Method for preparing component gradient type melt electric writing support with assistance of magnetic force

Abstract

The invention belongs to the technical field of tissue engineering scaffold materials, and particularly relates to a method for preparing a component gradient type melt electric writing scaffold under the assistance of magnetic force. The preparation method comprises the following steps: drying a dispersion liquid of magnetic hydroxyapatite (Fe3O4 nanoparticles and a hydroxyapatite shell layer coated on the surface of the Fe3O4 nanoparticles) containing a core-shell structure, polycaprolactone and dichloromethane, heating an obtained mHA-PCL composite raw material to a molten state to form a composite melt, treating the composite melt by a linear gradient magnetic field, and then performing melt electric writing printing. And obtaining the component gradient type melt electric writing bracket. According to the method, the content of hydroxyapatite (HA) in the melt electrowriting stent is distributed in a continuous linear gradient manner through directional migration of magnetic hydroxyapatite with a core-shell structure in a linear gradient magnetic field, a natural calcification gradient structure existing in an osteochondral tissue is simulated, the bionic performance of the stent is improved, and the stent has a good application prospect. The problems that the HA concentration in a stent prepared by a traditional melt electric writing method suddenly changes, and gradient change cannot be achieved are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering scaffold materials, and particularly relates to a method for preparing a component gradient melt electrowriting scaffold with the assistance of magnetic force. Background Art

[0002] Tissue engineering scaffolds have broad application scenarios in the field of osteochondral tissue repair due to their advantages of personalized customization and good biocompatibility. There is a natural calcification gradient structure inside the osteochondral tissue, and the content of hydroxyapatite (HA) gradually increases from the cartilage side to the bone side. This gradual calcification characteristic makes osteochondral tissue show unique advantages in bearing mechanical loads and realizing biological functions. Therefore, in order to better simulate the structure and function of natural osteochondral tissue, tissue engineering scaffolds need to have a continuous composition gradient of HA. However, traditional melt electrowriting (MEW) technology can only achieve changes in HA concentration by changing the melt batch, and cannot achieve a gradual change in HA concentration gradient in the prepared scaffold, which is significantly different from the gradual distribution of HA in natural tissue. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for magnetically assisted preparation of a composition-gradient melt electrowriting scaffold. This method realizes a continuous linear gradient distribution of hydroxyapatite content in the melt electrowriting scaffold through the directional migration of core-shell structured magnetic hydroxyapatite in a linear gradient magnetic field, simulates the natural calcification gradient structure existing inside bone and cartilage tissue, improves the bionic performance of the scaffold, and solves the problem of sudden change of HA concentration in the scaffold prepared by traditional melt electrowriting method and inability to achieve gradient change.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a composition-gradient melt electrowriting support with magnetic assistance, comprising the following steps:

[0006] The dispersion containing core-shell structured magnetic hydroxyapatite, polycaprolactone and dichloromethane is dried to obtain a mHA-PCL composite material;

[0007] The core-shell structured magnetic hydroxyapatite comprises Fe3O4 nanoparticles and a hydroxyapatite shell layer coated on the surface of the Fe3O4 nanoparticles;

[0008] The mHA-PCL composite raw material is heated to a molten state to form a composite melt. The composite melt is treated with a linear gradient magnetic field and then subjected to melt electrowriting printing to obtain a composition gradient melt electrowriting stent.

[0009] Preferably, the particle size of the Fe3O4 nanoparticles is 200-300 nm; the thickness of the hydroxyapatite shell is 10-50 nm.

[0010] Preferably, the number average molecular weight of the polycaprolactone is 25k-50k; the mass of the core-shell structured magnetic hydroxyapatite is 10-30% of the mass of the polycaprolactone.

[0011] Preferably, the linear gradient magnetic field treatment is to place the composite melt in a linear gradient magnetic field generated by passing current through a magnetic field generator.

[0012] Preferably, the magnetic field generator includes a Helmholtz coil or an anti-Helmholtz coil.

[0013] Preferably, the Helmholtz coil is composed of a pair of coaxially placed circular coils, and the two circular coils are fed with current in the same direction; the structure of the anti-Helmholtz coil is the same as that of the Helmholtz coil, but the two circular coils are fed with current in opposite directions; the radius of the two circular coils is R = 100 to 300 nm, and the spacing d = R.

[0014] Preferably, the temperature of the linear gradient magnetic field treatment is 110-130° C., and the time is 15-45 minutes.

[0015] Preferably, the parameters of the melt electrowriting printing include: heating temperature of 150-170°C; air supply pressure of 0.2-0.3 MPa; nozzle-collection plate voltage of 3.0-4.0 kV; nozzle-collection plate spacing of 2-8 mm; and collection plate movement speed of 5-15 mm / s.

[0016] Preferably, the heating temperature is 110-130° C. and the heating time is 8-10 h.

[0017] Preferably, the input current is 5-10A.

[0018] The present invention provides a method for preparing a composition-gradient melt electrowriting stent with magnetic assistance, comprising the following steps: drying a dispersion containing core-shell magnetic hydroxyapatite, polycaprolactone and dichloromethane to obtain an mHA-PCL composite raw material; the core-shell magnetic hydroxyapatite comprises Fe3O4 nanoparticles and a hydroxyapatite shell layer coated on the surface of the Fe3O4 nanoparticles; heating the mHA-PCL composite raw material to a molten state to form a composite melt; and subjecting the composite melt to linear gradient magnetic field treatment and melt electrowriting printing to obtain a composition-gradient melt electrowriting stent. In the present invention, the Fe3O4 nanoparticle core in the core-shell structure magnetic hydroxyapatite (mHA) gives mHA magnetophoretic migration ability, and the hydroxyapatite (HA) shell retains biological activity, ensuring that the mHA particles do not lose their bone induction function when they move in a magnetic field; different from the traditional HA concentration mutation caused by "changing melt batches", the present invention drives the directional migration of mHA through a linear gradient magnetic field to achieve a continuous linear gradient distribution of HA content inside the scaffold, which is highly consistent with the calcification gradient of natural bone cartilage tissue. It can not only significantly improve the bionic performance, but also provide the possibility for subsequent magnetic hyperthermia. mHA can generate heat in an alternating magnetic field to promote bone repair, improve cell adhesion rate and osteogenic differentiation efficiency. The method provided by the present invention can form a HA gradient distribution in the composite melt formed by the mHA-PCL composite raw material through a single magnetic field treatment, and is combined with melt electrowriting technology for one-time printing and molding, which significantly improves production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a position diagram of the barrel containing the composite melt and the gradient magnetic field generated by the Helmholtz coil in Example 1;

[0020] Figure 2 Schematic diagram of melt electrowriting printing in an embodiment of the present invention;

[0021] Figure 3 This is a physical picture of the composition gradient melt electrowriting support prepared in Example 1;

[0022] Figure 4 This is a graph showing the measurement results of the change in magnetic induction intensity on the axis of the Helmholtz coil in Example 1;

[0023] Figure 5 This is a graph showing the change in hydroxyapatite (HA) concentration between the stent layers after the composition-gradient melt electrowriting stent prepared in Example 1 was treated with a Helmholtz coil;

[0024] Figure 6 A diagram showing the positions of the barrel containing the composite melt and the gradient magnetic field generated by the anti-Helmholtz coil in Example 2;

[0025] Figure 7 Graph showing the change in magnetic induction intensity on the axis of the anti-Helmholtz coil in Example 2;

[0026] Figure 8 This is a graph showing the change in HA concentration between the stent layers of the composition-gradient melt electrowriting stent prepared in Example 2 after treatment with an anti-Helmholtz coil;

[0027] Figure 9 This is a graph showing the change in HA concentration between the layers of the scaffold in Comparative Example 1 without magnetic field treatment;

[0028] Figure 10 TEM image of the core-shell magnetic hydroxyapatite (mHA) prepared in Example 1;

[0029] Figure 11 XRD pattern of mHA prepared in Example 1;

[0030] Figure 12 FTIR spectrum of mHA (Fe3O4@HA) prepared in Example 1;

[0031] Figure 13 Figure 3 is a stress-strain curve of the mHA-polycaprolactone composite scaffold with different HA concentrations, where 0Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Comparative Example 2, 10Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 1, 20Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 3, and 30Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 4. DETAILED DESCRIPTION

[0032] The present invention provides a method for preparing a composition-gradient melt electrowriting support with magnetic assistance, comprising the following steps:

[0033] The dispersion containing core-shell structured magnetic hydroxyapatite, polycaprolactone and dichloromethane is dried to obtain a mHA-PCL composite material;

[0034] The core-shell structured magnetic hydroxyapatite comprises Fe3O4 nanoparticles and a hydroxyapatite shell layer coated on the surface of the Fe3O4 nanoparticles;

[0035] The mHA-PCL composite raw material is heated to a molten state to form a composite melt. The composite melt is treated with a linear gradient magnetic field and then subjected to melt electrowriting printing to obtain a composition gradient melt electrowriting stent.

[0036] Unless otherwise specified, the present invention has no special requirements on the sources of the raw materials used, and commercially available products known to those skilled in the art can be used.

[0037] The present invention dries a dispersion containing core-shell structured magnetic hydroxyapatite, polycaprolactone and dichloromethane to obtain an mHA-PCL composite raw material.

[0038] As an embodiment, the core-shell structured magnetic hydroxyapatite (mHA) includes Fe3O4 nanoparticles and a hydroxyapatite (HA) shell layer coated on the surface of the Fe3O4 nanoparticles; the particle size of the Fe3O4 nanoparticles is 200-300 nm, and in a specific embodiment, it is 200-250 nm; the thickness of the hydroxyapatite shell layer is 10-50 nm, and in a specific embodiment, it is 20-40 nm.

[0039] As an embodiment, the preparation method of the core-shell structured magnetic hydroxyapatite (mHA) comprises the following steps:

[0040] Calcium salt, Fe3O4 nanoparticles, a phosphorus-containing compound and water are mixed, and the pH value of the obtained dispersion containing calcium, phosphorus and Fe3O4 cores is adjusted to 10-11. The obtained reaction solution containing calcium, phosphorus and Fe3O4 cores is subjected to a hydrothermal reaction to obtain core-shell structured magnetic hydroxyapatite.

[0041] As an embodiment, the calcium salt includes calcium hydroxide (Ca(OH)2) and / or calcium nitrate (CaNO3), specifically calcium hydroxide in the embodiment; the phosphorus-containing compound is phosphoric acid (H3PO4) and / or diammonium hydrogen phosphate ((NH4)2HPO4); the phosphoric acid is an 85wt% phosphoric acid solution; the molar ratio of the calcium salt to the phosphorus-containing compound is 4 to 6:3, specifically 0.02:0.012 in the embodiment; the mass ratio of the calcium salt to the Fe3O4 nanoparticles is 1.475:2 to 3, specifically 1.475:2 in the embodiment; the water is deionized water.

[0042] As an embodiment, the mixing is as follows: first dissolving the calcium salt in a first portion of water and performing a first stirring, mixing the obtained calcium ion solution and Fe3O4 nanoparticles, and sequentially performing ultrasonic dispersion and a second stirring to obtain a dispersion containing calcium and Fe3O4 cores; dissolving the phosphorus-containing compound in a second portion of water, and dropwise adding the obtained phosphorus-containing solution to the dispersion containing calcium and Fe3O4 cores to obtain a dispersion containing calcium, phosphorus and Fe3O4 cores.

[0043] As an embodiment, the first stirring is carried out at room temperature; the first stirring rate is 50 to 150 rpm, specifically 100 rpm, and the time is 5 to 10 minutes, specifically 10 minutes; the calcium concentration in the calcium ion solution is 0.4 to 0.7 mol / L, specifically 0.5 mol / L.

[0044] As an embodiment, the power of the ultrasonic dispersion is 150-200 W, specifically 180 W in a specific embodiment, the frequency is 30-50 kHz, specifically 40 kHz in a specific embodiment, the temperature is 40-60° C., specifically 40-50° C. in a specific embodiment, and the time is 5-10 min, specifically 5 min in a specific embodiment; the second stirring is carried out at room temperature; the second stirring rate is 300-500 rpm, specifically 400 rpm in a specific embodiment, and the time is 5-10 min, specifically 10 min in a specific embodiment; the phosphorus concentration in the phosphorus-containing solution is 0.8-1.5 mol / L, specifically 1.2 mol / L in a specific embodiment; the dropwise addition rate is 3-5 mL / min, specifically 4-5 mL / min in a specific embodiment.

[0045] As an embodiment, the pH value of the obtained dispersion containing calcium, phosphorus and Fe3O4 cores is adjusted to 10.5; the reagent used to adjust the pH value is NH3·H2O.

[0046] As an embodiment, the temperature of the hydrothermal reaction is 170-190°C, specifically 180°C in a specific embodiment; the time of the hydrothermal reaction is 8-12 hours, specifically 10 hours in a specific embodiment; the equipment used for the hydrothermal reaction is a hydrothermal reactor; the volume of the reaction liquid containing calcium, phosphorus and Fe3O4 nuclei is ≤70% of the volume of the hydrothermal reactor, specifically 70% in a specific embodiment; the material of the hydrothermal reactor is polytetrafluoroethylene.

[0047] In the hydrothermal reaction, calcium salt and phosphorus-containing compound react to form hydroxyapatite. When the calcium salt is calcium hydroxide and the phosphorus-containing compound is phosphoric acid, the reaction formula is as follows: 10Ca(OH)2+6H3PO4=Ca 10 (PO4)6(OH)2↓ + 18H2O. The resulting hydroxyapatite is coated on the surface of Fe3O4 nanoparticles to form a core-shell structure, resulting in core-shell magnetic hydroxyapatite (mHA). This invention uses a hydrothermal method to prepare core-shell particles with both magnetic responsiveness and biological activity, providing a controllable unit for subsequent gradient distribution.

[0048] As an embodiment, after the hydrothermal reaction is completed, it also includes: separating the solid-liquid product of the hydrothermal reaction, washing the obtained precipitate with water and alcohol in sequence, and freeze-drying to obtain core-shell structured magnetic hydroxyapatite; the solid-liquid separation is centrifugation; the centrifugation temperature is 20-25°C, 25°C in the specific embodiment, the rotation speed is 10,000-15,000 rpm, 10,000-13,000 rpm in the specific embodiment, and the time is 15-20 min, 15 min in the specific embodiment; the reagent used for the water washing is deionized water; the reagent used for the alcohol washing is ethanol; the number of water washing and alcohol washing is independently 3 to 5 times, 3 to 4 times in the specific embodiment; the freeze-drying temperature is -60 to -20°C, -60 to -40°C in the specific embodiment, and the time is 16 to 24 h, 24 h in the specific embodiment.

[0049] As an embodiment, the number average molecular weight of the polycaprolactone (PCL) is 25k~50k, and in a specific embodiment, it is 25~40k; the mass of the core-shell structured magnetic hydroxyapatite is 10~30% of the mass of the polycaprolactone, and in a specific embodiment, it is 10%, 20% or 30%; the ratio of the mass of the polycaprolactone to the volume of dichloromethane is (2~4)g:40mL, and in a specific embodiment, it is 2g:40mL. The number average molecular weight of PCL will affect the viscosity of the composite melt formed in the subsequent process. The present invention sets the number average molecular weight of PCL within the above range to avoid excessive viscosity that is not conducive to the movement of the prepared mHA therein, resulting in an unclear continuous gradient. Polycaprolactone has good solubility in dichloromethane, and dichloromethane is relatively low in toxicity and volatile, making it easy to remove.

[0050] As an embodiment, the preparation method of the dispersion containing core-shell structure magnetic hydroxyapatite, polycaprolactone and dichloromethane is as follows: polycaprolactone is dissolved in dichloromethane, core-shell structure magnetic hydroxyapatite is added to the obtained polycaprolactone solution, and ultrasonic diffusion and stirring are carried out in sequence to obtain a dispersion containing core-shell structure magnetic hydroxyapatite, polycaprolactone and dichloromethane; the concentration of the polycaprolactone solution is 5-10% (w / v), and in a specific embodiment, it is 5% (w / v); the power of the ultrasonic diffusion is 1 The power is 50 to 200 W, 180 W in a specific embodiment; the frequency is 30 to 50 kHz, 40 kHz in a specific embodiment; the temperature is 40 to 60° C., 50° C. in a specific embodiment; the time is 30 min to 2 h, 2 h in a specific embodiment; the stirring is mechanical stirring; the stirring rate is 400 to 600 rpm, 500 rpm in a specific embodiment; the temperature is 20 to 25° C., 25° C. in a specific embodiment; the time is 1 to 2 h, 1 h in a specific embodiment. The present invention uses ultrasonic diffusion to break up the agglomeration of magnetic hydroxyapatite particles containing a core-shell structure, and then uses mechanical stirring to uniformly disperse mHA in PCL to prepare a uniformly dispersed magnetic composite melt, providing a base material for subsequent linear gradient magnetic field treatment.

[0051] In one embodiment, the drying comprises sequentially performing room temperature drying and high-temperature drying; the room temperature drying is performed at a temperature of 20-25°C, specifically 25°C in a specific embodiment, for a time of 48-72 hours, specifically 72 hours in a specific embodiment; the high-temperature drying is performed at a temperature of 40-50°C, specifically 40°C in a specific embodiment, for a time of 16-24 hours, specifically 24 hours in a specific embodiment. The present invention uses room temperature drying to volatilize the dichloromethane, and then uses high-temperature drying to set the mHA-PCL composite material.

[0052] In one embodiment, prior to drying, the process further includes placing the dispersion containing the core-shell magnetic hydroxyapatite, polycaprolactone, and dichloromethane in a container. The container is not particularly limited in the present invention, and any container known in the art can be used. In a specific embodiment of the present invention, the container used is a glass culture dish.

[0053] In one embodiment, after drying, the method further includes removing a 1 mm thick bottom portion of the dried solid by grinding and polishing; the grinding and polishing equipment used is a metallographic grinding and polishing machine; the grinding and polishing speed is 40 to 50 r / min, specifically 50 r / min, and the grinding and polishing time is 20 to 30 minutes, specifically 30 minutes. The present invention removes uncoated Fe3O4 particles that may have settled at the bottom of the mHA-PCL composite material by grinding and polishing, thereby uniformly dispersing the mHA in the PCL to obtain a uniform mHA-PCL composite material.

[0054] The present invention ensures the uniform initial distribution of mHA in the mHA-PCL composite material through ultrasonic diffusion and polishing treatment, providing a stable basis for subsequent gradient migration under the action of a linear gradient magnetic field, avoiding gradient unevenness caused by mHA particle agglomeration, and further improving the reliability of the method.

[0055] After obtaining the mHA-PCL composite raw material, the present invention heats the mHA-PCL composite raw material to a molten state to form a composite melt. After the composite melt is treated with a linear gradient magnetic field, melt electrowriting is performed to obtain a composition gradient melt electrowriting stent.

[0056] In one embodiment, the heating temperature is 110-130°C, specifically 120°C, for 8-10 hours, specifically 10 hours. Prior to heating, the mHA-PCL composite is crushed, and the crushing is performed by shearing. The present invention does not specifically limit the size of the crushed mHA-PCL composite; sizes known in the art may be used.

[0057] As an embodiment, the temperature of the linear gradient magnetic field treatment is 110-130° C., specifically 120° C., and the time is 15-45 minutes, specifically 30 minutes.

[0058] In one embodiment, the linear gradient magnetic field treatment involves placing the composite melt in a linear gradient magnetic field generated by passing an electric current through a magnetic field generator. In a specific embodiment, a barrel containing the composite melt is assembled with a double-walled heating vessel, ensuring that the axis of the composite melt coincides with the axis of the magnetic field generator. The composite melt is then placed in the linear gradient magnetic field generated by the magnetic field generator. The current is 5 to 10A, and in a specific embodiment, 8A. The magnetic field generator includes a Helmholtz coil or an anti-Helmholtz coil, and in a specific embodiment, a Helmholtz coil. The alignment of the melt axis and the magnetic field generator axis facilitates simulation and measurement of magnetic field strength, and the fixed position facilitates comparison across multiple experiments.

[0059] In one embodiment, the Helmholtz coils comprise a pair of coaxial, parallel circular coils. Currents flowing in the same direction flow through the two coils, generating a magnetic field distribution in which the central 50% of the coil's axial length is a uniform magnetic field, while the 25% of the coil's axial length on either side is a linear gradient magnetic field. The radius R of the two coils is 100 to 300 nm, specifically 100 mm in the embodiment, and the spacing d = R ensures a uniform magnetic field in the central region. The current I in the same direction is 5 to 10 A, specifically 8 A in the embodiment. The spacing between the two coils is equal to the coil radius, ensuring a uniform magnetic field in the central region.

[0060] As an embodiment, the structure of the anti-Helmholtz coil is the same as that of the Helmholtz coil, but reverse currents are passed through the two circular coils to generate a linear gradient magnetic field that changes linearly along the axis without a uniform area; the reverse currents are currents in opposite directions in the two circular coils, specifically 5 to 10 A is passed through one circular coil and -5 to -10 A is passed through the other circular coil. In a specific embodiment, 8 A is passed through one circular coil and -8 A is passed through the other circular coil.

[0061] The present invention utilizes the magnetophoretic effect to drive the directional migration of mHA in a composite melt, forming a continuous concentration gradient of hydroxyapatite (HA). Under the action of the magnetophoretic force of the gradient magnetic field, the mHA particles in the composite melt are distributed in a gradient according to the magnetic induction intensity of the gradient magnetic field.

[0062] The formula for magnetophoretic force is:

[0063] in:

[0064] F is the magnetophoretic force, which drives the migration of mHA particles;

[0065] V is the volume of mHA particles, which is determined by the Fe3O4 core particle size (200–300 nm);

[0066] χ p is the magnetic susceptibility of mHA particles (Fe3O4 is ferrimagnetic, (χ p >0));

[0067] χ m is the magnetic susceptibility of PCL melt (non-magnetic medium χ m ≈0);

[0068] μ0 is the magnetic permeability of vacuum;

[0069] B is the magnetic induction intensity, measured by a Tesla meter. The gradient magnetic field region causes mHA to migrate in the direction of increasing magnetic induction intensity (positive magnetophoresis).

[0070] As an embodiment, the equipment used for melt electrowriting (MEW) includes an air compressor, a barrel, a syringe, a heating module, a nozzle, a collecting plate and a high-voltage power supply.

[0071] In one embodiment, the melt electrographing process comprises loading the composite melt treated with the linear gradient magnetic field into the barrel of the melt electrographing apparatus, connecting a nozzle, which is then connected to the positive terminal of a high-voltage power supply, and a collecting plate connected to the negative terminal of the high-voltage power supply and grounded. The collecting plate is then activated to perform printing. The composite melt jet is refined and deposited under the action of the electric field, and after cooling, a composition-gradient melt electrographing support is obtained.

[0072] As an embodiment, the parameters of the melt electrowriting printing include: a heating temperature of 150-170°C, 160°C in a specific embodiment; an air supply pressure of 0.2-0.3 MPa, 0.25 MPa in a specific embodiment; a nozzle-collection plate voltage of 3.0-4.0 kV, 3.5 kV in a specific embodiment; a nozzle-collection plate spacing of 2-8 mm, 5 mm in a specific embodiment; a moving speed of the collection plate of 5-15 mm / s, 10 mm / s in a specific embodiment; a diameter of the collection plate of 100-300 mm, 150 mm in a specific embodiment; the collection plate is a round copper sheet; the thickness of the collection plate is 3-20 mm, 5-10 mm in a specific embodiment; the nozzle is a 22G double-threaded nozzle.

[0073] The present invention sets the heating temperature within the above range to ensure the fluidity of the composite melt; sets the gas supply pressure within the above range to drive the extrusion of the composite melt; the nozzle-collecting plate voltage is within the above range to form a Taylor cone and stretch the jet; the collecting plate moving speed is within the above range to control the fiber deposition accuracy.

[0074] The present invention does not specifically limit the structure of the composition-gradient melt electrographing support, and can be selected according to actual application needs. In the present invention, the composition-gradient melt electrographing support is a square grid support, each grid side length is 3 to 7 mm, specifically 5 mm in the embodiment, and the number of layers is 3 to 7, specifically 5 layers in the embodiment.

[0075] In the present invention, the basic principle of melt electrowriting (MEW) is to apply an electric field to the composite melt jet, so that the molten polymer (PCL) is stretched into micron or submicron fibers under the action of the electric field force and deposited into a three-dimensional structural scaffold.

[0076] The present invention utilizes the magnetic response characteristics of the core-shell structure mHA: the Fe3O4 core gives mHA magnetophoretic migration ability, and the HA shell retains biological activity, ensuring that the mHA particles do not lose their bone induction function when they move in a direction in the magnetic field; through precise control of the gradient magnetic field: the "middle uniform magnetic field + two-side linear gradient magnetic field" generated by the Helmholtz coil or the "linear gradient magnetic field" of the anti-Helmholtz coil, the migration direction of mHA is controlled by magnetophoretic force, so that the HA concentration changes continuously along the thickness / length direction of the scaffold, avoiding the discrete gradient defects of traditional methods, thereby driving the directional migration of mHA through the magnetic field, and realizing a continuous linear gradient distribution of the HA content inside the scaffold, which is highly consistent with the calcification gradient of natural bone and cartilage tissue, and significantly improving the biomimetic performance.

[0077] The present invention utilizes the directional driving effect of an external magnetic field on mHA, without the need for physical separation or multiple mixing of the melt. By adjusting the magnetic field parameters (current, processing time), the composition gradient construction can be completed within 30 to 60 minutes. Compared with the "multiple material replacement-mixing-printing" process of the traditional electrowriting method (which requires multiple replacements of melts with different HA concentrations to prepare scaffolds with different HA concentrations, which is cumbersome and time-consuming), at least 2 to 3 melt replacement steps are reduced; the present invention can form a gradient distribution in the melt through a single magnetic field treatment, combined with melt electrowriting technology for one-time printing and molding, which simplifies the production steps and significantly improves production efficiency.

[0078] The present invention uses a hydrothermal method to in situ grow HA shell layer, so that the surface of mHA particles is rich in hydroxyl (-OH) and phosphate (PO4 3- ) groups, forming hydrogen bonds with PCL polymer chains, improving the compatibility of particles and melts, and avoiding particle agglomeration or shedding caused by traditional mechanical mixing; the melt electrowriting process does not introduce organic solvents, and the PCL and mHA used have good biocompatibility. The gradient of HA concentration gradually increasing from the cartilage side to the bone side is consistent with the mineralization gradient of the natural bone and cartilage interface, providing stem cells with a gradual microenvironment from "chondrogenic differentiation induction" to "osteogenic differentiation induction", and promoting the natural transition of the tissue repair interface. The component gradient melt electrowriting scaffold prepared by the present invention has dual optimization of biocompatibility and functionality. The core-shell structure mHA has both the magnetic responsiveness of Fe3O4 and the biological activity of HA. The gradient distribution not only simulates the natural tissue calcification structure, but also provides the possibility for subsequent magnetic hyperthermia. mHA can generate heat in an alternating magnetic field to promote bone repair. Compared with pure PCL scaffolds or discrete gradient scaffolds, it improves cell adhesion rate and osteogenic differentiation efficiency.

[0079] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention, but they should not be understood as limiting the scope of protection of the present invention.

[0080] Example 1

[0081] (1) Preparation of core-shell structured mHA

[0082] 1) Raw materials: Fe3O4 nanoparticles (particle size 200 nm, 2 g), Ca(OH)2 (1.475 g), H3PO4 solution (85 wt%, 1.372 g), NH3·H2O (to adjust pH), and deionized water (50 mL).

[0083] 2) Operation steps:

[0084] Ca(OH)2 was dissolved in 40 mL of deionized water and stirred at 100 rpm for 10 min at room temperature to form a white suspension - a calcium ion solution containing 0.5 mol / L of calcium; Fe3O4 nanoparticles were added, and ultrasonic dispersion was carried out at 40 kHz and 180 W for 5 min at 40°C, and then mechanically stirred at 400 rpm for 10 min at room temperature to form a uniform mixed solution; H3PO4 was diluted to 10 mL, and the resulting phosphoric acid solution containing 1.2 mol / L of phosphorus was slowly added dropwise to the dispersion containing calcium and Fe3O4 cores at a rate of 5 mL / min. At the same time, NH3·H2O was added dropwise to maintain the pH at 10.5 and the total volume was controlled at 60 mL. The resulting reaction solution containing phosphorus, calcium and Fe3O4 cores was transferred to a 100 mL polytetrafluoroethylene hydrothermal reactor, sealed, and heated at 180°C for 10 h. The precipitate was collected by centrifugation at 10,000 rpm at 25°C for 15 min, washed three times with deionized water and ethanol, and freeze-dried at -60°C for 24 h to obtain core-shell magnetic hydroxyapatite mHA (Fe3O4@HA, HA shell thickness of 30 nm, Fe3O4 nanoparticle size of 200 nm).

[0085] (2) Preparation of mHA-PCL composite materials

[0086] 1) Raw materials: PCL particles (2 g, number average molecular weight 25k), dichloromethane (40 mL), mHA (0.2 g, 10% of the mass of PCL particles).

[0087] 2) Operation steps:

[0088] PCL particles were dissolved in dichloromethane to form a 5% (w / v) solution, and mHA was added. After ultrasonic diffusion at 40 kHz and 180 W at 50°C for 2 h, the dispersion was mechanically stirred at 200 rpm at 25°C for 1 h. The resulting dispersion was dried at 25°C for 72 h and then at 40°C for 24 h. It was polished at 50 r / min for 30 min using a metallographic grinder to remove 1 mm of the bottom (to remove possible precipitated Fe3O4) to obtain a uniform mHA-PCL composite raw material.

[0089] 3) Helmholtz coil assisted linear gradient magnetic field processing

[0090] Magnetic field parameters:

[0091] The invention consists of a pair of coaxial and parallel circular coils with a coil radius of R = 100 mm and a spacing of d = R = 100 mm. A current of I = 8 A (current in the same direction) is passed through the coil to form a uniform magnetic field of 50 mm in the middle (magnetic induction intensity of 12.15 mT) and a linear gradient magnetic field of 25 mm on each side (increasing on the left and decreasing on the right). The mHA-PCL composite raw material is cut into pieces and loaded into a barrel. The raw material is heated in a blast oven at 120 ° C for 10 h to a molten state to form a composite melt. The barrel containing the composite melt is assembled with a double-walled heating container to ensure that the axis of the composite melt coincides with the axis of the coil and is placed in the right-segment decreasing linear gradient magnetic field (such as Figure 1 ), and then heated at 120°C for 30 min for linear gradient magnetic field treatment. mHA was driven by magnetophoretic force to migrate to the left in the right decreasing linear gradient magnetic field region and stopped migrating in the uniform magnetic field region.

[0092] 4) Melt electrowriting bracket printing

[0093] Melt electrowriting process Figure 2 As shown, specifically:

[0094] The mHA-PCL composite raw material after magnetic field treatment was loaded into the MEW equipment barrel and connected to a 22G double-threaded nozzle; the nozzle was connected to the positive electrode of the high-voltage power supply, and the collecting plate (a round copper sheet with a diameter of 150 mm and a thickness of ()) was connected to the negative electrode and grounded. The equipment parameters were as follows: heating temperature 160 ° C, gas supply pressure 0.25 MPa, nozzle-collecting plate voltage 3.5 kV, nozzle-collecting plate spacing 5 mm; the collecting plate was started (moving speed of 10 mm / s) to print a square grid bracket, and 5 layers of square grid brackets were printed, with 50 grids per layer and a side length of 5 mm for each grid. The deposition direction was perpendicular to the magnetic field axis. The melt jet was refined and deposited under the action of the electric field force, and a composition gradient melt electro-writing bracket was formed after cooling (the actual object is shown in FIG. Figure 3 shown).

[0095] Performance Testing

[0096] (1) The measurement results of the change of magnetic induction intensity on the axis of the Helmholtz coil are as follows Figure 4 shown.

[0097] from Figure 4 It can be concluded that the magnetic induction intensity on the axis of the Helmholtz coil is uniform in the middle region and changes gradually on both sides. Therefore, in the uniform magnetic field region (near the midpoint): the mHA particles are not affected by the magnetophoretic force and remain stationary.

[0098] In the left gradient magnetic field region (x<-R / 4): the magnetic induction intensity decreases as x increases, and the mHA migrates toward the direction of increasing magnetic induction intensity (toward the left coil) under the positive magnetophoretic force.

[0099] In the right gradient magnetic field region (x > +R / 4), the magnetic induction intensity decreases as x increases, and the mHA migrates toward the midpoint (the uniform magnetic field region) until the magnetophoretic force reaches zero. The magnetic induction intensity distribution along the axis of the Helmholtz coil provides a basis for precise magnetic field control in the preparation of composition-gradient scaffolds, ensuring that mHA particles migrate in the desired direction and amplitude, achieving a continuously controllable distribution of HA concentration.

[0100] (2) Composition gradient characterization

[0101] Detection method: The gradient melt electrowriting stent prepared in Example 1 was disassembled layer by layer, and the Ca concentration was measured by ICP-AES to convert the HA content. The change of HA concentration between the stent layers after Helmholtz coil treatment is as follows: Figure 5 shown.

[0102] from Figure 5 It can be concluded that the composition-gradient melt electrographed scaffold prepared in Example 1 has an HA concentration of 3.188% in the bottom layer (layer 1), 5.873% in the middle layer (layer 3), and 1.280% in the top layer (layer 5). The gradient morphology shows a symmetrical "low-high-low" linear gradient along the thickness direction, consistent with the expected magnetic field distribution. As can be seen, the HA concentration in the scaffold prepared in the Helmholtz coil treatment group increases linearly from 3.188% to 5.873% and then decreases to 1.280%, demonstrating that the present invention can achieve a continuous linear gradient distribution of HA content within the scaffold, which is highly consistent with the calcification gradient of natural osteochondral tissue and significantly improves biomimetic performance.

[0103] Example 2

[0104] Anti-Helmholtz coil-assisted fabrication of unidirectional compositionally gradient scaffolds

[0105] 1) Preparation of core-shell structured mHA

[0106] Raw materials and steps: Same as Example 1, the particle size of Fe3O4 nanoparticles is 200nm, and the thickness of HA shell is 40nm.

[0107] 2) Preparation of mHA-PCL composite materials

[0108] Raw materials and steps: Same as Example 1, except that the mass of mHA was 10% of the mass of PCL particles. The uniformity of the composite raw material was verified by scanning electron microscopy (SEM), and the dispersion spacing of mHA particles was ≤5 μm.

[0109] 3) Anti-Helmholtz coil assisted linear gradient magnetic field processing

[0110] Magnetic field parameters:

[0111] The coil structure is the same as the Helmholtz coil, but the current is reversed (8A in the left coil and -8A in the right coil), forming a linear gradient magnetic field that decreases from left to right along the axis (magnetic induction intensity is 9.96mT at the left end and 4.55mT at the right end);

[0112] The barrel filled with mHA-PCL composite material coincides with the coil axis (e.g. Figure 6 After heating at 120°C for 30 min, mHA moves to the right due to the magnetophoretic force.

[0113] 4) Melt electrowriting bracket printing

[0114] Equipment parameters: Same as Example 1, the number of printing layers is 5, and the deposition direction is parallel to the magnetic field axis (printing along the gradient direction).

[0115] Performance Testing

[0116] (1) The magnetic induction intensity changes on the axis of the anti-Helmholtz coil as follows Figure 7 shown.

[0117] from Figure 7 It can be seen that from left to right along the anti-Helmholtz coil axis, the magnetic induction intensity gradually decreases from positive (toward the left coil) to zero (near the midpoint), and then becomes negative (toward the right coil), showing a unidirectional linear gradient. Therefore, in the gradient magnetic field region, mHA particles migrate in the direction of increasing magnetic induction intensity (to the left) due to the magnetophoretic force.

[0118] (2) Changes in HA concentration between scaffold layers after anti-Helmholtz coil treatment Figure 8 As shown, the detection method is the same as in Example 1.

[0119] from Figure 8 It can be concluded that after treatment with the anti-Helmholtz coil in Example 2, the starting end of the stent (the left end of the first layer) has an HA concentration of 4.907%; the middle layer (the midpoint of the third layer) has an HA concentration of 4.104%; the ending end (the right end of the fifth layer) has an HA concentration of 3.765%; and the gradient morphology presents a unidirectional decreasing linear gradient along the printing direction, with a gradient amplitude of 1.142% / 10 mm, verifying the controllability of the anti-Helmholtz coil over the gradient direction.

[0120] Comparative Example 1

[0121] Bracket printed without magnetic field-assisted treatment

[0122] 1) Preparation of core-shell structured mHA

[0123] Raw materials and steps: Same as Example 1, the particle size of Fe3O4 nanoparticles is 200nm, and the thickness of the HA shell is 40nm.

[0124] 2) Preparation of mHA-PCL composite materials

[0125] Raw materials and steps: Same as Example 1, except that the mass of mHA was 10% of the mass of PCL particles. The uniformity of the composite raw material was verified by scanning electron microscopy (SEM), and the dispersion spacing of mHA particles was ≤5 μm.

[0126] 3) Control variable processing

[0127] As in Example 1, the barrel was placed in the Helmholtz coil, but no current was applied. The barrel and the coil axis coincided, and the barrel was heated at 120°C for 30 minutes.

[0128] 4) Melt electrowriting bracket printing

[0129] Equipment parameters: Same as Example 1, the number of printing layers is 5, and the deposition direction is parallel to the magnetic field axis (printing along the gradient direction).

[0130] Performance Testing

[0131] The change of HA concentration between the scaffold layers without magnetic field treatment in comparative example 1 is as follows: Figure 9 As shown, the detection method is the same as in Example 1. Figure 9 It can be seen that in comparative example 1, the starting end of the stent that has not been treated with a magnetic field (the left end of the first layer): HA concentration is 3.617%; the middle layer (the midpoint of the third layer): HA concentration is 3.788%; the ending end (the right end of the fifth layer): HA concentration is 3.722%; no gradient morphology: the stent formed by printing has no gradient morphology.

[0132] Comparative Example 2

[0133] The difference from Example 1 is that PCL particles are directly used as a melt and subjected to Helmholtz coil-assisted linear gradient magnetic field treatment before melt electrowriting stent printing. The rest of the content is the same as Example 1.

[0134] Example 3

[0135] The difference from Example 1 is that the mass of mHA is 20% of the mass of PCL particles, and the rest of the contents are the same as Example 1.

[0136] Example 4

[0137] The difference from Example 1 is that the mass of mHA is 30% of the mass of PCL particles, and the rest of the contents are the same as Example 1.

[0138] Performance Testing

[0139] (1) Figure 10 TEM image of mHA prepared in Example 1.

[0140] from Figure 10It can be seen that the Fe₃O₄ nanoparticles exhibit high contrast in TEM images due to their high electron density, making the particles appear relatively bright and clear. The HA shell, due to its lower electron density, appears as a darker layer surrounding the Fe₃O₄ nanoparticles. This figure confirms that the present invention has successfully prepared magnetic hydroxyapatite (mHA) with a core-shell structure.

[0141] (2) Figure 11 This is the XRD pattern of mHA prepared in Example 1.

[0142] from Figure 11 It can be concluded that the XRD diffraction pattern of the prepared sample (mHA) shows characteristic peaks consistent with the diffraction peaks of standard HA, mainly appearing at positions such as 25.9° and 31.8° 2θ, corresponding to the (002) and (221) crystal planes of HA. The appearance of these characteristic diffraction peaks and their corresponding intensities and widths indicate that the synthesized sample contains substances with crystal structures matching those of standard HA.

[0143] (3) Figure 12 This is the FTIR spectrum of mHA (Fe3O4@HA) prepared in Example 1.

[0144] from Figure 12 It can be concluded that the FTIR spectrum shows absorption peaks related to the characteristic functional groups of HA molecules. 3- The characteristic absorption peak of the group appears at 1030~1093cm -1 , 960cm -1 and 556cm -1 OH - The group at 3570 cm -1 There is a strong absorption peak on the left and right, corresponding to OH - stretching vibration, at 626cm -1 A weak absorption peak will appear near - The appearance of these characteristic absorption peaks indicates that the sample contains HA functional groups, further verifying the successful synthesis of mHA.

[0145] (4) Figure 13 Figure 3 is a stress-strain curve of the mHA-polycaprolactone composite scaffold with different HA concentrations, where 0Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Comparative Example 2, 10Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 1, 20Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 3, and 30Hap-PCL refers to the mHA-polycaprolactone composite scaffold prepared in Example 4.

[0146] from Figure 13It can be concluded that HA, as a rigid filling material, can significantly improve the mechanical properties and elastic modulus of the material when added to the polymer: when the HA doping amount is increased from 0wt% to 30wt%, the fracture stress of the mHA-polycaprolactone composite scaffold increases from 7.3MPa to 13.5MPa, but the elongation at break decreases from 47% to 19%. It can be seen that the incorporation of HA will promote the orderly arrangement of PCL molecules, improve the crystallinity of the mHA-polycaprolactone composite scaffold material, and thus enhance its tensile strength.

[0147] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a composition gradient melt electrowriting support with magnetic assistance, characterized in that: The following steps are involved: The dispersion containing core-shell structured magnetic hydroxyapatite, polycaprolactone and dichloromethane is dried to obtain a mHA-PCL composite material; The core-shell structured magnetic hydroxyapatite comprises Fe3O4 nanoparticles and a hydroxyapatite shell layer coated on the surface of the Fe3O4 nanoparticles; The mHA-PCL composite raw material is heated to a molten state to form a composite melt. The composite melt is treated with a linear gradient magnetic field and then subjected to melt electrowriting printing to obtain a composition gradient melt electrowriting stent.

2. The method according to claim 1, characterized in that The particle size of the Fe3O4 nanoparticles is 200-300 nm; the thickness of the hydroxyapatite shell is 10-50 nm.

3. The method according to claim 1, characterized in that The number average molecular weight of the polycaprolactone is 25k-50k; the mass of the core-shell structured magnetic hydroxyapatite is 10-30% of the mass of the polycaprolactone.

4. The method according to claim 1, wherein The linear gradient magnetic field treatment is to place the composite melt in a linear gradient magnetic field generated by passing current through a magnetic field generator.

5. The method according to claim 4, characterized in that The magnetic field generator includes a Helmholtz coil or an anti-Helmholtz coil.

6. The method according to claim 5, characterized in that The Helmholtz coil is composed of a pair of coaxial and parallel circular coils, and the two circular coils are fed with current in the same direction; the structure of the anti-Helmholtz coil is the same as that of the Helmholtz coil, but the two circular coils are fed with current in opposite directions; the radius of the two circular coils is R = 100 to 300 nm, and the spacing d = R.

7. The method according to claim 1, characterized in that The temperature of the linear gradient magnetic field treatment is 110-130° C., and the time is 15-45 minutes.

8. The method according to claim 1, characterized in that The parameters of the melt electrowriting printing include: a heating temperature of 150 to 170° C.; a gas supply pressure of 0.2 to 0.3 MPa; a nozzle-collection plate voltage of 3.0 to 4.0 kV; a nozzle-collection plate spacing of 2 to 8 mm; and a collection plate movement speed of 5 to 15 mm / s.

9. The method according to claim 1, characterized in that The heating temperature is 110-130° C. and the heating time is 8-10 hours.

10. The method according to claim 4, characterized in that The input current is 5 to 10A.

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