Photocuring bio-ink, digital light processing 3D printing product, preparation method of digital light processing 3D printing product and application of digital light processing 3D printing product in preparation of skull repair material

Abstract

The invention provides light-cured bio-ink, a digital light processing 3D printing product, a preparation method of the digital light processing 3D printing product and application of the digital light processing 3D printing product in preparation of a skull repair material, and belongs to the technical field of biological medicine. The material provided by the invention can continuously release HO under different environmental conditions. Particularly, in a simulated in-vivo environment, the GHDP hydrogel keeps stable HO release for a long time, so that a signal channel related to angiogenesis is continuously activated, and in the skull defect repair process, the GHDP hydrogel can promote neovascularization and osteogenic differentiation by regulating and controlling the HO level, so that the skull defect repair effect is improved. And a new strategy and thought are provided for promoting tissue regeneration by utilizing redox signal regulation and control.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to photocurable bio-ink, digital light processing 3D printing products, their preparation methods, and their applications in preparing skull repair materials. Background Art

[0002] Skull defect repair has long been a key research area in bone tissue engineering. With the increasing incidence of various types of craniocerebral injuries in modern society, such as traffic accidents, sports injuries, and tumor resections, skull defects have become a major public health challenge. Due to the unique structure and physiological functions of the skull, skull defects not only affect facial contour and appearance but can also lead to serious consequences such as abnormal intracranial pressure and brain tissue damage, potentially endangering the patient's life. Therefore, effective skull defect repair is not only of great clinical significance but also helps improve patients' social function and quality of life. Developing efficient skull repair strategies is crucial for functional recovery and improved quality of life. Currently, autologous bone transplantation, allogeneic bone transplantation, and synthetic bone materials are the main methods used clinically to repair skull defects. However, autologous bone transplantation faces challenges with donor site damage and limited bone stock, while allogeneic bone transplantation carries the risk of immune rejection and infection. Traditional synthetic bone materials often lack good bioactivity and vascularization, making them inadequate for efficient bone repair. Therefore, the development of novel scaffold materials with excellent biocompatibility, angiogenesis, and osteoinduction has become a research priority.

[0003] During bone regeneration, vascularization is crucial for successful bone repair. Blood vessels not only provide oxygen and nutrients to newly formed tissue but also regulate the osteogenesis process by secreting a variety of signaling molecules. In the absence of adequate vascular support, osteoblast activity decreases significantly, leading to impeded or even failed bone regeneration. Secondly, vascularization plays a key role in regulating the bone regeneration microenvironment. New blood vessels not only provide structural support to the repair area but also regulate osteoblast behavior by secreting a variety of growth factors. For example, vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) secreted by vascular endothelial cells promote osteoblast proliferation, differentiation, and mineralization by activating signaling pathways such as Wnt / β-catenin and BMP / Smad. Furthermore, vascularization suppresses excessive inflammation by secreting anti-inflammatory factors and scavenging inflammatory mediators, thereby creating a favorable environment for bone regeneration. Summary of the Invention

[0004] The purpose of the present invention is to provide photocurable bio-ink, digital light processing 3D printing products and their preparation methods and applications in the preparation of skull repair materials, providing an effective means for the development of bionic bone repair materials.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a photocurable bio-ink, wherein the raw materials of the bio-ink include the following components in parts by weight: 10-20 parts of gelatin methacrylate, 2-4 parts of dopamine-modified hyaluronic acid, 3-8 parts of cross-linking agent, 0.3-0.8 parts of photoinitiator and 0.03-0.08 parts of light-blocking agent.

[0006] Preferably, the cross-linking agent is polyethylene glycol diacrylate.

[0007] Preferably, the gelatin methacrylate is obtained by a preparation method comprising the following steps: Gelatin is dissolved in a buffer solution, methacrylic anhydride is added to carry out methacrylation reaction, and gelatin methacrylate is obtained through dialysis, centrifugation and freeze-drying.

[0008] Preferably, the dopamine-modified hyaluronic acid is obtained by a preparation method comprising the following steps: Hyaluronic acid is dissolved in a buffer solution, the carboxyl group is activated by EDC / NHS, and then reacted with dopamine hydrochloride. Dopamine-modified hyaluronic acid is obtained after dialysis and freeze-drying.

[0009] Preferably, the molecular weight of the hyaluronic acid is 200-400 kDa; In the EDC / NHS activated carboxyl group, the molar ratio of EDC·HCl to NHS is HA:EDC·HCl:NHS=1:3~5:1.5~2.5; The pH of the reaction is controlled at 4.5-5.5, and the reaction time is 10-15 hours.

[0010] Preferably, the photoinitiator is selected from one or more of LAP, Irgacure 2959 or TPO-L; The light-blocking agent is selected from one or more of lemon yellow, carmine or nano titanium dioxide.

[0011] The present invention also provides a method for preparing the above-mentioned photocurable bio-ink, comprising the following steps: Gelatin methacrylate, dopamine-modified hyaluronic acid, a cross-linking agent, a photoinitiator and a light-blocking agent are mixed and dissolved to obtain the photocurable biological ink.

[0012] The present invention also provides a method for preparing a digital light processing 3D printed product, comprising the following steps: The above-mentioned photocurable bio-ink is used as a printing material, and the product is printed by performing photocuring layer by layer through digital light processing technology; The total number of layers of the layer-by-layer photocuring is 4 to 6 layers, the light intensity used by the digital light processing technology is 5 to 20 mW / cm², and the exposure time is 5 to 20 seconds.

[0013] The present invention also provides a digital light processing 3D printing product prepared by the above preparation method.

[0014] The present invention also provides the use of the above-mentioned photocurable biological ink or digital light processing 3D printing product in the preparation of skull repair materials.

[0015] Beneficial effects of the present invention: The 3D-printed scaffold product prepared using the photocurable bio-ink provided by the present invention can generate low levels of H2O2 over a long period of time, successfully activating the signaling pathways associated with angiogenesis and promoting vascularization of skull defects. It has the following advantages: Precise ROS homeostasis regulation: The material's HAD (dopamine-modified hyaluronic acid) continuously releases low concentrations of H2O2 through the auto-oxidation reaction of the catechol groups under physiological conditions, significantly promoting endothelial cell migration and angiogenesis. Simultaneously, the catechol groups in HAD maintain local redox balance through their free radical scavenging ability, preventing cell damage caused by excessive H2O2 concentrations (cytotoxicity at >500 μM). This dynamic ROS regulation mechanism (synergistic generation and scavenging) overcomes the limitations of traditional "pure antioxidant" strategies, achieving efficient synergy between angiogenesis and bone regeneration, while also enhancing mechanical properties and microporous structure.

[0016] Excellent Mechanical Properties: The material exhibits excellent storage and loss moduli. The material exhibits minimal stiffness fluctuations, demonstrating excellent mechanical stability. This property is particularly important for bone repair scaffolds, as bone tissue must withstand external dynamic loads during repair. Therefore, the scaffolds must possess sufficient rigidity and mechanical strength to prevent deformation or breakage during use. Optimal strength, ductility, and impact resistance make it particularly suitable for skull repair in critical load-bearing areas.

[0017] Good biocompatibility: The catechol group maintains redox balance through free radical scavenging, preventing H2O2 toxicity. In cell and animal experiments, the material group showed high cell survival rate, low cell death, and no significant inflammatory response.

[0018] High-precision 3D printing capabilities: Digital light processing (DLP) layer-by-layer photocuring technology is used to create 3D-printed scaffolds. DLP, a layer-by-layer curing printing technology, uses light to induce precise cross-linking reactions in liquid photosensitive resins, creating materials with complex three-dimensional structures. These properties are optimized by adjusting cross-linking time, light intensity, and resin formulation, resulting in bone scaffolds with excellent mechanical properties, a suitable degradation rate, and high cytocompatibility.

[0019] Controllable degradation rate: The degradation characteristics are finely regulated by optimizing its PEGDA content and cross-linking degree. By adjusting the PEGDA content, the cross-linking degree of the material can be precisely controlled, which directly affects the material's degradation rate. Reasonable degradation rate control is one of the key factors in bone tissue engineering materials. Excessively rapid degradation may cause the scaffold to lose its load-bearing capacity, affecting the effectiveness of bone repair; while too slow degradation may hinder the growth and replacement of new bone tissue. The material's degradation characteristics have been optimized to match the regeneration process of bone tissue, ensuring that the material can be gradually replaced by new bone tissue during the repair process.

[0020] Microporous Structure: The dense pore structure of 3D printing promotes a self-reinforcement effect in the in vivo microenvironment, resulting in a higher modulus and slower degradation rate after oxidation. This change not only enhances the material's ability to resist severe deformation caused by external forces at the defect site but also promotes the long-term stability of the scaffold within the defect. It provides more space for cell attachment and diffusion, facilitating initial vascularization. The material's improved internal permeability may also facilitate deeper blood vessel growth.

[0021] Promote angiogenesis: Catechol groups can self-oxidize to generate H2O2. The material of the present invention can continuously release H2O2 under different environmental conditions. In particular, in a simulated in vivo environment, GHDP hydrogel maintains a stable release of H2O2 for a long time, thereby continuously activating the signal pathways related to angiogenesis. Observations on cell migration experiments, cell trans-pore migration experiments, and tube formation experiments show that the material has a significant effect in promoting HUVECs migration, tube formation, and vascular network establishment. The continuous release of H2O2 can enhance the migration and proliferation ability of cells, promote the aggregation of endothelial cells to form a vascular network, provide blood supply for bone repair, and provide essential nutrients and oxygen for bone tissue regeneration.

[0022] Enhanced skull repair: By continuously regulating local ROS levels, the material effectively activates angiogenesis-related signaling pathways, thereby accelerating blood vessel formation. Furthermore, during the bone repair process, the GHDP material gradually degrades, allowing new blood vessels to penetrate deeper into the scaffold. This degradation process not only prevents new blood vessels from being affected by the rapid disintegration of the scaffold but also provides physical support for the expansion and stability of the vessels. During the skull defect repair process, the material can promote new blood vessel formation and osteogenic differentiation by regulating H2O2 levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Photo of 3D printed porous scaffold; Figure 2 Fourier infrared spectra of the synthetic materials; Figure 3The swelling phenomenon and swelling rate-time curve of 3D printed porous scaffolds (A: diameter; B: thickness); Figure 4 This is the general view of the freeze-dried scaffold; Figure 5 Scanning electron microscopy observation and porosity analysis of freeze-dried scaffolds in each group (A: top view; B: side view); Figure 6 Time-concentration curves of H2O2 generation in vitro for different materials; Figure 7 These are the live-dead staining images of cells in each group of materials under laser confocal microscopy; Figure 8 The tube formation phenomenon of the extracts of each group of materials in HUVECs cells; Figure 9 Material implantation process for rat skull defect model (A) and rabbit skull defect model (B) Figure 10 Figure 4: Angiogenesis during skull repair. Angiographic modeling process (A) and images of vascular reconstruction in each group (B). Figure 11 HE-stained sections of rabbit skulls taken 12 weeks after repair. Arrows indicate the defect area, and the green, red, and black boxes (boxes in each image are green, red, and black from left to right, respectively; the rightmost image contains only green and red boxes) correspond to magnified images of the center and edge of the defect, respectively (N: new bone tissue; V: new blood vessels; F: fibrous tissue; S: scaffold); Figure 12 Immunofluorescence staining of CD31, 4-HNE and type I collagen and comparison of fluorescence intensity were performed 12 weeks after surgery; Figure 13 Micro-CT scan image 12 weeks after surgery. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Preparation of gelatin methacrylate (GelMA): (1) Preparation of carbonate buffer solution: Weigh 3.43 g of sodium bicarbonate (NaHCO3, Chengdu Kelong, China) and 1 g of sodium carbonate (Na2CO3, Chengdu Kelong, China) and dissolve them in 1 L of deionized water. Adjust the pH of the solution to approximately 10 and the concentration to 0.25 M. Stir using a magnetic stirrer (IKA, Germany) for 5 minutes to ensure complete dissolution and mixing, then seal and store. Pre-treat the dialysis bag: Dissolve 20.4 g of sodium bicarbonate and 0.3 g of ethylenediaminetetraacetic acid (EDTA, Guangzhou Saiguo Biotechnology Co., Ltd., China) in 2000 mL of deionized water. Place a dialysis bag (molecular weight cutoff range 8-14 kDa) in the above solution, boil for 10 minutes, and then remove for later use.

[0027] (2) After the above process is completed, 200 mL of carbonate buffer solution and 10 g of gelatin (Gelatin, VWR, USA) are added to the flask and stirred in a magnetic stirrer at 50°C and 400 rpm until completely dissolved. Then, 0.8 mL of methacrylic anhydride (MA, Huaxia, China) is slowly added and the reaction is maintained under the same conditions for 2 hours. The reaction solution is collected in four 50 mL centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant is transferred to a pre-treated dialysis bag, the bag mouth is sealed, and the dialysis bag is placed in a flask containing 3000 mL of deionized water for dialysis. The temperature is set at 40°C and the speed is set at 400 rpm. The dialysis time is 72 hours, and the dialysate is replaced every 6 hours. After the dialysis is completed, the liquid in the bag is divided into culture dishes and frozen at -20°C for 24 hours. Then, it is transferred to a freeze dryer (Boyikang Laboratory Instrument Co., Ltd., China) for freeze drying. The freeze drying process is continued for 72 hours. Finally, the completely freeze-dried samples were collected and stored in a sealed container at -20 °C for future use.

[0028] Preparation of dopamine-modified hyaluronic acid (HAD): (1) Dissolve 400 mg of hyaluronic acid (HA, molecular weight 300 kDa) in 35 mL of completely degassed phosphate buffered saline (PBS); (2) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 766 mg) and N-hydroxysuccinimide (NHS, 230 mg) were dissolved in deionized water and slowly added to the HA solution prepared in the first step, ensuring that the molar ratio of HA, EDC·HCl, and NHS was 1:4:2; (3) After stirring for 30 minutes, 1 mmol of dopamine hydrochloride (DA) was added, and the pH value was dynamically monitored using precision pH test paper to maintain the pH of the mixed solution at approximately 5. The reaction was carried out at this pH for 12 hours in an anaerobic environment, using a double-row tube for reaction control. (4) After the reaction is complete, the reaction mixture is dialyzed and purified using acidic deionized water for 72 hours. The product is then freeze-dried to obtain a white powder, HA-Dopamine (HAD).

[0029] Prepare printing ink: 150 mg of GelMA, 150 mg of GelMA and 30 mg of HA, 150 mg of GelMA and 30 mg of HAD, and an additional 150 mg of GelMA and 30 mg of HAD were completely dissolved in 1 mL of deionized water (UP water) to a concentration of 15 w / v%. Using a vortex mixer (IKA, vortex, Genius 3), 45 μL of a 5 w / v solution of polyethylene glycol diacrylate (PEGDA, molecular weight 700, Sigma) was added to each of the first three solutions and mixed thoroughly. Subsequently, 0.5 w / v% of the photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) phosphate lithium salt (LAP, Yinchang New Materials Co., Ltd., China) and 0.05 w / v% of the light absorber tartrazine (Shanghai Stone Brand) were added to each of the four solutions, ensuring complete dissolution. The solution was stirred with a vortex mixer (37°C) and further mixed using a mixing and defoaming device (THINKY, ARE-310, Japan) to remove bubbles. The solution was sealed and protected from light, stirred at 60°C until completely dissolved, and then brought to 37°C. Using the above method, four sets of photocurable inks were prepared with the following mass / volume ratios: GP (15% GelMA, 5% PEGDA, 0.5% LAP, 0.05% tartrazine), GHP (15% GelMA, 3% HA, 5% PEGDA, 0.5% LAP, 0.05% tartrazine), GHD (15% GelMA, 3% HAD, 0.5% LAP, 0.05% tartrazine), and GHDP (15% GelMA, 3% HAD, 5% PEGDA, 0.5% LAP, 0.05% tartrazine).

[0030] Example 2

[0031] This embodiment is based on the GHDP group of photocurable inks in embodiment 1.

[0032] The porous scaffold models for animal and cell experiments were designed using the computer-aided design software Magics (version 22.0, Materialise, BelgiμM). The models were pie-shaped and measured 10 × 2 mm and 5 × 1 mm. After design, the models were stored in the operating system of a stereolithography 3D printer (ADMAFLEX 130 plus, Admatec, the Netherlands) based on digital light processing (DLP) technology.

[0033] During the printing process, the model was sliced ​​using Magics software, and the design orientation was optimized using the print path. The sliced ​​files were then exported (SLC format). During printing, the DLP 3D printer projected and cured the material layer by layer (layer height: 100 μm, base layer: 5 layers, light intensity: 10 mW / cm²). To ensure sufficient gelation of the ink and avoid overexposure, the exposure time was tested multiple times, ultimately settling on a 10-second exposure time. After printing, the porous scaffolds were washed in 40°C PBS to remove any uncrosslinked ink. The scaffolds were then sealed and stored in a dark place until needed.

[0034] Finally, the 3D printed GHDP hydrogel scaffold was obtained.

[0035] Example 3

[0036] This embodiment is based on the GHP group photocurable ink in embodiment 1.

[0037] The porous scaffold models for animal and cell experiments were designed using the computer-aided design software Magics (version 22.0, Materialise, BelgiμM). The models were pie-shaped and measured 10 × 2 mm and 5 × 1 mm. After design, the models were stored in the operating system of a stereolithography 3D printer (ADMAFLEX 130 plus, Admatec, the Netherlands) based on digital light processing (DLP) technology.

[0038] During the printing process, the model was sliced ​​using Magics software, and the design orientation was optimized using the print path. The sliced ​​files were then exported (SLC format). During printing, the DLP 3D printer projected and cured the material layer by layer (layer height: 100 μm, base layer: 5 layers, light intensity: 10 mW / cm²). To ensure sufficient gelation of the ink and avoid overexposure, the exposure time was tested multiple times, ultimately settling on a 10-second exposure time. After printing, the porous scaffolds were washed in 40°C PBS to remove any uncrosslinked ink. The scaffolds were then sealed and stored in a dark place until needed.

[0039] Finally, the 3D printed GHP hydrogel scaffold was obtained.

[0040] Example 4

[0041] This embodiment is based on the GHD group of photocurable inks in embodiment 1.

[0042] The porous scaffold models for animal and cell experiments were designed using the computer-aided design software Magics (version 22.0, Materialise, BelgiμM). The models were pie-shaped and measured 10 × 2 mm and 5 × 1 mm. After design, the models were stored in the operating system of a stereolithography 3D printer (ADMAFLEX 130 plus, Admatec, the Netherlands) based on digital light processing (DLP) technology.

[0043] During the printing process, the model was sliced ​​using Magics software, and the design orientation was optimized using the print path. The sliced ​​files were then exported (SLC format). During printing, the DLP 3D printer projected and cured the printed parts layer by layer (layer height: 100 μm, base layer: 5 layers, light intensity: 10 mW / cm²). To ensure sufficient gelation of the ink and avoid overexposure, the exposure time was tested multiple times, ultimately settling on a 35-second exposure time for all material groups. After printing, the porous scaffolds were washed in 40°C PBS to remove any uncrosslinked ink. The scaffolds were then sealed and stored in a dark place until needed.

[0044] Finally, the 3D printed GHD hydrogel scaffold was obtained.

[0045] Example 5

[0046] This embodiment is based on the GP group photocurable ink in embodiment 1.

[0047] The porous scaffold models for animal and cell experiments were designed using the computer-aided design software Magics (version 22.0, Materialise, BelgiμM). The models were pie-shaped and measured 10 × 2 mm and 5 × 1 mm. After design, the models were stored in the operating system of a stereolithography 3D printer (ADMAFLEX 130 plus, Admatec, the Netherlands) based on digital light processing (DLP) technology.

[0048] During the printing process, the model was sliced ​​using Magics software, and the design orientation was optimized using the print path. The sliced ​​files were then exported (SLC format). During printing, the DLP 3D printer projected and cured the material layer by layer (layer height: 100 μm, base layer: 5 layers, light intensity: 10 mW / cm²). To ensure sufficient gelation of the ink and avoid overexposure, the exposure time was tested multiple times, ultimately settling on a 6-second exposure time. After printing, the porous scaffolds were washed in 40°C PBS to remove any uncrosslinked ink. The scaffolds were then sealed and stored in a dark place until needed.

[0049] Finally, the 3D printed GP hydrogel scaffold was obtained.

[0050] Experimental Example 1

[0051] The following experiments were performed on the hydrogel scaffolds prepared in Examples 2 to 5: (1) Printability and structural characterization In terms of hydrogel scaffold 3D printing accuracy, the porosity of GHDP is higher than that of the other groups ( Figure 1 ). Figure 2 The infrared spectra of three hydrogel scaffolds, GHP, GHD, and GHDP, are shown. Compared to the GHP group, the catechol-complexed GHD and GHDP groups exhibit characteristic absorption peaks of catechol at 1720 cm⁻¹ and 1235 cm⁻¹, indicating that GHD and GHDP retain the catechol amino group and CN-related structure.

[0052] (2) Stent swelling test Four sets of hydrogel inks were prepared according to the aforementioned protocol and thoroughly dissolved and mixed. After printing, the diameter (Do) and thickness (To) of each scaffold sample were measured and recorded, and photographed and recorded using a stereo microscope (Zeiss, Discover, V20). Next, the test scaffolds were immersed in PBS and placed on a shaker at 37°C. The diameter and thickness of each scaffold were measured at different time points (1 day, 3 days, 5 days, 7 days, 14 days, 21 days, and 28 days), and recorded as Ds and Ts. After 28 days, the scaffolds were removed and photographed and recorded under a stereo microscope. The swelling ratio of the samples at each time point was calculated using the following formula: diameter swelling ratio = (Ds - Do) / Do × 100%, thickness swelling ratio = (Ts - To) / To × 100%. After approximately 10 days of immersion in PBS, the surface of the GHD group showed significant swelling, completely destroying the material structure and making further testing impossible. For the other three groups of materials, after immersion for 28 days, the swelling rate of the GHDP group was about 10% in terms of diameter, and the swelling rate of the GP group was about 9%, which was slightly lower than the swelling rate of the GHP group (12%). In terms of thickness, the swelling rate of the GP group was slightly higher, about 20%. The swelling rate of the GHDP group was about 18%, and the swelling rate of the GP group was about 16% ( Figure 3After 28 days of immersion, observation under a stereomicroscope revealed that the structure of the GHDP group remained largely intact, with clearly visible pores. In contrast, the structures of the GHP and GP groups were partially damaged, with less pronounced pores. These results demonstrate that PEGDA plays a crucial role in maintaining the structural stability of the porous scaffold. The GHDP porous scaffold exhibited superior structural stability under conditions that simulated the in vivo microenvironment, enabling prolonged maintenance of oxygen, nutrients, and metabolic products, allowing for the in-growth of new blood vessels following skull defects and providing a solid structural foundation for promoting angiogenesis and bone repair at the defect site.

[0053] (III) Stereomicroscope observation of porous scaffolds Soak the printed scaffold in PBS and place it in a 4°C refrigerator overnight. The next day, transfer the scaffold to an EP tube, poke a hole in the tube cap, and freeze it in a -20°C refrigerator for 24 hours. Then, transfer it to a flask equipped with a freeze dryer and connect it to the freeze dryer for 12 hours of freeze drying to remove moisture from the material. After freeze drying, use a stereo microscope at different magnifications to observe the morphological characteristics of the test scaffold and record pictures. Figure 4 It can be seen that the GHDP group has a uniform porous structure with regular pore morphology and consistent pore size, which is conducive to the exchange of oxygen and nutrients in the body. Although the GHP group also has a porous structure, some pores are blocked, which may affect the efficiency of oxygen and nutrient exchange in the body. The GP group also has a porous structure, without obvious pore blockage, but compared with the GHDP group, its scaffold structure lacks mechanical support. In addition, the material of the GHD group without PEGDA lost its original structure after drying and had an irregular morphology, indicating that PEGDA plays an important role in the structural stability of the material.

[0054] (IV) Scanning electron microscopy (SEM) observation of porous scaffolds After the prepared scaffold is immersed in PBS, it is allowed to stand at 4°C overnight. The next day, the sample is transferred to a pre-punched EP tube and placed in a -20°C low-temperature environment for 24 hours. After that, the sample is moved into a special flask of a freeze dryer and connected to the equipment for 12 hours of freeze-drying to achieve the dehydration process of the material. After the freeze-drying is completed, the scaffold is broken with liquid nitrogen and a gold film is sprayed using an ion sputtering apparatus (SBC-12); the sample is fixed with conductive tape and placed on the sample stage, vacuumed for 1 minute, and then ion sputtering coating is started for 30 seconds. After completion, it is placed in a field emission scanning electron microscope (SEM, S-4800, Hitachi High-Tech, Japan) under high vacuum mode for observation, and the surface morphology characteristics and data of the scaffold are recorded using different magnifications. From the top view of the material ( Figure 5 A) It can be seen that all four groups of materials have porous structures. Except for the irregular pores in the GHD group, the pores in the other three groups are relatively regular. Figure 5 B) The microscopic morphological differences among the four groups of materials are even more pronounced. Compared with the other three groups, the GHDP group's material has more uniformly connected micropores, a structure that is more conducive to the exchange of oxygen and nutrients in the body.

[0055] (V) Detection of ROS levels in the microenvironment regulated by materials in vitro To test the H2O2 generation capacity and duration of the GHDP material, GHDP, GHP, GHD, and GP hydrogel disc samples were prepared using a mold (using the same method as above) and placed in 48-well plates, along with a blank control. 1 mL of PBS containing 1% double-streptomycin (a mixture of penicillin and streptomycin) was added to each well. Next, another 48-well plate was prepared, and each sample group was placed in a single well using the same method. A blank control was also included. 1 mL of PBS containing 1340 μM H2O2 and 1% double-streptomycin was added to each well. Both 48-well plates were incubated at 37°C in a 5% CO2 incubator. Samples (18 μL each) were taken on days 1, 2, 4, 8, and 16. After dilution 100-fold with PBS, H2O2 concentrations were measured using the hydrogen peroxide fluorescent probe Amplex Red (Shanghai Maokang Biotechnology). Dissolve Amplex Red in dimethyl sulfoxide (DMSO) in the dark to make a 10 mM stock solution. Dissolve horseradish peroxidase in PBS to make a 10 U / mL stock solution, and prepare 0.05 M PBS. Mix 50 μL of Amplex Red stock solution, 100 μL of horseradish peroxidase stock solution, and 4.85 mL of PBS to make a 5 mL working solution.

[0056] The working solution and sample solution (50 μL / time point) were mixed at a ratio of 1:1, incubated at room temperature for 30 minutes, and then the fluorescence intensity was measured using a fluorescence microplate reader (excitation light 530-560 nm, emission light 590 nm). Figure 6A standard curve of H2O2 concentration versus fluorescence intensity was constructed based on the results. H2O2 concentrations at each time point were calculated, and finally, time-concentration curves were plotted. Initially (the first two days), H2O2 production in the GHDP group ranged from 0 to 35 μM. The concentration then gradually decreased, reaching approximately 10 μM on day 8. It then began to slowly recover, reaching 40 μM on day 16, indicating that the GHDP group had a moderate H2O2 production capacity. The GHP and GP groups showed similar trends, with slower H2O2 production rates. Initially, H2O2 concentrations slowly increased, reaching approximately 10 μM on day 2, before decreasing to approximately 2 μM on day 4. Although concentrations recovered somewhat later, they remained at approximately 10 μM on day 16, significantly lower than those in the GHDP group, indicating limited H2O2 production capacity. H2O2 production in the GHD group increased significantly, particularly around day 8, rapidly rising to 80 μM and reaching 210 μM on day 16, the highest of all groups, demonstrating excellent long-term response capacity.

[0057] The results showed that the dopamine-containing materials had significantly better H2O2 generation capabilities than the dopamine-free materials, suggesting that dopamine can promote H2O2 generation. Furthermore, the GHD group had significantly higher H2O2 generation capabilities than the GHDP group, while all PEGDA-containing materials experienced a decrease in H2O2 concentration, likely due to the large number of double bonds in PEGDA consuming some H2O2, leading to a decrease in concentration.

[0058] (VI) Biocompatibility characterization Remove 10× Assay Buffer from the freezer and, under sterile conditions, dilute the appropriate amount to 1× with UP water to obtain the desired 1× Assay Buffer solution. Staining steps: Remove the cell and material well plates after 1 and 3 days of co-culture, aspirate the culture medium, and wash the samples twice with 1× Assay Buffer (pH 7.4). Add 1 mL of PBS to each well, along with 1 μL of calcein-AM stock solution, and incubate at 37°C in the dark for 20 minutes. Next, add 3 μL of PI stock solution, mix thoroughly, and continue incubating at room temperature in the dark for 5 minutes. Finally, observe using a laser scanning confocal microscope (CLSM, Zeiss, SM 800, Germany) (excitation wavelength: 490 nm and emission wavelength: 515 nm, respectively) to assess material toxicity, cell proliferation, and other aspects. Figure 7The results of the live-dead staining experiment are presented, showing the survival and death of cells under treatment with different materials. After the first day, the cell survival rate of the GHDP group was higher, with green cells accounting for the majority and fewer dead cells. The cell survival rates of the other three groups of materials were lower than that of the GHDP group, and there were more dead cells. After the third day of co-culture of the material and cells, the cell survival in the GHDP group was still good, with most green cells remaining alive and a low proportion of dead cells, indicating that the material continued to support cell growth. The number of dead cells in the GHP and GHD groups decreased, and the proportion of green cells increased compared to the first day, indicating that the material's support for cells was enhanced. The proportion of dead cells in the GP group also decreased, but the number of surviving cells was still small, indicating that the material's ability to support cell growth was limited. In summary, the GHDP material showed the strongest cell survival support effect in the cell co-culture experiment, with the highest number of surviving cells and the least dead cells, and had excellent biocompatibility.

[0059] (VII) In vitro tube formation experiment To evaluate the ability of each material group to promote HUVEC angiogenesis, the extracts of each material group were used for intervention, and the process of HUVEC angiogenesis was recorded dynamically under a microscope. The tube formation image at the 6th hour was randomly selected. Figure 8 The results showed that the GHDP group and the H2O2 group showed the highest cell grid numbers.

[0060] Experimental Example 2

[0061] A 3D-printed hydrogel scaffold was constructed for a critical-size skull defect model, demonstrating that the GHDP group could better promote skull vascularization and repair than the GHP group.

[0062] This experiment was approved by the Ethics Committee of Sichuan University (Ethics Approval No.: 20230302015). Male New Zealand rabbits (weighing 2.0-2.5 kg) and male Sprague-Dawley rats (weighing 220-260 g) were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. All animals were housed in a standard, specific pathogen-free animal room and fed a standard diet with free access to food and water. All surgical instruments were autoclaved, and all experimental procedures were performed in a sterile environment. Rabbits and rats were randomly divided into four groups, each containing 12 animals. The experimental groups included two material (3D-printed scaffold) groups: the GHDP group and the GHP group; the remaining two groups were a normal group and a blank control group. The Sprague-Dawley rat model primarily evaluated short-term (1 week) reactive oxygen species levels, inflammatory responses, and vascularization at the skull defect site; the rabbit model primarily evaluated long-term (4 and 12 weeks) bone formation at the skull defect site.

[0063] A short-term (1 week) skull defect animal model was established using SD rats to evaluate the level of reactive oxygen species and vascularization at the skull defect site. A long-term (4 and 12 weeks) skull defect animal model was established using rabbits to evaluate the formation of new bone at the skull defect site at two time points. The steps for constructing the rat and rabbit skull defect models were as described above, and the surgical implantation of the porous scaffolds was as follows. Figure 9 shown.

[0064] One week after surgery, rats with skull modeling were treated under general anesthesia, and the angiographic modeling process was as described previously ( Figure 10 A). Through Micro-CT image denoising, enhancement and 3D reconstruction algorithms, 3D reconstructed images of each group of blood vessels were obtained ( Figure 10 B). Results show that vascularization was observed within the burr hole area (indicated by the red circle) in all groups. In particular, the GHDP material group showed significantly higher vascular volume and density than the GHP group, indicating that the GHDP material group possesses a stronger ability to vascularize the skull defect.

[0065] The GHDP group showed the best ability to induce bone regeneration throughout the experimental period, which was significantly better than the GHP group and the blank control group. This shows that the GHDP group has potential advantages in promoting bone repair and its value in clinical application can be further explored. Figure 11 It showed that at week 12, a small amount of new bone tissue could be observed in the blank control group, and the defect was still mainly composed of fibrous connective tissue. The defect area of ​​the GHDP and GHP groups showed a mixture of new bone tissue and undegraded material. Local magnification showed that new bone tissue was generated around the material. In addition, compared with the GHP and blank control groups, the GHDP group showed more abundant new blood vessels, which is very important for bone tissue regeneration in vivo. On the other hand, the new blood vessels observed in the GHDP and GHP groups fused with the undegraded material, indicating that it may further promote the formation of new bone tissue.

[0066] like Figure 12 It was shown that 12 weeks after surgery, immunofluorescence staining was performed on rabbit skull samples (including porous scaffolds) in the GHDP group and GHP material group, as well as blank control group samples (including the skull drilling area). CD31 (R&D, AF3628), 4-hydroxynonenoic acid (4-HNE, ab46545) and type I collagen (ARG21965) primary antibodies were used for labeling, and the samples were observed and images were taken under a fluorescence microscope. The results showed that 12 weeks after surgery, the fluorescence intensity of the GHDP group was higher than that of the GHP group in terms of angiogenesis, in vivo ROS expression and osteogenesis. At week 12, Micro-CT analysis found that the bone tissue content in the GHDP group was relatively higher than that in the GHP and blank control groups, and showed a trend of growing towards the center of the defect ( Figure 13 ).

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A photocurable bio-ink, characterized in that: The raw materials of the bio-ink include the following components in parts by weight: 10-20 parts of gelatin methacrylate, 2-4 parts of dopamine-modified hyaluronic acid, 3-8 parts of cross-linking agent, 0.3-0.8 parts of photoinitiator and 0.03-0.08 parts of light-blocking agent.

2. The photocurable biological ink according to claim 1, characterized in that: The cross-linking agent is polyethylene glycol diacrylate.

3. The photocurable biological ink according to claim 1, characterized in that: The gelatin methacrylate is obtained by a preparation method comprising the following steps: Gelatin is dissolved in a buffer solution, methacrylic anhydride is added to carry out methacrylation reaction, and gelatin methacrylate is obtained through dialysis, centrifugation and freeze-drying.

4. The photocurable biological ink according to claim 1, characterized in that The dopamine-modified hyaluronic acid is obtained by a preparation method comprising the following steps: Hyaluronic acid is dissolved in a buffer solution, the carboxyl group is activated by EDC / NHS, and then reacted with dopamine hydrochloride. Dopamine-modified hyaluronic acid is obtained after dialysis and freeze-drying.

5. The photocurable biological ink according to claim 4, characterized in that: The molecular weight of the hyaluronic acid is 200-400 kDa; In the EDC / NHS activated carboxyl group, the molar ratio of EDC·HCl to NHS is HA:EDC·HCl:NHS=1:3~5:1.5~2.5; The pH of the reaction is controlled at 4.5-5.5, and the reaction time is 10-15 hours.

6. The photocurable biological ink according to claim 1, characterized in that: The photoinitiator is selected from one or more of LAP, Irgacure 2959 or TPO-L; The light-blocking agent is selected from one or more of lemon yellow, carmine or nano titanium dioxide.

7. The method for preparing the photocurable bio-ink according to any one of claims 1 to 6, characterized in that: The following steps are involved: Gelatin methacrylate, dopamine-modified hyaluronic acid, a cross-linking agent, a photoinitiator and a light-blocking agent are mixed and dissolved to obtain the photocurable biological ink.

8. A method for preparing a digital light processing 3D printed product, characterized in that: The following steps are involved: The product is obtained by printing the photocurable bio-ink according to any one of claims 1 to 6 as a printing material and performing layer-by-layer photocuring by digital light processing technology; The total number of layers of the layer-by-layer photocuring is 4 to 6 layers, the light intensity used by the digital light processing technology is 5 to 20 mW / cm², and the exposure time is 5 to 20 seconds.

9. A digital light processing 3D printed product produced by the preparation method according to claim 8.

10. Use of the photocurable bio-ink according to any one of claims 1 to 6 or the digital light processing 3D printing product according to claim 9 in the preparation of skull repair materials.

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