An injectable 3D printed magnesium porous scaffold with anti-infection, pro-angiogenesis and osteogenesis functions and a preparation method thereof

Abstract

The application discloses a kind of water gel embedded 3D printing magnesium porous support with anti-infection, promote blood vessel and osteogenic function and preparation method thereof.The preparation includes:1) using metal 3D printing magnesium porous support;2) using template method to obtain MSN, load osteogenic polypeptide by combining impregnation adsorption method, and in-situ encapsulation is carried out to channel by DA in-situ self-polymerization process to obtain drug-loaded MSN-PDA;3) preparation GelMA and QCS mixed solution, then the solution is cooled and kept at 40 DEG C condition, add drug-loaded MSN-PDA, promote blood vessel polypeptide and AgNPs and mix uniformly, centrifugal defoaming;4) the obtained solution is transferred to transparent mould, magnesium porous support is also placed in above-mentioned mould;In the dark condition, ultrasonic treatment is carried out in 40 DEG C water bath;5) using spin-ultraviolet in-situ photocuring combination processing.The obtained support can release anti-infection, promote blood vessel and promote osteogenic active ingredient according to time gradient, and can effectively prevent and treat infection and promote bone tissue repair.

Description

Technical Field

[0001] This invention relates to the field of bone defect repair scaffold preparation, specifically to a hydrogel-embedded 3D printed magnesium porous scaffold with anti-infection, angiogenesis and osteogenic functions, and its preparation method. Background Technology

[0002] With the increasing number of patients suffering from various orthopedic traumas and other orthopedic diseases each year, the clinical demand for bone repair materials is growing. Autologous bone or allogeneic bone grafting is the gold standard for treating bone defects. However, autologous bone grafting has problems such as additional trauma and insufficient supply; allogeneic bone grafting has problems such as rejection and high cost. At the same time, implant-associated infections (IAI) are the most common cause of defect repair failure, with an incidence of about 5%, and a high recurrence rate, high treatment cost, poor treatment effect, and high mortality rate. Therefore, the development of a new bone implant material to address the above problems is urgent.

[0003] In recent years, with the rapid development of tissue engineering technology, materials such as scaffolds and hydrogels have gradually shown great application potential in the field of bone repair. However, many tissue engineering materials currently have relatively limited functions and generally suffer from insufficient bioactivity, poor anti-infection effects, inability to promote osteogenesis, or even delayed osteogenesis. In addition, simple drug-loaded scaffolds have the problem of initial drug burst release, manifested as excessively high early drug concentrations and shortened duration of action, leading to severe early cytotoxicity and late-stage secondary infections.

[0004] To address the aforementioned issues, this invention presents a hydrogel-embedded 3D-printed magnesium porous scaffold that combines anti-infection, angiogenesis-promoting, and osteogenic functions. The customizable scaffold can adapt to various defect shapes, and through the physical shielding effect of the porous scaffold and the multiple sustained-release functions of mesoporous silica and hydrogel, it achieves intelligent sustained-release of anti-infection and osteogenic active components, thereby achieving the goals of postoperative infection prevention and efficient repair of bone defects. Summary of the Invention

[0005] The purpose of this invention is to address clinical problems such as low osteogenic efficiency, poor anti-infection ability, and difficulty in achieving individualized customization of conventional implants. By combining the characteristics of materials such as mesoporous silica, hydrogel, and 3D printed scaffolds, this invention provides a hydrogel-embedded 3D printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions, as well as its preparation method.

[0006] This invention is achieved using the following technical solution:

[0007] A hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions is fabricated using magnesium, methacrylic anhydride gelatin (GelMA), quaternized chitosan (QCS), osteogenic peptides, nano-silver AgNPs, angiogenesis-promoting peptides, cetyltrimethylammonium bromide (CTAB), tetrasilane (TEOS), and dopamine (DA) as main raw materials. It is prepared using a combination of processes including metal 3D printing, template method, in-situ self-polymerization, adsorption method, blending method, mold method, ultrasonic process, and rotation-UV in-situ photocuring.

[0008] The specific fabrication steps of this hydrogel-embedded 3D-printed magnesium porous scaffold include the following:

[0009] 1) Magnesium was melted into a molten material using a vacuum induction melting furnace and placed in an atomizing device to form powder with a diameter of approximately 40-120 micrometers. A porous magnesium scaffold with a specific pore size was then prepared using metal 3D printing technology. 2) Mesoporous silica (MSN) was prepared using a template method and then impregnated in a PBS solution containing osteogenic peptides to load the peptides into the pores of the MSN, thus obtaining drug-loaded MSN. The drug-loaded MSN was further impregnated in a dopamine (DA) / Tris-HCl solution (pH = 8.5), and the drug-loaded MSN was encapsulated through in-situ self-polymerization of DA, thus obtaining drug-loaded MSN-PDA.

[0010] 3) Under light-protected and 60°C conditions, dissolve GelMA and QCS in PBS solution containing photoinitiator; when the completely dissolved solution is cooled to 40°C, transfer it to a 40°C water bath, then add drug-loaded MSN-PDA, AgNPs and angiogenic peptides, mix well and centrifuge at low speed to remove bubbles.

[0011] 4) Under light-protected conditions, the solution from step 3) is transferred into a transparent mold, and the porous magnesium scaffold from step 1) is also placed in the mold. Combined with ultrasonic technology, the mixed solution is allowed to penetrate into the pores of the porous scaffold. 5) The solution in the mold from step 4) is completely gelled using a rotation-in-situ ultraviolet curing process. After demolding, the gel outside the scaffold body is removed. After irradiation sterilization and encapsulation, the desired hydrogel embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions is obtained.

[0012] In the above technical solution, further, the parameters for magnesium powder printing in step 1) are: electron beam velocity of 4000-5000 mm / s, electron beam intensity of 8-15 mA, velocity parameter of 40-45, and vacuum degree in the printing chamber less than 10. -4The argon pressure was 0.02-0.06 MPa, and the sample preheating temperature was 600℃. The pore size of the 3D-printed magnesium porous scaffold was between 500-1000 micrometers to ensure that the subsequent GelMA / QCS mixed solution could smoothly enter the pores of the scaffold. At the same time, the appropriate pore size facilitated the rapid growth of cells into the scaffold. However, if the pore size was too large, the mechanical support performance of the scaffold would be difficult to guarantee.

[0013] Further, the MSN preparation process parameters in step 2) are as follows: CTAB, water, and a certain amount of sodium hydroxide are added sequentially to the reaction vessel, and stirred for 2-4 hours in a water bath at 70-80℃ with mechanical stirring at 300-500 rpm; then TEOS is quickly added, and after reacting for 12-24 hours, the precipitate is obtained by high-speed centrifugation. The precipitate is washed repeatedly with ethanol and water 3-5 times and then dried at 40-45℃. The dried product is then dispersed in ethanol to form a 5-40 mg / ml dispersion, and a certain amount of APTES is added. The mixture is stirred for 6-12 hours, and the secondary precipitate is separated by high-speed centrifugation. After washing repeatedly with ethanol 3-5 times, the product is lyophilized to obtain the desired MSN. In the above process, the amount of CTAB is 0.5-2 g / L, the amount of sodium hydroxide is to adjust the pH of the solution to 10-11, the amount of TEOS is 4-10 g / L, the amount of APTES is 5-12 ml / L, and the speed of the two high-speed centrifugations is 10000-15000 rpm, and the centrifugation time is 5-15 minutes.

[0014] Furthermore, in step 2), the osteogenic peptide is one or more of BMP-2, osteogrowth peptide (OGP), BMP-7, TGF-β, etc., and the concentration of the osteogenic peptide in the PBS solution is 30-80 μg / ml; magnetic stirring is used during impregnation at a rate of 100-200 rpm, and the impregnation time is 4-6 hours; the MSN concentration is 5-40 mg / ml, the DA concentration is 6-12 mg / ml, and the reaction time is 4-6 hours.

[0015] Furthermore, in step 3), the degree of substitution of GelMA is 60-90%, and the concentration is 50-200 g / L; the number-average molecular weight of QCS is 3000-12000, the degree of quaternization is 85-90%, and the concentration is 50-200 g / L; the photoinitiator is one or more of LAP, I2959, Irgacure, etc., and the concentration is 5-25 g / L; the amount of drug-loaded MSN-PDA is 50-100 mg / ml GelMA / QCS solution; the particle size of AgNPs is 30-50 nm, and the amount added is 30-60 mg / ml GelMA / QCS solution; the angiogenic peptide is one or more of α-FGF, β-FGF, PD-ECGF, TGF, TNF, VEGF, and the amount added is 10-40 μg / ml GelMA / QCS solution.

[0016] Furthermore, in step 4), the transparent mold can be customized to closely resemble the shape of the magnesium porous scaffold, but slightly larger in size, so that the scaffold can fit perfectly into the mold, resulting in less solution loss during subsequent in-situ UV curing. The ultrasonic process involves an ultrasonic power of 80-100W and an ultrasonic time of 15-60 minutes, determined specifically based on the scaffold's shape and volume. Larger or more complex scaffolds may require a longer ultrasonic time.

[0017] Furthermore, in step 5), the rotation-in-situ UV curing process parameters are as follows: rotation speed 10-20 rpm; UV wavelength 405 nm; UV curing time 15-60 minutes. Due to the opacity and shielding effect of the external magnesium porous scaffold, a rotation-in-situ UV curing process was specifically designed to address these issues, enabling comprehensive curing of the scaffold material. Compared to the conventional hydrogel photocuring time (tens of seconds to several minutes), this invention extends the UV curing time to 15-60 minutes, ensuring complete curing of the hydrogel within the pores of the scaffold material. This process is specifically designed and customized to the structural characteristics of the material in this invention.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1) The scaffold in this invention uses magnesium, GelMA, QCS, osteogenic polypeptide, AgNPs, angiogenic polypeptide, cetyltrimethylammonium bromide (CTAB), n-silane (TEOS), DA, etc. as main raw materials, and is prepared by a combination of processes such as metal 3D printing, template method, in-situ self-polymerization, adsorption method, blending method, mold method, ultrasonic process, and rotation-ultraviolet in-situ photocuring. The above raw material selection and combination process are all designed separately according to the material preparation, which together ensures the successful preparation of the scaffold material.

[0020] 2) The scaffold in this invention has a triple composite structure, consisting of a drug-loaded MSN-PDA and a single nanocomposite structure of silver nanoparticles, a double structure of composite hydrogel, and a triple structure of magnesium porous scaffold. The magnesium porous scaffold triple structure provides a framework structure with high mechanical properties; the double structure of composite hydrogel is embedded within the pores of the magnesium scaffold, allowing for drug loading; the drug-loaded MSN-PDA and the single nanocomposite structure of silver nanoparticles can slowly release silver nanoparticles under the encapsulation of the hydrogel, and the osteogenic peptides within the MSN can be released during the later stages of bone repair.

[0021] 3) The metal scaffold body of the present invention is prepared by 3D printing, which allows for flexible adjustment of the scaffold shape and pore size, making it suitable for different drug delivery schemes and different bone defect morphologies. By controlling the microstructure of the pores, the mechanical properties of the scaffold can be improved, avoiding the stress shielding effect that may occur with traditional scaffolds. The present invention combines centrifugal degassing process with ultrasonic loading process, so that the hydrogel is evenly distributed in the pores of the scaffold, avoiding the problem of local infection caused by uneven distribution. Compared with the traditional in-situ ultraviolet curing method, the rotation-ultraviolet curing method is more conducive to the uniform and complete curing of hydrogel.

[0022] 4) This invention innovatively integrates a metal scaffold, MSN, and hydrogel materials to form the following four-fold sustained-release system: ① The hydrogel has a nanoporous structure, and the AgNPs loaded within it have excellent anti-infective effects, releasing to the local area early to produce highly effective antibacterial activity; the angiogenic peptides promote local angiogenesis, improve blood supply, and exert their effects early; ② The magnesium scaffold has a porous structure, forming a physical shielding effect and prolonging the action time of the hydrogel; ③ The magnesium scaffold degrades slowly in vivo, and the released magnesium ions have an osteogenic effect, with scaffold degradation occurring throughout the entire osteogenic process; ④ The MSN-PDA micropores loaded in the hydrogel contain osteogenic peptides that significantly stimulate osteoblast activity and accelerate bone deposition at the defect site; due to the simultaneous influence of PDA encapsulation, hydrogel pore blockage, and the physical shielding effect of the metal scaffold, the release of the osteogenic peptides within the micropores is slow, resulting in a longer action time that can be maintained throughout the entire osteogenic process. The above four-fold sustained-release structure works synergistically, effectively preventing infection while promoting bone formation. Compared to traditional implants, this invention avoids the problems of explosive drug release in the early stage and the resulting cytotoxicity and short duration of action caused by traditional drug-loaded implants.

[0023] 5) The drug-loaded magnesium scaffold designed and prepared in this invention simultaneously possesses excellent biocompatibility, anti-infection ability, osteopromoting ability, long-lasting effect, biodegradability, and customizability: Biocompatibility is a basic requirement for implantable devices; the AgNPs loaded in the hydrogel have good antibacterial activity and can exert their effects early; the loaded angiogenic peptides can significantly improve blood supply to the affected area early on, providing a guarantee for later osteoogenesis; the osteopromoting polypeptides are affected by multiple sustained-release structures, resulting in a long-lasting effect that can be maintained throughout the entire osteogenic process; biodegradability avoids secondary surgery, and the magnesium ions released by the scaffold degradation have an osteopromoting effect, while the hydrogel degradation products are common biological components; the selectivity of 3D-printed scaffolds and drugs makes the scaffold highly customizable, which can meet various defect morphologies and individual patient conditions; during the process of new bone formation, the material degrades slowly at the same time, and the two work synergistically and match each other to meet the requirements of various bone defect repairs. Attached Figure Description

[0024] Figure 1a , Figure 1b and Figure 1c Photographs of magnesium porous supports with different pore sizes and shapes;

[0025] Figure 2 Photographs from different angles of the hydrogel-embedded 3D-printed magnesium porous scaffold of the present invention, which has anti-infection, angiogenesis-promoting and osteogenic functions.

[0026] Figure 3 This is a partial enlarged view of the hydrogel-embedded 3D-printed magnesium porous scaffold of the present invention, which has anti-infection, angiogenesis-promoting and osteogenic functions. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] The present invention provides a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions. The specific preparation process is as follows: 1) Magnesium is melted into a molten mass using a vacuum induction melting furnace and placed in an atomizing device to form powder with a diameter of approximately 40-120 micrometers. A porous magnesium scaffold with a specific pore size is then prepared using metal 3D printing technology; 2) MSN is prepared using a template method and impregnated in a PBS solution containing osteogenic peptides to achieve peptide loading within the MSN pores; further, the drug-loaded MSN is impregnated in a DA / Tris-HCl solution (pH = 8.5), and the drug-loaded MSN is encapsulated through in-situ self-polymerization of DA to obtain drug-loaded MSN-PDA; 3) Under light-protected conditions and at 60°C, GelMA and... QCS is dissolved in a PBS solution containing a photoinitiator; after the completely dissolved solution is cooled to 40°C, it is transferred to a 40°C water bath, and then drug-loaded MSN-PDA, AgNPs and angiogenic peptides are added. After mixing evenly, the mixture is centrifuged at low speed to remove bubbles; 4) Under light-protected conditions, the solution in step 3) is transferred to a customized transparent mold, and the porous magnesium scaffold in step 1) is also placed in the mold. Combined with ultrasonic technology, the mixed solution is allowed to permeate into the pores of the porous scaffold; 5) The solution in the mold in step 4) is completely gelled using a rotation-in-situ UV curing process. After demolding, the gel outside the scaffold body is removed. After irradiation sterilization and encapsulation, the desired hydrogel embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenic and osteogenic functions is obtained.

[0029] Example 1:

[0030] 1) First, metallic magnesium is melted in a vacuum induction melting furnace, and then placed in an atomizing device to form powder with a diameter of 60μm. The magnesium powder is then placed in a 3D printer, with the electron beam velocity set to 4000mm / s, the electron beam intensity to 10mA, the velocity parameter to 42, and the vacuum degree inside the printing chamber to <10. -4 Pa, argon pressure potential is 0.02MPa, sample preheating temperature is 600℃, magnesium porous scaffold with pore size of 500μm was printed layer by layer, ready for use;

[0031] 2) Prepare a 0.5 g / L CTAB solution in a flask, and then adjust the pH of the solution to 10 using sodium hydroxide. Place the above reaction system in a 70°C water bath and stir at 400 rpm for 2 h. Then, quickly add 5 g / L TEOS, react for 24 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate. Wash the precipitate three times alternately with anhydrous ethanol and ultrapure water, and dry it at 40°C. Take 10 mg / ml of the dried product, disperse it with anhydrous ethanol, add 7 ml / L APTES, stir for 6 h, centrifuge at 12000 rpm for 10 min, take the precipitate, wash it three times with anhydrous ethanol, freeze dry to obtain MSN; add BMP-2 to PBS solution to prepare a solution with a concentration of 40 ug / ml, then add 10 mg / ml MSN, and impregnate for 4 h under magnetic stirring at 200 rpm for drug loading; place the drug-loaded MSN in 6 mg / ml DA / Tris-HCl (pH=8.5) and react for 4 h to obtain drug-loaded MSN-PDA;

[0032] 3) Add GelMA with a degree of substitution of 60% and QCS (number average molecular weight of 5000 and degree of quaternization of 87%) to a PBS solution containing 5 g / L LAP, where the concentration of GelMA is 100 g / L and the concentration of QCS is 50 g / L; place the above solution in a 60°C water bath under light-protected conditions until it is completely dissolved, then cool to 40°C, transfer to a 40°C water bath, add 50 mg / ml drug-loaded MSN-PDA; add 30 mg / ml AgNPs with a particle size of 30 nm and 10 μg / ml VEGF, mix well, and centrifuge at low speed to remove bubbles, then set aside for use;

[0033] 4) Under light-protected conditions, the obtained solution and magnesium scaffold were transferred to a custom mold and placed under an 80W ultrasonic environment for 15 minutes for loading. After loading, the mold was placed on a rotating UV curing stage and cured under 405nm UV light for 20 minutes at a speed of 10 rpm. After demolding, excess gel was removed from the magnesium scaffold, and the scaffold was encapsulated and sterilized by irradiation to obtain the desired hydrogel-embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions.

[0034] 5) The above-mentioned scaffold material has a bending strength of 286.5 MPa and an infection resistance rate of 81%. After 7 days of MTT assay, the cell survival rate was found to be 89% compared with the blank control group.

[0035] Example 2:

[0036] 1) First, metallic magnesium is melted in a vacuum induction melting furnace, and then placed in an atomizing device to form powder with a diameter of 60μm. The magnesium powder is then placed in a 3D printer, with the electron beam velocity set to 4000mm / s, the electron beam intensity to 10mA, the velocity parameter to 42, and the vacuum degree inside the printing chamber to <10. -4 Pa, argon pressure potential is 0.02MPa, sample preheating temperature is 600℃, magnesium porous scaffold with pore size of 1000μm was printed layer by layer, ready for use;

[0037] 2) Prepare a 0.5 g / L CTAB solution in a flask, and then adjust the pH of the solution to 10 using sodium hydroxide. Place the above reaction system in a 70°C water bath and stir at 400 rpm for 2 h. Then, quickly add 5 g / L TEOS, react for 24 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate. Wash the precipitate three times alternately with anhydrous ethanol and ultrapure water, and dry it at 40°C. Take 10 mg / ml of the dried product, disperse it with anhydrous ethanol, add 7 ml / L APTES, stir for 6 h, centrifuge at 12000 rpm, take the precipitate, wash it three times with anhydrous ethanol, freeze dry to obtain MSN; add BMP-2 to PBS solution to prepare a solution with a concentration of 40 μg / ml, then add 10 mg / ml MSN, and impregnate for 4 h under magnetic stirring at 200 rpm for drug loading; place the drug-loaded MSN in 6 mg / ml DA / Tris-HCl (pH = 8.5) and react for 4 h to obtain drug-loaded MSN-PDA;

[0038] 3) Add GelMA with a degree of substitution of 60% and QCS (molecular weight of 5000, degree of quaternization of 87%) to a PBS solution containing 5 g / L LAP, where the concentration of GelMA is 100 g / L and the concentration of QCS is 50 g / L; place the above solution in a 60°C water bath under light-protected conditions until it is completely dissolved, then cool to 40°C, transfer to a 40°C water bath, add 50 mg / ml drug-loaded MSN-PDA; add 30 mg / ml AgNPs with a particle size of 30 nm and 10 μg / ml VEGF, mix well, and centrifuge at low speed to remove bubbles, then set aside for use;

[0039] 4) Under light-protected conditions, the obtained solution and magnesium scaffold were transferred to a custom mold and placed under an 80W ultrasonic environment for 15 minutes for loading. After loading, the mold was placed on a rotating UV curing stage and cured under 405nm UV light for 20 minutes at a speed of 10 rpm. After demolding, excess gel was removed from the magnesium scaffold, and the scaffold was encapsulated and sterilized by irradiation to obtain the desired hydrogel-embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions.

[0040] 5) Compared with Example 1, the pore size of the magnesium porous scaffold was increased. The scaffold material has a bending strength of 246.1 MPa and an anti-infection rate of 84%. After 7 days of MTT assay, the cell survival rate was found to be 87% compared with the blank control group.

[0041] Example 3:

[0042] 1) First, metallic magnesium is melted in a vacuum induction melting furnace, and then placed in an atomizing device to form powder with a diameter of 60μm. The magnesium powder is then placed in a 3D printer, with the electron beam velocity set to 4000mm / s, the electron beam intensity to 10mA, the velocity parameter to 42, and the vacuum degree inside the printing chamber to <10. -4 Pa, argon pressure potential is 0.02MPa, sample preheating temperature is 600℃, magnesium porous scaffold with pore size of 500μm was printed layer by layer, ready for use;

[0043] 2) Prepare a 0.5 g / L CTAB solution in a flask, and then adjust the pH of the solution to 10 using sodium hydroxide. Place the above reaction system in a 70°C water bath and stir at 400 rpm for 2 h. Then, quickly add 5 g / L TEOS, react for 24 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate. Wash the precipitate three times alternately with anhydrous ethanol and ultrapure water, and dry it at 40°C. Take 10 mg / ml of the dried product, disperse it with anhydrous ethanol, add 7 ml / L APTES, stir for 6 h, centrifuge at 12000 rpm, take the precipitate, wash it three times with anhydrous ethanol, freeze dry to obtain MSN; add BMP-2 to PBS solution to prepare a solution with a concentration of 40 μg / ml, then add 10 mg / ml MSN, and impregnate for 4 h under magnetic stirring at 200 rpm for drug loading; place the drug-loaded MSN in 6 mg / ml DA / Tris-HCl (pH = 8.5) and react for 4 h to obtain drug-loaded MSN-PDA;

[0044] 3) Add GelMA with a degree of substitution of 60% and QCS (molecular weight of 5000, degree of quaternization of 90%) to a PBS solution containing 5 g / L LAP, where the concentration of GelMA is 100 g / L and the concentration of QCS is 200 g / L; place the above solution in a 60°C water bath under light-protected conditions until it is completely dissolved, then cool to 40°C, transfer to a 40°C water bath, add 50 mg / ml drug-loaded MSN-PDA; add 30 mg / ml AgNPs with a particle size of 30 nm and 10 μg / ml VEGF, mix well, and centrifuge at low speed to remove bubbles, then set aside for use;

[0045] 4) Under light-protected conditions, the obtained solution and magnesium scaffold were transferred to a custom mold and placed under an 80W ultrasonic environment for 15 minutes for loading. After loading, the mold was placed on a rotating UV curing stage and cured under 405nm UV light for 20 minutes at a speed of 10 rpm. After demolding, excess gel was removed from the magnesium scaffold, and the scaffold was encapsulated and sterilized by irradiation to obtain the desired hydrogel-embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions.

[0046] 5) Compared with Example 1, the degree of quaternization of QCS and the amount of QCS were increased. The bending strength of the above scaffold material was 282.9 MPa, the anti-infection rate was 91%, and the cell survival rate was 86% compared with the blank control group after 7 days of MTT assay.

[0047] Example 4:

[0048] 1) First, metallic magnesium is melted in a vacuum induction melting furnace, and then placed in an atomizing device to form powder with a diameter of 60μm. The magnesium powder is then placed in a 3D printer, with the electron beam velocity set to 4000mm / s, the electron beam intensity to 10mA, the velocity parameter to 42, and the vacuum degree inside the printing chamber to <10. -4 Pa, argon pressure potential is 0.02MPa, sample preheating temperature is 600℃, magnesium porous scaffold with pore size of 500μm was printed layer by layer, ready for use;

[0049] 2) Prepare a 0.5 g / L CTAB solution in a flask, and then adjust the pH of the solution to 10 using sodium hydroxide. Place the above reaction system in a 70°C water bath and stir at 400 rpm for 2 h. Then, quickly add 5 g / L TEOS, react for 24 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate. Wash the precipitate three times alternately with anhydrous ethanol and ultrapure water, and dry it at 40°C. Take 10 mg / ml of the dried product, disperse it with anhydrous ethanol, add 7 ml / L APTES, stir for 6 h, centrifuge at 12000 rpm, take the precipitate, wash it three times with anhydrous ethanol, freeze dry to obtain MSN; add BMP-2 to PBS solution to prepare a solution with a concentration of 40 μg / ml, then add 10 mg / ml MSN, and impregnate for 4 h under magnetic stirring at 200 rpm for drug loading; place the drug-loaded MSN in 6 mg / ml DA / Tris-HCl (pH = 8.5) and react for 4 h to obtain drug-loaded MSN-PDA;

[0050] 3) Add GelMA with a degree of substitution of 90% and QCS (molecular weight of 5000, degree of quaternization of 87%) to a PBS solution containing 10 g / L LAP, where the concentration of GelMA is 200 g / L and the concentration of QCS is 50 g / L; place the above solution in a 60°C water bath under light-protected conditions until it is completely dissolved, then cool to 40°C, transfer to a 40°C water bath, add 50 mg / ml drug-loaded MSN-PDA; add 30 mg / ml AgNPs with a particle size of 30 nm and 10 μg / ml VEGF, mix well, and centrifuge at low speed to remove bubbles, then set aside for use;

[0051] 4) Under light-protected conditions, the obtained solution and magnesium scaffold were transferred to a custom mold and placed under an 80W ultrasonic environment for 15 minutes for loading. After loading, the mold was placed on a rotating UV curing stage and cured under 405nm UV light for 20 minutes at a speed of 10 rpm. After demolding, excess gel was removed from the magnesium scaffold, and the scaffold was encapsulated and sterilized by irradiation to obtain the desired hydrogel-embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions.

[0052] 5) Compared with Example 1, by increasing the degree of substitution of GelMA and the amount of GelMA used, the flexural strength of the above scaffold material was 282.4 MPa, the anti-infection rate was 76%, and the cell survival rate was 92% compared with the blank control group after 7 days of MTT assay.

[0053] Example 5:

[0054] 1) First, metallic magnesium is melted in a vacuum induction melting furnace, and then placed in an atomizing device to form powder with a diameter of 60μm. The magnesium powder is then placed in a 3D printer, with the electron beam velocity set to 4000mm / s, the electron beam intensity to 10mA, the velocity parameter to 42, and the vacuum degree inside the printing chamber to <10. -4 Pa, argon pressure potential is 0.02MPa, sample preheating temperature is 600℃, magnesium porous scaffold with pore size of 500μm was printed layer by layer, ready for use;

[0055] 2) Prepare a 0.5 g / L CTAB solution in a flask, and then adjust the pH of the solution to 10 using sodium hydroxide. Place the above reaction system in a 70°C water bath and stir at 400 rpm for 2 h. Then, quickly add 5 g / L TEOS, react for 24 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate. Wash the precipitate three times alternately with anhydrous ethanol and ultrapure water, and dry it at 40°C. Take 10 mg / ml of the dried product, disperse it with anhydrous ethanol, add 7 ml / L APTES, stir for 6 h, centrifuge at 12000 rpm, take the precipitate, wash it three times with anhydrous ethanol, freeze dry to obtain MSN; add BMP-2 to PBS solution to prepare a solution with a concentration of 40 μg / ml, then add 10 mg / ml MSN, and impregnate for 4 h under magnetic stirring at 200 rpm for drug loading; place the drug-loaded MSN in 6 mg / ml DA / Tris-HCl (pH = 8.5) and react for 4 h to obtain drug-loaded MSN-PDA;

[0056] 3) Add GelMA with a degree of substitution of 60% and QCS (molecular weight of 5000, degree of quaternization of 90%) to a PBS solution containing 5 g / L LAP, where the concentration of GelMA is 100 g / L and the concentration of QCS is 200 g / L; place the above solution in a 60°C water bath under light-protected conditions until it is completely dissolved, then cool to 40°C, transfer to a 40°C water bath, add 50 mg / ml drug-loaded MSN-PDA; add 60 mg / ml AgNPs with a particle size of 30 nm and 10 μg / ml VEGF, mix well, and centrifuge at low speed to remove bubbles, then set aside for use;

[0057] 4) Under light-protected conditions, the obtained solution and magnesium scaffold were transferred to a custom mold and placed under an 80W ultrasonic environment for 15 minutes for loading. After loading, the mold was placed on a rotating UV curing stage and cured under 405nm UV light for 20 minutes at a speed of 10 rpm. After demolding, excess gel was removed from the magnesium scaffold, and the scaffold was encapsulated and sterilized by irradiation to obtain the desired hydrogel-embedded 3D printed magnesium porous scaffold material with anti-infection, angiogenesis and osteogenic functions.

[0058] 5) Compared with Example 1, the degree of quaternization of QCS was increased, and the amount of AgNPs and QCS was also increased. The bending strength of the above scaffold material was 284.3 MPa, the anti-infection rate was 97%, and the cell survival rate was 84% ​​compared with the blank control group after 7 days of MTT assay.

Claims

1. A method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions, characterized in that: Includes the following steps: 1) Magnesium is melted into a molten material using a vacuum induction melting furnace and placed in an atomizing device to form powder with a diameter of 40-120 micrometers. A porous magnesium scaffold with a pore size between 500-1000 micrometers is then prepared using metal 3D printing technology. 2) Mesoporous silica MSN was prepared using a template method and then impregnated in a PBS solution containing osteogenic peptides to load the peptides into the MSN pores, thus obtaining drug-loaded MSN. The drug-loaded MSN was further impregnated in a dopamine DA / Tris-HCl solution with a pH of 8.5, and the drug-loaded MSN was encapsulated by the in-situ self-polymerization of DA to obtain drug-loaded MSN-PDA. 3) Under light-protected and 60°C conditions, methacrylic anhydride gelatin (GelMA) and quaternized chitosan (QCS) were dissolved in a PBS solution containing a photoinitiator. After the completely dissolved solution was cooled to 40°C, it was transferred to a 40°C water bath, and then drug-loaded MSN-PDA, nano-silver AgNPs, and angiogenic peptides were added. After mixing evenly, the mixture was centrifuged to remove bubbles. 4) Under light-protected conditions, transfer the solution from step 3) into a transparent mold, and then place the porous magnesium scaffold from step 1) into the mold. Combine this with an ultrasonic process to allow the mixed solution to permeate into the pores of the porous scaffold. 5) The solution in the mold from step 4) is completely gelled using a rotation-in-situ UV curing process. After demolding, the gel outside the scaffold body is removed. After irradiation sterilization and encapsulation, the desired hydrogel-embedded 3D printed magnesium porous scaffold with anti-infection, angiogenesis-promoting and osteogenic functions is obtained. Rotation-in-situ UV curing process parameters: rotation speed is 10-20 rpm; UV wavelength is 405 nm; UV curing time is 15-60 minutes.

2. The method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions according to claim 1, characterized in that: Step 1) Magnesium powder printing parameters: electron beam velocity 4000-5000 mm / s, electron beam intensity 8-15 mA, velocity parameter 40-45, vacuum degree in the printing chamber less than 10. -4 The argon pressure was 0.02-0.06 MPa, the sample preheating temperature was 600℃, and the pore size of the 3D printed magnesium porous scaffold was between 500-1000 micrometers.

3. The method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions according to claim 1, characterized in that: MSN in step 2) The preparation process parameters are as follows: CTAB, water, and sodium hydroxide are added sequentially to a reaction vessel and stirred for 2-4 hours in a water bath at 70-80℃ with mechanical stirring at 300-500 rpm; then TEOS is added, and after reacting for 12-24 hours, the precipitate is obtained by high-speed centrifugation. The precipitate is washed repeatedly with ethanol and water 3-5 times, dried at 40-45℃, and then the dried product is dispersed with ethanol to form a 5-40 mg / ml dispersion. APTES is then added, and the reaction is stirred for 6-12 hours. The secondary precipitate is then separated by high-speed centrifugation, washed repeatedly with ethanol 3-5 times, and then freeze-dried to obtain the desired MSN. In the above process, the amount of CTAB is 0.5-2 g / L, the amount of sodium hydroxide is used to adjust the pH of the solution to 10-11, the amount of TEOS is 4-10 g / L, and the amount of APTES is 5-12 ml / L. The speed of both high-speed centrifugations is 10000-15000 rpm, and the centrifugation time is 5-15 minutes.

4. The method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions according to claim 1, characterized in that: The osteogenic peptide in step 2) is one or more of BMP-2, bone growth peptide OGP, BMP-7, and TGF-β, and the concentration of the osteogenic peptide in the PBS solution is 30-80 μg / ml; magnetic stirring is used during impregnation at a rate of 100-200 rpm for 4-6 hours; the MSN concentration is 5-40 mg / ml, the DA concentration is 6-12 mg / ml, and the reaction time is 4-6 hours.

5. The method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions according to claim 1, characterized in that: In step 3), the degree of substitution of GelMA is 60-90%, and the concentration is 50-200 g / L; the number-average molecular weight of QCS is 3000-12000, the degree of quaternization is 85-90%, and the concentration is 50-200 g / L; the photoinitiator is one or more of LAP, I2959, and Irgacure, and the concentration is 5-25 g / L; the amount of drug-loaded MSN-PDA is 50-100 mg / ml; the particle size of AgNPs is 30-50 nm, and the amount added is 30-60 mg / ml; the angiogenic peptide is one or more of α-FGF, β-FGF, PD-ECGF, TGF, TNF, and VEGF, and the amount added is 10-40 μg / ml.

6. The method for preparing a hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions according to claim 1, characterized in that: In step 4), the ultrasonic process is performed with an ultrasonic power of 80-100W and an ultrasonic time of 15-60 minutes.

7. A hydrogel-embedded 3D-printed magnesium porous scaffold with anti-infection, angiogenesis-promoting, and osteogenic functions, characterized in that, It is prepared by the method described in any one of claims 1-6.

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