Bone organ chip loaded with microelectrode array and use method

Abstract

The invention discloses a bone organ chip loaded with a microelectrode array and a use method, and relates to the technical field of organ chips, the bone organ chip is made of a polydimethylsiloxane material, and the bone organ chip comprises a three-layer structure: a top layer, a middle layer and a bottom layer; the middle layer comprises two blood vessel micro-channels, two osteoblast micro-channels and a neuron micro-channel, the channels are separated through columns and micro-columns, and cell interaction in a bone micro-environment is simulated; a microelectrode array is arranged on the top layer and is used for monitoring electrical activity of neurons in real time; the bottom layer is provided with a pneumatic valve which can control opening and closing of the micro-channel. According to the invention, nerve and blood vessel channels are introduced, a nerve-blood vessel-immunoregulation bone microenvironment is constructed, physiological processes such as osteogenesis-blood vessel formation coupling and the like are embodied, intervals of different sizes can enhance intercellular crosstalk and allow blood vessels and neuron axons to selectively pass through, so that the chip can simulate a complex microenvironment of bone tissues, and the microenvironment of the bone tissues can be simulated; and an efficient and accurate in-vitro model is provided for bone disease mechanism research and drug development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organ chip, in particular to a bone organ chip loaded with a microelectrode array and a use method thereof. BACKGROUND

[0002] In the bone tissue microenvironment, osteoblasts and osteoclasts are mutually regulated, and there is a complex osteoclast-osteoblast coupling process, which is called "bone remodeling". Studies have shown that angiogenesis has a significant promoting effect on the osteogenesis process, i.e. "osteogenesis-angiogenesis coupling", in addition, the role of nerve regulation cannot be ignored. Any disorder in the above-mentioned aspects may lead to imbalance of homeostasis, and further lead to the occurrence of diseases such as osteoporosis and osteosclerosis.

[0003] Osteoporosis is a systemic bone disease characterized by low bone mass, damage to bone tissue microstructure, and increased bone fragility and susceptibility to fractures. It is more common in postmenopausal women and elderly men. In recent years, the prevalence of osteoporosis has risen rapidly, and it has become an important public health problem. M-CSF and RANKL are key cytokines in the differentiation process of osteoclasts, and a series of pro-osteoclast factors, interleukins and prostaglandin E2 can induce the expression of M-CSF and RANKL, stimulate osteoclasts, and cause bone loss, leading to osteoporosis.

[0004] Although some progress has been made in understanding the bone remodeling process and osteoporosis, the regulatory pathways and pathogenic mechanisms of certain factors and new drug screening still need to be further explored. Traditional animal models and two-dimensional cell culture models have significant limitations, such as large species differences, high cost, ethical controversy, lack of three-dimensional microenvironment spatial structure and cell-cell interactions, and inability to simulate the "vascular-nerve-osteogenesis" multi-coupling mechanism in bone tissue. Organ chips, as a new research model, can meet the above requirements under certain conditions and are expected to help researchers further elucidate the mechanism of bone remodeling.

[0005] Therefore, the present application provides a bone organ chip loaded with a microelectrode array and a use method thereof, which can eliminate the drawbacks of the prior art. SUMMARY

[0006] The present application aims to provide a bone organ chip loaded with a microelectrode array and a use method thereof to solve the problem of insufficient research methods due to the limitations of traditional models in the background art.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions: A bone organ-on-a-chip loaded with microelectrode array, the chip substrate is made of polydimethylsiloxane material, the chip comprises a three-layer structure: a top layer provided with a microelectrode array for recording and stimulating cell electrical activity; an intermediate layer composed of five microfluidic channels, five liquid inlets and five liquid outlets arranged in parallel and in sequence, the microfluidic channels include two blood vessel microchannels, two osteoblast microchannels and one neuron microchannel, each of the microfluidic channels is connected with one liquid inlet and one liquid outlet, the two osteoblast microchannels are respectively arranged on the left and right sides of the neuron microchannel, the two blood vessel microchannels are respectively arranged on the left and right sides of the osteoblast microchannels, the blood vessel microchannels and the osteoblast microchannels are separated by a plurality of uniformly arranged first pillars, the first pillars include spacer pillars A and B, the first pillars are arranged inside the blood vessel microchannels, the neuron microchannel and the osteoblast microchannels on both sides thereof are separated by a plurality of second pillars arranged in a certain ratio of density, the second pillars include spacer pillars C and D, a plurality of uniformly arranged micro pillars are arranged in the osteoblast microchannels, the micro pillars include micro pillars E and F; a bottom layer provided with pneumatic valves for connecting external gas controllers, each of the microfluidic channels is connected with one pneumatic valve at the inlet and the outlet for controlling the opening and closing of the microchannels, the bottom layer is used to realize the sealing effect of the intermediate layer in cooperation with the top layer.

[0008] Preferably, the diameter of the blood vessel microchannel is 400 μm, which is used for inoculating umbilical vein endothelial cells in the early stage, and mononuclear cells are inoculated after the blood vessels are formed and grow into adjacent channels, the diameter of the osteoblast microchannel is 200 μm, which is used for inoculating mesenchymal stem cells, and the diameter of the neuron microchannel is 300 μm, which is used for inoculating embryonic dorsal root ganglion and simulating neurons in the bone microenvironment.

[0009] Preferably, the cross-sectional shape of the first pillar is isosceles trapezoidal, the upper base size of the isosceles trapezoid is 50 μm, the lower base size is 150 μm, and the height is 150 μm, the distance between adjacent two first pillars is 100 μm, the osteoblast microchannel is provided with micro pillars arranged uniformly along the 100 μm interval holes, the diameter of the micro pillar is 20 μm, the interval is 30 μm, and the height is 30 μm, the micro pillars are used to guide the blood vessels to grow into the neuron microchannel along the channel surrounded by the micro pillars.

[0010] Preferably, the second columnar section shape is rectangular, the length of the rectangle is 150 μm, the width is 40 μm, every 3 adjacent second columns form a column group, the interval between the two adjacent second columns in the column group is 15 μm, the interval between the two adjacent column groups is 100 μm, the ratio of the number of the interval of 15 μm to the interval of 100 μm is 2:1, the interval of 15 μm is used to define the neuron axon passing, and the interval of 100 μm is used for blood vessels and neuron axons to pass and grow into the neuron microchannel.

[0011] Preferably, the microelectrode array comprises a plurality of microelectrode sites for directly contacting biological tissues and detecting extracellular electrical activity, the microelectrode sites comprising first microelectrode sites and second microelectrode sites, wherein the first microelectrode sites are located in the neuron microchannel for stimulating the growth of axon initial, and the second microelectrode sites are located in the osteoblast microchannel for detecting the electrical physiological signals of the growth of neuron axon and cell body, and the microelectrode sites are connected with connecting wires for transmitting electrical signals, and the other end of the connecting wires is connected to an integrated circuit matrix.

[0012] Preferably, the chip is filled with 3D printed biological ink, which is prepared by mixing methacrylic acid gelatin, methacrylate alginate and hydroxyapatite.

[0013] A use method of a bone organ chip loaded with a microelectrode array, specifically comprising the following steps: S1, cleaning the top layer, the middle layer and the bottom layer, modifying the surface of each layer by plasma, and then assembling; S2, after cleaning the five microfluidic channels with an alcohol solution, sterilizing by ultraviolet irradiation; S3, mesenchymal stem cells and umbilical vein endothelial cells are inoculated into the osteoblast microchannel and the blood vessel microchannel respectively, and corresponding cell culture solution is added for culture, after culture, dorsal root ganglion cells are inoculated into the neuron microchannel, and corresponding culture medium is added, and after culture, mononuclear cells are inoculated into the blood vessel microchannel; S4, after the cells in the five microfluidic channels grow, mature and produce crosstalk, the activity, matrix composition and morphology of various cells in the chip are monitored to simulate the bone remodeling process.

[0014] Preferably, the cell culture solution of the mesenchymal stem cells comprises 10% fetal bovine serum and 1% penicillin or streptomycin culture medium, supplemented with 10 nM dexamethasone, 100 μM ascorbic acid and 10 mM β-glycerophosphate.

[0015] Preferably, the cell culture solution of the umbilical vein endothelial cells comprises a basal medium of 10% fetal bovine serum, 1% penicillin or streptomycin, 90 μg / mL heparin and 15 μg / mL endothelial cell growth supplement, the cell culture solution of the monocytes comprises a culture medium of 10% fetal bovine serum and 1% penicillin or streptomycin, and is supplemented with 50 ng / mL RANKL and 25 ng / mL M-CSF. Preferably, the cell culture solution of the umbilical vein endothelial cells comprises a basal medium of 10% fetal bovine serum, 1% penicillin or streptomycin, 90 μg / mL heparin and 15 μg / mL endothelial cell growth supplement, the cell culture solution of the monocytes comprises a culture medium of 10% fetal bovine serum and 1% penicillin or streptomycin, and is supplemented with 50 ng / mL RANKL and 25 ng / mL M-CSF.

[0016] Preferably, the cell culture solution of the embryonic dorsal root ganglion cells comprises a neural basal medium of B-27 serum-free additive, 60 μM 5-fluoro-2'-deoxyuridine, 25 mM glucose, 1 mM sodium pyruvate, 50 ng / mL 7S nerve growth factor, 2 mM glutamine and 1% penicillin-streptomycin, and the volume ratio of the B-27 serum-free additive in the final solution is 2%.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a bone organ chip loaded with a microelectrode array, introduces nerve channels and blood vessel channels, constructs a bone microenvironment with nerve and immune regulation, embodies physiological processes such as osteogenesis-angiogenesis coupling, and influences osteogenesis-osteoclast balance by controlling the growth and differentiation of vascular endothelial cells and nerve cells, thereby inducing related disease models; 2. The present application designs micro-holes of different sizes between different micro-channels to appropriately enhance the crosstalk between cells, uses a first column to separate the interface between the blood vessel micro-channels and the osteoblast micro-channels on both sides to allow the blood vessels to grow in, uses another size of a second column to separate the adjacent interfaces between the neuron micro-channels and the osteoblast micro-channels on both sides, and the micro-holes separated by the second column are of inconsistent sizes, so that the substances allowed to pass are different, thereby allowing the blood vessels on both sides to grow together into the neuron micro-channels in the middle through the adjacent osteoblast micro-channels, and embodying a bone microenvironment with nerve-vascular-immune multi-element regulation; 3. The present application is loaded with a microelectrode array, which overcomes the signal monitoring defects of two-dimensional models, can monitor neuron electrophysiological signals in real time, is composed of multiple microelectrodes, can record or stimulate the electrical activity of cells, and has good adaptation performance with microfluidic chips. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of the overall structure of the chip of the present application.

[0019] Figure 2 It is a schematic diagram of the internal channels of the chip of the present application.

[0020] Figure 3 It is a schematic diagram of the channel structure of the organ chip of the present application.

[0021] Figure 4 Structure diagram of microelectrode array of the present application.

[0022] Figure 5 Flow diagram of the use method of the present application.

[0023] Figure mark annotation: liquid inlet 1~5; liquid outlet 6~10; pneumatic valve 11~20; first microelectrode site 21; second microelectrode site 22; integrated circuit matrix 23; connecting wire 24; blood vessel microchannel 25, 29; osteoblast microchannel 26, 28; neuron microchannel 27; top layer 30; middle layer 31; bottom layer 32; microelectrode array 33. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples.

[0025] In this embodiment, as shown in Figure 1 - Figure 5 A bone-like organ chip carrying a microelectrode array, the chip substrate is made of polydimethylsiloxane material, which has good biocompatibility, gas permeability and optical transparency, facilitating microscopic observation, and the chip includes a three-layer structure: The top layer 30 integrates the microelectrode array 33 and the electronic interface, which is used for real-time monitoring and intervention of cell electrical activity, and can partially close the middle layer 31, the top layer 30 is provided with a microelectrode array 33 for recording and stimulating cell electrical activity, covering most of the central area of the chip, the microelectrode array 33 includes a plurality of microelectrode sites for directly contacting biological tissues and detecting extracellular electrical activity, the microelectrode sites include first microelectrode sites 21 and second microelectrode sites 22, wherein the first microelectrode sites 21 are located in the neuron microchannel 27 for stimulating the growth of the initial part of the axon, and the second microelectrode sites 22 are located in the osteoblast microchannels 26, 28 for detecting the electrophysiological signals of the growth of the neuron axon and the cell body, the microelectrode sites are connected with connecting wires 24 for transmitting electrical signals, the other end of the connecting wires 24 is connected to the integrated circuit matrix 23; The intermediate layer 31 is a core reaction zone, and is internally provided with a microfluidic channel network, simulating different functional regions in bone tissue. The intermediate layer 31 is composed of five microfluidic channels arranged in parallel and in sequence, five liquid inlets 1-5 and five liquid outlets 6-10. The microfluidic channel includes two blood vessel microchannels 25, 29, two osteoblast microchannels 26, 28 and one neuron microchannel 27. Each microfluidic channel is connected with one liquid inlet and one liquid outlet. The two osteoblast microchannels 26, 28 are respectively arranged on the left and right sides of the neuron microchannel 27. The two blood vessel microchannels 25, 29 are respectively arranged on the left and right sides of the osteoblast microchannels 26, 28. The above layout simulates the spatial relationship between the nerve blood vessel bundle and the bone unit in the natural bone tissue. The blood vessel microchannels 25, 29 and the osteoblast microchannels 26, 28 are separated by a plurality of uniformly arranged first columns. The first column includes a spacing column A and a spacing column B. The first column is arranged in the interior of the blood vessel microchannel 25, 29. The neuron microchannel 27 and the osteoblast microchannels 26, 28 located on the two sides thereof are separated by a plurality of second columns arranged at a certain ratio of density. The second column includes a spacing column C and a spacing column D. A plurality of micro columns are arranged in the osteoblast microchannels 26, 28. The micro column includes a micro column E and a micro column F. The bottom layer 32 is provided with pneumatic valves 11-20 for connecting an external gas controller. Each microfluidic channel is connected with one pneumatic valve at the inlet and the outlet, respectively, for controlling the opening and closing of the microchannel. The bottom layer 32 is used to realize the sealing effect of the intermediate layer 31 in cooperation with the top layer 30. Specifically, the chip is essentially a kind of bionic system, aiming to accurately reproduce the cell composition, spatial structure and functional characteristics in bone tissue in vitro. Compared with traditional models, it can integrate multi-element cell networks and dynamic monitoring functions, thereby providing a more comprehensive bone remodeling research platform. As shown in Figure 2 and Figure 3 The diameter of the blood vessel microchannel 21 is 400 μm, which is used for inoculating umbilical vein endothelial cells in the early stage. After the blood vessels are formed and grow into the adjacent channels, mononuclear cells are inoculated. The diameters of the osteoblast microchannels 26, 28 are 200 μm, which are used for inoculating mesenchymal stem cells. The diameter of the neuron microchannel 27 is 300 μm, which is used for inoculating embryonic dorsal root ganglion and simulating neurons in the bone microenvironment. Specifically, the blood vessel microchannels 25, 29 with a diameter of 400 μm are optimized by fluid dynamics to ensure that the endothelial cells form a stable three-dimensional vascular network under shear force. After the umbilical vein endothelial cells are inoculated in the early stage, vascularization is promoted by perfusing VEGF (50 ng / mL). After about 7 days, the blood vessels can be observed to extend to the adjacent osteoblast microchannels 26, 28. After the blood vessels mature, mononuclear cells are inoculated and RANKL and M-CSF are added to induce them to differentiate into osteoclasts, simulating the "blood vessel-osteoclast coupling". Osteoblast microchannels 26 and 28, with a diameter of 200 μm, allow mesenchymal stem cells to differentiate into osteoblasts within 14 days through osteogenic induction medium, exhibiting increased alkaline phosphatase activity and calcium nodule deposition (positive Alizarin Red staining). The 100 μm gap between these microchannels and vascular microchannels 25 and 29 allows for vascular ingrowth, promoting osteogenic-angiogenic coupling. Simultaneously, osteoclast-osteogenic coupling is achieved through co-culturing osteoclasts. The 300μm diameter neuronal microchannel 27 optimizes the axonal growth space of the embryonic dorsal root ganglion, simulating the neurovascular interaction; Among them, such as Figure 2 As shown, the cross-sectional shape of the first column is set as an isosceles trapezoid, with the upper base dimension set to 50μm, the lower base dimension set to 150μm, and the height set to 150μm. The spacing between two adjacent first columns is set to 100μm, which allows vascular endothelial cell migration and blood vessel ingrowth. Spacing column A and spacing column B are fabricated using soft lithography. Osteoblast microchannels 26 and 28 are provided with several microcolumns evenly arranged along 100μm spacing pores. The microcolumns have a diameter of 20μm, a spacing of 30μm, and a height of 30μm. The microcolumns are used to guide blood vessels to grow into neuronal microchannels 27 along the channels formed by the microcolumns. Microcolumns E and F are fabricated using soft lithography. Among them, such as Figure 2 As shown, the cross-sectional shape of the second column is set to rectangular, with a length of 150 μm and a width of 40 μm. Every three adjacent second columns form a column group. The spacing between two adjacent second columns within a column group is set to 15 μm, which effectively simulates the physical barrier of a neural synapse, allowing only neuronal axons with a diameter less than 15 μm to pass through, thereby promoting the directional extension of nerve fibers. The spacing between two adjacent column groups is set to 100 μm, which simulates the neurovascular anastomosis structure in bone tissue. The ratio of 15μm intervals to 100μm intervals is set to 2:1. This interval ratio conforms to the density gradient of nerve-blood vessel distribution in natural bone tissue. The 15μm intervals are used to limit the passage of neuronal axons, while the 100μm intervals are used to allow blood vessels and neuronal axons to pass through and grow into the neuronal microchannel 27. The column group between the neuronal microchannel 27 and the osteoblast microchannels 26 and 28 is achieved by two-photon laser engraving. Its surface can be further modified with neurotrophic factors such as laminin to ensure selective growth of neuronal axons. Among them, such as Figure 1As shown, the chip is filled with 3D-printed bio-ink, which is composed of gelatin methacrylate, alginate methacrylate, and hydroxyapatite. The mixture of gelatin methacrylate (10% w / v), alginate methacrylate (2% w / v), and hydroxyapatite (5% w / v) is filled into the microfluidic channels of the chip using 3D printing technology. The porosity is approximately 80%, and the pore size is set at 50~100 μm. This provides mechanical support and simulates the chemical composition and microstructure of the natural bone matrix. The microporous structure design of the bio-ink works synergistically with the layout of the first and second pillars in the chip to ensure that cells in different channels can permeate each other through the pores of the bio-ink scaffold, achieving multi-dimensional coupling of nerve-blood vessel-osteogenes, thereby simulating the complex cell network in bone tissue. In addition, the transparency and non-conductivity of the bio-ink do not affect the function of the top microelectrode array 33, nor do they affect the detection of electrical signals, ensuring stable monitoring of electrophysiological signals. Among them, such as Figure 5 As shown, the cell culture medium for mesenchymal stem cells contains 10% fetal bovine serum and 1% penicillin or streptomycin. The culture medium was supplemented with 10 nM dexamethasone, 100 μM ascorbic acid and 10 mM β-glycerophosphate. Specifically, dexamethasone, as a glucocorticoid, can promote the differentiation of mesenchymal stem cells into osteoblasts; ascorbic acid is an essential cofactor for collagen synthesis and can promote the deposition of extracellular matrix; β-glycerophosphate provides a source of phosphate, which combines with calcium ions to form hydroxyapatite crystals, mimicking the bone mineralization process. This culture medium combination has been optimized to efficiently induce osteogenic differentiation of mesenchymal stem cells, manifested by increased alkaline phosphatase activity, calcium nodule formation, and upregulation of osteogenic-related gene expression. Furthermore, the addition of penicillin / streptomycin to the culture medium can effectively prevent bacterial contamination and ensure the stability of cell culture. Among them, such as Figure 5 As shown, the cell culture medium for umbilical vein endothelial cells contained 10% fetal bovine serum, 1% penicillin or streptomycin, 90 μg / mL heparin and 15 μg / mL endothelial cell growth supplement. The cell culture medium for monocytes contained 10% fetal bovine serum and 1% penicillin or streptomycin, supplemented with 50 ng / mL RANKL and 25 ng / mL LM-CSF. Specifically, by inoculating umbilical vein endothelial cells into the microchannels 25 and 29 on both sides, a functional three-dimensional vascular network can be formed. The blood vessels will grow into the osteogenic channel through the channels formed by the designed micropores and micropillars E and F, reproducing the "osteogenic-angiogenic coupling" process, improving the nutritional supply of deep cells, and actively participating in the regulation of bone metabolism by secreting factors such as VEGF and Angiopoietin-1. After adding circulating monocytes, the system can further simulate the interaction between blood vessels and immune cells under inflammatory conditions, which is helpful for osteoporosis research. Among them, such as Figure 5 As shown, the cell culture medium for embryonic dorsal root ganglion cells contained B-27 serum-free additive, 60 μM 5-fluoro-2'-deoxyuridine, 25 mM glucose, 1 mM sodium pyruvate, 50 ng / mL 7S nerve growth factor, 2 mM glutamine, and 1% penicillin-streptomycin as the basic nerve medium. The volume ratio of B-27 serum-free additive in the final solution was 2%. Specifically, by inoculating embryonic dorsal root ganglion cells, a functional neural network can be formed in the chip. Neurons extend axons through micropores to interact with osteoblasts, simulating the nerve innervation in bone tissue. The microelectrode array 33 can monitor the electrical activity of neurons and their effects on osteoblast / osteoclast function in real time, which helps to explore the association between neuropathy and osteoporosis. like Figure 5 As shown, a method for using a bone organoid chip carrying a microelectrode array specifically includes the following steps: S1. Clean the top layer 30, the middle layer 31 and the bottom layer 32, and use plasma to modify the surface of each layer before assembling. S2. After cleaning the above five microfluidic channels with alcohol solution, perform disinfection by ultraviolet irradiation. S3. Mesenchymal stem cells and umbilical vein endothelial cells were seeded into osteoblast microchannels 26 and 28 and vascular microchannels 25 and 29, respectively. The corresponding cell culture media were added for culture. After culture, embryonic dorsal root ganglion cells were seeded into neuronal microchannel 27. The corresponding culture medium was added and cultured. After culture, monocytes were seeded into vascular microchannels 25 and 29. S4. After the cells inside the five microfluidic channels grow, mature, and generate crosstalk, monitor the activity, matrix composition, and morphology of various cells in the chip to simulate the bone remodeling process. Specifically, S1, the chip body adopts a polydimethylsiloxane material (PDMS), and is formed by molding. After each layer structure (top layer 30, middle layer 31, and bottom layer 32) is prepared, the surface is treated by oxygen plasma, the power of the plasma machine is set to 50 W, and the time is set to 30 seconds to enhance the hydrophilicity and interlayer adhesion of the PDMS. After plasma treatment, the layers are immediately assembled to avoid surface energy decay. The layers are bonded layer by layer using a PDMS adhesive to ensure no leakage; The microelectrode array 33 is embedded in the top layer 30, and the electrode material is gold or platinum. The diameter and spacing can be adjusted according to actual needs. Assuming the diameter is 20 μm and the spacing is 100 μm, the electrodes are prepared by photolithography technology. The electrode leads 24 are connected to an external signal acquisition system or device through a flexible printed circuit board; S2, fill the microfluidic channel with a 75% ethanol solution, and after standing for 10 minutes, rinse with sterile PBS several times, and then irradiate with ultraviolet light for about 30 minutes, with a wavelength of 254 nm and an intensity of 30 mW / cm² to ensure a sterile environment. Fill the 3D printed bio-ink and crosslink it under ultraviolet light to form a scaffold; S3, seed umbilical vein endothelial cells in the vascular microchannels 25 and 29 at a density of 2×10 6 cells / mL, and perfuse the umbilical vein endothelial cell culture medium. Culture for 48 hours. Verify whether a preliminary vascular network has been formed by CD31 immunofluorescence. Seed mesenchymal stem cells in the osteoblast microchannels 26 and 28 at a density of 1×10 6 cells / mL, and the mesenchymal stem cell culture medium is medium. Culture for 72 hours. Confirm whether osteogenic differentiation has started by alkaline phosphatase activity detection method. Seed embryonic dorsal root ganglion cells in the neuron microchannel 27 at a density of 5×10 5 cells / mL, and the embryonic dorsal root ganglion cell culture medium is neural basal medium. After 24 hours of culture, observe axon extension by β-lllTubulin staining method. Add monocytes to the vascular microchannels 25 and 29 at a density of 1×10 6 cells / mL, and the monocyte culture medium is supplemented with 50 ng / mL RANKL and 25 ng / mL M-CSF. After 5 days of culture, confirm whether osteoclasts have differentiated by TRAP staining method; During this process, the microfluidic pump maintains the flow rate of the culture medium according to the actual environmental requirements to simulate a physiological fluid environment; S4, record the electrical activity signals of the neuron axons in real time through the microelectrode array 33, analyze the action potential frequency and peak potential interval, and calculate the neuron activity quantitatively by the following formula: where N is the number of recorded peak potentials, and T is the recording time. The area of calcium nodules was quantified using alizarin red staining, and the degree of osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity detection, with the formula The number of osteoclasts was counted by tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity detection, which can be observed by microscopic image analysis software. After immunofluorescence staining, angiogenesis was evaluated by the number of vascular branch points and the total length of blood vessels, with the formula ; Calcium yellow-green fluorescence staining combined with fluorescence microscope imaging was used to analyze the mineralization area ratio by gray value qPCR was used to detect the expression levels of osteogenic-related genes and osteoclastic-related genes, and the relative expression level was calculated by the 2−ΔΔCt method. All data were expressed as mean ± standard deviation, and ANOVA was used to analyze the differences between groups. The three-dimensional model of the bone-like structure within the chip was reconstructed by micro-CT scanning, and the results verified the effectiveness of the chip in simulating bone microenvironment, monitoring cell interaction, and constructing disease models.

[0026] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A bone organoid chip carrying a microelectrode array, characterized in that, The chip substrate is made of polydimethylsiloxane material, and the chip comprises a three-layer structure: The top layer (30) is provided with a microelectrode array (33) for recording and stimulating cellular electrical activity. The intermediate layer (31) consists of five parallel and sequentially arranged microfluidic channels, five inlets (1-5), and five outlets (6-10). The microfluidic channels include two vascular microchannels (25, 29), two osteoblast microchannels (26, 28), and one neuron microchannel (27). Each microfluidic channel is connected to one inlet and one outlet. The two osteoblast microchannels (26, 28) are respectively located on the left and right sides of the neuron microchannel (27), and the two vascular microchannels (25, 29) are respectively located on the left and right sides of the osteoblast microchannels (26, 28). On both sides, the vascular microchannels (25, 29) and the osteoblast microchannels (26, 28) are separated by a number of uniformly arranged first columns, each first column including a spacer column A and a spacer column B. The first columns are located inside the vascular microchannels (25, 29). The neuronal microchannel (27) is separated from the osteoblast microchannels (26, 28) located on both sides by a number of second columns arranged in a certain ratio of density. The second columns include a spacer column C and a spacer column D. The osteoblast microchannels (26, 28) are provided with a number of uniformly arranged microcolumns, each microcolumn including a microcolumn E and a microcolumn F. The bottom layer (32) is provided with pneumatic valves (11~20) for connecting to an external gas controller. Each microfluidic channel is connected to a pneumatic valve at the inlet and outlet to control the opening and closing of the microchannel. The bottom layer (32) is used to cooperate with the top layer (30) to achieve the sealing effect of the middle layer (31).

2. The bone organoid chip with a microelectrode array according to claim 1, characterized in that, The diameter of the vascular microchannels (25, 29) is set to 400 μm, which is used for the initial inoculation of umbilical vein endothelial cells. After the blood vessels are formed and grow into the adjacent channels, mononuclear cells are inoculated. The diameter of the osteoblast microchannels (26, 28) is set to 200 μm, which is used for the inoculation of mesenchymal stem cells. The diameter of the neuronal microchannel (27) is set to 300 μm, which is used for the inoculation of embryonic dorsal root ganglia and to simulate neurons in the bone microenvironment.

3. A bone organoid chip with a microelectrode array according to claim 2, characterized in that, The first column has an isosceles trapezoidal cross-section. The upper base of the isosceles trapezoid is 50 μm, the lower base is 150 μm, and the height is 150 μm. The distance between two adjacent first columns is 100 μm. The osteoblast microchannels (26, 28) are provided with microcolumns evenly arranged along 100 μm intervals. The diameter of the microcolumns is 20 μm, the interval is 30 μm, and the height is 30 μm. The microcolumns are used to guide blood vessels to grow into the neuronal microchannels (27) along the channel formed by the microcolumns.

4. A bone organoid chip with a microelectrode array according to claim 3, characterized in that, The second column is rectangular in shape, with a length of 150 μm and a width of 40 μm. Every three adjacent second columns form a column group. The interval between two adjacent second columns in a column group is 15 μm, and the interval between two adjacent column groups is 100 μm. The ratio of the number of intervals with a distance of 15 μm to the number of intervals with a distance of 100 μm is 2:

1. The intervals with a distance of 15 μm are used to limit the passage of neuronal axons, and the intervals with a distance of 100 μm are used to allow blood vessels and neuronal axons to pass through and grow into the neuronal microchannel (27).

5. A bone organoid chip with a microelectrode array according to claim 4, characterized in that, The microelectrode array (33) includes multiple microelectrode sites for direct contact with biological tissue and detection of extracellular electrical activity. The microelectrode sites include a first microelectrode site (21) and a second microelectrode site (22). The first microelectrode site (21) is located in the neuronal microchannel (27) and is used to stimulate the growth of the axon origin. The second microelectrode site (22) is located in the osteoblast microchannel (26, 28) and is used to detect the electrophysiological signals of neuronal axon and cell body growth. The microelectrode sites are connected to connecting wires (24) for transmitting electrical signals. The other end of the connecting wires (24) is connected to the integrated circuit matrix (23).

6. A bone organoid chip with a microelectrode array according to claim 5, characterized in that, The chip is filled with 3D-printed bio-ink, which is composed of gelatin methacrylate, methacrylate alginate, and hydroxyapatite.

7. A method of using a bone organoid microarray according to any one of claims 1-6, characterized in that, Specifically, the following steps are included: S1. Clean the top layer (30), middle layer (31) and bottom layer (32), and use plasma to modify the surface of each layer before assembling. S2. After cleaning the above five microfluidic channels with alcohol solution, perform disinfection by ultraviolet irradiation. S3. Mesenchymal stem cells and umbilical vein endothelial cells were seeded into osteoblast microchannels (26, 28) and vascular microchannels (25, 29), respectively. The corresponding cell culture medium was added for culture. After culture, embryonic dorsal root ganglion cells were seeded into neuronal microchannels (27). The corresponding culture medium was added. After culture, mononuclear cells were seeded into vascular microchannels (25, 29). S4. After the cells inside the five microfluidic channels grow, mature, and generate crosstalk, monitor the activity, matrix composition, and morphology of various cells in the chip to simulate the bone remodeling process.

8. The method of using the bone organoid chip according to claim 7, characterized in that, The cell culture medium for the mesenchymal stem cells contained 10% fetal bovine serum and 1% penicillin or streptomycin. The culture medium was supplemented with 10 nM dexamethasone, 100 μM ascorbic acid and 10 mM β-glycerophosphate.

9. The method of using the bone organoid microarray according to claim 7, characterized in that, The cell culture medium for the umbilical vein endothelial cells contained 10% fetal bovine serum, 1% penicillin or streptomycin, 90 μg / mL heparin, and 15 μg / mL endothelial cell growth supplement. The cell culture medium for the monocytes contained 10% fetal bovine serum and 1% penicillin or streptomycin. Culture medium, supplemented with 50 ng / mL RANKL and 25 ng / mL LM-CSF.

10. The method of using the bone organ-on-a-chip according to claim 7, characterized in that, The cell culture medium for the embryonic dorsal root ganglion cells contained B-27 serum-free additive, 60 μM 5-fluoro-2'-deoxyuridine, 25 mM glucose, 1 mM sodium pyruvate, 50 ng / mL 7S nerve growth factor, 2 mM glutamine, and 1% penicillin-streptomycin as the basic nerve medium, with the B-27 serum-free additive comprising 2% of the final solution by volume.

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