A method for constructing a patterned multilayer structure based on immiscible phase-assisted filling

Abstract

The present invention relates to the field of advanced manufacturing technology, and discloses a method for constructing a patterned multilayer structure based on immiscible phase auxiliary filling. The method for constructing a patterned multilayer structure comprises the following steps: S1, dispersing assembly units in a curable aqueous phase to obtain a pre-assembly liquid; S2, adding the pre-assembly liquid to the bottom layer of the assembly chamber, adding an immiscible phase to the upper layer until it is flush with the upper edge of the assembly chamber, applying a standing wave, so that the assembly units form a pattern in the pre-assembly liquid, and curing the pre-assembly liquid to obtain a first structural layer; S3, removing the immiscible phase, adding assembly liquid to the surface of the first structural layer, adding an immiscible phase again until it is flush with the upper edge of the assembly chamber, applying a standing wave, so that the assembly units in the pre-assembly liquid form a pattern, and curing the pre-assembly liquid to obtain a second structural layer; S4, repeating step S3 to obtain a patterned multilayer structure.

Description

Technical Field

[0001] The present invention relates to the field of advanced manufacturing technology, and in particular to a method for constructing a patterned multilayer structure based on immiscible phase auxiliary filling. Background Art

[0002] Acoustic bioassembly refers to the use of acoustic waves to assemble pre-formed modular units (including cells or cell aggregates, cells and biomaterial complexes) to create biomimetic multi-level structures. As an emerging bioassembly technology, acoustic bioassembly has many advantages and great application potential.

[0003] As a type of sound wave, Faraday wave is defined as a nonlinear standing wave caused by the acceleration generated by the vertical oscillation of the liquid layer. The Faraday wave bio-assembly method can realize the assembly of modules in the field within a few seconds through the acoustic radiation force or fluid force generated by sound waves. It has the characteristics of non-contact, non-invasive, and efficient molding, which can minimize damage to cells and show unique technical advantages and huge application potential in the engineering construction of tissues and organs.

[0004] The Faraday wave multilayer assembly method refers to using the solidified upper layer as the base for assembling the next layer, and constructing it layer by layer to obtain a multilayer structure. However, during the Faraday wave assembly process, the assembly chamber must be filled with the assembly liquid, that is, the liquid level of the assembly liquid must be flush with the outer edge of the assembly chamber, in order to achieve the driving of the assembly unit located at the solid-liquid interface. Therefore, the thickness of a single layer in the existing Faraday wave assembly depends on the thickness of the chamber, so the precise control of the thickness of each layer depends on the manufacturing process of the assembly chamber, resulting in the thickness of each layer being difficult to control, low precision, and large interlayer spacing. In addition, the thickness of the assembly chamber of the existing Faraday wave assembly is concentrated in the range of 1.0-4.5 mm, so the thickness of the single-layer structure prepared by the manufacturing process of the assembly chamber is generally greater than 1.0 mm. CN 114603846 A discloses a method for preparing a three-dimensional multilayer structure. The method forms a first layer structure in a first assembly chamber, transfers the first layer structure to another assembly chamber with a deeper depth as a bottom layer, and constructs a second layer structure on the surface of the first layer structure. The steps are repeated to achieve the preparation of the three-dimensional multilayer structure. Although the method can make the thickness of each layer independently 0.5-5 mm, it actually still depends on the manufacturing process of the assembly chamber. During the preparation process, chambers of different depths need to be continuously replaced, and the manufacturing process has high requirements, the steps are complicated, and the manufacturing cost is high. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, one of the purposes of the present invention is to provide a method for constructing a patterned multilayer structure based on immiscible phase auxiliary filling; the second purpose of the present invention is to provide an application of the method for constructing a patterned multilayer structure.

[0006] The basic principle of the present invention is described as follows:

[0007] The present invention utilizes the characteristics that the immiscible phase and the curable aqueous phase are immiscible and non-reactive, disperses the assembly units in the curable aqueous phase to form a pre-assembly liquid, adds the pre-assembly liquid into the assembly chamber as the bottom layer according to the desired single layer thickness, adds the immiscible phase into the upper layer to fill the assembly chamber, and adjusts the volume of the immiscible phase and the aqueous phase to achieve the regulation of the single layer thickness of the structural layer; applies standing waves to form a pattern of the assembly units in the lower aqueous phase assembly liquid, and solidifies the lower layer under specific curing conditions to form a structural layer, while the upper immiscible phase does not change, and after taking out and cleaning the surface of the structural layer, the immiscible phase remaining, and the next structural layer can be directly constructed on its surface, and the immiscible phase can be reused; repeating the above steps can obtain a patterned multilayer structure with controllable single layer thickness, small interlayer spacing and high precision in the same assembly chamber.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a method for constructing a patterned multilayer structure based on immiscible phase-assisted filling, comprising the following steps:

[0010] S1, dispersing the assembly unit in a curable aqueous phase to obtain a pre-assembly liquid;

[0011] S2, adding the pre-assembly liquid to the bottom layer of the assembly chamber, adding the immiscible phase to the upper layer until it is flush with the upper edge of the assembly chamber, applying standing waves to make the assembly units form patterns in the pre-assembly liquid, and curing the pre-assembly liquid to obtain a first structural layer;

[0012] S3, removing the immiscible phase, adding a pre-assembly liquid to the surface of the first structural layer, adding the immiscible phase again until it is flush with the upper edge of the assembly chamber, applying a standing wave to form a pattern of assembly units in the pre-assembly liquid, and curing the pre-assembly liquid to obtain a second structural layer;

[0013] S4, repeating step S3 to obtain a patterned multilayer structure;

[0014] The immiscible phase is a liquid that is immiscible with water and does not chemically react with water.

[0015] In some embodiments of the present invention, the curable aqueous phase is selected from one of a photocurable liquid, a thermosensitive hydrogel, a thrombin and prothrombin system, and an ionically cross-linked hydrogel.

[0016] In some embodiments of the present invention, the photocurable liquid comprises a methacrylic anhydride-modified biomaterial.

[0017] In some specific embodiments of the present invention, the photocurable liquid is selected from at least one of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), and methacrylated extracellular matrix.

[0018] In some embodiments of the present invention, the curing condition of the photocurable liquid is irradiation with 400-410 nm blue light for 10-120 s.

[0019] In some embodiments of the present invention, the assembly units are micron-sized particles that are water-insoluble and do not react with the curable aqueous phase.

[0020] In some specific embodiments of the present invention, the assembly unit is selected from at least one of polypropylene microspheres, chitosan powder, cell spheres, cell-loaded microspheres, and microgels.

[0021] In some embodiments of the present invention, in the pre-assembly solution, the concentration of the assembly unit is 20-50 mg / mL.

[0022] In some specific embodiments of the present invention, in the pre-assembly solution, the concentration of the assembly unit is 20-40 mg / mL.

[0023] In some embodiments of the present invention, the immiscible phase is selected from one of an oil phase and a hydrophobic organic solvent.

[0024] In some specific embodiments of the present invention, the oil phase is selected from at least one of vegetable oil, animal oil, fluorinated oil and mineral oil.

[0025] In some specific embodiments of the present invention, the hydrophobic organic solvent includes chloroform, carbon tetrachloride, benzene, toluene, xylene, n-hexanol, octanol, and petroleum ether.

[0026] In some embodiments of the present invention, the immiscible phase further includes an emulsifier; the content of the emulsifier is 0.3-10% (w / v).

[0027] In some specific embodiments of the present invention, the emulsifier includes Span 80.

[0028] In some embodiments of the present invention, the frequency of the standing wave is 10-500 Hz.

[0029] In some specific embodiments of the present invention, the frequency of the standing wave is 20-100 Hz.

[0030] In some embodiments of the present invention, the standing wave includes at least one of a linear standing wave and a nonlinear standing wave.

[0031] In some embodiments of the present invention, the waveform of the standing wave includes at least one of a sine wave, a cosine wave, and a square wave.

[0032] In some embodiments of the present invention, the applied standing wave drives the pre-assembly liquid to vibrate vertically up and down.

[0033] In some embodiments of the present invention, before applying the standing wave, the assembly unit is deposited at the bottom of the pre-assembly liquid.

[0034] The second aspect of the present invention provides the use of the patterned multilayer structure construction method described in the first aspect of the present invention in the preparation of tissue and organ models and tissue repair materials.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1) The method for constructing a patterned multilayer structure based on immiscible phase-assisted filling provided by the present invention can achieve precise control of the thickness of a single layer by adjusting the amount of the immiscible phase and the aqueous phase of the pre-assembly liquid added; the assisted filling of the immiscible phase is used to realize the assembly of a multilayer patterned structure in the same assembly chamber, and the method is simple to operate without the need to stack or adjust the assembly chamber;

[0037] 2) The method for constructing a patterned multilayer structure based on immiscible phase-assisted filling provided by the present invention can reuse the immiscible phase, and significantly reduces the usage of expensive assembly fluids such as methacrylylated gelatin and thrombin, thereby greatly reducing the amount of raw materials and manufacturing costs;

[0038] 3) The patterned multilayer structure provided by the present invention can achieve a single layer thickness of 0.4 mm, which overcomes the problem of too large spacing between two adjacent layers of assembly patterns in the prior art and significantly improves the manufacturing accuracy of the standing wave multilayer assembly;

[0039] 4) The method for constructing a patterned multilayer structure based on immiscible phase-assisted filling provided by the present invention has simple steps, low cost, and high precision, and can be applied to the preparation of tissue and organ models and tissue repair materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A technical roadmap of a method for constructing a patterned multilayer structure based on immiscible phase-assisted filling in an embodiment;

[0041] Figure 2 This is a physical picture of the patterned double-layer structure in Example 1;

[0042] Figure 3 This is a physical picture of the patterned double-layer structure in Example 2;

[0043] Figure 4 This is a physical picture of the patterned double-layer structure in Example 3;

[0044] Figure 5 This is a physical picture of the patterned three-layer structure in Example 4;

[0045] Figure 6 It is the thickness of the three-layer concentric circle structure in Example 4. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0047] Figure 1 The technical roadmap of the method for constructing a patterned multilayer structure based on immiscible phase-assisted filling in the embodiment, in which the oil phase is used as the immiscible phase to assist in constructing the patterned multilayer structure, will be referred to below Figure 1 The process of constructing a patterned multilayer structure in the embodiment is described:

[0048] Example 1

[0049] This embodiment constructs a patterned double-layer structure, and the steps are as follows:

[0050] S11, dispersing chitosan powder at a concentration of 25 mg / mL in a methacrylated gelatin solution at a concentration of 8% (w / v), and mixing well to obtain a pre-assembly solution;

[0051] S21, take 500 μL of pre-assembly liquid and add it to an assembly chamber with a diameter of 20 mm and a thickness of 3.0 mm, and add about 500 μL of peanut oil to which 0.5% (w / v) Siban 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber; after the chitosan settles to the bottom of the assembly chamber, it is driven to move by a standing wave with a frequency of 20 Hz to form a cross patterned structure, and the methacrylylated gelatin aqueous solution is solidified into a gel by irradiating with blue light with a wavelength of 405 nm for 100 s to obtain the first structural layer;

[0052] S31. Aspirate the oil phase, clean the residual oil phase on the surface of the first structural layer with deionized water, and continue to add about 500 μL of pre-assembly liquid, that is, a methacryloyl gelatin aqueous solution in which chitosan is evenly dispersed, into the assembly chamber, so that the aqueous phase interface is flush with the upper edge of the assembly chamber. After the chitosan is precipitated to the surface of the first structural layer, it is driven by a standing wave with a frequency of 20 Hz to move to form a cross patterned structure. The methacryloyl gelatin aqueous solution is solidified into a gel by irradiating it with blue light with a wavelength of 405 nm for 100 s to obtain a double-layer cross structure.

[0053] Example 2

[0054] This embodiment constructs a patterned double-layer structure, and the steps are as follows:

[0055] S11, dispersing chitosan powder at a concentration of 25 mg / mL in a methacrylated gelatin solution at a concentration of 8% (w / v), and mixing well to obtain a pre-assembly solution;

[0056] S21, take 500 μL of pre-assembly liquid and add it to an assembly chamber with a diameter of 20 mm and a thickness of 3.0 mm, and add about 500 μL of peanut oil to which 0.5% (w / v) Siban 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber; after the chitosan settles to the bottom of the assembly chamber, it is driven to move by a standing wave with a frequency of 25 Hz to form a concentric circle patterned structure, and the methacrylated gelatin aqueous solution is solidified into a gel by irradiating with blue light with a wavelength of 405 nm for 100 s to obtain the first structural layer;

[0057] S31. Aspirate the oil phase, clean the residual oil phase on the surface of the first structural layer with deionized water, and continue to add about 500 μL of pre-assembly liquid, that is, a methacryloyl gelatin aqueous solution in which chitosan is evenly dispersed, into the assembly chamber, so that the aqueous phase interface is flush with the upper edge of the assembly chamber. After the chitosan is precipitated to the surface of the first structural layer, it is driven by a standing wave with a frequency of 25 Hz to move to form a concentric circle patterned structure. The methacryloyl gelatin aqueous solution is solidified into a gel by irradiating it with blue light with a wavelength of 405 nm for 100 s to obtain a double-layer concentric circle structure.

[0058] Example 3

[0059] This embodiment constructs a patterned double-layer structure, and the steps are as follows:

[0060] S11, dispersing chitosan powder at a concentration of 25 mg / mL in a methacrylated gelatin solution at a concentration of 8% (w / v), and mixing well to obtain a pre-assembly solution;

[0061] S21, take 500 μL of pre-assembly liquid and add it to an assembly chamber with a diameter of 20 mm and a thickness of 3.0 mm, and add about 500 μL of peanut oil to which 0.5% (w / v) Siban 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber; after the chitosan settles to the bottom of the assembly chamber, it is driven to move by a standing wave with a frequency of 40 Hz to form a petal-shaped patterned structure, and the methacrylated gelatin aqueous solution is solidified into a gel by irradiating with blue light with a wavelength of 405 nm for 100 s to obtain the first structural layer;

[0062] S31. Aspirate the oil phase, clean the residual oil phase on the surface of the first structural layer with deionized water, and continue to add about 500 μL of pre-assembly liquid, that is, a methacryloyl gelatin aqueous solution in which chitosan is evenly dispersed, into the assembly chamber, so that the aqueous phase interface is flush with the upper edge of the assembly chamber. After the chitosan is precipitated to the surface of the first structural layer, it is driven by a standing wave with a frequency of 40 Hz to move to form a petal-shaped patterned structure. The methacryloyl gelatin aqueous solution is solidified into a gel by irradiating it with blue light with a wavelength of 405 nm for 100 s to obtain a double-layer petal-shaped structure.

[0063] Example 4

[0064] This embodiment constructs a patterned three-layer structure, and the steps are as follows:

[0065] S11, dispersing chitosan powder at a concentration of 30 mg / mL in a methacryloyl gelatin solution at a concentration of 8% (w / v), mixing well, and obtaining a pre-assembly solution;

[0066] S21, take 150 μL of pre-assembly liquid and add it to an assembly chamber with a diameter of 20 mm and a thickness of 3.0 mm, and add about 850 μL of peanut oil to which 0.5% (w / v) Siban 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber; after the chitosan settles to the bottom of the assembly chamber, it is driven to move by a standing wave with a frequency of 45 Hz to form a concentric circle patterned structure, and the methacrylylated gelatin aqueous solution is solidified into a gel by irradiating it with blue light with a wavelength of 405 nm for 100 s to obtain the first structural layer;

[0067] S31, aspirate the oil phase, clean the residual oil phase on the surface of the first structural layer with deionized water, continue to add about 150 μL of pre-assembly liquid, i.e., a methacryloyl gelatin aqueous solution in which chitosan is evenly dispersed, into the assembly chamber, and then add about 700 μL of peanut oil to which 0.5% (w / v) Siban 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber, and after the chitosan settles to the surface of the first structural layer, it is driven by a standing wave with a frequency of 45 Hz to move to form a concentric circle patterned structure, and irradiated with blue light with a wavelength of 405 nm for 100 s to solidify the methacryloyl gelatin aqueous solution into a gel, thereby obtaining a second structural layer;

[0068] S41. Aspirate the oil phase, and use deionized water to clean the residual oil phase on the surface of the second structural layer. Continue to add about 150 μL of pre-assembly liquid, that is, a methacryloyl gelatin aqueous solution in which chitosan is evenly dispersed, into the assembly chamber. Then, add about 550 μL of peanut oil to which 0.5% (w / v) Span 80 has been added, so that the oil phase interface is flush with the upper edge of the assembly chamber. After the chitosan is precipitated to the surface of the second structural layer, it is driven to move by a standing wave with a frequency of 45 Hz to form a concentric circle patterned structure. The methacryloyl gelatin aqueous solution is solidified into a gel by irradiating it with blue light with a wavelength of 405 nm for 100 s to obtain a three-layer concentric circle structure.

[0069] Figure 2 This is a physical picture of the patterned double-layer structure in Example 1, wherein: Figure 2 (a) is a physical picture of the first structural layer in Example 1, Figure 2 (b) is a physical picture of the double-layer cross structure in Example 1; Figure 3 This is a physical picture of the patterned double-layer structure in Example 2, wherein: Figure 3 (a) is a physical picture of the first structural layer in Example 2, Figure 3 (b) is a physical picture of the double-layer concentric circle structure in Example 2; Figure 4 This is a physical picture of the patterned double-layer structure in Example 3, wherein: Figure 4 (a) is a physical picture of the first structural layer in Example 3, Figure 4 (b) is a physical picture of the double-layer petal structure in Example 3; Figure 5 This is a physical picture of the patterned three-layer structure in Example 4, wherein: Figure 5 (a) is a physical picture of the first structural layer in Example 4, Figure 5 (b) is a physical picture of the second structural layer in Example 4, Figure 5 (c) is a physical picture of the three-layer concentric circle structure in Example 4. Figure 2 , Figure 3 , Figure 4 and Figure 5 It can be seen that each layer of the multi-layer patterned structure constructed by the method provided by the present invention can form a specific pattern. Figure 6 is the thickness of the three-layer concentric circle structure in Example 4, Figure 6 It can be seen that the thickness of the three-layer concentric circle structure in Example 4 is about 1.2 mm, and the thickness of a single layer can reach 0.4 mm.

[0070] The method for constructing a patterned multilayer structure provided by the present invention is based on the auxiliary filling of an oil phase or other water-immiscible phase, and can construct a multilayer patterned structure in the same assembly chamber, and can make the single layer thickness reach 0.4 mm. The construction of double-layer and triple-layer structures is provided in the embodiments, and the assembly of more hierarchical patterned structures can be achieved by adjusting the amount of the immiscible phase and the curable aqueous phase added.

Claims

1. A method for constructing a patterned multilayer structure based on immiscible phase-assisted filling, characterized in that: The following steps are involved: S1, dispersing the assembly unit in a curable aqueous phase to obtain a pre-assembly liquid; S2, adding the pre-assembly liquid to the bottom layer of the assembly chamber, adding the immiscible phase to the upper layer until it is flush with the upper edge of the assembly chamber, applying standing waves to make the assembly units form patterns in the pre-assembly liquid, and curing the pre-assembly liquid to obtain a first structural layer; S3, removing the immiscible phase, adding a pre-assembly liquid on the surface of the first structural layer, adding the immiscible phase again until it is flush with the upper edge of the assembly chamber, applying a standing wave to form a pattern of assembly units in the pre-assembly liquid, and curing the pre-assembly liquid to obtain a second structural layer; S4, repeating step S3 to obtain a patterned multilayer structure; The immiscible phase is a liquid that is immiscible with water and does not chemically react with water.

2. The method for constructing a patterned multilayer structure according to claim 1, characterized in that: The curable aqueous phase is selected from one of a photocurable liquid, a thermosensitive hydrogel, a thrombin and prothrombin system, and an ion-crosslinked hydrogel.

3. The method for constructing a patterned multilayer structure according to claim 2, characterized in that: The photocurable liquid includes a methacrylic anhydride-modified biomaterial.

4. The method for constructing a patterned multilayer structure according to claim 3, characterized in that: The curing condition of the photocurable liquid is 400-410 nm blue light irradiation for 10-120 s.

5. The method for constructing a patterned multilayer structure according to claim 2, characterized in that: The assembly units are micron-sized particles that are water-insoluble and do not react with the curable aqueous phase.

6. The method for constructing a patterned multilayer structure according to claim 1, characterized in that: The immiscible phase is selected from one of an oil phase and a hydrophobic organic solvent.

7. The method for constructing a patterned multilayer structure according to claim 1, characterized in that: The frequency of the standing wave is 10-500 Hz.

8. Use of the method for constructing a patterned multilayer structure according to any one of claims 1 to 7 in preparing tissue and organ models and tissue repair materials.

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