Microscopic bone lacuna-bone canaliculus system mass transfer chip and experimental method

Abstract

This invention relates to a microscopic lacunar-canal mass transfer chip and experimental method. The microscopic lacunar-canal mass transfer chip includes an upper cover plate, a micropore design, and a lower cover plate. The micropore design has several lacunae arranged sequentially, and the lacunae are connected sequentially by corresponding canals. The three longitudinal canals are Haver tubes, and the transverse canals are Folkmann tubes. The micropores are etched onto the lower cover plate. The upper cover plate is made of polydimethylsiloxane (PDMS), and the lower cover plate is made of quartz glass. The micropores are deepened to the desired depth to form a pore network etched chip. The upper cover plate and the lower cover plate with the etched micropore structure are bonded together to form the microscopic chip. This invention provides a lacunar-canal microscopic mass transfer chip that allows for clear observation of the transport patterns of the lacunar-canal system, improving the understanding of intraosseous mass transfer.

Description

Technical fields:

[0001] This invention relates to the application of microfluidic chip technology to the field of bone mass transfer technology, and in particular to a microscopic bone lacunar-canal system mass transfer chip and experimental method. Background technology:

[0002] Nutrient transport and waste removal are the basic conditions for the metabolism of organisms. Blood circulation and lymphatic circulation can meet the needs of organism growth and development. In addition to the normal vascular transport pathway for substances in bone tissue, bone tissue has a unique load-induced interstitial transport process in the lacunae-tubule system. The process from capillaries to bone cells requires transport through the lacunae-tubule system to achieve substance exchange.

[0003] To investigate the mass transfer mechanisms of the lacunar inguinal canaliculus system, researchers conducted in vivo and in vitro experiments, using fluorescent tracers to simulate mass transfer and then observing the results with a laser scanning confocal microscope. However, laser scanning confocal microscopy can only observe fluorescence at a depth of tens of micrometers and cannot observe the entire mass transfer process within bone units.

[0004] In recent years, with the development of microfabrication technology, microfluidic chip technology has gradually become a major tool for studying tissues and organs. Common microfluidic chips are mostly used in research on the lungs, liver, kidneys, and intestines, yielding more effective results compared to traditional in vitro and in vivo experiments, and without raising ethical concerns.

[0005] Therefore, microfluidic chip technology is applied to the study of mass transfer experiments in the lacunar-canal system. This microscopic mass transfer chip can clearly observe the transport process of the lacunar-canal system and the overall structure of bone units. Establishing a microscopic lacunar-canal system mass transfer chip can solve the problem of observation in mass transfer experiments. Summary of the Invention:

[0006] The technical problem to be solved by the present invention is to provide a microscopic lacunar-canal system mass transfer chip and experimental method. The mass transfer chip is easy to observe, simple to use, and has good light transmittance, which can clearly observe the flow and distribution of fluid in the lacunar-canal system.

[0007] The technical solution adopted by the present invention to solve its technical problem is: to construct a microscopic bone lacunar-bone canal system mass transfer chip, including an upper cover plate, a microscopic pore structure design and a lower cover plate; the upper cover plate and the lower cover plate are both made of transparent materials, the upper cover plate is made of polydimethylsiloxane (PDMS) and the lower cover plate is made of quartz glass.

[0008] Microscopic pore structure design: Based on the structure of lacunae and canaliculi in real bone tissue, microscopic structure etching is performed on the lower cover plate. The dimensions of Haver tubes, Falkmann tubes, canaliculi, and lacunae on the quartz glass are the same as those in the real bone structure. The three Haver tubes are connected longitudinally, and the canaliculi are connected to the Haver tubes. The lacunae are connected to the canaliculi. The Falkmann tubes are connected to the Haver tubes laterally, forming a complete lacunae-canaliculi transport system.

[0009] The upper and lower cover plates are connected by a bonding mechanism to ensure that, except for the liquid flowing inside the designed pipe, the rest of the surface is sealed.

[0010] The upper and lower cover plates are trapezoidal in shape with an upper base of 5mm and a lower base of 3mm, and a thickness of 2mm.

[0011] The beneficial effects of this invention are that the model size is close to the actual size of the lacunae-canaliculi, thus achieving a more realistic and accurate simulation of the lacunae-canaliculi structure. Its transparent material allows for clear observation of the flow and distribution of liquids, thereby providing reliable experimental evidence for subsequent mass transfer experiments of the lacunae-canaliculi system.

[0012] This invention provides an experimental method for mass transfer in the aforementioned microscopic lacunar infarct-canal system, comprising the following steps:

[0013] (a) The upper cover plate and the lower cover plate are connected together by bonding to form a whole microchip.

[0014] (ii) The micro mass transfer chip is placed in a transparent silicone sleeve. The transparent silicone sleeve has a horizontal through hole and is the same size as the micro mass transfer chip. The injection end of the transparent silicone sleeve is connected to the latex tube, and the middle part of the latex tube is connected to the syringe. The syringe is fixed in the horizontal direction, and the rod of the syringe is in contact with the cam.

[0015] (iii) Place the fluorescent tracer into the syringe, connect one end of the latex tube to the precision digital electronic pressure gauge, and connect the other end to the silicone sleeve.

[0016] (iv) By adjusting the motor speed to control the infusion and loading of fluorescent tracer, the value of the precision digital display electronic pressure gauge is observed to simulate the pulsating pressure of the human body. The fluorescent tracer is injected from the bottom of the microchip for 20 minutes and then the injection is stopped.

[0017] (v) Remove the microfluidic chip, place it under a fluorescence microscope for observation, take images, and record experimental data. Attached image description:

[0018] The present invention will now be further described with reference to the accompanying drawings and examples, in which:

[0019] Figure 1 This is a schematic diagram of the microscopic mass transfer chip structure of the present invention;

[0020] Figure 2 This is an enlarged schematic diagram of the mass transfer chip of the microscopic bone lacunae-bone canaliculi system of the present invention;

[0021] Figure 3 This is a schematic diagram of the microscopic bone lacunae and bone canaliculi structures of the present invention;

[0022] Figure 4 This is a schematic diagram of the experimental apparatus of the present invention;

[0023] In the diagram: 1. Upper cover plate; 2. Microscopic mass transfer chip main body pattern; 3. Lower cover plate; 4. Haver tube; 5. Folkmann tube; 6. Bony lacuna-canal system; 7. Bony lacuna; 8. Transparent silicone sleeve; 9. Latex tube; 10. Precision digital display electronic pressure gauge; 11. Compression spring; 12. Injector; 13. Cam; 14. Motor; 15. Detailed implementation method:

[0024] To provide a clearer understanding of the objectives and effects of the present invention, specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0025] Implementation of List 1

[0026] This invention provides a visualized microscopic model of the lacunae and canaliculi, the main structure of which is as follows: Figure 1 As shown, it includes: an upper cover plate (1), a micro-mass transfer chip body pattern (2), and a lower cover plate (3).

[0027] The upper and lower cover plates are both trapezoidal in shape, with dimensions of 5mm for the upper base, 3mm for the lower base, and a thickness of 2mm. The upper cover plate is made of polydimethylsiloxane (PDMS), and the lower cover plate is made of quartz glass. The microscopic lacunar-canal model is etched onto the lower cover plate, and the upper and lower cover plates are connected by bonding. The upper cover plate is a smooth plane, and the lacunar foci on the lower cover plate are connected sequentially by the canals. There are three Haver canals (4) in the longitudinal direction and Falkmann canals (5) in the transverse direction. The Falkmann canals (5) are connected to the Haver canals (4), forming a complete lacunar-canal system model (6).

[0028] The upper cover plate (1) is made of transparent polydimethylsiloxane (PDMS), which allows for clear observation of the fluid flow and distribution in the lacunae and canaliculi.

[0029] The main pattern of the micro-mass transfer chip is a trapezoid with an upper base of 5mm, a lower base of 3mm, and a thickness of 2mm, which is a 3D pore model. The upper base of the micro-chip is the liquid inlet, and the lower base is the liquid outlet. The micro-light chip is etched onto the lower cover plate (3) by etching.

[0030] On the lower cover plate (3), there are three longitudinal Haversian tubes (4) with a diameter of 50 μm and a depth of 25 μm, and a transverse Falkmann tube (5) with a diameter of 30 μm and a depth of 15 μm. The Falkmann tube (5) is connected to the Haversian tubes (4). Bone lacunae (7) and bone canaliculi (8) are distributed on the left and right sides of the Haversian tubes (4). There are 6 bone lacunae (7) transversely and 16 bone lacunae (7) longitudinally on the left side. Each bone lacunae (7) is connected to each other by bone canaliculi (8). The bone canaliculi (8) is 20 μm long, 0.7 μm wide and 0.35 μm deep. The major axis of the bone lacunae (7) is 10 μm, the minor axis is 5 μm and the depth is 5 μm.

[0031] This invention provides an experimental method for visualizing mass transfer in a microscopic model of the lacunar infarct-canal system, specifically including the following steps:

[0032] (i) Connect the upper cover plate (1) and the lower cover plate (3) together by bonding to form an integral micro mass transfer chip; insert the micro mass transfer chip horizontally into the transparent silicone sleeve (9) and connect the injection end of the transparent silicone sleeve (9) to the latex tube (10).

[0033] (ii) Connect the middle part of the latex tube (10) to the syringe (13), and connect the other end of the latex tube (10) to the precision digital display electronic pressure gauge (11); the channels of the variable diameter tee connector are connected to the syringe (13), the precision digital display electronic pressure gauge (11) and the micro mass transfer chip through latex tubes of different diameters.

[0034] (III) Fluorescent tracer is drawn into the syringe. The DC variable speed motor (15) drives the cam (14) to rotate, which causes the piston rod of the syringe (13) to reciprocate. The compression spring (12) assists the piston rod of the syringe to form a timely return motion, so that the syringe can be circulated and filled with liquid. While adjusting the position of the syringe (13), observe the reading of the precision digital display electronic pressure gauge (11). When the syringe is fixed in the appropriate position, it can generate a pressure of 120 mmHg systolic and 80 mmHg diastolic, with a pulsation frequency of 1.1 Hz, which can simulate the situation of human bone tissue being subjected to pulsating pressure when at rest under normal gravity.

[0035] (iv) Replace the appropriate cam (14) to set the pulsation pressure to 200 mmHg systolic pressure and 80 mmHg diastolic pressure, with a pulsation frequency of 3.3 Hz. This can simulate the situation of human bone tissue doing high-intensity exercise under normal gravity. By observing the mass transfer of the lacunar-canal micro-model through pulsation pressure of different frequencies, the mass transfer law of the lacunar-canal system can be summarized.

[0036] (V) After 20 minutes of the experiment, the microfluidic chip was placed under a fluorescence microscope to observe the mass transfer between the lacunae and canaliculi. After taking the images, the fluorescence intensity was quantitatively expressed using ImageJ software, and the data were statistically analyzed to summarize the patterns.

Claims

1. A micro-osteocrypt-osteocanalicular mass transfer chip, characterized in that, The micro bone lacuna-bone canaliculus system mass transfer chip comprises an upper cover plate (1), a bone lacuna-bone canaliculus system micro pattern (2) and a lower cover plate (3), the upper cover plate (1) and the lower cover plate (3) are both made of transparent material, the upper cover plate (1) is made of polydimethylsiloxane (PDMS) and the lower cover plate (3) is made of quartz glass; the bone lacuna-bone canaliculus system micro pattern (2) is etched on the lower cover plate (3), the micro bone lacuna-bone canaliculus system mass transfer chip has a 3D pore, the upper cover plate (1) and the lower cover plate (3) are connected by bonding, the size of the Haversian canal (4), the Volkmann's canal (5), the bone lacuna (7) and the bone canaliculus (8) is the size of the corresponding size in the real bone cell, the micro bone lacuna-bone canaliculus system mass transfer chip is convenient to observe, the use method is simple, the light transmission is good, the liquid flow and distribution of the bone lacuna-bone canaliculus can be clearly observed, the mass transfer experiment method of the micro bone lacuna-bone canaliculus system mass transfer chip is that the upper cover plate (1) and the lower cover plate (3) are connected by bonding to form a complete micro bone lacuna-bone canaliculus system mass transfer chip, the micro bone lacuna-bone canaliculus system mass transfer chip is horizontally placed in a transparent silica gel sleeve (9), the transparent silica gel sleeve (9) is placed in one end of a latex tube (10), the middle part of the latex tube (10) is connected with a syringe (13), the other end is connected with a precise digital display electronic pressure gauge (11), the precise digital display electronic pressure gauge (11) is fixed on a vertical plate by glue, a motor (15) and the syringe (13) are fixed on a horizontal plate by a fixed block, a fluorescent tracer is placed in the syringe (13), the injection of the fluorescent tracer is controlled by adjusting the rotating speed of the motor (15), the value of the precise digital display electronic pressure gauge (11) is observed to simulate the pulsatile pressure of the human body, the fluorescent tracer is injected from the bottom of the micro bone lacuna-bone canaliculus system mass transfer chip, the injection is stopped after 20 min, the micro bone lacuna-bone canaliculus system mass transfer chip is taken out and placed under a fluorescence microscope for observation, the image is shot and the experimental data is recorded.

Images