Mechanical loading device for cartilage tissue layering research and use method thereof

Abstract

The invention discloses a mechanical loading device for cartilage tissue layering research and a use method of the mechanical loading device, and belongs to the technical field of microphysiology devices. Comprising a flow channel layer and a tissue layer which are arranged up and down, the lower surface of the flow channel layer is provided with an 8-shaped culture solution flow channel, the upper surface of the tissue layer is symmetrically provided with two semicircular gel chambers, and cylindrical cavities are formed in the tissue layer and located on the inner sides of the gel chambers; hydrogel mixed with cartilage cells is conveyed to the gel chamber on one side to simulate an articular cartilage layer; hydrogel mixed with bone cells, osteoblasts and osteoclasts is conveyed to the gel chamber on the other side to simulate a subchondral bone layer; a culture solution circularly flows in the culture solution flow channel; continuously introducing gas at a constant speed through a guide pipe; the device can simulate the mechanical stimulation suffered by real cartilage tissues, and is more in line with the real physiological environment of articular cartilage; the method can be used for researching substance exchange and signal conduction in the articular cartilage.

Description

Technical Field

[0001] The present invention belongs to the technical field of microphysiological devices, and particularly relates to a mechanical loading device for cartilage tissue stratification research and a using method thereof. Background Art

[0002] The incidence of osteoarthritis is second only to cardiovascular diseases and can lead to long-term disability, seriously affecting the health and self-care ability of middle-aged and elderly people. However, the pathogenesis of osteoarthritis is still unclear, and the current clinical treatment methods for osteoarthritis are only limited to pain relief and delaying the progression of the disease. Existing experimental devices are difficult to simulate the activities of cartilage tissue in joints, which is not convenient for the mechanism research and drug screening of osteoarthritis.

[0003] The prior patent technology with the publication number of CN117025359A is a mechanically stretchable annular organ chip and method. Cells are planted in a novel uniform strain chamber of an adherent cell loading device. Four positioning holes of the chamber are placed on fixed piles, and the fixed piles are connected to a stator. A controller controls a stepping motor, and the rotational motion of the motor is converted into a linear motion through a transmission lead screw and transmitted to a mover, thereby indirectly applying strain stimulation to the cells in the chamber cavity. This invention provides a mechanically stretchable annular organ chip and method for forming experimental groups and control groups in the same chip. In the control group chip and the experimental group chip, it is avoided that the experimental results are affected due to uneven mixing of cell suspensions; the cells are uniformly stretched to observe the cell growth trend. The object studied by this device is cells, and the cells grow on a thin film layer. However, the true mechanical microenvironment of cartilage is at the tissue level rather than the cell level. This invention only realizes the stretching of cells without considering the actual physiological environment. Moreover, most chondrocytes actually receive compression rather than tensile deformation. Secondly, this device also needs to place the chip in an incubator for a period of time before applying force stimulation to it, resulting in low efficiency.

[0004] Journal: Nature Biomedical Engineering; Publication Date: June 3, 2019; Article Title: Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model; discloses a device for applying uniform and limited mechanical pressure to a three-dimensional cartilage microstructure, belonging to the field of biomechanical engineering technology; the device includes two chambers separated by a flexible membrane: an upper culture chamber for accommodating a 3D microconstruct, and a lower chamber serving as an actuation chamber. The culture chamber consists of a 300-meter-wide central channel for accommodating the 3D microconstruct, surrounded by two side channels for supplementing the culture medium. The central channel is defined by two parallel arranged hanging columns (T-shaped columns). In the static position, a gap separates the hanging columns from the flexible membrane, keeping the 3D microstructure in a relaxed state. When the actuation compartment is pressurized, the flexible membrane bends upward until it abuts against the bottom end of the T-shaped column, resulting in restricted compression of the 3D microconstruct. When the pressure is released, elastic recoil causes the membrane and the 3D microconstruct to relax to their original static configuration. Two T-shaped column sizes are designed to achieve 10% physiological compression and 30% hyperphysiological compression levels respectively. At the same time, cells and the culture medium come into contact at the gap of the T-shaped column. However, the biggest problem with this technology is the relatively large error; the heights of the T-shaped columns are designed to be 10% and 30% respectively after cell compression, and two devices need to be processed separately for comparative experiments, which inevitably causes errors during the processing and the cost is too high.

[0005] Journal: Lab on a Chip; Publication Year: 2022; Article Title: Emulating the chondrocyte; is based on emulating the in vivo environment to study how different types of mechanical stimuli affect the phenotype of chondrocytes and the production of the extracellular matrix (ECM). The system uses a thin deformable membrane and three separately addressable actuation chambers, allowing well-defined compression and multi-directional mechanical stimuli to be applied to an agarose hydrogel loaded with 3D chondrocytes; however, the same as the above-mentioned prior art, columns are used to separate the gel and the culture medium, and the processing is rather cumbersome; and it is also compressed up and down, which does not conform to the actual physiological environment.

[0006] Most existing studies are based on cell-level stimuli and responses, and most studies in traditional biology can only conduct single-factor stimuli; while the true mechanical microenvironment of cartilage is at the tissue level rather than the cell level, and cartilage tissue is mainly subjected to compressive force and shear force in the physiological environment, so the prior art is very different from the true physiological environment of cartilage. Summary of the Invention

[0007] The present invention overcomes the deficiencies of the prior art. At the tissue level, the present invention proposes a mechanical loading device for cartilage tissue stratification research and its usage method, which applies circumferential compressive force and fluid shear force to cartilage through uniform compression and an eight-shaped flow channel.

[0008] The present invention is realized through the following technical solutions: A mechanical loading device for cartilage tissue stratification research, including a flow channel layer located above and a tissue layer located below the flow channel layer, and the flow channel layer is connected to the tissue layer; an "8"-shaped culture medium flow channel is arranged on the lower surface of the flow channel layer, and two semi-circular gel chambers are symmetrically arranged on the upper surface of the tissue layer. A cylindrical cavity is arranged on the tissue layer and inside the gel chamber, and an elastic catheter is arranged in the cavity, and the outer wall of the catheter is attached to the inner side of the gel chamber; both the gel chamber and the culture medium flow channel are groove-shaped open structures; The culture medium flow channel is located directly above the gel chamber, and the two ends of the culture medium flow channel away from each other are semi-circular arc structures; the two gel chambers are respectively opposite to the two semi-circular arc structures of the culture medium flow channel; a hydrogel mixed with chondrocytes is transported to one gel chamber to simulate the articular cartilage layer; a hydrogel mixed with osteocytes, osteoblasts, and osteoclasts is transported to the other gel chamber to simulate the subchondral bone layer; the culture medium circulates in the culture medium flow channel; gas is continuously and uniformly introduced through the catheter to expand the catheter space area and achieve circumferential uniform extrusion of the hydrogel in the gel chamber, simulating mechanical stimulation of cartilage tissue. A bottom layer is connected below the tissue layer, and a gas buffer chamber is arranged in the bottom layer, and the bottom of the catheter is communicated with the gas buffer chamber.

[0009] Preferably, the materials of both the flow channel layer and the tissue layer are PDMS.

[0010] More preferably, the flow channel layer and the tissue layer are connected by bonding or the PDMS glue method.

[0011] Preferably, the two ends of the culture medium flow channel away from each other are connected with a culture medium delivery channel for delivering the culture medium; the gel chamber is connected with a gel delivery channel for delivering the hydrogel.

[0012] Preferably, the culture medium delivery channel, the gel delivery channel, and the catheter all pass through the flow channel layer and communicate with the outside.

[0013] Preferably, a microcirculation pump and a culture medium storage bin are connected between the two culture medium delivery channels.

[0014] A usage method of a mechanical loading device for cartilage tissue stratification research includes the following steps: Step 1, inject the hydrogel wrapped with chondrocytes into one gel chamber to simulate the articular cartilage layer, and then inject the hydrogel wrapped with osteocytes, osteoblasts, and osteoclasts into the other gel chamber to simulate the subchondral bone layer; Step 2: When the hydrogel changes from the liquid state to the solidified state, introduce the culture medium into the culture medium flow channel to make the culture medium circulate in the culture medium flow channel; the culture medium flows through the surface of the solidified hydrogel to generate a fluid shear force on the articular cartilage layer and the subchondral bone layer. Step 3: Introduce gas into the two conduits to apply a continuous and constant circumferential force stimulus or a dynamic circumferential force stimulus to the articular cartilage layer and the subchondral bone layer.

[0015] The beneficial effects of the present invention compared with the prior art are as follows: Based on the influence of mechanical stimulation on cartilage tissue, the present invention conducts a hierarchical study on articular cartilage tissue and explores biological problems at the tissue level. Through the hierarchical structure, the mechanical transmission, substance exchange (such as metabolites, cytokines), and intercellular signal transduction between cartilage and subchondral bone can be studied, and it can also be used to simulate the pathogenesis of diseases such as osteoarthritis (OA). For example: the relationship between cartilage degeneration and subchondral bone remodeling; the transmission of inflammatory factors between cartilage and subchondral bone and their effects on tissues.

[0016] The present invention can mimic the mechanical stimulation received by real cartilage tissue by applying circumferential compressive force and fluid shear force to the cartilage through uniform compression and the eight-shaped flow channel; the semi-circular structure compression is more in line with the real physiological environment of articular cartilage; it is used to study problems such as substance exchange and signal conduction inside articular cartilage. Brief Description of the Drawings

[0017] Figure 1 It is a schematic external structure diagram of the mechanical loading device described in the present invention; Figure 2 It is a front view of the mechanical loading device described in the present invention; Figure 3 is Figure 2 the sectional view taken along the line A-A in Figure 4 is Figure 2 the sectional view taken along the line B-B in Figure 5 is Figure 2 the sectional view taken along the line C-C in Figure 6 It is a schematic external structure diagram of the overall flow channel layer, tissue layer, and bottom layer; Figure 7 It is a perspective view of the bottom structure of the flow channel layer; Figure 8 It is an axonometric view of the top structure of the tissue layer; Figure 9 It is an axonometric view of the bottom layer; Figure 10 It is a top view of the overall flow channel layer, tissue layer, and bottom layer; Figure 11For Figure 10 D-D sectional view in the figure; Reference numerals in the figure: 1 is the flow channel layer, 2 is the tissue layer, 3 is the culture medium flow channel, 4 is the culture medium delivery channel, 5 is the gel chamber, 6 is the gel delivery channel, 7 is the catheter, 8 is the microcirculation pump, 9 is the culture medium storage bin, 10 is the bottom layer, 11 is the gas buffer chamber. Specific embodiments

[0018] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention will be further described in detail in conjunction with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention will be described in detail below in conjunction with embodiments and drawings, but the protection scope is not limited by this.

[0019] Embodiment 1 See Figures 1 to 11 , this embodiment proposes a mechanical loading device for cartilage tissue stratification research, including a flow channel layer 1 located above and a tissue layer 2 located below the flow channel layer 1. The tissue layer 2 is connected to a bottom layer 10 below. The flow channel layer 1, the tissue layer 2 and the bottom layer 10 are all horizontally arranged and relatively fixed. The flow channel layer 1 and the tissue layer 2, as well as the tissue layer 2 and the bottom layer 10, are connected by bonding or PDMS glue method; the materials of the flow channel layer 1, the tissue layer 2 and the bottom layer 10 are all PDMS.

[0020] The lower surface of the flow channel layer 1 is provided with an "8"-shaped groove as the culture medium flow channel 3. The two ends of the culture medium flow channel 3 that are far away from each other are symmetrically connected to the culture medium delivery channel 4; the upper surface of the tissue layer 2 is symmetrically provided with two semi-circular grooves as the gel chambers 5. The gel chambers 5 are connected to the gel delivery channel 6; a cylindrical cavity is provided inside the tissue layer 2 at the inner side of the gel chamber 5, and a cylindrical catheter 7 is provided in the cavity. The catheter 7 is made of an elastic material, and the outer wall of the catheter 7 is closely attached to the gel chamber 5.

[0021] After the flow channel layer 1 and the tissue layer 2 are connected up and down, the culture medium flow channel 3 is located directly above the gel chamber 5, and the two ends of the culture medium flow channel that are far away from each other are in a semi-circular arc structure; the two gel chambers 5 are respectively opposite to the two semi-circular arc structures of the culture medium flow channel 3.

[0022] The culture medium delivery channel 4, the gel delivery channel 6, and the catheter 7 all pass through the flow channel layer 1 and communicate with the outside; a gas buffer chamber 11 is provided in the bottom layer 10, and the bottom of the catheter 7 communicates with the gas buffer chamber 11. The catheter 7 vertically passes through the inside of the gel chamber 5 of the tissue layer 2 and extends to the gas buffer chamber 11 of the bottom layer 10 to make the gel chamber 5 receive a uniform circumferential force. The gas buffer chamber 11 can buffer the gas flowing in from the catheter 7, so that the gas in the catheter 7 can more evenly extrude the gel chamber 5.

[0023] Among them, the hydrogel mixed with chondrocytes is delivered to the corresponding gel chamber 5 through the gel delivery channel 6 on one side. After solidification, it simulates the articular cartilage layer; the hydrogel mixed with osteocytes, osteoblasts, and osteoclasts is delivered to the corresponding gel chamber 5 through the gel delivery channel 6 on the other side. After solidification, it simulates the subchondral bone layer.

[0024] A microcirculation pump 8 and a culture medium storage bin 9 are connected between the two culture medium delivery channels 4. Through the microcirculation pump 8, a closed loop of circulating flow is formed between the two culture medium delivery channels 4 and the culture medium flow channel 3. The microcirculation pump 8 transports the liquid nutrient substances in the culture medium storage bin 9 through one culture medium delivery channel 4 to the culture medium flow channel 3, and then flows out through the other culture medium delivery channel 4, so that a culture medium circulating flow body is formed inside the culture medium flow channel 3. The culture medium flows between the surfaces of the articular cartilage layer and the subchondral bone layer. On the one hand, it can carry out material and information exchange between the two gel chambers 5, and on the other hand, it also has an effect of generating fluid shear force on the simulated articular cartilage layer and subchondral bone layer.

[0025] By continuously and uniformly introducing gas through the catheter 7 and the buffering effect of the gas buffer chamber 11, the space area of the catheter 7 expands to evenly extrude the gel chamber 5 from all directions, realizing circumferential uniform extrusion of the hydrogel in the two gel chambers 5 on both sides, and simulating the application of mechanical stimulation to cartilage tissue.

[0026] In this embodiment, the diameter of chondrocytes is between 10 and 20 microns and is evenly distributed in the hydrogel; the hydrogel is PEG or alginate, and the inner diameter of the gel delivery channel 6 is between 100 and 500 microns, which can not only ensure the smooth passage of the hydrogel but also facilitate the uniform distribution of cells.

[0027] The gel chamber 5 can withstand cyclic mechanical loads and support mechanical stimuli (such as compression and shear). The inner diameter of the gel chamber 5 is between 500 microns and 1 mm, which is more suitable for applying mechanical stimuli.

[0028] The inner diameters of the culture medium flow channel 3 and the culture medium delivery channel 4 are between 100 and 300 microns, which is more conducive to the flow and diffusion of nutrient substances and metabolites.

[0029] Example 2 The usage method of a mechanical loading device for cartilage tissue stratification research proposed in this embodiment is as follows: Step 1: Ensure that all components are clean and undamaged. Correctly connect the culture medium storage tank 9 and the microcirculation pump 8 to the culture medium delivery channel 4 through connecting pipelines, and ensure that the interfaces are well sealed to avoid liquid leakage. Set parameters such as the flow rate and pressure of the microcirculation pump 8 according to experimental requirements.

[0030] Step 2: Inject the hydrogel encapsulating chondrocytes into the corresponding gel chamber 5 through one gel delivery channel 6, and then inject the hydrogels encapsulating osteocytes, osteoblasts, and osteoclasts into the corresponding gel chambers 5 through another gel delivery channel 6. The amount of hydrogel injected is obtained based on the volume of the two gel chambers 5.

[0031] Step 3: When the hydrogel changes from a liquid state to a solid state, inject the culture medium into the culture medium storage tank 9 (the culture medium storage tank 9 is the central node for liquid storage and distribution. Injecting the culture medium starting from the culture medium storage tank 9 can ensure that the culture medium evenly fills the entire culture medium flow channel 3 and avoid air bubble residues); the culture medium flows through one culture medium delivery channel 4 into the culture medium flow channel 3, and then flows back to the culture medium storage tank 9 through another culture medium delivery channel 4 to form a cycle. The culture medium flows through the surface of the solidified hydrogel to simulate the fluid shear force formed on the articular cartilage layer and the subchondral bone layer.

[0032] Step 4: Connect the air inlet at the top of the catheter 7 to a gas supply device or manually inject gas, and apply a continuous and constant circumferential force (the circumferential force is mainly pressure) or a dynamic circumferential force stimulus to the gel chamber 5 on the upper surface of the tissue layer 2 by introducing gas into the bottom layer 10 through the two catheters 7.

[0033] Example 3 The preparation method of a mechanical loading device for cartilage tissue stratification research proposed in this embodiment is as follows: Step 1: Fabricate a photolithography mask: Design a three-dimensional diagram of the mechanical loading device, and fabricate the designed three-dimensional diagram into a photolithography mask (usually using a transparent film or a chromium plate).

[0034] Step 2: Prepare a silicon wafer mold: Clean the silicon wafer successively with acetone, isopropyl alcohol, and deionized water to remove surface contaminants. Spin-coat a layer of photoresist (such as SU-8) on the silicon wafer, and the thickness is determined according to the height requirements of the flow channel layer 1, the tissue layer 2, and the bottom layer 10. Use the photolithography mask to perform ultraviolet exposure on the photoresist, and then develop it to form an internal structural pattern. Bake the developed silicon wafer to completely cure the photoresist and form a silicon wafer mold.

[0035] Step 3, preparing the PDMS layer: Mix the PDMS prepolymer and the curing agent in a mass ratio of 10:1 and stir evenly. Put the mixed PDMS into a vacuum desiccator for degassing to remove air bubbles. Pour the degassed PDMS onto the silicon wafer mold to ensure complete coverage of the mold surface. Place the mold in an oven and cure at 80 °C for 2 hours to completely cure the PDMS. Carefully peel the cured PDMS from the mold to obtain the flow channel layer 1, the tissue layer 2, and the bottom layer 10.

[0036] Step 4, interlayer bonding method: Treat the bonding surfaces of each layer with oxygen plasma, and the treatment time is usually 30 - 60 seconds, 60 seconds in this embodiment. Quickly align and bond the treated flow channel layer 1, tissue layer 2, and bottom layer 10 to ensure accurate positions of the channels and chambers. After bonding, apply slight pressure on the top layer, place it in an oven, and heat at 80 °C for 20 minutes to enhance the bonding strength.

[0037] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific embodiments of the present invention are limited thereto. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the patent protection scope determined by the claims submitted for the present invention.

Claims

1. A mechanical loading device for cartilage tissue stratification research, characterized in that It includes a flow channel layer (1) located above and a tissue layer (2) located below the flow channel layer (1), and the flow channel layer (1) is connected to the tissue layer (2); an "8"-shaped culture medium flow channel (3) is arranged on the lower surface of the flow channel layer (1), and two semi-circular gel chambers (5) are symmetrically arranged on the upper surface of the tissue layer (2), and the culture medium flow channel (3) is completely attached to the gel chamber (5); a cylindrical cavity is arranged on the tissue layer (2) and inside the gel chamber (5), and an elastic catheter (7) is arranged in the cavity, and the outer wall of the catheter (7) is attached to the inner side of the gel chamber (5); both the gel chamber (5) and the culture medium flow channel (3) are groove-shaped open structures; The culture medium flow channel (3) is located directly above the gel chamber (5), and the two ends of the culture medium flow channel (3) away from each other are semi-circular arc structures; the two gel chambers (5) are respectively opposite to the two semi-circular arc structures of the culture medium flow channel (3); a hydrogel mixed with chondrocytes is transported to one side of the gel chamber (5) to simulate the articular cartilage layer; a hydrogel mixed with osteocytes, osteoblasts, and osteoclasts is transported to the other side of the gel chamber (5) to simulate the subchondral bone layer; the culture medium circulates in the culture medium flow channel (3); gas is continuously and uniformly introduced through the catheter (7) to expand the spatial area of the catheter (7) to achieve circumferential uniform extrusion of the hydrogel in the gel chamber (5), simulating mechanical stimulation of the cartilage tissue; A bottom layer (10) is connected below the tissue layer (2), and a gas buffer chamber (11) is arranged in the bottom layer (10), and the bottom of the catheter (7) is communicated with the gas buffer chamber (11).

2. The mechanical loading device for cartilage tissue stratification research according to claim 1, wherein The two ends of the culture medium flow channel (3) away from each other are connected with a culture medium delivery channel (4) for delivering the culture medium; the gel chamber (5) is connected with a gel delivery channel (6) for delivering the hydrogel.

3. The mechanical loading device for cartilage tissue stratification research according to claim 2, characterized in that, The culture medium delivery channel (4), the gel delivery channel (6), and the catheter (7) all pass through the flow channel layer (1) and communicate with the outside world.

4. The mechanical loading device for cartilage tissue stratification research according to claim 2, wherein A microcirculation pump (8) and a culture medium storage bin (9) are connected between the two culture medium delivery channels (4).

5. The method of using a mechanical loading device for cartilage tissue stratification research according to any one of claims 1-4, characterized in that, It includes the following steps: Step 1, inject the hydrogel wrapped with chondrocytes into one gel chamber (5) to simulate the articular cartilage layer, and then inject the hydrogel wrapped with osteocytes, osteoblasts, and osteoclasts into the other gel chamber (5) to simulate the subchondral bone layer; Step 2, when the hydrogel changes from a liquid state to a solid state, introduce the culture medium into the culture medium flow channel (3) to make the culture medium circulate in the culture medium flow channel (3); The culture medium flows through the surface of the solidified hydrogel, generating a fluid shear force on the articular cartilage layer and the subchondral bone layer; Step 3, introduce gas into the two catheters (7) to apply a continuous and constant circumferential force stimulation or a dynamic circumferential force stimulation to the articular cartilage layer and the subchondral bone layer.

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