3D printing hydrogel stent for treating bone defect and preparation method thereof

Abstract

The invention discloses a 3D printing hydrogel stent for treating bone defect and a preparation method thereof, fish gelatin and fishbone powder are extracted from cold water fish, the fish gelatin and the fishbone powder are mixed as bio-ink, and vascular endothelial growth factors are loaded in the bio-ink to prepare the 3D printing hydrogel stent for treating bone defect. Preparing a methacrylated fish gelatin scaffold doped with fishbone powder by adopting a 3D printing technology; the preparation method comprises the following steps: preparing methylacryloylated fish gelatin with BMP2 and alginic acid microspheres by adopting a micro-fluidic technology; after the microspheres are assembled into the fishbone powder mixed hydrogel stent, methacrylic acid groups on the surfaces of the microspheres and in the stent are polymerized and crosslinked under the action of a light curing agent through ultraviolet irradiation, so that the microspheres and the frame are fixed to form the composite stent. The problem of balance among porosity, mechanical stability and active ingredient retention of the stent is solved, and a personalized and multifunctional repair scheme is provided for complex bone defects.

Description

Technical Field

[0001] This invention relates to the field of human tissue engineering technology, specifically to a 3D-printed hydrogel scaffold for treating bone defects and its preparation method. Background Technology

[0002] As one of the largest organ systems in the human body, the skeleton accounts for 15%-20% of total body weight and provides mechanical support for movement. Bone defects are a common and refractory condition in clinical orthopedics, often induced by various factors such as tumors, trauma, and infection. Due to the importance of the skeletal system for normal physiological movement, bone defects not only severely impair patients' motor function and reduce their quality of life, but also bring heavy medical resource consumption and socioeconomic burdens, becoming a pressing problem in orthopedics. Although bone has the potential for self-healing, critical-sized bone injuries are difficult to repair spontaneously and require bone fillers to promote bridging of bone ends. Currently, various repair strategies have been explored and applied in clinical and research fields. Among them, biomaterial-based repair schemes have become a core research direction in the field of bone repair and have been widely used due to their good biocompatibility and tissue affinity. These biomaterials are mainly divided into two categories: collagen materials and natural bone-derived materials, both of which have achieved certain repair effects in clinical applications. Collagen materials include animal-derived gelatin and plant collagen extracts, while natural bone-derived materials include human bone grafts and animal-derived bone tissue derivatives such as bovine and porcine bone tissue. However, both types of materials have inherent defects that significantly limit their clinical application prospects. On the one hand, both types of materials generally involve safety risks and ethical controversies. Animal-derived materials may carry species-specific pathogens, increasing the risk of cross-infection, while human-derived materials face the dual limitations of donor shortage and ethical considerations. On the other hand, these materials generally lack reasonable structural design during preparation and application, making it difficult to accurately match the porous structure and mechanical support properties of natural bone tissue, and failing to provide an ideal microenvironment for osteoblast adhesion, proliferation, and differentiation. These defects collectively restrict the clinical application value of existing biomaterials. Developing novel bone repair materials that combine biosafety, excellent structural properties, and bioactivity has significant academic research value and an urgent need for clinical translation.

[0003] The existing technology currently has the following limitations and shortcomings:

[0004] (1) Existing bone repair materials have the dual defects of potential biosafety risks (such as cross-infection) and insufficient mechanical properties, and a single material is difficult to simultaneously meet the structural requirements for bone cell growth and the supply of osteogenic activity.

[0005] (2) The release of growth factors in traditional bone repair scaffolds lacks temporality and cannot match the natural process of bone repair, which is “early angiogenesis and later osteogenic differentiation”.

[0006] (3) The existing composite scaffold structure design is not well adapted to the microenvironment of bone defects, and it is difficult to meet the comprehensive needs of cell adhesion, nutrient exchange and preservation of active factors. Summary of the Invention

[0007] To address the problems in the prior art, the present invention provides a 3D-printed hydrogel scaffold for treating bone defects and a method for preparing the same, thereby solving or at least alleviating some or all of the technical problems in the prior art.

[0008] To achieve the above objectives, the present invention is specifically implemented through the following technical solutions: The first aspect of the present invention provides a 3D-printed hydrogel scaffold for treating bone defects, comprising a fish gelatin scaffold prepared by 3D printing from the main raw materials fish methacrylamide gelatin and fish bone powder, and mixed microspheres containing fish methacrylamide gelatin and methacrylamide alginate filled in the scaffold, wherein the mass ratio of the fish gelatin scaffold to the mixed microspheres is (8-15):1.

[0009] In a preferred embodiment, the mass ratio of fish gelatin scaffold to mixed microspheres is 10:1.

[0010] In a preferred embodiment, the fish methacrylamide gelatin is obtained by methacrylating fish skin gelatin.

[0011] In a preferred embodiment, the fish bone powder is obtained by sieving through a 1000-mesh filter.

[0012] A second aspect of the present invention provides a method for preparing a 3D-printed hydrogel scaffold for treating bone defects as described above, comprising the following steps: (1) Methacrylamide treatment of gelatin derived from fish skin; (2) Collect fresh cold-water fish bones, process them, grind them into powder, and then decellularize them for later use; (3) The fish methacrylamide gelatin obtained in step (1) and the fish bone powder obtained in step (2) are used as the main raw materials to prepare bio-ink, which is then printed using a 3D printer; (4) Fish methacrylamide gelatin and methacrylamide alginate were used as the main raw materials to prepare mixed microspheres. The microspheres were collected with calcium chloride solution and crosslinked by photoirradiation with 405nm blue-violet light. The collected microspheres were washed with water and anhydrous ethanol. (5) The 3D printed scaffold prepared in step (3) and the mixed microspheres prepared in step (4) are assembled at low temperature by photocrosslinking and then irradiated with 405nm blue-violet light. After cleaning, the product is obtained.

[0013] As a preferred embodiment, step (1) includes completely dissolving fish skin gelatin at a concentration of 10% in cell phosphate buffer (PBS) solution within 3 hours at 50°C, then slowly adding methacrylic anhydride to the previous solution at a ratio of 5-10% and stirring vigorously, reacting at 50°C for 3 hours, removing unreacted methacrylic anhydride by dialysis with pure water, and lyophilizing the obtained reaction product at -40°C.

[0014] As a preferred embodiment, step (2) includes collecting fresh cold-water fish bones, rinsing the fish bones with PBS, and removing blood vessels, muscles, and periosteum from the surface of the fish bones; washing the fish bones successively with a 10% sodium chloride solution and a 3% sodium bicarbonate solution to remove fat residue. Grinding the frozen fish bones in liquid nitrogen at -80°C and sieving the powder using a 1000-mesh filter; the decellularization process includes the following steps: treating with a 1% polyethylene glycol octylphenyl ether (Triton X-100) solution to disrupt cell membranes, followed by rinsing successively with a Tris-HCl buffer containing 0.1% EDTA and 0.1% sodium dodecyl sulfate (SDS) to remove cellular components, and finally lyophilizing the treated fish bone powder at -40°C and storing it at -20°C for subsequent use.

[0015] In a preferred embodiment, step (3) includes dissolving the fish methacrylamide gelatin prepared in step (1) and the fish bone powder obtained in step (2) (0-2%) with an optimal mass fraction of 1% using deionized water, and adding 0.5% by mass of light curing agent LAP, 0.1% by mass of light stabilizer tartrazine, and vascular endothelial growth factor (VEGF) (2 μg / mL). 1 The scaffold was prepared into a bio-ink for printing. A 3D printer was used to print each layer with a thickness of 10 micrometers. Each layer was cross-linked with 405nm light for 10s. The total height of the scaffold was 3mm. After printing, the scaffold was rinsed with PBS and stored at 4°C for later use.

[0016] As a preferred embodiment, step (4) includes assembling a microfluidic device in a concentric circle structure using capillary glass tubes, dissolving 15% fish methacrylamide gelatin, 5% sodium alginate and 0.5% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in deionized water as the aqueous phase, using paraffin oil as the oil phase, and controlling the flow rate ratio of the aqueous phase to the oil phase to be 1:10 using a test injection pump to prepare microspheres; collecting the microspheres with 5% calcium chloride solution and crosslinking them with 405nm blue-violet light for 5 minutes, and washing the collected microspheres sequentially with water and anhydrous ethanol.

[0017] As a preferred embodiment, step (5) includes assembling the 3D printed scaffold prepared in step (3) and the mixed microspheres prepared in step (4) at 4°C by photocrosslinking, using a microsyringe to draw up the microspheres and inject them into the gaps of the 3D printed scaffold, with the weight ratio of the 3D printed scaffold to the microspheres being (8-15):1, irradiating the composite scaffold with 405nm blue-violet light for 5 minutes, and then washing away the uncrosslinked microspheres in the scaffold with PBS to obtain the final product.

[0018] Compared with the prior art, the present invention has the following advantages: (1) This invention optimizes the composition ratio of fish gelatin and fish bone powder, and combines it with the precise structural construction characteristics of 3D printing to reveal the core mechanism by which natural derivative materials synergistically enhance the biocompatibility and mechanical support performance of scaffolds.

[0019] (2) This invention achieves targeted loading and sustained release design of BMP2 through microfluidic technology, and combines the rapid release regulation of VEGF by 3D printed scaffold to establish a dual-factor time-sequential synergistic release system, elucidating the role of time-sequential matching of angiogenesis and osteogenic differentiation in improving bone repair efficiency.

[0020] (3) This invention solves the problem of balancing the porosity, mechanical stability and retention of active ingredients of the scaffold by using the innovative composite structure of “3D printed scaffold frame + microfluidic microspheres”, providing a personalized and multifunctional repair solution for complex bone defects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the fabrication process of the 3D printed hydrogel scaffold of the present invention; Figure 2 Microscopic morphology of the 3D-printed hydrogel scaffold prepared in this invention; Figure 3 This is a schematic diagram illustrating the rheological properties of embodiments and comparative examples of the present invention; Figure 4 This is a schematic diagram of the pressure compression curves of the embodiments and comparative examples of the present invention; Figure 5 This is a schematic diagram of the drug release curve of the stent in Embodiment 1 of the present invention. Detailed Implementation

[0022] To better understand the technical solutions of the embodiments of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] This invention utilizes 3D printing and microfluidic technology to construct a composite scaffold of fish gelatin and bone powder for bone defect repair. Gelatin, typically extracted from mammalian skin, is widely used in food processing, drug delivery systems, and tissue engineering. Compared to mammalian-derived gelatin, fish gelatin offers advantages such as easy availability, low production cost, and high biocompatibility. Its core advantage lies in the significant species differences between fish and humans, eliminating the risk of fish-human genetic diseases and effectively avoiding potential cross-infection issues associated with mammalian-derived materials. Furthermore, fish gelatin possesses excellent biodegradability and bioactivity, enabling it to participate in the regulation of the bone repair microenvironment. Fish bone powder, a natural byproduct of the fish processing industry, also features low cost and easy availability. Its main component is hydroxyapatite, which can precisely replenish the calcium needs of bone defect areas. The natural organic components it contains enhance the bioavailability of calcium ions and impart certain osteogenic induction activity to the material. 3D printing technology, with its high precision and excellent repeatability, can accurately fabricate scaffold materials with personalized structures, controllable porosity, and mechanical properties matching bone tissue, providing an ideal microenvironment for bone cell adhesion and proliferation. It has become the preferred technology for constructing scaffolds in bone tissue engineering. Similarly, microfluidic technology, capable of producing stable and tunable-sized microspheres, has attracted significant attention in tissue engineering. Microspheres can load cells, carry drugs, and construct tissue repair scaffolds, finding wide application in bone repair.

[0025] Example 1 This embodiment provides a 3D-printed hydrogel scaffold for treating bone defects and its preparation method, such as Figure 1 As shown, it includes the following steps: (1) Synthesis of fish methacrylamide gelatin (fish GelMA): Fish skin gelatin was completely dissolved in PBS solution at a concentration of 10% over 3 hours at 50°C. Then, methacrylic anhydride was slowly added to the solution at a ratio of 6% with vigorous stirring, and the reaction was carried out at 50°C for 3 hours. Unreacted methacrylic anhydride was removed by dialysis with pure water, and the resulting reaction product was lyophilized at -40°C.

[0026] (2) Preparation of fish bone powder: Fresh cold-water fish bones were collected, rinsed with PBS, and the blood vessels, muscles, and periosteum on the surface of the bones were removed. The fish bones were then washed successively with 10% sodium chloride solution and 3% sodium bicarbonate solution to remove fatty residues. The fish bones, frozen in liquid nitrogen, were cryogenically ground at -80°C, and the powder was sieved through a 1000-mesh sieve. The powder was then treated with 1% Triton X-100 solution to disrupt cell membranes, followed by rinsing with Tris-HCl buffer containing 0.1% EDTA and 0.1% SDS to remove cellular components. Finally, the treated fish bone powder was freeze-dried at -40°C and stored at -20°C for later use.

[0027] (3) 3D printed fish gelatin scaffold The prepared 20% fish gelMA and 1% fish bone powder were dissolved in deionized water, and 0.5% light curing agent LAP, 0.1% light stabilizer tartrazine, and VEGF (2 μg / mL) were added. 1 The prepared bio-ink was then used to prepare the scaffold model for printing. The scaffold structure was dissected layer by layer at a thickness of 10 micrometers using slicing software. The prepared bio-ink was then introduced into an ink tank and printed using a 3D printer (nanoArch® S140 Pro) at a thickness of 10 micrometers per layer. Each layer was photocrosslinked at 405 nm for 10 seconds, resulting in a total scaffold height of 3 mm. After printing, the scaffold was rinsed with PBS and stored at 4°C for later use.

[0028] (4) Microfluidic control to produce mixed microspheres of GelMA and methacrylamide alginate (AlgMa) Microfluidic devices were assembled using capillary glass tubes in a concentric circle structure. 15% fish gelMA, 5% AlgMa, and 0.5% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) were dissolved in deionized water as the aqueous phase, and paraffin oil was used as the oil phase. Microspheres were fabricated using a test syringe pump at a flow rate ratio of 1:10. The microspheres were collected with a 5% calcium chloride solution and crosslinked under 405 nm blue-violet light for 5 minutes. The collected microspheres were then washed with water and anhydrous ethanol.

[0029] (5) Assembly of 3D printed scaffold and microspheres 3D-printed fish gelatin scaffolds and microspheres were assembled together at 4°C using photocrosslinking technology. Microspheres were injected into the voids of the 3D-printed scaffold using a microsyringe, with a scaffold-to-microsphere weight ratio of 10:1. The composite scaffold was irradiated with 405nm blue-violet light for 5 minutes. Uncrosslinked microspheres were then washed away with PBS.

[0030] The resulting hydrogel scaffold has the following microstructure: Figure 2 As shown, the red markings represent 3D printed scaffolds, and the yellow markings represent microspheres.

[0031] Comparative Example 1 The difference between this comparative example and Example 1 is that no microspheres are added, and no fish bone powder is added to the 3D printed scaffold; only a 3D fish gelatin scaffold is printed. The steps include: (1) Synthesis of fish methacrylamide gelatin (fish GelMA): Fish skin gelatin was completely dissolved in PBS solution at a concentration of 10% over 3 hours at 50°C. Then, methacrylic anhydride was slowly added to the solution at a ratio of 6% with vigorous stirring, and the reaction was carried out at 50°C for 3 hours. Unreacted methacrylic anhydride was removed by dialysis with pure water, and the resulting reaction product was lyophilized at -40°C.

[0032] (3) 3D printed fish gelatin scaffold The 20% fish gelMA was prepared by dissolving it in deionized water, and 0.5% light curing agent LAP, 0.1% light stabilizer tartrazine, and VEGF (2 μg / mL) were added. 1 The prepared bio-ink was then used to prepare the scaffold model for printing. The scaffold structure was dissected layer by layer at a thickness of 10 micrometers using slicing software. The prepared bio-ink was then introduced into an ink tank and printed using a 3D printer (nanoArch® S140 Pro) at a thickness of 10 micrometers per layer. Each layer was photocrosslinked using 405nm light for 10 seconds, resulting in a total scaffold height of 3mm. After printing, the scaffold was rinsed with PBS and stored at 4°C.

[0033] Comparative Example 2 The difference between this comparative example and Example 1 is that microspheres are not added; only a 3D-printed fishbone powder scaffold is performed, including the following steps: (1) Synthesis of fish methacrylamide gelatin (fish GelMA): Fish skin gelatin was completely dissolved in PBS solution at a concentration of 10% over 3 hours at 50°C. Then, methacrylic anhydride was slowly added to the solution at a ratio of 6% with vigorous stirring, and the reaction was carried out at 50°C for 3 hours. Unreacted methacrylic anhydride was removed by dialysis with pure water, and the resulting reaction product was lyophilized at -40°C.

[0034] (2) Preparation of fish bone powder: Fresh cold-water fish bones were collected, rinsed with PBS, and the blood vessels, muscles, and periosteum on the surface of the bones were removed. The fish bones were then washed successively with 10% sodium chloride solution and 3% sodium bicarbonate solution to remove fatty residues. The fish bones, frozen in liquid nitrogen, were ground at -80°C, and the powder was sieved through a 1000-mesh sieve. The powder was then treated with 1% Triton X-100 solution to disrupt cell membranes, followed by rinsing with Tris-HCl buffer containing 0.1% EDTA and 0.1% SDS to remove cellular components. Finally, the treated fish bone powder was freeze-dried at -40°C and stored at -20°C for later use.

[0035] (3) 3D printed fish gelatin scaffold The prepared 20% fish gelMA and 1% fish bone powder were dissolved in deionized water, and 0.5% light curing agent LAP, 0.1% light stabilizer tartrazine, and VEGF (2 μg / mL) were added. 1 The prepared bio-ink was then used to prepare the scaffold model for printing. The scaffold structure was dissected layer by layer at a thickness of 10 micrometers using slicing software. The prepared bio-ink was then introduced into an ink tank and printed using a 3D printer (nanoArch® S140 Pro) at a thickness of 10 micrometers per layer. Each layer was photocrosslinked using 405nm light for 10 seconds, resulting in a total scaffold height of 3mm. After printing, the scaffold was rinsed with PBS and stored at 4°C.

[0036] The composite hydrogel scaffold prepared in this invention, after being implanted into the bone defect area, effectively provides support for cell attachment due to its interconnected porous and multi-channel structure, allowing cells to grow freely along the scaffold wall. The addition of fish bone powder effectively improves the mechanical strength of the scaffold. This invention tested the effects of adding fish bone powder and microspheres on the mechanical properties of the scaffold. Figure 3 As shown, in the rheological tests, Comparative Example 2 exhibited higher storage modulus and loss modulus than Comparative Example 1, while Example 1 showed the largest G' and G''. In the compression test, Comparative Example 1 had the lowest compression modulus, while Comparative Example 2 had a significantly higher compression modulus than Comparative Example 1, indicating that the addition of fish bone powder increased the maximum pressure the scaffold could withstand. Example 1 possessed a significantly higher compression modulus than both Comparative Examples 1 and 2, indicating that the assembly of microspheres further increased the compression modulus of the scaffold. According to... Figure 4 As shown, compared with Comparative Example 1 and Comparative Example 2, Example 1 has the highest Young's modulus. These results collectively demonstrate that the addition of fish bone powder and microspheres enhances the mechanical properties of the composite scaffold.

[0037] The mechanism behind this phenomenon is that fish bones are mainly composed of hydroxyapatite / calcium phosphate inorganic phases and collagen organic phases, which are highly similar to bone components. As a rigid inorganic filler, it is uniformly dispersed in the GelMA matrix, bearing loads and resisting deformation; it forms a strong interfacial bond with the polymer chains, transferring stress, inhibiting crack propagation, and significantly improving the strength and modulus of the scaffold.

[0038] Simultaneously, the microspheres fill the scaffold pores, forming a filled-interlocking structure; filling internal voids reduces stress concentration and improves overall density; AlgMA-GelMA is a bivalent covalent cross-linked network, with a dense structure, resistance to swelling and deformation, and superior mechanical properties compared to pure GelMA; the microspheres adhere tightly to the inner wall of the scaffold, synergistically bearing force, thus improving overall compressive and shear resistance. Furthermore, as the scaffold degrades, osteogenic components from the fish bone powder are slowly released, promoting bone repair. The dual-module structure encapsulating microspheres within the scaffold enables differentiated release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF), such as... Figure 5 As shown, in Embodiment 1 of the present invention, VEGF is rapidly released in the initial stage, while BMP2 is gradually released in the later stage. This sequential release mimics the natural processes of angiogenesis and late-stage osteoogenesis during early bone repair.

[0039] Loading and controlled release of VEGF onto 3D-printed scaffolds can promote vascular remodeling in the damaged area, thereby improving the transport of oxygen and nutrients in the early stages of bone repair. Simultaneously, the incorporation of microspheres effectively improves the mechanical properties of the scaffold and enables differential release of growth factors, further enhancing the osteogenic activity and angiogenesis-promoting effects of the composite scaffold.

[0040] The scaffold body is composed of Fish-GelMA hydrogel, supplemented with FBP inorganic phase doping. A moderate cross-linking design is employed to meet the requirements of 3D printing moldability and cell infiltration. The degradation mode involves simultaneous surface dissolution and specific enzymatic hydrolysis: fish gelatin, a collagen hydrolysis product, can be specifically hydrolyzed by collagenases and matrix metalloproteinases secreted by cells in the bone defect area; simultaneously, the scaffold swells rapidly under physiological conditions, allowing tissue fluid to fully penetrate into the matrix, accelerating the non-specific dissolution of the hydrogel network. The overall scaffold degradation cycle is concentrated within 1-4 weeks post-surgery, with significant matrix dissolution and network relaxation occurring as early as 0-14 days. Correspondingly, VEGF embedded in the matrix is ​​rapidly released simultaneously with scaffold degradation, with a cumulative release of over 80% of the total load within 0-14 days, perfectly matching the pro-angiogenic needs in the early stages of bone repair.

[0041] In this invention, the mixed microspheres are a GelMA / ALG composite system, prepared via microfluidic monodispersion and photocuring crosslinking. A high-crosslinking densification design is employed, and the degradation mode is slow ionic decrosslinking + delayed enzymatic hydrolysis. The alginate component in the microspheres is degraded via Ca²⁺.+ A dense ionic cross-linked network is formed. Mammals lack specific glycosidases that degrade alginate; they can only degrade it through Na+ in body fluids. + With cross-linked Ca² + The slow ion exchange facilitates the decrosslinking of the microsphere network; simultaneously, the dense alginate network significantly inhibits the penetration of degrading enzymes into the microspheres. Only after the alginate network is decrosslinked and relaxed can the gelatin component fully contact the enzymes and undergo degradation, resulting in a significant degradation lag effect. The overall degradation cycle of the microspheres is extended to 4–12 weeks post-surgery, with no significant bulk degradation in the early stage (0–14 days). Correspondingly, BMP2 embedded in the microsphere matrix is ​​continuously released only as the microspheres gradually degrade, with a burst release rate of less than 10% from 0–14 days and a stable, long-lasting sustained release characteristic from 14–84 days, perfectly matching the physiological needs of osteogenic differentiation and matrix mineralization in the mid-to-late stages of bone repair.

[0042] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A 3D-printed hydrogel scaffold for treating bone defects, characterized in that, It includes a fish gelatin scaffold prepared by 3D printing from the main raw materials fish methacrylamide gelatin and fish bone powder, and mixed microspheres containing fish methacrylamide gelatin and methacrylamide alginate filled in the scaffold, wherein the mass ratio of fish gelatin scaffold to mixed microspheres is (8-15):

1.

2. The 3D-printed hydrogel scaffold for treating bone defects according to claim 1, characterized in that, The mass ratio of fish gelatin scaffold to mixed microspheres is 10:

1.

3. The 3D-printed hydrogel scaffold for treating bone defects according to claim 1, characterized in that, The fish methacrylamide gelatin is obtained by methacrylating fish skin gelatin.

4. The 3D-printed hydrogel scaffold for treating bone defects according to claim 1, characterized in that, The fish bone powder is obtained by sieving through a 1000-mesh filter.

5. A method for preparing a 3D-printed hydrogel scaffold for treating bone defects according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Methacrylamide treatment of gelatin derived from fish skin; (2) Collect fresh cold-water fish bones, process them, grind them into powder, and then decellularize them for later use; (3) The fish methacrylamide gelatin obtained in step (1) and the fish bone powder obtained in step (2) are used as the main raw materials to prepare bio-ink, which is then printed using a 3D printer; (4) Fish methacrylamide gelatin and methacrylamide alginate were used as the main raw materials to prepare mixed microspheres. The microspheres were collected with calcium chloride solution and crosslinked by photoirradiation with 405nm blue-violet light. The collected microspheres were washed with water and anhydrous ethanol. (5) The 3D printed scaffold prepared in step (3) and the mixed microspheres prepared in step (4) are assembled at low temperature by photocrosslinking and then irradiated with 405nm blue-violet light. After cleaning, the product is obtained.

6. The preparation method according to claim 5, characterized in that, Step (1) involves completely dissolving fish skin gelatin at a concentration of 10% in cell phosphate buffer (PBS) solution within 3 hours at 50°C, then slowly adding methacrylic anhydride to the previous solution at a ratio of 5-10% and stirring vigorously, reacting at 50°C for 3 hours, removing unreacted methacrylic anhydride by dialysis with pure water, and lyophilizing the resulting reaction product at -40°C.

7. The preparation method according to claim 5, characterized in that, Step (2) includes collecting fresh cold-water fish bones, rinsing the fish bones with PBS, and removing blood vessels, muscles, and periosteum from the surface of the fish bones; washing the fish bones successively with a 10% sodium chloride solution and a 3% sodium bicarbonate solution to remove fat residue. The frozen fish bones are ground in liquid nitrogen at -80°C, and the powder is sieved using a 1000-mesh filter; the decellularization process includes the following steps: treatment with a 1% polyethylene glycol octylphenyl ether (Triton X-100) solution to disrupt cell membranes, followed by rinsing with a 0.1% EDTA-containing Tris-HCl buffer and a 0.1% sodium dodecyl sulfate (SDS) solution to remove cellular components, and finally lyophilizing the treated fish bone powder at -40°C and storing it at -20°C for subsequent use.

8. The preparation method according to claim 5, characterized in that, Step (3) involves dissolving the fish methacrylamide gelatin prepared in step (1) and the fish bone powder obtained in step (2) (0-2%) with an optimal mass fraction of 1% using deionized water, and adding 0.5% by mass of light curing agent LAP, 0.1% by mass of light stabilizer tartrazine, and vascular endothelial growth factor (VEGF) (2 μg / mL). 1) Prepare bio-ink for printing; use a 3D printer to print at a thickness of 10 micrometers per layer, crosslink each layer with 405nm light for 10s, the total height of the scaffold is 3mm, after printing, rinse the scaffold with PBS and store at 4°C for later use.

9. The preparation method according to claim 5, characterized in that, Step (4) involves assembling a microfluidic device in a concentric circle structure using capillary glass tubes, dissolving 15% fish methacrylamide gelatin, 5% sodium alginate and 0.5% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in deionized water as the aqueous phase, using paraffin oil as the oil phase, and controlling the flow rate ratio of the aqueous phase to the oil phase to be 1:10 using a test injection pump to prepare microspheres; collecting the microspheres with 5% calcium chloride solution and crosslinking them with 405nm blue-violet light for 5 minutes, and washing the collected microspheres sequentially with water and anhydrous ethanol.

10. The preparation method according to claim 5, characterized in that, Step (5) involves assembling the 3D printed scaffold prepared in step (3) and the mixed microspheres prepared in step (4) at 4°C by photocrosslinking. Microspheres are injected into the gaps of the 3D printed scaffold using a microsyringe. The weight ratio of the 3D printed scaffold to the microspheres is (8-15):

1. The composite scaffold is irradiated with 405nm blue-violet light for 5 minutes, and then the uncrosslinked microspheres in the scaffold are washed away with PBS to obtain the final product.

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