Multi-material 3D printing bionic gradient soft-hard integrated hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to multi-material 3D printing bionic gradient soft-hard integrated hydrogel as well as a preparation method and application thereof. Based on a copolymerization system of high-strength supramolecular N-acryloylglycinamide and vinyl phosphoric acid, an NAGA aqueous solution and an NAGA-VPA aqueous solution are printed into hydrogel of a gradient structure by adopting a DLP multi-material printing technology, then the hydrogel is sequentially soaked in a calcium-phosphorus mixed aqueous solution (Ca / P = 1.67), ammonia water, a calcium chloride aqueous solution and deionized water, and the hydrogel is prepared through a hydrothermal method. Therefore, in-situ gradient deposition and curing of hydroxyapatite (HAP) are realized. The multi-material 3D printing bionic gradient soft-hard integrated hydrogel prepared by the method has a continuous biochemical-mechanical gradient, a surface layer low-modulus area is matched with cartilage performance, a deep layer high-mineralization area is integrated with bone tissue, the HAP crystallinity of a transition area is in spatial dependence distribution, and the hydrogel is suitable for osteochondral defect repair and manufacturing of bionic joint prostheses.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel, its preparation method, and its application. Background Technology

[0002] As a key biomechanical transmission structure in the joint system, osteochondral tissue achieves a stable connection between flexible cartilage and rigid bone through its unique layered gradient characteristics. The natural osteochondral interface contains an ultrathin (20-30 micrometers) calcified transition layer, in which the hydroxyapatite (HAP) content exhibits a continuous spatial gradient distribution from the cartilage surface (nearly 0%) to the subchondral bone (>20%). This gradual change in biochemical composition and physical properties endows osteochondral tissue with two core functions: first, to efficiently disperse the mechanical load during joint movement, avoiding fatigue damage caused by stress concentration; and second, to maintain structural integrity under long-term physiological loads, achieving stable integration of cartilage and bone.

[0003] However, the repair of osteochondral defects still faces severe challenges. Full-thickness defects caused by disease or trauma can disrupt the continuity of gradient structures and lead to degenerative diseases such as osteoarthritis. Existing repair materials are difficult to replicate the gradient characteristics of natural tissues, mainly due to the following three technical bottlenecks: (1) Gradient construction failure: As the main inorganic phase, the large particle size and low fluidity of HAP hinder the formation of continuous gradient structures. After mineralization, the HAP of traditional homogeneous hydrogels is uniformly distributed and cannot simulate the gradual change from flexible to rigid. Stress concentration is easily generated at the soft-hard interface, leading to material delamination or fracture. (2) Mechanical property mismatch: The compressive modulus of natural subchondral bone is much higher than that of traditional hydrogels. Although homogeneous mineralization materials can improve strength, the sudden change in rigidity exacerbates the mechanical incompatibility between the implant and the host tissue. (3) Lubrication-bearing contradiction: Existing technologies improve friction performance through double-layer structures or the addition of lubricating components, but the surface layer is too thin and is prone to wear failure, while the addition of lubricating components will weaken the mechanical strength of the material.

[0004] Some progress has been made in exploring hydrogels as cartilage substitutes. Their high water content and biocompatibility can mimic the lubricating environment of natural cartilage, but traditional modification methods can only adjust mechanical properties to a limited extent and cannot simultaneously replicate the integration of gradient structure and function. For example, although homogeneous P(NAGA-VPA) mineralized hydrogels improve HAP deposition efficiency through phosphate groups, it is still difficult to establish a continuous biochemical-mechanical gradient. This structural defect leads to interface fatigue failure under long-term loads, failing to meet the service requirements of load-bearing joints. Furthermore, the superior performance of the human osteocartilage interface stems from its multi-scale ordered structure: HAP crystals are oriented along collagen fibers within the micron-scale calcification layer, and the non-uniform distribution at the nanoscale, in synergy with elastin, forms a progressive mechanical response. In current synthetic materials, the fluidity limitation of HAP makes it difficult for it to migrate directionally within polymer networks; and abrupt connections at the soft-hard interface are more prone to stress concentration. Therefore, overcoming the physical limitations of gradient construction and achieving multi-scale biomimetic design from molecular cross-linked networks to macroscopic mechanical properties is a key path to solving the problem of osteocartilage regeneration. Related technologies attempt to construct gradients through multi-step processes, such as layered injection of prepolymer solutions of different concentrations or surface modification with inorganic coatings. However, the problems of weak interlayer bonding and abrupt transitions remain unresolved. Scanning electron microscopy observations show that such materials are prone to phase separation at the interface, and the accumulation of mineralized particles leads to pore blockage, disrupting the swelling balance and load-bearing synergy of the hydrogel. The smooth transition characteristics of natural tissues require molecular design to endow the material with intrinsic gradient-forming capabilities, rather than relying on external superposition. Furthermore, there is an intrinsic relationship between the mechanical and lubrication properties of hydrogels. While high water content can reduce the coefficient of friction, it sacrifices the material's stiffness; conversely, increased stiffness may reduce the thickness of the interfacial hydration layer, exacerbating wear. Traditional homogeneous materials cannot simultaneously meet the requirements of high load-bearing capacity at the deep layer and super-lubrication at the surface. Therefore, there is an urgent need to develop integrated hydrogels that combine continuous gradient structures, high strength and toughness, and durable lubrication properties. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel, its preparation method, and its applications. The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared by this invention possesses a continuous gradient structure, high strength and toughness, and durable lubrication properties.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel, comprising the following steps: N-Acryloylglycine, a first photoinitiator, and water were mixed to obtain an aqueous solution of NAGA; N-Acryloylglycine, vinyl phosphoric acid, a second photoinitiator, and water were mixed to obtain an aqueous solution of NAGA-VPA; The NAGA aqueous solution is subjected to a first DLP printing to obtain several P(NAGA) layers. The NAGA-VPA aqueous solution is then subjected to a second DLP printing on the surface of the P(NAGA) layers to form several P(NAGA-VPA) layers, resulting in a P(NAGA)-P(NAGA-VPA) gradient structure hydrogel. The P(NAGA)-P(NAGA-VPA) gradient structure hydrogel was subjected to a first immersion treatment in a calcium-phosphorus mixed aqueous solution, a second immersion treatment in ammonia water, a aging and stabilization treatment in a calcium chloride aqueous solution, and a third immersion treatment in water to obtain a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel; the Ca / P molar ratio in the calcium-phosphorus mixed solution was 1.67.

[0007] Preferably, the mass concentration of the first N-acryloylglycine in the NAGA aqueous solution is 25-30%; The mass of the first photoinitiator is 0.3~0.5% of the mass of N-acryloylglycamide; The first photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0008] Preferably, the mass concentration of the second N-acryloylglycine in the NAGA-VPA aqueous solution is 25-30%; The mass ratio of the second N-acrylglycolamide to vinyl phosphoric acid is 6~12:1; The mass of the second photoinitiator is 0.3%-0.5% of the total mass of the second N-acryloylglycine and vinyl phosphate; The second photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0009] Preferably, the printing parameters for the first DLP printing and the second DLP printing independently include: the bottom layer exposure time is 10000~12000ms, the exposure time for each layer is 8000~10000ms, the wavelength is 405nm, and the slice thickness is 0.05~0.1mm; The number of layers in the P(NAGA) layer is 5 to 100. The P(NAGA-VPA) layer has 5 to 100 layers.

[0010] Preferably, in the mixed reaction solution, the calcium ions are derived from calcium chloride, and their concentration can be 0.5~1 mol / L; the phosphate ions are derived from phosphoric acid, and their concentration can be 0.3-0.6 mol / L; the molar ratio of calcium ions to phosphate ions (Ca / P) is 1.67.

[0011] The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to the calcium-phosphorus mixed solution is 1:25~30; The temperature of the first soaking treatment is 18~30℃, and the time is 24h.

[0012] Preferably, the mass concentration of the ammonia solution is 15%; The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to ammonia is 1:25~30; The second soaking treatment is carried out at a temperature of 18~30℃ for 4 hours.

[0013] Preferably, the concentration of the calcium chloride aqueous solution is 0.75 mol / L; The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to the calcium chloride aqueous solution is 1:25~30; The aging and stabilization temperature is 18~30℃, and the time is 24h; The third soaking treatment is carried out at a temperature of 18~30℃ for 3~7 days.

[0014] This invention also provides a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by the preparation method described above.

[0015] This invention also provides the application of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel described above in the preparation of biological soft tissue substitutes.

[0016] Preferably, the biological soft tissue substitute includes osteochondral defect repair bodies or biomimetic joint prostheses.

[0017] This invention utilizes a copolymer system of high-strength supramolecular N-acryloylglycamide (NAGA) and vinyl phosphate (VPA) to prepare a multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel using a synergistic strategy of multi-material gradient printing and ion coordination step mineralization. Specifically, using DLP (Digital Light Processing) technology, a P(NAGA) layer is printed first, followed by a P(NAGA-VPA) layer, constructing a gradient network layer by layer from the bottom up. This structure retains the lubricating properties of the surface layer while providing diffusion channels for deep mineralization. If the P(NAGA-VPA) layer is printed first, VPA will be mixed into the NAGA layer, leading to mineralization of the upper layer. The added vinyl phosphate (VPA) phosphate groups chelate calcium ions to form mineralization nucleation sites, and individual hydroxyapatite (HAP) particles are cross-linked with multiple phosphate groups, enhancing the inorganic phase strengthening effect while retaining the hydrogen bond network. After the calcium-phosphorus mixed aqueous solution (Ca / P=1.67) permeates into the gradient network, ammonia water triggers heterogeneous nucleation of HAP, and calcium chloride aqueous solution ripens and enhances crystallinity, achieving directional deposition of HAP along the crosslinking gradient. Using the preparation method of this invention, customized bio-soft tissue substitutes with continuous biochemical-mechanical gradients (e.g., osteochondral defect repairs, biomimetic joint prostheses) can be constructed using 3D printing technology. The surface low-modulus region matches cartilage properties, the deep high-mineralization region integrates bone tissue, and the HAP crystallinity in the transition region exhibits a spatially dependent distribution. This method is suitable for osteochondral defect repair and the manufacture of biomimetic joint prostheses.

[0018] The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared in this invention utilizes DLP printing technology (especially DLP multi-material printing technology) combined with VPA-Ca 2+ The coordination mineralization mechanism successfully reproduced the three-layered gradient structure of natural osteochondral.

[0019] The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared by this invention has a surface region that can form a fluid lubrication layer with high water content to match cartilage performance, a deep high-mineralization zone that integrates bone tissue to induce osteogenic regeneration, and a transition zone that effectively disperses interfacial stress. This avoids the risk of sudden soft-hard transitions in traditional bilayer materials and achieves an integrated function of "flexible lubrication - gradient transition - rigid support".

[0020] The multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by this invention can complete the gradient deposition and maturation of HAP in a short time (within 2 days) through a step mineralization process of calcium phosphorus infiltration → ammonia water triggering → calcium ion maturation. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the process and mechanism of multi-material 3D printing of biomimetic gradient soft-hard integrated hydrogel in this invention; Figure 2The images show the physical images of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11, where a is Example 8, b is Example 9, c is Example 10, and d is Example 11. Figure 3 This is an image of a joint bone sample of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel in this invention; Figure 4 Comparison of the tensile properties of P(NAGA-VPA) hydrogels prepared in Examples 1-4; Figure 5 Comparison of the tensile properties of P(NAGA-VPA) hydrogels prepared in Examples 1, 3, and 5-6; Figure 6 The interfacial tensile properties of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel prepared in Example 7; Figure 7 Comparison of the compressibility of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11; Figure 8 Comparison of lubrication properties of multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11; Figure 9 Scanning electron microscope images of the upper P(NAGA) layer and the lower P(NAGA-VPA)HAP layer in the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel prepared in Example 11. Figure 10 Scanning electron microscope image of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel P(NAGA) layer prepared in Example 11; Figure 11 Scanning electron microscope image of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel P(NAGA-VPA) HAP layer prepared in Example 11. Detailed Implementation

[0022] This invention provides a method for preparing a multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel (the preparation method and mechanism are as follows). Figure 1 (As shown), including the following steps: N-Acryloylglycine, a first photoinitiator, and water were mixed to obtain an aqueous solution of NAGA; N-Acryloylglycine, vinyl phosphoric acid, a second photoinitiator, and water were mixed to obtain an aqueous solution of NAGA-VPA; The NAGA aqueous solution is subjected to a first DLP printing to obtain several P(NAGA) layers. The NAGA-VPA aqueous solution is then subjected to a second DLP printing on the surface of the P(NAGA) layers to obtain a P(NAGA)-P(NAGA-VPA) gradient structure hydrogel. The P(NAGA)-P(NAGA-VPA) gradient structure hydrogel was subjected to a first immersion treatment in a calcium-phosphorus mixed aqueous solution, a second immersion treatment in ammonia water, a aging and stabilization treatment in a calcium chloride aqueous solution, and a third immersion treatment in water to obtain a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel; the Ca / P molar ratio in the calcium-phosphorus mixed solution was 1.67.

[0023] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0024] This invention involves mixing N-acrylylglycamide, a first photoinitiator, and water to obtain an aqueous NAGA solution. In this invention, the mass concentration of the first N-acrylylglycamide in the NAGA aqueous solution can be 25-30%, specifically 30%. In this invention, the first photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). The LAP used in this invention is water-soluble and low in toxicity, and the prepared material can be used for bone and cartilage repair. In this invention, the mass of the first photoinitiator can be 0.3-0.5% of the mass of N-acrylylglycamide, specifically 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%.

[0025] This invention involves mixing N-acrylylglycine, vinyl phosphoric acid, a second photoinitiator, and water to obtain an aqueous solution of NAGA-VPA. In this invention, the mass concentration of the second N-acrylylglycine in the NAGA-VPA aqueous solution can be 25-30%, specifically 30%. In this invention, the mass ratio of the second N-acrylylglycine to vinyl phosphoric acid can be 6-12:1, specifically 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In this invention, the second photoinitiator may include lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP). In this invention, the mass of the second photoinitiator can be 0.3-0.5% of the total mass of N-acrylylglycine and vinyl phosphoric acid, specifically 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%.

[0026] After obtaining the NAGA aqueous solution and the NAGA-VPA aqueous solution, the present invention performs a first DLP printing on the NAGA aqueous solution to obtain several P(NAGA) layers, and performs a second DLP printing on the surface of the P(NAGA) layers with the NAGA-VPA aqueous solution to obtain a P(NAGA)-P(NAGA-VPA) gradient structure hydrogel.

[0027] In this invention, the printing parameters for the first and second DLP printing can independently include: a bottom layer exposure time of 10000~12000ms, specifically 10000ms, 10500ms, 11000ms, 11500ms, or 12000ms; an exposure time per layer of 8000~10000ms, specifically 8000ms, 8500ms, 9000ms, 9500ms, or 10000ms; a wavelength of 405nm; and a slice thickness (thickness per layer) of 0.05~0.1mm, specifically 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0.09mm, or 0.1mm. In this invention, the first and second DLP printing can include digital light processing photopolymerization 3D printing.

[0028] In this invention, the number of P(NAGA) layers can be 5 to 100, specifically 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 layers.

[0029] In this invention, the number of P(NAGA-VPA) layers can be 5 to 100, specifically 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 layers.

[0030] After obtaining the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel, the present invention places the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel in a calcium-phosphorus mixed aqueous solution for a first immersion treatment, in ammonia water for a second immersion treatment, in calcium chloride aqueous solution for aging and stabilization, and in water for a third immersion treatment to obtain a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel; the Ca / P molar ratio in the calcium-phosphorus mixed solution is 1.67.

[0031] In this invention, the calcium-phosphorus aqueous solution comprises calcium chloride, phosphoric acid, and water. The concentration of calcium chloride in the calcium-phosphorus aqueous solution can be 0.5~1 mol / L, specifically 0.594 mol / L, 0.792 mol / L, or 0.990 mol / L; the concentration of phosphoric acid can be 0.3~0.6 mol / L, specifically 0.356 mol / L, 0.474 mol / L, or 0.593 mol / L. The Ca / P molar ratio in the calcium-phosphorus aqueous solution is 1.67. In this invention, the mass ratio of the P(NAGA)-P(NAGA-VPA) gradient hydrogel to the calcium-phosphorus aqueous solution can be 1:25~30, specifically 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30. In this invention, the temperature of the first soaking treatment can be 18~30℃; the time of the first soaking treatment is 24 hours.

[0032] In this invention, the mass concentration of the ammonia water can be 15%. In this invention, the mass ratio of the P(NAGA)-P(NAGA-VPA) gradient hydrogel to the ammonia water can be 1:25~30, specifically 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30. The temperature of the second immersion treatment can be 18~30℃; the time of the second immersion treatment can be 4 hours.

[0033] In this invention, the concentration of the calcium chloride aqueous solution is 0.75 mol / L. In this invention, the mass ratio of the P(NAGA)-P(NAGA-VPA) gradient hydrogel to the calcium chloride aqueous solution can be 1:25~30, specifically 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30. In this invention, the aging and stabilization temperature is 18~30℃; the aging and stabilization time is 24 h.

[0034] In this invention, the mass ratio of the P(NAGA)-P(NAGA-VPA) gradient hydrogel to the water used in the third immersion treatment can be 1:25~30, specifically 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30; the water can include one or more of deionized water, tap water, and purified water. In this invention, the temperature of the third immersion treatment is 18~30℃; the duration of the third immersion treatment can be 3~7 days, specifically 3 days.

[0035] This invention is based on a high-strength supramolecular N-acryloylglycine and vinyl phosphate copolymer system. Using DLP multi-material printing technology, NAGA aqueous solution and NAGA-VPA aqueous solution are printed into a gradient-structured hydrogel. This hydrogel is then sequentially immersed in a calcium-phosphorus mixed aqueous solution (Ca / P=1.67), ammonia, calcium chloride aqueous solution, and deionized water to achieve in-situ gradient deposition and maturation of hydroxyapatite (HAP). The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared by this invention possesses a continuous biochemical-mechanical gradient. The surface low-modulus region matches cartilage properties, the deep high-mineralization region integrates bone tissue, and the HAP crystallinity in the transition region exhibits a spatially dependent distribution. This makes it suitable for repairing osteochondral defects and manufacturing biomimetic joint prostheses.

[0036] This invention also provides a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by the preparation method described above. The multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by this invention (such as...) Figure 2 As shown, there is a clear boundary.

[0037] The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared in this invention utilizes DLP printing technology (especially DLP multi-material printing technology) combined with VPA-Ca 2+ The coordination mineralization mechanism successfully reproduced the three-layered gradient structure of natural osteochondral.

[0038] The multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel prepared by this invention has a surface region that can form a fluid lubrication layer with high water content to match cartilage performance, a deep high-mineralization zone that integrates bone tissue to induce osteogenic regeneration, and a transition zone that effectively disperses interfacial stress. This avoids the risk of sudden soft-hard transitions in traditional bilayer materials and achieves an integrated function of "flexible lubrication - gradient transition - rigid support".

[0039] The multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by this invention can complete HAP gradient deposition in a short time (within 2 days) through a step mineralization process of calcium phosphorus infiltration → ammonia water triggering → calcium ion ripening.

[0040] This invention also provides the application of the multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogel described above in the preparation of biological soft tissue substitutes. In this invention, the biological soft tissue substitute may include osteochondral defect repair bodies or biomimetic joint prostheses.

[0041] In this invention, the difference between the preparation method of the biological soft tissue substitute and the preparation method of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel is only that the mold corresponding to the biological soft tissue substitute is used in the 3D printing process.

[0042] In this invention, the shape of the biological soft tissue substitute can be as follows: Figure 3 As shown.

[0043] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel, its preparation method, and its application, is provided but should not be construed as limiting the scope of protection of the present invention.

[0044] In the following embodiments, unless otherwise specified, the ambient temperature is room temperature (18~30℃). The tensile breaking strength test method is a uniaxial tensile test, referring to ISO 527. A dumbbell-shaped specimen is stretched at a constant tensile speed until fracture; the maximum stress is the breaking strength. The breaking elongation test method is to record the change in length of the specimen at fracture during the tensile test, calculated as (gauge length at fracture - original gauge length) / original gauge length × 100%. The elastic modulus test method is to calculate the ratio of stress increment to strain increment in the initial linear elastic stage of the stress-strain curve during the tensile test, i.e., the slope of the straight line segment. The compressive strength test method is a compression test, referring to ISO 604. A cylindrical specimen is subjected to a compressive load at a constant speed until a specific deformation is achieved, and the maximum stress is recorded. The compressive modulus test method is to calculate the ratio of stress increment to strain increment in the initial linear segment of the stress-strain curve during the compression test, which is taken as the compressive elastic modulus.

[0045] Example 1 Photoinitiators LAP, NAGA, and VPA were dissolved in water to obtain a NAGA-VPA aqueous solution. This solution was poured into a square mold and cured using light at a wavelength of 405 nm to obtain a P(NAGA-VPA) 9:1 hydrogel. The mass ratio of LAP to (NAGA+VPA) was 0.3%, the mass ratio of NAGA to VPA was 9:1, and the total concentration of NAGA and VPA in the NAGA-VPA aqueous solution was 30 wt%.

[0046] The tensile breaking strength of the P(NAGA-VPA) 9:1 hydrogel prepared in this embodiment is 0.56 MPa, the elongation at break is 477.7%, and the elastic modulus is 0.071 MPa.

[0047] Example 2 P(NAGA-VPA) 9:1 hydrogel was prepared according to Example 1. The P(NAGA-VPA) 9:1 hydrogel was immersed in a calcium-phosphorus mixed aqueous solution (Ca / P molar ratio = 1.67) for 24 h, then transferred to 15 wt% ammonia water for 4 h, matured in a 0.75 mol / L CaCl2 aqueous solution for 24 h, and then immersed in deionized water for 3 days to remove excess calcium ions, yielding P(NAGA-VPA) 9:1 HAP3X hydrogel. The calcium-phosphorus mixed aqueous solution had a CaCl2 concentration of 0.594 mol / L. The mass ratio of P(NAGA-VPA) 9:1 hydrogel, calcium-phosphorus mixed aqueous solution, ammonia water, CaCl2 aqueous solution, and deionized water (for immersion) was 1:30:30:30.

[0048] The tensile breaking strength of the P(NAGA-VPA)9:1HAP3X hydrogel prepared in this embodiment is 0.76±0.021MPa, the elongation at break is 679.9±13.58%, and the elastic modulus is 0.22±0.0066MPa.

[0049] Example 3 The only difference from Example 2 is that the CaCl2 concentration in the calcium-phosphorus mixed aqueous solution (Ca / P molar ratio = 1.67) is 0.792 mol / L, resulting in P(NAGA-VPA)9:1HAP4X hydrogel.

[0050] The tensile breaking strength of the P(NAGA-VPA)9:1HAP4X hydrogel prepared in this embodiment is 0.94±0.023MPa, the elongation at break is 752.4±22.5%, and the elastic modulus is 0.43±0.0065MPa.

[0051] Example 4 The only difference from Example 2 is that the CaCl2 concentration in the calcium-phosphorus mixed aqueous solution (Ca / P molar ratio = 1.67) is 0.990 mol / L; thus, P(NAGA-VPA)9:1HAP5X hydrogel is obtained.

[0052] The tensile breaking strength of the P(NAGA-VPA)9:1HAP5X hydrogel prepared in this embodiment is 0.99±0.04MPa, the elongation at break is 777.4±26.16%, and the elastic modulus is 0.41±0.0156MPa.

[0053] Figure 4 The tensile properties of the P(NAGA-VPA) hydrogels prepared in Examples 1-4 were determined by... Figure 4It can be seen that the tensile properties of P(NAGA-VPA) hydrogel gradually increase with the increase of calcium and phosphorus concentration in the calcium-phosphorus mixed aqueous solution. The P(NAGA-VPA)9:1HAP5X hydrogel has the best performance. Since the properties of P(NAGA-VPA)9:1HAP4X hydrogel and P(NAGA-VPA)9:1HAP5X hydrogel are basically the same, a calcium-phosphorus mixed aqueous solution with a CaCl2 concentration of 0.792 mol / L and a Ca / P molar ratio of 1.67 is selected as the soaking solution.

[0054] Example 5 The only difference from Example 3 is that the mass ratio of NAGA to VPA is 6:1, resulting in P(NAGA-VPA)6:1HAP4X hydrogel.

[0055] The tensile breaking strength of the P(NAGA-VPA)6:1HAP4X hydrogel prepared in this embodiment is 0.81±0.029MPa, the elongation at break is 699.3±81.9%, and the elastic modulus is 0.3±0.0136MPa.

[0056] Example 6 The only difference from Example 3 is that the mass ratio of NAGA to VPA is 12:1, resulting in P(NAGA-VPA)12:1HAP4X hydrogel.

[0057] The tensile breaking strength of the P(NAGA-VPA)12:1HAP4X hydrogel prepared in this embodiment is 0.89±0.05MPa, the elongation at break is 701±39.43%, and the elastic modulus is 0.10±0.015MPa. Figure 5 The tensile properties of the P(NAGA-VPA) hydrogels prepared in Examples 1, 3, and 5-6 were determined by... Figure 5 It can be seen that the tensile properties of P(NAGA-VPA) hydrogel gradually increase with the decrease of VPA content, and the optimal mass ratio of NAGA:VPA is 9:1.

[0058] Example 7 Photoinitiator LAP and NAGA were dissolved in water to obtain an aqueous NAGA solution. The mass ratio of LAP to NAGA was 0.3%, and the concentration of NAGA in the aqueous NAGA solution was 30 wt%.

[0059] Photoinitiators LAP, NAGA, and VPA were dissolved in water to obtain a NAGA-VPA aqueous solution. The mass ratio of LAP to (NAGA + VPA) was 0.3%, the mass ratio of NAGA to VPA was 9:1, and the total concentration of NAGA and VPA in the NAGA-VPA aqueous solution was 30 wt%.

[0060] A NAGA aqueous solution was 3D printed with 100 layers of P(NAGA) (top layer) using a 405nm wavelength DLP printing system. A NAGA-VPA aqueous solution was then 3D printed with 100 layers of P(NAGA-VPA) (bottom layer) on the surface of the P(NAGA) layers using the same 405nm wavelength DLP printing system, resulting in a P(NAGA)-P(NAGA-VPA) gradient hydrogel structure. The 3D printing parameters were as follows: the bottom layer exposure time was 10000ms, the exposure time for each layer was 8000ms, and the height of each single P(NAGA) layer was 0.1mm.

[0061] The P(NAGA)-P(NAGA-VPA) gradient hydrogel was soaked in a calcium-phosphorus mixed aqueous solution for 24 hours, then transferred to 15wt% ammonia water for 4 hours, matured in 0.75mol / L CaCl2 aqueous solution for 24 hours, and soaked in deionized water for 3 days to remove excess calcium ions, resulting in a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel (denoted as 100P(NAGA)-100P(NAGA-VPA)HAP). The calcium-phosphorus mixed aqueous solution had a CaCl2 concentration of 0.792mol / L and a Ca / P molar ratio of 1.67. The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient hydrogel, the calcium-phosphorus mixed aqueous solution, ammonia water, CaCl2 aqueous solution, and deionized water (for soaking) was 1:30:30:30.

[0062] The tensile fracture strength of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel prepared in this embodiment is 0.93 MPa, the elongation at break is 796.3%, and the elastic modulus is 0.095 MPa.

[0063] Figure 6 The interfacial tensile properties of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel prepared in Example 7 were determined by... Figures 5-6 It can be seen that in Example 7, the bonding strength between P(NAGA) hydrogel and P(NAGA-VPA)HAP mineralized hydrogel is close to the tensile properties of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel, which meets the requirements of integrated hydrogel.

[0064] Example 8 The only difference from Example 7 is that the number of P(NAGA) layers is 5, and the number of P(NAGA-VPA) layers is 35.

[0065] In this embodiment, the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel (denoted as 5P(NAGA)-35P(NAGA-VPA)HAP) exhibits a compressive strength of 0.12±0.02MPa and a compressive modulus of 0.064±0.00185MPa under a compression of 50%, and the friction coefficient of the P(NAGA) layer is 0.314±0.0256.

[0066] Example 9 The only difference from Example 7 is that the number of P(NAGA) layers is 10, and the number of P(NAGA-VPA) layers is 30.

[0067] The multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel (denoted as 10P(NAGA)-30P(NAGA-VPA)HAP) prepared in this embodiment has a compressive strength of 0.14±0.0049MPa and a compressive modulus of 0.094±0.013MPa under a compression of 50%, and the friction coefficient of the P(NAGA) layer is 0.345±0.0169.

[0068] Example 10 The only difference from Example 7 is that the number of P(NAGA) layers is 15 and the number of P(NAGA-VPA) layers is 25.

[0069] The multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel (denoted as 15P(NAGA)-25P(NAGA-VPA)HAP) prepared in this embodiment has a compressive strength of 0.16±0.014MPa and a compressive modulus of 0.084±0.011MPa under a compression of 50%, and the friction coefficient of the P(NAGA) layer is 0.365±0.00105.

[0070] Example 11 The only difference from Example 7 is that the number of P(NAGA) layers is 20 and the number of P(NAGA-VPA) layers is 20.

[0071] The multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel (denoted as 20P(NAGA)-20P(NAGA-VPAHAP)) prepared in this embodiment has a compressive strength of 0.185±0.015MPa and a compressive modulus of 0.13±0.0073MPa under a compression of 50%, and the friction coefficient of the P(NAGA) layer is 0.389±0.0114.

[0072] Figure 2These are physical images of the multi-material 3D-printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11, where a is Example 8, b is Example 9, c is Example 10, and d is Example 11. Figure 2 It can be seen that the multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by the present invention has obvious boundaries.

[0073] Figure 7 The compressibility of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11 was determined by... Figure 7 It can be seen that the compressibility of multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels with different layer heights increases progressively, indicating that mineralization is effective in modifying hydrogels.

[0074] Figure 8 The lubrication properties of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels prepared in Examples 8-11 were determined by... Figure 8 It can be seen that the lubrication performance of multi-material 3D printed biomimetic gradient soft-hard integrated hydrogels with different layer heights increases progressively, indicating that gradient soft-hard integration is effective.

[0075] Figure 9 This is a scanning electron microscope (SEM) image of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel prepared in Example 11, consisting of an upper P(NAGA) layer and a lower P(NAGA-VPA)HAP layer. Figure 9 It can be seen that there is a boundary in the middle of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel.

[0076] Figure 10 This is a scanning electron microscope (SEM) image of the P(NAGA)-P(NAGA-VPA) HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel P(NAGA) layer prepared in Example 11. Figure 10 It can be seen that the P(NAGA) layer of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel does not show obvious mineralization.

[0077] Figure 11 This is a scanning electron microscope (SEM) image of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel P(NAGA-VPA)HAP layer prepared in Example 11. Figure 11It can be seen that the P(NAGA)-VPA)HAP layer of the P(NAGA)-P(NAGA-VPA)HAP multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel has obvious mineralization phenomenon.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel, characterized in that, Includes the following steps: N-Acryloylglycine, a first photoinitiator, and water were mixed to obtain an aqueous solution of NAGA; N-Acryloylglycine, vinyl phosphoric acid, a second photoinitiator, and water were mixed to obtain an aqueous solution of NAGA-VPA; The NAGA aqueous solution is subjected to a first DLP printing to obtain several P(NAGA) layers. The NAGA-VPA aqueous solution is then subjected to a second DLP printing on the surface of the P(NAGA) layers to form several P(NAGA-VPA) layers, resulting in a P(NAGA)-P(NAGA-VPA) gradient structure hydrogel. The P(NAGA)-P(NAGA-VPA) gradient structure hydrogel was subjected to a first immersion treatment in a calcium-phosphorus mixed aqueous solution, a second immersion treatment in ammonia water, a aging and stabilization treatment in a calcium chloride aqueous solution, and a third immersion treatment in water to obtain a multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel; the Ca / P molar ratio in the calcium-phosphorus mixed solution was 1.

67.

2. The preparation method according to claim 1, characterized in that, The mass concentration of the first N-acryloylglycine in the NAGA aqueous solution is 25-30%; The mass of the first photoinitiator is 0.3~0.5% of the mass of N-acryloylglycamide; The first photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

3. The preparation method according to claim 1, characterized in that, The mass concentration of the second N-acryloylglycine in the NAGA-VPA aqueous solution is 25-30%; The mass ratio of the second N-acrylglycolamide to vinyl phosphoric acid is 6~12:1; The mass of the second photoinitiator is 0.3-0.5% of the total mass of the second N-acryloylglycine and vinyl phosphate; The second photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

4. The preparation method according to claim 1, characterized in that, The printing parameters for the first DLP printing and the second DLP printing independently include: the bottom layer exposure time is 10000-12000ms, the exposure time for each layer is 8000-10000ms, the wavelength is 405nm, and the slice thickness is 0.05mm-0.1mm. The number of layers in the P(NAGA) layer is 5 to 100. The P(NAGA-VPA) layer has 5 to 100 layers.

5. The preparation method according to claim 1, characterized in that, In the mixed reaction solution, calcium ions are derived from calcium chloride, and their concentration can be 0.5~1 mol / L; phosphate ions are derived from phosphoric acid, and their concentration can be 0.3-0.6 mol / L; the molar ratio of calcium ions to phosphate ions (Ca / P) is 1.

67. The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to the calcium-phosphorus mixed solution is 1:25~30; The temperature of the first soaking treatment is 18~30℃, and the time is 24h.

6. The preparation method according to claim 1, characterized in that, The mass concentration of the ammonia solution is 15%. The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to ammonia is 1:25~30; The second soaking treatment is carried out at a temperature of 18~30℃ for 4 hours.

7. The preparation method according to claim 1, characterized in that, The concentration of the calcium chloride aqueous solution is 0.75 mol / L; The mass ratio of the P(NAGA)-P(NAGA-VPA) gradient structure hydrogel to the calcium chloride aqueous solution is 1:25~30; The aging and stabilization temperature is 18~30℃, and the time is 24h; The third soaking treatment is carried out at a temperature of 18~30℃ for 3~7 days.

8. The multi-material 3D printing biomimetic gradient soft-hard integrated hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the multi-material 3D printed biomimetic gradient soft-hard integrated hydrogel as described in claim 8 in the preparation of biological soft tissue substitutes.

10. The application according to claim 9, characterized in that, The biological soft tissue substitutes include osteochondral defect repairs or biomimetic joint prostheses.

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