Multi-nozzle 3D printing composite scaffold capable of promoting bone regeneration as well as preparation method and application of multi-nozzle 3D printing composite scaffold

Abstract

The invention provides a multi-nozzle 3D printing composite scaffold capable of promoting bone regeneration as well as a preparation method and application thereof, the multi-nozzle 3D printing composite scaffold is prepared by adopting multi-nozzle collaborative printing of a 3D printing system, the multi-nozzle 3D printing composite scaffold comprises a synthetic polymer material printed by a high-temperature nozzle and a hydrogel material printed by a low-temperature nozzle, and microfilament patterns formed by the two materials are alternately arranged. Wherein the synthetic polymer material is prepared from polycaprolactone and magnesium oxide nano particles; the hydrogel material is prepared from methacrylated gelatin, methacrylated gilan gum and double drug loading nano particles. The 3D printing composite scaffold provided by the invention has excellent mechanical properties, the double-drug-loaded nanoparticle sequence released osteogenesis promoting and vascular promoting drug coupling osteogenesis-vascularization promoting effects, released magnesium ions synergistically enhance osteogenesis and vascularization performance, and the 3D printing composite scaffold has a good bone defect repair effect and has application potential in the field of bone tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a multi-nozzle 3D printed composite scaffold that promotes bone regeneration, its preparation method, and its application. Background Technology

[0002] Bone defects are a common orthopedic condition, often caused by infection, trauma, or tumor resection. Bone defects not only lead to pain, deformity, and limited mobility, but can also cause complications such as infection and muscle atrophy, resulting in long-term suffering for patients. With the development of tissue engineering, 3D-printed bone defect repair scaffolds have shown great potential as bone repair materials due to their high degree of customization and excellent biocompatibility. However, existing 3D-printed scaffolds still suffer from problems such as insufficient vascularization and poor osteogenic induction. Furthermore, in treating bone defects, 3D bioprinted scaffolds also exhibit insufficient mechanical strength, mismatched degradation characteristics, and insufficient osteogenic capacity. Therefore, developing highly active repair scaffolds that can effectively adapt to the bone regeneration process using 3D printing technology remains a challenge that urgently needs to be overcome.

[0003] Extrusion-based 3D printing technology can rapidly fabricate scaffolds with complex geometries and enhance bioactivity by loading cells, making it a common technique for preparing bone tissue engineering scaffolds. However, scaffolds with a single hydrogel component often have inherent drawbacks, such as weak mechanical strength and a lack of versatility. The application of such scaffolds in the repair of load-bearing bone defects requiring high mechanical strength is limited by their insufficient mechanical properties. While increasing the hydrogel concentration can enhance mechanical strength, excessively high concentrations can inhibit cell migration, proliferation, and angiogenesis. Furthermore, hydrogel scaffolds exhibit poor chemical stability. Their degradation behavior can affect in vivo repair efficacy, potentially leading to premature or delayed degradation that is mismatched with the rate of new bone formation. Researchers have developed a new approach to overcome these limitations by synergistically 3D printing two different materials to create bone defect repair scaffolds that combine improved mechanical properties with good bioactivity.

[0004] During bone remodeling, various growth factors play an indispensable role in angiogenesis and osteogenic processes. Among osteogenic factors, bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor β (TGF-β) superfamily, is a potent osteogenic and chondrogenic inducing factor. BMP-4 deficiency can lead to skeletal deformities, especially in BMP-4 and BMP-2 double knockout mice, which exhibit severe osteogenic impairment. BMP-4 exerts its osteogenic induction effect through the BMP-4 / Smad pathway: this pathway forms a transcriptional complex by binding to membrane receptors, initiating the expression of osteogenic-related genes and activating downstream Smad signaling, thereby promoting bone matrix deposition. Furthermore, BMP-4 promotes chondrocyte differentiation and hypertrophy, ultimately driving endochondral ossification. Integrating BMP-4 into biological scaffolds can significantly improve the efficiency of bone defect repair.

[0005] Studies have shown that the degree of vascularization has a profound impact on the therapeutic effect of implants. New blood vessels can deliver nutrients and oxygen, thereby maintaining cell survival and differentiation. Previous studies have identified hypoxia-inducible factor (HIF) as a key signaling molecule in angiogenesis, and dimethyloxaloylglycine (DMOG) can stabilize HIF-1α expression and enhance VEGF expression under normoxic conditions. The application of DMOG can significantly promote vascularized bone regeneration. Therefore, by combining osteogenic and angiogenic functions in a 3D-printed scaffold to synergistically deliver osteogenic and angiogenic factors, osteogenic-angiogenic coupling can be achieved, resulting in superior vascularized bone regeneration. Summary of the Invention

[0006] To integrate bioactivity and mechanical properties, this invention provides a method for preparing a 3D-printed composite scaffold that combines mechanical properties and bioactivity, as well as its application.

[0007] A multi-nozzle 3D-printed composite scaffold promoting bone regeneration was fabricated using a dual-nozzle synergistic 3D printing technique. It comprises a hydrogel material printed by a low-temperature nozzle and a synthetic polymer material printed by a high-temperature nozzle. The hydrogel material consists of methacrylamide gelatin, methacrylamide gluconate gum, and dual-drug-loaded nanoparticles, while the synthetic polymer material consists of polycaprolactone and magnesium oxide nanoparticles. The dual-drug-loaded nanoparticles in the hydrogel material can sequentially release osteogenic and angiogenic drugs, while the synthetic polymer material can continuously release magnesium ions. The synergistic effect of these multiple factors promotes osteogenic-angiogenic coupling. The synthetic polymer material exhibits good mechanical strength and, due to its slow degradation, can provide long-term mechanical support.

[0008] A method for fabricating a multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties includes the following steps: Step S1: Synthesize nanoparticles with a mesoporous structure. Mix the nanoparticles with a solution of angiotensin-promoting drugs to obtain single-drug-loaded nanoparticles with angiotensin-promoting drugs loaded in the lumen. Immerse the single-drug-loaded nanoparticles in polyelectrolytes with opposite charge properties to obtain polyelectrolyte-modified single-drug-loaded nanoparticles. Then mix them with bone-promoting protein molecules to obtain dual-drug-loaded nanoparticles.

[0009] Step S2: Methacrylated gelatin, methacrylated glycan gum, dual-drug-loaded nanoparticles and photoinitiator are thoroughly stirred and dissolved in phosphate buffer solution to obtain the 3D printing ink of the hydrogel material part.

[0010] Step S3: Place polycaprolactone in tetrahydrofuran, and after dissolving, add magnesium oxide nanoparticles, stir thoroughly to mix, and evaporate the solvent to obtain the 3D printing ink of the synthesized polymer material part.

[0011] Step S4: The hydrogel material 3D printing ink prepared in step S2 is loaded into the low-temperature nozzle barrel of the pneumatic extrusion 3D printer, and the synthetic polymer material 3D printing ink prepared in step S3 is loaded into the high-temperature nozzle barrel of the temperature-controlled screw extrusion. The composite scaffold is obtained by dual-nozzle collaborative printing technology, and then the hydrogel part is crosslinked by light irradiation.

[0012] A multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties is used as a biological scaffold for bone defect repair.

[0013] Technical principle of the invention: A biocomposite scaffold was fabricated using a dual-nozzle synergistic 3D printing technique. The dual nozzles can load different printing materials, and through extrusion printing and fused deposition modeling, microfilaments of the two materials are alternately arranged. By loading dual drug-loaded nanoparticles into a hydrogel material, sequential drug release is achieved, while simultaneously maintaining osteogenic and angiogenic properties, effectively enhancing the osteogenic activity of the composite. Furthermore, the synergistic printing of polycaprolactone loaded with magnesium oxide nanoparticles further improves the scaffold's mechanical properties, maintaining long-term mechanical support and promoting cell or tissue ingrowth into the scaffold. Simultaneously, the released magnesium ions further enhance osteogenic and angiogenic effects. This biocomposite scaffold exhibits excellent drug release and osteogenic and angiogenic activity, while also achieving enhanced mechanical strength, improving its applicability in bone defect repair.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses dual-nozzle collaborative 3D printing technology to prepare a composite scaffold combining multiple materials. The hydrogel part provides biocompatibility and bioactivity, while the synthetic polymer material part provides mechanical support, thus taking into account both bioactivity and mechanical properties.

[0015] 2. The dual-drug-loaded nanoparticles in the hydrogel material of this invention are modified by surface modification of chitosan / hyaluronic acid polyelectrolyte, which endows the internal pores with dimethyl oxaloyl glycine and adsorbs cytokines through the polyelectrolyte layer on the outside, thus realizing a time-differentiated drug delivery strategy.

[0016] 3. The biocomposite scaffold of the present invention can promote osteogenic differentiation and subsequent vascular invasion of bone marrow mesenchymal stem cells by sequentially delivering osteogenic and angiogenic drugs, thereby achieving osteogenic-angiogenic coupling at the bone defect site and accelerating vascularized bone regeneration.

[0017] 4. The hydrogel material in the biocomposite scaffold of this invention degrades rapidly, which leads to the rapid loss of the loaded drug. However, the loaded magnesium oxide nanoparticles have a long-term magnesium ion release effect, which can play a supplementary role, continuously maintain osteogenic-angiogenic effects, and synergistically enhance the bone repair effect at the bone defect site. Attached Figure Description

[0018] Figure 1 Schematic diagram of the fabrication of a composite scaffold printed by dual nozzles.

[0019] Figure 2 Characterization and concentration ratio screening of printing ink materials. (AB) NMR and Fourier transform infrared spectra of GelMA and GGMA; (C) Printability test of inks with different concentration ratios; (DF) Rheological property test of printing inks; (GI) Mechanical property test of different hydrogels; *P<0.05, **P<0.01, one-way ANOVA.

[0020] Figure 3 Characterization of 3D-printed composite scaffolds. (A) Photographs of scaffolds printed with different filament spacings; (B) Photographs of scaffolds printed with different compositions; (C) Three-view diagrams of the composite scaffold; (D) Scanning electron microscope images of scaffolds with different components; (EG) Mechanical property tests of the composite scaffold; *P<0.05, one-way ANOVA.

[0021] Figure 4 Characterization of the in vitro angiogenic properties of the composite scaffold. (AB) Microscopic images and quantitative analysis of scratch healing rate of HUVECs cells; (CD) Microscopic images and quantitative analysis of HUVECs cell migration results; (EF) Microscopic images and quantitative analysis of tubular structure formation in HUVECs cells; *P<0.05, **P<0.01, one-way ANOVA.

[0022] Figure 5 Characterization of the osteogenic properties of the composite scaffold in vitro. (A) Alkaline phosphatase staining results after 14 days of BMSCs culture; (B) Quantitative analysis of alkaline phosphatase activity after 3, 7 and 14 days of culture; (CD) Alizarin Red S staining results and quantitative analysis after 14 days of culture; *P<0.05, **P<0.01, one-way ANOVA. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0024] A multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties is disclosed. The scaffold is prepared by a multi-nozzle collaborative printing device of a 3D printing system. The synthetic polymer material printed by the high-temperature nozzle includes polycaprolactone and magnesium oxide nanoparticles, while the hydrogel material printed by the low-temperature nozzle includes methacrylamide gelatin, methacrylamide gluconate gum, and dual-drug-loaded nanoparticles. The microfilament patterns formed by the two printed materials are arranged alternately.

[0025] The dual-drug-loaded nanoparticles in the multi-nozzle 3D-printed composite scaffold of the present invention exhibit sequential release behavior of bone-promoting and angiogenesis-promoting drugs, and also release magnesium ions.

[0026] like Figure 1 As shown, a method for preparing a multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties includes the following steps: Step S1: Synthesize nanoparticles with mesoporous structure, mix the nanoparticles with a drug solution that promotes angiogenesis to obtain single-drug-loaded nanoparticles with an intraluminal loading of the drug; immerse the single-drug-loaded nanoparticles in polyelectrolytes with opposite charge properties to obtain polyelectrolyte-modified single-drug-loaded nanoparticles; then mix the single-drug-loaded nanoparticles with bone-promoting protein molecules to obtain dual-drug-loaded nanoparticles. Step S2: Methacrylated gelatin, methacrylated glycan gum, the dual-drug-loaded nanoparticles prepared in step S1, and the photoinitiator are thoroughly stirred and dissolved in a phosphate buffer solution to obtain the 3D printing ink of the hydrogel material portion. Step S3: Place polycaprolactone in tetrahydrofuran, and after dissolving, add magnesium oxide nanoparticles, stir thoroughly to mix, and evaporate the solvent to obtain the 3D printing ink of the synthesized polymer material part; Step S4: The hydrogel material 3D printing ink prepared in step S2 is loaded into the low-temperature nozzle barrel of the pneumatic extrusion 3D printer, and the synthetic polymer material 3D printing ink prepared in step S3 is loaded into the high-temperature nozzle barrel of the temperature-controlled screw extrusion. The composite scaffold is obtained by dual-nozzle collaborative printing technology, and then the hydrogel part is crosslinked by light irradiation.

[0027] The dual-drug-loaded nanoparticles of the present invention are modified with polyelectrolytes, and the polyelectrolytes used are at least one selected from chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, and polylysine. Preferably, the polyelectrolytes with opposite charge properties used in step S1 are chitosan and hyaluronic acid, with a chitosan solution concentration of 5 mg / mL and a hyaluronic acid solution concentration of 5 mg / mL.

[0028] The dual-drug-loaded nanoparticles of the present invention contain at least one of the following drugs with pro-angiogenic properties: dimethyloxaloylglycine, deferoxamine, and sphingosine 1-phosphate. The externally adsorbed drug is at least one of the following bone morphogenetic proteins: bone morphogenetic protein 2, bone morphogenetic protein 4, and bone morphogenetic protein 7. Preferably, the pro-angiogenic drug used is dimethyloxaloylglycine. The bone morphogenetic protein molecule used is bone morphogenetic protein 4.

[0029] The mass ratio of methacrylated gelatin to methacrylated glucon in this invention is 7:0.5~3. Preferably, the mass ratio of methacrylated gelatin to methacrylated glucon is 7:2.

[0030] The mass ratio of the dual-drug-loaded nanoparticles in this invention is 0.01 to 0.1:1. Preferably, the mass ratio of the dual-drug-loaded nanoparticles in the hydrogel material is 0.1:1.

[0031] The mass ratio of magnesium oxide nanoparticles in this invention is 0.05~0.2:1. Preferably, the mass ratio of magnesium oxide nanoparticles in the synthetic polymer material is 0.1:1.

[0032] The temperature control temperature of the low-temperature nozzle of this invention is 4~25℃, and the temperature control temperature of the high-temperature nozzle is 60~120℃. Preferably, the temperature control temperature of the low-temperature nozzle is 20℃, and the temperature control temperature of the high-temperature nozzle is 90~110℃.

[0033] Preferably, the nanoparticles in step S1 are mesoporous silica.

[0034] Preferably, in step S3, the microfilament patterns printed by the hydrogel material and the synthetic polymer material are arranged alternately.

[0035] Preferably, in step S3, the bone-promoting drug release rate of the dual-drug-loaded nanoparticles in the 3D-printed composite scaffold is faster than that of the angiogenesis-promoting drug, and it also has a continuous magnesium ion release.

[0036] A multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties is used as a biological scaffold for bone defect repair.

[0037] Example 1: A method for preparing a multi-nozzle 3D-printed composite scaffold with bone regeneration promotion, comprising the following steps: 1. Preparation of dual-drug-loaded nanoparticles: In a flask, 24 mL of cetyltrimethylammonium chloride (25 wt%), 180 mg of triethylamine, and 36 mL of deionized water were added. The mixture was stirred slowly at 60 °C for 1 hour. Then, 20 mL of 10% tetraethyl orthosilicate solution was slowly added to the mixture. The mixture was stirred slowly at 60 °C for 60 hours, followed by centrifugation to collect the product. After washing with ethanol and centrifugation again, 2 g of sodium chloride and 100 mL of ethanol were added, and the mixture was stirred at 60 °C to remove the template. This process was repeated 2 to 3 times. Finally, the product was freeze-dried to obtain mesoporous silica nanoparticles (bMSN). 100 mg of bMSN was added to a 3 mg / mL DMOG solution, stirred in the dark for 24 hours, followed by centrifugation at 11,000 rpm for 15 minutes. After freeze-drying, DMOG-loaded nanoparticles (DMOG@bMSN) were obtained.

[0038] A 5 mg / mL hyaluronic acid (HA) solution and a 5 mg / mL chitosan (Chi) solution were prepared. DMOG@bMSN nanoparticles were added to the Chi solution and stirred for 30 minutes. The supernatant was removed by centrifugation. Then, HA solution was added and stirred for 30 minutes. After two cycles, the mixture was finally transferred to the Chi solution and stirred for 30 minutes to achieve Chi modification of the outermost layer of the particles. The nanoparticles were then washed with deionized water and centrifuged to obtain polyelectrolyte-modified nanoparticles. These nanoparticles were placed in a 1 μg / mL BMP-4 solution and stirred at room temperature in the dark for 12 hours. The product was collected by centrifugation and freeze-dried to obtain dual-drug-loaded nanoparticles (BD@bMSN).

[0039] 2. Dual printheads work together to print composite supports: (1) Synthesis and preparation of hydrogel-based printing ink 140 mg of methacrylamide gelatin (GelMA) and 40 mg of methacrylamide glycan (GGMA) were dissolved in 2 mL of phosphate-buffered saline (PBS), and the solution was stirred in a water bath at 37°C. Subsequently, 10% (w / w) of the dry weight of the hydrogel containing dual-drug-loaded nanoparticles (BD@bMSN) was dispersed in the hydrogel solution. After thorough stirring, 0.25% (w / w) of the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) was added to obtain the 3D printing ink (BD@GG) for the hydrogel portion.

[0040] (2) Preparation of synthetic materials for printing 20 g of polycaprolactone (PCL) was placed in a sieve-sealed glass bottle, and an appropriate amount of tetrahydrofuran (THF) was added. The mixture was stirred at room temperature until completely dissolved. Then, 1-4 g of magnesium oxide nanoparticles were added, and the mixture was stirred thoroughly. The solution was then poured into a dish and the solvent was evaporated in a fume hood. After drying, magnesium oxide-doped polycaprolactone (Mg@PCL) was obtained.

[0041] (3) Fabrication of a composite scaffold for dual-nozzle synergistic 3D printing The BD@GG printing ink for the hydrogel portion was placed in the cryogenically controlled barrel of a 3D printer, while Mg@P was placed in a cryogenically controlled screw barrel. A Mg@P / BD@GG composite scaffold was fabricated using extrusion 3D printing in conjunction with fused deposition modeling (FDM) 3D printing. The parameters for the hydrogel portion were set as follows: nozzle diameter 0.31 mm, barrel temperature 20 °C. The parameters for the composite material portion were set as follows: nozzle diameter 0.30 mm, nozzle temperature 90–110 °C, filament spacing 1.2 mm, layer height 0.3 mm.

[0042] 3. Characterization of printing ink and support (1) Characterization of printing ink nuclear magnetic resonance hydrogen spectrum ( 1 H NMR and Fourier transform infrared (FTIR) spectra showed that GelMA exhibited a new vinyl proton peak for methacrylates in the range of 5.3–5.6 ppm. Figure 2 (A) Compared to pure gelatin (Gel) samples, the proton peak of the lysine methylene group on the GelMA molecular chain decreased at ~2.9 ppm, indicating that some amino groups were successfully substituted by methacrylate. The acrylic proton peak of GGMA appeared at 5.6-6.1 ppm, and a new signal peak of methacrylate methyl groups (1.9 ppm) appeared, confirming the successful synthesis of GGMA. FTIR spectra of gelatin and GelMA ( Figure 2 Both GelMA and GGMA exhibit a C=O stretching vibration peak at 1632 cm⁻¹ for amide I, an NH bending vibration peak at 1529 cm⁻¹ for amide II, and a CN bending vibration peak at 1232 cm⁻¹ for amide III. GelMA and GGMA both show an asymmetric COO stretching vibration peak at 1603 cm⁻¹, a symmetric COO stretching vibration peak at 1405 cm⁻¹, and a CO stretching vibration peak at 1022 cm⁻¹. GGMA shows a C=O stretching vibration peak characteristic of methacrylate structures at 1706 cm⁻¹. These results indicate the successful synthesis of GelMA and GGMA.

[0043] The printability of inks with different ratios of GelMA / GGMA was tested. Studies show that adding GGMA broadens the gelation temperature range of the ink, making it more suitable for 3D printing with loaded cells. Figure 2As shown in Figure C, low-concentration GelMA / GGMA (gray area) exhibits poor gelation ability at 20°C, forming droplets at the nozzle and thus is unsuitable for 3D printing. High-concentration GelMA / GGMA (orange area), due to its higher concentration and stronger gelation properties, shows over-gelling, requiring higher air pressure (above 0.2 MPa) for extrusion, and is therefore unsuitable for cell-borne bioprinting. In contrast, medium-concentration GelMA / GGMA (blue area) demonstrates superior gel formation and extrusion performance at lower pressures, providing better conditions for cell-borne 3D printing. Therefore, the combination of 7% GelMA and 0.5%–2% GGMA shows the most ideal printing performance.

[0044] To determine the optimal concentration ratio of GelMA to GGMA, four different bio-inks (7% GelMA, 7% GelMA / 1% GGMA, 7% GelMA / 2% GGMA, and 7% GelMA / 3% GG-MA) were selected for rheological testing. The gel-sol transition temperatures of the four bio-inks were obtained by detecting the changes in storage modulus (G′) and loss modulus (G″) with temperature. Figure 2 (D). The gelation temperature of 7% GelMA was 19.7℃. The gelation temperature of 7% GelMA / 1% GGMA was even higher, reaching 21.4℃. 7% GelMA / 2% GGMA exhibited a wider gelation temperature range due to the increased GGMA content, reaching 30.4℃. 7% GelMA / 3% GGMA, due to its excessively high GGMA concentration, had a gelation temperature exceeding 35℃. This indicates that the addition of GGMA significantly affects the gelation temperature of GelMA / GGMA bio-inks, possibly because GGMA enhances hydrogen bonding in the hydrogel network. Figure 2 Figure E shows the viscosity changes of GelMA / GGMA bio-inks at different temperatures. All bio-inks showed a trend of decreasing viscosity with increasing temperature. Figure 2 The results show that the viscosity of GelMA / GGMA bio-inks decreases with increasing shear rate, indicating that all bio-inks exhibit shear-thinning properties, suggesting that these bio-inks can be used for extrusion printing.

[0045] Hydrogels were then prepared by photocrosslinking the different bio-inks mentioned above, and their compressive mechanical properties were tested. The strain-stress curve results showed that the compressive strength of the GelMA / GGMA hydrogel was improved compared to that of the GelMA hydrogel. Figure 2 (G). Figure 2 H and Figure 2Figure I shows the compressive stress and compressive modulus data. Both compressive stress and compressive modulus increase with increasing GGMA content in the hydrogel. The maximum stress of the 7% GelMA / 2% GGMA hydrogel is significantly higher than that of the 7% GelMA hydrogel, while the maximum stress of the 7% GelMA / 3% GGMA hydrogel is significantly better than that of all other hydrogels. Figure 2 The results also showed that adding GGMA significantly improved the compressive modulus of the hydrogel, with 7% GelMA / 2% GGMA and 7% GelMA / 3% GGMA exhibiting even higher compressive moduli. Considering printability, rheology, and mechanical properties, 7% GelMA / 2% GGMA was ultimately selected as the printing ink for the hydrogel portion.

[0046] (2) Characterization of the printing bracket Figure 3 Figure A shows different filament pitches for PCL / GelMA / GGMA (P / GG) scaffolds. To ensure good performance of the printing scaffolds, 1.2 mm was selected as the pitch for the printing scaffolds. Figure 3 The image in section B shows actual photos of P / GG, Mg@P / GelMA / GGMA (Mg@P / GG) and Mg@P / bMSN@GelMA / GGMA (Mg@P / bMSN@GG) composite scaffolds, each exhibiting excellent 3D printing performance. Figure 3 Image C shows a three-view diagram of a Mg@P / bMSN@GG composite scaffold with a height and width of 5 mm, indicating that the composite scaffold structure is intact and the filaments are evenly distributed. These results demonstrate that 3D-printed composite scaffolds can be prepared using synergistic 3D printing technology, with bMSN@GelMA / GGMA as the hydrogel component and MgO@PCL as the synthetic polymer, providing feasibility for the manufacture of large-size scaffolds.

[0047] 3D-printed scaffolds with different components were observed using scanning electron microscopy (SEM). Figure 3 As shown in Figure D, each type of 3D printed scaffold exhibits a mesh-like structure. The GG hydrogel scaffold shows obvious porous characteristics in its filaments, while the bMSN@GG scaffold has a coarse filament structure, indicating the presence of nanoparticles. The PCL and Mg@PCL scaffolds are of uniform thickness, with obvious particles visible on the surface of the Mg@PCL scaffold filaments, confirming the incorporation of MgO nanoparticles. The Mg@P / bMSN@GG composite scaffold prepared based on bMSN@GG hydrogel and Mg@PCL exhibits both of the above-mentioned fiber structures, with bMSN@GG filaments uniformly distributed among the Mg@PCL filaments.

[0048] Hydrogel scaffolds are inherently brittle and have relatively weak mechanical strength. Therefore, using PCL material as the support structure will significantly improve the mechanical properties of the scaffold. Figure 3In the figures, E, G, and E represent the stress-strain curves, maximum stress, and compressive modulus of the composite scaffold, respectively. The results show that the mechanical properties of the Mg@P / bMSN@GG scaffold are superior to those of the P / GG scaffold. The compressive modulus of the Mg@P / bMSN@GG scaffold (27.28 ± 1.31 MPa) is higher than that of the P / GG scaffold (22.51 ± 0.68 MPa), indicating that the addition of MgO has a certain effect on improving compressive strength. This study suggests that scaffolds with high mechanical strength can provide better mechanical support for structural maintenance and are suitable for the repair of weight-bearing bone defects.

[0049] 3. Biocompatibility Testing of Composite Scaffolds (1) In vitro angiogenesis properties The recruitment capacity of different scaffolds for human umbilical vein endothelial cells (HUVECs) was evaluated using scratch assays and Transwell migration assays. 5 × 10⁶ cells were seeded per well. 4 After collecting HUVECs cells, 24-well plates were placed in an incubator at 37 ℃. When the cell confluence reached 80%, scratches were prepared using a 200 μL pipette tip. FBS-free medium containing scaffold extract was then added to the wells, and the plates were incubated at 37 ℃ and images were taken under a microscope. Wound closure rate was calculated using ImageJ software. The scratch experiment results showed that cell migration varied at 24 and 48 hours, exhibiting a scratch healing trend. The Mg@P / BD@GG group showed the most significant healing effect. Figure 4 (A). Quantitative analysis showed that the Mg@P / BD@GG group exhibited the highest healing rate ( Figure 4 (B)

[0050] The ability of the Mg@P / BD@GG scaffold to recruit HUVECs was further verified by Transwell migration experiments. 200 μL of Mg@P / BD@GG scaffold was used to recruit HUVECs. 4 A suspension of HUVECs cells was added to the lower chamber of a 24-well Transwell plate, along with 600 μL of scaffold extraction buffer. After culturing for 24 hours, the cells were fixed on the membrane with 4% paraformaldehyde. After gently wiping away unmigrated cells, the cells were incubated with 1% crystal violet solution for 10 minutes. The surface of the lower chamber was observed and photographed under an inverted fluorescence microscope. Quantitative analysis was performed by dissolving crystal violet in 10% acetic acid and measuring absorbance at 590 nm to assess cell migration ability. The number of cells migrating to the lower chamber in the Mg@P / BD@GG group was significantly higher than that in other groups. Figure 4 (C). Quantitative analysis showed that the Mg@P / BD@GG group had a higher absorbance value ( Figure 4 (D).

[0051] The ability of the scaffold to promote tubular structure formation in HUVECs was evaluated using a tubular structure formation assay. Matrigel cells were thawed at 4°C and transferred to 48-well plates on ice, followed by incubation at 37°C for 30 minutes. Two × 10⁶ seeds were seeded onto the Matrigel surface in each well. 4 HUVECs were cultured in a medium containing different scaffold extracts. After 6 hours of culture, images were taken using a microscope, and vascular parameters in the images were calculated using ImageJ software. The results are as follows: Figure 4 As shown in Figure E, HUVECs formed tubular structures in all groups. Among them, the Mg@P / BD@GG group exhibited a richer tubular network. Quantitative analysis of the number of grids, trunks, and nodes showed that ( Figure 4 Compared to other groups, the Mg@P / BD@GG group had the highest vascular parameter values, indicating that DMOG and Mg ion release have a positive effect on inducing angiogenesis.

[0052] (2) In vitro osteogenic properties The in vitro osteogenic properties of the scaffold were evaluated using alkaline phosphatase (ALP) staining and ALP activity assays. Bone marrow mesenchymal stem cells (BMSCs) were used at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of 24-well plates and cultured for 24 hours, then the medium was replaced with different scaffold extracts. After 3, 7, and 14 days of culture, cells were washed with PBS and lysed using RIPA lysis buffer. Lysis buffer was collected by centrifugation. ALP activity was measured using an ALP detection kit, and total protein concentration in the lysate was determined using a BCA protein assay kit. ALP activity was calculated according to the kit instructions. In addition, cells cultured for 14 days were fixed with paraformaldehyde and stained using an ALP staining kit; the stained cells were photographed under a microscope. Results showed that after 14 days of culture, all groups exhibited significant ALP expression staining, with the Mg@P / BD@GG group showing higher staining intensity. Figure 5 (A). Quantitative analysis showed that ALP activity gradually increased with prolonged culture time. Figure 5 (Middle B). After 7 and 14 days of culture, the Mg@P / B@GG and Mg@P / BD@GG groups showed higher ALP activity compared to other scaffold groups, which was attributed to the presence of Mg ions and BMP-4 as stimulators of osteogenic differentiation.

[0053] Alizarin Red S (ARS) staining was used to detect the ability of scaffolds to induce BMSCs to form a mineralized matrix. The BMSC culture procedure was the same as that for ALP activity assay. Cells cultured for 14 days were fixed with 4% paraformaldehyde solution. After removing the paraformaldehyde, 2% (w / v) ARS staining solution was added to each well for 10 minutes. The ARS was then washed away with distilled water, and the culture plate was observed and photographed under a microscope. Quantitative ARS analysis was performed by adding 10% acetic acid solution to the wells and incubating at room temperature. After centrifugation, the supernatant was collected and 10% ammonia was added. 100 μL of sample was placed in a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader. The results are as follows: Figure 5 As shown in Figure C, all Mg@P groups showed significant calcareous deposition, with a marked increase in the number of mineralized nodules in the Mg@P / B@GG and Mg@P / BD@GG groups. Quantitative analysis using ARS staining was consistent with the staining results. Figure 5 (D). The Mg@P / B@GG and Mg@P / BD@GG groups showed higher calcium deposition, indicating that the composite scaffold has stronger osteogenic capacity.

[0054] This invention provides a multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties, prepared through dual-nozzle synergistic printing. The hydrogel portion is printed using methacrylamide gelatin / methacrylamide gluconate printing ink and dual-drug-loaded nanoparticles, while the synthetic polymer portion is prepared using polycaprolactone composites containing magnesium oxide nanoparticles. The composite scaffold exhibits excellent integration properties and demonstrates enhanced mechanical strength and bioactivity. Through the release of BMP-4, DMOG, and Mg ions, the scaffold promotes osteogenic differentiation of bone marrow mesenchymal stem cells and simultaneously promotes angiogenesis. The 3D-printed composite scaffold provided by this invention possesses excellent mechanical properties, controllable drug molecule release, good biocompatibility, promotes angiogenesis, and couples osteogenic-angiogenic bone regeneration, demonstrating its application potential in the field of bone tissue engineering scaffold repair.

Claims

1. A multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties, characterized in that, The 3D printed composite scaffold is prepared by a multi-nozzle collaborative printing device of a 3D printing system. The synthetic polymer material printed by the high-temperature nozzle includes polycaprolactone and magnesium oxide nanoparticles, while the hydrogel material printed by the low-temperature nozzle includes methacrylamide gelatin, methacrylamide gluconate gum, and dual-drug-loaded nanoparticles. The microfilament patterns composed of the two printed materials are arranged alternately.

2. The multi-nozzle 3D-printed composite scaffold with bone regeneration promotion as described in claim 1, characterized in that, In multi-nozzle 3D printed composite scaffolds, dual-drug-loaded nanoparticles exhibit sequential release behavior of osteogenic and angiogenic drugs, along with the release of magnesium ions.

3. A method for preparing the multi-nozzle 3D-printed composite scaffold with bone regeneration promotion as described in claim 1, characterized in that, Includes the following steps: Step S1: Synthesize nanoparticles with mesoporous structure, mix the nanoparticles with a drug solution that promotes angiogenesis to obtain single-drug-loaded nanoparticles with an intraluminal loading of the drug; immerse the single-drug-loaded nanoparticles in polyelectrolytes with opposite charge properties to obtain polyelectrolyte-modified single-drug-loaded nanoparticles; then mix the single-drug-loaded nanoparticles with bone-promoting protein molecules to obtain dual-drug-loaded nanoparticles. Step S2: Methacrylated gelatin, methacrylated glycan gum, the dual-drug-loaded nanoparticles prepared in step S1, and the photoinitiator are thoroughly stirred and dissolved in a phosphate buffer solution to obtain the 3D printing ink of the hydrogel material portion. Step S3: Place polycaprolactone in tetrahydrofuran, and after dissolving, add magnesium oxide nanoparticles, stir thoroughly to mix, and evaporate the solvent to obtain the 3D printing ink of the synthesized polymer material part; Step S4: The hydrogel material 3D printing ink prepared in step S2 is loaded into the low-temperature nozzle barrel of the pneumatic extrusion 3D printer, and the synthetic polymer material 3D printing ink prepared in step S3 is loaded into the high-temperature nozzle barrel of the temperature-controlled screw extrusion. The composite scaffold is obtained by dual-nozzle collaborative printing technology, and then the hydrogel part is crosslinked by light irradiation.

4. The preparation method according to claim 3, characterized in that, The dual-drug-loaded nanoparticles are modified with polyelectrolytes, and the polyelectrolytes used are at least one of chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, and polylysine.

5. The preparation method according to claim 3, characterized in that, The drug loaded in the inner cavity of the dual-drug-loaded nanoparticles is at least one of the following: dimethyloxaloylglycine, deferoxamine, and sphingosine 1-phosphate, which promote angiogenesis; and the drug adsorbed on the outside is at least one of the following: bone morphogenetic protein 2, bone morphogenetic protein 4, and bone morphogenetic protein 7, which promote bone formation.

6. The preparation method according to claim 3, characterized in that, The mass ratio of methacrylated gelatin to methacrylated glycan is 7:0.5~3.

7. The preparation method according to claim 3, characterized in that, The mass ratio of dual-drug-loaded nanoparticles in the hydrogel material is 0.01~0.1:

1.

8. The preparation method according to claim 3, characterized in that, The mass ratio of magnesium oxide nanoparticles in the synthetic polymer material is 0.05~0.2:

1.

9. The preparation method according to claim 3, characterized in that, The temperature control temperature of the low-temperature nozzle is 4~25℃, and the temperature control temperature of the high-temperature nozzle is 60~120℃.

10. A multi-nozzle 3D-printed composite scaffold with bone regeneration-promoting properties, obtained by the preparation method according to any one of claims 3-9, is used as a biological scaffold for bone defect repair.

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