Bone fossa-tubule organ chip and experimental method

Abstract

The invention discloses a bone fossa-tubule organ-like chip and an experimental method, and particularly relates to the field of organ-like chip technology and bone mass transfer. Comprising an upper cover plate and a lower cover plate which are made of transparent materials, liquid inlet grooves and liquid outlet grooves are formed in the two sides of the lower cover plate respectively, an artificial bone tissue located between the two liquid inlet grooves is etched on the lower cover plate, the artificial bone tissue comprises six longitudinally-arranged Harvard tubes and a plurality of transversely-arranged Forkman tubes, and the Harvard tubes and the Forkman tubes are arranged in a staggered mode. The liquid inlet groove and the liquid outlet groove are communicated through the Harvard tubes, all the formkman tubes are connected between every two adjacent Harvard tubes respectively, and a plurality of bone fossa and bone tubules which are communicated with each other are arranged on the two sides of each Harvard tube. By adopting the technical scheme of the invention, the problems that the experimental sample preparation process is tedious and only the limited depth can be observed in the existing in-vitro experimental method are solved, and the mass transfer law between bone cells is explored.

Description

Technical Field

[0001] The present invention relates to the field of organoid chip technology and bone mass transfer, and in particular to a bone lacuna-tubule organoid chip and an experimental method. Background Art

[0002] Nutritional transport and waste excretion are basic conditions for the metabolism of organisms. Blood circulation and lymphatic circulation can meet the needs of organisms' growth and development, while bone tissue needs to pass through the lacunae-canaliculi interstitial fluid to achieve material exchange. The Haversian canals are surrounded by 4-20 layers of mineralized bone plates arranged in concentric circles. Osteocytes are located in or between the lamina. The elliptical small cavity they occupy is the lacunae. The space where the protrusions of osteocytes are located is the canaliculus. The canaliculus connects adjacent lacunae. Both contain tissue fluid, and osteocytes obtain nutrients from this. The Haversian canals-canaliculi-lacuna-canaliculi in the bone unit form a three-dimensional spatial pathway for material exchange between osteocytes, which is the basic structure of mass transfer in bones. Therefore, it is necessary to explore the mass transfer rules of the lacunae-canaliculus system.

[0003] At present, the in vitro experiment method is usually used to explore the mass transfer law of the lacunae-canaliculi system. However, the technology used in the in vitro experiment to prepare the experimental samples has limitations. The process of preparing the experimental samples is cumbersome, and when observing the samples with a laser confocal microscope, only a limited depth can be observed, which is not easy to explore the mass transfer law of the lacunae-canaliculi system. Summary of the invention

[0004] The present invention aims to provide a bone lacuna-tubule organoid chip and an experimental method, which solves the problem that the experimental sample preparation process in the existing in vitro experimental method is cumbersome and only a limited depth can be observed.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a bone lacuna-tubule organ chip, comprising an upper cover plate and a lower cover plate made of transparent material, a liquid inlet groove and a liquid outlet groove are respectively provided on both sides of the lower cover plate, artificial bone tissue located between the two liquid inlets is etched on the lower cover plate, the artificial bone tissue comprises six longitudinally arranged Haversian tubes and a plurality of transversely arranged Volkmann tubes, the Haversian tubes connect the liquid inlet groove and the liquid outlet groove, all the Volkmann tubes are respectively connected between two adjacent Haversian tubes, and a plurality of mutually connected bone lacunas and bone tubules are provided on both sides of each of the Haversian tubes.

[0006] Furthermore, the width of the Haversian duct and Volkmann duct is 40-60 μm and the depth is 20-30 μm.

[0007] Through the above settings, the accuracy and authenticity of the experiment of this scheme are improved by setting micro-channels of different sizes to simulate the structure of micro-channels in the bones of children, young people, middle-aged people and the elderly.

[0008] Furthermore, the width of the bone canaliculus is 10-20 μm in length, 0.2-0.6 μm in width, and 1-2 μm in depth.

[0009] Furthermore, there are 6 bone lacunas distributed laterally and 16 bone lacunas distributed longitudinally on both sides of the Haversian canal.

[0010] Furthermore, the bone lacuna has a long diameter of 5-10 μm, a short diameter of 4-8 μm, and a depth of 5 μm.

[0011] Furthermore, the upper cover plate is made of polydimethylsiloxane, and the lower cover plate is made of quartz glass.

[0012] Through the above arrangement, the flow and distribution of the fluid in the bone lacuna-canaliculus can be clearly observed.

[0013] Furthermore, the experimental method of the bone lacuna-tubule organ chip is as follows:

[0014] S1. Place three organoid chips in a loading box, which has three grooves for placing the organoid chips, and a liquid collection tank connected to all liquid inlet tanks on the same side, and two of the liquid collection tanks are respectively connected to a liquid inlet pipe and a liquid outlet pipe arranged in the loading box;

[0015] S2, the liquid inlet pipe and the liquid outlet pipe are both connected with latex tubes through external joints, and the latex tubes are provided with peristaltic pumps for driving the liquid in the liquid inlet pipe and the liquid outlet pipe to flow, and a fluorescent tracer is added to the liquid;

[0016] S3, control the perfusion loading of fluorescent tracer by peristaltic pump to simulate the situation that bone tissue is subjected to pulsating pressure, that is, blood pressure is about 120 mmHg systolic pressure, 80 mmHg diastolic pressure, pulsation frequency is about 1.1 Hz, and stop loading after 20 minutes;

[0017] S4. Take out the organoid chip, place the organoid chip under a fluorescence microscope for observation, take images, and record experimental data.

[0018] Through the above settings, the fluid is isolated in the pump tube, the pump tube can be quickly replaced, the fluid is reversible, dry operation is possible, and low maintenance costs constitute the main competitive advantages of peristaltic pumps.

[0019] Organoid chip technology has great potential in studying the regularity of the lacunae-tubule system. Organoid chips can simulate the real physiological environment and simulate the flow of human body fluids through perfusion loading with a peristaltic pump to control the experimental conditions, providing a new means for studying the regularity of the lacunae-tubule system.

[0020] Furthermore, the peristaltic pump is loaded in a manner of constant energy flow, sinusoidal pulsating flow or triangular wave flow.

[0021] Compared with the prior art, this solution has the following beneficial effects:

[0022] This scheme provides a bone lacuna-tubule organoid chip and experimental method, which can solve the problem that the experimental sample preparation process in the existing in vitro experimental methods is cumbersome and can only observe a limited depth. The size of this organoid chip is close to the size of the real bone lacuna-tubule system, thus achieving the purpose of simulating the bone lacuna-tubule structure more realistically and accurately; the loading box and the organoid chip are both made of transparent materials, which can clearly observe the flow and distribution of the liquid; the peristaltic pump provides power loading, which can simulate the real pulsating pressure in the body, thereby providing a reliable experimental basis for subsequent mass transfer experiments of the bone lacuna-tubule system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the structure of a bone lacuna-tubule organ-on-a-chip of the present invention;

[0024] Figure 2 Schematic diagram of the structure of the lower cover plate in this embodiment;

[0025] Figure 3 is a partial enlarged schematic diagram of the structure simulating the Haversian system in this embodiment;

[0026] Figure 4 It is a schematic diagram of a device for an experimental method of a bone lacuna-tubule organ-on-a-chip according to the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below through specific embodiments:

[0028] The figure marks in the drawings of the specification include: upper cover plate 1, lower cover plate 2, liquid inlet groove 3, Haversian duct 4, Volkmann duct 5, liquid outlet groove 6, bone lacuna 7, bone canaliculus 8, loading box 9, liquid inlet tube 10, liquid outlet tube 11, liquid accumulation groove 12, external joint 13, latex tube 14, peristaltic pump 15.

[0029] Example

[0030] like Figures 1 to 4As shown, a bone lacuna-tubule organ chip includes an upper cover plate 1 and a lower cover plate 2 made of transparent material, and the upper cover plate 1 and the lower cover plate 2 are sealed and connected together by bonding. In this embodiment, the shape of the upper cover plate 1 and the lower cover plate 2 are both rectangular, and the dimensions are 10 mm long, 6 mm wide, and 2 mm thick. Among them, the upper cover plate 1 is made of polydimethylsiloxane, and the lower cover plate 2 is quartz glass, which can clearly observe the flow and distribution of liquid in the bone lacuna-tubule. The upper cover plate 1 is a smooth flat plate, and the two sides of the lower cover plate 2 are respectively provided with a liquid inlet trough 3 and a liquid outlet trough 6. The lower cover plate 2 is etched with artificial bone tissue located between the two liquid inlet troughs 3. The artificial bone tissue includes six longitudinally arranged Haversian tubes 4 and thirteen transversely arranged Volkman tubes 5. In this embodiment, the width of the Haversian tubes 4 and Volkman tubes 5 is 40-60 μm and the depth is 20-30 μm. Different sizes are used to simulate the structure of the micro-channels in the bones of different populations. The Haversian tube 4 connects the liquid inlet trough 3 and the liquid outlet trough 6. All Volkmann tubes 5 are connected between two adjacent Haversian tubes 4. A plurality of interconnected bone lacunas 7 and bone canaliculi 8 are provided on the left and right sides of each Haversian tube 4. There are 6 bone lacunas 7 distributed transversely and 16 bone lacunas 7 distributed longitudinally on the left and right sides of the Haversian tube 4. Each bone lacuna 7 is connected by a bone canaliculi 8. The long diameter of the bone lacuna 7 is 5-10 μm, the short diameter is 4-8 μm, and the depth is 5 μm. The width of the bone canaliculi 8 is 10-20 μm, 0.2-0.6 μm, and 1-2 μm, thus forming a complete bone lacuna-canaliculus system model.

[0031] In order to simulate the flow of tissue fluid in the microscopic pores of bone, fluorescent tracers were added to the tissue fluid. The experimental method of the bone lacuna 7-tubule organoid chip is as follows:

[0032] S1. Place three organoid chips in a loading box 9. The loading box 9 is provided with three grooves for placing the organoid chips. The loading box 9 is also provided with a liquid storage tank 12 connected to all the liquid inlet tanks 3 on the same side, so as to ensure that the liquid flow conditions of each organoid chip are the same. The two liquid storage tanks 12 are respectively connected to a liquid inlet pipe 10 and a liquid outlet pipe 11 arranged in the loading box 9.

[0033] S2, the liquid inlet pipe 10 and the liquid outlet pipe 11 are connected to a latex tube 14 through an external joint 13. The latex tube 14 is provided with a peristaltic pump 15 for driving the flow of liquid in the liquid inlet pipe 10 and the liquid outlet pipe 11. Fluorescent tracers, such as sodium fluorescein, rhodamine tracers, etc., are added to the liquid.

[0034] S3. The perfusion loading of the fluorescent tracer is controlled by the peristaltic pump 15. In this embodiment, the loading method of the peristaltic pump 15 is such as constant flow, the pressure is 20 mmHg; the pressure amplitude of the pulsating sine is 600 mmHg, and the situation where the bone tissue is subjected to pulsating pressure can also be realized, that is, the blood pressure is about 120 mmHg systolic pressure, 80 mmHg diastolic pressure, the pulsating frequency is about 1.1 Hz, and the loading stops after 20 minutes; the loading method of the present embodiment includes but is not limited to the constant flow, and the loading methods such as sinusoidal pulsating flow or triangular wave flow can also be used. Through different loading methods, the flow conditions of bone tissue in various situations can be truly simulated, which is conducive to exploring the working law of the present bone lacuna-tubule system model.

[0035] S4. Take out the organoid chip, place the organoid chip under a fluorescence microscope for observation, take images, and record experimental data.

[0036] The above are only embodiments of the present invention, and the common knowledge such as the known specific structures and / or characteristics in the scheme are not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A bone lacuna-tubule organ-on-a-chip, characterized in that: The invention comprises an upper cover plate and a lower cover plate made of transparent material, wherein a liquid inlet groove and a liquid outlet groove are respectively provided on both sides of the lower cover plate, and artificial bone tissue located between the two liquid inlets is etched on the lower cover plate, wherein the artificial bone tissue comprises six longitudinally arranged Haversian tubes and a plurality of transversely arranged Volkmann tubes, wherein the Haversian tubes connect the liquid inlet groove and the liquid outlet groove, and all the Volkmann tubes are respectively connected between two adjacent Haversian tubes, and a plurality of bone lacunas and bone canaliculi which are interconnected are provided on both sides of each Haversian tube.

2. The bone lacuna-tubule organ-on-a-chip according to claim 1, characterized in that: The Haversian and Volkmann ducts have a width of 40-60 μm and a depth of 20-30 μm.

3. The bone lacuna-tubule organ-on-a-chip according to claim 1, characterized in that: The width of the bone canaliculus is 10-20 μm in length, 0.2-0.6 μm in width, and 1-2 μm in depth.

4. The bone lacuna-tubule organ-on-a-chip according to claim 3, characterized in that: There are 6 bone lacunas distributed laterally and 16 bone lacunas distributed longitudinally on both sides of the Haversian canal.

5. The bone lacuna-tubule organ-on-a-chip according to claim 4, characterized in that: The bone lacuna has a long diameter of 5-10 μm, a short diameter of 4-8 μm, and a depth of 5 μm.

6. The bone lacuna-tubule organ-on-a-chip according to claim 1, characterized in that: The upper cover plate is made of polydimethylsiloxane, and the lower cover plate is made of quartz glass.

7. The bone lacuna-tubule organ-on-a-chip according to any one of claims 1 to 6, characterized in that: The experimental method of the bone lacuna-tubule organ chip is as follows: S1. Place three organoid chips in a loading box, which has three grooves for placing the organoid chips, and a liquid collection tank connected to all liquid inlet tanks on the same side, and two of the liquid collection tanks are respectively connected to a liquid inlet pipe and a liquid outlet pipe arranged in the loading box; S2, the liquid inlet pipe and the liquid outlet pipe are both connected with latex tubes through external joints, and the latex tubes are provided with peristaltic pumps for driving the liquid in the liquid inlet pipe and the liquid outlet pipe to flow, and a fluorescent tracer is added to the liquid; S3, control the perfusion loading of fluorescent tracer by peristaltic pump to simulate the situation that bone tissue is subjected to pulsating pressure, that is, blood pressure is about 120 mmHg systolic pressure, 80 mmHg diastolic pressure, pulsation frequency is about 1.1 Hz, and stop loading after 20 minutes; S4. Take out the organoid chip, place the organoid chip under a fluorescence microscope for observation, take images, and record experimental data.

8. The bone lacuna-tubule organ-on-a-chip according to claim 7, characterized in that: The peristaltic pump is loaded in a manner of constant energy flow, sinusoidal pulsating flow or triangular wave flow.

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