3D printed porous titanium alloy stent loaded with self-assembled BMP2 shell-core microsphere sustained release system and preparation method thereof

Abstract

The invention discloses a 3D printing porous titanium alloy stent loaded with a self-assembly BMP2 shell-core microsphere slow release system and a preparation method thereof.The 3D printing porous titanium alloy stent comprises a 3D printing porous titanium alloy stent body and BMP2 shell-core microspheres, the BMP2 shell-core microspheres are laid on the 3D printing porous titanium alloy stent body, the 3D printing porous titanium alloy stent body is of an X-shaped structure, and the BMP2 shell-core microspheres are laid on the 3D printing porous titanium alloy stent body. And the X-shaped structure is formed by stacking round ball heads and is arranged in an array. The BMP2-PLA-Alg shell-core microspheres prepared by the method have the characteristic of uniform and controllable particle size, can realize effective adsorption and good slow release of BMP2, are physically embedded with a 3D printed titanium alloy stent, are effectively bonded with a PDA coating, and can be used as a fixed interface to promote bone growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system and a preparation method thereof. Background Art

[0002] With the aging of the population, sports or accidents, osteoarthritis has become one of the world's top disabling diseases. At present, the most effective surgical method for treating osteoarthritis is artificial joint replacement, which can eliminate joint pain, improve and restore joint function, and improve the quality of life of osteoarthritis patients. Common fixation methods for artificial joint prostheses mainly include bone cement fixation and biological fixation. The traditional bone cement fixation method is to inject bone cement to produce a mechanical noose and volume filling between the prosthesis and the bone cavity to achieve immediate fixation after surgery. However, after bone cement is implanted into the human body, bone cement leakage will occur, causing bone dissolution and bone absorption around the prosthesis, and eventually leading to loosening and failure of the prosthesis. In addition, it is easy to cause bone cement reaction syndrome, accompanied by a certain sudden death rate; the new biological fixation is to construct a tiny pore structure to allow bone tissue to gradually grow into the prosthesis, which is a more reliable fixation method. However, if biological fixation is only achieved by the autonomous growth of bone tissue into the prosthesis, there is a slow fixation process, weak early bonding, and a greater risk of falling off. Therefore, it is necessary to study and design a biological fixation method that can shorten the bone ingrowth time, enhance the bonding strength of the fixation interface, and promote bone growth.

[0003] The porosity of bone implants should be above 50%, and the pore size should be between 100-1000μm. Ti6Al4V (Ti4 material) has excellent strength, hardness, corrosion resistance, long service life, lighter weight, can withstand the complex movement and load of human joints, and is more comfortable to use. It has highly controllable molding performance through 3D printing laser melting technology (SLM) and is widely used in joint prostheses. However, compared with ceramic materials and polymer materials, metal materials have poor biological activity and need to introduce active substances that promote bone growth to promote cell recognition and adhesion and accelerate the bone ingrowth process.

[0004] Different stages of bone tissue repair and reconstruction are regulated by different growth factors. The most important osteogenic factor is bone morphogenetic proteins-2 (BMP2). BMP2 is widely involved in the maintenance of the basic physiological functions of bone tissue and various stages of bone tissue repair, and can transform bone marrow mesenchymal stem cells into osteocytes. The concentration and activity of BMP2 directly affect the division and differentiation rate of osteocytes. However, as a protein, the activity of BMP2 is easily affected by the external environment. It will be inactivated under excessively high temperatures and extreme acid-base conditions. Free BMP2 cannot effectively promote bone repair, but will cause heterotopic ossification, bone cysts and bone tumors. Excessive BMP2 will trigger the defense mechanism of the immune system and cause inflammation. Therefore, BMP2 needs to be fixed with an embedding system with a stable and sustained release effect, and organically combined with a 3D printed porous scaffold to achieve the purpose of shortening bone ingrowth time, enhancing the bonding strength of the fixed interface, and promoting bone growth. Therefore, in order to solve the above problems, the present application proposes a 3D printed porous titanium alloy scaffold loaded with a self-assembled BMP2 core-shell microsphere sustained-release system and a preparation method thereof. Summary of the invention

[0005] Technical problem to be solved: In view of the above technical problem, the present invention provides a 3D printed porous titanium alloy scaffold loaded with a self-assembled BMP2 core-shell microsphere sustained-release system and a preparation method thereof, which specifically includes two parts: (1) based on the emulsified solvent volatilization method, polylactic acid microspheres with uniform particle size and good monodispersity are obtained, and BMP2 is grafted onto the microspheres after heparinization modification to obtain BMP2-PLA microspheres; based on the electrostatic spraying technology, the above microspheres are physically encapsulated to obtain BMP2-PLA-Alg core-shell microspheres with sustained-release function; (2) based on SolidWorks software and a metal 3D printer, an X-shaped porous titanium alloy scaffold is designed and prepared, and a PDA coating is performed to obtain a PDA-X-Ti4 scaffold whose mechanical properties and porosity match those of human bone tissue; BMP2-PLA-Alg core-shell microspheres and porous PDA-X-Ti4 scaffolds have potential application value in the field of promoting bone growth at the artificial joint fixation interface.

[0006] Technical solution: In the first aspect, the present invention provides a 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system, comprising a 3D printed porous titanium alloy stent and BMP2 core-shell microspheres, wherein the BMP2 core-shell microspheres are spread flat on the 3D printed porous titanium alloy stent, and the 3D printed porous titanium alloy stent is an X-shaped structure, wherein the X-shaped structure is composed of stacked circular ball heads and arranged in an array.

[0007] Preferably, the BMP2 core-shell microspheres are BMP2-PLA-Alg core-shell microspheres, which include core microspheres made of polylactic acid and shell microspheres made of calcium alginate.

[0008] Preferably, the 3D printed porous titanium alloy stent is a porous PDA-X-Ti4 stent, the surface of which is modified with a polydopamine coating.

[0009] In a second aspect, the present invention provides a method for preparing a 3D printed porous titanium alloy scaffold loaded with a self-assembled BMP2 core-shell microsphere sustained-release system as described in the first aspect, comprising the following steps:

[0010] S1, preparation of porous PDA-X-Ti4 scaffold;

[0011] S2, preparation of BMP2-PLA-Alg core-shell microspheres;

[0012] S3. Spread the BMP2-PLA-Alg core-shell microspheres prepared in step S2 on the porous PDA-X-Ti4 scaffold, drip PBS buffer solution, allow the core-shell microspheres to penetrate into the porous PDA-X-Ti4 scaffold, and swell by absorbing liquid, finally obtaining a 3D printed porous titanium alloy scaffold loaded with a self-assembled BMP2 core-shell microsphere sustained-release system.

[0013] Preferably, step S1 includes the following specific steps:

[0014] S1-1, using Solidworks software to create a three-dimensional model of the X-shaped cell structure, and using selective laser melting technology to obtain the X-Ti4 bracket;

[0015] S1-2, performing stress relief annealing on the X-Ti4 bracket obtained in step S1-1 by a heat treatment process, the annealing temperature is 750° C. to 980° C., and then cooling to room temperature with the furnace;

[0016] S1-3, using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 5 to 10 minutes until the unmelted titanium alloy powder on the stent surface is removed;

[0017] S1-4, ultrasonically clean the acid-etched X-Ti4 stent with analytically pure acetone, ethanol and deionized water in turn for 10 minutes, repeatedly clean until the ultrasonic liquid no longer becomes turbid, and then dry and store;

[0018] S1-5. Dissolve dopamine in Tris buffer to prepare a dopamine solution with a mass concentration of 1.0-2 mg / mL. Place the cleaned X-Ti4 scaffold in the dopamine solution for 10-12 hours of constant temperature shaking for polymerization to obtain a porous PDA-X-Ti4 scaffold, which is then dried at 37-40°C for storage and set aside.

[0019] Preferably, step S2 includes the following specific steps:

[0020] S2-1, polylactic acid PLA and dichloromethane solution are mixed at a mass ratio of 1:19-99 as a continuous phase; polyvinyl alcohol and pure water are heated and dissolved at a mass ratio of 1:19-99, and the obtained aqueous solution is used as a dispersed phase; after the continuous phase is fully emulsified, a mechanical stirring device is used for low-speed stirring, and the low-speed stirring speed is 600-800rpm, and 1 volume part of the continuous phase is slowly added drop by drop into 10 volume parts of the dispersed phase at room temperature, and high-speed stirring emulsification is started, and the high-speed stirring speed is 6000-9000rmp;

[0021] S2-2, after the dichloromethane is completely evaporated, the mixture is collected and washed by centrifugal separation technology, and sieved with a 900-mesh cell sieve to obtain PLA microspheres;

[0022] S2-3, adding ethylenediamine to a 0.6 mg / mL PDA / Tris solution to prepare a 0.3 mg / mL PDA / EDA / Tris solution, adding the PLA microspheres obtained in step S2-2, the mass ratio of PLA microspheres to PDA / EDA / Tris solution being 1:1-20, stirring at low speed at room temperature for 2-3 h, the stirring speed being 200-300 rpm, to obtain PDA / EDA-PLA microspheres;

[0023] S2-4, dissolving heparin sodium, 1-ethyl-(3-dimethylaminopropyl) and N-hydroxysuccinimide in MES buffer at a mass ratio of 12:4:1 to prepare a heparin sodium solution with a mass concentration of 1%, adding the PDA / EDA-PLA microspheres obtained in step S2-3, wherein the mass ratio of PDA / EDA-PLA microspheres to heparin sodium solution is 1:1-20, stirring at room temperature for 24-36 hours, to obtain Hep-PDA / EDA-PLA microspheres;

[0024] S2-5, prepare a solution with BMP2 and pure water in a mass ratio of 1:1-20, add the Hep-PDA / EDA-PLA microspheres obtained in step S2-4, wherein the mass ratio of Hep-PDA / EDA-PLA microspheres to BMP2 solution is 1:1-20, stir at low speed at room temperature for 2-3 hours to obtain BMP2-PLA microspheres, and freeze-dry for storage;

[0025] S2-6, mixing sodium alginate and pure water at a mass ratio of 1:45-99 to obtain a sodium alginate aqueous solution, adding the BMP2-PLA microspheres obtained in step S2-5 to prepare an electrostatic spray solution with a mass percentage of 0.5% to 1.5%; preparing calcium chloride and pure water at a mass ratio of 1:9 to 99 to prepare a collection solution for standby use;

[0026] S2-7, loading the electrostatic spray solution into a syringe, wherein the syringe is provided with a right-angle needle and is placed on a push pump; loading the collected solution into a disposable culture dish, and placing the disposable culture dish under the needle, and adjusting the distance between the needle tip and the collected solution;

[0027] S2-8, install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed, start the voltage control device and adjust the voltage; 10 minutes after the injection, collect the microspheres by centrifugation, wash with pure water and freeze-dry to obtain BMP2-PLA-Alg core-shell microspheres with reduced particle size.

[0028] Furthermore, in step S2-7, the distance between the needle tip and the collected solution should be 10 to 15 cm, and the needle diameter should be 20G, 25G or 30G.

[0029] Furthermore, in step S2-8, the injection speed of the injection pump is 1-2 mm / min, and the positive electrode voltage is 5-15 kV.

[0030] Beneficial effects: 1) BMP2-PLA-Alg core-shell microspheres are composed of core microspheres of polylactic acid and shell microspheres of calcium alginate. A safer physical encapsulation and covalent binding strategy is used to slow down the release and degradation of BMP2. Heparin is fixed on the surface of polylactic acid microspheres by covalent binding with BMP2, and then physically encapsulated in a calcium alginate matrix, thereby preventing BMP2 from being free in the bone growth environment and causing side effects such as ectopic ossification, bone cysts, and bone tumors; preventing excessive BMP2 from triggering the immune system's defense mechanism and causing inflammation; and avoiding chemical modifications that may damage the activity of BMP2.

[0031] 2) All materials used are biosafe materials with the characteristics of promoting bone growth and being biodegradable: Alginate is a natural polysaccharide polymer derived from brown algae, which is non-toxic, harmless, biodegradable, and widely used in food processing and biomaterials; Polylactic acid can be degraded in the human body, the intermediate metabolite is lactic acid, and the final metabolite is water and CO2, and its metabolites will not accumulate in important organs;

[0032] 3) The X-shaped Ti6Al4V porous scaffold (X-Ti4) was designed and prepared using SolidWorks and selective laser melting technology. It has high precision and basically maintains the surface morphology after acid etching. Compression tests show that its compressive strength and elastic modulus meet the requirements for implantation in the human body. At the same time, it retains a high porosity for osteoblast growth. The polydopamine (PDA) coating on the surface of the scaffold has good adhesion, hydrophilicity and biocompatibility. While adhering and fixing the core-shell microspheres, it provides cell adhesion recognition sites for the Ti4 scaffold, improving its biological performance.

[0033] 4) Taking advantage of the fact that the morphology of calcium alginate microspheres remains unchanged and the particle size decreases after freeze-drying, the freeze-dried microspheres are infiltrated into the porous scaffold; taking advantage of the fact that the calcium alginate microspheres swell after absorbing liquid, PBS buffer is injected to allow the microspheres to swell in situ and embed inside the porous scaffold; taking advantage of the adhesion property of the polydopamine coating, the microspheres are adhered to the porous scaffold and the titanium alloy material is modified for bioactivity; without changing the pore structure of the porous scaffold, the surface of the titanium alloy without bioactivity is modified, and at the same time, the active substance BMP2 is introduced by mechanical embedding and chemical bonding methods to retain the sustained release effect of the core-shell structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The three-dimensional model of the porous X-Ti4 stent of the present invention;

[0035] Figure 2 The scanning electron microscope images of the surface morphology of the PDA-X-Ti4 bracket of the present invention, wherein a is a scanning electron microscope image of the PDA-X-Ti4 bracket magnified 100 times, b is a scanning electron microscope image of the PDA-X-Ti4 bracket magnified 5k times, and c is a scanning electron microscope image of the PDA-X-Ti4 bracket magnified 50k times;

[0036] Figure 3 The compressive performance of X-Ti4 scaffold a is the stress-strain curve, and b is the compression modulus diagram;

[0037] Figure 4 Light microscopic images of shell-core microspheres with different BMP2-PLA contents;

[0038] Figure 5 An inverted microscope image of BMP2-PLA-Alg core-shell microspheres;

[0039] Figure 6 Scanning electron micrographs of Alg microspheres (a) and PLA-Alg microspheres (b) after freeze-drying;

[0040] Figure 7 The physical picture of the assembled BMP2-PLA-Alg core-shell microspheres and porous PDA-X-Ti4 scaffold;

[0041] Figure 8 is the sustained release curve of BMP2-PLA-Alg core-shell microspheres;

[0042] Fig. 9 This is a graph showing the in vitro mineralization ability of porous PDA-X-Ti4 scaffolds loaded with Alg microspheres, PLA-Alg core-shell microspheres and BMP2-PLA-Alg core-shell microspheres. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0044] Example 1

[0045] A method for preparing a 3D printed scaffold for self-assembling sustained-release BMP2 core-shell microspheres comprises the following steps:

[0046] (1) Use Solidworks software to create a three-dimensional model of the X-shaped cell structure, and obtain the X-Ti4 bracket through selective laser melting (SLM) technology;

[0047] (2) using a heat treatment process to perform stress relief annealing at 800° C. on the X-Ti4 bracket obtained in the above step (1);

[0048] (3) using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 10 minutes until the unmelted titanium alloy powder on the stent surface is removed;

[0049] (4) ultrasonically cleaning the stent for 10 minutes using analytically pure acetone, analytically pure ethanol, and deionized water, and then drying and storing;

[0050] (5) dissolving dopamine in Tris buffer (pH=8.5) to prepare a dopamine solution with a mass concentration of 2 mg / mL, placing the scaffold obtained in step (4) in the dopamine solution for 12 hours of constant temperature shaking polymerization to obtain a porous PDA-X-Ti4 scaffold, and drying and storing at 37°C;

[0051] (6) preparing a 3% solution with a mass ratio of 3:100 for polylactic acid and dichloromethane solution as a continuous phase; heating and dissolving polyvinyl alcohol and pure water at a mass ratio of 1:100 to prepare a 1% aqueous solution as a dispersed phase; using a mechanical stirring device to stir at a low speed of 800 rpm, slowly adding 1 volume part of the continuous phase dropwise to 10 volume parts of the dispersed phase at room temperature, and starting high-speed stirring for emulsification at a stirring speed of 8000 rpm;

[0052] (7) After the dichloromethane is completely evaporated, the microspheres are collected and washed by centrifugal separation technology, and sieved with a 900-mesh cell sieve to obtain PLA microspheres;

[0053] (8) Adding ethylenediamine to a 0.6 mg / mL PDA / Tris solution to prepare a 0.3 mg / mL PDA / EDA / Tris solution, adding the PLA microspheres obtained in step (7), wherein the mass ratio of PLA microspheres to PDA / EDA / Tris solution is 1:10, stirring at low speed for 2 h at room temperature, and the stirring speed is 300 rpm to obtain PDA / EDA-PLA microspheres;

[0054] (9) dissolving heparin sodium, 1-ethyl-(3-dimethylaminopropyl) and N-hydroxysuccinimide in MES buffer at a mass ratio of 12:4:1 to prepare a heparin sodium solution with a mass concentration of 1%, adding the PDA / EDA-PLA microspheres obtained in the above step (8), wherein the mass ratio of PDA / EDA-PLA microspheres to the heparin sodium solution is 1:10, stirring at room temperature for 24 hours, and obtaining Hep-PDA / EDA-PLA microspheres;

[0055] (10) BMP2 and pure water were prepared into a 1% BMP2 solution at a mass ratio of 1:10, and the Hep-PDA / EDA-PLA microspheres obtained in step S2-4 were added, wherein the mass ratio of Hep-PDA / EDA-PLA microspheres to BMP2 solution was 1:10, and the mixture was stirred at low speed for 2 h at room temperature at a stirring speed of 300 rpm to obtain BMP2-PLA microspheres, which were freeze-dried for storage;

[0056] (11) sodium alginate and pure water were uniformly mixed at a mass ratio of 3:197 to prepare a sodium alginate aqueous solution with a mass concentration of 1.5%, and then BMP2-PLA microspheres were added to prepare an electrostatic spray solution with a mass percentage concentration of 1%; calcium chloride and pure water were prepared at a mass ratio of 1:99 to prepare a 1% collection solution;

[0057] (12) The electrostatic spray solution in step (11) is loaded into a 5 mL syringe, and a right-angle needle with an inner diameter of 25G is installed and placed on a push pump; 5 mL of the collection solution prepared in step (11) is loaded into a disposable culture dish and placed under the needle tip, and the distance between the needle tip and the collection solution is adjusted to 15 cm;

[0058] (13) Install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed to 1 mm / min, start the voltage control device and adjust the voltage to 10 kV; after the injection is completed and calcified for 10 min, the microspheres are collected by centrifugation, washed with pure water and freeze-dried to obtain BMP2-PLA-Alg core-shell structure microspheres with reduced particle size;

[0059] (14) The dried BMP2-PLA-Alg core-shell structure microspheres obtained in step (13) are spread on the PDA-coated 3D printed porous titanium alloy scaffold (porous PDA-X-Ti4 scaffold), and PBS buffer is dripped into the porous PDA-X-Ti4 scaffold to allow the microspheres to penetrate into the interior of the porous PDA-X-Ti4 scaffold and swell by absorbing the liquid, thereby finally obtaining a 3D printed scaffold of self-assembled sustained-release BMP2 core-shell microspheres.

[0060] Example 2

[0061] The method for preparing a 3D printed scaffold for self-assembled sustained-release BMP2 core-shell microspheres is different from that in Example 1 in that: in step (11), sodium alginate and pure water are prepared in a mass ratio of 1:49 to prepare a sodium alginate aqueous solution with a mass concentration of 2%, and the remaining operations are the same as in Example 1.

[0062] Example 3

[0063] The method for preparing a 3D printed scaffold for self-assembling sustained-release BMP2 core-shell microspheres is different from that in Example 1 in that the inner diameter of the right-angle needle installed in step (12) is 30G, and the remaining operations are the same as in Example 1.

[0064] Example 4

[0065] The method for preparing a 3D printed scaffold for self-assembled sustained-release BMP2 core-shell microspheres is different from that in Example 1 in that: in step (11), sodium alginate and pure water are prepared into a sodium alginate aqueous solution with a mass concentration of 2% in a mass ratio of 1:49; in step (13), the voltage control device is started and the voltage is adjusted to 15 kV; and the remaining operations are the same as in Example 1.

[0066] Example 5

[0067] The method for preparing a 3D printed scaffold for self-assembled sustained-release BMP2 core-shell microspheres is different from that in Example 1 in that: the inner diameter of the right-angle needle installed in step (12) is 30G; in step (13), the voltage control device is started and the voltage is adjusted to 15 kV; and the remaining operations are the same as in Example 1.

[0068] Example 6

[0069] The method for preparing a 3D printed scaffold for self-assembled sustained-release BMP2 core-shell microspheres is different from that in Example 1 in that: in step (13), the calcification time after injection is 30 minutes, and the remaining operations are the same as in Example 1.

[0070] Comparative Example 1

[0071] A method for preparing a 3D printed scaffold for self-assembling sustained-release BMP2 core-shell microspheres comprises the following steps:

[0072] (1) Use Solidworks software to create a three-dimensional model of the X-shaped cell structure, and obtain the X-Ti4 bracket through selective laser melting (SLM) technology;

[0073] (2) using a heat treatment process to perform stress relief annealing at 800° C. on the X-Ti4 bracket obtained in the above step (1);

[0074] (3) using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 10 minutes until the unmelted titanium alloy powder on the stent surface is removed;

[0075] (4) ultrasonically cleaning the stent for 10 minutes using analytically pure acetone, analytically pure ethanol, and deionized water, and then drying and storing;

[0076] (5) dissolving dopamine in Tris buffer (pH=8.5) to prepare a dopamine solution with a mass concentration of 2 mg / mL, placing the scaffold obtained in step (4) in the dopamine solution for 12 hours of constant temperature shaking polymerization to obtain a porous PDA-X-Ti4 scaffold, and drying and storing at 37°C;

[0077] (6) preparing a 3% solution with a mass ratio of 3:100 for polylactic acid and dichloromethane solution as a continuous phase; heating and dissolving polyvinyl alcohol and pure water at a mass ratio of 1:100 to prepare a 1% aqueous solution as a dispersed phase; using a mechanical stirring device to stir at a low speed, setting the stirring speed to 800 rpm, slowly adding 1 volume part of the continuous phase dropwise to 10 volume parts of the dispersed phase at room temperature, and starting high-speed stirring for emulsification at a stirring speed of 8000 rpm;

[0078] (7) After the dichloromethane is completely evaporated, the microspheres are collected and washed by centrifugal separation technology, and sieved with a 900-mesh cell sieve to obtain PLA microspheres;

[0079] (8) sodium alginate and pure water were uniformly mixed at a mass ratio of 3:197 to prepare a sodium alginate aqueous solution with a mass concentration of 1.5%, and PLA microspheres were added to prepare an electrostatic spray solution with a mass percentage concentration of 1%; calcium chloride and pure water were prepared at a mass ratio of 1:99 to prepare a 1% collection solution;

[0080] (9) The electrostatic spray solution in step (8) is loaded into a 5 mL syringe, and a right-angle needle with an inner diameter of 25G is installed and placed on a push pump; 5 mL of the collection solution prepared in step (8) is loaded into a disposable culture dish and placed under the needle tip, and the distance between the needle tip and the collection solution is adjusted to 15 cm;

[0081] (10) Install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed to 1 mm / min, start the voltage control device and adjust the voltage to 10 kV; after the injection is completed and calcified for 10 min, the microspheres are collected by centrifugation, washed with pure water and freeze-dried to obtain PLA-Alg core-shell structure microspheres with reduced particle size;

[0082] (11) The dried PLA-Alg core-shell structure microspheres obtained in step (10) are spread on a 3D printed porous titanium alloy scaffold modified with a PDA coating (porous PDA-X-Ti4 scaffold), and PBS buffer is dripped into the porous PDA-X-Ti4 scaffold to allow the microspheres to penetrate into the interior of the porous PDA-X-Ti4 scaffold and swell by absorbing the liquid, thereby finally obtaining a 3D printed scaffold loaded with core-shell microspheres.

[0083] Comparative Example 2

[0084] The method for preparing a 3D printed scaffold for self-assembled sustained-release BMP2 core-shell microspheres is different from that of comparative example 1 in that: steps (6) and (7) are removed, and the electrostatic spray solution in step (8) is replaced by uniformly mixing sodium alginate and a 1% BMP2 aqueous solution at a mass ratio of 1:99 to prepare a sodium alginate-BMP2 aqueous solution with a mass concentration of 1%. The specific steps are:

[0085] (1) Use Solidworks software to create a three-dimensional model of the X-shaped cell structure, and obtain the X-Ti4 bracket through selective laser melting (SLM) technology;

[0086] (2) using a heat treatment process to perform stress relief annealing at 800° C. on the X-Ti4 bracket obtained in the above step (1);

[0087] (3) using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 10 minutes until the unmelted titanium alloy powder on the stent surface is removed;

[0088] (4) ultrasonically cleaning the stent for 10 minutes using analytically pure acetone, analytically pure ethanol, and deionized water, and then drying and storing;

[0089] (5) dissolving dopamine in Tris buffer (pH=8.5) to prepare a dopamine solution with a mass concentration of 2 mg / mL, placing the scaffold obtained in step (4) in the dopamine solution for 12 hours of constant temperature shaking polymerization to obtain a porous PDA-X-Ti4 scaffold, and drying and storing at 37°C;

[0090] (6) BMP2 and pure water are mixed at a mass ratio of 1:10 to prepare a 1% BMP2 solution; sodium alginate and BMP2 solution are uniformly mixed at a mass ratio of 3:197 to prepare a sodium alginate-BMP2 aqueous solution with a mass concentration of 1%, i.e., an electrostatic spray solution; calcium chloride and pure water are mixed at a mass ratio of 1:99 to prepare a collection solution;

[0091] (7) The electrostatic spray solution in step (6) is loaded into a 5 mL syringe, and a right-angle needle with an inner diameter of 25G is installed and placed on a push pump; 5 mL of the collection solution prepared in step (6) is loaded into a disposable culture dish and placed under the needle tip, and the distance between the needle tip and the collection solution is adjusted to 15 cm;

[0092] (8) Install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed to 1 mm / min, start the voltage control device and adjust the voltage to 10 kV; after the injection is completed and calcified for 10 min, the microspheres are collected by centrifugation, washed with pure water and freeze-dried to obtain BMP2-Alg core-shell structure microspheres with reduced particle size;

[0093] (9) The dried BMP2-Alg core-shell structure microspheres obtained in step (8) are spread on the 3D printed porous titanium alloy scaffold modified with a PDA coating (porous PDA-X-Ti4 scaffold), and PBS buffer is dripped into the porous PDA-X-Ti4 scaffold to allow the microspheres to penetrate into the interior of the porous PDA-X-Ti4 scaffold and swell by absorbing the liquid, thereby finally obtaining a 3D printed scaffold loaded with BMP2 microspheres.

[0094] Comparative Example 3

[0095] The method for preparing a 3D printed scaffold for self-assembling sustained-release BMP2 core-shell microspheres is different from that of comparative example 1 in that: step (6) and step (7) are removed, and the sodium alginate and pure water in step (8) are uniformly mixed at a mass ratio of 1:99 to prepare a sodium alginate aqueous solution with a mass concentration of 1%, which is used as an electrostatic spray solution. The specific steps are as follows:

[0096] (1) Use Solidworks software to create a three-dimensional model of the X-shaped cell structure, and obtain the X-Ti4 bracket through selective laser melting (SLM) technology;

[0097] (2) using a heat treatment process to perform stress relief annealing at 800° C. on the X-Ti4 bracket obtained in the above step (1);

[0098] (3) using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 10 minutes until the unmelted titanium alloy powder on the stent surface is removed;

[0099] (4) ultrasonically cleaning the stent for 10 minutes using analytically pure acetone, analytically pure ethanol, and deionized water, and then drying and storing;

[0100] (5) dissolving dopamine in Tris buffer (pH=8.5) to prepare a dopamine solution with a mass concentration of 2 mg / mL, placing the scaffold obtained in step (4) in the dopamine solution for 12 hours of constant temperature shaking polymerization to obtain a porous PDA-X-Ti4 scaffold, and drying and storing at 37°C;

[0101] (6) sodium alginate and pure water were uniformly mixed in a mass ratio of 1:99 to prepare a sodium alginate aqueous solution with a mass concentration of 1%, which was used as an electrostatic spray solution; calcium chloride and pure water were prepared in a mass ratio of 1:99 to prepare a collection solution;

[0102] (7) The electrostatic spray solution in step (6) is loaded into a 5 mL syringe, and a right-angle needle with an inner diameter of 25G is installed and placed on a push pump; 5 mL of the collection solution prepared in step (6) is loaded into a disposable culture dish and placed under the needle tip, and the distance between the needle tip and the collection solution is adjusted to 15 cm;

[0103] (8) Install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed to 1 mm / min, start the voltage control device and adjust the voltage to 10 kV; after the injection is completed and calcified for 10 minutes, the microspheres are collected by centrifugation, washed with pure water and freeze-dried to obtain Alg core-shell structure microspheres with reduced particle size;

[0104] (9) The dried Alg core-shell structure microspheres obtained in step (8) are spread on the 3D printed porous titanium alloy scaffold modified with PDA coating (porous PDA-X-Ti4 scaffold), and PBS buffer is dripped into the porous PDA-X-Ti4 scaffold to allow the microspheres to penetrate into the interior of the porous PDA-X-Ti4 scaffold and swell by absorbing liquid, finally obtaining a 3D printed scaffold loaded with microspheres.

[0105] All parameters of Examples 1-6 and Comparative Examples 1-3 are summarized in Table 1 below:

[0106] Table 1 Summary of all parameters of Examples 1-6 and Comparative Examples 1-3

[0107]

[0108] The surface morphology and performance of the BMP2-PLA-Alg core-shell microspheres obtained in Examples 1 to 6 and Comparative Examples 1 to 3 were tested, and the results were as follows:

[0109] like Figure 1As shown, the present invention discloses an X-shaped porous structure for an artificial joint fixation interface, which is composed of round ball heads stacked into an X-shaped structure and an array; a polydopamine (PDA) coating is generated on the surface of the porous bracket by an oxidative self-polymerization method, Figure 2 It can be seen that spherical PDA particles are formed on the surface of the scaffold, indicating that dopamine undergoes spontaneous oxidative polymerization on the substrate surface to form PDA.

[0110] In order to evaluate the effect of adding different contents of BMP2-PLA particles on the performance of core-shell microspheres, the light microscopy images of 0.5 mg / mL, 1 mg / mL and 1.5 mg / mL BMP2-PLA particles were compared. Figure 2 and Figure 4 As shown, it can be seen that the content of BMP2-PLA particles has a certain influence on the performance of core-shell microspheres. The content of BMP2-PLA particles at 0.5 mg / mL is too little, and the content of BMP2-PLA particles at 1.5 mg / mL is too much, which causes aggregation in the Alg microspheres and is not conducive to distribution. The content of BMP2-PLA particles at 1 mg / ml is more appropriate, and they are evenly distributed in the Alg microspheres without aggregation.

[0111] In order to evaluate the mechanical properties of the X-Ti4 stent, compression tests were performed on the stent before and after acid etching. Figure 3 Figures a and b are the stress-strain curve and compression modulus results of the porous Ti6Al4V scaffold with an X-shaped structure. The results show that the elastic modulus of the X-shaped porous Ti6Al4V scaffold before and after acid etching is 6.497 GPa and 6.155 GPa, respectively, which meets the requirements of being a load-bearing implant in the body and avoids the stress shielding effect to a large extent.

[0112] The appearance and surface morphology of the microspheres obtained in Example 1 and Comparative Example 2 were characterized by scanning electron microscopy. Figure 6 As shown in a, the pure Alg microspheres without adding PLA obtained in Comparative Example 2 have a smooth surface after freeze-drying, a shape close to a sphere, and a complete structure; Figure 6 As can be seen in b, polylactic acid particles occasionally protrude from the surface of the BMP2-PLA-Alg core-shell microspheres obtained in Example 1, but the surface is not damaged and has no effect on the microsphere structure, indicating that the addition of polylactic acid particles has no effect on the overall structure of the microspheres while ensuring the construction of a sustained-release system.

[0113] In order to evaluate the sustained release ability of BMP2, the two groups of microspheres in Example 1 and Comparative Example 1 were immersed in PBS buffer for 200 h, and the BMP2 release curves were measured and plotted using an ultraviolet spectrophotometer. Figure 8It can be seen that both groups of microspheres showed a burst release phenomenon in the early stage, and then entered a relatively stable stage. BMP2-PLA-Alg core-shell microspheres had a relatively good sustained release ability, with less than 50% released in 200 hours.

[0114] In order to evaluate the in vitro mineralization ability of the microsphere-scaffold system, the three groups of microsphere-scaffold system samples of Example 1, Comparative Example 1 and Comparative Example 3 were immersed in simulated body fluid for 7 days, and the apatite deposition on the surface of the samples was observed by scanning electron microscopy, and the calcium and phosphorus content on the surface of the samples was analyzed by EDS. Figure 8 As shown: calcium and phosphorus elements are deposited on the surface of the three groups of microsphere-scaffold systems. The apatite growth on the surface of the sample of Example 1 is the best, showing better in vitro mineralization ability, which is beneficial to the integration of artificial joint prosthesis and human bone, and has great potential in promoting bone growth.

[0115] Therefore, the BMP2-PLA-Alg core-shell microspheres prepared in the present invention have the characteristics of uniform and controllable particle size, can achieve effective adsorption and good sustained release of BMP2, and are physically embedded with the 3D printed titanium alloy scaffold and effectively bonded with the PDA coating, and can serve as a fixed interface to promote bone growth.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system, characterized in that: It includes a 3D printed porous titanium alloy stent and BMP2 core-shell microspheres, wherein the BMP2 core-shell microspheres are laid flat on the 3D printed porous titanium alloy stent, and the 3D printed porous titanium alloy stent is an X-shaped structure, wherein the X-shaped structure is formed by stacking circular ball heads and arranged in an array.

2. A 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system according to claim 1, characterized in that: The BMP2 core-shell microspheres are BMP2-PLA-Alg core-shell microspheres, which include core microspheres of polylactic acid and shell microspheres of calcium alginate.

3. A 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system according to claim 1, characterized in that: The 3D printed porous titanium alloy stent is a porous PDA-X-Ti4 stent, the surface of which is modified with a polydopamine coating.

4. A method for preparing a 3D printed porous titanium alloy stent loaded with a self-assembled BMP2 core-shell microsphere sustained-release system according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, preparation of porous PDA-X-Ti4 scaffold; S2, preparation of BMP2-PLA-Alg core-shell microspheres; S3. Spread the BMP2-PLA-Alg core-shell microspheres prepared in step S2 on the porous PDA-X-Ti4 scaffold, drip PBS buffer solution, allow the core-shell microspheres to penetrate into the porous PDA-X-Ti4 scaffold, and swell by absorbing liquid, finally obtaining a 3D printed porous titanium alloy scaffold loaded with a self-assembled BMP2 core-shell microsphere sustained-release system.

5. The method according to claim 4, characterized in that Step S1 includes the following specific steps: S1-1, using Solidworks software to create a three-dimensional model of the X-shaped cell structure, and using selective laser melting technology to obtain the X-Ti4 bracket; S1-2, performing stress relief annealing on the X-Ti4 bracket obtained in step S1-1 by a heat treatment process, the annealing temperature is 750°C to 980°C, and then cooling to room temperature with the furnace; S1-3, using 3% by volume hydrofluoric acid and 25% by volume nitric acid to sequentially etch the stress-relieved X-Ti4 stent for 5 to 10 minutes until the unmelted titanium alloy powder on the stent surface is removed; S1-4, ultrasonically clean the acid-etched X-Ti4 stent with analytically pure acetone, ethanol and deionized water in turn for 10 minutes, repeatedly clean until the ultrasonic liquid no longer becomes turbid, and then dry and store; S1-5. Dissolve dopamine in Tris buffer to prepare a dopamine solution with a mass concentration of 1.0-2 mg / mL. Place the cleaned X-Ti4 scaffold in the dopamine solution and shake it at a constant temperature for 10-12 hours to polymerize to obtain a porous PDA-X-Ti4 scaffold, which is then dried at 37-40°C for storage.

6. The preparation method according to claim 4, characterized in that: Step S2 includes the following specific steps: S2-1, mix polylactic acid PLA and dichloromethane solution at a mass ratio of 1:19-99 as a continuous phase; heat and dissolve polyvinyl alcohol and pure water at a mass ratio of 1:19-99, and the obtained aqueous solution is used as a dispersed phase; after the continuous phase is fully emulsified, use a mechanical stirring device to stir at a low speed of 600-800 rpm, and slowly add 1 volume part of the continuous phase drop by drop into 10 volume parts of the dispersed phase at room temperature, and start high-speed stirring emulsification at a speed of 6000-9000 rmp; S2-2, after the dichloromethane is completely evaporated, the mixture is collected and washed by centrifugal separation technology, and sieved with a 900-mesh cell sieve to obtain PLA microspheres; S2-3, adding ethylenediamine to a 0.6 mg / mL PDA / Tris solution to prepare a 0.3 mg / mL PDA / EDA / Tris solution, adding the PLA microspheres obtained in step S2-2, the mass ratio of PLA microspheres to PDA / EDA / Tris solution being 1:1-20, stirring at low speed at room temperature for 2-3 hours at a speed of 200-300 rpm, to obtain PDA / EDA-PLA microspheres; S2-4, dissolving heparin sodium, 1-ethyl-(3-dimethylaminopropyl) and N-hydroxysuccinimide in MES buffer at a mass ratio of 12:4:1 to prepare a heparin sodium solution with a mass concentration of 1%, adding the PDA / EDA-PLA microspheres obtained in step S2-3, wherein the mass ratio of PDA / EDA-PLA microspheres to heparin sodium solution is 1:1-20, stirring at room temperature for 24-36 hours, to obtain Hep-PDA / EDA-PLA microspheres; S2-5, prepare a solution of BMP2 and pure water in a mass ratio of 1:1-20, add the Hep-PDA / EDA-PLA microspheres obtained in step S2-4, wherein the mass ratio of Hep-PDA / EDA-PLA microspheres to BMP2 solution is 1:1-20, stir at low speed at room temperature for 2-3 hours to obtain BMP2-PLA microspheres, and freeze-dry for storage; S2-6, mixing sodium alginate and pure water at a mass ratio of 1:45-99 to obtain a sodium alginate aqueous solution, adding the BMP2-PLA microspheres obtained in step S2-5 to prepare an electrostatic spray solution with a mass percentage of 0.5% to 1.5%; preparing calcium chloride and pure water at a mass ratio of 1:9 to 99 to prepare a collection solution for standby use; S2-7, loading the electrostatic spray solution into a syringe, wherein the syringe is provided with a right-angle needle and is placed on a push pump; loading the collected solution into a disposable culture dish, and placing the disposable culture dish under the needle, and adjusting the distance between the needle tip and the collected solution; S2-8, install a voltage control device, ground the negative electrode, start the injection pump and adjust the injection speed, start the voltage control device and adjust the voltage; 10 minutes after the injection, collect the microspheres by centrifugation, wash with pure water and freeze-dry to obtain BMP2-PLA-Alg core-shell microspheres with reduced particle size.

7. The preparation method according to claim 6, characterized in that In step S2-7, the distance between the needle tip and the collected solution should be 10-15 cm, and the needle diameter should be 20G, 25G or 30G.

8. The preparation method according to claim 6, characterized in that In step S2-8, the injection speed of the injection pump is 1-2 mm / min, and the positive electrode voltage is 5-15 kV.

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