Multi-component synergistic 3D printing multi-structure long-acting anti-infection bone repair composite scaffold and preparation method thereof

Abstract

According to the multi-component synergistic 3D printing multi-structure long-acting anti-infection bone repair composite scaffold and the preparation method thereof, the method comprises the following steps: preparing a PVA aqueous solution as a slurry adhesive, and mixing HA, beta-TCP, the PVA aqueous solution and glycerol as a printing slurry; 3D printing parameters are set, a model is imported, a printing structure, discharging and wiring speeds are set, and a customized support is obtained through 3D printing; drying and sintering the scaffold to obtain a porous bone scaffold without organic matters; preparing porous microspheres ABX coated PLGA loaded with antibiotics by taking gelatin as a pore-foaming agent; and coating the sintered porous bone scaffold with a gelatin coating, and loading the microspheres on the porous bone scaffold to obtain the drug-loaded composite bone scaffold. The bone repair scaffold disclosed by the invention is combined with an antibiotic local drug delivery system to prevent infectious bone defects, can overcome the defects of systemic drug delivery of antibiotics, realizes controllable release of drugs in the whole process in a bone regeneration period, has good antibacterial performance while repairing bone defects in vivo, and also has relatively good compressive strength and biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of bone repair, specifically to a multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold with hydroxyapatite, β-tricalcium phosphate and antibiotics as the main materials, and its preparation method. Background Technology

[0002] Bone defect repair is a significant challenge in clinical medicine, commonly seen in the treatment of trauma, tumor resection, infection, and congenital malformations. With the increasing aging population and growing demand for orthopedic surgery, the development of bone repair materials with good biocompatibility, osteoconductivity, mechanical properties, and anti-infection capabilities has become a research hotspot. Currently, commonly used bone repair materials in clinical practice mainly include autologous bone, allogeneic bone, and synthetic bone substitutes. Among these, autologous bone transplantation is considered the "gold standard" due to its superior biocompatibility and osteoinductive properties, but it suffers from limited donor availability, bone harvesting trauma, and postoperative complications. Allogeneic bone transplantation faces risks of immune rejection and disease transmission, limiting its widespread application. Therefore, the development of synthetic bone repair materials has become a key direction for solving these problems.

[0003] Calcium phosphate-based materials (such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP)) are widely used in bone repair due to their excellent biocompatibility, osteoconductivity, and degradability, as their chemical composition is similar to the inorganic components of human bone tissue. However, single calcium phosphate-based materials suffer from insufficient mechanical properties (especially porous scaffolds) and lack of anti-infection function, making it difficult to meet the needs of repairing complex bone defects. The development of 3D printing technology has provided a new approach for the precise preparation of bone repair scaffolds. By controlling printing parameters, scaffolds with specific porous structures can be constructed, which is beneficial for cell infiltration, nutrient transport, and new bone formation. However, simple 3D-printed calcium phosphate-based scaffolds also have shortcomings: for example, the porous structure leads to lower compressive strength, making it difficult to match the mechanical requirements of human cancellous bone, and they are prone to fracture under stress after implantation; they lack anti-infection capabilities, and bone defect areas (especially open wounds or postoperative infections) are prone to bacterial growth leading to repair failure; the surface of a simple calcium phosphate-based scaffold has limited effect on promoting cell adhesion and proliferation, affecting the efficiency of bone integration.

[0004] Therefore, existing technologies need to be improved by rationally controlling the ratio of calcium phosphate-based materials, 3D printing parameters, gelatin coating process, and drug-loaded microsphere preparation methods to prepare porous calcium phosphate-based drug-loaded composite bone repair scaffolds with excellent comprehensive performance, aiming to solve the above-mentioned problems in existing technologies. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, we now provide a multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold and its preparation method. It can maintain precise targeted anti-infection performance and ensure appropriate drug release characteristics, thus promoting bone differentiation while fighting infection.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing a 3D-printed gelatin-modified porous phospho-calcium-based drug-loaded composite bone repair scaffold, comprising the following steps: Adhesive preparation: Weigh PVA powder and deionized water according to a PVA mass fraction of 6-12 wt%, stir in an oil bath at 80-95℃ until a transparent solution is formed, add 0.5-1 mL of glycerol and stir to obtain a colorless, homogeneous, and transparent solution; 3D printing slurry preparation: Weigh out calcium phosphate-based material, which is hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), with a mass ratio of hydroxyapatite to β-tricalcium phosphate of 3:7-7:3; Slowly add the calcium phosphate-based material to the binder solution and stir, wherein the mass ratio of PVA aqueous solution to the total mass of calcium phosphate-based material is 10:5-10, to obtain a uniform printing slurry; Create the printing model: Import the .stl format cylindrical model into the printing software, set the printing table temperature to 20-25℃, the model bottom diameter to 6-12mm, the layer height to 0.3-0.6mm, the line spacing to 1-2mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the printing model; Sample drying and sintering: The printed sample is left to stand in a dry environment for 24-48 hours. The dried scaffold is placed in a muffle furnace and heated to 1100℃-1200℃ at a heating rate of 2-5℃ / h. It is held for 2-6 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare ABX@PLGA porous microspheres resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight of 100,000-120,000 Mw was weighed, wherein the mass ratio of L-lactolactone (LLA) to glycolate (GA) was 50:50; it was added to 10 mL of CH2Cl2 solution, and 20-40 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved to obtain the oil phase O. ABX@PLGA ; Prepare 8% W Gel Add 2.5 mL of the aqueous solution to the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX@PLGA The emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, ABX@PLGA porous microspheres were obtained. Preparation of gelatin coating: Dissolve gelatin in deionized water at 40-60℃ to prepare a 2-10wt% gelatin solution; immerse the sintered bone scaffold in the gelatin solution, vacuum treat for 5-15 min, and vacuum dry at 40-60℃ for 24 h; after drying, immerse the scaffold in a 2-3wt% glutaraldehyde solution and crosslink the gelatin layer for 20-40 min, then immerse in ethanol, wash with distilled water, and dry. Preparation of drug-loaded composite scaffold: Drug-loaded microsphere powder was added to deionized water and ultrasonically dispersed for 5-10 min. The sintered bone scaffold was then soaked and shaken for 10-30 min and dried at a constant temperature for 5-10 h to obtain a 3D-printed gelatin-modified porous phospho-calcium-based drug-loaded composite bone repair scaffold.

[0007] Furthermore, in the preparation of the adhesive, the oil bath temperature is 85°C and the PVA mass fraction is 6-9 wt%.

[0008] Furthermore, in the preparation of 3D printing slurry, the total mass of calcium-phosphorus-based materials is 7-10g, and the mass ratio of hydroxyapatite to β-tricalcium phosphate is 7:3.

[0009] Furthermore, during the process of creating the printing model, the printing table temperature was 23℃, the bottom diameter of the model was 8-12mm, the layer height was 0.45mm, and the line spacing was 1mm.

[0010] Furthermore, in the sample drying and sintering process, the drying time was 30 h, the heating rate was 2-3.5 °C / h, the sintering temperature was 1100 °C, and the holding time was 3-6 h.

[0011] Furthermore, in the preparation of the drug-loaded microspheres, the antibiotics are vancomycin, roxithromycin, doxycycline, or levofloxacin, the mass of PLGA is 0.4 g, and the volume of dichloromethane is 10 mL.

[0012] Furthermore, in the preparation of the gelatin coating, the gelatin solution temperature is 50℃, the gelatin mass fraction is 2-10wt%, the glutaraldehyde mass fraction is 2-3wt%, and the crosslinking time is 25min.

[0013] Furthermore, in the preparation of the drug-loaded composite scaffold, the ultrasonic dispersion time was 5 min, the shaking time was 20 min, and the drying time was 5 h.

[0014] The present invention also discloses a 3D printed gelatin-modified porous phospho-calcium-based drug-loaded composite bone repair scaffold, characterized in that the scaffold has a porosity of 55%-61%, a compressive strength of 7.9-9.3 MPa, a bottom diameter shrinkage rate of about 27.7% and a height shrinkage rate of about 20.6% after sintering.

[0015] Furthermore, the scaffold comprises a gelatin coating and drug-loaded PLGA microspheres containing antibiotics.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of this invention uses PVA aqueous solution as a binder to formulate a printing slurry, 3D prints structures with controllable porosity, dries and sinters, and then coats the sintered porous bone scaffold with a gelatin coating. Antibiotics are then encapsulated in PLGA microspheres and loaded onto the bone scaffold to obtain a gelatin-modified porous phospho-calcium-based drug-loaded composite bone repair scaffold. The bone repair scaffold prepared by this method, combined with a local antibiotic delivery system, prevents infectious bone defects, effectively overcoming the drawbacks of systemic antibiotic administration. It achieves full-cycle control of drug release, enabling the scaffold to repair bone defects in vivo while also possessing good antibacterial properties, as well as good compressive strength and biocompatibility. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the preparation method of the multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold of the present invention; Figure 2 A macroscopic view of the bone scaffold prepared in Example 1 of the present invention; Figure 3 The images shown are SEM images of the bone scaffold prepared according to Embodiment 1 of the present invention. (A) is an SEM image of the bone scaffold at different magnifications (50×, 2000×, 6000×), (B) is an SEM image of the Gel bone scaffold at different magnifications (50×, 100×, 200×), and (C) is an SEM image of the GeIPLGA bone scaffold at different magnifications (50×, 100×, 200×). Figure 4 XRD pattern of the bone scaffold prepared in Example 1 of the present invention; Figure 5 This is an in vitro drug release diagram of the drug-loaded composite bone scaffold prepared in Example 1 of the present invention; Figure 6 This is an image showing the antibacterial effect of the drug-loaded composite bone scaffold prepared in Example 1 of the present invention; Figure 7 This is an in vitro osteogenic differentiation diagram of the drug-loaded composite bone scaffold prepared in Example 1 of the present invention. Detailed Implementation

[0018] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0019] Antibiotics are widely used as antibacterial drugs in the traditional treatment of bone infections. They can inhibit bacterial cell wall synthesis, interact with cell membranes, interfere with protein synthesis, and inhibit nucleic acid replication and transcription. PLGA is a polylactic acid-derived polymer that degrades into lactic acid and glycolic acid. Lactic acid can be further metabolized in the body to ultimately produce CO2 and H2O; glycolic acid can be excreted through the kidneys without accumulating toxic side effects in the body. PLGA has been approved by the FDA and has become a sustained-release carrier for various drugs. It has good biocompatibility, mechanical strength, and controllable biodegradability. Its sustained-release rate can be adjusted by regulating different polymerization ratios, polymerization rate, and polymer length.

[0020] Gelatin, a polymer with good biocompatibility, is a product of collagen thermal denaturation or physical and chemical decomposition. It can be completely absorbed in the body. Calcium-phosphorus-based ceramics are generally hydrophilic. During the coating process, the gelatin solution, due to its good wettability with the ceramic, is adsorbed onto the ceramic body and even penetrates into the micropores of the ceramic surface, forming a gelatin coating that tightly bonds with the ceramic, further enhancing the mechanical properties of the scaffold. Gelatin is a partially denatured derivative of collagen, containing a large number of functional side groups that promote cell adhesion and growth. When combined with porous calcium-phosphorus-based scaffolds, it maintains the good biocompatibility of the porous scaffold and allows for a tighter bond between drug-loaded microspheres and the bone scaffold.

[0021] This invention discloses a multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold and its preparation method. (Screenshot) Figures 1 to 7 As shown, the method includes the following steps: Adhesive preparation: Weigh PVA powder with a mass fraction of 6-12 wt% and deionized water into a beaker, seal it with plastic wrap, stir in an oil bath at 80-95℃ until a transparent solution is formed, add 0.5-1 mL of glycerin and stir to obtain a colorless, homogeneous, and transparent solution. 3D printing slurry preparation: Weigh out the calcium-phosphorus-based material, which consists of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), with a mass ratio of HA to β-TCP of 3:7-7:3. Slowly add the calcium-phosphorus-based material to the binder solution and stir. The total mass ratio of PVA aqueous solution to the calcium-phosphorus-based material is 10:5-10 to obtain a uniform printing slurry. Combining nano-hydroxyapatite with β-tricalcium phosphate helps to solve the problem of excessively rapid degradation of tricalcium phosphate and can also fully utilize the osteoinductive effect of nano-hydroxyapatite. Create the printing model: Import the .stl format cylindrical model into the printing software, set the printing table temperature to 20-25℃, adjust the bottom diameter of the model to 6-12mm, the layer height to 0.3-0.6mm, the line spacing to 1-2mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the printing model; Sample drying and sintering: The printed sample is left to stand in a dry environment for 24-48 hours. After the sample is completely dried, it is placed in a muffle furnace and heated to 1100℃-1200℃ at a heating rate of 2-5℃ / h. The temperature is held for 2-6 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as the pore-forming agent to prepare porous microspheres ABX@PLGA resistant to antibiotic loading; At room temperature, weigh 0.2 g of PLGA raw material with a weight-average molecular weight (Mw) of 100,000-120,000, wherein the mass ratio of LLA to GA is 50:50; add it to 10 mL of CH2Cl2 (dichloromethane) solution, and simultaneously weigh 20-40 mg of ABX and add it to the CH2Cl2 solution, mixing and stirring until dissolved, thus controlling the PLGA:ABX ratio to be 10:1; obtain oil phase O ABX@PLGA ; Prepare 2.5 nL of an 8% gel aqueous solution, record it as WGel (gelatin aqueous solution) for later use. Gel Add the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX@PLGA The emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, the final ABX@PLGA porous microspheres were obtained. Figure 3The white, uniform porous microspheres shown in Figure B are used to achieve a spatially and temporally long-acting release of the antibiotic ABX, extending the duration of action of ABX from 24 hours to 72 hours. Preparation of gelatin coating: Dissolve gelatin in deionized water at 40-60℃ to prepare a 2-10wt% gelatin solution; immerse the sintered bone scaffold in the gelatin solution and vacuum treat for 5-15 minutes; remove the sample and place it in a vacuum drying oven and dry at 40-60℃ for 24 hours; after drying, immerse the scaffold in a 2-3wt% glutaraldehyde solution and crosslink the gelatin layer for 20-40 minutes, then immerse it in ethanol 3 times, wash it with distilled water, and dry it. Preparation of drug-loaded composite scaffold: PLGA microsphere powder loaded with antibiotics was added to deionized water and ultrasonically dispersed for 5-10 min. The sintered bone scaffold was then soaked and shaken for 10-30 min. The sample was then placed in a constant temperature oven to dry for 5-10 h to obtain a multi-component synergistic 3D printed multi-structure long-lasting anti-infection bone repair composite scaffold. Example 1:

[0022] Adhesive preparation: Weigh PVA powder and deionized water according to a PVA mass fraction of 6% and put them into a beaker. Seal the beaker with plastic wrap and stir in an oil bath at 85°C to obtain a homogeneous and transparent solution. Then add 0.4 mL of glycerin and stir to obtain a colorless, homogeneous and transparent solution. 3D printing paste preparation: Weigh 4.9g of HA and 2.1g of β-TCP (total 7g) of two calcium phosphate-based materials, and slowly add the weighed materials to the binder solution while stirring to obtain a uniform printing paste; Create the printing model: Import the .stl format cylindrical printing model into the printing software, set the printing table temperature to 23℃, adjust the bottom diameter of the model to 8mm, the layer height to 0.45mm, the line spacing to 1mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the final printing model. Sample drying and sintering: The printed sample was left to stand in a dry environment for 30 hours. The completely dried scaffold was then placed in a muffle furnace for sintering. The temperature was increased to 1100℃ at a rate of 2℃ / h and held for 3 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare porous microspheres ABX@PLGA resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight (Mw) of 100,000 was weighed, wherein the mass ratio of LLA to GA was 50:50; it was added to 10 mL of CH2Cl2 (dichloromethane) solution, and 40 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved, thus controlling the PLGA:ABX ratio to be 5:1; the oil phase OABX@PLGA was obtained. Prepare 2.5 nL of an 8% Gel aqueous solution, recorded as W. Gel (Gelatin aqueous solution) is ready for use. Prepare the W... Gel Add the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX@PLGA The emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, the ABX@PLGA porous microspheres were finally obtained. The white and uniform porous microsphere particles have a spatial and temporal long-acting release effect on the antibiotic ABX, which can extend the action time of ABX from 24h to 72h. Preparation of the gelatin coating: Dissolve gelatin in deionized water at 50℃ to prepare a 2wt% gelatin solution. Immerse the bone scaffold in the gelatin solution and vacuum treat for 10 min. After removal, place the sample in a vacuum drying oven and dry at 50℃ for 24 h. Immerse the dried scaffold in a 2wt% glutaraldehyde solution to crosslink the gelatin layer for 25 min. Then, soak in ethanol three times, and finally wash and dry repeatedly with distilled water. Preparation of drug-loaded composite scaffold: Vancomycin-loaded PLGA microsphere powder was added to deionized water and ultrasonically dispersed for 5 min. Then, the sintered bone scaffold was soaked and shaken for 20 min. The sample was placed in a constant temperature drying oven and dried for 5 h to obtain the final multi-component synergistic 3D printed multi-structure long-lasting anti-infection bone repair composite scaffold. Example 2:

[0023] Adhesive preparation: Weigh PVA powder and deionized water according to a PVA mass fraction of 7% and put them into a beaker. Seal the beaker with plastic wrap and stir in an oil bath at 85°C to obtain a homogeneous and transparent solution. Then add 0.4 ml of glycerin and stir to obtain a colorless, homogeneous and transparent solution. 3D printing paste preparation: Weigh 4.0g of HA and 4.0g of β-TCP (total 8g) of two calcium phosphate-based materials, and slowly add the weighed materials to the binder solution while stirring to obtain a uniform printing paste; Create the printing model: Import the .stl format cylindrical printing model into the printing software, set the printing table temperature to 23℃, adjust the bottom diameter of the model to 8mm, the layer height to 0.45mm, the line spacing to 1mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the final printing model. Sample drying and sintering: The printed sample was left to stand in a dry environment for 30 hours. The completely dried scaffold was then placed in a muffle furnace for sintering. The temperature was increased to 1100℃ at a rate of 2.5℃ / h and held for 4 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare porous microspheres ABX@PLGA resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight (Mw) of 110,000 was weighed, wherein the mass ratio of LLA to GA was 50:50; it was added to 10 mL of CH2Cl2 (dichloromethane) solution, and 25 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved, thus controlling the PLGA:ABX ratio to be 8:1; the oil phase OABX@PLGA was obtained. Prepare 2.5 nL of an 8% Gel aqueous solution, recorded as W. Gel (Gelatin aqueous solution) is ready for use. Prepare the W... Gel Add the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX@PLGA The emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, the ABX@PLGA porous microspheres were finally obtained. The white and uniform porous microsphere particles have a spatial and temporal long-acting release effect on the antibiotic ABX, which can extend the action time of ABX from 24h to 72h. Preparation of gelatin coating: Dissolve gelatin in deionized water at 50℃ to prepare a 5wt% gelatin solution. Immerse the bone scaffold in the gelatin solution and vacuum treat for 10 min. After removal, place the sample in a vacuum drying oven and dry at 50℃ for 24 h. Immerse the dried scaffold in a 2.5wt% glutaraldehyde solution to crosslink the gelatin layer for 25 min. Then, immerse it in ethanol three times, and then wash and dry it multiple times with distilled water. Preparation of drug-loaded composite scaffold: PLGA microsphere powder loaded with roxithromycin was added to deionized water and ultrasonically dispersed for 5 min. Then, the sintered bone scaffold was soaked and shaken for 20 min. The sample was placed in a constant temperature drying oven and dried for 5 h to obtain the final multi-component synergistic 3D printed multi-structure long-lasting anti-infection bone repair composite scaffold. Example 3:

[0024] Adhesive preparation: Weigh PVA powder (8% PVA by mass) and deionized water into a beaker, seal with plastic wrap, and stir in an 85°C oil bath to obtain a homogeneous and transparent solution. Then add 0.4 mL of glycerin and stir to obtain a colorless, homogeneous, and transparent solution. 3D printing paste preparation: Weigh 5.4 g of HA and 3.6 g of β-TCP (total 9 g) of two calcium-phosphorus-based materials, and slowly add the weighed materials to the adhesive solution while stirring to obtain a uniform printing paste. Create the printing model: Import the .stl format cylindrical printing model into the printing software, set the printing table temperature to 23℃, adjust the bottom diameter of the model to 11mm, the layer height to 0.45mm, the line spacing to 1mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the final printing model. Sample drying and sintering: The printed sample was left to stand in a dry environment for 30 hours. The completely dried scaffold was then placed in a muffle furnace for sintering. The temperature was increased to 1100℃ at a rate of 3℃ / h and held for 5 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare porous microspheres ABX@PLGA resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight (Mw) of 120,000 was weighed, wherein the mass ratio of LLA to GA was 50:50; it was added to 10 mL of CH2Cl2 (dichloromethane) solution, and 20 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved, thus controlling the PLGA:ABX ratio to be 10:1; the oil phase OABX@PLGA was obtained. Prepare 2.5 nL of an 8% Gel aqueous solution, recorded as W. Gel (Gelatin aqueous solution) is ready for use. Prepare the W... Gel Add the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX@PLGA The emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / WPVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, the final ABX@PLGA porous microspheres were obtained. Preparation of gelatin coating: Dissolve gelatin in deionized water at 50℃ to prepare a 7wt% gelatin solution. Immerse the bone scaffold in the gelatin solution and vacuum treat for 10 min. After removal, place the sample in a vacuum drying oven and dry at 50℃ for 24 h. Immerse the dried scaffold in a 2.5wt% glutaraldehyde solution to crosslink the gelatin layer for 25 min. Then immerse it in ethanol 3 times, wash it multiple times with distilled water, and dry it. Preparation of drug-loaded composite scaffold: PLGA microsphere powder loaded with doxycycline was added to deionized water and ultrasonically dispersed for 5 min. Then, the sintered bone scaffold was soaked and shaken for 20 min. The sample was placed in a constant temperature drying oven and dried for 5 h to obtain the final multi-component synergistic 3D printed multi-structure long-lasting anti-infection bone repair composite scaffold. Example 4:

[0025] Adhesive preparation: Weigh PVA powder and deionized water according to a PVA mass fraction of 9% and put them into a beaker. Seal the beaker with plastic wrap and stir in an oil bath at 85°C to obtain a homogeneous and transparent solution. Then add 0.4 ml of glycerin and stir to obtain a colorless, homogeneous and transparent solution. 3D printing paste preparation: Weigh 3g of HA and 7g of β-TCP (total 10g) of two calcium phosphate-based materials, and slowly add the weighed materials to the binder solution while stirring to obtain a uniform printing paste; Create the printing model: Import the .stl format cylindrical printing model into the printing software, set the printing table temperature to 23℃, adjust the bottom diameter of the model to 12mm, the layer height to 0.45mm, the line spacing to 1mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the final printing model. Sample drying and sintering: The printed sample was left to stand in a dry environment for 30 hours. The completely dried scaffold was then placed in a muffle furnace for sintering. The temperature was increased to 1100℃ at a rate of 3.5℃ / h and held for 6 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare porous microspheres ABX@PLGA resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight (Mw) of 110,000 was weighed, wherein the mass ratio of LLA to GA was 50:50; it was added to 10 mL of CH2Cl2 (dichloromethane) solution, and 20 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved, thus controlling the PLGA:ABX ratio to be 10:1; an oil phase O was obtained. ABX@PLGA ; Prepare 2.5 mL of an 8% gel aqueous solution, recorded as WGel (gelatin aqueous solution), for later use. Gel Add the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; The WGel / OABX@PLGA emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, the final ABX@PLGA porous microspheres were obtained. Preparation of gelatin coating: Dissolve gelatin in deionized water at 50℃ to prepare a 10wt% gelatin solution. Immerse the bone scaffold in the gelatin solution and vacuum treat for 10 min. After removal, place the sample in a vacuum drying oven and dry at 50℃ for 24 h. Immerse the dried scaffold in a 3wt% glutaraldehyde solution to crosslink the gelatin layer for 25 min. Then, immerse it in ethanol three times. Finally, wash and dry it multiple times with distilled water. Preparation of drug-loaded composite scaffold: PLGA microsphere powder loaded with levofloxacin was added to deionized water and ultrasonically dispersed for 5 min. Then, the sintered bone scaffold was soaked and shaken for 20 min. The sample was placed in a constant temperature drying oven and dried for 5 h to obtain the multi-component synergistic 3D printed multi-structure long-lasting anti-infection bone repair composite scaffold.

[0026] Comparative Example 1 (containing only phosphorus-calcium based materials): Adhesive preparation: Weigh PVA powder (9% PVA by mass) and deionized water into a beaker, seal with plastic wrap, stir in an oil bath at 85°C to obtain a homogeneous and transparent solution, then add 0.4 mL of glycerin and stir to obtain a colorless, homogeneous and transparent solution. 3D printing paste preparation: Weigh 4.9g of HA and 2.1g of β-TCP (total 7g) of two calcium phosphate-based materials, and slowly add the weighed materials to the binder solution while stirring to obtain a uniform printing paste; Create the printing model: Import the .stl format cylindrical printing model into the printing software, set the printing table temperature to 23℃, adjust the bottom diameter of the model to 8mm, the layer height to 0.45mm, the line spacing to 1mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the final printing model. Sample drying and sintering: The printed sample was left to stand in a dry environment for 30 hours. The completely dried scaffold was then placed in a muffle furnace for sintering. The temperature was increased to 1100℃ at a rate of 2℃ / h and held for 3 hours to obtain the sintered bone scaffold. Characterization and performance testing of the stent: The scaffold samples prepared in Examples 1-4 were used to characterize the microstructure and elemental distribution of the blank scaffold, microspheres, and drug-loaded composite scaffold using scanning electron microscopy. Their porosity, compressive strength, and dimensional shrinkage before and after sintering were also tested. The specific testing method is as follows: Different groups of scaffolds were immersed in a certain volume (V1) of ethanol for 5 minutes, and the total volume after immersion (V2) was recorded. Then, the scaffolds were removed, and the remaining ethanol volume (V3) was recorded. Finally, the porosity of different groups of scaffolds was calculated using the following formula:

[0027] Calculations showed that the average porosity of the samples in Examples 1-4 were 55.3%, 58.2%, 56.8%, and 60.1%, respectively. Compressive strength was tested using a universal testing machine. Five sample scaffolds from Examples 1-4 and Comparative Example 1 were tested for compressive strength. The compressive strengths were 7.906 MPa, 8.887 MPa, 9.285 MPa, 8.457 MPa, and 6.568 MPa, respectively. This indicates that the bone scaffold samples prepared using this process meet the mechanical property standards. Furthermore, the gelatin coating enhances the mechanical properties of the bone scaffold, providing compressive strength similar to that of human cancellous bone, ensuring stability during implantation. The shrinkage rate of the samples before and after sintering was tested. After sintering, the shrinkage rate of the bottom diameter was approximately 27.7%, and the shrinkage rate of the height was approximately 20.6%.

[0028] Based on the controllable 3D printing structure of this invention, the shape of the scaffold can be adjusted arbitrarily, and the drug-loaded microspheres are evenly distributed in the gaps of the scaffold, which can precisely control the infected site and provide long-lasting antibacterial protection, while simultaneously repairing bone defects.

[0029] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold, characterized in that, Includes the following steps: Adhesive preparation: Weigh PVA powder and deionized water according to a PVA mass fraction of 6-12 wt%, stir in an oil bath at 80-95℃ until an adhesive solution is formed, add 0.5-1 mL of glycerol and stir to obtain a colorless and homogeneous adhesive solution. 3D printing slurry preparation: Weigh out calcium phosphate-based material, which is hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), with a mass ratio of hydroxyapatite to β-tricalcium phosphate of 3:7-7:3; Slowly add the calcium phosphate-based material to the binder solution and stir, wherein the total mass ratio of the binder solution to the calcium phosphate-based material is 10:5-10, to obtain a uniform printing slurry; Create the printing model: Import the .stl format cylindrical model into the printing software, set the printing table temperature to 20-25℃, the model bottom diameter to 6-12mm, the layer height to 0.3-0.6mm, the line spacing to 1-2mm, the pore structure to 0-90° filling, and the printing path to Z-shape to obtain the printing model; Sample drying and sintering: The printed sample is left to stand in a dry environment for 24-48 hours. The dried scaffold is placed in a muffle furnace and heated to 1100℃-1200℃ at a heating rate of 2-5℃ / h. It is held for 2-6 hours to obtain the sintered bone scaffold. Preparation of drug-loaded microspheres: Gelatin was selected as a pore-forming agent to prepare ABX@PLGA porous microspheres resistant to antibiotic loading; At room temperature, 0.2 g of PLGA raw material with a weight-average molecular weight of 100,000-120,000 Mw was weighed, wherein the mass ratio of L-lactolactone (LLA) to glycolate (GA) was 50:50; it was added to 10 mL of CH2Cl2 solution, and 20-40 mg of ABX was weighed and added to the CH2Cl2 solution. The mixture was stirred until dissolved to obtain the oil phase O. ABX@PLGA ; Prepare 8% W Gel Add 2.5 mL of the aqueous solution to the above oil phase; stir using a homogenizer at 10000 rpm / min for 60 s to obtain a uniformly dispersed W. Gel / O ABX@PLGA Emulsion; W Gel / O ABX The PLGA emulsion was slowly transferred to 100 mL of 0.1% PVA solution at a stirring speed of 400 rpm / min to obtain W. Gel / O ABX@PLGA / W PVA Re-emulsion; The solution was transferred to a 50°C warm water bath and stirred at low speed to remove excess gel from the microspheres. After centrifugation, washing, and freeze-drying, ABX@PLGA porous microspheres were obtained. Preparation of gelatin coating: Dissolve gelatin in deionized water at 40-60℃ to prepare a 2-10wt% gelatin solution; immerse the sintered bone scaffold in the gelatin solution, vacuum treat for 5-15 min, and vacuum dry at 40-60℃ for 24 h; after drying, immerse the scaffold in a 2-3wt% glutaraldehyde solution and crosslink the gelatin layer for 20-40 min, then immerse in ethanol, wash with distilled water, and dry. Preparation of drug-loaded composite scaffold: Drug-loaded microsphere powder was added to deionized water and ultrasonically dispersed for 5-10 min. The sintered bone scaffold was then soaked and shaken for 10-30 min and dried at a constant temperature for 5-10 h to obtain a 3D-printed gelatin-modified porous phospho-calcium-based drug-loaded composite bone repair scaffold.

2. The preparation method according to claim 1, characterized in that, In the adhesive formulation, the oil bath temperature is 85℃, and the PVA mass fraction is 6-9wt%.

3. The preparation method according to claim 1, characterized in that, In the preparation of 3D printing slurry, the total mass of calcium-phosphorus-based materials is 7-10g, and the mass ratio of hydroxyapatite to β-tricalcium phosphate is 7:

3.

4. The preparation method according to claim 1, characterized in that, During the process of creating the printing model, the printing table temperature was 23℃, the bottom diameter of the model was 8-12mm, the layer height was 0.45mm, and the line spacing was 1mm.

5. The preparation method according to claim 1, characterized in that, In the sample drying and sintering process, the drying time was 30 h, the heating rate was 2-3.5 ℃ / h, the sintering temperature was 1100 ℃, and the holding time was 3-6 h.

6. The preparation method according to claim 1, characterized in that, In the preparation of drug-loaded microspheres, the antibiotics were vancomycin, roxithromycin, doxycycline, or levofloxacin, the mass of PLGA was 0.4 g, and the volume of dichloromethane was 10 mL.

7. The preparation method according to claim 1, characterized in that, In the preparation of the gelatin coating, the gelatin solution temperature is 50℃, the gelatin mass fraction is 2-10wt%, the glutaraldehyde mass fraction is 2-3wt%, and the crosslinking time is 25min.

8. The preparation method according to claim 1, characterized in that, In the preparation of the drug-loaded composite scaffold, the ultrasonic dispersion time was 5 min, the shaking time was 20 min, and the drying time was 5 h.

9. A multi-component synergistic 3D-printed multi-structure long-lasting anti-infection bone repair composite scaffold prepared by the method of any one of claims 1-8, characterized in that, The porosity of the support is 55%-61%, the compressive strength is 7.9-9.3 MPa, the bottom diameter shrinkage rate after sintering is about 27.7%, and the height shrinkage rate is about 20.6%.

10. The bone repair composite scaffold according to claim 9, characterized in that, The scaffold comprises a gelatin coating and drug-loaded PLGA microspheres containing antibiotics.

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