Elastic cavity and six-degree-of-freedom multi-axis loading nucleus pulposus biological reactor system

Abstract

This application discloses an elastic cavity and a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system. The elastic cavity includes a hollow structure having a receiving cavity, an inlet, and an outlet, both of which are connected to the receiving cavity. The volume ratio of the cavity wall to the receiving cavity is (0.6-0.7):1, and the elastic modulus of the hollow structure is 0.1 MPa to 10 MPa. This application precisely matches the volume ratio of the annulus fibrosus and nucleus pulposus of the human intervertebral disc, while simultaneously adjusting the overall elastic modulus to adapt to the mechanical properties of the human intervertebral disc. It effectively mimics the biomechanical properties of the human intervertebral disc, accurately reproducing the physiological and mechanical environment of the nucleus pulposus under load, and constructing an in vitro experimental platform that closely resembles the physiological state of the human body. This provides reliable experimental support for research on the physiological mechanisms of the intervertebral disc and nucleus pulposus, disease investigation, and drug development.

Description

Technical Field

[0001] This invention relates to the field of biological experimental equipment technology, specifically to an elastic cavity and a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system. Background Technology

[0002] Intervertebral discs are the core buffering and load-bearing structures between adjacent vertebrae in the human spine, widely distributed in spinal segments such as the cervical, thoracic, lumbar, and coccygeal vertebrae. Figure 1 As shown, the intervertebral disc is mainly composed of a dense outer annulus fibrosus, a central gel-like nucleus pulposus, and cartilaginous endplates on both sides. The unique layered composite structure of the intervertebral disc, with its "annulus fibrosus enveloping the nucleus pulposus," gives the spine excellent resistance to compression, torsion, and shear forces, effectively buffering the mechanical loads generated by bodily movement. The annulus fibrosus primarily bears compressive, tensile, and torsional loads, while the nucleus pulposus mainly bears compressive loads and can evenly transmit stress to the annulus fibrosus. Together, they ensure the stable conduct of normal physiological activities such as flexion, extension, and rotation of each segment of the spine.

[0003] Lower back pain is a prevalent public health problem worldwide, severely impacting people's quality of life. Intervertebral disc degeneration is the leading pathological factor inducing lower back pain, and it can also cause functional abnormalities in multiple spinal segments, including the cervical and thoracic spine. Extensive clinical and basic research has confirmed that imbalances in the biomechanical microenvironment and abnormal biomechanical load stimulation are the core causes that induce and accelerate intervertebral disc degeneration, especially the nucleus pulposus. Therefore, accurately replicating the in vivo physiological and biomechanical environment of the human nucleus pulposus and constructing an in vitro experimental system that closely matches physiological conditions are the core prerequisites and key foundations for in-depth research into the mechanisms of nucleus pulposus degeneration, the development of intervertebral disc repair technologies, and the performance evaluation of related medical devices.

[0004] Bioreactors are core equipment for simulating the in vivo physiological microenvironment in vitro and for conducting tissue and cell culture and pathological mechanism research. Their biomimetic performance and mechanical control precision directly determine the authenticity and reliability of in vitro experimental data, as well as the clinical translational value of related research results. Currently, existing bioreactors used for nucleus pulposus research still have significant technical shortcomings, failing to achieve precise biomimetic replication of the intervertebral disc's physiological and mechanical environment. Specific technical bottlenecks are as follows:

[0005] First, the mechanical simulation results deviate significantly from the actual physiological environment of the human body. Existing nucleus pulposus bioreactors often use rigid materials such as stainless steel, hard plastics, and glass for their chambers. Under physiological loads, these chamber structures are almost unable to undergo elastic deformation, completely losing the elastic characteristics and dynamic deformation capabilities of the human intervertebral disc's annulus fibrosus. During physiological activities, the human intervertebral disc continuously withstands multiple combined loads, including axial compression, circumferential tension, and torsion. The annulus fibrosus can adapt to changes in load and regulate the stress distribution and deformation state within the nucleus pulposus through interfacial coupling. However, rigid chambers impose rigid constraints on the internal nucleus pulposus tissue, cells, or experimental instruments, failing to simulate the dynamic mechanical coupling relationship between the annulus fibrosus and the nucleus pulposus. This results in a severe disconnect between the in vitro constructed mechanical environment and the actual physiological state in vivo.

[0006] Secondly, structural defects in the cavity lead to distorted stress distribution and abnormal physiological responses in nucleus pulposus cells. Under normal physiological conditions, the nucleus pulposus, after bearing pressure loads, can uniformly transfer stress to the surrounding annulus fibrosus, forming a stable and balanced mechanical microenvironment. This microenvironment is crucial for maintaining the normal proliferation, differentiation, and physiological homeostasis of nucleus pulposus cells. Existing rigid cavities disrupt the natural mechanical coupling interface between the annulus fibrosus and the nucleus pulposus, causing disordered stress distribution and imbalanced stress distribution in cultured nucleus pulposus cells. Ultimately, this results in significant differences between cell physiological function and tissue morphology and the normal in vivo state, failing to accurately reflect the physiological characteristics and pathological evolution of nucleus pulposus tissue, thus offering extremely low experimental reference value.

[0007] Finally, the structural parameters of existing reactor chambers do not match the actual physiological parameters of human intervertebral discs. The volume ratio of the annulus fibrosus to the nucleus pulposus is a crucial physiological parameter that determines the overall mechanical conduction and stress distribution of the intervertebral disc. However, existing bioreactor chambers are mostly generic structures that have not been precisely designed for the actual volume ratio of the annulus fibrosus and nucleus pulposus of the human intervertebral disc. The volume ratio of the chamber wall to the containment cavity deviates significantly from human physiological parameters, causing mechanical biomimicry distortion at its structural root. Summary of the Invention

[0008] To address one of the aforementioned technical deficiencies, this application provides an elastic cavity and a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system.

[0009] The present invention adopts the following technical solution:

[0010] In a first aspect, embodiments of this application provide an elastic cavity, comprising: a hollow structure having a receiving cavity, an inlet and an outlet, the inlet and outlet being connected to the receiving cavity, the ratio of the volume of the cavity wall of the hollow structure to the volume of the receiving cavity being (0.6 to 0.7):1, and the elastic modulus of the hollow structure being 0.1 MPa to 10 MPa.

[0011] Optionally, the hollow structure is made of polymer material, and by adjusting the composition parameters and / or preparation process parameters of the polymer material, combined with the geometric design of the hollow structure, the elastic modulus of the hollow structure is made to be 0.1 MPa to 10 MPa.

[0012] The compositional parameters of the polymer material being controlled include any one or more of the following: the ratio of hard and soft segments, the blending ratio with the flexible polymer, and the degree of crystallinity.

[0013] Optionally, the hollow structure may be manufactured in any of the following ways:

[0014] The hollow structure is made using 3D printing technology. By adjusting the composition parameters of the polymer material and / or adjusting the filling density of the 3D printer, the elastic modulus of the hollow structure is made to be 0.1MPa to 10MPa.

[0015] The hollow structure is prepared by casting molding process. By adjusting the composition parameters of the polymer material and / or adjusting the curing parameters of the casting process, the elastic modulus of the hollow structure is made to be 0.1MPa to 10MPa.

[0016] Optionally, the hollow structure is prepared by a 3D printer, and the printing filling path of the 3D printer is a concentric ring, so that the formed hollow structure forms a concentric fiber arrangement structure consistent with the natural intervertebral disc annulus fibrosus.

[0017] Optionally, the hollow structure is made of medical-grade PEBA material, and the PEBA material meets the following mechanical property parameters:

[0018] The Shore hardness is 75A to 90A, the elongation at break is 500% to 800%, the tensile strength is 15MPa to 25MPa, and the elastic recovery rate under 100% strain conditions is not less than 90%.

[0019] Optionally, the hollow structure is cylindrical;

[0020] The hollow structure has a wall thickness of 4mm to 6mm, an inner diameter of 32 to 38mm, and an axial length of 8 to 12mm.

[0021] Optionally, the hollow structure includes:

[0022] The main cylinder has the receiving cavity, the liquid inlet, the liquid outlet, and the port, the port being connected to the receiving cavity;

[0023] Top cover, the top cover being used to close or open the port;

[0024] A sealing ring is disposed between the main cylinder and the upper cover to seal the gap between the upper cover and the main cylinder.

[0025] Fasteners are provided through the upper cover and the main cylinder to fix the upper cover to the main cylinder.

[0026] Secondly, this application provides a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system, comprising:

[0027] Elastic cavity;

[0028] A six-degree-of-freedom mechanical loading module is disposed at both ends of the hollow structure along the length direction and is respectively connected to the elastic cavity. The six-degree-of-freedom mechanical loading module is used to apply external force along the first axis, external force around the first axis, external force along the second axis, external force around the second axis, external force along the third axis, and external force around the third axis to the elastic cavity.

[0029] The first axis is the central axis of the hollow structure, and the second and third axes both extend radially along the hollow structure. The angle between any two of the first, second, and third axes is 90 degrees.

[0030] Secondly, the six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system includes a real-time physicochemical index detection component, which includes:

[0031] A circulation pipeline, the two ends of which are respectively connected to the inlet and outlet of the hollow structure;

[0032] A peristaltic pump, wherein the peristaltic pump is disposed in the circulation pipeline;

[0033] A physicochemical index detection device, which is connected to the circulation pipeline, has multiple parameter detection modules;

[0034] A syringe that can be connected to the circulation tubing.

[0035] Optionally, each of the parameter detection modules includes one or more of a pH sensor, a dissolved oxygen sensor, a temperature sensor, and a pressure sensor.

[0036] By adopting the above technical solution, this application has the following beneficial effects:

[0037] This application achieves high-precision synergistic replication of the intervertebral disc anatomical structure and biomechanical environment through dual optimization of structural proportion biomimicry and mechanical property biomimicry. It overcomes several technical deficiencies of existing bioreactors, such as low biomimicry accuracy, distorted mechanical simulation, and inability to simulate dynamic degeneration processes. It can provide in vitro experimental conditions that are closer to the physiological state of the human body for the study of the physiological mechanism of the nucleus pulposus, the prevention and treatment of intervertebral disc degeneration diseases, the development of repair technology, and the evaluation of related medical devices. It has important scientific research value and broad prospects for clinical translational application. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0039] Figure 1 A schematic diagram of the nucleus pulposus tissue structure in the human body is shown.

[0040] Figure 2 This diagram shows a structural schematic of the main cylinder of the elastic cavity provided in an embodiment of the present disclosure;

[0041] Figure 3 This diagram illustrates the structure of the elastic cavity provided in an embodiment of the present disclosure.

[0042] Figure 4 An exploded view of the elastic cavity provided in an embodiment of this disclosure is shown;

[0043] Figure 5 This diagram illustrates the structure of a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system provided in an embodiment of this disclosure.

[0044] Figure 6 The flowchart shows the cell seeding and static culture process;

[0045] Figure 7 This diagram shows a flowchart of real-time monitoring of physicochemical parameters of a six-DOF multiaxial loading nucleus pulposus bioreactor system;

[0046] Figure 8 This diagram illustrates the self-regulating control flow of a six-DOF multi-axis loaded nucleus pulposus bioreactor system.

[0047] Figure 9 The diagram shows data obtained from a mechanical loading experiment on an elastic cavity.

[0048] In the diagram, 1. Nucleus pulposus tissue; 2. Annulus fibrosus; 3. Hollow structure; 31. Main cylinder; 311. Inlet; 312. Outlet; 32. Top cover; 33. Sealing ring; 34. Fastener; 4. Peristaltic pump; 5. Physicochemical index detection device; 51. pH sensor; 52. Temperature sensor; 53. Dissolved oxygen sensor; 6. Circulation pipeline; 7. Syringe; 8. Six-degree-of-freedom mechanical loading module. Detailed Implementation

[0049] To make the technical solutions and advantages of the embodiments of this application clearer, the exemplary embodiments of this application will be described in further detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not an exhaustive list of all embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0050] like Figures 2 to 8 As shown, this application embodiment provides an elastic cavity (or 3D bionic cavity), including: a hollow structure 3, the hollow structure 3 having a receiving cavity, a liquid inlet 311 and a liquid outlet 312, the liquid inlet 311 and the liquid outlet 312 being connected to the receiving cavity, the ratio of the volume of the cavity wall of the hollow structure 3 to the volume of the receiving cavity being (0.6~0.7):1, and the elastic modulus of the hollow structure 3 being 0.1MPa~10MPa.

[0051] This application precisely replicates the physiological volume ratio of the annulus fibrosus and nucleus pulposus of the human intervertebral disc by limiting the volume ratio of the hollow structure's three-cavity wall to the internal cavity to (0.6-0.7):1, achieving a high degree of biomimicry of the original anatomical structure of the human intervertebral disc at the structural level. Compared with the shortcomings of existing general-purpose cavity structure parameters that deviate significantly from human physiological dimensions and have low structural biomimicry, this application optimizes the design based on the actual structural parameters of the human intervertebral disc, ensuring that the ratio of the elastic structure of the cavity wall to the internal internal cavity space is highly consistent with the physiological state. This fundamentally eliminates the mechanical transmission deviation caused by structural proportion mismatch, laying a structural foundation for accurately replicating the stress distribution within the nucleus pulposus.

[0052] This application employs an elastic hollow structure 3 with an elastic modulus of 0.1 MPa to 10 MPa to replace the traditional rigid cavity, effectively simulating the elastic mechanical properties and dynamic deformation behavior of the human annulus fibrosus. Compared to existing rigid cavities made of stainless steel, hard plastic, etc., which cannot deform and suffer from rigid constraints, the elastic cavity of this application can produce adaptive elastic deformation consistent with the human annulus fibrosus under physiological composite loads such as axial compression, torsion, and shear. It truly restores the dynamic mechanical coupling interface between the annulus fibrosus and the nucleus pulposus, accurately replicates the physiological processes of load transfer and stress redistribution in the intervertebral disc, and significantly improves the biomimetic accuracy of the in vitro mechanical microenvironment.

[0053] This application effectively solves the problems of distorted stress distribution and abnormal physiological responses in nucleus pulposus cells in existing technologies. Based on an elastic cavity structure matching physiological proportions, this application achieves the physiological and mechanical characteristics of uniform stress diffusion and orderly transmission after the nucleus pulposus is compressed, avoiding the drawbacks of local stress concentration and disordered cell stress caused by rigid cavity constraints. This makes the stress state, proliferation and differentiation behavior, and physiological function characteristics of in vitro cultured nucleus pulposus tissue 1 and cells more closely resemble the actual in vivo levels, effectively improving the authenticity, reliability, and reproducibility of in vitro experimental data, and significantly enhancing the experimental precision of nucleus pulposus physiological and pathological studies.

[0054] This application can realistically simulate the dynamic mechanical evolution of intervertebral disc degeneration, meeting the needs of degeneration mechanism research. The elastic cavity in this application can simulate the dynamic mechanical changes of decreased elastic modulus and reduced deformation capacity of the annulus fibrosus during degeneration through mechanical parameter adjustment. This overcomes the technical bottleneck of traditional rigid cavities with fixed mechanical properties, which cannot simulate the dynamic mechanical evolution of degeneration. It can construct an in vitro pathological model that more closely resembles the characteristics of progressive in vivo degeneration, providing a high-quality experimental platform for in-depth understanding of the degeneration mechanism of the intervertebral disc nucleus pulposus and for evaluating the effects of repair drugs and regeneration technologies.

[0055] In summary, this application achieves high-precision synergistic replication of the intervertebral disc anatomical structure and biomechanical environment through dual optimization of structural proportion biomimicry and mechanical property biomimicry. It overcomes several technical shortcomings of existing bioreactors, such as low biomimicry accuracy, distorted mechanical simulation, and inability to simulate dynamic degeneration processes. It can provide in vitro experimental conditions that are closer to the physiological state of the human body for the study of the physiological mechanism of the nucleus pulposus, the prevention and treatment of intervertebral disc degeneration diseases, the development of repair technology, and the evaluation of related medical devices. It has important scientific research value and broad prospects for clinical translational application.

[0056] The hollow structure 3 is roughly cylindrical, and the ratio of its cavity wall volume to the volume of the containing cavity is consistent with the ratio of the volume of the annulus fibrosus to the volume of the nucleus pulposus in a human intervertebral disc. This is the geometric basis for achieving mechanical simulation, ensuring that the relative deformation ratio of the cavity wall and the contents of the cavity is consistent with that of a natural intervertebral disc under stress.

[0057] In some possible implementations, the hollow structure 3 is made of polymer material. By adjusting the composition parameters and / or preparation process parameters of the polymer material, combined with the geometric design of the hollow structure, the elastic modulus of the hollow structure 3 is made to be 0.1 MPa to 10 MPa. The adjustment of the composition parameters of the polymer material includes changing one or more of the following: the ratio of soft and hard segments, the blending ratio with the flexible polymer, and the degree of crystallinity.

[0058] The hollow structure of this application is integrally prepared using biocompatible polymer materials. Through multi-dimensional collaborative design and control of the intrinsic composition parameters of the polymer materials, the preparation process parameters, and the geometric structure of the hollow structure, the overall elastic modulus of the hollow structure can be precisely and stably controlled within a large biomimetic range of 0.1 MPa to 10 MPa. This allows it to match the mechanical properties of the annulus fibrosus of the intervertebral disc at different spinal segments, ages, and degrees of degeneration, achieving a high-precision, adaptable, and gradient-based biomimetic mechanical effect.

[0059] Meanwhile, the overall elasticity and stiffness of the material can be further refined by blending the matrix polymer with different types and proportions of flexible polymers. By changing the doping and blending ratio of the flexible polymer, the flexibility, deformation resistance, and stress relaxation characteristics of the composite material can be continuously controlled without changing the main material system. This allows the mechanical properties of the material to smoothly cover the modulus range of 0.1 MPa to 10 MPa, adapting to the mechanical gradient changes from healthy, highly elastic fiber rings to degenerated, low-elasticity, high-stiffness fiber rings.

[0060] In addition to adjusting material composition parameters, this application also allows for synergistic optimization by combining fabrication process parameters with the geometric design of the hollow structure. By adjusting the aforementioned process parameters such as 3D printing infill density, printing path, curing process, and molding temperature, and combining them with the integrated matching design of geometric structural parameters such as cavity wall thickness, inner diameter, and cavity volume ratio, a second precise correction of the macroscopic equivalent elastic modulus of the hollow structure can be achieved. Compared to single material control methods, this application employs a multi-dimensional coupled control strategy involving intrinsic material modification, process control, and biomimetic geometric structure, enabling continuous, precise, and wide-range adjustable mechanical properties of the cavity. This effectively avoids the shortcomings of single control methods, such as narrow modulus adjustment range, large dispersion of mechanical parameters, and low biomimetic accuracy.

[0061] In some possible implementations, the hollow structure 3 is made using 3D printing technology, and the elastic modulus of the hollow structure 3 is made to be 0.1 MPa to 10 MPa by adjusting the composition parameters of the polymer material and / or adjusting the infill density of the 3D printer.

[0062] The hollow structure 3 of this application can be fabricated using a 3D printing integrated molding process. Compared with traditional methods such as separate assembly and bonding, the 3D printing process can achieve the integral molding of the hollow structure 3 without the need for secondary bonding, assembly and fixing. This can effectively avoid the structural seams, weak mechanical areas and assembly errors that exist in traditional manufacturing processes, and significantly improve the integrity and mechanical consistency of the overall structure.

[0063] In this application, the elastic modulus of the hollow structure 3 can be precisely controlled to the biomimetic range of 0.1 MPa to 10 MPa within the human intervertebral disc biomimetic range by synergistically controlling the composition parameters of the polymer material and the 3D printing process parameters. The polymer material composition parameters can include key parameters such as the ratio of hard and soft segments, the blending ratio with flexible polymers, and crystallinity. By altering the intrinsic mechanical properties of the material, precise control of the cavity's basic elastic properties can be achieved. Simultaneously, by adjusting process parameters such as printing infill density, printing path, layer thickness, and printing rate during the 3D printing process, the porosity and density of the internal support structure can be further controlled without changing the material formulation. This achieves a secondary precise adjustment of the overall macroscopic stiffness and elasticity, ultimately ensuring that the overall elastic modulus of the formed hollow structure 3 precisely matches the mechanical parameter range of the human intervertebral disc annulus fibrosus.

[0064] In some possible implementations, the hollow structure 3 can also be prepared by casting molding. By adjusting the composition parameters of the polymer material and / or adjusting the curing parameters of the casting process, the elastic modulus of the hollow structure 3 can be made to be 0.1 MPa to 10 MPa.

[0065] Hollow structure 3 can also be prepared using a casting molding process. This process offers advantages such as high molding efficiency, strong structural integrity, and compatibility with various polymer materials. It enables the integrated molding of the elastic cavity, avoiding the mechanical defects inherent in modular assembly structures. This approach can precisely control the elastic modulus of the cast hollow structure 3 to the biomimetic range of 0.1 MPa to 10 MPa, similar to that of a human intervertebral disc, by synergistically controlling the composition parameters of the polymer material and the curing parameters of the casting process. The polymer material composition parameters include key parameters such as the ratio of soft to hard segments in the polymer system, the blending ratio with flexible polymers, and crystallinity. By optimizing the material composition ratio, the fundamental elasticity and mechanical stiffness of the matrix can be controlled at the intrinsic level, providing a basic guarantee for the target elastic modulus range of the cavity. Meanwhile, the molding curing process parameters can specifically include curing temperature, curing time, segmented curing rate, vacuum degassing conditions, and curing environment. By precisely adjusting the curing parameters, the degree of polymer crosslinking reaction, the density of molecular chain arrangement, and the microscopic pore structure inside the molded cavity can be effectively controlled. Under the premise of fixed material formulation, the overall mechanical properties of the structure can be further refined, avoiding problems such as mechanical property deviations, structural embrittlement, and insufficient elasticity caused by uneven curing, over-curing, or under-curing. Through the bidirectional adjustable design of material composition and curing process, an integrated hollow structure 3 with an elastic modulus precisely matching the mechanical properties of the annulus fibrosus of the human intervertebral disc can be stably prepared. This adapts to the biomimetic experimental needs of intervertebral discs in different physiological and degenerative states, enriches the preparation process path of the cavity, and improves the engineering feasibility and versatility of the solution.

[0066] In some possible implementations, the hollow structure 3 is fabricated using a 3D printer, with the printing and filling path of the 3D printer being a concentric ring, so that the formed hollow structure 3 forms a concentric fiber arrangement structure consistent with the natural intervertebral disc annulus fibrosus.

[0067] When using 3D printing technology, a ring-shaped infill path can be designed to simulate the concentric circular fiber orientation of natural fibrous rings. By adjusting the printing infill density (e.g., 20%–80%), cavities with different elastic moduli can be fabricated to simulate fibrous rings ranging from normal to different degrees of degradation.

[0068] The hollow structure 3 of this application is fabricated using a 3D printer, and specifically employs a concentric circular ring printing filling path for structural forming. The natural annulus fibrosus of the human intervertebral disc is not an isotropic homogeneous structure, but rather composed of multiple layers of fiber bundles arranged in a regular concentric ring pattern. This unique concentric fiber orientation is the structural basis for the intervertebral disc's excellent circumferential tensile, torsional, and stress circumferential transmission capabilities, directly determining the deformation characteristics and mechanical transmission laws of the intervertebral disc under physiological loads. Traditional cavity printing often uses conventional paths such as cross-shaped or unidirectional parallel filling, resulting in chaotic internal textures and poor uniformity of anisotropic mechanical properties. It fails to replicate the natural anisotropic mechanical characteristics of the annulus fibrosus, leading to low biomimetic accuracy.

[0069] This implementation scheme employs a dedicated concentric ring-shaped printing and filling path to create a concentric ring-shaped biomimetic fiber arrangement within the three-cavity wall of the formed hollow structure, mirroring the height of the natural intervertebral disc annulus fibrosus. This precisely replicates the original tissue topology of the human annulus fibrosus at the microscopic level. This structural design ensures that the circumferential tensile, torsional, and annular stress transmission characteristics of the biomimetic cavity closely match those of the real annulus fibrosus, effectively replicating the anisotropic mechanical response of the intervertebral disc under physiological conditions. It overcomes the technical shortcomings of conventionally printed structures, which exhibit isotropic mechanical properties and do not conform to the mechanical behavior of the real annulus fibrosus, further enhancing the overall biomimeticity and mechanical simulation accuracy of the reactor.

[0070] Furthermore, based on the aforementioned concentric ring-shaped filling path, the elastic modulus of the cavity can be gradient-adjusted by precisely controlling the infill density of 3D printing. The controllable range of the infill density is set to 20%–80%. Specifically, under the premise of a fixed polymer material formulation and printing path, changing the ring-shaped infill density can directly control the internal density, porosity, and interlayer bonding strength of the cavity wall structure, thereby achieving continuous adjustment of the overall elastic modulus. Among them, a lower infill density corresponds to higher porosity, lower stiffness, and better elasticity of the cavity structure, which can simulate the mechanical properties of a healthy annulus fibrosus with high elasticity and high toughness under normal physiological conditions. As the infill density gradually increases, the density of the cavity structure increases, the elastic modulus gradually increases, and the structural toughness and deformation capacity gradually decrease. This can accurately simulate the progressive pathological characteristics of decreased elasticity, abnormal stiffness, and deterioration of mechanical properties of the annulus fibrosus during mild, moderate, and severe degeneration of the intervertebral disc.

[0071] This implementation scheme, through the combined design of "concentric circular annular biomimetic printing path and gradient filling density control", not only achieves precise biomimicry of the microscopic topology of the annulus fibrosus, but also enables the batch production of biomimetic cavities adapted to the mechanical properties of intervertebral discs under normal physiological conditions and at different stages of degeneration through simple adjustment of process parameters. It can construct a gradient in vitro experimental model of intervertebral disc degeneration, greatly improving the applicability and experimental accuracy of this bioreactor in scenarios such as nucleus pulposus degeneration mechanism research, pathological simulation of different degrees of degeneration, and evaluation of the effects of repair drugs.

[0072] In some possible implementations, the hollow structure 3 is made of medical-grade PEBA material, which meets the following mechanical property parameters: Shore hardness of 75A to 90A, elongation at break of 500% to 800%, tensile strength of 15MPa to 25MPa, and elastic recovery rate of not less than 90% under 100% strain conditions.

[0073] The hollow structure 3 of this application is preferably made of medical-grade PEBA material. Medical-grade PEBA is a thermoplastic elastomer material with excellent biocompatibility, physicochemical stability and fatigue deformation resistance. It is non-cytotoxic and non-irritating to tissues, and can meet the biosafety requirements of long-term in vitro tissue culture and dynamic mechanical loading experiments. It can be stably applied in in vitro biomimetic research scenarios related to intervertebral disc nucleus pulposus.

[0074] Specifically, the Shore A hardness range of 75A to 90A gives the material the flexibility and supporting stiffness to match the human annulus fibrosus. This avoids excessive rigidity and insufficient deformation capacity due to excessive hardness, and excessive softness and insufficient mechanical support due to insufficient hardness, allowing for precise simulation of the flexible load-bearing characteristics of the annulus fibrosus under different physiological conditions. The high elongation at break of 500% to 800% endows the cavity with excellent tensile deformation properties, completely replicating the large deformation behavior of the human intervertebral disc under combined physiological loads such as tension, torsion, and bending, thus avoiding failure problems such as structural cracking and damage during experiments.

[0075] Meanwhile, the tensile strength range of 15MPa to 25MPa ensures the structural stability of the hollow structure 3 under long-term periodic dynamic mechanical loading conditions. It can withstand repeated compression, torsion, and shear loads, and is not prone to fatigue damage and plastic deformation, meeting the experimental requirements for constructing long-term in vitro degeneration models. A high elastic recovery rate of no less than 90% ensures that the cavity can quickly and efficiently recover its initial structural morphology after undergoing large strain deformation, with extremely low residual deformation. This closely matches the reversible elastic physiological characteristics of the human body's natural annulus fibrosus under stress deformation and unloading reset, effectively replicating the dynamic mechanical response mechanism of the intervertebral disc.

[0076] By selecting medical-grade PEBA material within the aforementioned mechanical property range, the elastic cavity of this application can achieve a high degree of matching with the mechanical properties of the human intervertebral disc annulus fibrosus at the intrinsic material level. Combined with the aforementioned volume ratio structure design and molding process control, it can accurately reproduce the elastic deformation law and stress transmission characteristics of the natural intervertebral disc, effectively improving the authenticity and experimental reliability of the in vitro mechanical biomimetic model of the intervertebral disc.

[0077] In some possible implementations, the hollow structure 3 is cylindrical, with a wall thickness of 4mm to 6mm, an inner diameter of 32 to 38mm, and an axial length of 8 to 12mm. Its dimensions can match the macroscopic dimensions of the human L4-L5 intervertebral disc.

[0078] This set of size ranges is designed based on the conventional physiological size range of the human lumbar intervertebral disc, which can effectively simulate the overall spatial configuration and volume ratio of the human intervertebral disc. By adopting the above-mentioned interval structural size design, this embodiment can not only highly replicate the macroscopic anatomical structure of the human intervertebral disc, ensuring a high degree of consistency between the in vitro model and the in vivo physiological structure, but also adapt to the biomimetic simulation needs of different human body sizes and different spinal segments, further improving the overall accuracy and experimental versatility of the reactor's mechanical biomimetic design.

[0079] In some possible implementations, the hollow structure 3 can be manufactured as a single piece or as separate parts. Taking a separate part configuration as an example, such as... Figure 3 and Figure 4As shown, the hollow structure 3 includes: a main cylinder 31, a top cover 32, a sealing ring 33, and a fastener 34. The main cylinder 31 has the receiving cavity, the liquid inlet 311, the liquid outlet 312, and a port, the port communicating with the receiving cavity. The top cover 32 is used to close or open the port. The sealing ring 33 is disposed between the main cylinder 31 and the top cover 32 to close the gap between the top cover 32 and the main cylinder 31. The fastener 34 passes through the top cover 32 and the main cylinder 31, fixing the top cover 32 to the main cylinder 31.

[0080] The hollow structure 3 of this application can be flexibly molded into various forms according to experimental conditions and usage requirements. It can be manufactured as a single, sealed structure using an integral molding process to meet the needs of long-term steady-state mechanical culture and uninterrupted dynamic loading experiments; or it can be designed as a detachable, modular structure to suit experiments requiring frequent sampling, implantation, cavity cleaning, and consumable replacement, effectively improving the adaptability and versatility of the device. This detachable hollow structure 3 mainly includes a main cylinder 31, a top cover 32, a sealing ring 33, and fasteners 34. The main cylinder 31 serves as the core support and main structure for culture, with an internal cavity for placing nucleus pulposus tissue 1, cell slides, cell scaffolds, or experimental samples. The sidewalls of the cylinder have independent inlet ports 311 and outlet ports 312, allowing for the circulation and dynamic replacement of culture medium or drug solutions within the cavity. Simultaneously, the upper end of the main cylinder 31 has a port connecting to the cavity, providing an operational channel for sample insertion, removal, and internal maintenance. The upper cover 32 is designed to match the port size of the main cylinder 31, allowing for flexible opening and closing of the port. When the cavity is open, operators can easily implant and install experimental samples such as tissue engineering constructs and large-size repair scaffolds. Samples can also be removed from the cavity without damage after the experiment. When the cavity is closed, the upper cover 32 completely covers and seals the port of the main cylinder 31. Combined with subsequent sealing and locking structures, a sealed biomimetic culture cavity is formed, ensuring the stability of the physical, chemical, and mechanical environments within the cavity.

[0081] To address the issues of gaps, liquid leakage, and air leakage at the joint surfaces of the split structure, this application incorporates a sealing ring 33 between the mating surfaces of the main cylinder 31 and the upper cover 32. The sealing ring 33 can be made of a flexible material with excellent biocompatibility and elastic sealing performance, such as silicone rubber. The sealing ring 33 effectively fills the assembly gaps between the upper cover 32 and the main cylinder 31, eliminating leakage channels caused by structural assembly gaps and achieving a full circumferential seal at the joint surfaces. This effectively ensures the liquid and air tightness of the cavity, preventing problems such as culture medium leakage, external bacterial intrusion, and pressure loss during experiments, thus providing a fundamental guarantee for a closed-loop in vitro culture environment.

[0082] Fastener 34, as a locking and fixing component of the split structure, can adopt various detachable locking structures such as bolts, snaps, and threaded caps. Fastener 34 passes through the corresponding assembly holes of the upper cover 32 and the main cylinder 31, realizing precise alignment, compression fixing, and detachable connection between the upper cover 32 and the main cylinder 31. Through the locking action of fastener 34, a uniform clamping force can be applied to the mating surface of the upper cover 32 and the main cylinder 31, so that the sealing ring 33 continues to maintain a compressed sealing state, further improving the sealing stability of the cavity; at the same time, fastener 34 can be repeatedly disassembled and locked, realizing multiple opening and closing and reuse of the cavity.

[0083] The modular, detachable structural design employed in this application overcomes the technical limitations of integrated, sealed chambers that hinder convenient sample implantation and retrieval. It is a core structural guarantee for achieving the implantation of large-volume tissue engineering constructs, mechanical evaluation of large-size intervertebral disc repair scaffolds, and non-destructive removal of experimental samples after culture. This structure significantly reduces the difficulty of sample loading / unloading and chamber maintenance while ensuring the reliability of the sealed culture environment. It is adaptable to various complex in vitro intervertebral disc experimental conditions, significantly improving the operational convenience and experimental applicability of the bioreactor. Furthermore, its reusable design effectively reduces the cost of experimental consumables.

[0084] This application allows for targeted optimization of the fluid interface, inner surface of the cavity, and cavity filling medium of the hollow structure 3, further enhancing the biomimetic culture performance and experimental adaptability of the device. Specifically, the inlet 311 and outlet 312 on the side wall of the main cylinder 31 can adopt a standard Luer connector structure. This type of connector is a universal standard interface for biological experimental equipment, with strong adaptability and versatility. It can quickly connect to external perfusion pipelines, fluid circulation equipment, and cell inoculation devices, and can stably realize the closed-loop circulation and replacement of culture medium or drug solution in the cavity, ensuring the dynamic steady-state supply of the physicochemical environment during the culture process. At the same time, it can assist in the quantitative inoculation of cell suspension in the cavity, meeting the experimental needs of dynamic inoculation and long-term perfusion culture of nucleus pulposus cells. The interface is easy to assemble and disassemble, and has good sealing performance, which can effectively avoid problems such as leakage and contamination during fluid transportation.

[0085] Furthermore, this application allows for plasma grafting modification of the inner surface of the cavity. By altering the physicochemical properties of the cavity substrate surface through plasma surface modification technology, bioactive molecules such as type I collagen and hyaluronic acid can be effectively grafted and fixed onto the inner surface of the cavity. These bioactive molecules are highly similar to the components of the extracellular matrix of human intervertebral discs, which can significantly improve the biocompatibility of the inner surface of the cavity, optimize the microenvironment for cell adhesion, spreading, and proliferation, improve the interaction between the cell and cavity interface, and effectively solve the problems of weak cell adhesion and poor cell colonization on the surface of polymer cavity materials. This provides excellent interfacial support conditions for the stable culture of nucleus pulposus cells and tissue engineering constructs.

[0086] In addition, the cavity can be selectively filled with flexible biomimetic media such as sodium alginate hydrogel or hyaluronic acid hydrogel, depending on experimental needs. These hydrogels possess high water content, excellent rheological properties, and adjustable mechanical flexibility, closely resembling the gel-like physiological morphology and fluid dynamics of the human nucleus pulposus. They can accurately simulate pressure transmission, fluid flow, and load buffering characteristics within the nucleus pulposus, further compensating for the shortcomings of pure cavity structures in biomimetic nucleus pulposus fluid dynamics. By matching the elastic cavity structure with the biomimetic hydrogel medium, a highly biomimetic extracorporeal microenvironment of the intervertebral disc in multiple dimensions—structure, mechanics, fluidity, and extracellular matrix—can be constructed, significantly improving the experimental realism and accuracy of degeneration model construction, mechanical mechanism research, and drug efficacy evaluation.

[0087] Figure 9 The figure shows data obtained from a mechanical loading experiment on the elastic cavity of this application. The horizontal axis represents displacement (mm), and the vertical axis represents load (N). As can be clearly seen from the figure, the mechanical load on the elastic cavity increases linearly within the range of approximately 0-4 mm. After exceeding 4 mm, the data decreases, indicating that the elastic cavity has been damaged. The curve shows that the modulus of the elastic cavity is approximately 10 MPa, which falls within the range of elastic modulus of the annulus fibrosus tissue of the human intervertebral disc.

[0088] like Figure 5 As shown, this application also provides a six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system, including: the aforementioned elastic cavity and a six-degree-of-freedom mechanical loading module 8. The six-degree-of-freedom mechanical loading module 8 is disposed at both ends of the hollow structure 3 along its length and is respectively connected to the elastic cavity. The six-degree-of-freedom mechanical loading module 8 is used to apply external forces along a first axis, circumferentially around the first axis, along a second axis, circumferentially around the second axis, along a third axis, and circumferentially around the third axis to the elastic cavity. The first axis is the central axis of the hollow structure 3, and the second and third axes both extend radially along the hollow structure 3. The angle between any two of the first, second, and third axes is ninety degrees.

[0089] Thanks to the dual matching of volume ratio and elastic modulus mentioned above, the elastic cavity can highly simulate the "bulging" behavior of the human annulus fibrosus under physiological load by the pressure and / or displacement applied by the six-degree-of-freedom mechanical loading module in the axial and circumferential directions, thereby making the mechanical environment inside the cavity (such as pressure distribution and hydrostatic pressure) close to the mechanical microenvironment of the nucleus pulposus tissue 1 in the human body.

[0090] The loading parameters of the six-degree-of-freedom mechanical loading module are programmable and adjustable. The axial compressive stress ranges from 0.1 to 1.0 MPa at a frequency of 0.5 to 2 Hz, the torsional angle ranges from ±1.5° to ±3.0° at a frequency of 0.2 to 1 Hz, the lateral bending angle ranges from ±2.0° to ±5.0° at a frequency of 0.2 to 1 Hz, and the forward and backward flexion-extension angle ranges from ±3.0° to ±8.0° at a frequency of 0.2 to 1 Hz.

[0091] In some possible implementations, the six-degree-of-freedom multiaxially loaded nucleus pulposus bioreactor system includes a real-time physicochemical parameter detection component. This component comprises a circulation pipeline 6, a peristaltic pump 4, a physicochemical parameter detection device 5, and a syringe 7. The two ends of the circulation pipeline 6 are connected to the inlet 311 and outlet 312 of the hollow structure 3, respectively. The peristaltic pump 4 is located within the circulation pipeline 6. The physicochemical parameter detection device 5 is connected to the circulation pipeline 6 and has multiple parameter detection modules. The syringe 7 can be connected to the circulation pipeline 6. Each parameter detection module includes one or more of a pH sensor 51, a dissolved oxygen sensor 53, a temperature sensor 52, and a pressure sensor.

[0092] The six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system of this application is equipped with a real-time physicochemical index detection component for continuous monitoring and closed-loop control of the dynamic culture environment inside the chamber. This ensures that key physiological parameters such as temperature, dissolved oxygen, pH, and fluid pressure of the in vitro nucleus pulposus culture system are maintained within the standard range for human physiological parameters. This avoids fluctuations in the physicochemical environment during prolonged dynamic mechanical loading and culture medium circulation, which can lead to decreased cell activity, model distortion, and poor experimental reproducibility. The real-time physicochemical index detection component mainly includes a circulation pipeline 6, a peristaltic pump 4, a physicochemical index detection device 5, and a syringe 7 auxiliary module, forming an integrated monitoring system with closed-loop circulation, real-time sampling, and dynamic detection.

[0093] Specifically, the two ends of the circulation pipeline 6 are respectively connected to the inlet 311 and outlet 312 of the hollow structure 3, forming a complete closed-loop circulation pathway for the culture medium together with the internal cavity. The peristaltic pump 4, as the power source for fluid circulation, is arranged in the circulation pipeline 6, providing a stable, continuous, and low-shear driving force for the culture medium, buffer solution, or drug working solution in the pipeline. This meets the perfusion culture requirements while avoiding damage to fragile nucleus pulposus cells and tissue structures caused by high-shear fluid. The physicochemical index detection device 5 is connected in series in the circulation pipeline 6, which can collect various physiological parameters of the circulating fluid in real time. The syringe 7 can be detachably connected to the reserved interface of the circulation pipeline 6 for cell seeding, fluid replenishment, drug addition, sampling, and pipeline venting in the early stage of the experiment, improving the operational flexibility of the system.

[0094] The physicochemical index detection device 5 integrates a multi-parameter detection module, which can be equipped with any one or more combinations of pH sensor 51, dissolved oxygen sensor 53, temperature sensor 52, and pressure sensor according to experimental needs, to achieve simultaneous monitoring of multi-dimensional environmental parameters. To minimize interference from invasive detection on the flow field, stress field, and tissue samples, all sensors in this application adopt a non-invasive optical sensing detection structure, eliminating the need for probes to penetrate the fluid and preserving the stability of the pipeline flow field and the microenvironment of the cavity's mechanics, making it fully adaptable to high-precision biomimetic mechanical experimental scenarios. Simultaneously, this system possesses ultra-high detection accuracy, specifically: pH detection accuracy up to ±0.01, dissolved oxygen detection accuracy up to ±0.5% air saturation, temperature detection accuracy up to ±0.1℃, and fluid pressure detection accuracy up to ±0.01MPa. It can accurately capture minute fluctuations in physicochemical parameters during cultivation, realistically reproducing subtle changes in the dynamic microenvironment within the body, greatly improving the accuracy and reliability of in vitro experimental data.

[0095] Furthermore, the real-time physicochemical index detection component of this application is equipped with an independent parameter control component, forming a closed-loop control system of "real-time monitoring - data comparison - automatic intervention - parameter stabilization" with the multi-parameter detection device, thereby realizing intelligent dynamic correction of the culture environment. Specifically, the control component may include a drug supply unit, a gas supply unit, a temperature regulation unit, a pressure stabilization unit, and corresponding electronically controlled valve groups and intelligent control modules, enabling precise and zoned intervention and control for deviations in different physicochemical parameters.

[0096] For example, when pH sensor 51 detects that the pH value of the circulating culture medium deviates from the physiological standard range, the intelligent control module can automatically identify the deviation value and drive the drug supply unit to work. By precisely pushing acidic or alkaline adjusting solution, the pH of the system in the circulating pipeline 6 is finely adjusted, neutralizing acidic metabolic products or buffering system fluctuations in real time, so that the pH in the cavity quickly returns to the normal physiological range, avoiding acid-base imbalance that induces apoptosis and metabolic abnormalities in nucleus pulposus cells. When dissolved oxygen sensor 53 detects that the dissolved oxygen concentration in the culture medium is lower than the physiological threshold, the system automatically starts the gas supply unit. Through micro-nano aeration, oxygen-enriched pipeline osmosis, or precise oxygen introduction, the dissolved oxygen content of the culture medium is increased to maintain the normal aerobic metabolic level of nucleus pulposus cells and adapt to the oxygen supply environment of the intervertebral disc tissue in the body.

[0097] Correspondingly, the temperature control unit can compensate for system temperature fluctuations in real time through pipeline temperature tracing, water bath heat exchange, or semiconductor temperature control structures, ensuring that cell culture remains in a constant physiological temperature state. The pressure stabilization unit can work with the pipeline valve group to dynamically balance the fluid pressure in the circulation pipeline 6 and the cavity, simulating the physiological pressure fluctuation range of the intervertebral disc in vivo, avoiding distortion of the mechanical microenvironment caused by excessively high or low pressure. This application, through the integrated design of detection and control, enables the entire bioreactor culture system to have self-detection, self-correction, and self-adaptation capabilities, effectively solving the defects of traditional in vitro intervertebral disc culture systems such as unstable physicochemical environment, large parameter drift, and inability to dynamically intervene, further ensuring the experimental stability and reproducibility of degeneration model construction, drug screening, and mechanical mechanism research.

[0098] A method for preparing an elastic cavity for in vitro culture of intervertebral discs may include the following steps:

[0099] A three-dimensional model of the elastic cavity is established, wherein the ratio of the elastic cavity wall volume to the cavity volume of the hollow structure 3 is consistent with the ratio of the annulus fibrosus volume to the nucleus pulposus volume of the human intervertebral disc.

[0100] Choose biocompatible polymer materials;

[0101] By adjusting the composition or preparation process parameters of the polymer material, the overall elastic modulus of the molded cavity can be matched with that of human intervertebral disc tissue.

[0102] The elastic cavity is fabricated using a one-piece molding technique.

[0103] The present application will be described in detail below with reference to specific embodiments:

[0104] Example 1: Fabrication of an elastic cavity using 3D printing (adjusting the filler density)

[0105] This embodiment details a preparation method.

[0106] 1. Modeling and Volume Ratio Design: A cavity model was created using SolidWorks, with an outer diameter of 45 mm, an inner diameter of 35 mm, a height of 10 mm, and a wall thickness of 5 mm. The calculated volume of the elastic cavity wall is approximately π × (45 mm). 2 -35 2 ) / 4×10 ≈ 6283 mm 3 The internal volume of the cavity is approximately π × 35 2 / 4×10 ≈ 9621 mm 3 The volume ratio is approximately 0.65:1. This is consistent with the reported volume ratio of the annulus fibrosus to the nucleus pulposus in the human L4-L5 intervertebral disc (approximately 0.6-0.7:1 in the literature).

[0107] 2. Materials and Printing: Medical-grade 85A PEBA material is used. Printing parameters: nozzle 0.4 mm, layer height 0.1 mm, nozzle temperature 240℃, heated bed temperature 60℃, printing speed 40 mm / s. The printing infill path adopts a concentric circular ring design with an interlayer rotation angle of 0° to simulate the lamellar fiber orientation of natural fiber rings.

[0108] 3. Adjusting the elastic modulus: Three cavities were prepared with filling densities of 80%, 50%, and 20%, respectively, corresponding to the mechanical properties of normal, moderately degenerated, and severely degenerated fiber rings.

[0109] 4. Post-processing: Soak in 75% ethanol for 30 min, rinse with PBS, and irradiate with ultraviolet light for 30 min.

[0110] Example 2: Fabrication of an elastic cavity using hot-melt molding technology (adjusting material parameters)

[0111] 1. Mold preparation:

[0112] The male mold is fabricated according to the target intervertebral disc structure dimensions, and a high-temperature resistant female mold (aluminum or high-temperature silicone mold can be used) is prepared to ensure the accuracy of the cavity structure.

[0113] 2. Material selection and pretreatment:

[0114] Medical-grade PEBA thermoplastic elastomer particles are selected. The mechanical properties of the molded cavity can be altered by adjusting the material parameters according to the elasticity of the target cavity, such as selecting different hardness grades (e.g., Shore A 35–85) or adjusting the porosity / filling rate within the particles.

[0115] 3. Melt casting:

[0116] Heat PEBA granules to a molten state (180–230°C, specific temperature depends on the model), remove air bubbles or air bubbles, and then inject them into the mold cavity. Ensure complete filling of the structure and avoid air bubbles or defects.

[0117] 4. Cooling and demolding

[0118] The material is cooled to room temperature or by a programmed process until it is fully shaped (by crystallization), and then demolded to obtain a one-piece elastic cavity.

[0119] 5. Mechanical property control

[0120] The elastic modulus of the intervertebral disc cavity can be continuously adjusted by changing the PEBA hardness rating, porosity, or structural parameters to simulate the mechanical characteristics of intervertebral discs with different degrees of degeneration. Experimental tests show that increasing the proportion of soft segments or porosity reduces the cavity modulus, while increasing hardness or density increases the modulus.

[0121] Example 3: Assembly and Sterilization of the Reactor System

[0122] Please see Figure 6 As shown, the elastic cavity prepared in Example 1 or Example 2 is installed in a biomechanical reactor.

[0123] If an integrated elastic cavity is used, during installation, first fit the elastic cavity onto the positioning mandrel of the reactor to ensure a tight fit; then, make contact with the upper loading head and lock it in place to ensure uniform force distribution.

[0124] If a split-type elastic cavity is used, the assembly and sealing steps are as follows:

[0125] The sterilized sealing ring 33 (such as a medical-grade silicone O-ring) is precisely placed in the sealing groove on the port of the main cylinder. The compression deformation of the sealing ring 33 is precisely controlled by a torque wrench with a torque value of 0.5-0.8 N·m.

[0126] Inside a clean bench, tissues to be cultured, cell-hydrogel complexes, or repair scaffolds to be evaluated are implanted into the cavity. For example, sodium alginate hydrogel loaded with nucleus pulposus cells can be directly injected or placed into the receiving cavity.

[0127] Replace the top cover 32, ensuring that the pressure ring of the top cover 32 is in uniform contact with the sealing ring 33. Use a torque wrench to tighten the circumferentially distributed locking bolts in a diagonal sequence (or engage the integrated clips) to cause the sealing ring 33 to undergo 20%-30% compression deformation, thereby achieving reliable liquid and air tightness. This sealing structure has been tested to withstand an internal pressure of at least 0.2 MPa without leakage under subsequent six-degree-of-freedom mechanical loading and circulating perfusion conditions.

[0128] After sealing, the liquid circulation pipeline 6 is connected through the upper cover 32 and the Luer port integrated on the cavity.

[0129] The chamber is connected to the external peristaltic pump 4 and culture medium reservoir via an integrated Luer port, forming a closed-loop circulation circuit. The initial flow rate is set to 1–5 mL / min.

[0130] After assembly, the entire device is sterilized with ethylene oxide gas. Following sterilization, the culture medium reservoir and peristaltic pump 4 are connected inside the laminar flow hood, and the airtightness is checked. Peristaltic pump 4 is then started, and the tubing and interior of the chamber are flushed with sterile culture medium for 3-5 minutes to remove air bubbles and pre-humidify.

[0131] Example 4: Seeding and Static Culture of Nucleus Pulp Cells

[0132] Primary isolated nucleus pulposus cells were digested and resuspended in DMEM / F12 complete medium, with the density adjusted to 1×10⁶ cells / day. 6 -5×10 6 cell / mL.

[0133] Inside the clean bench, the cell suspension is injected into the elastic cavity through an integrated port. In this embodiment, the elastic cavity is pre-filled with sodium alginate hydrogel to simulate the nucleus pulposus matrix; the cell suspension is mixed with the hydrogel before injection. Alternatively, the inner surface of the cavity is plasma-modified, and cells are directly seeded onto the inner surface. Driven by a peristaltic pump 4 at a low flow rate (approximately 0.5 mL / min), the cells can be evenly distributed within the cavity. After seeding, the peristaltic pump 4 is turned off, and the reactor is placed in a CO2 incubator for 4-6 hours to allow the cells to adhere fully and grow.

[0134] After cell adhesion, start peristaltic pump 4 and circulate the medium at a flow rate of 1-3 mL / min to begin static culture. Change the medium twice a week, 30-50 mL each time. Observe cell growth under a microscope regularly and detect proliferation activity using a CCK-8 assay kit. Live / dead cell staining (Calcein-AM / PI) results showed that the cells were evenly distributed within the cavity with no significant regional differences.

[0135] Example 5: Bionic Cultivation under Six Degrees of Freedom Mechanical Loading

[0136] The elastic cavity containing cells is mounted on a six-degree-of-freedom mechanical loading module. Its standard interface ensures direct compatibility with commercially available systems.

[0137] The loading program is configured to simulate the biomechanical environment under daily human activities, with typical parameters as follows:

[0138] Axial compression: stress 0.1-1.0MPa, frequency 0.5-2 Hz, single loading 1000-5000 cycles;

[0139] Torsion: Angle ±1.5° to ±3.0°, frequency 0.2-1 Hz;

[0140] Lateral bending: Angle ±2.0° to ±5.0°, frequency 0.2-1 Hz;

[0141] Forward and backward flexion / extension: angle ±3.0° to ±8.0°, frequency 0.2-1 Hz.

[0142] The loading waveform should preferably be a sine wave to simulate the smooth mechanical properties of human physiological activities; if impact loads are required, square or triangular waves can be used, with continuous loading for 2-8 hours / day and continuous culture for 7-28 days. Simultaneously with mechanical loading, a circulating perfusion system (flow rate 0.5-2 mL / min) should be started to provide nutrients to the cells and remove waste.

[0143] To simulate the degeneration process, cavities with different filling densities (i.e. different stiffness) can be compared and the differences in cell survival rate, extracellular matrix synthesis (such as COL2A1 and ACAN gene expression), and inflammatory factor secretion (such as IL-1β and TNF-α) can be observed under the same loading conditions.

[0144] Example 6: Real-time online monitoring of physicochemical indicators

[0145] Please see Figure 7 and Figure 8 An online sensor module (parameter detection module) is connected to the circulation pipeline 6 to monitor dissolved oxygen, pH, temperature, and pressure in real time. Preferably, the pH and dissolved oxygen sensors 53 adopt optical sensing, integrating a fluorescent indicator patch into the inner wall of the port, and measuring non-contactly through a fiber optic probe.

[0146] All sensor signals are conditioned, amplified, and converted from analog to digital before being transmitted to the host computer for real-time display and recording of parameter changes. Automatic alarms are triggered when thresholds are exceeded. Typical monitoring range: dissolved oxygen 40%-80% air saturation, pH 7.2-7.4, temperature 36.5℃-37.5℃.

[0147] Example 7: Sample processing and detection after culture

[0148] After culture, disassemble the reactor, remove the cell spreaders or cell scaffolds from the elastic cavity, and rinse with sterile PBS. The following assays can then be performed:

[0149] (1) Cell viability: Calcein-AM / PI staining and confocal microscopy observation showed that cell viability was usually >80% under this system.

[0150] (2) Extracellular matrix: After paraffin sectioning, Sirius red staining (collagen) and Alcian blue staining (proteoglycan) were performed.

[0151] (3) Gene expression: Total RNA was extracted and the expression of genes such as COL1A1, COL2A1, ACAN, MMP-3, and MMP-13 was detected by RT-qPCR.

[0152] (4) Mechanical properties: The cavity was compressed before and after culture using a universal testing machine to evaluate the effects of cell growth and mechanical loading on the mechanical properties of the cavity itself.

[0153] (5) Sterility and biocompatibility testing: Sterility tests should be performed on culture media periodically. In accordance with ISO 10993, cytotoxicity, cell adhesion and hemolysis tests should be performed on the materials before formal cell experiments, and the results should meet the requirements for medical materials.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the scope of the concept and principles of the present invention should be included within the protection scope of the present invention.

[0155] (6) Histological observation: Frozen sections were prepared and immunofluorescence staining was performed to detect the expression of type II collagen, aggregates, and SOX9, and to assess the maintenance of the phenotype of nucleus pulposus cells.

[0156] The foregoing disclosure provides many different embodiments or examples for implementing different structures of this application. To simplify the disclosure, specific examples of components and arrangements are described above. Of course, these are merely examples and are not intended to limit the scope of this application. Furthermore, reference numerals and / or letters may be repeated in different examples; such repetition is for simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed. In addition, examples of various specific processes and materials are provided in this application, but those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0157] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0158] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. An elastomeric chamber characterized by, include: A hollow structure having a receiving cavity, an inlet and an outlet, the inlet and outlet being connected to the receiving cavity, the ratio of the volume of the cavity wall to the volume of the receiving cavity being (0.6-0.7):1, and the elastic modulus of the hollow structure being 0.1 MPa to 10 MPa.

2. The elastic cavity according to claim 1, characterized in that, The hollow structure is made of polymer material. By adjusting the composition parameters and / or preparation process parameters of the polymer material, combined with the geometric design of the hollow structure, the elastic modulus of the hollow structure is made to be 0.1 MPa to 10 MPa. The compositional parameters of the polymer material being controlled include any one or more of the following: the ratio of hard and soft segments, the blending ratio with the flexible polymer, and the degree of crystallinity.

3. The elastic cavity according to claim 2, characterized in that, The hollow structure is manufactured using any of the following methods: The hollow structure is made using 3D printing technology. By adjusting the composition parameters of the polymer material and / or adjusting the filling density of the 3D printer, the elastic modulus of the hollow structure is made to be 0.1 MPa to 10 MPa. The hollow structure is prepared by casting molding process. By adjusting the composition parameters of the polymer material and / or adjusting the curing parameters of the casting process, the elastic modulus of the hollow structure is made to be 0.1 MPa to 10 MPa.

4. The elastic cavity according to claim 3, characterized in that, The hollow structure is fabricated using a 3D printer. The printing and filling path of the 3D printer is a concentric ring, which makes the formed hollow structure form a concentric fiber arrangement structure consistent with the natural intervertebral disc annulus fibrosus.

5. The elastic cavity according to claim 1, characterized in that, The hollow structure is made of medical-grade PEBA material, and the PEBA material meets the following mechanical property parameters: The Shore hardness is 75A to 90A, the elongation at break is 500% to 800%, the tensile strength is 15MPa to 25MPa, and the elastic recovery rate under 100% strain conditions is not less than 90%.

6. The elastic cavity according to claim 1, characterized in that, The hollow structure is cylindrical; The hollow structure has a wall thickness of 4mm to 6mm, an inner diameter of 32 to 38mm, and an axial length of 8 to 12mm.

7. The elastic cavity according to any one of claims 1-6, characterized in that, The hollow structure includes: The main cylinder has the receiving cavity, the liquid inlet, the liquid outlet, and the port, the port being connected to the receiving cavity; Top cover, the top cover being used to close or open the port; A sealing ring is disposed between the main cylinder and the upper cover to seal the gap between the upper cover and the main cylinder. Fasteners are provided through the upper cover and the main cylinder to fix the upper cover to the main cylinder.

8. A six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system, characterized in that, include: The elastic cavity as described in any one of claims 1-7; A six-degree-of-freedom mechanical loading module is disposed at both ends of the hollow structure along the length direction and is respectively connected to the elastic cavity. The six-degree-of-freedom mechanical loading module is used to apply external force along the first axis, external force around the first axis, external force along the second axis, external force around the second axis, external force along the third axis, and external force around the third axis to the elastic cavity. The first axis is the central axis of the hollow structure, and the second and third axes both extend radially along the hollow structure. The angle between any two of the first, second, and third axes is 90 degrees.

9. The six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system according to claim 8, characterized in that, The system includes a real-time detection component for physicochemical indicators, which comprises: A circulation pipeline, the two ends of which are respectively connected to the inlet and outlet of the hollow structure; A peristaltic pump, wherein the peristaltic pump is disposed in the circulation pipeline; A physicochemical index detection device, which is connected to the circulation pipeline, has multiple parameter detection modules; A syringe that can be connected to the circulation tubing.

10. The six-degree-of-freedom multiaxial loading nucleus pulposus bioreactor system according to claim 9, characterized in that, Each of the parameter detection modules includes one or more of a pH sensor, a dissolved oxygen sensor, a temperature sensor, and a pressure sensor.

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