Micro-fluidic chip, system and method for osteochondral organs

Abstract

The invention provides a microfluidic chip, system and method for osteochondral organs, and relates to the technical field of organoid microfluidic chips, the chip comprises a bone microvascular endothelial cell channel, a bone compartment, a cartilage compartment and a synovial fluid channel which are arranged in parallel and in sequence, and permeation areas between the channel and the compartments and between the compartments. According to the scheme, the complex structure and function of a human osteochondral system are effectively simulated, the physiological and pathological processes of human bone joints can be simulated more truly in an in-vitro environment, and a method for establishing a highly-simulated femoral head necrosis model and osteoarthritis model based on the chip is provided. The problems that a traditional model is insufficient in physiological environment simulation, lack of control over dynamic culture conditions, tedious and time-consuming in the construction process, difficult to support high-throughput experiments and the like are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid microfluidic chips, and in particular to a microfluidic chip, system and method for osteochondral organoids. Background Art

[0002] Organoids are three-dimensional cell clusters formed by in vitro culture of stem cells or pluripotent stem cells, which simulate the specific characteristics of human organs in structure and function. The emergence of organoid technology has provided an ideal model and tool for research in the fields of human development, organ function, disease mechanism, drug screening, personalized treatment, etc. Although significant progress has been made, the culture of traditional organoids is usually limited to a single organ type and lacks the ability to simulate the interaction of multiple organ systems.

[0003] Microfluidics is a technology that manipulates fluids at the micrometer scale, with the advantages of high precision, low consumption and repeatability. In the biomedical field, microfluidics has a wide range of applications, including cell analysis, molecular diagnosis, drug screening, etc. Microfluidics systems can provide a highly precise cell culture environment, allowing researchers to control the microenvironment at the cell level, simulate the microenvironment conditions in the body, and achieve long-term culture and functional testing of organoids.

[0004] Femoral head necrosis is a serious hip disease with complex and diverse causes. It is mainly due to interruption or damage to the blood supply to the femoral head, which leads to the death of bone marrow components, osteocytes and adipocytes, and then causes structural changes, collapse and joint dysfunction of the femoral head. Osteoarthritis is a common chronic disease, and its core pathological change is the degeneration of articular cartilage. Articular cartilage is a layer of smooth, elastic tissue covering the surface of the joint. Its main function is to reduce friction and impact during joint movement and protect the joint from damage. Degenerative changes in articular cartilage are manifested as degradation of cartilage matrix, reduction of chondrocytes, cartilage fibrosis and eventual cartilage wear. However, the current treatment of osteoarthritis is still relatively scarce, mainly due to the lack of accurate preclinical osteoarthritis models.

[0005] Intra-articular drug injection is a non-invasive or minimally invasive treatment for bone and joint diseases. It increases the distribution of drug concentration in the diseased tissue by directly administering the drug to the lesion area in the joint cavity, thereby more effectively relieving pain, promoting bone repair or inhibiting disease progression. However, the selection of drugs, dosage, and optimization of administration methods all need to be based on in-depth basic research and scientific efficacy evaluation.

[0006] In the research and treatment of the above diseases, the construction technology of traditional models has the following defects: it usually relies on animal experiments or in vitro simulations, and the physiological environment simulation is insufficient. It is difficult to accurately simulate the microenvironment in the body and cannot fully reproduce the complex physiological reactions in the disease process, which limits the in-depth study of the disease mechanism; there is usually a lack of control over dynamic culture conditions, such as the supply of nutrients and the removal of metabolic waste, which leads to the inability to stably maintain the physiological state of cells or tissues during long-term experiments, affecting the reliability of experimental results; the construction process is cumbersome and time-consuming, and it is difficult to support high-throughput drug screening and pathological research, which to a certain extent limits the extensive exploration of treatment methods. New technologies are urgently needed to improve the accuracy and functionality of experimental models. Summary of the invention

[0007] The object of the present invention is to provide a microfluidic chip, system and method for osteochondral organs to solve at least one technical problem existing in the above-mentioned background technology.

[0008] In the first aspect, the present invention provides a microfluidic chip of osteochondral organoids, comprising a first microfluidic channel 1, a first gel compartment 2, a second gel compartment 3 and a second microfluidic channel 4 arranged in parallel and in sequence; a first permeation zone 5 is arranged between the first microfluidic channel 1 and the first gel compartment 2; a second permeation zone 6 is arranged between the first gel compartment 2 and the second gel compartment 3; a third permeation zone 7 is arranged between the second gel compartment 3 and the second microfluidic channel 4; bone microvascular endothelial cells are loaded in the first microfluidic channel 1 to simulate the vascular environment; bone cell hydrogel is loaded in the first gel compartment 2 to simulate bone tissue; chondrocyte hydrogel is loaded in the second gel compartment 3 to simulate cartilage tissue; synovial fluid is loaded in the second microfluidic channel 4 to simulate the synovial fluid environment; through the above chip, the microenvironment of bone joints can be better simulated in vitro, and a bone joint-related disease model that is closer to the pathological characteristics of the human body can be constructed, thereby providing a reliable platform for dynamic monitoring, systematic research and treatment development of bone joint diseases.

[0009] Preferably, the width of the first microfluidic channel 1 is set to 200 μm; this is convenient for manufacturing and practical; The widths of the first gel compartment 2 , the second gel compartment 3 , and the second microfluidic channel 4 are all set to 400 μm; this facilitates the injection of relatively viscous synovial fluid.

[0010] Furthermore, liquid storage areas are provided at the outlet and inlet of the microfluidic channel and / or the gel compartment to prevent contamination caused by overflow of the corresponding fluid when the single chip is used.

[0011] Furthermore, the liquid storage area is circular to facilitate processing and manufacturing; Preferably, the diameters of the outlet and inlet of the gel compartment and the liquid storage area at the inlet of the microfluidic channel are all set to 1 mm; the diameter of the liquid storage area at the outlet of the microfluidic channel is set to 4 mm; this facilitates the injection of fluids such as culture medium or synovial fluid into the microfluidic channel or gel compartment, while a large amount of overflow fluids such as culture medium or synovial fluid can be stored at the outlet.

[0012] Furthermore, a plurality of first permeation holes 8 are evenly distributed in the first permeation zone 5 and the third permeation zone 7; the opening size of the first permeation hole 8 on one side close to the microfluidic channel is smaller than the opening size at the other end of the first permeation hole 8; thus, it is convenient for the culture medium and other fluids in the microfluidic channel to penetrate and diffuse into the gel compartment, and it is also possible to reduce the leakage of the gel in the gel compartment into the microfluidic channel.

[0013] Preferably, the first permeation zone 5 and the third permeation zone 7 are formed by arranging trapezoidal columns 9 with uniform spacing, the upper and lower bases of the isosceles trapezoids on the bottom surface of the trapezoidal columns 9 are 56 μm and 160 μm respectively, and the distance between the lower bases of adjacent trapezoids is 90 μm, thereby forming a first permeation hole 8 with a small opening size on the microfluidic channel side and a large opening size on the gel chamber side for photolithography processing.

[0014] Furthermore, a plurality of second permeable holes 10 are evenly distributed in the second permeable area 6; the opening sizes of both ends of the second permeable holes 10 are the same, which is beneficial to the interaction between adjacent organoids or tissues.

[0015] Preferably, the second permeation zone 6 is formed by rectangular columns 11 arranged at uniform intervals, the length and width of the bottom rectangle of the rectangular column are 160 μm and 90 μm respectively, the long side of the rectangle is on the chamber side, and the interval between two adjacent rectangles is 90 μm, thereby forming second permeation holes 10 with the same opening size on both sides of the gel compartment, which is convenient for photolithography processing.

[0016] Furthermore, the Navier-Stokes equation of incompressible fluid in each microfluidic channel is used to model and calculate the shear stress applied to the inner wall of the channel, and the shear stress is controlled by the fluid flow rate. The specific formula is: ; in, represents the fluid density, represents the fluid flow rate, Indicates time, represents the dynamic viscosity of the fluid, represents the fluid pressure, represents the gradient operator, represents the matrix transpose, represents the external force applied to the fluid, represents shear stress; And, the mass conservation continuity equation of incompressible fluid is satisfied at the same time: .

[0017] Furthermore, the modeling conditions for injecting culture medium into the first microfluidic channel 1 include: The culture medium density is 1000 kg / m 3 , the dynamic viscosity of the culture medium is 0.00082Pa·s; The boundary conditions of the culture medium include: In the microfluidic channel, the culture medium pressure is 0 Pa and the culture medium flow rate is 0 m / s; The laminar flow inflow at the inlet of the microfluidic channel was 5, 10, 15, and 30 μl / h; The culture medium pressure at the outlet of the microfluidic channel was 0 Pa.

[0018] In a second aspect, based on the same inventive concept, the present invention further provides a method for preparing the chip as described in the first aspect, specifically comprising: Step a1: construct a chip structure model using mapping software; Step a2: using vector graphics processing software, obtain an optical mask pattern of the chip structure model and print it on a polyester film using a photolithography machine; transferring the optical mask pattern on the polyester film to a negative photoresist (such as SU-8) spin-coated on a silicon wafer to obtain a master mold; Preferably, the optical mask of the chip structure model is printed on a polyester film using the software Adobe Illustrator CC (Adobe Systems Inc.); Step a3, based on the master mold, replicating the semi-finished microfluidic chip of the osteochondral organoid by polydimethylsiloxane, specifically comprising: mixing the silicone base and the curing agent in a ratio of 10:1, pouring the mixture onto the master mold, and then curing the master mold at 65° C. for at least 3 hours; Step a4, stamping and manufacturing a liquid storage area on the semi-finished microfluidic chip to obtain a finished microfluidic chip; Step a5: Bond the finished microfluidic chip to a glass slide by plasma bonding.

[0019] In a third aspect, based on the same inventive concept, the present invention further provides a microfluidic system using the chip as described in the first aspect, specifically comprising a control module, a dynamic fluid transmission module and a visual monitoring module; The control module includes a first microfluidic channel control unit and a second microfluidic channel control unit, which respectively configure microenvironment parameters according to different microfluidic channel requirements, including fluid flow rate, chemical gradient, temperature and oxygen concentration; The dynamic fluid transmission module includes a micro pump unit and a valve unit, which are used to pump fluid into the microfluidic channel, wherein the fluid includes nutrient solution and / or medicine, etc.; The visual monitoring module includes a microscope unit and an environmental sensor unit, which are used to monitor the growth status, physiological parameters, and environmental parameters of the osteochondral organoids in real time.

[0020] In a fourth aspect, based on the same inventive concept, the present invention further provides an application method of the chip as described in the first aspect, specifically comprising: Step b1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment 2 and the second gel compartment 3 respectively; Step b2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel 1 and allowed to stand for at least 1 hour; Step b3, injecting bone microvascular endothelial cells into the first microfluidic channel 1, and culturing the chip model for at least 24 hours to form a single layer of endothelial cells; at the same time, injecting synovial fluid into the second microfluidic channel 4; Step b4: continuously inject alcohol or glucocorticoid into the first microfluidic channel 1 to construct a femoral head necrosis model.

[0021] In a fifth aspect, based on the same inventive concept, the present invention further provides another application method of the chip as described in the first aspect, specifically comprising: Step c1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment 2 and the second gel compartment 3 respectively; Step c2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel 1 and allowed to stand for at least 1 hour; Step c3, injecting bone microvascular endothelial cells into the first microfluidic channel 1, and culturing the chip model for at least 24 hours to form a single layer of endothelial cells; at the same time, injecting synovial fluid into the second microfluidic channel 4; Step c4: injecting a preset dose of inflammatory factors or experimental drugs into the second microfluidic channel 4 to construct an osteoarthritis model.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention provides a microfluidic chip, system and method for osteochondral organoids. By introducing microfluidic technology, the construction and preparation of osteochondral organoid chips are realized, and multiple key functional units of the osteochondral system are integrated on the same chip, effectively simulating the complex structure and function of the human osteochondral system, and can more realistically simulate the physiological and pathological processes of human bone joints in an in vitro environment. A method for establishing a highly simulated femoral head necrosis model and an osteoarthritis model based on the chip is provided, which overcomes many problems existing in traditional models, such as insufficient simulation of the physiological environment, lack of control over dynamic culture conditions, cumbersome and time-consuming construction process, and difficulty in supporting high-throughput experiments. It provides a more reliable platform for the development of research, treatment, drug screening and other fields of diseases such as femoral head necrosis and osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 A schematic diagram of the working principle of the osteochondral organoid provided by an embodiment of the present invention; Figure 2 A top view of a microfluidic chip for osteochondral organoids provided in an embodiment of the present invention; Figure 3 An enlarged structural illustration of a microfluidic channel and a gel compartment in a microfluidic chip of an osteochondral organoid provided in an embodiment of the present invention; Figure 4 A schematic three-dimensional cross-sectional view of a microfluidic channel and a gel compartment in a microfluidic chip of an osteochondral organoid provided in an embodiment of the present invention; Figure 5 A side view of the three-dimensional structure of a microfluidic chip of an osteochondral organoid provided in an embodiment of the present invention; Figure 6 A flow chart of a method for preparing a bone cartilage organ chip provided by an embodiment of the present invention; Figure 7 A flow chart of an application method for constructing a femoral head necrosis model using the osteochondral organoid chip provided by an embodiment of the present invention; Figure 8 The present invention is a flowchart of an application method for constructing an osteoarthritis model using the osteochondral organ-on-chip provided by an embodiment of the present invention.

[0025] Reference numerals: 1-first microfluidic channel; 2-first gel compartment; 3-second gel compartment; 4-second microfluidic channel; 5-first permeation zone; 6-second permeation zone; 7-third permeation zone; 8-first permeation hole; 9-trapezoidal column; 10-second permeation hole; 11-rectangular column; 12-inlet of the first microfluidic channel; 13-inlet of the first gel compartment; 14-inlet of the second gel compartment; 15-inlet of the second microfluidic channel; 16-outlet of the first microfluidic channel; 17-outlet of the first gel compartment; 18-outlet of the second gel compartment; 19-outlet of the second microfluidic channel. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The present invention is further explained below in conjunction with specific implementation modes.

[0028] It should also be noted that the following specific embodiments or specific implementations are a series of optimized settings listed in the present invention to further explain the specific content of the invention, and these settings can be used in combination or in association with each other.

[0029] The structural design principle of the osteochondral organoid provided by the present invention is as follows Figure 1 As shown. The original intention of the design of this scheme is to simulate the microenvironment of cartilage tissue, build synovial fluid channels, cartilage hydrogel compartments, bone hydrogel compartments and endothelial cell channels, and simulate the interaction between chondrocytes, osteoblasts and endothelial cells, the interaction between cells and matrix, and the mechanical stimulation of cells. Through microfluidic control and "layered" structure, the microenvironment of chondrocytes in the body is simulated, including continuous nutrient supply and specific mechanical stimulation, so that the biological behavior and pathological process of chondrocytes can be studied more accurately. It is helpful to construct disease models and drug screening platforms in the future to study the pathogenesis of osteoarthritis, evaluate the efficacy and toxic side effects of drugs, etc. This scheme has the advantages of high throughput, high efficiency, low cost and high precision.

[0030] Embodiment 1: like Figure 2-5 FIG. 1 is a top view of a bone cartilage organ chip structure provided by an embodiment of the present invention. The bone cartilage organ microfluidic chip provided by this embodiment comprises a first microfluidic channel 1, a first gel compartment 2, a second gel compartment 3 and a second microfluidic channel 4 arranged in parallel and in sequence.

[0031] In this embodiment, the first microfluidic channel 1 is used as a bone microvascular endothelial cell channel to load a monolayer of bone microvascular endothelial cells to simulate the vascular environment. Since the vascular system is crucial to the nutrient supply and waste removal of bone tissue, by introducing bone microvascular endothelial cells into the first microfluidic channel 1, the formation and function of the vascular network can be reproduced, providing continuous nutrition for the microfluidic chip and removing metabolic waste. In addition, the microfluidic channel can also simulate changes in the vascular system under pathological conditions, such as vascular lesions and the formation of new blood vessels, which can be used for subsequent research on vascular-related diseases.

[0032] In this embodiment, the first gel compartment 2 is used as a bone compartment to load osteoblast hydrogel to simulate bone tissue. Since bone tissue is an important structure that supports and protects the body, it has high mechanical strength and biological activity, and osteoblasts play a key role in the formation and repair of bone tissue. Therefore, implanting osteoblast hydrogel in the bone compartment can effectively simulate the generation process and functional characteristics of bone tissue, which can be used for subsequent research on the growth, repair and related diseases of bone tissue.

[0033] In this embodiment, the second gel compartment 3 is used as a cartilage compartment, loaded with chondrocyte hydrogel, and used to simulate cartilage tissue. Since cartilage is a tissue covering the surface of the joint, it plays a role in buffering and shock absorption. Therefore, by implanting chondrocyte hydrogel in the cartilage compartment, the chip provided by this embodiment can generate cartilage tissue with elasticity and pressure bearing capacity, providing a more realistic biomechanical environment. In addition, the use of chondrocyte hydrogel makes the growth and functional performance of cartilage tissue closer to the actual situation in the body.

[0034] In this embodiment, the second microfluidic channel 4 is used as a synovial fluid channel to load synovial fluid to simulate the synovial fluid environment in the joint. Since synovial fluid not only lubricates the joints but also provides necessary nutrients for the joints, the microfluidic chip effectively reproduces the physical and chemical properties of synovial fluid through the synovial fluid channel, ensuring that the constructed osteochondral organoid model has lubrication and nutrient supply functions similar to the in vivo environment during the experiment.

[0035] The two microfluidic channels and the two gel compartments are arranged in parallel in the above order to simulate the positional relationship of the tissues and organs in the bone joint. A permeation zone is also provided between the microfluidic channel and the gel compartment to simulate the interaction and influence relationship between adjacent tissues or organs in the bone joint. Figure 2 As shown, a first permeation zone 5 is provided between the first microfluidic channel 1 and the first gel compartment 2 ; a second permeation zone 6 is provided between the first gel compartment 2 and the second gel compartment 3 ; and a third permeation zone 7 is provided between the second gel compartment 3 and the second microfluidic channel 4 .

[0036] In order to simulate and restore the bone joint microenvironment more realistically, this embodiment provides a detailed structural design of each channel, gel compartment and permeation zone. Figure 3-4 As shown, a plurality of first permeation holes 8 are evenly distributed in the first permeation area 5 and the third permeation area 7; the opening size of the first permeation hole 8 close to the microfluidic channel is smaller than the opening size of the other end of the first permeation hole 8.

[0037] More specifically, trapezoidal columns 9 are arranged with uniform spacing on one side of the two gel compartments close to the microfluidic channel. The gaps between the trapezoidal columns 9 form permeation holes 8 with a small opening on the microfluidic channel side and a large opening on the gel compartment side. The height of the trapezoidal columns 9 is consistent with the height of the microfluidic channel and the gel compartment. The width of the two gel compartments is preferably set to 400 μm. The purpose of this design is to minimize gel leakage during the injection process during the experiment and to facilitate processing and manufacturing.

[0038] The size of the first microfluidic channel 1, i.e., the bone microvascular endothelial cell channel, is preferably set to a width of 200 μm, taking into account the size of post-capillary venules (Ø: 20–50 μm) and to strike a balance between manufacturing constraints and ease of use.

[0039] The second microfluidic channel 2 is preferably set to have a width of 400 μm in consideration of the viscosity of the synovial fluid and to facilitate synovial fluid injection.

[0040] In order to simulate the permeation between tissues / organs more realistically, in this embodiment, the bottom surface of the trapezoidal column 9 is preferably set to an isosceles trapezoid, the upper and lower bases of the isosceles trapezoid are set to 56 μm and 160 μm respectively, and the distance between the lower bases of adjacent trapezoids is set to 90 μm. Figure 3 shown.

[0041] In this embodiment, a plurality of second permeable holes 10 are evenly distributed in the second permeable area 6 , and the opening sizes of both ends of the second permeable holes 10 are the same.

[0042] More specifically, the second permeable area 6 is formed by arranging rectangular columns 11 with uniform spacing. The length and width of the rectangle on the bottom of the rectangular column are 160 μm and 90 μm respectively. The long side of the rectangle is on the gel compartment side. The spacing between two adjacent rectangles is 90 μm, thereby forming a second permeable hole 10 with an opening size of 90 μm on both sides of the gel compartment. Figure 3 shown.

[0043] Through the second microfluidic channel 2, i.e., the synovial fluid channel, the intra-articular injection process can be simulated, and the drug to be evaluated can be delivered directly to the lesion area. This process not only ensures that the drug can act on the target tissue in the most realistic way, but also greatly shortens the experimental cycle and improves research efficiency. In the subsequent stage of exploring the efficacy of drugs, the high-throughput screening capability of this chip can be used to quickly evaluate a variety of potential therapeutic drugs. By monitoring key indicators such as cell survival rate, proliferation ability, apoptosis, and expression changes of bone repair-related genes after drug action, the efficacy and safety of the drug can be comprehensively and accurately evaluated. This can not only shorten the research and development process of new drugs, but also provide a basis for the optimization of clinical treatment plans.

[0044] In this embodiment, a liquid storage area is further provided at the outlet of the microfluidic channel, and the liquid storage area is circular.

[0045] More specifically, if Figure 2 As shown, the outlets and inlets of each microfluidic channel and gel compartment are punched into different sizes: The inlet 13 of the first gel compartment, the inlet 14 of the second gel compartment, the outlet 17 of the first gel compartment, and the outlet 18 of the second gel compartment are all punched into circular holes with a diameter of 1 mm; The inlet 12 of the first microfluidic channel and the inlet 15 of the second microfluidic channel are also punched into circular holes with a diameter of 1 mm; The outlet 16 of the first microfluidic channel and the outlet 19 of the second microfluidic channel are punched into circular holes with a diameter of 4 mm, serving as corresponding liquid storage areas; The inlets are used to directly deliver fluids such as culture medium, gel or synovial fluid into the microfluidic channel or gel compartment, or to connect corresponding pumping pipelines; while the reservoir area is used to serve as a reservoir for fluids such as culture medium or synovial fluid.

[0046] In this embodiment, for the first microfluidic channel 1, the shear stress acting on the inner wall of the channel is calculated by fluid dynamics (CFD) simulation (simulation software COMSOL Multiphysics 4.3b, AB). Specifically, the shear stress applied to the inner wall of the channel is modeled and calculated by the Navier-Stokes equation of incompressible fluid, and the shear stress is controlled by the fluid flow rate. The specific formula can be: ; in, represents the density of the culture medium, represents the medium flow rate, Indicates time, represents the dynamic viscosity of the culture medium, represents the medium pressure, represents the gradient operator, represents the matrix transpose, represents the external force applied to the culture medium; represents shear stress.

[0047] At the same time, it satisfies the mass conservation continuity equation of incompressible fluid: ; Substituting the continuity equation into the Navier-Stokes equations, we can simplify and solve for the shear stress.

[0048] Shear stress refers to the stress caused by relative displacement when different parts of an object are subjected to forces in parallel directions. In blood vessels, shear stress is caused by the friction between endothelial cells and blood vessels.

[0049] There is micron-level shear stress (fluid flow shear stress FFSS) in osteocytes and chondrocytes. Low levels of shear stress will promote anabolic reactions, while high levels of shear stress will produce more decomposition reactions. It also affects the degree of attachment of endothelial cells, the degree of morphological changes, and the secretion of biological signal factors.

[0050] The present invention can simulate shear stress. Unlike static culture, the fluid can form shear stress on the side wall, which is similar to blood vessels under physiological conditions. The purpose of calculating shear stress in this embodiment is to simulate the shear stress and control the shear stress through the relationship between shear stress and the flow rate of the fluid in the channel. When the flow rate is too low, there is no shear stress. When the flow rate is too high, it will cause tearing of the attached vascular endothelial cells and affect the attachment.

[0051] In this embodiment, the first microfluidic channel 1, i.e., the bone microvascular endothelial channel, can be modeled as a rectangular domain through computer simulation, and its numerical grid can contain 2165 240 tetrahedral elements. In finite element calculation, the denser the grid density, the more accurate the calculation result, but it will reduce the calculation speed. The grid density, i.e., the number of tetrahedral elements, can be determined according to the actual computer conditions.

[0052] It should be noted that the modeling of the bone microvascular endothelial channel by a rectangular domain is an approximate representation, that is, the gel deformation caused by the gap between adjacent columns is ignored. This is because the gel deformation mainly depends on the contraction of fibrin and is difficult to predict. Therefore, the simulation modeling of the bone microvascular endothelial channel with a rectangular domain is the most reliable method to obtain the range of shear stress levels imposed on endothelial cells under different flow conditions.

[0053] Exemplarily, the modeling conditions of the culture medium in the bone microvascular endothelial channel include: the culture medium density is 1000 kg / m 3, the dynamic viscosity of the culture medium is 0.00082Pa·s; the boundary conditions of modeling include: before starting modeling, in the first microfluidic channel 1, the culture medium pressure is 0Pa, and the culture medium flow rate is 0m / s; the laminar inflow at the inlet of the microfluidic channel is 5, 10, 15 and 30μl / h respectively; the culture medium pressure at the outlet of the microfluidic channel is 0Pa.

[0054] By controlling the flow rate of the culture medium in the bone microvascular endothelial cell channel to simulate shear stress, we can observe and analyze the effects of different shear stresses, that is, different flow rates, on a series of biological factor components secreted by bone microvascular endothelial cells.

[0055] This chip integrates a single-layer endothelial cell channel specifically used to simulate capillaries, a bone compartment, a cartilage compartment, and a synovial fluid channel used to simulate the microenvironment in the joint cavity, successfully simulating the synergistic effect of multiple organ systems. Thus, multiple key functional units of the bone and cartilage system are integrated on the same chip, making it possible to more realistically simulate the physiological and pathological processes of human bone and joints in an in vitro environment. This sequentially arranged channel design not only optimizes the interaction between various organs, but also ensures the effective supply of nutrients and waste removal under experimental conditions, thereby improving the reliability and repeatability of subsequent experiments, which is beneficial to the treatment and research of bone and joint related diseases.

[0056] Embodiment 2: like Figure 6 As shown, this embodiment provides a method for preparing a microfluidic chip as described in Example 1, and the specific steps thereof include: Step a1: construct a chip structure model using mapping software; In this embodiment, the chip structure model is designed and constructed using Solidworks software; Step a2: obtaining an optical mask pattern of the chip structure model by using vector graphics processing software and printing it on a polyester film by using a photolithography machine; transferring the optical mask pattern on the polyester film to a SU-8 negative photoresist spin-coated on a silicon wafer to obtain a master mold; In this embodiment, the vector graphics processing software used is Adobe Illustrator CC (Adobe Systems Inc.); Step a3, based on the master mold, replicating the semi-finished microfluidic chip of the osteochondral organoid by polydimethylsiloxane, specifically comprising: mixing the silicone base and the curing agent in a ratio of 10:1, pouring the mixture onto the master mold, and then curing the master mold at 65° C. for 3 hours; In this embodiment, the chip is made of a polymer with high biocompatibility, such as polydimethylsiloxane (PDMS), to ensure that the organoids will not be affected by the toxicity of the material during the culture process. These materials are not only non-toxic to cells, but also have good air permeability and plasticity, and are suitable for the manufacture of microfluidic chips. PDMS material has the characteristics of high transparency, which is convenient for microscopic observation and imaging. In addition, the flexibility and plasticity of PDMS material make it easy to produce complex microfluidic channels and chamber structures to meet the needs of different experiments. The application of biocompatible materials ensures that organoids can maintain a good growth state during long-term culture, reducing experimental errors and data fluctuations. These materials can also be surface modified to increase their functionality, such as improving cell adhesion through surface modification, or realizing specific biological functions through coatings; Step a4, stamping and manufacturing a liquid storage area on the semi-finished microfluidic chip to obtain a finished microfluidic chip; The ports of the microfluidic channels and gel compartments are punched into different sizes: the inlet 13 of the first gel compartment, the inlet 14 of the second gel compartment, the outlet 17 of the first gel compartment, and the outlet 18 of the second gel compartment are all punched into circular holes with a diameter of 1 mm; the inlet 12 of the first microfluidic channel and the inlet 15 of the second microfluidic channel are also punched into circular holes with a diameter of 1 mm; the outlet 16 of the first microfluidic channel and the outlet 19 of the second microfluidic channel are punched into circular holes with a diameter of 4 mm, serving as reservoirs for culture medium / synovial fluid; Step a5: Bond the finished microfluidic chip to a glass slide using a Harrick plasma bonding device.

[0057] Embodiment three: This embodiment provides a microfluidic system using the chip described in the first embodiment, including a control module, a dynamic fluid transmission module and a visual monitoring module; The control module includes a first microfluidic channel control unit and a second microfluidic channel control unit, which respectively configure microenvironment parameters according to different microfluidic channel requirements, including fluid flow rate, chemical gradient, temperature and oxygen concentration; The dynamic fluid transmission module includes a micro pump unit and a valve unit, which are used to pump nutrient solution and / or medicine into the microfluidic channel; The visual monitoring module includes a microscope unit and an environmental sensor unit, which are used to monitor the growth status, physiological parameters and environmental parameters of osteochondral organoids in real time.

[0058] Specifically, through the control module, each channel / compartment can be independently and accurately controlled, and environmental parameters can be customized according to the needs of different organoids to achieve precise regulation of these conditions, ensuring that each organoid grows and develops under the most suitable conditions. This not only improves the repeatability and reliability of the experiment, but also allows multiple organoids to be cultured and studied simultaneously in the same experiment; Through the micropump unit and valve unit, precise delivery of nutrient solution and drugs can be achieved. This can simulate the blood circulation in the body, provide a continuous supply of nutrients and remove metabolic waste, which is conducive to long-term culture and observation of interactions between organoids. The micropump unit can adjust the flow rate and direction of the fluid according to the preset program or the results of real-time monitoring to meet the needs of different organoids. For example, the flow rate can be adjusted to simulate different states of blood flow, so as to study the impact of fluid dynamics on cell behavior. The valve unit can also quickly switch between different flow paths to switch and compare multiple experimental conditions; The visual monitoring module can dynamically observe cell growth and activity status through a high-resolution microscope, and then use sensors to monitor environmental parameters in real time, such as temperature, pH value, oxygen concentration, etc., to ensure that organoids grow in the most suitable environment. The sensor can also monitor the metabolites and signal molecules of the cells. By analyzing these data, it is helpful to gain a deeper understanding of the interactions between cells and the signal transmission mechanism. The visualization and real-time monitoring functions facilitate the adjustment and optimization of culture conditions at any time during the experiment, thereby improving the success rate of subsequent experiments and the reliability of data.

[0059] Embodiment 4: like Figure 7 As shown, this embodiment provides an application method of the chip as described in Embodiment 1, including: Step b1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment 2 and the second gel compartment 3 respectively; Step b2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel 1 and allowed to stand for 1 hour; Step b3, injecting bone microvascular endothelial cells into the first microfluidic channel 1, and culturing the chip model for 24 hours to form a single layer of endothelial cells; at the same time, injecting synovial fluid into the second microfluidic channel 4; Step b4: continuously inject alcohol or glucocorticoid into the first microfluidic channel 1 to construct a femoral head necrosis model.

[0060] In this embodiment, by introducing microfluidic technology, the microenvironment of the femoral head can be better simulated in vitro, dynamic monitoring of disease progression can be achieved, and combined with advanced bioengineering methods, a model close to the pathological characteristics of the human body is constructed, which facilitates subsequent research and treatment development of femoral head necrosis.

[0061] Embodiment five: like Figure 8 As shown, this embodiment provides an application method of the chip as described in Embodiment 1, including: Step c1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment 2 and the second gel compartment 3 respectively; Step c2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel 1 and allowed to stand for 1 hour; Step c3, injecting bone microvascular endothelial cells into the first microfluidic channel 1, and culturing the chip model for 24 hours to form a single layer of endothelial cells; at the same time, injecting synovial fluid into the second microfluidic channel 4; Step c4: injecting a preset dose of inflammatory factors or experimental drugs into the second microfluidic channel 4 to construct an osteoarthritis model.

[0062] The osteochondral organ chip and microfluidic technology provided by the present invention can precisely simulate the microenvironment of human joints, including key elements such as the bone microvascular endothelial cell network, synovial tissue and synovial fluid circulation system, and successfully build a highly simulated osteoarthritis model on the organ chip. This model can not only reproduce the pathological and physiological process of the occurrence and development of the disease, but also facilitate in-depth research on the mechanism of action of the drug.

[0063] This protocol can accurately construct a femoral head necrosis and osteoarthritis model that is close to the physiological environment. It has the advantages of high throughput and rapid response, which can effectively accelerate the subsequent scientific research process, greatly reduce the use of experimental animals, and reduce research costs. It also reflects the respect and protection of life ethics. This protocol is efficient and accurate, which is helpful for the subsequent formulation of personalized medical plans.

[0064] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A microfluidic chip of osteochondral organoids, characterized in that: The invention comprises a first microfluidic channel (1), a first gel compartment (2), a second gel compartment (3) and a second microfluidic channel (4) which are arranged in parallel and in sequence; a first permeation zone (5) is arranged between the first microfluidic channel (1) and the first gel compartment (2); a second permeation zone (6) is arranged between the first gel compartment (2) and the second gel compartment (3); and a third permeation zone (7) is arranged between the second gel compartment (3) and the second microfluidic channel (4); The first microfluidic channel (1) is loaded with bone microvascular endothelial cells to simulate a vascular environment; The first gel compartment (2) is loaded with bone cell hydrogel for simulating bone tissue; The second gel compartment (3) is loaded with chondrocyte hydrogel for simulating cartilage tissue; The second microfluidic channel (4) is loaded with synovial fluid to simulate the synovial fluid environment.

2. The chip according to claim 1, characterized in that: Liquid storage areas are also provided at the outlet and inlet of the microfluidic channel and / or the gel compartment.

3. The chip according to claim 1, characterized in that: A plurality of first permeation holes (8) are evenly distributed in the first permeation zone (5) and the third permeation zone (7); the opening size of the first permeation hole (8) close to one end of the microfluidic channel is smaller than the opening size of the other end of the first permeation hole (8).

4. The chip according to claim 1, characterized in that: A plurality of second permeable holes (10) are evenly distributed in the second permeable area (6); the opening sizes of both ends of the second permeable holes (10) are the same.

5. The chip according to claim 1, characterized in that , the shear stress applied to the inner wall of each microfluidic channel is modeled and calculated, and the shear stress is controlled by the fluid flow rate. The specific formula is: ； in, represents the fluid density, represents the fluid flow rate, Indicates time, represents the dynamic viscosity of the fluid, represents the fluid pressure, represents the gradient operator, represents the matrix transpose, represents the external force applied to the fluid, represents shear stress; And satisfy the equation: 。 6. The chip according to claim 5, characterized in that: The modeling conditions for injecting culture medium into the first microfluidic channel (1) include: The culture medium density is 1000 kg / m3, and the dynamic viscosity of the culture medium is 0.00082 Pa·s; The boundary conditions of the culture medium include: In the microfluidic channel, the culture medium pressure was 0 Pa and the culture medium flow rate was 0 m / s; The laminar flow inflow at the inlet of the microfluidic channel was 5, 10, 15, and 30 μl / h; The culture medium pressure at the outlet of the microfluidic channel was 0 Pa.

7. A method for preparing a chip as claimed in any one of claims 1 to 6, characterized in that: include: Step a1, constructing a chip structure model; Step a2, drawing an optical mask pattern of the chip structure model and printing it on a polyester film through a photolithography machine; transferring the optical mask pattern on the polyester film to a negative photoresist spin-coated on a silicon wafer to obtain a master mold; Step a3, based on the master mold, replicating the semi-finished microfluidic chip of the osteochondral organoid by polydimethylsiloxane, specifically comprising: mixing the silicone base and the curing agent in a ratio of 10:1, pouring the mixture onto the master mold, and then curing the master mold at 65° C. for at least 3 hours; Step a4, stamping and manufacturing a liquid storage area on the semi-finished microfluidic chip to obtain a finished microfluidic chip; Step a5: Bond the finished microfluidic chip to a glass slide by plasma bonding.

8. A microfluidic system using a chip as claimed in any one of claims 1 to 6, characterized in that: It includes a control module, a dynamic fluid transmission module and a visual monitoring module; The control module includes a first microfluidic channel control unit and a second microfluidic channel control unit, which respectively configure microenvironment parameters according to different microfluidic channel requirements, including fluid flow rate, chemical gradient, temperature and oxygen concentration; The dynamic fluid transmission module includes a micro pump unit and a valve unit, which are used to pump fluid into the microfluidic channel; The visual monitoring module includes a microscope unit and an environmental sensor unit, which are used to monitor the growth status, physiological parameters and environmental parameters of osteochondral organoids in real time.

9. An application method of the chip according to any one of claims 1 to 6, characterized in that: include: Step b1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment (2) and the second gel compartment (3), respectively; Step b2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel (1) and allowed to stand for at least 1 hour; Step b3, injecting bone microvascular endothelial cells into the first microfluidic channel (1), and culturing the chip model for at least 24 hours to form a monolayer of endothelial cells; At the same time, synovial fluid is injected into the second microfluidic channel (4); Step b4: continuously injecting alcohol or glucocorticoid into the first microfluidic channel (1) to construct a femoral head necrosis model.

10. An application method of the chip according to any one of claims 1 to 6, characterized in that: include: Step c1, embedding osteoblasts and articular chondrocytes into respective hydrogels, and injecting the hydrogels into the first gel compartment (2) and the second gel compartment (3), respectively; Step c2, after the gel is completely polymerized, fibronectin is coated in the first microfluidic channel (1) and allowed to stand for at least 1 hour; Step c3, injecting bone microvascular endothelial cells into the first microfluidic channel (1), and culturing the chip model for at least 24 hours to form a monolayer of endothelial cells; At the same time, synovial fluid is injected into the second microfluidic channel (4); Step c4: injecting a preset dose of inflammatory factors or experimental drugs into the second microfluidic channel (4) to construct an osteoarthritis model.

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