Multi-stage vascular structure muscular tissue 3D printing perfusion stretching culture equipment and method

Abstract

The invention relates to multi-stage vascular structure muscular tissue 3D printing perfusion stretching culture equipment and method.A stretching mechanism in a stretching module comprises two stretching sliding seats, the inner end of each stretching sliding seat is provided with a positioning cavity, the outer end of each stretching sliding seat is provided with a perfusion head, a positioning pile is arranged in each positioning cavity, and multi-stage vascular muscular tissue comprises a pressure-bearing shell; positioning grooves are formed in the two ends of the pressure-bearing shell, the corresponding positioning piles are sleeved with the positioning grooves, a culture medium bottle in the culture medium module is connected with a port A in the four-way valve through a connecting pipeline, and the pouring module is connected with a port D in the four-way valve through a pouring pipeline; a port B on the four-way valve is connected with the filling head on the stretching sliding seat on the corresponding side through a liquid inlet pipeline, the filling head on the stretching sliding seat on the other side is connected with a waste liquid bottle in the culture medium module through a liquid outlet pipeline, and a port C on the four-way valve is connected with an exhaust pipeline. According to the invention, continuous nutrition supply and metabolism excretion can be provided for the skeletal muscle tissue subjected to 3D printing in vitro.

Description

Technical Field

[0001] The present invention relates to the field of 3D printing of muscle tissue, and more specifically to a perfusion stretching culture device and method for 3D printing muscle tissue with a multi-level vascular structure. Background Art

[0002] Skeletal muscle tissue is crucial for human activities. It has a certain self-repair ability, but it is difficult to self-repair after severe trauma. Therefore, it is particularly important to develop a muscle tissue regeneration strategy that can simulate the structure and function of natural skeletal muscle. With the rapid development of biomaterial science, cell biology, and 3D printing technology, 3D printing of skeletal muscle cells, as a new means of regenerative medicine, provides new hope for repairing and replacing damaged skeletal muscle.

[0003] The 3D printed vascular muscle tissue can be perfused and cultured in vitro, and by applying appropriate stretching and compression stimuli, it can simulate the mechanical environment in vivo, thereby promoting the maturation, differentiation, and functional expression of skeletal muscle cells. Existing research shows that the traditional static culture method cannot simulate the dynamic environment in vivo, so it is difficult for cells to obtain sufficient nutrients and oxygen during in vitro culture. However, under long-term dynamic perfusion and mechanical stimulation, the degree of orientation and contractility of muscle fibers can be significantly improved, thereby promoting the elongation and thickening of skeletal muscle tissue and enhancing the functional characteristics of skeletal muscle.

[0004] For example, Patent CN104673668B discloses a chamber, a soft tissue perfusion culture system, and a stretching test system for soft tissue. The chamber includes an upper structure and a lower structure made of an elastic material. The two ends of the upper structure and the two ends of the lower structure are respectively embedded in the second grooves of the corresponding side fixing blocks. The soft tissue is clamped between the upper structure and the lower structure, and a fixing component fixedly connects the fixing blocks, the upper structure, and the lower structure. The upper structure is provided with a liquid inlet, and the lower structure is provided with a liquid outlet to achieve perfusion culture of the soft tissue. When performing a stretching test, the fixing blocks at both ends of the chamber are respectively fixed on the first fixture fixing plate and the second fixture fixing plate, and then the first fixture fixing plate drives the movement of the chamber through the driving of the motor, thereby driving the movement of the soft tissue. However, since the liquid inlet and the liquid outlet are respectively arranged on the upper structure and the lower structure that are stretched together in this system, the liquid inlet and the liquid outlet need to be kept closed during the stretching test, and it cannot provide continuous nutrient supply and metabolic discharge for the 3D printed skeletal muscle tissue. Summary of the Invention

[0005] The object of the present invention is to provide a 3D printing perfusion stretching culture device and method for multi-level vascular structure muscle tissue, which has the functions of automatically supplementing culture medium and automatically exhausting air, and can provide continuous nutrient supply and metabolic discharge for the 3D printed skeletal muscle tissue in vitro, so as to simulate the blood circulation function in vivo.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A 3D printing perfusion stretching culture device for multi-level vascular structure muscle tissue, comprising a multi-level vascular muscle tissue, a culture medium module, a perfusion module and a stretching module. The stretching module includes a stretching mechanism, and the stretching mechanism includes two stretching sliders. A positioning cavity is provided at the inner end of each stretching slider, and a perfusion head is provided at the outer end. A positioning pile is provided in the positioning cavity. The multi-level vascular muscle tissue includes a pressure-bearing housing, and positioning grooves are provided at both ends of the pressure-bearing housing. The two ends of the multi-level vascular muscle tissue are respectively fixed in the corresponding positioning cavities, and the positioning grooves are sleeved on the corresponding positioning piles. The culture medium module internally includes a culture medium bottle, a four-way valve and a waste liquid bottle. The culture medium bottle is connected to port A on the four-way valve through a connecting pipeline with a connecting control valve. The perfusion module is connected to port D on the four-way valve through a perfusion pipeline. Port B on the four-way valve is connected to the perfusion head on the corresponding side stretching slider through a liquid inlet pipeline with a liquid inlet control valve. The perfusion head on the other side stretching slider is connected to the waste liquid bottle through a liquid outlet pipeline. Port C on the four-way valve is connected to an exhaust pipeline.

[0008] The two stretching sliders of the stretching mechanism are a first stretching slider and a second stretching slider. A first positioning protrusion is provided on the first stretching slider, and a first positioning cavity is provided inside the first positioning protrusion. A first cover plate is provided at the upper end of the first positioning protrusion. A first channel communicating the first perfusion head and the liquid inlet of the multi-level vascular muscle tissue is provided in the first positioning protrusion, and the first perfusion head is connected to the liquid inlet pipeline. A second positioning protrusion is provided on the second stretching slider, and a second positioning cavity is provided inside the second positioning protrusion. A second cover plate is provided at the upper end of the second positioning protrusion. A second channel communicating the second perfusion head and the liquid outlet of the multi-level vascular muscle tissue is provided in the second positioning protrusion, and the second perfusion head is connected to the liquid outlet pipeline. Positioning piles are provided in both the first positioning cavity and the second positioning cavity.

[0009] The stretching mechanism includes a base and a stretching lead screw. A first slider is provided on the lower side of the first stretching slider and is slidably connected to the base. A second slider is provided on the lower side of the second stretching slider and is slidably connected to the base. A first lead nut is provided inside the first slider, and a second lead nut is provided inside the second slider. One end of the stretching lead screw is inserted into the first lead nut, and the other end is inserted into the second lead nut. The stretching lead screw is driven to rotate by a stretching driving device.

[0010] The stretching module includes a stretching housing. A driving cavity for accommodating the stretching driving device is provided on one side of the stretching housing, and a stretching cavity for accommodating the stretching mechanism is provided on the other side. A stretching displacement sensor for real-time detecting the displacement of the stretching slide seat is provided in the driving cavity.

[0011] The culture medium module, the perfusion module and the stretching module are all arranged on an installation base plate, and a control module is provided on the installation base plate; the pressure-bearing housing of the multi-stage vascular muscle tissue is internally filled with artificial muscle tissue, and multi-stage vascular channels are provided in the artificial muscle tissue.

[0012] The perfusion module includes a perfusion housing, a connection joint, a syringe, a moving seat and a moving seat driving mechanism. The syringe and the connection joint are both arranged on the upper side of the perfusion housing. The front end of the syringe is connected to the perfusion pipeline through the connection joint. A syringe piston rod is provided at the rear end of the syringe and is connected to the moving seat. The moving seat is driven to move by a moving seat driving mechanism arranged inside the perfusion housing. A moving slot for the moving seat to move is provided on the upper side of the perfusion housing. A gas-liquid sensor is provided at the front end of the syringe.

[0013] The moving seat driving mechanism includes a perfusion lead screw and a perfusion driving device. A nut seat is provided at the lower end of the moving seat and is sleeved on the perfusion lead screw. The perfusion lead screw is driven to rotate by the perfusion driving device. In addition, a perfusion displacement sensor for real-time detecting the displacement of the nut seat is provided inside the perfusion housing.

[0014] The front end of the syringe is threadedly connected to a joint nut arranged on the connection joint. The rear end of the syringe piston rod is fixed to the moving seat through a fixing bolt.

[0015] A method for 3D printing a perfusion stretching culture device for a multi-stage vascular structure muscle tissue as described above is specifically as follows: First, both ends of the multi-stage vascular muscle tissue are respectively installed on the stretching slide seats on the corresponding sides in the stretching mechanism. Then, the stretching mechanism is started and applies periodic stretching and compression stimuli to the multi-stage vascular muscle tissue according to preset parameters. During this process, the perfusion module automatically extracts the culture medium from the culture medium bottle of the culture medium module and perfuses and supplements it into the multi-stage vascular muscle tissue. Before perfusion, the connection control valve is opened, the liquid inlet control valve is kept in a closed state, and the syringe piston rod at the rear end of the syringe moves backward to suck the culture medium in the culture medium bottle into the syringe. During perfusion, the connection control valve is closed, the liquid inlet control valve is opened, and the syringe piston rod at the rear end of the syringe moves forward to input the culture medium in the syringe into the multi-stage vascular muscle tissue.

[0016] The gas-liquid sensor detects in real time whether air enters the injector. If gas is detected, the connection control valve and the liquid inlet control valve remain closed, and at the same time, the exhaust pipeline is activated to drain the gas in the injector and the perfusion pipeline until the gas-liquid sensor passes the detection.

[0017] The advantages and positive effects of the present invention are as follows:

[0018] 1. Through the culture medium module and the perfusion module, the present invention can achieve long-term dynamic perfusion of the culture medium, that is, it can automatically supplement the culture medium and discharge the air in the culture system. It solves the problem of the in vivo dynamic environment that cannot be simulated by the traditional static culture method, and can provide continuous nutrient supply and metabolic discharge for the 3D printed skeletal muscle tissue in vitro, and then simulate the blood circulation function in vivo. The two stretching sliding seats of the stretching module of the present invention can automatically apply periodic stretching and compression stimuli according to the preset parameters, ensuring that the frequency, amplitude and duration of the mechanical stimuli are precisely controlled, avoiding the errors caused by manual operation, and improving the repeatability and reliability of the experiment.

[0019] 2. Considering that the main body of the artificial muscle tissue in the multi-stage vascular muscle tissue is hydrogel, which is a soft material and is not easy to be connected to the clamping device, and it is also easy to be damaged and leak during perfusion, and excessive external force will also cause serious damage. Therefore, the present invention sets positioning grooves at both ends of the pressure-bearing shell of the multi-stage vascular muscle tissue, and uses the positioning grooves to cooperate with the positioning piles in the stretching sliding seats on both sides of the stretching mechanism, so as to realize the stretching and fixing of both ends of the multi-stage vascular muscle tissue. In this way, when the stretching power acts on both ends of the pressure-bearing shell during the test of the present invention, it will not damage the internal artificial muscle tissue. At the same time, the matching structure of the positioning groove and the positioning pile can also ensure that the liquid inlet and outlet of the multi-stage vascular muscle tissue are automatically aligned with the through-hole positions of the corresponding perfusion heads on the corresponding side. And because the front end of the perfusion head abuts against the pressure-bearing shell, it will not damage the internal artificial muscle tissue either.

[0020] 3. The multi-stage vascular muscle tissue of the present invention has a multi-stage vascular structure and an outer pressure-bearing system, which can simulate the vascular structure and mechanical environment of natural skeletal muscle, ensure the supply of oxygen and nutrients inside the tissue, and enhance the mechanical properties of the muscle tissue. Description of the Drawings

[0021] Figure 1 is a three-dimensional structural schematic diagram of the device of the present invention,

[0022] Figure 2 is Figure 1 the top view of the device of the present invention in

[0023] Figure 3 is Figure 2Schematic diagram of the medium module and the perfusion module

[0024] Figure 4 is Figure 3 Cross-sectional view of the perfusion module in

[0025] Figure 5 is Figure 2 Schematic diagram of the control module and the stretching module in

[0026] Figure 6 is Figure 5 Schematic diagram of the three-dimensional structure of the stretching mechanism in

[0027] Figure 7 is Figure 6 Top view of the first stretching slide and the second stretching slide in

[0028] Figure 8 is Figure 7 View A-A in

[0029] Figure 9 is Figure 7 Schematic diagram of the multi-level vascular muscle tissue in

[0030] Among them, 1 is the control module, 101 is the control module housing, 102 is the first controller, 103 is the second controller, 2 is the culture medium module, 201 is the culture medium bottle, 202 is the connecting pipeline, 2021 is the connecting control valve, 203 is the exhaust pipeline, 204 is the perfusion pipeline, 205 is the four-way valve, 206 is the liquid inlet pipeline, 2061 is the liquid inlet control valve, 207 is the waste liquid bottle, 208 is the culture medium module housing, 209 is the liquid outlet pipeline, 3 is the perfusion module, 301 is the connecting joint, 3011 is the joint nut, 302 is the gas-liquid sensor, 303 is the injector, 3031 is the injector piston rod, 304 is the fixing bolt, 305 is the moving seat, 306 is the perfusion housing, 3061 is the moving slot, 3062 is the housing positioning column, 307 is the nut seat, 308 is the perfusion lead screw, 309 is the perfusion displacement sensor, 310 is the perfusion driving device, 311 is the first support of the lead screw, 312 is the second support of the lead screw, 4 is the stretching module, 401 is the driving cavity, 402 is the stretching cavity, 5 is the stretching mechanism, 501 is the base, 5011 is the first limit block, 5012 is the second limit block, 502 is the first stretching slide, 5021 is the first perfusion head, 5022 is the first positioning protrusion, 50221 is the first cover plate, 5023 is the first cover plate bolt, 5024 is the first mounting bolt, 5025 is 507 is the stretching lead screw, 508 is the guide rod, 6 is the stretching driving device, 601 is the control cable, 602 is the coupling, 7 is the mounting base plate, 8 is the multi-stage vascular muscle tissue, 801 is the vascular channel, 802 is the liquid outlet, 803 is the pressure-bearing housing, 8031 is the positioning groove, 804 is the liquid inlet, 805 is the artificial muscle tissue. Detailed implementation mode

[0031] The present invention will be further described in detail below with reference to the accompanying drawings.

[0032] As Figures 1-9 shown, the present invention includes a multi-stage vascular muscle tissue 8, a culture medium module 2, a perfusion module 3 and a stretching module 4. Among them, as Figures 6-8 shown, the stretching module 4 includes a stretching mechanism 5, and the stretching mechanism 5 includes two stretching slides. A positioning cavity is provided at the inner end of each stretching slide, and a perfusion head is provided at the outer end. A positioning pile 5033 is provided in the positioning cavity. As Figure 9 shown, the multi-stage vascular muscle tissue 8 includes a pressure-bearing housing 803, and positioning grooves 8031 are provided at both ends of the pressure-bearing housing 803. Both ends of the multi-stage vascular muscle tissue 8 are respectively fixed in the corresponding positioning cavities, and the positioning groove 8031 is sleeved on the corresponding positioning pile 5033. As Figures 1-3As shown, inside the culture medium module 2, there are a culture medium bottle 201, a four-way valve 205, and a waste liquid bottle 207. Among them, the culture medium bottle 201 is connected to port A on the four-way valve 205 through a connecting pipeline 202 with a connecting control valve 2021. The perfusion module 3 is connected to port D on the four-way valve 205 through a perfusion pipeline 204. Port B on the four-way valve 205 is connected to the perfusion head on the corresponding side stretching slide through a liquid inlet pipeline 206 with a liquid inlet control valve 2061. The perfusion head on the other side stretching slide is connected to the waste liquid bottle 207 through a liquid outlet pipeline 209. Port C on the four-way valve 205 is connected to an exhaust pipeline 203.

[0033] Through the above-mentioned culture medium module 2 and perfusion module 3, the present invention can achieve long-term dynamic perfusion of the culture medium, that is, it can automatically supplement the culture medium and discharge the air in the system, solving the problem of the in vivo dynamic environment that cannot be simulated by the traditional static culture method. It can provide continuous nutrient supply and metabolic discharge for the 3D printed skeletal muscle tissue in vitro, and then simulate the blood circulation function in vivo. The two stretching slides of the stretching module 4 can automatically apply periodic stretching and compression stimuli according to preset parameters, ensuring that the frequency, amplitude, and duration of the mechanical stimuli are precisely controlled, avoiding errors caused by manual operation, and improving the repeatability and reliability of the experiment.

[0034] In addition, as Figure 9 shown, in the multi-stage vascular muscle tissue 8 targeted by the present invention, the main body of the artificial muscle tissue 805 is a hydrogel. As a soft material, it is not easy to connect with the clamping device, and it is also easy to be damaged and leak during perfusion. Excessive external force will also cause serious damage. Therefore, the present invention sets positioning grooves 8031 at both ends of the pressure-bearing housing 803, and uses the positioning grooves 8031 to cooperate with the positioning piles 5033 to realize the stretching and fixing of both ends of the multi-stage vascular muscle tissue 8. In this way, when the present invention is tested, the stretching force acts on both ends of the pressure-bearing housing 803 and will not affect the internal artificial muscle tissue 805. At the same time, the matching structure of the above-mentioned positioning grooves 8031 and positioning piles 5033 can also ensure that the liquid inlet 804 and liquid outlet 802 of the multi-stage vascular muscle tissue 8 are automatically aligned with the through-hole positions of the corresponding side perfusion heads, and the front end of the perfusion head abuts against the pressure-bearing housing 803 and will not affect the internal artificial muscle tissue 805.

[0035] As Figures 6-9As shown, in this embodiment, the stretching mechanism 5 includes a first stretching slide 502 and a second stretching slide 503. The inner end of the first stretching slide 502 is provided with a first positioning cavity 5025, and the outer end is provided with a first perfusion head 5021. The inner end of the second stretching slide 503 is provided with a second positioning cavity 5035, and the outer end is provided with a second perfusion head 5031. Positioning piles 5033 are provided inside both the first positioning cavity 5025 and the second positioning cavity 5035. One end of the multi-stage blood vessel muscle tissue 8 is fixed in the first positioning cavity 5025, and the other end is fixed in the second positioning cavity 5035. Moreover, the first perfusion head 5031 is communicated with the liquid inlet 804 of the multi-stage blood vessel muscle tissue 8, the liquid outlet 802 of the multi-stage blood vessel muscle tissue 8 is communicated with the second perfusion head 5031, the liquid inlet pipeline 206 is connected to the first perfusion head 5021, and the second perfusion head 5031 is connected to the liquid outlet pipeline 209.

[0036] As Figures 7-8 shown, in this embodiment, a first positioning protrusion 5022 is provided on the first stretching slide 502, and the first positioning cavity 5025 is provided inside the first positioning protrusion 5022. A first cover plate 50221 that seals and covers the first positioning cavity 5025 is provided at the upper end of the first positioning protrusion 5022. A first channel that communicates the first perfusion head 5021 and the liquid inlet 804 of the multi-stage blood vessel muscle tissue 8 is provided inside the first positioning protrusion 5022. A second positioning protrusion 5032 is provided on the second stretching slide 503, and the second positioning cavity 5035 is provided inside the second positioning protrusion 5032. A second cover plate 50321 that seals and covers the second positioning cavity 5035 is provided at the upper end of the second positioning protrusion 5032. A second channel that communicates the second perfusion head 5031 and the liquid outlet 802 of the multi-stage blood vessel muscle tissue 8 is provided inside the second positioning protrusion 5032.

[0037] As Figure 7 shown, in this embodiment, the first stretching slide 502 is fixed on the base 501 through a first mounting bolt 5024, the first cover plate 50221 is fixed on the first positioning protrusion 5022 through a first cover plate bolt 5023, the second stretching slide 503 is fixed on the base 501 through a second mounting bolt 5034, and the second cover plate 50321 is fixed on the second positioning protrusion 5032 through a second cover plate bolt.

[0038] As Figure 6As shown in the figure, in this embodiment, the stretching mechanism 5 includes a base 501 and a stretching lead screw 507. A first slider 506 is provided on the lower side of the first stretching slider 502 and is slidably connected to the base 501. A second slider 505 is provided on the lower side of the second stretching slider 503 and is slidably connected to the base 501. A first nut is provided inside the first slider 506, and a second nut is provided inside the second slider 505. One end of the stretching lead screw 507 is inserted into the first nut, and the other end is inserted into the second nut. The stretching lead screw 507 is driven to rotate by a stretching driving device 6, thereby driving the two stretching sliders to move to achieve stretching and compressing actions. The stretching driving device 6 can be a servo motor or other devices as required.

[0039] As Figure 6 shown in the figure, in this embodiment, a first limit block 5011 is provided at one end of the base 501 to limit the displacement of the first stretching slider 502, and a second limit block 5012 is provided at the other end to limit the displacement of the second stretching slider 503.

[0040] As Figures 1-2 shown in the figure, in this embodiment, the stretching module 4 includes a stretching housing. A driving cavity 401 for accommodating the stretching driving device 6 is provided on one side of the stretching housing, and a stretching cavity 402 for accommodating the stretching mechanism 5 is provided on the other side. The front end of the stretching driving device 6 is connected to the stretching lead screw 507 through a coupling 602. A stretching displacement sensor 504 for real-time detection of the displacements of the two stretching sliders is provided in the driving cavity 401. The stretching displacement sensor 504 is a commercially available product.

[0041] As Figure 4 shown in the figure, in this embodiment, the perfusion module 3 includes a perfusion housing 306, a connection joint 301, a sampler 303, a moving seat 305, and a moving seat driving mechanism. The sampler 303 and the connection joint 301 are both provided on the upper side of the perfusion housing 306. The front end of the sampler 303 is connected to the perfusion pipeline 204 through the connection joint 301. A sampler piston rod 3031 is provided at the rear end of the sampler 303 and is connected to the moving seat 305. The moving seat 305 is linked by a moving seat driving mechanism provided inside the perfusion housing 306, thereby realizing the insertion and extraction movement of the sampler piston rod 3031. A moving slot 3061 for the movement of the moving seat 305 is provided on the upper side of the perfusion housing 306. A gas-liquid sensor 302 for detecting whether gas flows into the sampler 303 is provided at the front end of the sampler 303. The gas-liquid sensor 302 is a commercially available product.

[0042] As Figure 4As shown, in this embodiment, the moving seat driving mechanism includes a perfusion lead screw 308 and a perfusion driving device 310. A nut seat 307 is provided at the lower end of the moving seat 305 and sleeved on the perfusion lead screw 308. The perfusion lead screw 308 is driven to rotate by the perfusion driving device 310. In addition, a perfusion displacement sensor 309 for real-time detection of the displacement of the nut seat 307 is provided inside the perfusion housing 306. The perfusion displacement sensor 309 is a commercially available product.

[0043] As Figure 4 shown, in this embodiment, a first lead screw support 311 and a second lead screw support 312 for supporting the rotation of the perfusion lead screw 308 are provided inside the perfusion housing 306. A housing positioning post 3062 is provided on the lower side of the perfusion housing 306 for Figure 1 positioning and installing with the mounting base plate 7 shown in

[0044] As Figure 4 shown, in this embodiment, the front end of the sampler 303 is threadedly connected to a joint nut 3011 provided on the connection joint 301. The rear end of the sampler piston rod 3031 is fixed to the moving seat 305 by a fixing bolt 304. This structure facilitates the replacement of the sampler 303 with different volume specifications according to actual needs. When replacing, first remove the fixing bolt 304 to separate the rear end of the sampler piston rod 3031 from the moving seat 305, and then rotate the sampler 303 and the joint nut 3011 relative to each other to separate the front end of the sampler 303 from the joint nut 3011.

[0045] As Figure 1 shown, in this embodiment, the culture medium module 2 includes a culture medium module housing 208. The culture medium bottle 201, the connecting pipeline 202, the four-way valve 205, and the waste liquid bottle 207 are all provided in the culture medium module housing 208. The perfusion pipeline 204 extends out of the culture medium module housing 208 and is connected to the perfusion module 3. The liquid inlet pipeline 206 and the liquid outlet pipeline 209 extend out of the culture medium module housing 208 and are connected to the stretching mechanism 5 in the stretching module 4.

[0046] As Figure 1 shown, in this embodiment, the culture medium module 2, the perfusion module 3, and the stretching module 4 are all provided on a mounting base plate 7. And a control module 1 for controlling the actions of each mechanism is also provided on the mounting base plate 7. In this embodiment, the control module 1 includes a control module housing 101 and a first controller 102 and a second controller 103 provided in the control module housing 101. The rear end of the stretching driving device 6 is connected to the corresponding controller in the control module 1 through a control cable 601.

[0047] AsFigure 9 As shown, in this embodiment, the pressure-bearing housing 803 of the multi-stage vascular muscle tissue 8 is filled with artificial muscle tissue 805, and a multi-stage vascular channel 801 is provided in the artificial muscle tissue 805, which can simulate the vascular structure and mechanical environment of natural skeletal muscle, ensure the supply of oxygen and nutrients inside the tissue, and enhance the mechanical properties of the muscle tissue. In this embodiment, the material of the pressure-bearing housing 803 is high-concentration GelMA hydrogel, which can construct an artificial muscle tissue with a layered structure. The artificial muscle tissue 805 is printed with a bio-ink configured by low-concentration GelMA hydrogel and skeletal muscle cells. The low-concentration GelMA hydrogel can provide a suitable growth environment for cells and support cell proliferation and differentiation. The multi-stage vascular channels 801 are located inside the artificial muscle tissue 805 and are printed with gelatin hydrogel. There are primary blood vessels on both sides and a secondary vascular network in the middle. The number of channels of the secondary blood vessels is relatively large, which can increase the specific surface area of the blood vessels, and then simulate the microstructure of natural skeletal muscle and improve the transport flux of nutrients and metabolic wastes.

[0048] In addition, in this embodiment, the first positioning cavity 5025 and the second positioning cavity 5035 are sealed with high-concentration GelMA hydrogel, and ultraviolet light is used to crosslink the poured GelMA hydrogel to prevent the leakage of the culture medium during the perfusion process.

[0049] The working principle of the present invention is as follows:

[0050] When the present invention works, first, both ends of the multi-stage vascular muscle tissue 8 are respectively installed on the stretching sliders on the corresponding sides of the stretching mechanism 5. Then, the stretching mechanism 5 is started and automatically applies periodic stretching and compression stimuli to the multi-stage vascular muscle tissue 8 according to preset parameters. During this process, the perfusion module 3 automatically extracts the culture medium from the culture medium bottle 201 of the culture medium module 2 and perfuses and supplements it into the multi-stage vascular muscle tissue 8. Before perfusion, the connection control valve 2021 is opened, and the liquid inlet control valve 2061 is kept closed. The piston rod 3031 at the rear end of the injector 303 moves backward to suck the culture medium in the culture medium bottle 201 into the injector 303. During perfusion, the connection control valve 2021 is closed, and the liquid inlet control valve 2061 is opened. The piston rod 3031 at the rear end of the injector 303 moves forward to input the culture medium in the injector 303 into the multi-stage vascular muscle tissue 8.

[0051] In addition, during the above process, the gas-liquid sensor 302 detects in real time whether air enters the sampler 303. If gas is detected, the connection control valve 2021 and the liquid inlet control valve 2061 remain closed. At the same time, the exhaust pipeline 203 is activated to exhaust the gas in the sampler 303 and the perfusion pipeline 204 until the gas-liquid sensor 302 passes the detection. The exhaust pipeline 203 can be connected to an air extraction device to achieve the exhaust function, which is a well-known technology in the art.

[0052] The present invention uses the perfusion displacement sensor 309 to detect the lead screw seat 307 to determine whether the culture medium in the sampler 303 is completely output. After the complete output, the sampler 303 repeats the above-mentioned action of sucking the culture medium. After the skeletal muscle cells in the multi-stage vascular muscle tissue 8 complete the absorption of nutrients and the excretion of metabolic wastes, the waste liquid is output from the second perfusion head 5031 and enters the waste liquid bottle 207.

Claims

1. A multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device, characterized by: The invention comprises a multi-level vascular muscle tissue (8), a culture medium module (2), a perfusion module (3) and a stretching module (4), wherein the stretching module (4) comprises a stretching mechanism (5), and the stretching mechanism (5) comprises two stretching slides, each of which is provided with a positioning cavity at the inner end and a perfusion head at the outer end, and a positioning pile (5033) is provided in the positioning cavity, the multi-level vascular muscle tissue (8) comprises a pressure-bearing shell (803), and both ends of the pressure-bearing shell (803) are provided with positioning grooves (8031), the two ends of the multi-level vascular muscle tissue (8) are respectively fixed in the positioning cavity on the corresponding side, and the positioning grooves (8031) are sleeved on the corresponding positioning piles (5033), the culture medium The module (2) is provided with a culture medium bottle (201), a four-way valve (205) and a waste liquid bottle (207), wherein the culture medium bottle (201) is connected to port A on the four-way valve (205) via a connecting pipeline (202) with a connecting control valve (2021), the perfusion module (3) is connected to port D on the four-way valve (205) via a perfusion pipeline (204), port B on the four-way valve (205) is connected to the perfusion head on the stretching slide on the corresponding side via a liquid inlet pipeline (206) with a liquid inlet control valve (2061), the perfusion head on the stretching slide on the other side is connected to the waste liquid bottle (207) via a liquid outlet pipeline (209), and port C on the four-way valve (205) is connected to the exhaust pipeline (203).

2. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 1, characterized in that: The two stretching slides of the stretching mechanism (5) are a first stretching slide (502) and a second stretching slide (503), wherein the first stretching slide (502) is provided with a first positioning protrusion (5022), and a first positioning cavity (5025) is provided inside the first positioning protrusion (5022), a first cover plate (50221) is provided at the upper end of the first positioning protrusion (5022), a first channel connecting the first perfusion head (5021) and the liquid inlet (804) of the multi-level vascular muscle tissue (8) is provided in the first positioning protrusion (5022), and the first perfusion head (5021) and the liquid inlet pipeline (206) are connected. The second stretching slide (503) is provided with a second positioning protrusion (5032), and a second positioning cavity (5035) is provided inside the second positioning protrusion (5032), a second cover plate (50321) is provided at the upper end of the second positioning protrusion (5032), a second channel connecting the second perfusion head (5031) and the liquid outlet (802) of the multi-level vascular muscle tissue (8) is provided in the second positioning protrusion (5032), and the second perfusion head (5031) is connected to the liquid outlet pipeline (209), and positioning piles (5033) are provided in both the first positioning cavity (5025) and the second positioning cavity (5035).

3. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 2, characterized in that: The stretching mechanism (5) comprises a base (501) and a stretching screw (507), wherein a first slider (506) is provided on the lower side of the first stretching slide (502) and is slidably connected to the base (501), and a second slider (505) is provided on the lower side of the second stretching slide (503) and is slidably connected to the base (501), a first nut is provided inside the first slider (506), and a second nut is provided inside the second slider (505), and one end of the stretching screw (507) is inserted into the first nut and the other end is inserted into the second nut, and the stretching screw (507) is driven to rotate by a stretching drive device (6).

4. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 3, characterized in that: The stretching module (4) comprises a stretching shell, and a driving chamber (401) for accommodating the stretching driving device (6) is provided on one side of the stretching shell, and a stretching chamber (402) for accommodating the stretching mechanism (5) is provided on the other side, and a stretching displacement sensor (504) for real-time detection of stretching slide displacement is provided in the driving chamber (401).

5. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 1, characterized in that: The culture medium module (2), the perfusion module (3) and the stretching module (4) are all arranged on a mounting base plate (7), and a control module (1) is arranged on the mounting base plate (7); the pressure-bearing shell (803) of the multi-level vascular muscle tissue (8) is filled with artificial muscle tissue (805), and the artificial muscle tissue (805) is provided with a multi-level vascular channel (801).

6. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 1, characterized in that: The perfusion module (3) comprises a perfusion shell (306), a connecting joint (301), an injector (303), a movable seat (305) and a movable seat driving mechanism, wherein the injector (303) and the connecting joint (301) are both arranged on the upper side of the perfusion shell (306), and the front end of the injector (303) is connected to the perfusion pipeline (204) through the connecting joint (301), the rear end of the injector (303) is provided with an injector piston rod (3031) connected to the movable seat (305), the movable seat (305) is driven to move by the movable seat driving mechanism arranged inside the perfusion shell (306), the upper side of the perfusion shell (306) is provided with a movable slot (3061) for the movable seat (305) to move, and the front end of the injector (303) is provided with a gas-liquid sensor (302).

7. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 6, characterized in that: The movable seat driving mechanism comprises an injection screw (308) and an injection driving device (310); a nut seat (307) is provided at the lower end of the movable seat (305) and is sleeved on the injection screw (308); the injection screw (308) is driven to rotate by the injection driving device (310); and an injection displacement sensor (309) for detecting the displacement of the nut seat (307) in real time is provided inside the injection housing (306).

8. The multi-level vascular structure muscle tissue 3D printing perfusion stretching culture device according to claim 6, characterized in that: The front end of the sample injector (303) is threadedly connected to a joint nut (3011) provided on the connecting joint (301), and the rear end of the sample injector piston rod (3031) is fixed to the movable seat (305) via a fixing bolt (304).

9. A method for 3D printing perfusion stretching culture equipment for multi-level vascular structure muscle tissue according to claim 6, characterized in that: First, the two ends of the multi-level vascular muscle tissue (8) are respectively mounted on the stretching slide seats on the corresponding sides of the stretching mechanism (5), and then the stretching mechanism (5) is started and periodic stretching and compression stimulation is applied to the multi-level vascular muscle tissue (8) according to preset parameters. In this process, the perfusion module (3) automatically extracts culture medium from the culture medium bottle (201) of the culture medium module (2) and perfuses and replenishes it into the multi-level vascular muscle tissue (8), wherein before perfusion, the connection control valve (221) is opened. During perfusion, the connection control valve (2021) is closed, the liquid inlet control valve (2061) is opened, and the injector piston rod (3031) at the rear end of the injector (303) moves backward to suck the culture medium in the culture medium bottle (201) into the injector (303). During perfusion, the connection control valve (2021) is closed, the liquid inlet control valve (2061) is opened, and the injector piston rod (3031) at the rear end of the injector (303) moves forward to input the culture medium in the injector (303) into the multi-stage vascular muscle tissue (8).

10. The method for 3D printing perfusion stretching culture equipment of multi-level vascular structure muscle tissue according to claim 9, characterized in that: The gas-liquid sensor (302) detects in real time whether air enters the sample injector (303). If gas is detected, the connection control valve (2021) and the liquid inlet control valve (2061) remain in a closed state, and at the same time, the exhaust pipeline (203) is started to exhaust the gas in the sample injector (303) and the perfusion pipeline (204) until the gas-liquid sensor (302) passes the test.

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