An anisotropic supramolecular polymer hydrogel-based meniscus scaffold and its preparation method

Abstract

This invention discloses an anisotropic supramolecular polymer hydrogel-based meniscus scaffold and its preparation method. N-acryloylurea is dissolved in a solvent and polymerization is initiated to obtain poly(N-acryloylurea). Pre-stretching and solvent displacement are then performed to obtain a pre-stretched hydrogel. 3D printing ink is formulated based on NNASC (N-acryloylglycine) and printed according to a pre-designed meniscus model with radially and circumferentially arranged fibers to obtain a PNASC meniscus structure. A pre-loaded meniscus skeleton is obtained through pre-loading and solvent displacement and placed in a meniscus-shaped mold. An aqueous solution containing N-acryloylglycine and an initiator is added to initiate polymerization. This invention constructs a scaffold with a simulated collagen fiber orientation structure, performs pre-loading and hydrogen bond reconstruction, and fills the skeleton with an elastic, hydrogen-crosslinked supramolecular polymer hydrogel, simulating the buffering and water-retention functions of glycosaminoglycans. This achieves the simulation of the biological structure and mechanics of the natural meniscus and has application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically to an anisotropic supramolecular polymer hydrogel-based meniscus scaffold and its preparation method. Background Technology

[0002] Meniscus tears are a common type of meniscus injury. When an irreparable meniscus tear occurs, partial or total meniscectomy is the gold standard for relieving pain and slowing cartilage degeneration. However, after meniscectomy, the contact stress within the knee joint increases dramatically, accelerating the progression of osteoarthritis. To address this challenge, meniscus replacements have been developed in hopes of restoring the biomechanics of the knee joint. However, existing meniscus replacements on the market are limited by their homogeneous structure and insufficient biomechanical properties, making it difficult to achieve long-term restoration of meniscus function.

[0003] The combination of 3D printing technology and high-strength hydrogels is expected to overcome the above challenges. 3D printing technology can accurately reproduce the structure and function of various biological tissues. The 3D printing of high-strength hydrogels is of great significance for simulating the anisotropic structure and excellent comprehensive mechanical properties of mechanically supported tissues. Summary of the Invention

[0004] This invention overcomes the shortcomings of existing technologies. Most meniscus substitutes are limited by homogeneous structures and insufficient biomechanical properties, making it difficult to achieve long-term recovery of meniscus function. This invention proposes an anisotropic supramolecular polymer hydrogel-based meniscus scaffold and its preparation method. The proposed meniscus scaffold simultaneously simulates the anisotropic structure and function of the natural meniscus. Rigid hydrogel fibers are 3D printed to simulate the radial and circumferential orientation structure of collagen fibers. After "preloading-hydrogen bond reconstruction", an anisotropic meniscus skeleton is obtained. After filling with highly elastic flexible hydrogel, the scaffold is endowed with energy dissipation function, which initially confirms its application potential in constructing meniscus tissue substitutes.

[0005] The objective of this invention is achieved through the following technical solution.

[0006] An anisotropic supramolecular polymer hydrogel-based meniscus scaffold and its preparation method are disclosed, comprising the following steps:

[0007] Step 1: According to the pre-designed meniscus model with radially and circumferentially arranged fibers, 3D printing ink is printed using DLP 3D printing technology to construct the polyN-acryloylurea meniscus structure; in the 3D printing ink, the mass percentage of N-acryloylurea is 10-30wt%, the mass percentage of the initiator is 0.1-0.5wt%, and the mass percentage of the light blocker is 0.05-0.2wt%;

[0008] In step 1, the mass percentage of N-acryloylurea is 30 wt%, the mass percentage of the initiator is 0.25-0.5 wt%, and the mass percentage of the light blocker is 0.05-0.15 wt%.

[0009] In step 1, the light blocker is lemon yellow.

[0010] In step 1, the initiator is a photoinitiator, such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or 2-hydroxy-2-methylphenylacetone (1173).

[0011] In step 1, the solvent in the 3D printing ink is a mixture of dimethyl sulfoxide and water in a volume ratio of 7:3.

[0012] In step 1, DLP 3D printing technology (such as the DLP 3D printer EFL-BP8600) is used for 3D printing. While printing, polymerization is initiated, and a light-blocking agent is added to control the degree of polymerization.

[0013] Step 2: Preloading-solvent replacement is performed on the polyN-acryloylurea meniscus structure obtained in Step 1.

[0014] A poly(N-acryloylurea) meniscus structure was preloaded using a loading head. After orientation, it was immersed in deionized water for solvent displacement to rebuild hydrogen bonds and remove unreacted impurities, thus constructing a preloaded poly(N-acryloylurea) meniscus framework.

[0015] In step 2, the preloading time is 20-24 hours.

[0016] In step 2, the strain induced by preloading is 50%–150%.

[0017] Step 3: The preloaded poly(N-acryloylurea) meniscus skeleton is surface-treated with an ethanol solution of benzophenone, followed by rinsing the skeleton with ethanol and air drying.

[0018] In step 3, the concentration of benzophenone is 5-15 wt%, and the treatment time is 1-10 min.

[0019] In step 3, the concentration of benzophenone is 10-15 wt%, and the treatment time is 5-10 min.

[0020] Step 4: The preloaded poly(N-acrylamide) meniscus skeleton obtained in Step 3 is placed in a meniscus-shaped mold. An aqueous solution containing N-acrylamide and an initiator is added to the mold to initiate the free radical polymerization reaction of N-acrylamide to obtain poly(N-acrylamide). After dialysis to remove unreacted impurities, an anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) is obtained. In the aqueous solution containing N-acrylamide and an initiator, the mass percentage of N-acrylamide is 10-30 wt%, and the amount of initiator is 0.5-2 wt% of the monomer concentration (i.e., the mass of N-acrylamide).

[0021] In step 4, the initiator is a photoinitiator, such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or 2-hydroxy-2-methylphenylacetone (1173).

[0022] In step 4, free radical polymerization is initiated under ultraviolet light for 30-60 minutes, preferably 40-60 minutes.

[0023] min.

[0024] In step 4, the mass percentage of N-acryloylglycine is 30 wt%, and the amount of initiator is 1-2 wt% of the monomer concentration.

[0025] In step 4, the preloaded polyN-acrylamide meniscus skeleton is placed in a meniscus-shaped mold. An aqueous solution containing N-acrylamide and an initiator is added to the mold to fill the pores of the meniscus skeleton. After polymerization is initiated, a preloaded polyN-acrylamide meniscus skeleton embedded with polyN-acrylamide is obtained. The entire scaffold is dialyzed with deionized water to remove unreacted impurities, and then the pl-PNASC / PNAGA scaffold obtained by combining the two is obtained.

[0026] The meniscus solution of the present invention is based on an improved preloading-solvent replacement scheme for poly(N-acryloylurea) (PNASC) hydrogels, through which orientation and hydrogen bond reconstruction are expected to be formed in the hydrogel.

[0027] The poly(N-acryloylurea) hydrogel was preloaded to induce and maintain strain, thus forming an orientation. It was then immersed in deionized water for solvent displacement to rebuild hydrogen bonds and remove unreacted impurities, resulting in the preloaded poly(N-acryloylurea) hydrogel.

[0028] With preloading, the strain of the poly(N-acryloylurea) hydrogel is 50%-150%, and the strain is maintained for 20-24 hours.

[0029] N-Acryloylurea was dissolved in a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3), and a photoinitiator, such as 2-hydroxy-2-methylphenylacetone (1173), was added to form a polymerization solution. After complete dissolution and deoxygenation, the solution was poured into a mold and free radical polymerization was initiated under ultraviolet light to obtain poly(N-acryloylurea) (PNASC)DMSO / hydrogel. The mass percentage of N-acryloylurea in the polymerization solution was 10-30 wt%, the mass percentage of 1173 in the polymerization solution was 0.5-2 wt%, and the ultraviolet irradiation time was 30-60 min.

[0030] In this field, the preloading / prestretching method is generally only applicable to gels composed of rigid or semi-rigid molecular chains. This is because rigid molecular chains are more likely to respond to external forces and become oriented, and are not suitable for preparing anisotropic hydrogels composed of ordinary flexible chains. This is because after the preloading force is removed, the straightened oriented polymer chains tend to revert to a random coil conformation due to entropic elasticity, and also lack an effective mechanism to freeze orientation, thus losing their oriented structure. Therefore, the orientation and fixation of flexible molecular chains has always been a challenge in constructing anisotropic hydrogels. The PNASC molecular chain used in this invention is a flexible molecular chain. The amide and urea groups on its side groups can form high-density hydrogen bonds. These high-density hydrogen bonds can act as reinforcing factors to strongly fix the orientation of the molecular chain. The molecular chain does not require a rigid structure or potentially toxic ionic crosslinking, and is not limited to crystalline polymers. The PNASC molecular chain, with the above advantages, can achieve the preparation of anisotropic hydrogels through a simple "preloading + solvent replacement" strategy, and this strategy is used to construct meniscus substitutes.

[0031] The present invention has a simple formulation and an easy preparation method. On the one hand, 3D printing of high-rigidity hydrogen-bonded cross-linked supramolecular polymer hydrogel is used to simulate the radial and circumferential orientation structure of collagen fibers, and the "preloading-hydrogen bond reconstruction" strategy further enhances its mechanical strength and anisotropy. On the other hand, elastic hydrogen-bonded cross-linked supramolecular polymer hydrogel is filled into the skeleton to simulate the buffering and water-retaining functions of glycosaminoglycans, thereby obtaining an anisotropic biomimetic meniscus substitute. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation process of the present invention, wherein (a) is the preparation process of pre-stretched PNASC hydrogel, and (b) is the preparation process of anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA).

[0033] Figure 2These are scanning electron microscope images of PNASC hydrogels without pre-stretching and with pre-stretching in this invention. Among them, (a) is the SEM of PNASC hydrogel without pre-stretching (scale bar: 5 μm), (b) is the SEM of 50% pre-stretched PNASC hydrogel (scale bar: 5 μm), and (c) is the SEM of 100% pre-stretched PNASC hydrogel (scale bar: 5 μm). The direction of the white arrow indicates the pre-stretching direction.

[0034] Figure 3 This is a statistical chart of the tensile strength and Young's modulus of PNASC hydrogel without pre-stretching, PNASC hydrogel with 50% pre-stretching, and PNASC hydrogel with 100% pre-stretching in this invention.

[0035] Figure 4 This is a photograph of the anisotropic supramolecular polymer hydrogel-based meniscus scaffold of this invention.

[0036] Figure 5 This is a load-displacement curve of the 3D printed meniscus skeleton scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) in the suture strength test of the present invention.

[0037] Figure 6 These are load-displacement curves of the 3D-printed meniscus skeleton scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) in this invention during 1000 suture fatigue tests, where (a) corresponds to pl-PNASC; and (b) corresponds to pl-PNASC / PNAGA.

[0038] Figure 7 This is a graph showing the final load change of the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) in this invention during 1000 cycles of compression testing.

[0039] Figure 8 These are cell activity diagrams of the 3D-printed meniscus scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) in this invention.

[0040] Figure 9 These are photographs of the implantation process of anisotropic supramolecular polymer hydrogel-based meniscus scaffold in a rabbit model during animal experiments of this invention.

[0041] Figure 10 These are gross observation images of the femoral condyle (FC), tibial plateau (TP), and meniscus scaffold in a rabbit model after implantation of an anisotropic supramolecular polymer hydrogel-based meniscus scaffold in animal experiments according to the present invention, at 4, 8, and 12 weeks.

[0042] Figure 11 These are histological evaluation images of the femoral condyle (FC) and tibial plateau (TP) after 4, 8, and 12 weeks of implantation of an anisotropic supramolecular polymer hydrogel-based meniscus scaffold in a rabbit model in animal experiments according to the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described below through specific embodiments.

[0044] The overall implementation strategy is as follows: Figure 1 As shown: N-acryloylurea (NASC) was dissolved in a mixed solvent of dimethyl sulfoxide (DMSO) and water. After adding the photoinitiator 2-hydroxy-2-methylphenylacetone (1173), the solution was fully dissolved and deoxygenated before being poured into a mold. Free radical polymerization was initiated under ultraviolet light to obtain poly(N-acryloylurea) (PNASC)DMSO / hydrogel. The gel was subjected to "pre-stretching-solvent displacement" and fully dialyzed in deionized water to obtain pre-stretched PNASC hydrogel. The pre-stretched hydrogel was tested and showed orientation properties. Furthermore, this feature was applied to the meniscus scaffold. NASC was dissolved in a mixed solvent of DMSO and water, and a photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and a light-blocking agent, lemon yellow, were added to prepare a 3D printing ink. Using digital light processing 3D printing technology, a pre-designed meniscus model with radially and circumferentially arranged fibers was printed to obtain a PNASC meniscus structure. Through a "preloading-solvent replacement" strategy, a preloaded PNASC meniscus skeleton (pl- Finally, the pl-PNASC meniscus skeleton was surface-treated with an ethanol solution of benzophenone, rinsed with ethanol and air-dried, and placed in a meniscus-shaped mold. An aqueous solution containing N-acryloylglycamide and photoinitiator 1173 was added to the mold, and a free radical polymerization reaction was initiated by ultraviolet light to obtain poly(N-acryloylglycamide) (PNAGA). After dialysis of the scaffold with deionized water, an anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) was obtained.

[0045] Model Design: NNASC ink was printed using an EFL-BP8600 digital light processing 3D printer. The 3D printed model design referenced Chinese patent "A 3D Printed Scaffold for Meniscus Replacement and Its Preparation Method" (Application No. 2022101338931, Application Date February 14, 2022). To construct a rigid hydrogen-bonded cross-linked supramolecular polymer hydrogel network as the scaffold framework, inspired by the microstructure of the meniscus, radially and circumferentially oriented PNASC fibers were 3D printed layer by layer to simulate the arrangement of collagen fibers in the natural meniscus. In the natural meniscus, due to the presence of circumferential and radial fibers, the axial compressive load from the femur and tibia is converted into circumferential tensile stress. Therefore, each layer of the model consists of a series of aligned radial fibers and a series of circumferentially oriented fibers. To create the wedge-shaped appearance of the meniscus, the number of fibers in each layer decreased with increasing model height. It is worth noting that the outer region of the meniscus has a higher collagen fiber density to resist circumferential tensile stress, while the inner region has a lower collagen fiber density to resist compressive loads. To simulate this characteristic, the fiber spacing (the center-to-center distance between two adjacent annular fibers) of the scaffold is also adjusted to a gradient mode in the model, i.e., denser on the outside and sparser on the inside. Therefore, a PNASC scaffold model with a fine microstructure is obtained through the above design.

[0046] Example 1

[0047] Step 1: Weigh 0.3g of N-acryloylurea (NASC) into a centrifuge tube using an analytical balance, add 1mL of a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3), vortex to completely dissolve, add 3μL of photoinitiator 2-hydroxy-2-methylphenylacetone (1173), purge with nitrogen for 10min to remove oxygen, and obtain a homogeneous mixed solution. After fully dissolving and removing oxygen, pour the solution into a mold and irradiate under ultraviolet light for 60min to initiate the free radical polymerization of NASC to obtain poly(N-acryloylurea) (PNASC)DMSO / hydrogel.

[0048] Step 2: Perform "pre-stretching-solvent replacement" on the PNASC DMSO / hydrogel from Step 1. Use a self-made fixture to pre-stretch the PNASC DMSO / hydrogel to 50% strain and hold for 24 hours. Then, immerse it in deionized water for 5 days to perform solvent replacement to rebuild hydrogen bonds and remove unreacted impurities. Change the deionized water once a day until swelling equilibrium is reached to obtain the pre-stretched PNASC 50% hydrogel.

[0049] Example 2

[0050] Step 1: Weigh 0.3g of N-acryloylurea (NASC) into a centrifuge tube using an analytical balance, add 1mL of a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3), vortex to completely dissolve, add 3μL of photoinitiator 2-hydroxy-2-methylphenylacetone (1173), purge with nitrogen for 10min to remove oxygen, and obtain a homogeneous mixed solution. After fully dissolving and removing oxygen, pour the solution into a mold and irradiate under ultraviolet light for 60min to initiate the free radical polymerization of NASC to obtain poly(N-acryloylurea) (PNASC)DMSO / hydrogel.

[0051] Step 2: Perform "pre-stretching-solvent replacement" on the PNASC DMSO / hydrogel from Step 1. Use a self-made fixture to pre-stretch the PNASC DMSO / hydrogel to 100% strain and hold for 24 hours. Then, immerse it in deionized water for 5 days to perform solvent replacement to rebuild hydrogen bonds and remove unreacted impurities. Change the deionized water once a day until swelling equilibrium is reached to obtain pre-stretched PNASC 100% hydrogel.

[0052] Example 3

[0053] Step 1: Weigh 0.3g of N-acryloylurea (NASC) into a centrifuge tube using an analytical balance, add 1mL of a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3), vortex to completely dissolve, add 3μL of photoinitiator 2-hydroxy-2-methylphenylacetone (1173), purge with nitrogen for 10min to remove oxygen, and obtain a homogeneous mixed solution. After fully dissolving and removing oxygen, pour the solution into a mold and irradiate under ultraviolet light for 60min to initiate the free radical polymerization of NASC to obtain poly(N-acryloylurea) (PNASC)DMSO / hydrogel.

[0054] Step 2: Immerse the PNASC DMSO / hydrogel from Step 1 in deionized water for 5 days to perform solvent replacement to rebuild hydrogen bonds and remove unreacted impurities. Change the deionized water once a day until swelling equilibrium is reached to obtain a PNASC hydrogel without pre-stretching as a control.

[0055] Characterization was performed on Examples 1-3 above—(1) Scanning electron microscopy (SEM) tests; (2) Mechanical tensile property tests. The surface morphology of the prepared unstretched PNASC, 50% prestretched PNASC, and 100% prestretched PNASC hydrogels was observed using a scanning electron microscope (Hitachi, Japan). SEM images of the hydrogels are shown below. Figure 2As shown, the surface of unstretched PNASC exhibits random morphology, while both 50% and 100% prestretched PNASC show oriented fiber structures along the prestretching direction. The tensile mechanical properties of the unstretched, 50% prestretched, and 100% prestretched PNASC hydrogels were tested using an electronic universal testing machine (Instron, USA). Rectangular tensile specimens (length: 30 mm, width: 4 mm, thickness: 1 mm) were cut and tensile tests were performed at a strain rate of 50 mm / min. The tensile strength and Young's modulus of the unstretched and prestretched PNASC hydrogels are shown in the figures below. Figure 3 As shown, pre-stretched PNASC hydrogels exhibit higher tensile strength and Young's modulus than unstretched PNASC hydrogels [(a) Tensile strength of unstretched, 50% prestretched, and 100% prestretched PNASC hydrogels; (b) Young's modulus of unstretched, 50% prestretched, and 100% prestretched PNASC hydrogels]. This indicates that pre-stretched PNASC hydrogels undergo molecular chain orientation under external force and rebuild hydrogen bonds during solvent displacement, resulting in higher mechanical properties. Regardless of whether parallel to ( / / ) or perpendicular to (⊥) the prestretching direction, 100% prestretched PNASC hydrogels exhibit higher tensile strength and Young's modulus than 50% prestretched PNASC hydrogels, indicating that higher prestretching strain leads to stronger mechanical reinforcement. Furthermore, pre-stretched hydrogels exhibit better mechanical properties parallel to the prestretching direction than perpendicular to it, indicating that pre-stretched hydrogels possess anisotropic mechanical properties.

[0056] The meniscus material was prepared and characterized using the above-mentioned pre-stretching-solvent replacement scheme as follows:

[0057] Example 4

[0058] Step 1: Weigh 0.3g of N-acryloylurea (NASC) into a centrifuge tube using an analytical balance. Add 1mL of a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3). Vortex to dissolve completely. Then add 5mg of photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and 1mg of light blocker lemon yellow. Purge with nitrogen for 10min to remove oxygen and obtain a homogeneous mixed solution. Prepare the digital light processing (DLP) 3D printing ink.

[0059] Step 2: Transfer the ink from Step 1 into the printer's ink tank. Using a DLP 3D printer (EFL-BP8600), print the ink according to a pre-designed meniscus model with radially and circumferentially arranged fibers to construct a poly(N-acryloylurea) (PNASC) meniscus structure. Input the STL file of the meniscus model designed using Pro / Engineer WildFire 5.0 software into the 3D printer's computer. Set the printing parameters (layer thickness: 50μm, layer exposure time: 5s) and begin printing. During printing, the ink is exposed to blue light (405nm, 10mW / cm²). 2 Irradiation triggers ink polymerization, and layer-by-layer printing yields a solidified gel, resulting in a PNASC meniscus-shaped structure.

[0060] Step 3: Using a "preloading-solvent replacement" strategy, the PNASC meniscus structure from Step 2 is preloaded for 24 hours using a loading head. Then, it is immersed in deionized water for 5 days for solvent replacement to rebuild hydrogen bonds and remove unreacted impurities. The deionized water is replaced daily until swelling equilibrium is reached to construct the preloaded PNASC meniscus skeleton (pl-PNASC).

[0061] Since the 3D-printed PNASC has a meniscus shape, a hemispherical loading head is used to apply a force of 26N and a displacement of 1.6mm for 24 hours for mechanical preloading. The compressive load applied by the loading head can be converted into a circumferential tensile load, thereby achieving the preloading effect. After 24 hours of preloading, the sample is placed in deionized water for solvent replacement to rebuild the hydrogen bonds in the network, thereby restoring its mechanical properties and obtaining the "preloaded PNASC meniscus skeleton" (pl-PNASC).

[0062] Step 4: Immerse the pl-PNASC meniscus stent from Step 3 in a 10wt% benzophenone (BP) ethanol solution for surface treatment. After 5 minutes, remove the stent, rinse the surface with ethanol, air dry, and place it in a meniscus-shaped mold.

[0063] Step 5: Weigh 0.03 g of N-acryloylglycine (NAGA) into a centrifuge tube using an analytical balance, add 100 μL of deionized water, vortex to dissolve completely, then add 0.3 μL of photoinitiator 2-hydroxy-2-methylphenylacetone (1173), purge with nitrogen for 10 min to remove oxygen, and obtain a homogeneous mixed solution. Then add this solution to the mold from Step 4, irradiate under ultraviolet light for 60 min to initiate the free radical polymerization reaction of NAGA to obtain poly(N-acryloylglycine) (PNAGA). Dialyze the obtained scaffold with deionized water for 5 days to remove unreacted impurities, and change the deionized water once a day until swelling equilibrium is reached to obtain an anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA), which is then stored under low temperature and sterile conditions.

[0064] Example 5

[0065] Step 1: Weigh 0.3g of N-acryloylurea (NASC) into a centrifuge tube using an analytical balance. Add 1mL of a mixed solvent of dimethyl sulfoxide and water (volume ratio 7:3). Vortex to dissolve completely. Then add 5mg of photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and 1mg of light blocker lemon yellow. Purge with nitrogen for 10min to remove oxygen and obtain a homogeneous mixed solution. Prepare the digital light processing (DLP) 3D printing ink.

[0066] Step 2: Transfer the ink from Step 1 to the printer ink tank, and print the ink using a DLP 3D printer EFL-BP8600 according to the pre-designed meniscus model with radially and circumferentially arranged fibers to construct a poly(N-acryloylurea) (PNASC) meniscus structure.

[0067] Step 3: Using a "preloading-solvent replacement" strategy, the PNASC meniscus structure from Step 2 was preloaded for 24 hours using a loading head. Subsequently, it was immersed in deionized water for 5 days for solvent replacement to rebuild hydrogen bonds and remove unreacted impurities. The deionized water was changed daily until swelling equilibrium was reached to construct the preloaded PNASC meniscus framework (pl-PNASC). The pl-PNASC meniscus framework was not filled as a control and was preserved under low-temperature aseptic conditions.

[0068] like Figure 4The images shown are of the anisotropic supramolecular polymer hydrogel-based meniscus scaffold of this invention, where (a) corresponds to the 3D-printed meniscus skeleton scaffold (pl-PNASC) (scale bar: 10 mm); and (b) corresponds to the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) (scale bar: 10 mm). Using the mechanical testing methods described in Examples 1-3 above, the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) underwent 1000 cyclic compression tests at a strain rate of 10 mm / min, with a maximum strain of 12%. Figure 7 As shown, during 1000 cycles of compression, the final load remained stable at around 3.6N, demonstrating the fatigue resistance of the pl-PNASC / PNAGA meniscus support.

[0069] The suture resistance of the scaffolds was tested using an electronic universal testing machine (Instron, USA). The suture pull-out strength of the 3D-printed meniscus skeleton scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) was tested using 4-0 sutures at a strain rate of 50 mm / min. The final failure load was defined as the suture pull-out strength, with a suture pass-through point 2 mm from the scaffold edge. To test the suture fatigue resistance of the meniscus scaffolds, the samples were placed in a water bath, and loads ranging from 2 to 5 N were applied at room temperature for 1000 tensile load-unload tests, ultimately leading to failure. The load-displacement curves for the suture pull-out tests of the 3D-printed meniscus skeleton scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) are shown below. Figure 5 As shown, the pl-PNASC / PNAGA exhibits a suture pull-out strength as high as 12.36 ± 0.20 N, higher than that of pl-PNASC (10.53 ± 0.32 N), and both are significantly higher than the suture pull-out strength of previously reported meniscal supports, indicating the excellent suture resistance of the pl-PNASC and pl-PNASC / PNAGA meniscal supports. Furthermore, in Figure 6 In the figures, (a) and (b) show that the pl-PNASC and pl-PNASC / PNAGA meniscal stents still exhibit high suture pull-out strengths, reaching 14.78 N and 14.92 N respectively, even after undergoing 1000 suture loading-unloading tests. This demonstrates the excellent suture fatigue resistance of the pl-PNASC and pl-PNASC / PNAGA stents, proving that the pl-PNASC and pl-PNASC / PNAGA meniscal stents can maintain accurate position and structural integrity under both static and dynamic loads.

[0070] Cytotoxicity assays—The CCK-8 assay kit was used to test the cytotoxicity of L929 mouse fibroblasts with 3D-printed meniscus scaffolds (pl-PNASC) and anisotropic supramolecular polymer hydrogel-based meniscus scaffolds (pl-PNASC / PNAGA). Figure 8 As shown, the cell viability results of L929 mouse fibroblasts on pl-PNASC and pl-PNASC / PNAGA meniscus scaffolds indicate that the scaffolds exhibit excellent cell compatibility and no cytotoxicity on both day 1 and day 3, and are expected to be further applied in vivo.

[0071] Animal experiments using a rabbit implantation model—Forty-two male rabbits weighing 2.5–3.0 kg were selected and subjected to experiments at 4, 8, and 12 weeks. All rabbits were randomly divided into four groups: sham surgery group (n=2 at each time point), meniscectomy group (n=4 at each time point), pl-PNASC group (experimental group, n=4 at each time point), and pl-PNASC / PNAGA group (experimental group, n=4 at each time point). The surgical procedure was as follows: Figure 9As shown, (a) corresponds to cutting the skin and fascia, (b) corresponds to cutting the medial collateral ligament and opening the knee joint capsule, (c) corresponds to meniscectomy, (d) corresponds to stent implantation, (e) corresponds to suturing the medial collateral ligament and closing the knee joint capsule, and (f) corresponds to suturing the skin and fascia. The meniscus support was soaked in 75% alcohol for one day and disinfected with ultraviolet light for one hour before surgery. All surgeries were performed under sterile conditions. Preoperative general anesthesia was administered via intravenous injection of 10% chloral hydrate (3.5 mL / kg) in the ear. Intraoperative local anesthesia was administered with seraphine hydrochloride (0.2 mL / kg). The rabbit was then fixed on the operating table, its leg hair was shaved, and the area was disinfected with iodine. Subsequently, a total medial meniscectomy was performed on both sides of the rabbit's knees. First, the skin and fascia were incised to form a 2 cm incision. Then, the medial collateral ligament was cut to expose the posterior horn of the medial meniscus. Subsequently, the joint capsule was opened and the meniscectomy was performed. The anterior and posterior horns of the meniscus were cut along the junction of the medial meniscus and the tibial plateau with a sharp scalpel. Then, the entire meniscus was removed. Afterward, through gross observation during the operation, a meniscus replacement of appropriate size was selected for implantation. The pl-PNASC / PNAGA and pl prepared in Examples 4 and 5 of this invention were used. - The PNASC meniscus stent was implanted into the knee joint, with its anterior horn sutured to the ligament and its posterior horn sutured to the joint capsule. The meniscus stent was horizontally fixed in the initial position of the medial meniscus. Rabbits with only the meniscus exposed were designated as the sham surgery group, while rabbits with meniscectomy were designated as the meniscectomy group. The medial collateral ligament was connected with non-absorbable 4-0 surgical sutures, the joint capsule was closed, and then the fascia and skin tissues were sutured. The surgical leg was cleaned with an alcohol pad. Finally, all rabbits were injected with penicillin for three days postoperatively to prevent infection. After the operation, the rabbits were returned to their cages and allowed free movement. The condition of the rabbit's knee joint and its activity were then observed and recorded. All rabbits were euthanized at 4, 8, and 12 weeks postoperatively, and their femurs, tibias, and the pl-PNASC and pl-PNASC / PNAGA meniscus stents were harvested for gross observation and histological evaluation to assess the articular cartilage degeneration and the articular cartilage protection effect of the stents.

[0072] Gross observations were performed on the aforementioned femoral condyle (FC) and tibial plateau (TP), as well as the pl-PNASC and pl-PNASC / PNAGA meniscal frameworks. Macroscopic photographs are shown below. Figure 10As shown, the group that only exposed the rabbit meniscus was designated as the sham surgery group, the group that only underwent meniscectomy was designated as the meniscectomy group, and the 3D-printed meniscus skeleton scaffold (pl-PNASC) group and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) group were the experimental groups. After meniscectomy of the medial side of both knees of rabbits, the 3D-printed meniscus skeleton scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscus scaffold (pl-PNASC / PNAGA) were implanted respectively. Scale bar: 10 mm.

[0073] In the meniscectomy group, large cracks and abnormal wear were observed on the femoral condyle and tibial plateau, which worsened cumulatively with prolonged implantation time. After 12 weeks, severe wear and matrix delamination were observed in some areas of the articular cartilage surface, even exposing the subchondral bone. This was due to the increased intra-articular contact stress following meniscectomy, which exacerbated cartilage wear and further accelerated the osteoarthritis process. In contrast, the implantation of the pl-PNASC meniscal scaffold alleviated wear on the femoral condyle and tibial plateau cartilage surface, reflected in the presence of only minor cracks and surface abrasion in the superficial areas of the cartilage. Furthermore, the position of the pl-PNASC meniscal scaffold remained unchanged throughout the 12 weeks of implantation, ensuring its effective load-bearing function. However, at 8 and 12 weeks post-implantation, the pNASC meniscal scaffold deformed, leading to a decrease in the articular cartilage contact area and an increase in contact stress, resulting in scattered cracks on the articular cartilage surface. In contrast, in the pl-PNASC / PNAGA implantation group, the femoral condyle and tibial plateau exhibited intact and smooth surfaces similar to the sham surgery group. Even 12 weeks after implantation, the articular cartilage surface showed only minor cracks and slight wear, exhibiting normal cartilage morphology without significant degenerative changes. Furthermore, the pl-PNASC / PNAGA meniscal scaffold did not deform or shift during implantation, maintaining accurate implantation position at all time points. In conclusion, the gross observations of the articular cartilage in the pl-PNASC / PNAGA meniscal scaffold group were closest to those in the sham surgery group, confirming that the pl-PNASC framework and PNAGA hydrogel respectively provide mechanical support and energy absorption, working together to protect the articular cartilage from wear.

[0074] Further microscopic histological evaluation of the aforementioned femoral condyle (FC) and tibial plateau (TP) was conducted, such as... Figure 11As shown, the cartilage degeneration of the femoral condyle and tibial plateau in all groups was assessed by hematoxylin-eosin (H&E) staining and safranin O-fast green (SOFG) staining. Among them, the group that only exposed the rabbit meniscus was designated as the sham surgery group, the group that only underwent meniscectomy was designated as the meniscectomy group, and the 3D-printed meniscal scaffold (pl-PNASC) group and the anisotropic supramolecular polymer hydrogel-based meniscal scaffold (pl-PNASC / PNAGA) group were the experimental groups. After meniscectomy of the medial side of both knees of rabbits, the 3D-printed meniscal scaffold (pl-PNASC) and the anisotropic supramolecular polymer hydrogel-based meniscal scaffold (pl-PNASC / PNAGA) were implanted respectively. Scale bar: 500 micrometers.

[0075] In the meniscectomy group, discontinuity of the matrix was observed in the superficial region of the femoral condyle and tibial plateau at 4 weeks postoperatively. However, vertical cracks appeared on the articular cartilage surface at 8 weeks postoperatively, and severe delamination and erosion of the surface matrix indicated increased wear and degeneration of the articular cartilage at 12 weeks. In contrast, at 4 weeks postoperatively, the femoral condyle and tibial plateau surface matrix in the pl-PNASC meniscus implantation group showed normal structure and an intact and continuous surface. However, at 8 and 12 weeks postoperatively, discontinuity and superficial fibrosis appeared on the articular cartilage surface due to the high rigidity of the pl-PNASC meniscus implantation group. Conversely, in the pl-PNASC / PNAGA meniscus implantation group, the articular cartilage surface showed an intact superficial matrix and normal cartilage morphology at all time points, most closely resembling the cartilage morphology of the sham surgery group. Based on the above results, the pl-PNASC / PNAGA meniscus brace can effectively protect the femoral condyle and tibial plateau from wear and tear, and delay the progression of osteoarthritis.

[0076] Adjusting the process parameters according to the present invention can achieve the preparation of the material of the present invention, and testing has shown that it exhibits performance substantially consistent with that of the present invention. The present invention has been described above as exemplary. It should be noted that any simple modifications, alterations, or other equivalent substitutions that can be made by those skilled in the art without creative effort, without departing from the core of the present invention, fall within the protection scope of the present invention.

Claims

1. An anisotropic supramolecular polymer hydrogel-based meniscus scaffold, characterized in that, Follow these steps: Step 1: Following a pre-designed meniscus model with radially and circumferentially arranged fibers, DLP 3D printing technology is used to print 3D printing ink to construct a polyN-acryloylurea meniscus structure. In the 3D printing ink, the mass percentage of N-acryloylurea is 10-30 wt%, the mass percentage of the initiator is 0.1-0.5 wt%, and the mass percentage of the light blocker is 0.05-0.2 wt%. The solvent in the 3D printing ink is a mixture of dimethyl sulfoxide and water, with a volume ratio of 7:

3. Step 2: Preloading and solvent replacement of the polyN-acryloylurea meniscus structure obtained in Step 1. The polyN-acryloylurea meniscus structure is preloaded using a loading head. After orientation, it is immersed in deionized water for solvent replacement to rebuild hydrogen bonds and remove unreacted impurities, thereby constructing the preloaded polyN-acryloylurea meniscus framework. Step 3: The preloaded poly(N-acryloylurea) meniscus skeleton is surface-treated with an ethanol solution of benzophenone, then rinsed with ethanol and air-dried. Step 4: The preloaded poly(N-acrylamide) meniscus skeleton obtained in Step 3 is placed in a meniscus-shaped mold. An aqueous solution containing N-acrylamide and an initiator is added to the mold to fill the pores of the meniscus skeleton, initiating the free radical polymerization reaction of N-acrylamide to obtain poly(N-acrylamide), i.e., the preloaded poly(N-acrylamide) meniscus skeleton embedded with poly(N-acrylamide). After dialysis to remove unreacted impurities, an anisotropic supramolecular polymer hydrogel-based meniscus scaffold is obtained. In the aqueous solution containing N-acrylamide and an initiator, the mass percentage of N-acrylamide is 10-30 wt%, and the amount of initiator is 0.5-2 wt% of the concentration of N-acrylamide.

2. The anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 1, characterized in that, In step 1, the mass percentage of N-acryloylurea is 30 wt%, the mass percentage of the initiator is 0.25-0.5 wt%, and the mass percentage of the light blocker is 0.05-0.15 wt%. The light blocker is lemon yellow, and the initiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate photoinitiator or 2-hydroxy-2-methylphenylacetone photoinitiator.

3. The anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 1, characterized in that, In step 2, the preloading time is 20-24 hours, and the strain induced by the preloading is 50%-150%; in step 3, the concentration of benzophenone is 5-15wt%, and the treatment time is 1-10min.

4. The anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 1, characterized in that, In step 4, the initiator is either lithium phenyl (2,4,6-trimethylbenzoyl) phosphate photoinitiator or 2-hydroxy-2-methylphenylacetone photoinitiator; free radical polymerization is initiated under ultraviolet light for 30-60 min; the mass percentage of N-acryloylglycamide is 30 wt%, and the amount of initiator is 1-2 wt% of the monomer concentration.

5. A method for preparing an anisotropic supramolecular polymer hydrogel-based meniscus scaffold, characterized in that, Follow these steps: Step 1: Following a pre-designed meniscus model with radially and circumferentially arranged fibers, DLP 3D printing technology is used to print 3D printing ink to construct a polyN-acryloylurea meniscus structure. In the 3D printing ink, the mass percentage of N-acryloylurea is 10-30 wt%, the mass percentage of the initiator is 0.1-0.5 wt%, and the mass percentage of the light blocker is 0.05-0.2 wt%. The solvent in the 3D printing ink is a mixture of dimethyl sulfoxide and water, with a volume ratio of 7:

3. Step 2: Pre-loading and solvent replacement of the polyN-acryloylurea meniscus structure obtained in Step 1. The polyN-acryloylurea meniscus structure is pre-loaded using a loading head. After orientation, it is immersed in deionized water for solvent replacement to rebuild hydrogen bonds and remove unreacted impurities, so as to construct the pre-loaded polyN-acryloylurea meniscus skeleton. Step 3: The preloaded poly(N-acryloylurea) meniscus skeleton is surface-treated with an ethanol solution of benzophenone, followed by rinsing the skeleton with ethanol and air drying. Step 4: The preloaded poly(N-acrylamide) meniscus skeleton obtained in Step 3 is placed in a meniscus-shaped mold. An aqueous solution containing N-acrylamide and an initiator is added to the mold to fill the pores of the meniscus skeleton, initiating the free radical polymerization reaction of N-acrylamide to obtain poly(N-acrylamide), i.e., the preloaded poly(N-acrylamide) meniscus skeleton embedded with poly(N-acrylamide). After dialysis to remove unreacted impurities, an anisotropic supramolecular polymer hydrogel-based meniscus scaffold is obtained. In the aqueous solution containing N-acrylamide and an initiator, the mass percentage of N-acrylamide is 10-30 wt%, and the amount of initiator is 0.5-2 wt% of the concentration of N-acrylamide.

6. The method for preparing an anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 5, characterized in that, In step 1, the mass percentage of N-acryloylurea is 30 wt%, the mass percentage of the initiator is 0.25-0.5 wt%, and the mass percentage of the light blocker is 0.05-0.15 wt%; the light blocker is lemon yellow; the initiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate photoinitiator or 2-hydroxy-2-methylphenylacetone photoinitiator.

7. The method for preparing an anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 5, characterized in that, In step 2, the preloading time is 20-24 hours, and the strain induced by the preloading is 50%-150%; in step 3, the concentration of benzophenone is 5-15wt%, and the treatment time is 1-10min.

8. The method for preparing an anisotropic supramolecular polymer hydrogel-based meniscus scaffold according to claim 5, characterized in that, In step 4, the initiator is either lithium phenyl (2,4,6-trimethylbenzoyl) phosphate photoinitiator or 2-hydroxy-2-methylphenylacetone photoinitiator; free radical polymerization is initiated under ultraviolet light for 30-60 min; the mass percentage of N-acryloylglycamide is 30 wt%, and the amount of initiator is 1-2 wt% of the monomer concentration.

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