A 3D printed bone-like biological scaffold and a preparation method thereof

Abstract

This invention relates to the field of bone repair and discloses a 3D-printed bone-like bioscaffold and its preparation method. First, ammonium bromide is added to a mixture of deionized water and anhydrous ethanol to obtain an ammonium bromide solution. Then, ammonia solution, tetraethyl orthosilicate, triethyl phosphate, calcium nitrate, and sodium vanadate are added sequentially, and the mixture is continuously stirred and centrifuged to collect the precipitate. After washing and aging, the precipitate is heated and then cooled, ground, pulverized, and sieved to obtain V-MBG powder. A TA solution and a V-MBG solution are prepared, mixed, and freeze-dried to obtain TA@V-MBG powder. A chloroform solution of PLGA is prepared, and V-MBG powder is added and stirred thoroughly. The chloroform solution is then dried to obtain a solid slurry. An inner scaffold and a hollow outer scaffold are printed to obtain a bone-like bioscaffold. This scaffold is then immersed in a TA solution to obtain a PLGA / TA@V-MBG composite scaffold. This invention solves the problems of poor bone repair effect and poor osteoid cell proliferation and differentiation in existing technologies, which prevent the repair of bone defects caused by bone tumors.

Description

Technical Field

[0001] This invention relates to the field of bone repair, and more particularly to a 3D-printed bone-like bioscaffold and its preparation method. Background Technology

[0002] Regeneration and repair of large bone defects caused by trauma, infection, tumors, etc., is a challenging problem in clinical medicine. Osteosarcoma (OS) is the most common primary malignant bone tumor. Currently, the clinical treatment model for OS combines surgical intervention with chemotherapy. Surgical resection inevitably leads to the formation of large-area bone defects, with some tumor cells remaining in the defect area. These residual tumor cells pose a significant risk of local recurrence. Simultaneously, chemotherapy-induced immunosuppression, infection, and insufficient blood supply can severely hinder bone defect healing. Constructing bioactive artificial bone using tissue engineering methods, incorporating composite seed cells, scaffold materials, and growth factors, is considered one of the most effective methods for bone defect repair.

[0003] Currently, the types and quantities of tissue-engineered products available for clinical use are very limited, and their functions and structures are relatively simple. Utilizing novel bone regeneration and repair materials with high bioactivity to prepare bone repair scaffolds with interconnected porous structures to repair bone defects exceeding critical sizes has become a hot research area in recent years. Bone regeneration and repair materials need to possess good biocompatibility, bioactivity, and a certain degree of biodegradability. Currently, synthetic bone regeneration and repair materials include inorganic non-metallic materials, such as bioactive ceramics (β-tricalcium phosphate, hydroxyapatite (HAP), and other calcium phosphate system materials), bioactive glass, and microcrystalline glass; polymeric materials include natural and synthetic polymers.

[0004] In the field of bone tissue engineering, 3D-printed scaffolds have attracted much attention due to their outstanding characteristics of personalized customization and high precision. Macroscopically, bone consists of two parts: cancellous bone and cortical bone. Microscopically, bone is mainly composed of various cells and abundant extracellular matrix (ECM). This ordered, layered structure enhances the mechanical strength and adaptability of the scaffold. Therefore, it is essential to develop a biomimetic 3D-printed scaffold with a complex gradient structure similar to natural bone for tumor removal and bone defect repair after osteosarcoma resection. Summary of the Invention

[0005] The present invention aims to provide a 3D printed bone-like biological scaffold and its preparation method, which solves the problems of poor bone repair effect and poor osteoid cell proliferation and differentiation effect in the prior art, and the inability to achieve bone defect repair caused by bone tumors.

[0006] To achieve the above objectives, the present invention provides the following method:

[0007] The present invention provides a method for preparing a 3D-printed bone-like bioscaffold:

[0008] S1: Add 1.2g of hexadecyltrimethylammonium bromide to a mixture of 60mL of deionized water and 40mL of anhydrous ethanol to obtain an ammonium bromide solution;

[0009] S2: Add ammonia solution as a catalyst to the ammonium bromide solution, then add tetraethyl orthosilicate, triethyl phosphate, calcium nitrate and sodium vanadate in sequence, and centrifuge after continuous stirring to collect the precipitate;

[0010] S3: The precipitate was washed alternately with deionized water and anhydrous ethanol, then aged, heated to a temperature of 3 hours and cooled, ground into powder and sieved to obtain V-MBG powder.

[0011] S4: Prepare a 10 mg / ml TA solution and a 30 mg / ml V-MBG solution with pure water, mix them in a 1:1 ratio, and freeze-dry to obtain TA@V-MBG powder;

[0012] S5: Weigh a mixture of PLA and PGA polymer PLGA, wherein the ratio of PLA to PGA is 3:1, and dissolve it in chloroform to prepare a chloroform solution of PLGA.

[0013] S6: Add the V-MBG powder to the PLGA chloroform solution, stir thoroughly overnight with a magnetic stirrer, and after thorough mixing, use an oil pump to drain the chloroform solution to obtain a solid slurry.

[0014] S7: The solid slurry is shredded and placed into a high-temperature barrel. A needle, a high-temperature pneumatic device, and a high-temperature platform are selected for melting to obtain a first composite material. After the first composite material is melted and extruded into filaments, it is extruded layer by layer. The nozzle of the needle moves along the XY axis first, and after each layer is completed, it moves along the Z axis to print out the inner layer support.

[0015] S8: The solid slurry is shredded and placed into a high-temperature barrel. A needle, high-temperature pneumatic and rotating platform are selected for melting to obtain a second composite material. After the second composite material is melted and extruded into filaments, a hollow tube outer support is printed on the rotating platform.

[0016] S9: After the inner scaffold and the outer scaffold are printed, the inner scaffold is nested in the outer scaffold to obtain a gradient structure of bone-like bio-scaffold with large pores in the outer layer and small pores in the inner layer. The bone-like bio-scaffold is then immersed in the TA solution to obtain the PLGA / TA@V-MBG composite scaffold.

[0017] Preferably, the steps of adding ammonia solution as a catalyst to the ammonium bromide solution, and then sequentially adding tetraethyl orthosilicate, triethyl phosphate, calcium nitrate, and sodium vanadate, followed by continuous stirring and centrifugation to collect the precipitate include: adding 0.8 mL of ammonia solution as a catalyst to the ammonium bromide solution; adding 3.60 mL of tetraethyl orthosilicate, 0.36 mL of triethyl phosphate, 1.98 g of calcium nitrate, and 100.0 mg of sodium vanadate every 30 min; and continuously stirring for 5.5 h before centrifugation to collect the precipitate.

[0018] Preferably, the steps of washing the precipitate alternately with deionized water and anhydrous ethanol, aging it, heating it to a certain temperature for 3 hours and then cooling it, grinding it into powder and sieving it with a sieve to obtain V-MBG powder include: washing the precipitate alternately with deionized water and anhydrous ethanol 3 times; aging it at room temperature for 3 days, aging it at 37 ℃ for 3 days, heating it to 650 ℃ at a rate of 2 ℃ / min and holding it at that temperature for 3 hours and then cooling it; grinding it into powder and sieving the powder to a particle size of about 500 nm with a sieve to obtain the final product V-MBG.

[0019] Preferably, the step of preparing a TA solution of 10 mg / ml and a V-MBG solution of 30 mg / ml with pure water, mixing them in a 1:1 ratio, and then freeze-drying them to obtain TA@V-MBG powder includes: preparing a TA solution of 10 mg / ml and a V-MBG solution of 30 mg / ml with pure water, mixing them in a 1:1 ratio, placing them at -20°C, and then freeze-drying them for 24 hours to obtain TA@V-MBG powder.

[0020] Preferably, the step of adding the V-MBG powder to the PLGA chloroform solution, stirring thoroughly overnight with a magnetic stirrer, and then evacuating the chloroform solution with an oil pump to obtain a solid slurry includes: adding the V-MBG powder with a particle size of approximately 500 nm to the PLGA chloroform solution at a PLGA:V-MBG (w:w) ratio of 4:1, so that the final V-MBG content is 20%, then stirring thoroughly overnight with a magnetic stirrer; and finally evacuating the chloroform solution with an oil pump to obtain a solid slurry.

[0021] Preferably, the solid slurry is shredded and placed into a high-temperature barrel, and melted using a needle, high-temperature pneumatics, and a high-temperature platform to obtain a first composite material. After the first composite material melts and is extruded into filaments, it is extruded layer by layer. The nozzle of the needle moves along the XY axis first, and then along the Z axis after each layer is completed to print the inner layer support. The steps include: shredding the solid slurry and placing it into a high-temperature barrel, selecting a needle with a diameter of 350 μm, selecting a high-temperature pneumatics and a high-temperature platform, and setting the barrel and needle temperatures to 1. The first composite material is obtained by melting at 60℃ and platform temperature at 40℃. After the composite material melts, it is extruded into filaments and then extruded layer by layer with a thickness of 250μm and a spacing of 600μm. The nozzle of the needle moves at a speed of 1000mm / min, the printing speed is 60mm / min, and the extrusion pressure is 100Kpa. The nozzle of the needle moves along the XY axis first, and after each layer is completed, it moves along the Z axis. The pore angle of the inner layer support is 60 degrees and the pore size is 600μm, thus printing out the inner layer support.

[0022] Preferably, the solid slurry is shredded and placed into a high-temperature barrel, and melted using a needle, high-temperature pneumatic system, and rotating platform to obtain a second composite material. After the second composite material melts and is extruded into filaments, the hollow tube outer support is printed on the rotating platform. This step includes: shredding the solid slurry and placing it into a high-temperature barrel; selecting a needle with a diameter of 350 μm; selecting a high-temperature pneumatic system and rotating platform; setting the barrel and needle temperatures to 160°C for melting to obtain the second composite material; after the second composite material is extruded into filaments, it is then extruded layer by layer with a thickness of 250 μm and a spacing of 600 μm; the nozzle movement speed of the needle is 1000 mm / min, the printing speed is 150 mm / min, and the rotation speed is 28 mm / min; the fiber angle is set to 60 degrees, and the cycle is 1, to print the hollow tube outer support.

[0023] Preferably, the bone-like bio-scaffold is immersed in the TA solution with a concentration of 5 mg / ml until it turns black to obtain the PLGA / TA@V-MBG composite scaffold.

[0024] This invention provides a 3D-printed bone-like bioscaffold prepared according to the 3D-printed bone-like bioscaffold preparation method described above.

[0025] The beneficial effects of this invention are as follows: This invention utilizes bioceramic materials with similar composition to bone and excellent photothermal performance and bone repair effects for research. TA@V-MBG and PLGA are mixed in a certain proportion to prepare a hollow tube composite material. By assembling smaller cylindrical structures into this composite material, the gradient structure of natural bone is better simulated. PLGA enhances the mechanical properties of the scaffold while rapidly degrading after four weeks, providing space for tissue growth within the scaffold. Simultaneously, the ceramic scaffold carries bioactive ions that release slowly during degradation, further enhancing its osteogenic and angiogenic properties. Furthermore, it can kill tumors under PTT (potentially toxic terephthalmic) action. This scaffold can achieve the repair of bone defects caused by bone tumors.

[0026] Structurally, this invention features a bone-mimicking structure—a cancellous bone-cortical bone structure—with a large outer pore size that facilitates early vascular ingrowth. In terms of material composition, it utilizes synthetic vanadium-doped bioactive glass capable of releasing ions or ionic groups such as V, Si, Ca, and P, promoting osteogenic differentiation. TA exhibits reducing properties and a strong affinity for high-valence metal ions, allowing V to bind with TA, further enhancing near-infrared absorption and achieving PTT (percutaneous transluminal radiation). PLGA, used as the substrate for 3D printing, is an FDA-approved bioabsorbable polymer with widespread clinical applications and good biocompatibility. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0028] Figure 1 A schematic flowchart illustrating a method for fabricating a 3D-printed bone-like bioscaffold according to an embodiment of the present invention;

[0029] Figure 2 Provided for embodiments of the present invention: a is a SEM image of V-MBG; b is a TEM image of V-MBG; c is a particle size distribution of V-MBG; d and e are EDS images of V-MBG; f is an XRD pattern of V-MBG;

[0030] Figure 3 Provided for embodiments of the present invention: a) is a schematic diagram of the hollow tube structure of the outer support; b) is a comparison diagram of the outer support before and after immersion in TA solution; c) is a schematic diagram of the inner small-pore support structure.

[0031] Figure 4 SEM image of the bionic scaffold provided in the embodiment of the present invention;

[0032] Figure 5Provided for embodiments of the present invention: a) is a schematic diagram comparing the mechanical properties of scaffolds with different bioactive glass contents; b) is a schematic diagram comparing the mechanical properties of different scaffolds (20% bioactive glass content);

[0033] Figure 6 For the purposes of this invention: a) is a schematic diagram of the in vitro degradation rate of the inner scaffold; b) is a schematic diagram of the in vitro degradation rate of the outer scaffold.

[0034] Figure 7 Provided for embodiments of the present invention: a) photothermal effect curves of different scaffolds; b) schematic diagram of dead / live double staining comparison (143B cells).

[0035] Figure 8 This is a schematic diagram of BMSC live / dead double staining assay provided in an embodiment of the present invention;

[0036] Figure 9 This is a schematic diagram of ALP staining provided in an embodiment of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or ends.

[0039] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0040] Currently, the types and quantities of tissue-engineered products available for clinical use are very limited, and their functions and structures are relatively simple. Utilizing novel bone regeneration and repair materials with high bioactivity to prepare bone repair scaffolds with interconnected porous structures to repair bone defects exceeding critical sizes has become a hot research area in recent years. Bone regeneration and repair materials need to possess good biocompatibility, bioactivity, and a certain degree of biodegradability. Currently, synthetic bone regeneration and repair materials include inorganic non-metallic materials, such as bioactive ceramics (β-tricalcium phosphate, hydroxyapatite (HAP), and other calcium phosphate system materials), bioactive glass, and microcrystalline glass; polymeric materials include natural and synthetic polymers.

[0041] In the field of bone tissue engineering, 3D-printed scaffolds have attracted much attention due to their outstanding characteristics of personalized customization and high precision. Macroscopically, bone consists of two parts: cancellous bone and cortical bone. Microscopically, bone is mainly composed of various cells and abundant extracellular matrix (ECM). This ordered, layered structure enhances the mechanical strength and adaptability of the scaffold. Therefore, it is essential to develop a biomimetic 3D-printed scaffold with a complex gradient structure similar to natural bone for tumor removal and bone defect repair after osteosarcoma resection.

[0042] The present invention aims to provide a 3D printed bone-like biological scaffold and its preparation method, which solves the problems of poor bone repair effect and poor osteoid cell proliferation and differentiation effect in the prior art, and the inability to achieve bone defect repair caused by bone tumors.

[0043] like Figure 1 As shown in the figure, a specific embodiment of the present invention provides a method for preparing a 3D-printed bone-like bioscaffold, comprising the following steps:

[0044] S1: Add 1.2g of cetyltrimethylammonium bromide to a mixture of 60mL of deionized water and 40mL of anhydrous ethanol to obtain an ammonium bromide solution.

[0045] S2: Add ammonia solution as a catalyst to ammonium bromide solution, then add tetraethyl orthosilicate, triethyl phosphate, calcium nitrate and sodium vanadate in sequence, and centrifuge after continuous stirring to collect the precipitate.

[0046] In this embodiment of the invention, 0.8 mL of ammonia solution was added to the ammonium bromide solution as a catalyst; 3.60 mL of tetraethyl orthosilicate, 0.36 mL of triethyl phosphate, 1.98 g of calcium nitrate, and 100.0 mg of sodium vanadate were added every 30 min; the mixture was stirred continuously for 5.5 h and then centrifuged to collect the precipitate.

[0047] S3: The precipitate was washed alternately with deionized water and anhydrous ethanol, then aged, heated to a holding temperature for 3 hours and then cooled. After being ground into powder, it was sieved to obtain V-MBG powder.

[0048] In this embodiment of the invention, the precipitate was washed three times alternately with deionized water and anhydrous ethanol; then aged at room temperature for 3 days, aged at 37 ℃ for 3 days, heated to 650 ℃ at a rate of 2 ℃ / min and held for 3 hours, and then cooled; after grinding and pulverizing the powder, the particle size of the powder was sieved to about 500 nm to obtain the final product V-MBG.

[0049] S4: Prepare a 10 mg / ml TA solution and a 30 mg / ml V-MBG solution with pure water, mix them in a 1:1 ratio, and freeze-dry to obtain TA@V-MBG powder.

[0050] In this embodiment of the invention, a TA solution of 10 mg / ml and a V-MBG solution of 30 mg / ml were prepared with pure water, mixed in a 1:1 ratio, placed at -20°C, and then freeze-dried for 24 hours to obtain TA@V-MBG powder.

[0051] S5: Weigh the mixture of PLA and PGA (PLGA polymer) in a ratio of 3:1 and dissolve it in chloroform to prepare a chloroform solution of PLGA.

[0052] S6: Add V-MBG powder to the chloroform solution of PLGA, stir thoroughly with a magnetic stirrer overnight, and after thorough mixing, use an oil pump to remove the chloroform solution to obtain a solid slurry.

[0053] In this embodiment of the invention, V-MBG powder with a particle size of about 500 nm was added to a chloroform solution of PLGA at a ratio of PLGA:V-MBG (w:w) of 4:1, so that the final content of V-MBG was 20%. Then, the mixture was stirred thoroughly overnight with a magnetic stirrer. After thorough mixing, the chloroform solution was drained with an oil pump to obtain a solid slurry.

[0054] S7: Shred the solid slurry and put it into a high-temperature barrel. Select the needle, high-temperature pneumatic and high-temperature platform to melt it to obtain the first composite material. After the first composite material melts, wait for it to be extruded into filaments, and then extrude it layer by layer. Move the nozzle of the needle along the XY axis first. After each layer is completed, move it along the Z axis to print out the inner support.

[0055] In this embodiment of the invention, solid slurry is shredded and placed into a high-temperature barrel. A needle with a diameter of 350 μm is selected, along with a high-temperature pneumatic system and a high-temperature platform. The barrel and needle temperatures are set to 160°C, and the platform temperature is set to 40°C for melting to obtain the first composite material. After the composite material melts and is extruded into filaments, it is then extruded layer by layer with a thickness of 250 μm and a spacing of 600 μm. The nozzle moving speed of the needle is 1000 mm / min, the printing speed is 60 mm / min, and the extrusion pressure is 100 kPa. The nozzle of the needle first moves along the XY axis, and after each layer is completed, it moves along the Z axis. The pore angle of the inner layer support is 60 degrees, and the pore size is 600 μm, thus printing out the inner layer support.

[0056] S8: The solid slurry is shredded and placed into a high-temperature barrel. A needle, a high-temperature pneumatic system, and a rotating platform are selected for melting to obtain a second composite material. After the second composite material is melted and extruded into filaments, a hollow tube outer support is printed on the rotating platform.

[0057] In this embodiment of the invention, solid slurry is shredded and placed into a high-temperature barrel. A needle with a diameter of 350 μm is selected, and a high-temperature pneumatic and rotating platform is chosen. The temperature of the barrel and the needle is set to 160°C for melting to obtain a second composite material. After the second composite material is extruded into filaments, it is then extruded layer by layer with a thickness of 250 μm and a spacing of 600 μm. The nozzle moving speed of the needle is 1000 mm / min, the printing speed is 150 mm / min, and the rotation speed is 28 mm / min. The fiber angle is set to 60 degrees, and the number of cycles is 1 to print the outer support of the hollow tube.

[0058] S9: After the inner and outer scaffolds are printed, the inner scaffold is nested in the outer scaffold to obtain a gradient structure of bone-like bio-scaffold with large pores in the outer layer and small pores in the inner layer. The bone-like bio-scaffold is then immersed in TA solution to obtain the PLGA / TA@V-MBG composite scaffold.

[0059] In this embodiment of the invention, the bone-like bio-scaffold is immersed in a TA solution with a concentration of 5 mg / ml until it turns black to obtain the PLGA / TA@V-MBG composite scaffold.

[0060] This invention provides a 3D-printed bone-like bioscaffold prepared according to the 3D-printed bone-like bioscaffold preparation method described above.

[0061] like Figure 2 As shown, Figure 2Provided for embodiments of the present invention: a) SEM image of V-MBG; b) TEM image of V-MBG; c) particle size distribution of V-MBG; d and e) EDS images of V-MBG; f) XRD pattern of V-MBG; Preparation and characterization of mesoporous bioactive glass: SEM images and particle size analysis of mesoporous bioactive glass are as follows Figure 2 As shown in a and c, the samples are spherical with smooth surfaces, relatively uniform size distribution, and a particle size of approximately 550 nm. TEM images are shown below. Figure 2 As shown in Figure b, this bioactive glass possesses an ordered mesoporous structure. Based on the EDS line spectrum and graph, as shown in Figure b... Figure 2 The results shown in d and e indicate the successful preparation of vanadium-doped mesoporous bioactive glass, with Si, Ca, P, and V as the main elements. Wide-angle XRD is shown below. Figure 2 The results show that neither MBG nor V-MBG has obvious diffraction peaks, indicating that both MBG and V-MBG particles exist in an amorphous phase, and the incorporation of vanadium hardly changes the phase structure of MBG.

[0062] like Figure 3 As shown, Figure 3 Provided for embodiments of the present invention: a) is a schematic diagram of the hollow tube structure of the outer support; b) is a comparison diagram of the outer support before and after immersion in TA solution; c) is a schematic diagram of the inner small-pore support structure; the outer support is a hollow tube structure as shown in the figure. Figure 3 As shown in a and b, the PLGA / V-MBG (PVM) hollow tube stent turns black after being immersed in TA solution, which is the PLGA / TA@V-MBG (PTVM) hollow tube stent. Figure 3 In the middle, 'c' represents the inner layer with a small aperture support; the two nested together form a biomimetic support.

[0063] like Figure 4 As shown, Figure 4 The image shown is an SEM image of the bionic scaffold provided in this embodiment of the invention. The bionic scaffold is characterized by scanning electron microscopy (SEM), which reveals the microscopic shape and pore structure of the scaffold. Figure 4 The first scaffold was made of pure PLGA with a smooth surface and no bioactive glass microspheres were observed. In contrast, the PM, PVM, and PTVM scaffolds showed successful embedding and printing of microspheres. This indicates that a bone-like bio-scaffold with a gradient structure was constructed using 3D printing high-temperature melting technology, and that the controllable fabrication of scaffolds with different structures can be successfully achieved. Furthermore, the pore size of the scaffold provides channels for nutrient delivery and metabolite discharge, promoting the growth of new tissue.

[0064] like Figure 5 As shown, Figure 5Provided for embodiments of the present invention: a) is a schematic diagram comparing the mechanical properties of scaffolds with different bioactive glass contents; b) is a schematic diagram comparing the mechanical properties of different scaffolds (20% bioactive glass content); according to the scaffold mechanical property results, the mechanical properties of scaffolds doped with bioactive glass are all higher than those of the pure PLGA group, such as... Figure 5 a, and the compressive strength increases with increasing content, such as Figure 5 b.

[0065] like Figure 6 As shown, Figure 6 For the embodiments of the present invention: a is a schematic diagram of the in vitro degradation rate of the inner scaffold; b is a schematic diagram of the in vitro degradation rate of the outer scaffold; the scaffold that can undergo corresponding biodegradation provides a void structure and support for the formation and growth of bone tissue. As the growth of bone tissue and surrounding tissues requires space, the scaffold begins to degrade and its strength begins to decrease.

[0066] like Figure 6 a. All four scaffolds underwent varying degrees of degradation within the first week, at a relatively slow rate. However, after two weeks of immersion, both PVM and PTVM experienced significant weight loss (13%), but compared to... Figure 6 Compared to the b-layer scaffold, the weight loss was smaller, while the hollow tube scaffold experienced a weight loss of 45%. This was because the overall structure of the scaffold began to partially disintegrate after immersion in PBS solution, leading to accelerated scaffold degradation. The hollow tube scaffold's greater weight loss than the inner layer scaffold was due to its less stable structure. By the fourth week, it was found that the composite scaffolds doped with bioactive glass showed significant degradation, while the pure PLGA scaffold degraded very slowly.

[0067] like Figure 7 As shown, Figure 7 Provided for embodiments of the present invention: a) photothermal effect curves of different scaffolds; b) schematic diagram of dead / live double staining comparison (143B cells); using a power of 0.4 W / cm². 2 The photothermal effect of laser on four types of scaffolds was investigated. The samples were sequentially classified as pure PLGA, PLGA / MBG (PM), PLGA / V-MBG (PV), and PLGA / TA@V-MBG (PTV). The figure shows that the equilibrium temperature of the scaffold PTV easily reaches above 58℃, sufficient to induce photothermal ablation of tumor cells. Further, we used human osteosarcoma cells (143B) to examine the photothermal effect of the material components, such as... Figure 7 b. Under 808 laser irradiation, it was found that the majority of cells in the TVM group were dead cells, indicating that its excellent photothermal ability is expected to enable further exploration of its biomedical applications.

[0068] like Figure 8 As shown, Figure 8This is a schematic diagram of the BMSC live-dead double staining assay provided in an embodiment of the present invention; the live-dead staining assay of bone marrow mesenchymal stem cells (BMSCs) shows that they are mainly live cells (green) with very few dead cells (red). This demonstrates that the material has good biocompatibility.

[0069] like Figure 9 As shown, Figure 9 This is a schematic diagram of ALP staining provided in an embodiment of the present invention; alkaline phosphatase, as a marker enzyme of osteoblasts, reflects the degree of differentiation of BMSCs into osteoblasts when its content is high, and the darker the ALP staining, the higher the ALP activity. Figure 9 As shown, all samples cultured in osteogenic induction medium showed positive ALP staining results after 7 days. Among them, the vanadium ion-containing group showed the strongest staining intensity, exhibiting the highest osteogenic capacity.

[0070] Bioactive glass exhibits excellent bioactivity, releasing ions or ionic groups such as Si, Ca, and P within the body. Through a mineralization deposition process, a low-crystallinity hydroxyapatite (HCA) layer is formed on the material surface. Because this mineralized layer is similar in structure and composition to the hydroxyapatite in bone, it is commonly referred to as a bone-like hydroxyapatite layer. This HCA layer can form a strong interfacial bond with the existing bone tissue, resulting in good bone repair.

[0071] Vanadium is a trace element in the human body, mainly existing in bones as tetravalent (IV) and pentavalent (V) ions. Although high concentrations of vanadium intake pose health risks, studies have shown that vanadium at concentrations of 10-26 μg / g can be safely present in human bone tissue. Within a certain concentration range, vanadium (V) compounds can also promote osteoblast-like cell proliferation and differentiation. Currently, there are few reports on the effects of V ions on osteogenic differentiation; most studies focus on the effects of V ions on osteoblast-like cell proliferation.

[0072] Tannic acid (TA) is a natural polyphenol, a hydrolyzable amphiphilic tannin derivative of gallic acid, with multiple galloyl groups in its structure. Tannic acid has been studied as a natural cross-linking agent with anti-inflammatory, antibacterial, and anticancer activities for various biomedical applications. TA possesses reducing properties and a good affinity for high-valence metal ions, further enhancing near-infrared absorption. Chen et al. discovered that TA@VOx NSs exhibits excellent NIR photomediated conversion capabilities, enabling PTT (phototransfer to tumor). Further in vitro and in vivo experiments demonstrated that TA@VOx NSs has a significant PTT / CDT synergistic inhibitory effect on tumors.

[0073] Bioactive glass-based composite scaffolds can be categorized into those with inorganic or organic materials. In organic composites, the addition of bioactive glass can effectively improve the mechanical properties of the scaffold, such as compressive and tensile strength. In bioactive glass composite scaffolds, the ratio of other materials to the bioactive glass significantly impacts the scaffold's mechanical strength, degradation rate, and repair efficacy. Lactic acid-glycolic acid copolymer (PLGA) is an FDA-approved bioabsorbable polymer widely used clinically. Bioactive glass, as a composite filler, can significantly improve the overall mechanical and biological properties of the scaffold. The calcium, silicate, and phosphate ions released from the bioactive glass in the composite scaffold can balance the acidity generated by PLGA polymer degradation by increasing the pH of the surrounding environment; this neutralization effect also slows down the polymer degradation rate.

[0074] The beneficial effects of this invention are as follows: This invention utilizes bioceramic materials with similar composition to bone and excellent photothermal performance and bone repair effects for research. TA@V-MBG and PLGA are mixed in a certain proportion to prepare a hollow tube composite material. By assembling smaller cylindrical structures into this composite material, the gradient structure of natural bone is better simulated. PLGA enhances the mechanical properties of the scaffold while rapidly degrading after four weeks, providing space for tissue growth within the scaffold. Simultaneously, the ceramic scaffold carries bioactive ions that release slowly during degradation, further enhancing its osteogenic and angiogenic properties. Furthermore, it can kill tumors under PTT (potentially toxic terephthalmic) action. This scaffold can achieve the repair of bone defects caused by bone tumors.

[0075] Structurally, this invention features a bone-mimicking structure—a cancellous bone-cortical bone structure—with a large outer pore size that facilitates early vascular ingrowth. In terms of material composition, it utilizes synthetic vanadium-doped bioactive glass capable of releasing ions or ionic groups such as V, Si, Ca, and P, promoting osteogenic differentiation. TA exhibits reducing properties and a strong affinity for high-valence metal ions, allowing V to bind with TA, further enhancing near-infrared absorption and achieving PTT (percutaneous transluminal radiation). PLGA, used as the substrate for 3D printing, is an FDA-approved bioabsorbable polymer with widespread clinical applications and good biocompatibility.

[0076] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for fabricating a 3D-printed bone-like bioscaffold, characterized in that, The method includes: S1: Add 1.2g of hexadecyltrimethylammonium bromide to a mixture of 60mL of deionized water and 40mL of anhydrous ethanol to obtain an ammonium bromide solution; S2: Add ammonia solution as a catalyst to the ammonium bromide solution, then add tetraethyl orthosilicate, triethyl phosphate, calcium nitrate and sodium vanadate in sequence, and centrifuge after continuous stirring to collect the precipitate; S3: The precipitate was washed alternately with deionized water and anhydrous ethanol, then aged, heated and kept at the temperature for 3 hours, then cooled, ground and pulverized with powder, and then sieved to obtain V-MBG powder. S4: Prepare a 10 mg / ml TA solution and a 30 mg / ml V-MBG solution with pure water, mix them in a 1:1 ratio, and freeze-dry to obtain TA@V-MBG powder; S5: Weigh a mixture of PLA and PGA and mix them to obtain PLGA polymer, wherein the ratio of PLA to PGA is 3:

1. Dissolve the PLGA polymer in chloroform to prepare a chloroform solution of PLGA. S6: Add the TA@V-MBG powder to the PLGA chloroform solution, stir thoroughly overnight with a magnetic stirrer, and after thorough mixing, use an oil pump to drain the chloroform solution to obtain a solid slurry; S7: The solid slurry is shredded and placed into a high-temperature barrel. A needle, a high-temperature pneumatic device, and a high-temperature platform are selected for melting to obtain a first composite material. After the first composite material is melted and extruded into filaments, it is extruded layer by layer. The nozzle of the needle moves along the XY axis first, and after each layer is completed, it moves along the Z axis to print out the inner layer support. S8: The solid slurry is shredded and placed into a high-temperature barrel. A needle, high-temperature pneumatic and rotating platform are selected for melting to obtain a second composite material. After the second composite material is melted and extruded into filaments, a hollow tube outer support is printed on the rotating platform. S9: After the inner scaffold and the outer scaffold are printed, the inner scaffold is nested in the outer scaffold to obtain a gradient structure of bone-like bio-scaffold with large pores in the outer layer and small pores in the inner layer. The bone-like bio-scaffold is then immersed in the TA solution to obtain the PLGA / TA@V-MBG composite scaffold.

2. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The step of adding ammonia solution as a catalyst to the ammonium bromide solution, then sequentially adding tetraethyl orthosilicate, triethyl phosphate, calcium nitrate, and sodium vanadate, and continuously stirring followed by centrifugation to collect the precipitate includes: 0.8 mL of ammonia solution was added to the ammonium bromide solution as a catalyst; 3.60 mL of tetraethyl orthosilicate, 0.36 mL of triethyl phosphate, 1.98 g of calcium nitrate and 100.0 mg of sodium vanadate were added every 30 min; the mixture was stirred continuously for 5.5 h and then centrifuged to collect the precipitate.

3. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The steps of washing the precipitate alternately with deionized water and anhydrous ethanol, aging it, heating it to a certain temperature for 3 hours and then cooling it, grinding it into powder and sieving it through a sieve to obtain V-MBG powder include: The precipitate was washed three times alternately with deionized water and anhydrous ethanol; then aged at room temperature for 3 days, aged at 37 °C for 3 days, heated to 650 °C at a rate of 2 °C / min and held for 3 hours, and then cooled. After grinding and pulverizing the powder, the particle size of the powder is sieved to about 500nm to obtain the final product V-MBG.

4. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The step of preparing a 10 mg / ml TA solution and a 30 mg / ml V-MBG solution with pure water, mixing them in a 1:1 ratio, and then freeze-drying to obtain TA@V-MBG powder includes: A TA solution of 10 mg / ml and a V-MBG solution of 30 mg / ml were prepared with pure water, mixed in a 1:1 ratio, and placed at -20℃. After freeze-drying for 24 hours, TA@V-MBG powder was obtained.

5. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The step of adding the TA@V-MBG powder to the PLGA chloroform solution, stirring thoroughly overnight with a magnetic stirrer, and then evacuating the chloroform solution with an oil pump to obtain a solid slurry includes: Add TA@V-MBG powder with a particle size of 500 nm to the PLGA chloroform solution at a ratio of PLGA:TA@V-MBG (w:w) of 4:1 to make the final content of TA@V-MBG 20%, and then stir thoroughly overnight with a magnetic stirrer. After thorough mixing, the chloroform solution was pumped out using an oil pump to obtain a solid slurry.

6. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The steps of cutting the solid slurry into small pieces and placing it into a high-temperature cylinder, selecting a needle, high-temperature pneumatics, and a high-temperature platform for melting to obtain a first composite material, waiting for the first composite material to melt and be extruded into filaments, and then extruding it layer by layer, with the nozzle of the needle first moving along the XY axis, and then moving along the Z axis after each layer is completed, to print out the inner layer support, include: The solid slurry was shredded and placed into a high-temperature barrel. A needle with a diameter of 350 μm was selected. High-temperature pneumatic and high-temperature platform were selected. The barrel and needle temperatures were set to 160°C, and the platform temperature was set to 40°C for melting to obtain the first composite material. After the first composite material is melted and extruded into filaments, it is extruded layer by layer with a thickness of 250 μm and a spacing of 600 μm. The nozzle of the needle moves at a speed of 1000 mm / min, the printing speed is 60 mm / min, and the extrusion pressure is 100 kPa. The nozzle of the needle first moves along the XY axis, and after each layer is completed, it moves along the Z axis. The pore angle of the inner layer support is 60 degrees and the pore size is 600 μm, thus printing out the inner layer support.

7. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that, The steps of cutting the solid slurry into small pieces and placing it into a high-temperature cylinder, then melting it using a needle, high-temperature pneumatic system, and a rotating platform to obtain a second composite material, and after the second composite material has melted and been extruded into filaments, printing a hollow tube outer support on the rotating platform, include: The solid slurry is shredded and placed into a high-temperature barrel. A needle with a diameter of 350 μm is selected, and a high-temperature pneumatic and rotating platform is chosen. The temperature of the barrel and the needle is set to 160°C for melting to obtain the second composite material. After the second composite material is extruded into filaments, it is extruded layer by layer with a thickness of 250μm and a spacing of 600μm. The nozzle of the needle moves at a speed of 1000mm / min, the printing speed is 150mm / min, and the rotating platform rotates at a speed of 28mm / min. The fiber angle is set to 60 degrees, and the number of cycles is 1, thus printing the outer support of the hollow tube.

8. The method for preparing a 3D-printed bone-like bioscaffold according to claim 1, characterized in that: The biomimetic bone scaffold was immersed in a TA solution with a concentration of 5 mg / ml until it turned black, thus obtaining the PLGA / TA@V-MBG composite scaffold.

9. A 3D-printed bone-like bioscaffold prepared according to any one of claims 1-8.

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