containing 2% double antibody, and the remaining tissue was further peeled off. Cutting two ends of the bone with a pair of surgical scissors to expose the marrow fluid in the cavity, fully flushing out the marrow fluid with a 5mL syringe filled with a culture medium, collecting the flushing fluid with a centrifuge tube, and centrifuging at 1000rpm for 5min to obtain cell sediment. Decanting the supernatant, resuspending it in DMEM/F12 complete medium, inoculating it in a flask, at 37 deg.C, 5% CO ₂ And (5) culturing under an environment. When the cells were cultured to reach 80% -90% confluence, passage was performed with pancreatin digestion (ratio 1. The SD rats used in the experiment are all from the experimental animal center of Zhongshan university.

(a) Cell proliferation

The CCK-8 method is adopted to detect the influence of the composite scaffold materials in the examples 1-2 and the comparative example 1 on the cell proliferation; the composite scaffold materials were first placed in a 24-well plate at 3X 10 for each composite scaffold material ⁵ Inoculating rBMSCs on the surface of a bracket at a density, culturing for 1, 3 and 5 days, preparing CCK-8 working solution according to the instruction of a CCK8 kit, adding the working solution into a 24-well plate, continuously culturing for 3 hours, transferring the working solution into a 96-well plate, and reading the absorbance at 450 nm. The specific test results are as follows:

FIG. 8 shows cell proliferation (OD) patterns of the surfaces of PEEK-1, ASP20 and ASP40 materials, wherein p <0.05, p <0.01, p <0.001, a shows cell proliferation pattern of PEEK-1, and b shows cell proliferation pattern of ASP 20; c is the cell increment map of ASP40. As can be seen from FIG. 8, after 1 day of culture, the OD value of the cells on the surfaces of ASP20 and ASP40 materials is significantly higher than that of PEEK-1, and the OD value of the cells on the surface of the composite scaffold material is increased along with the increase of the A-SrBG content. After 3 and 5 days of culture, the OD value of the cells on the ASP20 and ASP40 composite scaffold material is obviously higher than that of PEEK-1, and the OD value of ASP40 is the highest. The results show that the composite scaffold material added with the A-SrBG is more favorable for the proliferation of cells on the surface of the composite scaffold material compared with the PEEK-1 material.

FIG. 9 is SEM pictures of the cells on the surface of PEEK-1, ASP20 and ASP40 material after co-culture with the cells for 1, 3 and 5 days, wherein FIG. 9 (a), FIG. 9 (d) and FIG. 9 (g) are SEM pictures of the cells after co-culture of PEEK-1 with the cells for 1d, 3d and 5d, respectively; FIGS. 9 (b), 9 (e) and 9 (h) are SEM pictures of cells after co-culturing ASP20 with the cells for 1d, 3d and 5d, respectively; FIGS. 9 (c), 9 (f) and 9 (i) are SEM images of cells after co-culturing 1d, 3d and 5d with ASP40, respectively. As can be seen from fig. 9: the cells are adhered on the surfaces of PEEK-1, ASP20 and ASP40 materials, but the spreading appearance of the ASP20 and ASP40 composite scaffold material is better, and the cells are more spread along with the prolonging of the culture time. At the same time point, cells on the surface of the PEEK-1 material are circular, and the number of false feet of the cells is small; the ASP20 and ASP40 composite scaffold material has obviously better cell spreading appearance, more spread cells and more filamentous pseudo feet extending out and anchored on the surface of the composite scaffold material. Compared with PEEK-1, ASP20 and ASP40 composite scaffold material, the cell spreading form on the surface is better, and the cell affinity is better.

(b) Alkaline phosphatase (ALP) Activity and staining

2 x 10 of ⁴ Individual rBMSCs were seeded into 24-well plate wells and cultured for 24 hours, and then growth medium was replaced with osteogenic medium containing the leach solution of the composite scaffold material of example 1, example 2 or comparative example 1, respectively. After 7, 10 and 14 days of culture, the osteogenic medium was removed and 1mL of 0.2% Triton X-100 solution (polyethylene glycol octylphenyl ether) was added to each well and the cells lysed. The absorbance of the reaction solution at 405nm was measured on a microplate reader by performing the procedure according to the instructions of the alkaline phosphatase assay kit (Beyotime, china). rBMSCs cultured in osteogenic medium without composite scaffold extract were also used as controls. In addition, alkaline phosphatase staining was performed using BCIP/NBT alkaline phosphatase color development kit (Beyotime, china). In summary, rBMSCs were co-cultured with scaffolds in osteogenic induction medium for 14d, fixed with 4% paraformaldehyde, and then incubated with a mixture of BCIP and NBT solutions. Finally, cells were rinsed with deionized water and photographed using a microscope (OLYMPUS IX 71).

(c) Alizarin red staining quantification

To assess calcium deposition, rBMSCs were co-cultured with the composite scaffold materials of examples 1-2 and comparative example 1 in osteogenic induction medium at 21d,4% paraformaldehyde fixed cells were incubated with alizarin Red dye solution (2%, scienCell) at room temperature for 30And (5) min. The cells were then washed three times with deionized water to remove excess staining solution. The calcium nodules stained red spots were photographed under a microscope. Furthermore, cetylpyridinium chloride (100X 10) ^-3 M) quantifying calcium deposition. The absorbance of the dye was measured at 570 nm.

The specific test results are as follows:

fig. 10 is a graph of the effect of PEEK-1, ASP20, and ASP40 on osteoblasts, wherein, in fig. 10, p <0.05, p <0.01, p <0.001; FIG. 10 (a) is a graph showing the change in ALP activity (osteogenic differentiation) of cells after 7, 10, and 14 days of co-culture of PEEK-1, ASP20, and ASP40 with the cells; as can be seen from FIG. 10 (a), after 7 days of culture, ALP activity was significantly higher in the cells co-cultured with ASP20 and ASP40 than in the cells co-cultured with PEEK-1, and ALP activity was significantly higher in the cells co-cultured with ASP40 than in the cells co-cultured with ASP 20; after culturing for 10 days, the ALP activity of the cells co-cultured with the ASP20 and the ASP40 is obviously higher than that of the cells co-cultured with the PEEK-1, and the ALP activity of the cells co-cultured with the ASP40 is highest; after 14 days of culture, the ALP activity of the cells on the composite scaffold material is reduced along with the prolonging of the culture time, specifically: ASP40> ASP20> PEEK-1, ASP40 material surface cells with the highest ALP activity, indicating the best cell compatibility. FIGS. 10 (b), 10 (c) and 10 (d) show the ALP activity change staining of cells after 10 days of co-culture with PEEK-1, ASP20 and ASP40, showing that ALP staining was significantly darker in cells co-cultured with ASP20 and ASP40 than in PEEK-1 material, and the ALP staining was the deepest in cells co-cultured with ASP40 material.

Analyzing mineralization during osteogenic differentiation of rBMSCs, and staining cell samples co-cultured with PEEK-1, ASP20 and ASP40 by using alizarin red staining solution, wherein, FIG. 10 (f) shows cell staining of co-cultured PEEK-1 material; FIG. 10 (g) shows staining of cells co-cultured with ASP20 material; FIG. 10 (h) shows staining of cells co-cultured with ASP20 material. As a result, it was found that: the cells co-cultured with PEEK-1 material stained very little in alizarin red stain (see FIG. 10 (f)), while the cells co-cultured with ASP20 and ASP40 material showed significant staining after 21 days of osteogenic induction culture (see FIG. 10 (g) and FIG. 10 (h)). As shown in FIG. 10 (e), it can be seen from FIG. 10 (e) that the mineralization of ASP20 and ASP40 materials is significantly higher than that of PEEK-1 materials, and that the mineralization of ASP40 materials is stronger than that of ASP20 and PEEK-1, similar to the activity change and staining of ALP.

To further investigate the differentiation of rBMSCs at the gene level, mRNA expression of osteogenic associated genes, including ALP, run associated transcription factor 2 (Runx 2), osteocalcin (OCN), and collagen type I (COL I) was assessed using qPCR analysis, with specific results as shown in fig. 11, where p <0.05, > p <0.01, > p <0.001 in fig. 11; FIG. 11 (a) is a diagram showing the expression of ALP; FIG. 11 (b) is an expression diagram of Runx 2; FIG. 11 (c) is an expression diagram of OCN; FIG. 11 (d) is an expression diagram of COL I. As can be seen from fig. 11, the ALP gene showed the highest expression at day 10, and the expression of Runx2 and OCN genes increased with the lapse of time. Expression of COL I gradually increased until day 14 expression was highest. There was no significant difference between the control group and PEEK-1 for all these genes. However, osteogenic differentiation of rBMSCs was significantly enhanced in ASP20 and ASP40. Among them, ASP40 exhibits a stronger ability in the expression of the above genes.

(4) Effect of composite scaffold Material on osteoclasts

The effect of the composite scaffold materials in examples 1-2 and comparative example 1 on osteoclasts was evaluated using osteoclasts differentiated from a mouse monocyte-macrophage (RAW 264.7) cell line. RAW 264.7% CO at 37 ℃ in DMEM (high glucose, gibco, USA) and 10% FBS ₂ And (5) culturing under an environment. After 80% of the cells were obtained, the cells were scraped off from the flask using a cell scraper. To induce osteoclast differentiation, RAW264.7 cells were plated at 2 × 10 ⁴ The cells/mL were plated in 24-well plates. After 24 hours of culture, the culture was continued in an osteogenic differentiation medium containing RANKL (30 ng/mL, PEROTECH, USA).

(a) Anti-tartrate active phosphatase (TRAP) Activity

Tartrate-resistant acid phosphatase (TRAP) activity is a characteristic of osteoclasts and is detected using a tartrate-resistant acid phosphatase detection kit (bi yun tian, china) according to standard protocols. RAW264.7 was co-cultured with the composite scaffold leach solution of example 1, example 2 and comparative example 1 for 5 days, fixed with 4% paraformaldehyde for 30min, and lysed with 0.2% Triton-X-100 solution for 30min. mu.L of the cell lysate was added to a mixed solution of 40. Mu.L of the chromogenic substrate diluent and 5. Mu.L of tartaric acid solution, and incubated in a 96-well plate at 37 ℃ for 30min. The reaction was terminated with 160mol/L of a reaction termination solution, and the absorbance at 405nm was measured with a Gene5 microplate reader.

(b) Immunofluorescence staining

RAW264.7 cells were plated at 2X 10 ⁴ The density of cells/well was seeded onto 24-well plates. After 5 days of induction, cells were fixed with fixative and specimens were stained using F-actin staining kit (red fluorescence). Then, DAPI was added to stain the nuclei for 10min. Cells were observed using a confocal laser microscope. The specific test results are as follows:

FIG. 12 is a graph of TRAP activity of cells after 5 days of co-culture of PEEK-1, ASP20 and ASP40 material with RAW 264.7; as can be seen from fig. 12, both ASP20 and ASP40 significantly reduced the TRAP activity of RAW264.7 and the effect of ASP40 was more significant than PEEK-1. TRAP is a marker of osteoclast formation, and fig. 12 shows that both ASP20 and ASP40 can significantly inhibit osteoclast formation. Osteoclasts are multinucleated cells, and TRAP-positive multinucleated cells of three or more nuclei are considered osteoclasts. FIG. 13 is a laser Confocal (CLSM) profile of PEEK-1, ASP20 and ASP40 material after 5 days of co-culture with RAW264.7, wherein FIG. 13 (a) is a CLSM profile of PEEK-1 and RAW264.7 after 5 days of co-culture; FIG. 13 (b) is a CLSM map after 5 days of co-culture of ASP20 and RAW 264.7; FIG. 13 (c) is a CLSM plot after 5 days of co-culture of ASP40 with RAW 264.7; as can be seen from fig. 13, TRAP-positive multinucleated osteoclasts were reduced in ASP20 compared to PEEK-1, and multinucleated osteoclasts were hardly observed in ASP40. The results of fig. 12 and 13 show that ASP20 and ASP40 composite scaffold material hinders the maturation of osteoclasts, and the effect of ASP40 is more remarkable.

(5) Quantitative polymerase chain reaction (qPCR)

rBMSCs/RAW 264.7 cells at 2.0X 10 ⁵ The density of cells/well was seeded on 6-well plates and cultured in growth medium for 24 hours, then the growth medium was replaced with osteogenic/osteoclastic medium. Culture of 24, 72 and 120. Mu.lAfter this time, the osteogenic/osteoclastic medium was removed and total RNA was extracted using RNase erase (world Foregene, china). The concentration and purity of the extracted RNA was measured using a nanodrop spectrophotometer (thermoelectric science, usa). Then, the total RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (Takara, japan), and real-time PCR was performed using SYBR Green System (Takara, japan) in an Applied Biosystems 7500 Rapid real-time PCR system, and the relevant gene primers used in gene expression were as shown in Table 2 below.

TABLE 2 Gene primers of interest for use in Gene expression

To further investigate the differentiation of RAW264.7 at the gene level, mRNA expression of osteoclast-associated genes, including TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin K (cath-K), and nuclear factor of activated T cells (NFATc 1) genes, was assessed using qPCR analysis, as shown in particular in fig. 14, where p <0.05, p <0.01, p <0.001 in fig. 14; FIG. 14 (a) is an mRNA expression profile of the TRAP gene for PEEK-1, ASP20 and ASP40 material; FIG. 14 (b) is an mRNA expression profile of the MMP-9 gene for the PEEK-1, ASP20, and ASP40 materials; FIG. 14 (c) shows the mRNA expression profile of the cath-K gene from PEEK-1, ASP20 and ASP40 material; FIG. 14 (d) is the mRNA expression profile of the NFATc1 gene of PEEK-1, ASP20 and ASP40 materials. As can be seen from fig. 14, the expression of the above genes was significantly down-regulated in ASP20 and ASP40 compared to PEEK-1, and ASP40 material showed greater inhibitory effect than ASP20 material after 5 days of culture. These results indicate that the composite scaffold materials in ASP20 and ASP40 can significantly inhibit osteoclast differentiation of RAW264.7 cells.

Relevant studies have shown that Sr ions disrupt osteoclast formation by blocking RANKL-mediated activation of the NF- κ B pathway; but bone healing is more difficult in osteoporotic states. At present, the molecular mechanism of inhibiting bone resorption by using the nitrogen-containing diphosphate is considered to be a way of inhibiting mevalonic acid, and the application prospect in the aspect of treating osteoporosis is good, but the osteoporosis is treated by long-time intravenous injection of high-dose bisphosphonate, so that a lot of adverse reactions are caused. Therefore, the ALN and SrBG combined modified biological inert PEEK material provided by the invention can promote the proliferation and differentiation of osteogenesis and better inhibit the proliferation and differentiation of osteoclasts at the same time, and provides a new treatment way for bone healing in an osteoporosis state.

(6) Osteoblast and osteoclast coculture

Mixing 1.0X 10 ⁴ rBMSCs of each cell/hole are inoculated on a 24-hole plate and cultured in osteogenic induction liquid for 7 days; in addition, 4.0 × 10 ⁴ Rat bone marrow derived mononuclear cells, rBMMs, were seeded in a transwell chamber and cultured in osteoclast medium for 5 days; transwell chambers were then transferred to 24-well plates and co-cultured with rBMSCs in a mixture of rBMSCs osteogenic medium and rBMMs osteoclasts osteoclast medium at 1. After 7 days of co-culture, cell activity, differentiation and gene expression were analyzed. In addition, the ratio of Osteoprotegerin (OPG) to RANKL was determined using a rat OPG ELISA kit (Abcam, uk) and a mouse RANKL ELISA kit (Abcam, uk). Medium without extract was used as control.

To further evaluate the effect of examples 1-2 and comparative example 1 on promoting osteogenesis and inhibiting osteoclastogenesis in osteoblast/osteoclast co-culture systems, we also evaluated cell proliferation (OD), cell differentiation and OPG/RANKL (for measuring the anti-osteoclast potential of osteoblasts), and the results of the specific tests are shown in fig. 15, where p <0.05, <0.01, <0.001, and fig. 15 (ASP) is a cell proliferation map of rBMSCs from PEEK-1, ASP20, ASP40 groups, and it can be seen that: the ASP20 and ASP40 groups induced higher cell proliferation than the control and PEEK-1 groups, and the highest values were observed in the ASP40 group. Fig. 15 (b) is a cell proliferation map of rBMMs of PEEK-1, ASP20, ASP40 groups, and it can be seen that opposite trends were found in cell proliferation of rBMMs, ASP20 and ASP40 groups induced cell proliferation lower than control group and PEEK-1 group, and the highest value was observed in PEEK-1 group. FIG. 15 (c) shows ALP concentrations in osteoblasts of the PEEK-1, ASP20, and ASP40 groups, and ALP levels in osteoblasts of the ASP20 and ASP40 groups were significantly higher than those of the PEEK-1 group. In addition, the ALP activity was significantly higher in the ASP40 group than in the ASP20 group. FIG. 15 (d) is a TRAP activity profile for the PEEK-1, ASP20, ASP40 groups; TRAP activity was significantly down-regulated in ASP20 and ASP40 groups compared to the control and PEEK-1 groups. FIG. 15 (e) is a graph showing the OPG/RANKL ratio of PEEK-1, ASP20, and ASP40 groups; the OPG/RANKL levels were higher in the ASP20 and ASP40 groups than in the PEEK-1 group, and the OPG/RANKL levels were highest in the ASP40 group.

To further study the differentiation of osteoblasts and osteoclasts in co-culture systems at the gene level, the mRNA expression of the osteogenesis-related genes (ALP, runx2, OCN and COL I) and the osteoclast-related genes (TRAP, MMP-9, cath-K and NFATc 1) was determined, as shown in FIG. 16, in particular, where: p <0.05, p <0.01, p <0.001 in fig. 16 (a), 16 (b), 16 (c) and 16 (d) are mRNA expression profiles of ALP gene, runx2 gene, OCN gene and COL I gene of PEEK-1, ASP20, ASP40 group, respectively; FIG. 16 (e), FIG. 16 (f), FIG. 16 (g) and FIG. 16 (h) are the mRNA expression profiles of the TRAP gene, MMP-9 gene, cath-K gene and NFATc1 gene of PEEK-1, ASP20 and ASP40 groups, respectively. As can be seen in fig. 16: the mRNA levels of the osteogenesis related genes of the ASP20 and ASP40 groups were up-regulated on average, and rBMSCs of the ASP40 group were expressed at the highest level in all genes except COL I. The ASP20 and ASP40 groups had significantly lower mRNA levels of the rBMMs osteoclastogenesis associated gene than the PEEK-1 group, and the ASP40 group expressed the lowest. These results indicate that the composite scaffold materials in examples 1 to 2 and comparative example 1 can inhibit osteoclast differentiation not only by direct interaction, but even by osteoblast-mediated indirect regulation.

(7) In vivo experiments

(a) Establishing rat skull defect model

After 3 months of ovaries on both sides of the SD rat are removed, an osteoporosis model is established. Then, a critical dimension defect of 8mm in diameter and 2mm in depth was created, and the composite stent materials of examples 1 to 2 and comparative example 1 were used

And implanting the defect. After 6 and 12 weeks of surgery, animals were sacrificed by intraperitoneal injection of excess pentobarbital, and all cranium bones were fixed with 4% paraformaldehyde and further analyzed.

(b) Micro-CT analysis

To assess bone regeneration in a cranial defect, harvested cranium was scanned on a micro CT scanner. After three-dimensional reconstruction, new bone formation was calculated using CT analysis software using bone volume fraction (BV/TV) of the defect area.

FIG. 17 is a graph of Micro-CT after 6 weeks and 12 weeks of implantation of the composite scaffold materials of examples 1-2 and comparative example 1, wherein FIGS. 17 (a) and 17 (d) are graphs of Micro-CT after 6 weeks and 12 weeks of implantation of the composite scaffold material of PEEK-1, respectively; FIGS. 17 (b) and 17 (e) are graphs of Micro-CT after 6 and 12 weeks, respectively, of implantation of the composite scaffold material in ASP 20; FIGS. 17 (c) and 17 (f) are graphs of Micro-CT 6 and 12 weeks after implantation of the composite scaffold material in ASP40, respectively; at 6 and 12 weeks of implantation, the amount of new bone around the implant was significantly increased compared to PEEK-1, ASP20 and ASP40 implants. As the implantation time increases, the area of new bone around the ASP20 and ASP40 implant materials increases, and the area of new bone around the ASP40 is significantly larger than the ASP20. The area of new bone around PEEK-1 was minimal throughout the implant cycle (12 weeks) and there was no significant change in the new bone area at different time points. At the same implantation time point, the area of new bone around the ASP40 is the most, and more new bone appears around, showing the best osteogenetic performance. FIG. 18 is a BV/TV set of the composite stent material of examples 1-2 and comparative example 1 after 6 and 12 weeks of implantation in vivo, wherein FIG. 18 (a) is a BV/TV set of 6 weeks of implantation; FIG. 18 (b) is a BV/TV image at 12 weeks of implantation. As shown in FIG. 18, the new bone BV/TV around ASP40 was the highest and the new bone quality was the highest.

(c) Histological analysis

After micro-CT analysis, the specimens were decalcified, dehydrated and embedded in paraffin blocks. In the middle of the defect, a tissue section (5 μm thick) was made. Sections were stained with hematoxylin/eosin (H & E), masson's trichrome, according to the manufacturer's protocol. The specific operation is as follows:

HE staining

Staining with hematoxylin staining solution for 5-10 min, performing color separation with 0.5-1% hydrochloric acid alcohol, and performing microscopic examination until cell nucleus and chromatin in the cell nucleus are clear; flushing for half an hour by running water, and then short-time washing by distilled water; dyeing with 0.1-0.5% eosin dye liquor for 1-5 min; dehydrating with gradient ethanol, removing xylene twice, adding appropriate amount of neutral gum, and sealing with cover glass. The sections were analyzed for material residue, new bone matrix and inflammatory cells, etc. by observation using an inverted microscope (Nikon TE2000U, nikon, japan). The slices were placed under an inverted microscope and magnified 20 times, and 4 different fields of view were selected at the composite scaffold implantation site on each slice, with the center of the field of view being 0.5mm from the host bone. Using Image-Pro Plus 6.0 software, the area of new bone and the area of residual material were measured separately and averaged as the new bone mass and the material residual mass for each specimen.

Masson trichrome stain

Dyeing with hematoxylin dye solution for 5-10 min, separating with 0.5-1% hydrochloric acid alcohol, bluing with running water, and washing with distilled water; dyeing ponceau acid magenta solution for 5-8 minutes, and washing with distilled water; dyeing for 1-3 minutes by using 1% phosphomolybdic acid solution; directly immersing into aniline blue glacial acetic acid solution for 5 minutes; quickly washing with water, drying in 60 deg.C air-blowing drying box, making xylene transparent, and sealing with glass slide. And (4) analyzing the material residues, the new bone matrixes, the mature bone matrixes and the like in the slices by using an inverted microscope for observation. The sections were placed under an inverted microscope, magnified 20 times, and 4 different fields of view were selected at the composite scaffold implantation site on each section, with the center of the field of view being 0.5mm from the host bone. The residual material area, the new bone area and the mature bone area were measured using Image-Pro Plus 6.0 software, respectively, and the average values were taken as the material residual amount, the new bone amount and the mature bone amount of each specimen. The specific test results are as follows:

FIG. 19 is a sectional view of a bone defect repair tissue when PEEK-1, ASP20, ASP40 are implanted in vivo at 6 weeks and 12 weeks, with the small panel portion of FIG. 19 being a partial enlargement of the left large panel marked thereon. Wherein, FIG. 19 (a) is a bone defect repair tissue section image using HE staining; as can be seen from fig. 19, the three sets of composite scaffold materials all had a large amount of material residues, because of the good stability of polyetheretherketone, PEEK in the three sets of composite scaffold material samples can be stably present, after 6 weeks of implantation, PEEK-1 and ASP20 both had very little new bone formation, ASP40 had much new bone formation, and showed a random-shaped structure. At 12 weeks of implantation, more new bone tissue appeared inside the ASP20 and ASP40 scaffolds, and the most new bone appeared inside the ASP40 scaffold, showing the best osteogenesis effect. With the increase of the content of A-SrBG in the composite scaffold material, the new bone quantity in the material is increased. FIG. 19 (b) Masson trichrome stained bone defect repair tissue section. The Masson trichrome staining results were also complementary to the HE staining results. In fig. 19 (b), not only the formation of new bone but also the formation of mature bone in the defect region can be observed. Bone maturation is a progressive process involving a synergistic interaction between the implant and the bone cells/tissues. As can be seen in fig. 19, none of the three sets of composite scaffold samples degraded and ASP40 set had the best bone repair.

Researchers believe that a platform for the adhesion growth of osteocytes and tissues is crucial in the bone repair process, while porous implants have good results. The implant needs to have a through-going pore structure, high porosity and pore size (> 300 μm). In the present invention, the porosity of the composite scaffold material in examples 1 to 2 was about 70%. And has a mutually communicated macroporous structure of about 400 mu m, and the holes are sodium chloride leaching holes. In addition, srBG added into the composite scaffold material has a regular spherical shape, and the diameter of the SrBG is about 400 nm. Therefore, ASP20 and ASP40 have a multi-level pore structure of macropores and micropores, which have good connectivity between pores and are advantageous for the growth of tissues and cells, while micropores are advantageous for the transportation of nutrients and metabolites and for the adhesion of cells. The formed structure and the addition of the A-SrBG also enable the composite scaffold material to have higher porosity and better water absorption; the good water absorption can quickly absorb protein in tissue fluid, and the protein absorption as the first step of cell adhesion plays a key role in cell adhesion.

The ability of osteoid apatite to induce formation is an important indicator of the bioactivity of implant surfaces. In the present invention, after soaking in SBF solution for 10 days, osteoid apatite can form on the surfaces of ASP20 and ASP 40; whereas the PEEK-1 surface was not observed for apatite formation; and the amount of osteoid apatite formed on the ASP40 surface is much higher than the amount of surface energy formed by the ASP20. The formation of apatite is mainly due to ion exchange, and then the deposition and nucleation of Ca ions occur, and the formation of silanol groups on the surface can induce the generation of bone-like apatite. The PEEK-1 surface did not have the ability to ion exchange and apatite formation was not observed. Both ASP20 and ASP40 surfaces are able to efficiently exchange ions and hence apatite formation. However, the A-SrBG surface has a microporous structure, so that BG particles distributed on the surface have a higher specific surface area, and a more rapid and violent ion exchange effect is performed, so that surface bone-like apatite is formed more rapidly and effectively.

ASP20 and ASP40 can obviously promote the adhesion, proliferation and differentiation of rBMSCs cells on the surface of a material. On the one hand, the micropores present on the walls of the macropores of ASP20 and ASP40 favour the anchorage of cellular pseudopodia and the exchange and transport of nutrients and metabolites. On the other hand, the hydrophilic surface can effectively promote the adhesion and proliferation of cells; moreover, the composite scaffold material has more bioactive glass particles distributed on the surface, so that Si, ca and Sr ions can be released slowly, the three ions can promote the proliferation of cells and the osteogenic differentiation of the cells by promoting extracellular matrix, and therefore, the ASP20 and the ASP40 have the best effects of promoting the adhesion, proliferation and differentiation of the cells. Whereas the cell response of the ASP40 fraction was superior to that of the ASP20 fraction, it is considered that the change in ion concentration caused by the addition of A-SrBG plays a major role in promoting the cell response.

In addition, ASP20 and ASP40 can obviously inhibit TRAP activity of RAW264.7 cells and formation and maturation of multinuclear osteoclast. Sr ions not only promote osteoblast proliferation, but also disrupt osteoclast formation by blocking RANKL-mediated activation of the NF- κ B pathway. Bisphosphonates, particularly nitrogen-containing bisphosphonates, adsorb on mineral binding sites and interfere with attachment, leading to ultrastructural changes in osteoclasts. The molecular mechanism by which nitrogen-containing diphosphates inhibit bone resorption is the inhibition of the mevalonate pathway, which synthesizes cholesterol and prenyl esters, such as prenyl diphosphate, farnesyl pyrophosphate, and tallow pyrophosphate. Wherein the pyrophosphate farnesyl ester and the pyrophosphate diester participate in the post-translational modification of the small-molecule guanosine triphosphatase. The small molecule guanosine triphosphate is an important signal protein in cells, and regulates the formation of osteoclast ruffled edges and actin rings, the transport of endosomes, apoptosis and the like. The small-molecule guanosine triphosphate can be anchored on osteoclast only through prenylation, and promotes osteoclast to obtain the structure and function required by bone absorption. Thus, the nitrogenous diphosphate indirectly blocks prenylation of the small molecule guanosine triphosphatase by inhibiting the mevalonate pathway, resulting in loss of the ruffled margin of the osteoclast, interfering with the formation of the actin loop and inducing osteoclastogenesis. Therefore, both ASP20 and ASP40 can significantly inhibit osteoclast generation, and the effect is more significant because the content of Sr ions and ALN in ASP40 is higher.

After the composite scaffold material is implanted into the defects of the skull of a mouse, the composite scaffold material is found to have good stability, and no obvious degradation phenomenon occurs within 12 weeks. More mature bone tissue was generated in ASP40 by tissue section staining. Si and Sr ions can effectively enhance the synthesis of bone matrix and the activity of osteoblasts, ca ions are the most important mineral elements in bone structure, and can effectively promote mineralization and influence all stages of bone metabolism; the surface micropores can effectively promote the metabolic action of bone tissues adhered to the surface, can also promote Sr ions grown in the bone tissues and diphosphate drugs to inhibit the proliferation and differentiation of osteoclasts through different ways, balance the bone absorption generated by the osteoclasts and accelerate the recovery process of bone defect under osteoporosis; the combined action of ions, surface structure and small molecule drugs therefore resulted in the ASP40 sample having the best in vivo osteogenesis and bone ingrowth effects.

The invention selects the hot pressing-particle leaching method to prepare the composite stent material, and in vitro and in vivo researches prove that: the composite scaffold material (ASP 40) provided by the invention has a large number of through holes, can induce apatite mineralization when being soaked in SBF solution, and can promote cell adhesion, proliferation and differentiation when being co-cultured with rBMSCs; when the cells are cultured together with RAW264.7, the TRAP activity of the cells can be reduced, and the generation and the maturation of osteoclasts are influenced. In vivo implantation experiments prove that ASP40 can promote new bone regeneration. Therefore, the composite scaffold material has excellent bone-related biological performance and has potential development prospect in osteoporotic bone defect repair.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A composite stent material, characterized by: the composite scaffold material is composed of polyether-ether-ketone and drug-loaded metal-doped bioglass microspheres; the drug-loaded metal-doped bioglass microspheres are dispersed in the polyether-ether-ketone; the medicament carried on the medicament-carrying metal-doped bioglass microspheres is a bone metabolism regulator; the metals are strontium and calcium; the composite scaffold material contains interconnected pores; the composite support material contains a macropore with the aperture of 200-400 mu m and a micropore with the aperture of 0.1-2nm; the porosity of the composite bracket material is 60 to 80 percent; the mass ratio of the drug-loaded metal-doped bioglass microspheres to the polyether-ether-ketone is 1: (1 to 6); the bone metabolism regulator is at least one of alendronate sodium, pamidronate sodium and zoledronic acid.

2. The composite scaffold material of claim 1, wherein: the drug-loaded metal-doped bioglass microspheres meet at least one of the following conditions:

(1) The particle size of the drug-loaded metal-doped bioglass microspheres is 100 to 500nm;

(2) The drug loading amount of the metal-doped bioglass microspheres carrying the drug is 3-6%;

(3) The content of metal in the drug-loaded metal-doped bioglass microspheres is 10 to 20 percent.

3. The method for preparing the composite scaffold material of claim 1 or 2, wherein: the method comprises the following steps:

preparing metal-doped bioglass microspheres by a sol-gel method;

4. The method of preparing a composite scaffold material of claim 3, wherein: the preparation steps of the metal-doped bioglass microspheres are as follows:

5. The method for preparing the composite scaffold material according to claim 4, wherein: the sintering conditions are as follows: firstly, heating to a first temperature of 180-240 ℃ at a first heating speed of 0.5-2 ℃/min, and preserving heat for 1.5-2.5 h; and then heating to the second temperature of 600-700 ℃ at a second heating speed of 0.5-2 ℃/min, and keeping the temperature for 1.5-2.5 h.

6. The method of preparing a composite scaffold material of claim 4, wherein: the pore-foaming agent is sodium chloride.

7. Use of a composite scaffold material according to claim 1 or 2 in a material for repairing a bone defect.