Multiple bionic physiological gradient bone-cartilage organ chip and preparation method thereof

Abstract

The invention discloses a bone-cartilage organ chip with multiple bionic physiological gradients and a preparation method thereof, and belongs to the field of biomedical engineering.The preparation method comprises the following specific steps that 1, an upper-layer chip, a lower-layer chip, a middle-layer chip and a porous membrane are prepared; iI, performing trepanning and bonding on each prepared chip; iII, constructing a hydrogel bracket with a rigid gradient in the chip; iV, constructing an oxygen concentration gradient on the chip and performing cell culture; the highly bionic bone-cartilage interface model based on the micro-fluidic chip effectively fills the blank of an effective in-vitro model of mutual crosstalk of bone and cartilage tissues on a closely connected interface, the experiment cost can be greatly saved, the dispute of experiment ethics does not exist, and the model has a good application prospect. The experiment result shows that the organ chip model has good correlation with human beings, the chip and the experiment method have good repeatability, and batch bone-cartilage interface model construction and chip experiments can be carried out.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a bone-cartilage organ chip with multiple bionic physiological gradients and a preparation method thereof. Background Art

[0002] In both physiological and pathological processes, there is a rich and close exchange of materials and signal transmission between bone and cartilage. A deep understanding of the interaction between bone and cartilage is an important means to study the pathogenesis and treatment of osteoarthritis. At present, the pathogenesis of osteoarthritis is still unclear, and there is a lack of effective treatment methods. The main reason is the lack of effective in vitro models. Widely used animal models have obvious limitations and defects. In addition to ethical issues, there are huge physiological and genetic differences between the bone-cartilage interface of animals and humans that cannot be bridged. In addition, in clinical studies on the bone-cartilage interface, the sample size is too small, and there are huge differences in the age, gender, physiological condition, disease severity, diet and supplements of the subjects. The clinical samples are highly heterogeneous and the research time span is generally long. Therefore, constructing an effective bone-cartilage interface in vitro is the key to studying the physiology and diseases of joint development, degeneration, inflammation, etc.

[0003] In vivo, bone-cartilage tissue is a gradient structure formed by spontaneous differentiation. The smooth transition from cartilage to subchondral bone involves changes in oxygen concentration gradient, cell morphology and phenotype, extracellular matrix composition, collagen type and direction, and cytokines. First, in the skeletal system, oxygen is maintained at a high level in vascularized bone, while non-vascularized cartilage is close to an anaerobic state. Second, osteochondral tissue has different physiological and mechanical properties. Bone tissue is composed of a large amount of calcified intercellular matrix and several types of bone tissue cells. The calcified intercellular matrix is ​​called bone matrix. The bone matrix has high rigidity and small pores. The cartilage matrix is ​​composed of an amorphous matrix and fibers embedded in it. The main components of the amorphous matrix are proteoglycans and water. Proteoglycans form a molecular sieve structure, which makes the cartilage tissue have good permeability and the matrix stiffness is much less than that of the osteoblast. In addition, the osteoblast part is mainly enriched with growth factors related to osteogenesis, while the cartilage part is mainly enriched with growth factors related to cartilage. How to simulate the complex structure and characteristics of the bone-cartilage interface is the main consideration for in vitro modeling.

[0004] Organ chips based on microfluidic technology provide an effective means for the construction of in vitro bionic models. The present invention uses organ chip technology, combined with the application of microfluidics and fluid mechanics, to design and manufacture an organ chip that can construct multiple bionic physiological gradients for in vitro modeling and physiological and pathological research of the bone-cartilage interface. The organ chip should be able to create multiple bionic gradients, including oxygen concentration gradients, matrix stiffness gradients, and two growth factor gradients. Differentiation of the bone-cartilage interface is carried out in a multiple gradient environment. It provides an effective in vitro model for studying the interaction between bone and cartilage tissue in an arthritic state. Summary of the invention

[0005] The purpose of the present invention is to solve the defects in the prior art and to propose a bone-cartilage organ chip with multiple bionic physiological gradients and a preparation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Bone-cartilage organ chips with multiple bionic physiological gradients, including upper chip, middle chip and lower chip;

[0008] The microchannel width of the upper chip is 100 μm, the height is 100 μm, and the square structure size of the upper chip is 8 mm×6 mm, the height is 100 μm;

[0009] The square structure size of the intermediate layer chip is 8mm×6mm and the height is 3mm;

[0010] The microchannel of the lower chip has a width of 100 μm and a height of 200 μm, and the square structure size of the lower chip is 8 mm×6 mm and a height of 200 μm.

[0011] As a further solution of the present invention, the upper chip and the middle chip are separated by a porous membrane, the porous membrane has a size of 10 mm×8 mm, a pore size of 8 μm, and a pore spacing of 30 μm;

[0012] The upper chip and the lower chip each have a set of gradient generators, and the upper chip and the lower chip are structurally designed with multiple entrances and exits;

[0013] The inlets of the upper chip microchannel are a, b and c, and the outlets of the upper chip microchannel are d, e and f;

[0014] The inlets of the microchannels of the lower chip are g (g') and h (h'), and the outlets of the microchannels of the lower chip are i (i') and j (j').

[0015] A method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients, wherein the specific steps of the preparation method are as follows:

[0016] Ⅰ. Prepare an upper chip, a lower chip, an intermediate chip and a porous membrane;

[0017] II. Drilling and bonding the prepared chips;

[0018] III. Constructing a hydrogel scaffold with a stiffness gradient within the chip;

[0019] IV. Construct an oxygen concentration gradient on the chip and perform cell culture.

[0020] As a further solution of the present invention, the specific steps of preparing the upper chip, the lower chip, the middle chip and the porous membrane in step I are as follows:

[0021] S101: Design and manufacture masks for the upper chip and the lower chip respectively, and then prepare silicon wafers of the upper chip and the lower chip by a photolithography machine, and then pour PDMS prepolymer on the silicon wafers of the upper chip and the lower chip respectively, degas under -80 kPa until all bubbles are removed, and demold after curing at 60 ° C for 6 hours, and obtain the upper chip and the lower chip after demolding, the thickness of the upper chip and the lower chip are 3 mm and 2 mm respectively;

[0022] S102: Printing a positive mold of the middle layer chip using 3D printing technology, and then pouring PDMS prepolymer on the positive mold, the thickness of the middle layer chip is 3 mm;

[0023] S103: Prepare a chromium plate mask for the porous membrane according to the standard of microarray size of 8μm in diameter and 30μm in spacing, use a photolithography machine to prepare a silicon wafer for the porous membrane, then pour PDMS prepolymer on the microcolumn array of the silicon wafer, and cover the uncured prepolymer with a cured PDMS layer as a support layer, then place a weight of 3kg on the device, and cure the polymer at 60°C for 12 hours. After curing, peel off the PDMS porous membrane from the wafer and use a scalpel to cut it into a size of 10mm long and 8mm wide.

[0024] As a further solution of the present invention, the PDMS prepolymer in S101 and S103 specifically consists of PDMS and a curing agent in a mass ratio of 10:1.

[0025] As a further solution of the present invention, the specific steps of opening holes and bonding the prepared chips in step II are as follows:

[0026] S201: Bonding a porous membrane to the surface of the middle layer through plasma treatment, using a Punch tool to punch holes at a, b, c, d, e, and f of the upper chip, and then bonding the middle chip combined with the porous membrane to the upper chip. At this time, the microchannel structure of the upper chip faces downward, and the square structure of the upper chip and the square structure of the middle chip overlap in space. At this time, the porous membrane is located between the upper chip and the middle chip, separating the square structure of the upper chip and the square structure of the middle chip;

[0027] S202: Use a Punch tool to punch holes at the g(g'), h(h'), i(i'), and j(j') holes of the bonded upper and middle chips. The holes penetrate the upper chip and the middle chip. Through Plasma treatment, the lower chip is bonded to the combined upper and middle chips. At this time, the microchannel structure of the lower chip faces upward, and the square structure of the upper chip, the square structure of the middle chip, and the square structure of the lower chip overlap in space.

[0028] As a further solution of the present invention, the specific steps of constructing a hydrogel scaffold with a rigidity gradient in the chip in step III are as follows:

[0029] S301: Human bone marrow mesenchymal stem cells (BMSCs) were cultured. When the cells grew to 90% confluence, the cells were digested with trypsin, and the supernatant was removed by centrifugation. Gelma solutions with concentrations of 15% and 7.5% were prepared, and both Gelma solutions contained 0.15% of a photoinitiator (LAP, 365 nm);

[0030] S302: 5×10 6 cells / ml concentration as the standard, resuspend the cells with 15% and 7.5% Gelma solution, and then use a microinjection pump to inject 15% and 7.5% Gelma and cell mixtures into the two inlets g (g') and h (h') of the lower chip at a flow rate of 100 μL / min;

[0031] S303: When the two gelmas and the cell mixture fill the entire rectangular space of the middle layer chip, the injection is stopped, and then the bone-cartilage organ chip is placed under a 365nm wavelength ultraviolet lamp for 2 minutes for light curing.

[0032] As a further solution of the present invention, the specific steps of constructing an oxygen concentration gradient on the chip and performing cell culture as described in step IV are as follows:

[0033] S401: respectively preparing an osteogenic differentiation medium OG, a chondrogenic differentiation medium CG, and a common medium OC, and performing a deoxygenation treatment on the chondrogenic differentiation medium CG to reduce its oxygen concentration to about 1%, and performing a semi-deoxygenation treatment on the common medium OC to reduce its oxygen concentration to about 10%;

[0034] S402: Use a microinjection pump to inject OG, semi-deoxygenated OC, and deoxygenated CG into the bone-cartilage organ chip from inlets a, b, and c at a flow rate of 50 μL / h, respectively, and perform continuous perfusion culture by replacing the syringe connected to the injection pump.

[0035] As a further embodiment of the present invention, the osteogenic differentiation medium OG in S401 specifically comprises 89% DMEM, 10% FBS and 1% anti-anti, and is further supplemented with 0.1 μM Dexamethasone, 10 μM β-glycerophosphate, 50 μg / ml Ascorbate, 10 nM Vitamin D and 100 ng / mL BMP7;

[0036] The chondrogenic differentiation medium CG specifically comprises 99% DMEM and 1% anti-anti, and is further supplemented with 1 μg / ml ITS+, 0.1 μM Dexamethasone, 40 μg / ml Proline, 50 μg / ml Ascorbate, and 10 ng / ml TGF-β1;

[0037] The common culture medium OC specifically contained 99% DMEM and 1% anti-anti, and was additionally supplemented with 0.1 μM Dexamethasone and 50 μg / ml Ascorbate.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The bone-cartilage organ chip with multiple bionic physiological gradients uses organ chip technology, combined with the application of microfluidics and fluid mechanics, to design and manufacture an organ chip that can generate multiple gradients for in vitro modeling and physiological and pathological research of the bone-cartilage interface. The chip provides an effective model for the in vitro construction of a highly bionic bone-cartilage interface, effectively simulates the complex physiological structure and characteristics of the human bone-cartilage interface, and the experiment has good repeatability. The microfluidic chip-based, highly bionic bone-cartilage interface model constructed by the present invention effectively fills the gap in an effective in vitro model for the mutual crosstalk between bone and cartilage tissues at a closely connected interface, can greatly save experimental costs, and there is no controversy over experimental ethics. The experimental results show that the organ chip model has a good correlation with humans, the chip and experimental methods have good repeatability, and batch bone-cartilage interface model construction and chip experiments can be carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0041] Figure 1 This is a flowchart of the method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients proposed by the present invention;

[0042] Figure 2 This is a chip structure diagram of the bone-cartilage organ chip with multiple bionic physiological gradients proposed by the present invention;

[0043] Figure 3 This is a chip assembly diagram of the bone-cartilage organ chip with multiple bionic physiological gradients proposed by the present invention;

[0044] Figure 4 This is a schematic diagram of the upper chip simulation of the bone-cartilage organ chip with multiple bionic physiological gradients proposed by the present invention;

[0045] Figure 5 This is a schematic diagram of the simulation of the lower chip of the bone-cartilage organ chip with multiple bionic physiological gradients proposed in the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0047] Example 1

[0048] Reference Figure 2-3This embodiment discloses a bone-cartilage organ chip with multiple bionic physiological gradients, including an upper chip, a middle chip and a lower chip.

[0049] Specifically, according to Figure 2-3 It can be seen that the microchannel width of the upper chip is 100μm, the height is 100μm, and the square structure size of the upper chip is 8mm×6mm, the height is 100μm; the square structure size of the middle chip is 8mm×6mm, the height is 3mm; the microchannel width of the lower chip is 100μm, the height is 200μm, and the square structure size of the lower chip is 8mm×6mm, the height is 200μm.

[0050] It should be further explained that the upper chip and the middle chip are separated by a porous membrane with a size of 10 mm × 8 mm, a pore size of 8 μm, and a pore spacing of 30 μm; the upper chip and the lower chip each have a set of gradient generators, and the upper chip and the lower chip structure are designed with multiple entrances and exits; the entrances of the upper chip microchannel are a, b and c, and the exits of the upper chip microchannel are d, e and f; the entrances of the lower chip microchannel are g (g') and h (h'), and the exits of the lower chip microchannel are i (i') and j (j').

[0051] Example 2

[0052] Reference Figure 1-5 This embodiment discloses a method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients. The specific steps of the preparation method are as follows:

[0053] An upper chip, a lower chip, a middle chip and a porous membrane are prepared.

[0054] Specifically, masks are designed and made for the upper chip and the lower chip respectively, and then the silicon wafers of the upper and lower chips are prepared by a photolithography machine, and then the PDMS prepolymer is poured on the silicon wafers of the upper and lower chips respectively, degassed at -80 kPa to remove all bubbles, and then cured at 60 ° C for 6 hours before demolding. After demolding, the upper chip and the lower chip are obtained. The thickness of the upper chip and the lower chip are 3 mm and 2 mm respectively. The positive mold of the middle layer chip is printed using 3D printing technology, and then the PDMS prepolymer is poured on the positive mold. The thickness of the chip is 3mm. According to the standard of microarray size of 8μm in diameter and 30μm in spacing, a chromium plate mask of the porous membrane is prepared. A photolithography machine is used to prepare a silicon wafer of the porous membrane. Then, PDMS prepolymer is poured on the microcolumn array of the silicon wafer, and a cured PDMS layer is used as a support layer to cover the uncured prepolymer. Then, a weight of 3kg is placed on the device, and the polymer is cured at 60°C for 12 hours. After the curing is completed, the PDMS porous membrane is peeled off from the wafer and cut into a size of 10mm long and 8mm wide using a scalpel.

[0055] It should be further explained that the PDMS prepolymer is specifically composed of PDMS and a curing agent in a mass ratio of 10:1.

[0056] The prepared chips are opened and bonded.

[0057] Specifically, refer to Figure 2-3 It can be seen that the porous membrane is bonded to the surface of the middle layer through Plasma treatment, and the Punch tool is used to punch holes at the a, b, c, d, e, and f holes of the upper chip. Then the middle chip combined with the porous membrane is bonded to the upper chip. At this time, the microchannel structure of the upper chip faces downward, and the square structure of the upper chip and the square structure of the middle chip overlap in space. At this time, the porous membrane is located between the upper chip and the middle chip, separating the square structure of the upper chip and the square structure of the middle chip. The Punch tool is used to punch holes at the g(g'), h(h'), i(i'), and j(j') holes of the bonded upper and middle chips. The holes penetrate the upper chip and the middle chip. Through Plasma treatment, the lower chip is bonded to the combined upper and middle chips. At this time, the microchannel structure of the lower chip faces upward, and the square structure of the upper chip, the square structure of the middle chip, and the square structure of the lower chip overlap in space.

[0058] Constructing a hydrogel scaffold with a stiffness gradient within the chip.

[0059] Specifically, human bone marrow mesenchymal stem cells (BMSCs) were cultured. When the cells grew to 90% confluence, the cells were digested with trypsin, and the supernatant was removed by centrifugation. Gelma solutions with concentrations of 15% and 7.5% were prepared, and both Gelma solutions contained 0.15% of a photoinitiator (LAP, 365 nm). 6 Using the concentration of cells / ml as the standard, the cells were resuspended with 15% and 7.5% Gelma solutions, respectively. Then, a microinjection pump was used to inject 15% and 7.5% Gelma and cell mixtures into the two inlets g (g') and h (h') of the lower chip at a flow rate of 100 μL / min. When the two Gelma and cell mixtures filled the entire rectangular space of the middle chip, the injection was stopped. Then, the bone-cartilage organ chip was placed under a 365nm wavelength ultraviolet lamp for 2 minutes for light curing.

[0060] An oxygen concentration gradient was constructed on the chip and cells were cultured.

[0061] Specifically, osteogenic differentiation medium OG, chondrogenic differentiation medium CG and common medium OC were prepared respectively, and the chondrogenic differentiation medium CG was deoxygenated to reduce its oxygen concentration to about 1%, and the common medium OC was semi-deoxygenated to reduce its oxygen concentration to about 10%. OG, semi-deoxygenated OC and deoxygenated CG were injected into the bone-cartilage organ chip from inlets a, b and c at a flow rate of 50 μL / h using a micro-injection pump, and continuous perfusion culture was performed by replacing the syringe connected to the injection pump.

[0062] It should be further explained that the reference Figure 4-Figure 4 It can be seen that the fluid mechanics simulation of the bone-cartilage organ chip is Figure 4 In the schematic diagram of the oxygen concentration gradient of the middle and upper chips, the left part is red and the right part is blue, where red represents a concentration of 18% and blue represents a concentration of 0%. From left to right, the color gradually changes from red to blue, and there is a corresponding gradient change; Figure 5 In the schematic diagram of the hydrogel concentration gradient of the middle and lower layer chips, the left part is red and the right part is blue, where red represents a hydrogel concentration of 15% and blue represents a hydrogel concentration of 7.5%. The color gradually changes from red to blue from left to right, with a corresponding gradient change.

[0063] It should be further explained that the osteogenic differentiation medium OG specifically contains 89% DMEM, 10% FBS and 1% anti-anti, and is additionally supplemented with 0.1 μM Dexamethasone, 10 μM β-glycerophosphate, 50 μg / ml Ascorbate, 10 nM VitaminD and 100 ng / mL BMP7;

[0064] Chondrogenic differentiation medium CG specifically contained 99% DMEM and 1% anti-anti, and was additionally supplemented with 1 μg / ml ITS+, 0.1 μM Dexamethasone, 40 μg / ml Proline, 50 μg / ml Ascorbate, and 10 ng / ml TGF-β1;

[0065] Common medium OC specifically contained 99% DMEM and 1% anti-anti, and was additionally supplemented with 0.1 μM Dexamethasone and 50 μg / ml Ascorbate.

Claims

1. A bone-cartilage organ chip with multiple bionic physiological gradients, characterized in that: Including upper chip, middle chip and lower chip; The microchannel width of the upper chip is 100 μm, the height is 100 μm, and the square structure size of the upper chip is 8 mm×6 mm, the height is 100 μm; The square structure size of the intermediate layer chip is 8mm×6mm and the height is 3mm; The microchannel of the lower chip has a width of 100 μm and a height of 200 μm, and the square structure size of the lower chip is 8 mm×6 mm and a height of 200 μm.

2. The bone-cartilage organ chip with multiple bionic physiological gradients according to claim 1, characterized in that: The upper chip and the middle chip are separated by a porous membrane, the size of the porous membrane is 10 mm×8 mm, the pore size is 8 μm, and the pore spacing is 30 μm; The upper chip and the lower chip each have a set of gradient generators, and the upper chip and the lower chip are structurally designed with multiple entrances and exits; The inlets of the upper chip microchannel are a, b and c, and the outlets of the upper chip microchannel are d, e and f; The inlets of the microchannels of the lower chip are g (g') and h (h'), and the outlets of the microchannels of the lower chip are i (i') and j (j').

3. A method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients, which is used to realize the function of the bone-cartilage organ chip with multiple bionic physiological gradients as described in any one of claims 1-2, characterized in that: The specific steps of the preparation method are as follows: Ⅰ. Prepare an upper chip, a lower chip, an intermediate chip and a porous membrane; II. Drilling and bonding the prepared chips; III. Constructing a hydrogel scaffold with a stiffness gradient within the chip; IV. Construct an oxygen concentration gradient on the chip and perform cell culture.

4. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 3, characterized in that: The specific steps of preparing the upper chip, the lower chip, the middle chip and the porous membrane in step I are as follows: S101: Design and manufacture masks for the upper chip and the lower chip respectively, and then prepare silicon wafers of the upper chip and the lower chip by a photolithography machine, and then pour PDMS prepolymer on the silicon wafers of the upper chip and the lower chip respectively, degas under -80 kPa until all bubbles are removed, and demold after curing at 60 ° C for 6 hours, and obtain the upper chip and the lower chip after demolding, the thickness of the upper chip and the lower chip are 3 mm and 2 mm respectively; S102: Printing a positive mold of the middle layer chip using 3D printing technology, and then pouring PDMS prepolymer on the positive mold, the thickness of the middle layer chip is 3 mm; S103: Prepare a chromium plate mask for the porous membrane according to the standard of microarray size of 8μm in diameter and 30μm in spacing, use a photolithography machine to prepare a silicon wafer for the porous membrane, then pour PDMS prepolymer on the microcolumn array of the silicon wafer, and cover the uncured prepolymer with a cured PDMS layer as a support layer, then place a weight of 3kg on the device, and cure the polymer at 60°C for 12 hours. After curing, peel off the PDMS porous membrane from the wafer and use a scalpel to cut it into a size of 10mm long and 8mm wide.

5. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 4, characterized in that: The PDMS prepolymer described in S101 and S103 specifically consists of PDMS and a curing agent in a mass ratio of 10:

1.

6. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 4, characterized in that: The specific steps of opening holes and bonding the prepared chips in step II are as follows: S201: Bonding a porous membrane to the surface of the middle layer through plasma treatment, using a Punch tool to punch holes at a, b, c, d, e, and f of the upper chip, and then bonding the middle chip combined with the porous membrane to the upper chip. At this time, the microchannel structure of the upper chip faces downward, and the square structure of the upper chip and the square structure of the middle chip overlap in space. At this time, the porous membrane is located between the upper chip and the middle chip, separating the square structure of the upper chip and the square structure of the middle chip; S202: Use a Punch tool to punch holes at the g(g'), h(h'), i(i'), and j(j') holes of the bonded upper and middle chips. The holes penetrate the upper chip and the middle chip. Through Plasma treatment, the lower chip is bonded to the combined upper and middle chips. At this time, the microchannel structure of the lower chip faces upward, and the square structure of the upper chip, the square structure of the middle chip, and the square structure of the lower chip overlap in space.

7. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 3, characterized in that: The specific steps of constructing a hydrogel scaffold with a rigidity gradient in the chip as described in step III are as follows: S301: Human bone marrow mesenchymal stem cells (BMSCs) were cultured. When the cells grew to 90% confluence, the cells were digested with trypsin, and the supernatant was removed by centrifugation. Gelma solutions with concentrations of 15% and 7.5% were prepared, and both Gelma solutions contained 0.15% of a photoinitiator (LAP, 365 nm); S302: 5×10 6 cells / ml concentration as the standard, resuspend the cells with 15% and 7.5% Gelma solution, and then use a microinjection pump to inject 15% and 7.5% Gelma and cell mixtures into the two inlets g (g') and h (h') of the lower chip at a flow rate of 100 μL / min; S303: When the two gelmas and the cell mixture fill the entire rectangular space of the middle layer chip, the injection is stopped, and then the bone-cartilage organ chip is placed under a 365nm wavelength ultraviolet lamp for 2 minutes for light curing.

8. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 7, characterized in that: The specific steps of constructing an oxygen concentration gradient on the chip and culturing cells as described in step IV are as follows: S401: respectively preparing an osteogenic differentiation medium OG, a chondrogenic differentiation medium CG, and a common medium OC, and performing a deoxygenation treatment on the chondrogenic differentiation medium CG to reduce its oxygen concentration to about 1%, and performing a semi-deoxygenation treatment on the common medium OC to reduce its oxygen concentration to about 10%; S402: Use a microinjection pump to inject OG, semi-deoxygenated OC, and deoxygenated CG into the bone-cartilage organ chip from inlets a, b, and c at a flow rate of 50 μL / h, respectively, and perform continuous perfusion culture by replacing the syringe connected to the injection pump.

9. The method for preparing a bone-cartilage organ chip with multiple bionic physiological gradients according to claim 8, characterized in that: S401 The osteogenic differentiation medium OG specifically comprises 89% DMEM, 10% FBS and 1% anti-anti, and is further supplemented with 0.1 μM Dexamethasone, 10 μM β-glycerophosphate, 50 μg / ml Ascorbate, 10 nM Vitamin D and 100 ng / mL BMP7; The chondrogenic differentiation medium CG specifically comprises 99% DMEM and 1% anti-anti, and is further supplemented with 1 μg / ml ITS+, 0.1 μM Dexamethasone, 40 μg / ml Proline, 50 μg / ml Ascorbate, and 10 ng / ml TGF-β1; The common culture medium OC specifically comprises 99% DMEM and 1% anti-anti, and is further supplemented with 0.1 μM Dexamethasone and 50 μg / ml Ascorbate.

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