Meniscus-like organ based on voxelization variable wire diameter printing and construction method

Abstract

The invention discloses a meniscus organ construction method based on voxelization variable wire diameter printing and an obtained product. The method comprises the following steps: acquiring target meniscus medical image data and establishing a voxelization model; printing parameters are mapped according to mechanical distribution; the PCL stent is constructed by adopting a variable-wire-diameter melt extrusion technology, fibers are laid in a 0-degree / 60-degree / 120-degree three-axis staggered manner, the wire diameter is continuously adjustable in a range of 150-350 microns, and the porosity is from 80% to 10% in an inner region to 10% in an outer region; tGF-beta functional NorCol ink is injected into the inner area through a multi-nozzle system, and CTGF functional NorCol ink is injected into the outer area; packaging the hUC-MSCs at the density of 2 * 10 < 6 > cells / mL, performing cartilage differentiation culture for 28 days after 405nm light in-situ crosslinking, and applying periodic mechanical loading during the period. The invention also relates to the prepared meniscus organ and application of the meniscus organ in preparation of a graft. According to the scheme, continuous mechanical gradient reconstruction and regional biochemical microenvironment construction are realized, and the problems of insufficient mechanical bionics and limited regional induction in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and additive manufacturing technology, and in particular provides a meniscus organoid based on voxelization variable filament printing and its construction method. Background Technology

[0002] The meniscus is a crucial structure in the knee joint, responsible for cushioning, stress distribution, and lubrication. It exhibits significant regionalized mechanical and histological heterogeneity. Current meniscus repair and replacement methods suffer from the following technical limitations: First, current organoid manufacturing methods do not consider the mechanical environment during meniscus development. Most schemes only construct scaffolds based on the perspective of inducing stem cell differentiation through scaffold porosity, without considering the tensile and compressive stress fields actually experienced by the meniscus. As a result, the differentiated chondrocytes do not have obvious internal and external partitions and functions.

[0003] Secondly, the existing scaffolds have a homogeneous mechanical field and lack a continuous gradient. Most solutions use a single material or simple partition assembly, which cannot reproduce the continuous porosity and modulus gradient of the natural meniscus, which is "soft inside and hard outside, compressed inside and stretched outside". This leads to abnormal load transmission paths and stress concentration, which can easily aggravate the degeneration of adjacent articular cartilage.

[0004] Third, the fiber orientation and tensile load-bearing capacity are insufficient. Existing scaffolds have failed to effectively reconstruct the orientation and bundle structure of peripheral circumferential collagen fibers, resulting in low in-plane tensile modulus and fatigue life. They are prone to fixation point tearing, edge curling and deformation, and cannot bear the circumferential tension and "binding" function of the meniscus.

[0005] Fourth, the sustained release and regional induction effects of biochemical factors are poor. Commonly used physical mixing or adsorption methods lead to rapid diffusion and inactivation of growth factors, making it difficult to form a stable spatiotemporal concentration gradient between the inner and outer regions. This results in indistinct regional differentiation of stem cells and mismatch in extracellular matrix deposition.

[0006] Fifth, there are limitations in manufacturing precision. Traditional pneumatic extrusion printing methods suffer from unstable pressure-extrusion mapping due to the compressibility of gas, resulting in hysteresis. This can easily lead to problems such as under-extrusion at the beginning and over-extrusion at the end during start-up, shutdown, and cornering. The viscoelasticity and shear thinning properties of the material result in a larger and more fluctuating filament diameter, making it difficult to accurately reconstruct the continuous mechanical gradient field required for the meniscus at the three-dimensional voxel scale. Summary of the Invention

[0007] The purpose of this invention is to provide a meniscus organoid based on voxelization variable wire diameter printing and its construction method.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for constructing a meniscus organoid based on voxelization variable wire diameter printing includes the following steps: Acquire medical imaging data of the target meniscus; Three-dimensional reconstruction and voxel modeling are performed based on the aforementioned medical image data; Based on the mechanical distribution of the inner and outer regions of the meniscus, the voxel-level mechanical parameters are mapped to the printing path and wire diameter control parameters; Polycaprolactone scaffolds were constructed using a variable filament diameter melt extrusion printing module. The nozzle diameter of the printing module was 250 μm, and the fibers were laid out in an equiangular staggered pattern in three directions: 0°, 60°, and 120°. The fiber diameter was continuously adjusted within the range of 150 μm to 350 μm, and the porosity varied from 80% in the inner region to 10% in the outer region. The scaffold is injected with functionalized norbornene-modified type I collagen (NorCol) bio-ink loaded with transforming growth factor-β (TGF-β) into the inner region and with functionalized norbornene-modified type I collagen (NorCol) bio-ink loaded with connective tissue growth factor (CTGF) into the outer region using a multi-nozzle printing system. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were used at a rate of 2 × 10⁻⁶. 6 The density of cells / mL is encapsulated in the bio-ink; In-situ crosslinking was performed using 405nm wavelength light with an intensity of 10mW / cm². 2 The exposure time is 30 seconds; The cross-linked constructs were cultured in chondrocyte differentiation medium for 28 days. During the cultivation period, starting from day 7, axial periodic mechanical loading was applied, consisting of 5% to 15% compressive strain at a frequency of 0.5 Hz to 1.0 Hz, for 1 to 2 hours per day.

[0009] Furthermore, the concentration of the functionalized norbornene-modified type I collagen bio-ink is 0.6%.

[0010] Furthermore, the growth factor is covalently immobilized in the norbornene-modified type I collagen hydrogel network via a sulfur-ene click reaction.

[0011] Furthermore, the diffusion radius of the growth factor after incubation in phosphate-buffered saline for 24 hours does not exceed 50 μm.

[0012] Furthermore, the compressive modulus of the polycaprolactone scaffold transitions from 30 MPa in the inner region to 80 MPa in the outer region.

[0013] A meniscus organoid based on voxelization variable wire diameter printing, constructed using any one of the methods described above, comprising: The polycaprolactone-based fiber network is laid out in three directions of equal angles: 0°, 60° and 120°. The fiber diameter varies continuously in the range of 150μm-350μm, the porosity varies from 80% in the inner region to 10% in the outer region, and the compressive modulus transitions from 30MPa in the inner region to 80MPa in the outer region. A functionalized norbornene-modified type I collagen hydrogel, wherein the hydrogel covalently immobilizes growth factors via a sulfur-ene click reaction; Human umbilical cord mesenchymal stem cells are encapsulated in the hydrogel.

[0014] Furthermore, the growth factors include transforming growth factor-β and connective tissue growth factor, wherein transforming growth factor-β is immobilized in the inner region of norbornene-modified type I collagen hydrogel, and connective tissue growth factor is immobilized in the outer region of norbornene-modified type I collagen hydrogel.

[0015] Application of a meniscus organoid in the preparation of meniscus grafts.

[0016] This invention achieves the following beneficial technical effects by combining voxelized variable filament diameter printing technology with regionalized biofunctionalization: 1. A continuous and controllable structural-mechanical gradient was established. By precisely controlling the continuous variation of PCL fiber diameter within the range of 150-350μm, a gradient distribution of porosity from 80% in the inner region to 10% in the outer region was achieved, and the compressive modulus transitioned smoothly from 30MPa to 80MPa, effectively simulating the mechanical distribution characteristics of a natural meniscus.

[0017] 2. Regionalized and stable immobilization of growth factors was achieved. Using functionalized NorCol hydrogel, TGF-β and CTGF were covalently immobilized in the inner and outer regions respectively via a thio-ene click reaction. After 24 hours of incubation with PBS, the diffusion radius did not exceed 50 μm, forming a stable regionalized biochemical induction microenvironment.

[0018] 3. Improved manufacturing precision and controllability. By employing voxel modeling and viscoelastic compensation control methods, combined with a multi-nozzle printing system, continuous gradient control at the hundred-micron level was achieved, overcoming the precision limitations of traditional pneumatic extrusion printing.

[0019] 4. Promoted regional differentiation of cells. Through the synergistic effect of 0° / 60° / 120° triaxial staggered biomimetic fiber trajectory and region-specific growth factors, combined with periodic mechanical loading culture, hUC-MSCs were effectively induced to differentiate into hyaline cartilage-like tissue in the inner region and fibrocartilage-like tissue in the outer region. Attached Figure Description

[0020] Figure 1 An overview of the entire process of constructing a meniscus organoid. (Includes...) Figure 1 (a) is a three-dimensional reconstruction image. Figure 1 (b) is a voxel modeling diagram. Figure 1 (c) is the parameter mapping diagram. Figure 1 (d) is a multi-nozzle composite printing diagram. Figure 1 (e) is a meniscus organoid. Figure 1 (f) is a diagram of mechanical pressure culture.

[0021] Figure 2 A diagram showing the mapping relationship between voxelization modeling and printing parameters. Figure 2 (a) is a contour map of elastic modulus based on medical images of the meniscus. Figure 2 (b) is the mapping diagram of the modulus control of the voxelized wire diameter.

[0022] Figure 3 This is a schematic diagram of the fiber network structure of a variable diameter PCL scaffold. Figure 3 (a) is a design diagram of the voxelized gradient meniscus trajectory. Figure 3 (b) is a printed image of a voxelized gradient meniscus. Figure 3 (c) is the inner zone wire diameter of 150um - the middle zone of 250um - the outer zone of 350um.

[0023] Figure 4 This is a working diagram of a multi-nozzle printing and growth factor fixation system. Figure 4 (a) is a diagram of the growth factor immobilized in Norcol gel. Figure 4 (b) is a rapid gel hydrogel diagram based on the triple helix structure. Figure 4 (c) is a multi-nozzle printing system including a hot melt nozzle and two low-temperature bio-ink nozzles. Figure 4 (d) is a regionalized growth factor fixation diagram.

[0024] Figure 5 This is a schematic diagram of a mechanically loaded culture device.

[0025] Figure 6 This is a diagram of the organoid structure of the meniscus. Figure 6 (a) COL-1 staining of the internal region of the meniscus organoid. Figure 6 (b) is a merge diagram of the COL-1 / COL-2 / cytoskeleton region of the meniscus organoid. Figure 6 (c) COL-1 staining of the internal region of the meniscus organoid. Figure 6 (d) is a merge diagram of COL-1 / COL-2 / cytoskeleton in the inner region of the meniscus organoid.

[0026] Figure 7 This is a histological characterization diagram. Figure 7 (a) Physical diagram of the reconstructed meniscus in vivo. Figure 7 (b) HE staining of meniscus organoids. Figure 7(c) Sirius red staining of the inner meniscus. Figure 7 (d) Sirius red staining of the outer meniscus. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments and accompanying drawings, but is not limited thereto.

[0028] Example 1 Medical image data acquisition and voxel modeling Medical imaging data of the target meniscus is acquired through MRI or CT scans. Medical image processing software is used to perform 3D reconstruction of the image data, generating a 3D digital model of the meniscus. The model is then imported into voxelization software, discretized into a voxel mesh, with voxel sizes set to 100μm × 100μm × 100μm to ensure accuracy at the hundred-micrometer level.

[0029] Based on the mechanical properties of natural menisci, a compression modulus contour map was obtained through experimental measurements. The compression modulus was 19.09 MPa at 4.1 mm from the center, 28 MPa at 19.7 mm, 35.3 MPa at 33.5 mm, 61.13 MPa at 47.3 mm, and 74.93 MPa at 58.3 mm. The voxel-level mechanical parameters were mapped to printing path and filament diameter control parameters: the inner region corresponded to a lower modulus of 30-50 MPa with a filament diameter of 150-250 μm; the outer region corresponded to a higher modulus of 60-80 MPa with a filament diameter of 250-350 μm; and the filament diameter transitioned continuously in the middle region. The printing path used an equiangularly interlaced trajectory in three directions: 0°, 60°, and 120°, to simulate the travel direction of natural meniscus fibers.

[0030] like Figure 1 As shown, the construction process includes medical image acquisition: 3D reconstruction ( Figure 1 (a) Voxel modeling (Figure 1(b)), parameter mapping (Figure 1(c)), multi-nozzle composite printing ( Figure 1 (d) ), Meniscus organoids ( Figure 1 (e) Mechanical loading culture ( Figure 1 (f)). Figure 2 This demonstrates the specific process of voxelization modeling and parameter mapping, based on the elastic modulus cloud of meniscus medical images ( Figure 2 (a) and voxelized wire diameter control modulus mapping ( Figure 2 (b)).

[0031] Example 2 Variable wire diameter printing to construct polycaprolactone scaffolds A polycaprolactone scaffold was constructed using a variable filament diameter melt extrusion printing module. The material was medical-grade polycaprolactone with a molecular weight of 50,000. The printing temperature was set to 90℃, and the nozzle diameter was 250μm. By dynamically controlling the extrusion rate, the fill rate gradually increased from 20% in the inner zone to 90% in the outer zone, achieving continuous adjustment of the fiber diameter within the range of 150μm-350μm. During the printing process, a viscoelastic compensation control algorithm was employed to adjust the extrusion speed in real time based on the viscoelastic properties of polycaprolactone, eliminating flow hysteresis and filament diameter fluctuations.

[0032] (1) Variable filament diameter melt extrusion printing module The variable filament diameter melt extrusion printing module consists of a sealed hopper, a nozzle unit, a high-temperature melting unit, a precision extrusion unit, and other sealing and connecting components. The precision extrusion unit is the core drive structure, its function being to precisely control the speed of the extrusion motor (hereinafter referred to as the E-axis) to coordinate with the movement trajectory of the print head. Based on the principle of volume conservation in melt extrusion, the mathematical model for the control process of maintaining a constant extruded filament diameter is as follows: At any time t, the desired melt volumetric flow rate Q should be coordinated with the E-axis compression volume X(t) and the volume swept by the print head movement. This relationship is described by the following equation: Q = X(t) = π(D02) 2 vE=π(Dt2) 2 vh in: • D0 is the diameter of the printing cartridge; • vE is the velocity along the E-axis at time t; • Dt is the target extruded wire diameter at time t; • vh is the velocity of the printhead at time t.

[0033] (2) Viscoelastic dynamic compensation algorithm for variable diameter printing The viscoelastic dynamic compensation algorithm refers to the fact that, due to the significant viscoelasticity of polymer materials such as PCL in the molten state, the release of elastic potential energy within the material is slow during the actual printing process, leading to a large error between the actual output and input control signals, especially when the filament diameter changes dynamically. Therefore, a compensation algorithm is needed. The extrusion process of PCL in the extrusion printhead can be divided into two parts: elastic compression is dominant under high pressure and large volume conditions in the sealed hopper, while viscous flow is dominant under small diameter and high flow rate conditions in the nozzle. The control equation for elastic compression can be described as follows: Xt-YtV0K=P in: • Yt represents the actual output volume; • V0 is the volume of material in the silo; • K is the elastic modulus of PCL; • P represents the additional pressure generated during the compression process; The control process for viscous flow can be described as follows: Yt=πR48ηLP in: • R is the diameter of the printing nozzle; η is the viscosity of PCL; • L is the length of the nozzle tip; The solution can be obtained by combining the two equations. xt=yt+τyt τ=8ηLV0πR4K Where τ refers to the system transport relaxation parameter, with dimensions [s], representing the time required for the relaxation process to complete 63.21% during material transport. In G-code control, piecewise control is required, necessitating discretization. After processing using "time forward difference," the following can be obtained: Xn = Yn + τδ (Yn+1 - Yn) in: ·Xn is the E-axis compression volume in the nth extrusion cycle; • Yn is the actual output volume in the nth extrusion cycle; •Yn+1 represents the actual output volume over n+1 extrusion cycles; For different filament diameter variations and different printing conditions, the parameter τ is different, and the corresponding E-axis compression volume X will also change accordingly, thus completing the dynamic compensation under PCL variable diameter printing.

[0034] The resulting polycaprolactone scaffold has a continuous porosity gradient from 80% in the inner region to 10% in the outer region, and a compressive modulus gradient from 30 MPa in the inner region to 80 MPa in the outer region.

[0035] like Figure 3 As shown, the PCL fiber network adopts a triaxial staggered trajectory of 0° / 60° / 120°, with the filament diameter increasing from 150μm to 350μm from the inner zone to the outer zone, and the porosity decreasing from 80% to 10%, forming a continuous gradient structure.

[0036] Example 3 Preparation and regional immobilization of functionalized norbornene-modified type I collagen bio-ink Functionalized norbornene-modified type I collagen hydrogels were prepared. First, norbornene groups were chemically incorporated into type I collagen molecules. Acid-soluble type I collagen was dissolved in dilute hydrochloric acid to prepare a collagen solution of approximately 0.3% (w / v), which was then stirred in an ice bath. The pH of the system was adjusted to approximately 9.0 with NaOH solution and stirred at approximately 4°C. Then, norbornene carbonic anhydride was dissolved in a small amount of acetone (approximately 1 / 20 the volume of the collagen solution) and slowly added dropwise to the collagen solution at a mass ratio of approximately 1–6 mg norbornene carbonic anhydride / 1 mg collagen. During this process, the pH was maintained at approximately 9.0 by adding NaOH dropwise, and the reaction was carried out at 4°C for approximately 2 hours. After the reaction, the mixture was placed in a dialysis bag with a molecular weight cutoff of 8000–14000 Da and dialyzed against deionized water at 4°C for approximately 2 weeks, with the dialysate changed daily. The resulting product was then freeze-dried to obtain norbornene-modified type I collagen (NorCol) solid powder. No additional catalyst was used in the above modification process; only alkaline conditions were relied upon to promote the acylation reaction between norbornene carbonic anhydride and the free amino groups of collagen molecules, introducing norbornene groups. Norbornene-modified type I collagen was dissolved in PBS to prepare solutions of different concentrations, and the optimal concentration was determined to be 0.6% through cell compatibility testing. Growth factors TGF-β and CTGF were coupled to iFluor-555 or iFluor-488, respectively, via an acylhydrazine-carboxyl group reaction (for subsequent diffusion radius detection). The fluorescently labeled growth factors were then dissolved in MES buffer (pH=5.5) and incubated overnight at 4°C for later use.

[0037] Based on this, a 0.6% NorCol aqueous solution was prepared, and a photoinitiator (such as LAP, with a final concentration of approximately 0.05%–0.1%, w / v) and a fluorescently labeled growth factor with a thiol group (–SH) were added to uniformly disperse the mixture in the NorCol solution. The NorCol mixture was then injected into a mold or filled into a designated area of ​​a PCL gradient scaffold and irradiated with approximately 405 nm ultraviolet light (light intensity approximately 10 mW / cm²) at room temperature. 2 The photoclick reaction (approximately 30 seconds) triggers a thiol-ene photoclick reaction between the thiol group and the norbornene double bond on the NorCol molecule, causing the growth factor to covalently bind to the hydrogel network via the thiol group, ultimately forming a stable cross-linked structure. The immobilized growth factor was incubated in PBS for 24 hours, and the diffusion radius was measured to be ≤50 μm using a fluorescence microscope.

[0038] A multi-head printing system was used for zoned ink injection: printhead one was used for polycaprolactone printing at 90°C; printheads two and three were used for bio-ink printing at 20°C. Printhead two was loaded with TGF-β-functionalized norbornene-modified type I collagen bio-ink for the inner zone; printhead three was loaded with CTGF-functionalized norbornene-modified type I collagen bio-ink for the outer zone. Immediately after printing, in-situ crosslinking was performed using 405nm wavelength light at an intensity of 10mW / cm². 2 An exposure time of 30 seconds was used to form a stable hydrogel network.

[0039] like Figure 4 As shown, the multi-nozzle printing system includes a hot melt nozzle and two low-temperature bio-ink nozzles (Figure (4c)), which enable regional bio-ink injection and growth factor fixation with a diffusion radius of no more than 50 μm.

[0040] Example 4 Cell encapsulation and mechanical loading culture hUC-MSCs were used at 2×10 6 cells·mL -1 The density was resuspended in norbornene-modified type I collagen bioink and then injected into the corresponding regions of the polycaprolactone scaffold. The cross-linked constructs were placed in chondrocyte differentiation medium and cultured at 37°C and 5% CO2 for 28 days. Periodic mechanical loading was applied starting on day 7: compressive strain of 5%–15% was applied using a bioreactor at a frequency of 0.5 Hz–1.0 Hz for 1–2 hours per day. After culture, the regional differentiation effect was assessed by histological staining and gene expression analysis.

[0041] like Figure 5 As shown, the mechanical loading culture device includes a bioreactor, a culture chamber, and a loading head, applying a compressive strain of 5%-15% at a frequency of 0.5-1.0Hz for 1-2 hours per day.

[0042] Example 5 In vivo validation and mechanism study A medial meniscus defect model was established in New Zealand rabbits (defect size 3mm × 5mm × 2mm, located in the body of the medial meniscus), with 6 rabbits in each group. The experimental group received the meniscus organoid of this invention; the gradient scaffold group received a PCL gradient scaffold (without bio-ink and cells); the homogeneous scaffold group received a PCL homogeneous scaffold (250μm wire diameter, 45% porosity); and the blank control group only underwent modeling without material implantation. Histological analysis at 12 weeks post-operation showed that the organoid group was positive for Safranin-O staining and COLII immunohistochemistry in the inner region, and positive for COL-1 and toluidine blue staining in the outer region; the MMP13 staining intensity was significantly lower than that in the control group. Figure 6 and Figure 7As shown, after in vivo transplantation of the meniscus organoid, the actual image and HE staining showed that the regenerated meniscus structure was intact. Further Sirius red staining revealed that the cells in the inner region of the meniscus were round hyaline cartilage, while those in the outer region were spindle-shaped fibrocartilage, exhibiting good regional differentiation.

[0043] To further explore the mechanical regulation mechanism, gradient scaffold models under static and pressurized conditions were constructed in vitro, and whole transcriptome sequencing was performed after 28 days of culture. The results showed that mechanical loading activated the Piezo1 ion channel, triggering intracellular calcium signaling, which in turn activated mitochondrial metabolism and the Wnt / NFAT signaling pathway, promoting the expression of cartilage-related transcription factors such as SOX9 and COL-2. The expression level of SOX9 gene in the pressurized group was 2.3 times higher than that in the static group.

[0044] Key parameters Polycaprolactone printing parameters: molecular weight 50,000; printing temperature 90℃; nozzle diameter 250μm; fiber diameter 150μm-350μm; porosity 80%-10%; compressive modulus 30MPa-80MPa. Bio-ink parameters: norbornene-modified type I collagen concentration 0.6%; growth factor immobilization method: sulfur-ene click reaction; diffusion radius ≤50μm; cell density 2×10⁻⁶. 6 cells / mL. Culture period 28 days; mechanical loading parameters: compressive strain 5%-15%, frequency 0.5Hz-1.0Hz, 1h-2h per day.

[0045] The above examples are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a meniscus organoid based on voxelization variable wire diameter printing, characterized in that, Includes the following steps: Acquire medical imaging data of the target meniscus; Three-dimensional reconstruction and voxel modeling are performed based on the aforementioned medical image data; Based on the mechanical distribution of the inner and outer regions of the meniscus, the voxel-level mechanical parameters are mapped to the printing path and wire diameter control parameters; Polycaprolactone scaffolds were constructed using a variable filament diameter melt extrusion printing module. The nozzle diameter of the printing module was 250 μm, and the fibers were laid out in an equiangular staggered pattern in three directions: 0°, 60°, and 120°. The fiber diameter was continuously adjustable within the range of 150 μm to 350 μm, and the porosity varied from 80% in the inner region to 10% in the outer region. The inner region of the scaffold is injected with functionalized norbornene-modified type I collagen bio-ink loaded with transforming growth factor-β, and the outer region is injected with functionalized norbornene-modified type I collagen bio-ink loaded with connective tissue growth factor. Human umbilical cord mesenchymal stem cells were used at a rate of 2 × 10⁻⁶. 6 The density of cells / mL is encapsulated in the bio-ink; In-situ crosslinking was performed using 405nm wavelength light with an intensity of 10mW / cm². 2 The exposure time is 30 seconds; The cross-linked constructs were cultured in chondrocyte differentiation medium for 28 days. During the cultivation period, starting from day 7, axial periodic mechanical loading was applied, consisting of 5% to 15% compressive strain at a frequency of 0.5 Hz to 1.0 Hz, for 1 to 2 hours per day.

2. The method for constructing meniscus organoids based on voxelization variable wire diameter printing according to claim 1, characterized in that, The concentration of the functionalized norbornene-modified type I collagen bio-ink is 0.6%.

3. The method for constructing a meniscus organoid based on voxelization variable wire diameter printing according to claim 1, characterized in that, The growth factor is covalently immobilized in a norbornene-modified type I collagen hydrogel network via a sulfur-ene click reaction.

4. The method for constructing a meniscus organoid based on voxelization variable wire diameter printing according to claim 1, characterized in that, The diffusion radius of the growth factor after incubation in phosphate-buffered saline for 24 hours does not exceed 50 μm.

5. The method for constructing a meniscus organoid based on voxelization variable wire diameter printing according to claim 1, characterized in that, The compressive modulus of the polycaprolactone scaffold transitions from 30 MPa in the inner region to 80 MPa in the outer region.

6. A meniscus organoid based on voxelization variable wire diameter printing, characterized in that, Constructed using the method described in any one of claims 1-5, comprising: The polycaprolactone-based fiber network is laid out in three directions of equal angles: 0°, 60° and 120°. The fiber diameter varies continuously in the range of 150μm-350μm, the porosity varies from 80% in the inner region to 10% in the outer region, and the compressive modulus transitions from 30MPa in the inner region to 80MPa in the outer region. A functionalized norbornene-modified type I collagen hydrogel, wherein the hydrogel covalently immobilizes growth factors via a sulfur-ene click reaction; Human umbilical cord mesenchymal stem cells are encapsulated in the hydrogel.

7. The meniscus organoid according to claim 6, characterized in that, The growth factors include transforming growth factor-β and connective tissue growth factor, wherein transforming growth factor-β is immobilized in the inner region of norbornene-modified type I collagen hydrogel, and connective tissue growth factor is immobilized in the outer region of norbornene-modified type I collagen hydrogel.

8. The use of the meniscus organoid of claim 6 in the preparation of meniscus grafts.

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