CN110257243B

CN110257243B - Micro-fluidic chip printing nozzle and biological 3D printing system

Info

Publication number: CN110257243B
Application number: CN201910668437.5A
Authority: CN
Inventors: 弥胜利; 孙伟; 可鑫
Original assignee: Shenzhen Graduate School Tsinghua University
Current assignee: Shenzhen Graduate School Tsinghua University
Priority date: 2019-07-23
Filing date: 2019-07-23
Publication date: 2021-06-25
Anticipated expiration: 2039-07-23
Also published as: WO2021012534A1; CN110257243A

Abstract

A micro-fluidic chip nozzle comprises a micro-fluidic chip and a nozzle with a double-layer structure, wherein the micro-fluidic chip comprises a micro-fluidic chip substrate layer and a micro-fluidic chip upper layer, a plurality of flow channels and flow-mixing structure flow channels are formed on the micro-fluidic chip substrate layer, the flow-mixing structure flow channels are connected with a micro-fluidic chip outlet, micro valves corresponding to the flow channels are formed on the micro-fluidic chip upper layer, the micro valves can be controlled to be opened or closed to control liquid in different flow channels to enter the flow channels with the flow-mixing structure, the nozzle with the double-layer structure comprises an inner-layer micro needle and a shell, the inner-layer micro needle is connected with the micro-fluidic chip outlet, a cross-linking liquid inlet and a nozzle outlet are formed in the shell, mixed solution input from the inner-layer micro needle is mixed with cross. The micro-fluidic chip nozzle structure is particularly suitable for printing gradient tissue engineering cornea with a layered structure.

Description

Micro-fluidic chip printing nozzle and biological 3D printing system

Technical Field

The invention relates to a biological 3D printing and forming technology, in particular to a micro-fluidic chip printing nozzle and a biological 3D printing system.

Background

3D printing is a process of building three-dimensional objects by depositing material layer by layer on a platform using computer control. The term 3D printing technology was originally used to describe the process of depositing layers of raw material powder onto a platform via inkjet ejection under the action of a binder. In recent years, commercialization of cost-effective 3D printers has expanded the application range of this technology into the industries of architecture, art, automotive, biomedical, education, fashion, toys, etc. In the biomedical field, 3D printing is widely used in cell research, drug research, cancer research, medical device development, tissue engineering, and the like. Bioprinting combines 3D printing technology, cell biology and material science, combining a printing platform with a device capable of depositing bio-ink (biological materials are typically filled with active molecules and cells). Biological 3D printing technology enables the fabrication of a variety of biomaterials, such as synthetic or natural polymers as scaffolds, coupled with protein-containing serum, extracellular matrix (ECM), and the culturing of various cells in vitro, including stem cells and somatic cells. By selecting appropriate bioprinting materials and architectures, specific structural, physical, and biological properties can be tailored to mimic natural tissue function and provide the microenvironment required for cell growth, proliferation, and controlled differentiation. Furthermore, the parallel development of medical imaging, CAD and CAM enables tissue engineers to generate bioprinted tissue with a specific geometry of a patient's desired organ using common imaging modalities and reconstruction techniques.

Microfluidic technology, generally referred to as technology and science for manipulating micro-volume fluids in structures of micron and below dimensions, has unique characteristics, interface effects and heat conduction properties of fluids at micro-nano scale. The outstanding performance of the microfluidic prepared fiber shows the huge potential of micro-scale separation, and the appearance of the microfluidic chip further pushes a micro-nano fluidic system to a brand new height. The microfluidic chip can realize high integration of various functions, a microfluidic fiber manufacturing system is a typical representative, complete preparation processes such as sample pretreatment, reaction, multi-component loading and solidification and the like can be integrated on one chip through proper chip design, and the integrated chip is also called as a 'Lab-on-a-chip'. After more than twenty years of development, microfluidic technology has covered many fields such as chemistry, physics, biology, medicine, material science, optics and micro-electro-mechanical systems, and has become an important interdisciplinary subject.

Disclosure of Invention

The invention mainly aims to overcome the defects of the prior art and provide a micro-fluidic chip nozzle suitable for printing a gradient tissue engineering cornea and a biological 3D printing system suitable for constructing the gradient tissue engineering cornea.

In order to achieve the purpose, the invention adopts the following technical scheme:

a micro-fluidic chip nozzle is characterized by comprising a micro-fluidic chip and a nozzle with a double-layer structure, wherein the micro-fluidic chip comprises a micro-fluidic chip substrate layer and a micro-fluidic chip upper layer, a plurality of flow channels and inlets thereof as well as a mixed flow structure flow channel and a micro-fluidic chip outlet which are connected with the flow channels are formed on the micro-fluidic chip substrate layer, the mixed flow structure flow channel is connected with the micro-fluidic chip outlet, micro valves corresponding to the flow channels are formed on the micro-fluidic chip upper layer, the micro valves can be controlled to be opened or closed to control liquid in different flow channels to enter the mixed flow structure flow channel so as to control the concentration and the components of a mixed solution output from the micro-fluidic chip outlet, the nozzle with the double-layer structure comprises an inner micro needle head and a shell which surrounds the outer side of the inner micro needle head, and the inner micro needle head is, the mixed solution passes through inlayer micropipette head input in the shell, the flow state after the mixed solution gets into the nozzle of inlayer micropipette head provides the protection, be provided with cross-linking liquid input port and nozzle outlet on the shell, cross-linking liquid is after letting in the shell, through along the flow of a section length of inlayer micropipette outer wall, from circumference parcel follow the mixed solution that inlayer micropipette head flows out produces gel fibre with the mixed liquid cross-linking to utilize the flow direction of the gel fibre that the fluid focus effect control cross-linking of circumference produced, follow the nozzle outlet flows.

Further:

the shell is of an inverted cone structure, and the cross-linking liquid input port is arranged at the upper part, close to the outlet of the microfluidic chip, of the side wall of the shell.

The micro-fluidic chip is arranged in a vertical mode, the multiple flow channels comprise a main flow channel extending downwards along the vertical direction and at least one side flow channel connected with the main flow channel, the mixed flow structure flow channel is provided with two meandering structures extending downwards along the vertical direction, and the two meandering structures are crossed at least twice in the extending process.

The mixed flow structure flow channel respectively forms an inverted Y-shaped structure flow channel and a Y-shaped structure flow channel at the head end and the tail end, at least two X-shaped structure flow channels are formed in the middle, and the inverted Y-shaped structure flow channel, the at least two X-shaped structure flow channels and the Y-shaped structure flow channel are sequentially connected in series.

The outlet of the microfluidic chip is arranged into a round hole, and the top of the inner layer micro needle head is inserted into the round hole to form liquid sealing connection.

The micro-fluidic chip substrate layer and the micro-fluidic chip upper layer are both prepared from soft PDMS mixed with a curing agent, and the proportion of a main agent of the soft PDMS to the curing agent is 10: 1.

the micro-fluidic chip substrate layer and the micro-fluidic chip upper layer are bonded together through a plasma surface treatment technology.

A biological 3D printing system is provided with the microfluidic chip nozzle, a pipeline for conveying printing materials to the microfluidic chip nozzle, a pump for controlling the flow rate of solution conveyed to each flow channel on the microfluidic chip nozzle, and a circuit system for controlling the opening and closing of each micro valve on the microfluidic chip nozzle.

Further:

the 3D printing system adopts a printing mode of an annular path in the process of constructing the gradient tissue engineering cornea through route planning, and the gel fibers in the nozzles with the double-layer structure are deposited on the mould from inside to outside in a circle until the first layer is printed; after the first layer is printed, the nozzle returns to the center of the mold again, the gel fiber is deposited on the previous layer from inside to outside in a circle by circle on the basis of the first layer in the same way as the previous layer, and the like, until the whole cornea model is printed, the printed gradient tissue engineering cornea is divided into an inner layer, a middle layer and an outer layer according to the types of the contained cells, wherein the middle layer is printed by the gel fiber containing the corneal stroma cells, the inner layer and the outer layer are printed by the gel fiber without the cells, then the inner layer and the outer layer are respectively inoculated with the corneal endothelial cells and the corneal epithelial cells, the printed tissue engineering cornea is divided into an inner circle, a middle circle and an outer circle in the radial direction according to whether the printed tissue engineering cornea contains additional growth factors, wherein when the middle circle is printed, a micro valve of a flow passage of a supply liquid containing the high-concentration growth factors is opened, so that the printed gel fiber contains the high-concentration growth factors, finally, the construction of the gradient tissue engineering cornea with different growth factor concentration gradients in the radial direction and different cell components on each large layer is realized.

A method for printing a gradient tissue engineering cornea by using the biological 3D printing system.

The printed gradient tissue engineering cornea is divided into three layers, namely an inner layer, a middle layer and an outer layer, according to the types of cells contained in the gradient tissue engineering cornea, and is divided into three circles, namely an inner circle, a middle circle, an outer circle, and a middle circle, according to whether additional growth factors are contained or not, in the radial direction, the middle circle is printed by gel fibers containing high-concentration growth factors, the inner circle and the outer circle are printed by gel fibers containing no high-concentration growth factors, the middle layer is printed by gel fibers containing corneal stromal cells, the inner layer and the outer layer are printed by gel fibers containing no cells, then the inner layer and the outer layer are respectively inoculated with corneal endothelial cells and corneal epithelial cells, and the gradient tissue engineering cornea with different growth factor concentration gradients in the radial direction and different cell components in the different layers is formed.

The invention provides a micro-fluidic chip nozzle for biological three-dimensional printing by combining a biological 3D printing technology and a micro-fluidic technology, and the structural design of the micro-fluidic chip nozzle can provide fluid shear stress particularly suitable for printing gradient tissue engineering cornea with a layered structure. The micro-fluidic chip nozzle can accurately print the gradient tissue engineering cornea with a layered structure through the control of a micro-valve, so that the printed cornea has a three-layer structure of an epithelial layer, a matrix layer and an endothelial layer, and each layer can be inoculated with different cells.

The nozzle with the double-layer structure comprises an inner-layer micro-needle head and a shell surrounding the outer side of the inner-layer micro-needle head, wherein the inner-layer micro-needle head is connected with an outlet of a micro-fluidic chip and has a protection effect on the flowing state of a mixed solution after entering the nozzle, a cross-linking liquid inlet and a nozzle outlet are formed in the shell, the mixed solution input from the inner-layer micro-needle head enters the shell, and the cross-linking liquid can completely wrap the mixed solution flowing out of the inner-layer micro-needle head from the circumferential direction after flowing into the shell for a period of time along the length of the inner-layer micro-needle head, so that the cross-linking phenomenon with a good effect is realized, and the flow direction of gel fibers generated by cross-linking is controlled by using.

The microfluidic chip nozzle based on the microfluidic technology provided by the invention also has the following advantages:

1) the method can be used for 3D printing of the gradient tissue engineering cornea, and the gradient tissue engineering cornea can be prepared by stacking the gel fibers on the arc-shaped mold layer by layer in a circle from inside to outside manner through path planning;

2) the manufacturing technology of the microfluidic chip enables the nozzle to be manufactured simply, the weight and the volume of the nozzle are reduced, and the manufacturing cost is reduced;

3) the circulation condition of each side flow channel in the microfluidic chip can be controlled by controlling the valve on the microfluidic chip under the control of a computer, so that the response speed of the microfluidic chip is improved;

4) the time for each component fluid to enter the mixed flow channel can be controlled by controlling the micro valve switch, so that the components of the fiber prepared by the mixed flow channel can be controlled;

5) the upper limit on the amount of component material used in the printing process can be determined by controlling the number of component runners;

6) the multi-component gel fiber can be prepared without replacing the nozzle, and comprises the steps that the multi-component material exists in the same section of fiber at the same time, and the multi-section single-component material exists in a longer section of fiber at the same time;

7) the composition and proportion of the prepared fiber cross-section components can be controlled by controlling the number of component flow channels which flow simultaneously;

8) the increase of the cross-sectional area of the fiber can be achieved by simultaneously feeding and increasing the flow rate of the component flow path into the mixed flow path.

The invention combines the micro-fluidic chip technology and the 3D printing technology, fully utilizes the characteristic of continuity and stability when the micro-fluidic chip is used for preparing the fiber, combines the control valve in the invention, realizes the preparation of the fiber of various materials at the same outlet by multi-component regulation in the same hardware, and can accurately and flexibly prepare the gel fiber with different components in real time. The characteristics of simple operation, low cost and flexibility of the 3D printing technology are fully utilized, the fibers formed by the various component materials are directly printed on the mold for constructing the tissue engineering cornea, and the real-time and accurate construction of the multi-layer and multi-component gradient tissue engineering cornea is realized.

According to the embodiment of the invention, the micro-fluidic chip nozzle is used for preparing the tissue engineering cornea, the material of each section of fiber can be continuously switched according to the component corresponding to the position of the cornea in the printing process, the tissue engineering cornea can be prepared at one time by using a 3D printing technology, the nozzle does not need to be replaced in the whole printing process, the interruption is not needed, and the multi-layer and multi-component gradient tissue engineering cornea can be completed at one time.

The invention is realized based on the micro-fluidic chip technology and the 3D printing technology, is simple and easy to implement, has low cost and obvious effect, and has excellent advantages and commercial background.

The nozzle based on the micro-fluidic chip technology has the excellent characteristics of simple operation, wide material selection, higher manufacturing flexibility, high accuracy and the like, and provides important basis and premise for realizing the preparation of gradient tissue engineering cornea by the micro-fluidic chip nozzle.

Drawings

Fig. 1 is a schematic diagram of a micro-fluidic chip nozzle for preparing a gradient tissue engineering cornea according to an embodiment of the invention.

Fig. 2 is an exploded view of a microfluidic chip capable of controlling the composition of a printing material and a nozzle of a double-layer structure for generating gel fibers by cross-linking according to an embodiment of the present invention.

Fig. 3 is a schematic diagram of fiber preparation, radial variation of monolayer growth factor concentration gradient, and layer-by-layer deposition process of tissue engineered cornea in a method for controlling a multi-component fiber material through a micro-valve using a micro-fluidic chip nozzle according to an embodiment of the present invention.

FIG. 4 is a schematic diagram of a multi-layer cell and growth factor concentration structure of a gradient tissue engineered cornea according to an embodiment of the present invention.

Fig. 5 is a schematic structural diagram of a microfluidic chip in an embodiment of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.

Referring to fig. 1 to 5, in an embodiment, a microfluidic chip nozzle includes a microfluidic chip and a nozzle 2 having a double-layer structure, the microfluidic chip includes a microfluidic chip substrate layer 10 and a microfluidic chip upper layer 4, a plurality of

flow channels

5 and 8 and inlets 6 thereof are formed on the microfluidic chip substrate layer 10, a mixed flow structure flow channel 9 connecting the plurality of

flow channels

5 and 8 and a microfluidic chip outlet are formed on the microfluidic chip substrate layer 10, the mixed flow structure flow channel 9 is connected to the microfluidic chip outlet, a micro valve 7 corresponding to the plurality of flow channels is formed on the microfluidic chip upper layer 4, the micro valve 7 can be controlled to open or close to control liquids of different flow channels to enter the mixed flow structure flow channel 9, so as to control the concentration and the composition of a mixed solution output from the microfluidic chip outlet, the nozzle 2 having the double-layer structure includes an inner-layer micro needle 3 and a housing 11 surrounding the outer side of the inner-layer micro needle 3, the inner-layer micro needle head 3 is connected with the outlet of the micro-fluidic chip, a mixed solution is input into the shell 11 through the inner-layer micro needle head 3, the inner-layer micro needle head 3 has a protection effect on the flowing state of the mixed solution after entering the nozzle, the shell 11 is provided with a cross-linking liquid input port and a nozzle outlet, the cross-linking liquid can completely wrap the mixed solution flowing out of the inner-layer micro needle head 3 from the circumferential direction after being introduced into the shell 11 and flows along a section of length of the inner-layer micro needle head 3, the cross-linking liquid input from the cross-linking liquid input port is further mixed in the shell 11, so that the cross-linking phenomenon with a good effect is realized, the flow direction of gel fibers generated by cross-linking is controlled by the circumferential fluid focusing phenomenon, and the gel fibers finally flow out of. The structural design of the micro-fluidic chip nozzle can provide fluid shear stress suitable for printing the gradient tissue engineering cornea with the layered structure, and the gradient tissue engineering cornea with the layered structure can be accurately printed by using the micro-fluidic chip nozzle of the invention through controlling a micro valve.

Referring to fig. 1 and 2, in a preferred embodiment, the housing 11 has an inverted cone-shaped structure, and the cross-linking liquid inlet is disposed at an upper portion of a sidewall of the housing 11 near an outlet of the microfluidic chip.

Referring to fig. 1, 2 and 5, in a preferred embodiment, the microfluidic chip is arranged vertically, the multiple flow channels include a main flow channel extending downward in a vertical direction and at least one side flow channel connected to the main flow channel, the mixed flow structure flow channel 9 has two meandering structures extending downward in the vertical direction, and the two meandering structures meet at least twice during the extending process.

Referring to fig. 1, 2 and 5, in a more preferred embodiment, the mixed flow structure channel 9 forms an inverted Y-shaped structure channel and a Y-shaped structure channel at the head and tail ends, respectively, and forms at least two X-shaped structure channels in the middle, and the inverted Y-shaped structure channel, the at least two X-shaped structure channels and the Y-shaped structure channel are connected in series in sequence.

The mixed flow structure according to the preferred embodiment can destroy the laminar state of the fluid in the microfluidic chip, so that the solution is fully mixed before flowing out of the flow channel, and the mixed flow structure has high mixing efficiency and good effect. The mixed flow liquid is fully mixed and then reacts with the cross-linking liquid to generate high-quality gel fibers, which is beneficial to improving the quality of printing the gradient tissue engineering cornea.

In a preferred embodiment, the microfluidic chip outlet is provided as a circular hole, and the top of the inner layer micro-needle 3 is inserted into the circular hole and forms a liquid-tight connection.

In a preferred embodiment, the microfluidic chip substrate layer 10 and the microfluidic chip upper layer 4 are both prepared from soft PDMS mixed with a curing agent, and the ratio of the main agent of the soft PDMS to the curing agent is 10: 1.

in a preferred embodiment, the microfluidic chip base layer 10 and the microfluidic chip upper layer 4 are bonded together by a plasma surface treatment technique.

In another embodiment, the biological 3D printing system comprises the microfluidic chip nozzle, a pipeline (not shown) for delivering printing materials to the microfluidic chip nozzle, a pump (not shown) for controlling the flow rate of the solution delivered to each flow channel on the microfluidic chip nozzle, and a circuit system (not shown) for controlling the opening and closing of each micro valve on the microfluidic chip nozzle.

Referring to fig. 1, 3 and 4, in a preferred embodiment, the 3D printing system deposits the gel fibers from the two-layer nozzle onto the mold from inside to outside in a circle by a printing manner of a circular path in the process of constructing the gradient tissue engineering cornea through route planning until the first layer 12 is printed. After the first layer 12 is printed, the nozzle returns to the center of the mold again, and the gel fiber in the nozzle with the double-layer structure is deposited on the previous layer from inside to outside in the same way as before on the basis of the first layer 12, and so on, and the printing is carried out in a laminating way until the whole cornea model is printed. The printed gradient tissue engineering cornea is divided into an inner layer, a middle layer and an

outer layer

15, 14 and 16 according to the cell types, wherein the middle layer 14 is printed by gel fibers containing corneal stroma cells, and the inner layer 15 and the outer layer 16 are printed by gel fibers without cells, and then the corneal endothelium cells and the corneal epithelium cells are respectively inoculated. Meanwhile, the printed tissue engineered cornea is divided into an inner, a middle and an outer large circles in the radial direction according to whether additional growth factors are contained or not, wherein in printing the middle large circle 17, a micro valve for controlling a flow path of the supply liquid containing the growth factors with high concentration is opened, so that the printed fiber contains the growth factors with high concentration. Finally, gradient tissue engineering cornea construction with different growth factor concentration gradients 13 in the radial direction and different cell components on each large layer 14-16 is realized.

In another embodiment, as shown in fig. 3 and 4, a 3D printed gradient tissue engineered cornea is spatially divided into three layers, inner, middle and outer, according to the type of cells involved, and three circles, inner, middle and outer, according to whether additional growth factors are involved or not, the middle large ring 17 is printed by gel fibers containing high-concentration growth factors, the inner and outer large rings are printed by gel fibers containing no high-concentration growth factors, the middle large layer 14 is printed by gel fibers containing corneal stromal cells, the inner and outer

large layers

15 and 16 are printed by gel fibers containing no cells, then the inner and outer

large layers

15 and 16 are respectively inoculated with corneal endothelial cells and corneal epithelial cells to form a gradient tissue engineering cornea with different growth factor concentration gradients in the radial direction and different cell components on the different large layers.

Specific embodiments of the present invention are further described below with reference to the accompanying drawings.

Referring to fig. 1 to 5, in one embodiment, a microfluidic chip nozzle for printing gradient tissue engineering cornea includes a microfluidic chip base layer 10, a microfluidic chip upper layer 4 and a double-layer structure nozzle 2. A main flow channel 5, a plurality of side flow channels 8, inlets 6 thereof, a mixed flow structure flow channel 9 for connecting the various flow channels and fully mixing different solutions and an outlet are formed on the micro-fluidic chip substrate layer 10. The mixed flow structure 9 can destroy the laminar state of the fluid in the microfluidic chip so as to fully mix the solution before flowing out of the flow channel. The micro valve 7 corresponding to the side flow channels is formed on the upper layer 4 of the micro-fluidic chip, the micro valve 7 controls the flow and the closing of the flow channels through computer programming so as to control the liquid with different concentration components to enter the main flow channel 5, thereby controlling the concentration and the components of the solution of the object prepared by the mixed flow channel 9 and entering the inner layer micro needle head 3 of the double-layer structure nozzle. The double-layer structure nozzle 2 comprises an inner layer micro needle 3 and a shell 11 which can be filled with cross-linking liquid, and the cross-linking liquid is continuously filled into the shell 11 to be cross-linked with the solution of the inner layer micro needle 3, so that gel fiber with a specific concentration component is obtained and flows out from the outlet of the spray head.

In a preferred embodiment, the fluid outlets of the microfluidic chip substrate layer 10 and the microfluidic chip upper layer 4 are made into round holes by using a 0.5mm puncher, and the inner micro-needles 3 of the double-layer nozzle 2 are inserted into the round holes at the outlets and sealed by using glue.

In a preferred embodiment, the microfluidic chip substrate layer 10 and the microfluidic chip upper layer 4 are both prepared from soft PDMS mixed with a hardening agent.

In a preferred embodiment, the ratio of the base and curing agents of the soft PDMS is 10: 1.

in a preferred embodiment, the microfluidic chip substrate layer 10 and the microfluidic chip upper layer 4 are manufactured by using a mold, and the flow channel is manufactured by a soft lithography method or nanoimprint.

In a preferred embodiment, the microfluidic chip substrate layer 10 and the microfluidic chip upper layer 4 are bonded together by a plasma surface treatment technique.

In a preferred embodiment, the outer diameter of the inner layer micro needle 3 is 0.85mm, the inner diameter is 0.5mm, the material is 316 stainless steel, the double-layer structure nozzle 2 structure is composed of a side inlet flow channel and a conical shell 11, a liquid capable of generating cross-linking with the outlet solution of the microfluidic chip is introduced into the side inlet flow channel, and the cross-linking is generated at the outlet of the inner layer micro needle to generate gel fiber.

In another embodiment, the 3D printing system applied to the gradient tissue engineering cornea comprises the microfluidic chip, the nozzle with the double-layer structure, a channel inlet 6 for filling solution into the chip and the nozzle, a numerical control injection pump for controlling the flow rate of the solution in each channel, and a circuit system for controlling the opening and closing of the micro valve 7.

In a preferred embodiment, the 3D printing system deposits the gel fibers from the nozzle of the double-layer structure onto the mold from inside to outside in circles by a printing manner of a circular path in the process of constructing the gradient tissue engineering cornea through route planning until the first layer 12 is printed. After the first layer 12 is printed, the nozzle returns to the center of the mold again, and the gel fiber in the nozzle with the double-layer structure is deposited on the previous layer from inside to outside in the same way as before on the basis of the first layer 12, and so on, and the printing is carried out in a laminating way until the whole cornea model is printed. The printed gradient tissue engineering cornea is divided into an inner layer, a middle layer and an

outer layer

In a preferred embodiment, the gradient tissue engineered cornea 1 can be used for replacing the ocular surface of a rabbit, carrying out related animal experiments, and screening and toxicity testing of drugs.

In a preferred embodiment, the gradient tissue engineering cornea 1 can be used for researching the generation and development processes of corneal related pathological diseases.

The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention.

Claims

1. A method for constructing a gradient tissue engineering cornea by using a biological 3D printing system, wherein the biological 3D printing system comprises a micro-fluidic chip nozzle for printing gel fibers, a pipeline for conveying printing materials to the micro-fluidic chip nozzle, a pump for controlling the flow rate of solutions conveyed to each flow channel on the micro-fluidic chip nozzle, and a circuit system for controlling the opening and closing of each micro valve on the micro-fluidic chip nozzle, and is characterized in that the micro-fluidic chip nozzle comprises a micro-fluidic chip and a nozzle with a double-layer structure, the micro-fluidic chip comprises a micro-fluidic chip base layer and a micro-fluidic chip upper layer, a plurality of flow channels and inlets thereof, mixed flow structure flow channels connected with the plurality of flow channels and micro-fluidic chip outlets are formed on the micro-fluidic chip base layer, and the mixed flow structure flow channels are connected with the micro-fluidic chip, the micro valve corresponding to the multiple flow channels is formed on the upper layer of the microfluidic chip, the micro valve can be controlled to be opened or closed to control the liquid of different flow channels to enter the flow channel of the mixed flow structure, so as to control the concentration and the components of the mixed solution output from the outlet of the microfluidic chip, the nozzle of the double-layer structure comprises an inner-layer micro needle head and a shell surrounding the outer side of the inner-layer micro needle head, the inner-layer micro needle head is connected with the outlet of the microfluidic chip, the mixed solution is input into the shell through the inner-layer micro needle head, the inner-layer micro needle head provides a protection effect on the flowing state of the mixed solution after entering the nozzle, and the shell is provided with a cross-linked liquid input port and a nozzle outlet; the method for constructing the gradient tissue engineering cornea comprises the following printing processes: after the cross-linking liquid is introduced into the shell, the cross-linking liquid flows along a section of length of the outer wall of the inner-layer micro-needle, the mixed solution flowing out of the inner-layer micro-needle is wrapped in the circumferential direction, is cross-linked with the mixed liquid to generate gel fibers, the flow direction of the gel fibers generated by cross-linking is controlled by utilizing the circumferential fluid focusing effect, the gel fibers flow out of the nozzle outlet, and fluid shear stress suitable for printing a gradient tissue engineering cornea with a layered structure is generated, the biological 3D printing system deposits the gel fibers in the nozzle with the double-layer structure on a mold from inside to outside in a circle by adopting a printing mode of an annular path in the process of constructing the gradient tissue engineering cornea until a first layer is printed; after the first layer is printed, the nozzle returns to the center of the mold again, the gel fiber is deposited on the previous layer from inside to outside in a circle by circle on the basis of the first layer in the same way as the previous layer, and the like, until the whole cornea model is printed, the printed gradient tissue engineering cornea is divided into an inner layer, a middle layer and an outer layer according to the types of the contained cells, wherein the middle layer is printed by the gel fiber containing the corneal stroma cells, the inner layer and the outer layer are printed by the gel fiber without the cells, then the inner layer and the outer layer are respectively inoculated with the corneal endothelial cells and the corneal epithelial cells, the printed tissue engineering cornea is divided into an inner circle, a middle circle and an outer circle in the radial direction according to whether the printed tissue engineering cornea contains additional growth factors, wherein when the middle circle is printed, a micro valve of a flow passage of a supply liquid containing the high-concentration growth factors is opened, so that the printed gel fiber contains the high-concentration growth factors, finally, the construction of the gradient tissue engineering cornea with different growth factor concentration gradients in the radial direction and different cell components on each large layer is realized.

2. The method of gradient tissue engineering cornea of claim 1, wherein the housing is an inverted cone-shaped structure, and the cross-linking liquid input port is disposed at an upper portion of a side wall of the housing near the outlet of the microfluidic chip.

3. The method of gradient tissue engineering cornea of claim 1 or 2, wherein the microfluidic chip is vertically disposed, the plurality of flow channels include a main flow channel extending downward in a vertical direction and at least one side flow channel connected to the main flow channel, the mixed flow structure flow channel has two meandering structures extending downward in the vertical direction, and the two meandering structures meet at least twice during the extending process.

4. The method of gradient tissue engineering cornea of claim 3, wherein the mixed flow structure channel forms an inverted Y-shaped structure channel and a Y-shaped structure channel at the head and tail ends respectively, and forms at least two X-shaped structure channels in the middle, and the inverted Y-shaped structure channel, the at least two X-shaped structure channels and the Y-shaped structure channel are connected in series in sequence.

5. The method of gradient tissue engineering cornea of any one of claims 1 to 2, wherein the outlet of the microfluidic chip is configured as a circular hole, and the top of the inner layer micro needle is inserted into the circular hole and forms a liquid-tight connection.

6. The method of gradient tissue engineering cornea of any one of claims 1 to 2, wherein the base layer of the microfluidic chip and the upper layer of the microfluidic chip are both prepared from soft PDMS mixed with a curing agent, and the ratio of the main agent of the soft PDMS to the curing agent is 10: 1.