Method for constructing multi-scale early atherosclerosis in-vitro model

Abstract

The invention discloses a method for constructing a multi-scale early atherosclerosis in-vitro model, and belongs to the technical field of biomedical engineering and tissue engineering. The method comprises the following steps: providing an embedded suspension medium, wherein the suspension medium has adjustable yield stress, high optical transparency and reversible shear recovery performance; providing at least two kinds of bio-ink, wherein the bio-ink is respectively used for constructing different layers of the vascular tissue; using a multi-channel coaxial nozzle to programmably extrude the bio-ink in the embedded suspension medium so as to form a tubular tissue with a layered structure; after printing is completed, the suspension medium is removed through a physical or chemical method, and the tubular tissue is released. The model obtained through the method provides an in-vitro research platform which is highly bionic and high in controllability for disease mechanism research, drug screening and personalized medical treatment.

Description

Technical Field

[0001] This invention relates to a model construction method, and more particularly to a method for constructing a multi-scale in vitro model of early atherosclerosis, belonging to the fields of biomedical engineering, tissue engineering, and disease model construction technology. Background Technology

[0002] Cardiovascular disease is a leading cause of death worldwide, and atherosclerosis is the common underlying cause of many cardiovascular diseases. The occurrence and development of atherosclerosis are driven by a complex interplay of hemodynamic abnormalities such as oscillatory shear stress, systemic risk factors such as hyperlipidemia and chronic inflammation, and various cell types including vascular endothelium, smooth muscle, and fibroblasts. These multifactorial synergistic effects exhibit high heterogeneity both spatially and temporally, leading to key pathological events such as endothelial dysfunction, lipid retention and oxidation, inflammatory cell infiltration, and foam cell formation.

[0003] Existing in vivo and in vitro research methods have limitations: animal models can reproduce complex biological processes, but due to species differences and poor controllability of variables, they are difficult to directly extrapolate to human pathology; traditional two-dimensional culture, organoids, microfluidic chips, and three-dimensional bioprinting technologies can conveniently reveal single pathways or intercellular signals, but they lack the three-layer structure of natural arteries and macroscopic geometric features such as stenosis, aneurysm, and bifurcation. They cannot simultaneously reconstruct the pathological microenvironment at the microscopic level, such as interlayer interfaces, cell arrangement, and intercellular communication, and at the macroscopic level, such as hemodynamic abnormalities and accumulation of risk factors, thus limiting their ability to accurately simulate blood flow-structure-biochemical coupling pathological studies and drug screening.

[0004] Therefore, there is an urgent need in this field for an in vitro model construction method that can realize microscopic intima / media / adventitia layered tissue and ensure clear interlayer interfaces, as well as construct various controllable macroscopic vascular geometries and precisely regulate the biochemical and mechanical microenvironment under dynamic perfusion conditions.

[0005] To address the aforementioned separation issues, existing technologies have attempted to fabricate arterial organ-on-a-chips based on multi-material suspension bio-3D printing. Patent application number 202310075519.5, titled "Arterial Organ-on-a-chip and Preparation Method Based on Multi-material Suspension Bio-3D Printing," proposes a fabrication scheme for arterial organ-on-a-chips based on multi-material suspension printing. This scheme uses decellularized porcine skin matrix (dECM) as raw material, combined with a terpyridine ruthenium chloride / sodium persulfate photoinitiation system, to formulate a bio-ink with photo-irradiation crosslinking properties. Cell-containing dECM ink and sacrificial material PF-127 are sequentially printed in a support bath, followed by photo-crosslinking and removal of the sacrificial ink, resulting in an arterial organ-on-a-chip model with a multi-layered biomimetic structure. This method has certain innovations in achieving multi-layered vascular structure construction and pump-free perfusion. However, this technical solution also has shortcomings. First, due to limitations in the size of the printing needle during the printing process, the diameter of the final formed vascular channels is generally small, making it difficult to simulate the real structure of large-diameter blood vessels or different branch diameters. Secondly, this method cannot precisely control the thickness between different layers during the printing process, resulting in limitations in the layer transitions and physiological simulation of the obtained model. Therefore, existing technologies still face pressing technical challenges in achieving highly biomimetic arterial models with adjustable blood vessel sizes and controllable multilayer structural features. Summary of the Invention

[0006] To address the aforementioned technical deficiencies, the present invention aims to provide a method for constructing a multi-scale in vitro model of early atherosclerosis. This method aims to simultaneously achieve: microscopically, a clear replication of the three-layered vascular wall structure (intima / media / adventitia) while maintaining stable interlayer interfaces; macroscopically, the ability to fabricate normal and pathological vascular geometries (e.g., stenosis, aneurysm, bifurcation, etc.) as needed to generate a controllable pathological flow field; and the ability to induce and reproduce key early atherosclerotic events (endothelial activation, monocyte adhesion and infiltration, foam cell formation, etc.) under dynamic perfusion conditions, thereby providing a reproducible and controllable biomimetic platform for mechanism research and drug screening.

[0007] To achieve the above objectives, this invention first discloses a three-dimensional bioprinting method based on multi-channel coaxial on-demand extrusion, characterized by comprising the following steps:

[0008] An embedded suspension medium is provided, which has adjustable yield stress, high optical transparency and reversible shear recovery properties;

[0009] Provide at least two types of bio-inks, each of which is used to construct different layers of vascular tissue;

[0010] Using a multi-channel coaxial nozzle, the bio-ink is programmably extruded in the embedded suspension medium to form tubular tissue with a layered structure;

[0011] After printing, the suspended medium is removed by physical or chemical methods to release the tubular tissue.

[0012] Preferably, the embedded suspension medium comprises a combination of a block copolymer and a cellulose derivative; the block copolymer is Pluronic F-127 with a mass fraction of 8%-12% (w / v), and the cellulose derivative is hydrophobically modified hydroxypropyl methylcellulose with a mass fraction of 2%-4% (w / v); the suspension medium has a yield stress of 1-10 Pa, a light transmittance of greater than 90%, and recovers 50%-70% of its viscoelasticity within 1 second after shear unloading.

[0013] Preferably, the bio-ink comprises at least three types of functional formulations:

[0014] The outer membrane bio-ink uses a crosslinkable natural or semi-synthetic hydrogel as a substrate and loads fibroblasts or fibroblast-like cells; the hydrogel is methacrylamide gelatin (GelMA) with a mass fraction of 5%-15% (w / v);

[0015] The medium-film bio-ink uses a composite system of GelMA and polyethylene glycol diacrylate (PEGDA) as a base to load vascular smooth muscle cells; the PEGDA mass fraction is 1%-5% (w / v), and the cell concentration is 0.5×10^7~2×10^7 cells / mL.

[0016] Endometrial bio-ink is a printable low-viscosity endothelial bio-ink or endothelial cell suspension for direct printing or perfusion inoculation; the inoculation concentration of the endothelial cell suspension is 1×10^6~5×10^7 cells / mL.

[0017] Preferably, the substrate of the bio-ink supports alternative matrices, including collagen, hyaluronic acid methacrylamide, or silk fibroin; and / or supports cross-linking methods, including photocross-linking, ionic cross-linking, enzymatic cross-linking, or thermosensitive gelation.

[0018] Preferably, the multi-channel coaxial printhead includes at least two channels; each channel is loaded with different bio-inks, and programmable extrusion is achieved by rapid switching via valve control; printing is carried out in a pre-prepared suspension medium, and a low-osmotic or hydrophilic cosolvent-containing elution bath is used when removing the suspension medium.

[0019] Preferably, the tubular tissue has a three-layer structure of intima-media-adventitia, and can be constructed into macroscopic geometric shapes as needed, including stenosis, aneurysm, or bifurcation.

[0020] Based on the above-mentioned three-dimensional bioprinting method based on multi-channel coaxial on-demand extrusion, this invention also discloses a method for constructing a multi-scale in vitro model of early atherosclerosis, characterized by comprising the following steps:

[0021] A tubular tissue with a three-layered structure of inner membrane, middle membrane, and outer membrane was constructed using a three-dimensional bioprinting method.

[0022] The tubular tissue is endothelialized to form a continuous inner membrane layer;

[0023] Endothelialized tubular tissue was placed in a perfusion bioreactor and fluid shear stress was applied.

[0024] Introducing pro-atherosclerotic factors into the perfusion medium induces early atherosclerotic pathological events.

[0025] Preferably, the endothelialization treatment involves inoculating endothelial cells by perfusion or rotation, and using alternating rotation or periodic tilting to ensure uniform cell deposition; and pre-culturing under mild fluid shear conditions for 1-7 days after inoculation, with a shear force of 0.01-0.03 dyne / cm².

[0026] Preferably, the perfusion bioreactor includes a controllable pump, a pulse damper, and a programmable flow control unit to achieve a pulsating flow mode and precisely control the shear force, frequency, and waveform; the shear stress is 0.27-0.40 dyne / cm², and the frequency is 2Hz.

[0027] Preferably, the pro-atherosclerotic factors include inflammatory cytokines, low-density lipoprotein / oxidized low-density lipoprotein, and immune cells; the inflammatory cytokines are tumor necrosis factor-α (TNF-α) with a concentration of 10-100 ng / mL; the concentration of low-density lipoprotein / oxidized low-density lipoprotein is adjusted according to experimental needs; the immune cells are THP-1 cells or peripheral blood mononuclear cells with a cell density of 10^5~10^7 cells / mL.

[0028] Preferably, the early atherosclerotic pathological events include endothelial activation, mononuclear cell adhesion and infiltration, macrophage polarization, and foam cell formation.

[0029] Beneficial effects

[0030] Multi-channel coaxial printing enables the layered construction of the intima, media, and adventitia in a single operation, reproducing the anatomical structure of natural blood vessels. With the aid of embedded suspension media, complex morphologies such as stenosis, aneurysms, and bifurcations are stably printed, significantly improving the morphological controllability of the model. Different bio-inks are loaded with smooth muscle cells and fibroblasts, respectively, and a monolayer of endothelial cells is seeded on the inner surface, placing various cells in a microenvironment closer to physiological conditions, promoting their adhesion, proliferation, and functional maintenance. Under the action of a perfusion reactor, the model can receive physiologically or pathologically relevant fluid stimulation, thereby inducing key early events of atherosclerosis, including endothelial activation, monocyte adhesion and infiltration, macrophage polarization, and foam cell formation. Therefore, this invention not only reconstructs key processes of disease development in vitro but also has broad application value in mechanism research, drug screening, and the construction of patient-specific models. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the method for constructing a multi-scale in vitro model of early atherosclerosis according to the present invention;

[0032] Figure 2 The diagram shows the preparation process of the suspension medium and the bio-ink, where: (A) the preparation process of the suspension medium; (B) the preparation process of the bio-ink.

[0033] Figure 3 The following are schematic diagrams of arterial models with different macroscopic geometric structures printed by the present invention, wherein: (A) a programmable floating print illustration; (B) a printed uniform diameter double-layer tube; (C) a printed arterial stenosis model; (D) a printed aneurysm model; and (E) a printed bifurcation vessel model.

[0034] Figure 4 The diagram illustrates the in vitro induction method for an atherosclerosis model, including: (A) a schematic diagram of the development process of atherosclerosis; and (B) a schematic diagram of the process for constructing an atherosclerosis model. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0036] This invention discloses a three-dimensional bioprinting method based on multi-channel coaxial on-demand extrusion and a method for constructing a multi-scale in vitro model of early atherosclerosis using this printing method. This method constructs tubular tissue with a clear intima-media-adventitia three-layer structure in an embedded suspension medium by programmably switching between various bio-inks. It can also construct macroscopic geometries such as stenosis, aneurysm, and bifurcation as needed to generate a controllable pathological flow field. After dynamic perfusion culture and the introduction of biochemical and immunostimulatory factors, the model can repeatedly and tunably reproduce key pathological events related to early atherosclerosis (including endothelial activation, monocyte adhesion and infiltration, macrophage polarization, and foam cell formation) and their related mechanosensitive signaling pathways, thus providing an implementable and reproducible biomimetic platform for pathological mechanism research, drug screening, and the construction of patient-specific models. The embedded suspension medium comprises a combination of block copolymer components and cellulose derivatives. The preferred composition is approximately 8%–12% (w / v) of Pluronic F-127 (or a functionally equivalent component, such as a block copolymer like poloxamer) and approximately 2%–4% (w / v) of hydrophobically modified hydroxypropyl methylcellulose (or a functionally equivalent component, such as methylcellulose, hydroxyethylcellulose, or other cellulose derivatives that can form a reversible network through hydrophobic interactions). After formulation and process control, the suspension medium exhibits adjustable yield stress (preferably 1–10 Pa), high optical transparency (preferably transmittance >90%), and reversible shear recovery properties (preferably recovering ≥50%–70% viscoelasticity within 1 second after shear unloading), making it suitable for real-time monitoring of the printing process and supporting high-fidelity embedded printing. The bio-ink comprises at least three functional formulations to construct outer membranes, middle membranes, and inner membranes, or as endothelial inoculation media: the outer membrane ink is preferably based on GelMA or other crosslinkable natural / semi-synthetic hydrogels, with the carrier containing fibroblasts or fibroblast-like cells, and the GelMA mass fraction is preferably 5%–15% (w / v); the middle membrane ink is preferably a GelMA / polyethylene glycol diacrylate (PEGDA) composite system to balance biocompatibility and mechanical adjustability, and the PEGDA mass fraction is preferably 1%–5% (w / v), with the elastic modulus controlled by adjusting the PEGDA molecular weight and concentration; the middle membrane ink is loaded with vascular smooth muscle cells, with a cell concentration preferably 0.5 × 10⁻⁶. 7 –2×10 7 cells / mL; the inner membrane can be formed directly by printing with printable low-viscosity endothelial bio-ink, or preferably by perfusing / rotationally seeding an endothelial cell suspension after printing to form a continuous monolayer, with the preferred endothelial seeding concentration in the range of 1×10⁻⁶ cells / mL. 6 -5×10 7The printing equipment preferably supports alternative matrices (such as collagen (concentrations typically around 5-15 mg / mL), hyaluronic acid methacrylamide (concentrations typically around 2-8% w / v), silk fibroin (concentrations typically around 4-10% w / v), etc.) and various crosslinking methods (including but not limited to photocrosslinking, ionic crosslinking, pH crosslinking (e.g., collagen can be neutralized with sodium hydroxide and then induced to gel), enzymatic crosslinking (e.g., silk fibroin can be crosslinked with tyrosinase), or thermosensitive gelation) to cover equivalent implementations of different materials and processes. The printing equipment employs a multi-channel coaxial printhead (at least two channels, preferably three or more), with each channel loaded with different bio-inks; the printhead diameter and channel configuration are variable to form concentric layers (to achieve a double-layer tubular structure, a dual-channel coaxial printhead with inner / outer channels of 27G and 21G is preferred; to achieve a triple-layer tubular structure, a three-channel coaxial printhead with inner / middle / outer channels of 27G, 20G, and 16G is preferred). To reduce interfacial mixing and achieve rapid material switching, valve-controlled rapid switching is preferably employed, using on-demand gas supply in multiple channels to programmably extrude different bio-inks. The printing process takes place in a pre-prepared and sterilized suspension medium. After printing, the suspension medium is removed by physical or chemical methods to release the component. Preferred removal media include hypotonic elution baths or those containing hydrophilic solubilizers (such as PEG400 5% v / v in PBS), with removal conditions matched to the crosslinking strength to achieve suspension medium removal without damaging cells and structures. The endothelialization step involves inoculating endothelial cells via perfusion or rotation, using alternating rotation or periodic tilting to ensure uniform cell deposition within the lumen; pre-culturing is performed for 1–7 days post-inoculation under mild fluid shear (e.g., 0.01–0.03 dyne / cm²) to promote cell adhesion and monolayer formation. For tissue maturation and functional assessment, the construct is placed in an adjustable pulsatile perfusion bioreactor. The reactor preferably includes a controllable pump (such as a peristaltic pump or diaphragm pump), a pulse damper, and a programmable flow control unit to achieve precise control of instantaneous or mean shear force, frequency, and waveform. The early atherosclerosis induction method includes introducing pro-atherosclerotic factors into the perfusion medium to induce the pathological process. These factors include, but are not limited to, inflammatory cytokines (e.g., TNF-α, preferably 10-100 ng / mL), low-density lipoprotein / oxidized low-density lipoprotein (LDL / oxLDL, concentration adjustable according to experimental needs), and immune cells (e.g., THP-1 or peripheral blood mononuclear cells, cell density preferably 102). 5 -10 7(cells / mL). Preferably, the induction protocol can be continuous perfusion, intermittent perfusion, or gradient administration to simulate different disease stages and exposure conditions. It is worth noting that in the embodiments of the present invention, a uniform factor concentration is used for induction on arterial models with different macroscopic geometries such as normal, stenotic, aneurysmal, and bifurcation arteries, thereby simulating early atherosclerotic pathological events in vitro. Those skilled in the art can further optimize the above concentration by adjusting the factor exposure time, applying concentration gradients, or combining it with other stimuli.

[0037] Example 1

[0038] See attached document Figure 1-4 A method for constructing a multi-scale in vitro model of early atherosclerosis involves the preparation of a suspension medium. Pluronic F-127 (10%, w / v, No. 1) and hydroxypropyl methylcellulose (H-HPMC, 3%, w / v, No. 2) are added to 1×PBS solution (No. 3) and allowed to stand overnight at 4°C until completely dissolved. The solution is then stirred and mixed at 50°C and slowly cooled to room temperature to obtain a transparent suspension medium (No. 14) with yield stress. The preparation process and core components of this suspension medium can be found in [link to relevant documentation]. Figure 2 As shown in Figure A, this suspension medium maintains structural stability within the temperature range of 4-37℃, has a light transmittance of over 90%, and is suitable for embedded 3D printing.

[0039] Methacrylamide gelatin (GelMA, 10%, w / v, No. 4) and photoinitiator LAP (0.5%, w / v, No. 5) were dissolved in cell culture medium (No. 6) and kept in liquid state at 47°C. Fibroblasts (No. 7) were then added to a final concentration of 1×10⁻⁶. 7 Cells / mL, mixed well and set aside to form outer membrane bioink (No. 8). GelMA (5%, w / v), polyethylene glycol diacrylate (PEGDA, 3%, w / v, No. 9), and LAP (0.5%, w / v, No. 5) were dissolved in cell culture medium (No. 6) and kept liquid at 47°C. Vascular smooth muscle cells (No. 10) were added to a final concentration of 1×10⁻⁶ cells / mL. 7 Cells / mL, mixed thoroughly to form the middle membrane bioink (No. 11). Human vascular endothelial cells (No. 12) were resuspended in cell culture medium (No. 6) to prepare 3×10⁻⁶ cells / mL. 7 A suspension of cells / mL is used to form an endothelial cell suspension (number 13). The process can be found in [reference needed]. Figure 2 B.

[0040] A dual-channel coaxial nozzle (number 15) was used, with its inner and outer channels corresponding to 27G and 21G needles, respectively. Outer membrane bio-ink (number 7) and middle membrane bio-ink (number 11) were loaded into a 30cc syringe (number 16). Suspension medium (number 14) was added to the printing support groove (number 17), and printing was performed at a speed of 6 mm / s. By extruding only the outer membrane bio-ink in the outer layer and only the middle membrane bio-ink in the inner layer, a double-layered tubular structure (number 18) with an inner diameter of approximately 4 mm and an outer diameter of approximately 6 mm was obtained. A schematic diagram of this embedded printing process can be found in [reference needed]. Figure 3 A, and the specific shape of the uniform tubular structure obtained by printing is as follows: Figure 3 As shown in Figure B, the stability and fidelity of this method in constructing basic vascular structures are demonstrated. More importantly, by programmably controlling the printing path, this method can further construct structures such as... Figure 3 C (narrow) Figure 3 D (aneurysm) and Figure 3 The complex pathological geometric model shown in E (bifurcation) provides intuitive evidence for the core capability of this invention to achieve "multi-scale" construction. After printing, the structure was irradiated with 405 nm blue light (25 mW / cm², No. 19) for three minutes to crosslink and cure. The structure was then removed and residual material was washed away using a 5% PEG solution (No. 20) as the elution solution. This complete process from printing to crosslinking and curing follows... Figure 1 The path shown in the overall flowchart.

[0041] The solidified and cleaned double-layered tubular structure (No. 18) was placed in a culture dish; endothelial cell seeding suspension (No. 13) was perfused into the lumen, and the endothelial cells were uniformly adhered to the inner surface of the lumen by timed rotation and inversion (e.g., rotating 90° every 30 min, repeated several times); for 1–7 days after seeding, it was placed under low-shear pre-culture conditions (e.g., 0.01–0.03 dyne / cm² or a gentle shaker) to promote cell spread and the formation of a continuous inner membrane layer, thereby forming a three-layered tubular model. This endothelialization step is Figure 1 Process and Figure 4 B is a key step in the construction process, aimed at establishing a functional vascular intima barrier.

[0042] A mature, intimal-formed three-layered vascular model (No. 25) was placed in a perfusion reaction system and set to a pulsatile / oscillatory flow mode to simulate pathological blood flow (e.g., instantaneous mean shear stress 0.27–0.40 dyne / cm², frequency 2 Hz). Atherosclerotic pro-inflammatory factors: TNF-α (No. 22, example 50 ng / mL), LDL (No. 23, example 5 μg / mL), and monocyte THP-1 (No. 24, example 1 × 10⁻⁶) were added to the circulating culture medium. 6(cells / mL), continuous perfusion / co-culture for approximately 3 days to induce early pathological changes. This dynamic induction phase integrates both flow field and biochemical stimuli; its position and role in the overall methodology can be found in [reference needed]. Figure 1 The overall flowchart is as follows, and as a disease induction method, the expected pathological process and logical steps can be found separately in [reference needed]. Figure 4 A and Figure 4 B.

[0043] The model was characterized after induction using immunofluorescence staining, confocal microscopy, Oil Red O staining, and molecular biological assays. Exemplary observations included: upregulated expression of adhesion molecules such as VCAM-1; monocytes adhering to the endothelial surface and infiltrating into the subendothelial region; aggregation of Oil Red O-positive foam cells; and increased expression of mechanosensory pathway-related genes (such as Piezo1, YAP1, and β-catenin). These characterization results demonstrate that the model constructed using this method can reproduce key early pathological events in atherosclerosis.

[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A three-dimensional bioprinting method based on multi-channel coaxial on-demand extrusion, characterized in that, Includes the following steps: An embedded suspension medium is provided, which has adjustable yield stress, high optical transparency and reversible shear recovery properties; Provide at least two types of bio-inks, each of which is used to construct different layers of vascular tissue; Using a multi-channel coaxial nozzle, the bio-ink is programmably extruded in the embedded suspension medium to form tubular tissue with a layered structure; After printing, the suspended medium is removed by physical or chemical methods to release the tubular tissue.

2. The three-dimensional bioprinting method according to claim 1, characterized in that, The embedded suspension medium comprises a combination of a block copolymer and a cellulose derivative; the block copolymer is Pluronic F-127 with a mass fraction of 8%-12% w / v, and the cellulose derivative is hydrophobically modified hydroxypropyl methylcellulose with a mass fraction of 2%-4% w / v; the suspension medium has a yield stress of 1-10 Pa, a light transmittance greater than 90%, and recovers 50%-70% of its viscoelasticity within 1 second after shear unloading.

3. The three-dimensional bioprinting method according to claim 1, characterized in that, The bio-ink comprises at least three types of functional formulations: The outer membrane bio-ink uses a crosslinkable natural or semi-synthetic hydrogel as a substrate and loads fibroblasts or fibroblast-like cells; the hydrogel is methacrylamide gelatin (GelMA) with a mass fraction of 5%-15% w / v. The medium-film bio-ink uses a composite system of GelMA and polyethylene glycol diacrylate (PEGDA) as a base to load vascular smooth muscle cells; the PEGDA mass fraction is 1%-5% w / v, and the cell concentration is 0.5×10^7~2×10^7 cells / mL. Endometrial bio-ink is a printable low-viscosity endothelial bio-ink or endothelial cell suspension for direct printing or perfusion inoculation; the inoculation concentration of the endothelial cell suspension is 1×10^6~5×10^7 cells / mL.

4. The three-dimensional bioprinting method according to claim 3, characterized in that, The bio-ink's substrate supports alternative matrices, including collagen, hyaluronic acid methacrylamide, or silk fibroin; and / or supports cross-linking methods, including photocross-linking, ionic cross-linking, enzymatic cross-linking, or thermosensitive gelation.

5. The three-dimensional bioprinting method according to claim 1, characterized in that, The multi-channel coaxial printhead includes at least two channels; each channel is loaded with different bio-inks and programmable extrusion is achieved by rapid switching via valve control; printing is carried out in a pre-prepared suspension medium, and a low-osmotic or hydrophilic cosolvent-containing elution bath is used when removing the suspension medium.

6. The three-dimensional bioprinting method according to claim 1, characterized in that, The tubular tissue has a three-layer structure of intima-media-adventitia and can be constructed into macroscopic geometric shapes as needed, including stenosis, aneurysm, or bifurcation.

7. A method for constructing a multi-scale in vitro model of early atherosclerosis, characterized in that, Includes the following steps: A tubular tissue with a three-layer structure of inner membrane-middle membrane-outer membrane is constructed using the three-dimensional bioprinting method according to any one of claims 1-6; The tubular tissue is endothelialized to form a continuous inner membrane layer; Endothelialized tubular tissue was placed in a perfusion bioreactor and fluid shear stress was applied. Introducing pro-atherosclerotic factors into the perfusion medium induces early atherosclerotic pathological events.

8. The method according to claim 7, characterized in that, The endothelialization treatment involves inoculating endothelial cells by perfusion or rotation, and using alternating rotation or periodic tilting to ensure uniform cell deposition; the cells are pre-cultured under mild fluid shear conditions for 1-7 days after inoculation, with a shear force of 0.01-0.03 dyne / cm².

9. The method according to claim 7, characterized in that, The perfusion bioreactor includes a controllable pump, a pulse damper, and a programmable flow control unit to achieve a pulsating flow mode and precisely control the shear force, frequency, and waveform; the shear stress is 0.27-0.40 dyne / cm², and the frequency is 2 Hz.

10. The method according to claim 7, characterized in that, The pro-atherosclerotic factors include inflammatory cytokines, low-density lipoprotein / oxidized low-density lipoprotein, and immune cells; the inflammatory cytokines are tumor necrosis factor-α (TNF-α) with a concentration of 10-100 ng / mL; the concentration of low-density lipoprotein / oxidized low-density lipoprotein is adjusted according to experimental needs; the immune cells are THP-1 cells or peripheral blood mononuclear cells with a cell density of 10^5~10^7 cells / mL.

11. The method according to claim 7, characterized in that, The early atherosclerotic pathological events include endothelial activation, monocyte adhesion and infiltration, macrophage polarization, and foam cell formation.

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