A microfluidic 3D bionic tooth peripheral tissue organoid chip and its construction method

Abstract

The present invention discloses a microfluidic 3D bionic tooth peripheral tissue organoid chip and a construction method thereof, which belongs to the field of microfluidic chips, and the key points of its technical solution are: the organ chip includes a main layer, a PDMS film and a glass slide layer from top to bottom, the main layer includes an extracellular matrix channel, a microfluidic channel I and a microfluidic channel II; the extracellular matrix channel includes an injection section, a tissue culture chamber section and an outlet section arranged in sequence; the microfluidic channel I includes an inlet section I, a main section I and an outlet section I; the microfluidic channel II includes an inlet section II, a main section II and an outlet section II; the main section I and the main section II are both connected to the tissue culture chamber section. The present invention is mainly used to simulate the microstructure and microenvironment of organ tissues, and can present more corresponding microphysiological functions; and, compared with animal models, the organ chip directly uses cells as experimental objects, can save costs, and has the advantages of specificity, effectiveness and rapidity.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chips, and in particular to a microfluidic 3D bionic tooth peripheral tissue organoid chip and a construction method thereof. Background Art

[0002] Organ-on-a-chip is a revolutionary new technology that has emerged in recent years. It can establish a microtissue model in vitro that is closer to the physiological function of real organs. This technology uses microfabrication and tissue engineering to build a complex microfluidic system for three-dimensional tissue culture in vitro, and finally simulates and constructs an "organ" with a specific structure and function to study the daily function, mechanism of action and physiological response of the corresponding organ. Organ chips are not complete organs, but the smallest cell clusters required to replicate a certain function of a specific organ. Its advantages are: on the one hand, compared with the 2D cell model in the culture dish, organ chips are closer to the microstructure and microenvironment of organ tissues, so they can present more corresponding microphysiological functions; on the other hand, compared with animal models, because organ chips directly use cells / tissues as experimental objects, they can not only save costs, but also have the characteristics of specificity, effectiveness and rapidity. At the same time, in addition to being able to well realize fluid control for the collection of metabolic biomarkers, transparent microfluidic organ chips are also conducive to high-resolution and real-time imaging of cultured cells / tissues, thereby meeting the real-time detection needs of physiological / pathological processes.

[0003] Teeth are important masticatory organs for humans and many other animals. The teeth of humans and many higher mammals are complex alveolar teeth, which include the hard enamel and dentin of the teeth themselves and the cementum on the surface of the root. Together with the surrounding periodontal tissues such as the periodontal ligament, alveolar bone, and gingiva, they form an organ with complex biological functions. Cementum, alveolar bone tissue, and periodontal ligament are tissues with relatively simple cell components, mainly cementocytes and osteocytes in the lacuna of the bone matrix, and fibroblasts in the periodontal ligament. The root and the tissues around the teeth have active adaptation and reconstruction capabilities under the influence of physiological loading such as chewing or external factors such as orthodontic force. However, due to its very complex structure and the inability to completely separate multiple periodontal tissues, the current research on periodontal tissues is mostly limited to animal models or the culture of single cells, and it is impossible to achieve specific analysis of periodontal tissues such as alveolar bone, cementum, and periodontal ligament, or the study of the interaction between tissues.

[0004] In order to solve the above problems, a microfluidic 3D bionic tooth peripheral tissue organoid chip and a construction method thereof are provided on the basis of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide a microfluidic 3D bionic tooth surrounding tissue organ chip and a construction method thereof. A bionic tooth organ chip is designed based on the biological characteristics of periodontal tissue, which can simulate cementum, alveolar bone tissue, periodontal ligament and other tissues to form cementum organ chip, alveolar bone organ chip, periodontal ligament organ chip, and establish a periodontal tissue organoid with multiple adjustable microenvironment parameters; secondly, the organ chip can simulate the loading of physiological fluid shear force and compressive stress on various types of organ chips respectively, and observe the expression differences of periodontal tissue remodeling-related genes in cementum cells, bone cells and periodontal ligament cells under different loading conditions; thirdly, overloading of fluid shear force and compressive stress can also be loaded to simulate the excessive force in the process of tooth movement in clinical practice, and explore the possible mechanisms of pathological changes such as root resorption and periodontal ligament hyalinization in periodontal tissue; finally, several types of organ chips can be connected in parallel, and the mechanism of periodontal tissue remodeling can be explored by loading the same conditions; the biological response of periodontal tissue under different stimulation conditions can also be explored by changing the microenvironment (such as drugs or other loading conditions).

[0006] The above technical objectives of the present invention are achieved through the following technical solutions:

[0007] A microfluidic 3D bionic tooth peripheral tissue organ chip, the organ chip comprises a main layer, a PDMS film and a glass slide layer from top to bottom, the main layer comprises an extracellular matrix channel, a microfluidic channel I and a microfluidic channel II; the microfluidic channel I and the microfluidic channel II are symmetrically arranged with the extracellular matrix channel as the central axis; and the extracellular matrix channel, the microfluidic channel I and the microfluidic channel II are all open at the top;

[0008] The extracellular matrix channel comprises an injection section, a tissue culture chamber section and an outlet section which are arranged in sequence; a channel inlet 1 is provided at the end of the injection section, and a channel outlet 2 is provided at the end of the outlet section; the channel inlet 1 and the channel outlet 2 both extend to the outside of the top surface of the organ chip;

[0009] The microfluidic channel I comprises an inlet section I, a main section I and an outlet section I; a channel inlet three is provided at the end of the inlet section I, a channel outlet four is provided at the end of the outlet section I, and both the channel inlet three and the channel outlet four extend to the outside of the top surface of the organ chip;

[0010] The microfluidic channel II includes an inlet section II, a main section II and an outlet section II; a channel inlet five is provided at the end of the inlet section II, and a channel outlet six is ​​provided at the end of the outlet section II; the main section I and the main section II are both connected to the tissue culture chamber section.

[0011] Furthermore, the outer walls of the main body section I and the main body section II on the side close to the tissue culture chamber section are both provided with a plurality of bluff bodies at intervals.

[0012] Furthermore, a culture chamber is provided in the tissue culture chamber section, the height of the culture chamber is 200 μm, and the length of the culture chamber is 3 mm.

[0013] Furthermore, the size of the organ chip is 6mm×12mm.

[0014] Furthermore, the main body layer is prepared from polydimethylsiloxane.

[0015] Furthermore, the pore size of the PDMS film is 4 μm.

[0016] Furthermore, the organ chip includes a cementum organ chip, an alveolar bone organ chip or a periodontal ligament organ chip.

[0017] The present invention also provides a method for constructing a microfluidic 3D bionic tooth peripheral tissue organoid chip, the steps of which are as follows:

[0018] S1, injecting the extracellular matrix mixed with target cultured cells into the extracellular matrix channel from the channel inlet 1;

[0019] S2, injecting the cell culture solution from channel inlet 3 and channel inlet 5 respectively, and filling the microfluidic channel I and microfluidic channel II with the cell culture solution until droplets appear at the positions of channel outlet 4 and channel outlet 6;

[0020] S3. Block and seal channel outlet four and channel outlet five, and connect the injection pump to channel inlet three or channel inlet five; generate the required intercellular flow or fluid shear force in the extracellular matrix by adjusting the flow rate of the injection pump or adjusting the hydraulic pressure difference on both sides of microfluidic channel I and microfluidic channel II.

[0021] The present invention also provides another method for constructing a microfluidic 3D bionic tooth peripheral tissue organoid chip, the steps of which are as follows:

[0022] A. Inject the extracellular matrix mixed with target cultured cells into the extracellular matrix channel from the channel inlet 1;

[0023] B. injecting the cell culture fluid from channel inlet 3 and channel inlet 5 respectively, and filling the microfluidic channel I and microfluidic channel II with the cell culture fluid until droplets appear at the positions of channel outlet 4 and channel outlet 6;

[0024] C. Block and seal channel inlet five and channel outlet six, and connect the syringe pump to microfluidic channel inlet three or channel inlet five; by adjusting the flow rate of the syringe pump or adjusting the hydraulic pressure difference on both sides of microfluidic channel I and microfluidic channel II, the required intercellular flow or fluid shear force is finally generated in the extracellular matrix.

[0025] Wherein, the extracellular matrix in the above steps includes a single or mixed three-dimensional culture scaffold matrix composed of biomaterials such as hydrogel / microbeads / hydroxyapatite.

[0026] Among them, the periodontal tissue cells used in the above steps include but are not limited to periodontal tissue cells such as cementum cells, cementoblasts, osteocytes, periodontal ligament cells, etc.; their sources may be human, mouse, rat or other animal sources; their cell types may be cell lines or primary cells; their degree of differentiation may be different types such as stem cells or mature somatic cells.

[0027] Wherein, the extracellular matrix mixed with target culture cells in the above steps is a mixture of the above target culture cell type and a three-dimensional culture scaffold matrix in a certain proportion.

[0028] Through the above technical scheme, polydimethylsiloxane (PDMS) is used to make an organ chip, the chip size is 6mm×12mm, the culture chamber height is 200um, and the length is 3mm; the extracellular matrix mixed with specific periodontal tissue cells is injected from channel inlet one to channel outlet two, and under the action of the capillary passive valve, the extracellular matrix can be well confined in the tissue culture chamber; the cell culture fluid is injected from channel inlet three and channel inlet five respectively, and the cell culture fluid fills microfluidic channel I and microfluidic channel II until droplets appear at the positions of channel outlet four and channel outlet six, so as to ensure that there are no bubbles remaining in microfluidic channel I and microfluidic channel II.

[0029] The bottom surface of the chip adheres to the glass slide layer, and the glass slide layer is covered with a PDMS film with a diameter of 4μm, which helps to ensure that a 200μm cell culture layer can be formed between the PDMS film and the PDMS. Through the liquid penetration between the extracellular matrix channel, microfluidic channel I and microfluidic channel II, the use of microfluidic devices for cell culture fluid perfusion and multiple interval-arranged baffles to regulate the direction of fluid shear force, the regulation of different cell gap flow or shear force is finally achieved.

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1. The present invention constructs an organ chip that can simulate tissues such as cementum, alveolar bone tissue, and periodontal ligament, including a cementum organ chip, an alveolar bone organ chip, and a periodontal ligament organ chip, and establishes periodontal tissue organoids with adjustable multiple microenvironment parameters, using materials such as fibrin gel as a culture matrix to perform 3D culture of cementum cells / alveolar bone cells / periodontal ligament cells.

[0032] 2. The organ chip can simulate the loading of physiological fluid shear force and compressive stress on various types of organ chips, simulate the effects of physical factors such as physiological periodontal tissue stress in vivo, mechanical force in orthodontic tooth movement, abnormal occlusal stress, etc., and use microfluidic devices / fluid pressure difference and other methods to perfuse cell culture fluid to achieve in vitro 3D bionic culture of periodontal tissue cells such as cementum / alveolar bone / periodontal ligament, and observe the expression differences of periodontal tissue remodeling-related genes in cementum cells, bone cells and periodontal ligament cells under different loading conditions.

[0033] 3. This organ chip can also overload fluid shear force and compressive stress to simulate the excessive force in the process of tooth movement in clinical practice, explore the possible mechanisms of pathological changes such as root resorption and periodontal membrane hyaline degeneration in periodontal tissues, and simulate the effects of various chemical factors in clinical or scientific research by adding drugs or cytokines to the culture medium.

[0034] 4. By designing an elastic membrane and a perfusion chamber above the culture chamber, the cells can be stress-loaded by gas / liquid perfusion to construct a microfluidic 3D bionic tooth peripheral tissue organoid chip.

[0035] 5. The organ chip structure of the present invention can be used alone to construct an organ-like chip of a specific tissue around a tooth; at the same time, different organ-like chips can also be connected in parallel to observe and compare the differences in biological responses of cells in different parts by adjusting the loading of influencing factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the structure of the main layer of the microfluidic bionic tooth organ chip in Example 1 of the present invention Figure Ⅰ ;

[0037] Figure 2 Schematic diagram of the structure of the main layer of the microfluidic bionic tooth organ chip in Example 1 of the present invention Figure II ;

[0038] Figure 3 This is a simulation result diagram of the main layer of the microfluidic bionic tooth organ chip according to Example 1 of the present invention;

[0039] Figure 4 This is a three-layer unfolded structure diagram of the microfluidic bionic tooth organ chip according to Example 4 of the present invention;

[0040] Figure 5 This is a top view of the microfluidic bionic tooth organoid chip after bonding and assembly according to Example 4 of the present invention;

[0041] Figure 6 This is a three-layer unfolded structure diagram of the microfluidic bionic tooth organ chip according to Example 5 of the present invention;

[0042] Figure 7 It is a top view of the bonding assembly of the main layer of the microfluidic bionic tooth organ chip of Example 5 of the present invention;

[0043] Figure 8 A schematic diagram of a method for using the microfluidic bionic tooth organ chip according to Example 5 of the present invention;

[0044] Fig. 9 Schematic diagram of the force of the extracellular matrix in the main layer of the microfluidic bionic tooth organ chip of Example 1, Example 4, and Example 5 of the present invention;

[0045] Fig.10 The three-layer structure diagram, the overall structure diagram and the main layer structure diagram of the co-culture of cementum cells, osteoblasts and osteoclasts according to Example 6 of the present invention;

[0046] Fig.11 This is a schematic diagram of parallel application of Embodiment 7 of the present invention;

[0047] Fig.12 This is a schematic diagram of the expanded structure of the vertical positional relationship of the microfluidic bionic tooth organ chip of Example 1 of the present invention.

[0048] In the figure: 1. main body layer; 2. PDMS film; 3. glass slide layer; 4. extracellular matrix channel; 5. microfluidic channel I; 6. microfluidic channel II; 7. injection section; 8. tissue culture chamber section; 9. export section; 10. channel inlet one; 11. channel outlet two; 12. inlet section I; 13. main body section I; 14. outlet section I; 15. channel inlet three; 16. channel outlet four; 17. inlet section II; 18. main body section II; 19. outlet section II; 20. channel inlet five; 21. channel outlet six; 22. barrier body; 23. culture chamber. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0050] Example 1: A microfluidic 3D bionic tooth peripheral tissue organ chip, as shown in FIG12 , the organ chip comprises a main body layer 1, a PDMS film 2 and a glass slide layer 3 from top to bottom, such as Figures 1 to 3As shown, the main layer 1 includes an extracellular matrix channel 4, a microfluidic channel I 5 and a microfluidic channel II 6; the microfluidic channel I 5 and the microfluidic channel II 6 are symmetrically arranged with the extracellular matrix channel 4 as the central axis; and the extracellular matrix channel 4, the microfluidic channel I 5 and the microfluidic channel II 6 are all open at the top; the size of the organ chip is 6 mm × 12 mm; the main layer 1 is made of polydimethylsiloxane; the pore size of the PDMS film 2 is 4 μm.

[0051] The extracellular matrix channel 4 includes an injection section 7, a tissue culture chamber section 8 and an outlet section 9 which are arranged in sequence; a channel inlet 10 is provided at the end of the injection section 7, and a channel outlet 2 11 is provided at the end of the outlet section 9; both the channel inlet 10 and the channel outlet 2 11 extend to the outside of the top surface of the organ chip.

[0052] The microfluidic channel Ⅰ5 includes an inlet section Ⅰ12, a main section Ⅰ13 and an outlet section Ⅰ14; a channel inlet three 15 is provided at the end of the inlet section Ⅰ12, and a channel outlet four 16 is provided at the end of the outlet section Ⅰ14, and both the channel inlet three 15 and the channel outlet four 16 extend to the outside of the top surface of the organ chip.

[0053] The microfluidic channel II6 includes an inlet section II17, a main section II18 and an outlet section II19; a channel inlet five 20 is provided at the end of the inlet section II17, and a channel outlet six 21 is provided at the end of the outlet section II19; the main section I13 and the main section II18 are both connected to the tissue culture chamber section 8.

[0054] The outer walls of the main body section I 13 and the main body section II 18 close to the tissue culture chamber section 8 are both provided with a plurality of blocking bodies 22 at intervals; a culture chamber 23 is provided in the tissue culture chamber section 8, the height of the culture chamber 23 is 200 μm, and the length of the culture chamber 23 is 3 mm. Fig. 9 (a) is a schematic diagram of the force on the extracellular matrix in the organ chip, and the solid arrows indicate the flow direction of the intercellular flow.

[0055] Example 2: A method for constructing a microfluidic 3D bionic tooth peripheral tissue organoid chip, the steps are as follows:

[0056] S1, injecting the extracellular matrix mixed with target culture cells into the extracellular matrix channel 4 from the channel inlet 10;

[0057] S2, injecting the cell culture fluid from the channel inlet 3 15 and the channel inlet 5 20 respectively, and filling the microfluidic channel I and the microfluidic channel II with the cell culture fluid until droplets appear at the positions of the channel outlet 4 16 and the channel outlet 6 21;

[0058] S3. Block and seal the channel outlet four 16 and the channel outlet five, and connect the syringe pump to the channel inlet three 15; by adjusting the flow rate of the syringe pump, the required intercellular flow or fluid shear force is generated in the extracellular matrix.

[0059] Example 3: A method for constructing a microfluidic 3D bionic tooth peripheral tissue organoid chip, the steps are as follows:

[0060] A. Inject the extracellular matrix mixed with target culture cells into the extracellular matrix channel 4 from the channel inlet 10;

[0061] B. Inject the cell culture solution from the channel inlet 3 15 and the channel inlet 5 20 respectively, and fill the microfluidic channel I and the microfluidic channel II with the cell culture solution until droplets appear at the positions of the channel outlet 4 16 and the channel outlet 6 21;

[0062] C. Block and seal the channel inlet five 20 and the channel outlet six 21, and connect the syringe pump to the microfluidic channel inlet three 15; by adjusting the flow rate of the syringe pump, the desired intercellular flow or fluid shear force is finally generated in the extracellular matrix.

[0063] Another embodiment of the present invention is as follows:

[0064] Example 4: A microfluidic 3D bionic tooth surrounding tissue organoid chip. In order to study the effect of intercellular flow on cells in specific tooth surrounding tissue in three-dimensional space, the corresponding microfluidic chip adopts a three-layer structure design, such as Figures 4 to 5 As shown, it includes an upper microfluidic channel III, a middle porous film and a lower main body layer 1. The upper microfluidic channel III includes an inlet section III, a main body section III and an outlet section III, a channel inlet 7 is provided at the end of the inlet section III, a channel outlet 8 is provided at the end of the outlet section, and both the channel inlet 7 and the channel outlet 8 extend outside the top surface of the upper structure.

[0065] The main layer 1 includes an extracellular matrix channel 4, a microfluidic channel I 5 and a microfluidic channel II 6; the microfluidic channel I 5 and the microfluidic channel II 6 are symmetrically arranged with the extracellular matrix channel 4 as the central axis; the extracellular matrix channel 4 includes an injection section 7, a tissue culture chamber section 8 and an outlet section which are arranged in sequence; a channel inlet 10 is provided at the end of the injection section 7, and a channel outlet 2 11 is provided at the end of the outlet section; the channel inlet 10 and the channel outlet 2 11 both extend to the outside of the top surface of the organ chip.

[0066] The microfluidic channel Ⅰ5 includes an inlet section Ⅰ12, a main section Ⅰ13 and an outlet section Ⅰ14; a channel inlet three 15 is provided at the end of the inlet section Ⅰ12, a channel outlet four 16 is provided at the end of the outlet section Ⅰ14, and both the channel inlet three 15 and the channel outlet four 16 extend to the outside of the top surface of the organ chip; the microfluidic channel Ⅱ6 includes an inlet section Ⅱ17, a main section Ⅱ18 and an outlet section Ⅱ19; a channel inlet five 20 is provided at the end of the inlet section Ⅱ17, and a channel outlet six 21 is provided at the end of the outlet section Ⅱ19; the main section Ⅰ13 and the main section Ⅱ18 are both connected to the tissue culture chamber section 8. Fig. 9 (b) is a schematic diagram of the force on the extracellular matrix in the organ chip. The solid arrows indicate the flow direction of the intercellular flow.

[0067] The operation steps are as follows:

[0068] a. The extracellular matrix mixed with the target cultured cells is injected into the extracellular matrix channel 4 from the channel inlet 10;

[0069] b. injecting the cell culture medium from the channel inlet three 15 and the channel inlet five 20, respectively, and filling the microfluidic channel Ⅰ and the microfluidic channel Ⅱ with the cell culture medium until droplets appear at the channel outlet four 16 and the channel outlet six 21;

[0070] c. In order to generate vertical intercellular flow in the extracellular matrix, the cell culture medium is injected from the channel inlet 7 and fills the upper microfluidic channel III; then the channel outlet 4, the channel inlet 5 20 and the channel outlet 8 are blocked and sealed respectively;

[0071] d. Connect the injection pump to channel inlet three 15 and channel inlet seven. By applying a certain pressure, the cell culture fluid in the upper microfluidic channel III will flow into the extracellular matrix from top to bottom through the porous film and form a vertical intercellular flow. At the same time, combined with the horizontal intercellular flow, a three-dimensional intercellular flow will eventually be formed in the extracellular matrix.

[0072] By connecting the microfluidic device to channel inlet seven or channel inlet eight, gas / liquid is perfused into the channel, and the PDMS film will deform due to expansion and eventually convert into compressive stress on the extracellular matrix; when the gas pressure returns to the external atmospheric pressure, the PDMS film rebounds to a horizontal state; the amount of force applied can be controlled by the valve to control the perfusion flow and intensity, thereby achieving periodic or continuous mechanical stress stimulation of the extracellular matrix.

[0073] The microfluidic device is connected to channel inlet seven or channel inlet eight to extract the gas / liquid in microfluidic channel III to form a negative pressure, which will cause the PDMS film to deform and eventually convert into tensile stress on the extracellular matrix. The magnitude of the negative pressure can be controlled to achieve periodic or continuous tensile stress stimulation on the extracellular matrix.

[0074] Another embodiment of the present invention is as follows:

[0075] Example 5: A microfluidic 3D bionic tooth surrounding tissue organoid chip. In order to simultaneously study the effects of multiple different mechanical environments (such as fluid shear force and mechanical stress) on cells in tooth surrounding tissue, the corresponding microfluidic chip adopts a three-layer structure design, such as Figure 6 to Figure 8 As shown, it includes an upper mechanical stress control layer, a middle PDMS film and a lower main body layer 1. The upper mechanical stress control layer includes a microfluidic channel IV, and a channel inlet 9 is provided at the end of the microfluidic channel IV.

[0076] The main body layer 1 of the lower layer includes an extracellular matrix channel 4, a microfluidic channel I 5 and a microfluidic channel II 6; the microfluidic channel I 5 and the microfluidic channel II 6 are symmetrically arranged with the extracellular matrix channel 4 as the central axis; the extracellular matrix channel 4 includes an injection section 7, a tissue culture chamber section 8 and an outlet section fixed in sequence; the end of the injection section 7 is provided with a channel inlet 10, and the end of the outlet section is provided with a channel outlet 2 11; the channel inlet 10 and the channel outlet 2 11 both extend to the outside of the top surface of the organ chip; the microfluidic channel I 5 includes an inlet section I 12, The main body section Ⅰ13 and the outlet section Ⅰ14; the end of the inlet section Ⅰ12 is provided with a channel inlet three 15, and the end of the outlet section Ⅰ14 is provided with a channel outlet four 16, and the channel inlet three 15 and the channel outlet four 16 both extend to the outside of the top surface of the organ chip; the microfluidic channel Ⅱ6 includes an inlet section Ⅱ17, a main body section Ⅱ18 and an outlet section Ⅱ19; the end of the inlet section Ⅱ17 is fixedly connected with a channel inlet five 20, and the end of the outlet section Ⅱ19 is fixedly connected with a channel outlet six 21; the main body section Ⅰ13 and the main body section Ⅱ18 are both connected to the tissue culture chamber section 8. Fig. 9 (c) is a schematic diagram of the force on the extracellular matrix in the organ chip. The solid arrows indicate the flow direction of the intercellular flow, and the dotted arrows indicate the movement direction of the PDMS film.

[0077] The operation steps are as follows:

[0078] (1) Injecting the extracellular matrix mixed with target cultured cells into the extracellular matrix channel 4 from the channel inlet 10;

[0079] (2) injecting the cell culture fluid from the channel inlet 3 15 and the channel inlet 5 20 respectively, and filling the microfluidic channel I and the microfluidic channel II with the cell culture fluid until droplets appear at the positions of the channel outlet 4 16 and the channel outlet 6 21;

[0080] (3) The channel inlet 5 20 and the channel outlet 6 21 are blocked and sealed, and a syringe pump is connected to the microfluidic channel inlet 3 15; by adjusting the flow rate of the syringe pump, the desired intercellular flow or fluid shear force is finally generated in the extracellular matrix.

[0081] (4) In order to generate mechanical stress on the extracellular matrix, an air pump is connected to the inlet of channel 9. When the air pressure in microfluidic channel IV increases, the PDMS film will deform due to expansion and eventually transform into mechanical stress on the extracellular matrix; when the air pressure returns to the external atmospheric pressure, the PDMS film will rebound to a horizontal state; therefore, by periodically regulating the air pressure in the upper channel, regular mechanical stress stimulation can be generated on the extracellular matrix.

[0082] The basic principle is to cause the compression or stretching of the extracellular matrix (such as three-dimensional culture scaffolds made of hydrogels / microbeads / hydroxyapatite and other materials) through the deformation of the PDMS film, thereby causing changes in the voids of the extracellular matrix and ultimately generating tensile stress / compressive stress on the cells. At the same time, the film can be deformed at different frequencies through a gas pressure regulator, greatly increasing the flexibility of the experiment.

[0083] Another embodiment of the present invention is as follows:

[0084] Example 6: A microfluidic 3D bionic tooth peripheral tissue organoid chip, using a fluid perfusion culture mode, such as Fig. 9 As shown, the whole chip consists of a three-layer structure, which is an upper mechanical stress control layer, a middle layer is a PDMS film, and a lower layer is a main body layer 1. Figure 10 (a) is a schematic diagram of the three-layer structure of the organ chip. Fig.10 (b) is a schematic diagram of the overall structure of the organ chip. Fig.10 (c) is a schematic diagram of the structure of the main layer 1 of the organ chip.

[0085] By connecting the upper mechanical stress control layer to the air pump, the middle PDMS film can be deformed, thereby achieving mechanical stimulation to the tissue in the lower intermediate culture chamber 23. For the lower main body layer 1, both sides of the cementum tissue culture chamber 23 are cell culture fluid reservoirs, one side of which is a rectangular reservoir, and the other side is two independent square reservoirs, which can be used to culture effector cells such as osteoblasts and osteoclasts. Osteoblasts are pre-cultured on 6mm diameter glass slides in vitro, and osteoclasts are pre-cultured on 6mm diameter bone slices in vitro. Osteoblasts or osteoclasts are placed in the corresponding reservoirs at the right time. This structure can effectively solve many pain points in research, such as inconsistent differentiation time of osteoblasts and osteoclasts and inconsistent action conditions.

[0086] Through fluid perfusion and gas-driven microfluidic regulation, precise control of chip strain loading can be achieved, so that the effects of different strain environments on 3D cultured cells in the established chip can be studied, and the expression differences of important genes related to bone remodeling in effector cells under different microfluidic loading conditions can be observed.

[0087] Another embodiment of the present invention is as follows:

[0088] Example 7: Application of a microfluidic 3D bionic tooth peripheral tissue organoid chip, such as Fig.11 As shown, two organ chips are connected in parallel, and the mechanism of periodontal tissue remodeling is explored by loading the same conditions; and the biological response of periodontal tissue under different stimulation conditions can be explored by changing the microenvironment, such as drugs or other loading conditions.

[0089] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A microfluidic 3D bionic tooth peripheral tissue organoid chip, characterized by: The organ chip comprises a main body layer (1), a PDMS film (2) and a glass slide layer (3) from top to bottom, wherein the main body layer (1) comprises an extracellular matrix channel (4), a microfluidic channel I (5) and a microfluidic channel II (6); the microfluidic channel I (5) and the microfluidic channel II (6) are symmetrically arranged with the extracellular matrix channel (4) as the central axis, and the extracellular matrix channel (4), the microfluidic channel I (5) and the microfluidic channel II (6) are all open at the top; The extracellular matrix channel (4) comprises an injection section (7), a tissue culture chamber section (8) and an outlet section (9) which are arranged in sequence; a channel inlet 1 (10) is provided at the end of the injection section (7), and a channel outlet 2 (11) is provided at the end of the outlet section (9); the channel inlet 1 (10) and the channel outlet 2 (11) both extend to the outside of the top surface of the organ chip; The microfluidic channel I (5) comprises an inlet section I (12), a main section I (13) and an outlet section I (14); a channel inlet three (15) is provided at the end of the inlet section I (12), and a channel outlet four (16) is provided at the end of the outlet section I (14); the channel inlet three (15) and the channel outlet four (16) both extend to the outside of the top surface of the organ chip; The microfluidic channel II (6) comprises an inlet section II (17), a main section II (18) and an outlet section II (19); a channel inlet five (20) is provided at the end of the inlet section II (17), and a channel outlet six (21) is provided at the end of the outlet section II (19); the main section I (13) and the main section II (18) are both connected to the tissue culture chamber section (8); The outer walls of the main body section I (13) and the main body section II (18) on the side close to the tissue culture chamber section (8) are both provided with a plurality of bluff bodies (22) at intervals.

2. The microfluidic 3D bionic tooth peripheral tissue organoid chip according to claim 1, characterized in that: A culture chamber (23) is provided in the tissue culture chamber section (8), the height of the culture chamber (23) is 200 μm, and the length of the culture chamber (23) is 3 mm.

3. The microfluidic 3D bionic tooth peripheral tissue organoid chip according to claim 1, characterized in that: The size of the organ chip is 6 mm×12 mm.

4. The microfluidic 3D bionic tooth peripheral tissue organoid chip according to claim 1, characterized in that: The main body layer (1) is prepared from polydimethylsiloxane.

5. The microfluidic 3D bionic tooth peripheral tissue organoid chip according to claim 1, characterized in that: The pore size of the PDMS film (2) is 4 μm.

6. The microfluidic 3D bionic tooth peripheral tissue organoid chip according to claim 1, characterized in that: The organ chip includes a cementum organ chip, an alveolar bone organ chip or a periodontal ligament organ chip.

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