Microfiber arranging and assembling method

Abstract

The invention relates to the technical field of advanced manufacturing, and discloses a microfiber arranging and assembling method. The method comprises the following steps: S1, dispersing microfibers in a curable driving liquid to obtain a pre-assembly liquid; s2, the pre-assembly liquid is placed in an assembly chamber, and standing is performed to enable the microfibers to settle to the bottom of the chamber; s3, applying standing waves to enable the microfibers to be patterned and directionally arranged in the pre-assembly liquid, and curing the pre-assembly liquid; wherein the length-diameter ratio of the microfibers is greater than 2. According to the method, through Faraday wave sound field driving and the synergistic effect of non-contact acoustic radiation force and fluid mechanics, highly ordered arrangement of the microfibers with the length-diameter ratio larger than 2 in the curable driving liquid is achieved, and a method basis is provided for construction of a tissue and organ three-dimensional matrix fiber topological structure.

Description

Technical Field

[0001] This invention relates to the field of advanced manufacturing technology, and in particular to a method for arranging and assembling microfibers. Background Technology

[0002] In the fields of tissue engineering and regenerative medicine, the three-dimensional fibrous topology of the extracellular matrix (ECM) plays a crucial role in regulating cell migration, proliferation, differentiation, and tissue-specific functional expression. How to engineer the construction of natural tissue matrix fibrous topologies is an important topic in these fields. Current researchers have developed various microfiber arrangement and assembly methods, mainly including electrospinning for directional collection, 3D printing for fiber arrangement, microfluidic patterning, and mechanical stretching. However, existing methods generally suffer from low resolution, low assembly efficiency, and contact damage, failing to meet the needs of engineering natural tissue matrix fibrous topologies.

[0003] Among these technologies, 3D bioprinting utilizes molten electrospinning and near-field electrospinning to directly write fibers by controlling the deposition path of materials (such as polycaprolactone and collagen) using an electric field. However, it faces challenges such as resolution-velocity balance and cell co-printing damage. Electrospinning-guided collection involves stretching a polymer solution using a high-voltage electrostatic field to form submicron-sized fibers, which are then axially aligned using a rotating receiver. However, the high electric field environment (typically >1kV / cm) and residual organic solvents significantly inhibit cell activity, making it difficult to integrate living cells and construct cell-fiber synergistic systems. Microfluidic patterning uses laminar shear forces within microchannels to induce fiber orientation. However, the fixed geometry of the microchannels results in insufficient pattern flexibility, and fiber alignment efficiency decreases at low flow rates (flux <0.1mL / min), making it difficult to meet the needs of large-scale tissue construction. Mechanical stretching involves pre-stretching and relaxing the substrate or using an external stress field to force fibers to orient along the tension direction. However, the local strain (often >10%) generated during this process can disrupt cell membrane integrity and induce activation of apoptosis-related pathways.

[0004] Acoustic bioassembly, as an emerging non-contact manipulation technology, utilizes acoustic driving forces (such as acoustic radiation forces generated by standing wave fields) to achieve precise spatial arrangement of particulate assembly units (microspheres, cell spheres, etc.) at the micrometer scale. Its principle is based on the interaction between sound waves and particles, causing particles to directionally aggregate at acoustic pressure nodes or anti-nodes, offering advantages such as high assembly efficiency and no contact damage. Acoustic bioassembly technology has been used to construct patterned structures formed by the spatially specific aggregation of assembly units. For example, Faraday wave bioassembly drives the aggregation of cell-carrying microspheres to form concentric circles, petal-like patterns, and lattice-like patterns, further inducing the formation of biomimetic tissues and organs (Pu Chen, et al. Adv. Mater. 2014, 26, 5936–5941). However, research on the orientation and arrangement of the assembly units themselves in acoustic bioassembly is currently lacking. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Through in-depth analysis of the acoustic field potential energy distribution and the stress conditions of the assembly units, it can be predicted that under suitable process parameters, acoustic field bio-assembly can simultaneously drive the spatially specific aggregation of assembly units to form patterned structures, while achieving the oriented alignment and assembly of specific assembly units such as microfibers. Combining the advantages of acoustic field bio-assembly technology, such as its mildness and high efficiency, this innovative microfiber alignment and assembly method has promising application prospects in constructing natural tissue matrix fiber topologies. Therefore, one of the objectives of this invention is to provide a microfiber alignment and assembly method.

[0006] The second objective of this invention is to provide an application of this microfiber arrangement and assembly method.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a method for arranging and assembling microfibers, comprising the following steps:

[0009] S1. Disperse the microfibers in a curable driving liquid to obtain a pre-assembled liquid;

[0010] S2. Place the pre-assembly liquid in the assembly chamber and allow it to stand to allow the microfibers to settle to the bottom of the chamber.

[0011] S3. Apply a standing wave to pattern and orient the microfibers in the pre-assembly liquid, and then solidify the pre-assembly liquid.

[0012] The aspect ratio of the microfiber is greater than 2.

[0013] In some embodiments of the present invention, the concentration of the microfibers in the curable driving liquid is 1-100 mg / mL.

[0014] In some preferred embodiments of the present invention, the concentration of the microfibers in the curable driving liquid is 1-10 mg / mL.

[0015] In some embodiments of the present invention, the length of the microfiber is in the hundreds of nanometers, micrometers or millimeters.

[0016] In some preferred embodiments of the present invention, the length of the microfiber is on the micrometer or sub-millimeter scale.

[0017] In some preferred embodiments of the present invention, the microfibers have a length of 10-500 μm and a diameter of 3-50 μm.

[0018] In some embodiments of the present invention, the microfibers include at least one of natural fibers, naturally derived fibers, synthetic organic fibers, carbon-based fibers, and ceramic fibers.

[0019] In some preferred embodiments of the present invention, the microfibers are selected from at least one of collagen fibers, silk fibroin fibers, cellulose fibers, potassium titanate whiskers, polylactic acid fibers, and polycaprolactone fibers.

[0020] In some embodiments of the present invention, the curable driving fluid is selected from one of photocurable liquids, thermosensitive hydrogels, fibrinogen and thrombin systems.

[0021] In some embodiments of the present invention, the components of the photocurable liquid include a photocurable substrate, a photoinitiator, and a solvent.

[0022] In some embodiments of the present invention, the concentration of the photoinitiator in the solvent is 0.2%-0.5% (w / v).

[0023] In some preferred embodiments of the present invention, the concentration of the photoinitiator in the solvent is 0.2%-0.3% (w / v).

[0024] In some embodiments of the present invention, the concentration of the photocurable substrate in the photocurable liquid is 5%-10% (w / v).

[0025] In some preferred embodiments of the present invention, the concentration of the photocurable substrate in the photocurable liquid is 5%-8% (w / v).

[0026] In some embodiments of the present invention, the photocurable substrate comprises methacrylic anhydride-modified biomaterials.

[0027] In some preferred embodiments of the present invention, the methacrylic anhydride-modified biomaterial includes at least one of methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), methacrylamide decellularized matrix (dECMMA), methacrylamide chitosan (CSMA), methacrylamide carboxymethyl chitosan (CMCSMA), methacrylamide sodium alginate (AlgMA), methacrylamide silk fibroin (SilMA), methacrylamide dextran (DexMA), methacrylamide chondroitin sulfate (ChSMA), methacrylamide polylysine (PLMA), and acrylamide RGD peptide (Pep-RGDfKAC).

[0028] In some preferred embodiments of the present invention, the photocurable substrate is selected from at least one of methacrylamide gelatin (GelMA) and methacrylamide hyaluronic acid (HAMA).

[0029] In some embodiments of the present invention, the photoinitiator includes at least one of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (photoinitiator LAP), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone (photoinitiator I2959), and ethyl 2,4,6-trimethylbenzoylphenylphosphonate (photoinitiator TPO-L).

[0030] In some embodiments of the present invention, the solvent is selected from phosphate-buffered saline (PBS), water, or physiological saline.

[0031] In some embodiments of the present invention, the photocurable liquid is prepared by a method comprising the following steps:

[0032] The photoinitiator is heated and dissolved in a solvent, then a photocurable substrate is added, and the solution is heated in the dark to dissolve it, thus obtaining the photocurable liquid.

[0033] In some embodiments of the present invention, the photoinitiator is heated to dissolve at a temperature of 40-50°C for a time of 10-30 minutes.

[0034] In some embodiments of the present invention, the temperature for melting the photocurable substrate by heating in the dark is 60-70°C, and the time is 20-30 minutes.

[0035] In some embodiments of the present invention, the curing conditions of the photocurable liquid are irradiation with 400-410nm blue light for 10-150s.

[0036] In some preferred embodiments of the present invention, the curing conditions of the photocurable liquid are irradiation with 400-410nm blue light for 100-120s.

[0037] In some embodiments of the present invention, the thermosensitive hydrogel includes at least one of decellularized matrix solution, gelatin solution, methacrylamide gelatin, methacrylated decellularized matrix, and matrix gel.

[0038] In some embodiments of the present invention, the depth of the assembly chamber is 0.8-5 mm.

[0039] In some embodiments of the present invention, the shape of the assembly chamber is arbitrary.

[0040] In some embodiments of the present invention, the assembly chamber is made of a material selected from polytetrafluoroethylene (PTFE), glass, polymethyl methacrylate (PMMA), polystyrene (PS), or polydimethylsiloxane (PDMS).

[0041] In some embodiments of the present invention, the pre-assembly liquid is added to the assembly chamber until it is flush with the upper edge of the assembly chamber.

[0042] In some embodiments of the present invention, the pre-assembly liquid is left to stand in the assembly chamber for 10-60 seconds.

[0043] In some embodiments of the present invention, the frequency of the standing wave is 15-300Hz; the amplitude is 50-1500mV.

[0044] In some preferred embodiments of the present invention, the frequency of the standing wave is 45-300Hz.

[0045] In some embodiments of the present invention, the microfibers are oriented in the pre-assembly liquid for 5-60 seconds.

[0046] In some embodiments of the present invention, the standing wave is a nonlinear standing wave.

[0047] In some embodiments of the present invention, the standing wave drives the pre-assembled liquid to vibrate vertically up and down.

[0048] In some embodiments of the present invention, after the microfibers are oriented in the pre-assembly liquid, standing wave vibration is applied for 1-2 minutes.

[0049] The second aspect of the present invention provides the application of the microfiber arrangement and assembly method described in the first aspect of the present invention in simulating the construction of three-dimensional matrix fiber topology of tissues and organs.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] The microfiber arrangement and assembly method provided by this invention, driven by a Faraday wave acoustic field, achieves a highly ordered arrangement of microfibers with a certain aspect ratio (greater than 2) in a solidifiable driving liquid through the synergistic effect of non-contact acoustic radiation force and fluid dynamics. The fiber arrangement pattern can be adjusted in real time by controlling the acoustic field parameters (frequency / amplitude), avoiding physical contact damage and overcoming the defects of strain damage in existing 3D printing fiber arrangement and mechanical tensile methods. Acoustic field driving can complete microfiber arrangement and assembly within seconds, overcoming the drawbacks of long time consumption and low throughput of microfluidic patterning in 3D printing. Simultaneously, the diameter of the microfibers that can be arranged and assembled by acoustic field driving can be as low as hundreds of nanometers, approaching the size of natural tissue matrix fibers, overcoming the insufficient resolution of existing methods. Furthermore, this method retains the advantage of Faraday wave bioassembly without damaging cells, exhibits good biocompatibility, and the tight fiber arrangement promotes intercellular signal transduction, supporting differential arrangement from single cells to micro-tissue blocks (2-4900 μm), enabling the construction of heterogeneous structures. Attached Figure Description

[0052] Figure 1 The patterned and oriented arrangement structure of potassium titanate whiskers obtained by standing wave driving at different frequencies and amplitudes in Example 1;

[0053] Figure 2 The patterned and oriented arrangement structure of potassium titanate whiskers obtained at different vibration times in Example 2;

[0054] Figure 3 This describes the directional alignment effect of potassium titanate whiskers vibrating for 30 seconds in Example 2.

[0055] Figure 4 This is the patterned and oriented arrangement structure of decellularized matrix collagen fibers in Example 3. Detailed Implementation

[0056] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0057] Example 1

[0058] This embodiment provides a method for arranging and assembling microfibers, the steps of which are as follows:

[0059] S11. Preparation of curable driving fluid: Lithium phenyl (2,4,6-trimethylbenzoyl)phosphate is added to phosphate buffer and heated in a water bath at 40-50℃ for 15 min, with several oscillations during the process, to obtain an initiator solution with a concentration of 0.25% (w / v); then, methacrylamide gelatin is added to the initiator solution and heated in a water bath at 60-70℃ in the dark for 20-30 min, with several oscillations during the process, to obtain a curable driving fluid with a concentration of 5% (w / v);

[0060] S12. Disperse high-strength potassium titanate whiskers (diameter 3-5μm, length 10-30μm) in a curable driving liquid to a concentration of 2mg / mL, mix thoroughly to obtain a pre-assembled liquid;

[0061] S21. Add 530 μL of pre-assembly liquid to the prefabricated circular polymethyl methacrylate assembly chamber (20 mm in diameter and 1.5 mm in thickness) so that the liquid level is flush with the upper edge of the chamber. Let it stand for 10-60 seconds to allow the potassium titanate whiskers to settle to the bottom of the chamber.

[0062] S31. Set the combination of sound wave frequency and amplitude to 75Hz and 150-300mV, 50Hz and 80-200mV, 45Hz and 150-300mV, and 20Hz and 100-250mV respectively. Drive the liquid in the chamber to vibrate vertically up and down for 2 minutes using an acoustic biological assembly instrument. Stop the vibration and use blue light with a wavelength of 405nm to irradiate for 120s to solidify the pre-assembled liquid. Observe and photograph the directional arrangement structure of potassium titanate whiskers through a microscope.

[0063] Figure 1 The patterned and oriented arrangement structures of potassium titanate whiskers obtained by standing wave driving at different frequencies and amplitudes in Example 1 are shown below. Figure 1 (a), (b), (c), (d), and (e) in the image show the overall shooting effect before assembly, and at frequencies of 75Hz, 50Hz, 45Hz, and 20Hz, respectively. Figure 1 (f), (g), (h), (i), and (j) are magnified views of parts (1), (2), (3), (4), and (5) labeled in figures (a), (b), (c), (d), and (e), respectively. Figure 1It is known that before the directional alignment of potassium titanate whiskers driven by standing waves, the potassium titanate whiskers in the pre-assembly solution are randomly distributed and arranged without orientation. At low frequencies (20Hz and 100-250mV), the potassium titanate whiskers aggregate to form a cross-shaped pattern; at 45Hz and 150-300mV, the potassium titanate whiskers aggregate to form a circular inscribed cross-shaped pattern; at 50Hz and 80-200mV, the potassium titanate whiskers aggregate to form a double-layered concentric circle pattern; and at 75Hz and 150-300mV, the potassium titanate whiskers aggregate to form a wheel-shaped pattern. In addition, the potassium titanate whiskers have a certain directional alignment. In the straight areas of the pattern (75Hz, 45Hz), the fibers are arranged along the straight direction, and in the arc areas of the pattern (50Hz), the fibers are arranged along the tangent direction of the arc.

[0064] Example 2

[0065] This embodiment provides a method for arranging and assembling microfibers, the steps of which are as follows:

[0066] S11. Preparation of curable driving fluid: Lithium phenyl (2,4,6-trimethylbenzoyl)phosphate is added to phosphate buffer and heated in a water bath at 40-50℃ for 15 min, with several oscillations during the process, to obtain an initiator solution with a concentration of 0.25% (w / v); then, methacrylamide gelatin is added to the initiator solution and heated in a water bath at 60-70℃ in the dark for 20-30 min, with several oscillations during the process, to obtain a curable driving fluid with a concentration of 5% (w / v);

[0067] S12. Disperse high-strength potassium titanate whiskers (diameter 3-5μm, length 10-30μm) in a curable driving liquid to a concentration of 2mg / mL, mix thoroughly to obtain a pre-assembled liquid;

[0068] S21. Add 530 μL of pre-assembly liquid to the prefabricated circular polymethyl methacrylate assembly chamber (20 mm in diameter and 1.5 mm in thickness) so that the liquid level is flush with the upper edge of the chamber. Let it stand for 60 seconds to allow the potassium titanate whiskers to settle to the bottom of the chamber.

[0069] S31. Set the sound wave frequency to 75Hz and the amplitude to 150-300mV. Drive the liquid in the chamber to vibrate vertically up and down through an acoustic biological assembly instrument for 5s, 10s, 15s, 20s, 25s and 30s respectively. Stop the vibration and use 405nm blue light to irradiate the pre-assembled liquid for 120s to solidify it. Observe and photograph the directional arrangement structure of potassium titanate whiskers through a microscope.

[0070] Figure 2 The patterned and oriented structures of potassium titanate whiskers obtained at different vibration times in Example 2 are shown below. Figure 2In the image, (a), (b), (c), (d), (e), and (f) show the overall effects of shooting with vibrations for 5s, 10s, 15s, 20s, 25s, and 30s, respectively. Figure 2 (g), (h), (i), (j), (k), and (l) are enlarged views of parts (1), (2), (3), (4), (5), and (6) marked in (a), (b), (c), (d), (e), and (f), respectively. Figure 3 This refers to the directional alignment effect of potassium titanate whiskers vibrating for 30 seconds in Example 2. Figure 2 The magnified view of (i) in the diagram is... Figure 2 and Figure 3 It can be seen that when the assembly time of potassium titanate whiskers driven by standing waves is short (5s), the fiber units are slightly aggregated but do not form an obvious patterned structure, and the fibers are temporarily randomly arranged without orientation. When the driving assembly time is extended (10s and 15s), an obvious wheel-shaped patterned structure can be observed, and the fibers have a certain degree of orientation, but the degree of orientation is slightly low. When the driving assembly time reaches 20-30s, an obvious wheel-shaped pattern can still be observed, and the fibers have a very high degree of orientation.

[0071] Example 3

[0072] This embodiment provides a method for arranging and assembling microfibers, the steps of which are as follows:

[0073] S11. Preparation of curable driving fluid: Lithium phenyl (2,4,6-trimethylbenzoyl)phosphate is added to phosphate buffer and heated in a water bath at 40-50℃ for 15 min, with several oscillations during the process, to obtain an initiator solution with a concentration of 0.25% (w / v); then, methacrylamide gelatin is added to the initiator solution and heated in a water bath at 60-70℃ in the dark for 20-30 min, with several oscillations during the process, to obtain a curable driving fluid with a concentration of 5% (w / v);

[0074] S12. Disperse decellularized matrix collagen fibers (0.01-0.05 mm in diameter and 0.05-0.5 mm in length) in a curable driving liquid to a concentration of 2 mg / mL, mix thoroughly to obtain a pre-assembled solution;

[0075] S21. Add 530 μL of pre-assembly solution to the prefabricated circular polymethyl methacrylate assembly chamber (20 mm in diameter and 1.5 mm in thickness) until the liquid level is flush with the upper edge of the chamber. Let it stand for 60 seconds to allow the decellularized matrix collagen fibers to settle to the bottom of the chamber.

[0076] S31. Set the sound wave frequency to 50Hz and the amplitude to 80-200mV. Drive the liquid in the chamber to vibrate vertically up and down for 60s using an acoustic biological assembly instrument. Stop the vibration and use 405nm blue light to irradiate for 120s to solidify the pre-assembled liquid. Observe and photograph the directional arrangement structure of decellularized matrix collagen fibers through a microscope.

[0077] Figure 4 The patterned and oriented arrangement structure of decellularized matrix collagen fibers in Example 3, wherein, Figure 4 (a) in the image represents the overall result of the photograph. Figure 4 (b) in (a) is a magnified view of part (1) marked in (a). Figure 4 It can be seen that the decellularized matrix collagen fibers in the pre-assembled solution form a double-layered concentric circle patterned structure, and the fibers have a certain degree of directional alignment.

Claims

1. A method for arranging and assembling microfibers, characterized in that, Includes the following steps: S1. Disperse the microfibers in a curable driving liquid to obtain a pre-assembled liquid; S2. Place the pre-assembly liquid in the assembly chamber and allow it to stand to allow the microfibers to settle to the bottom of the chamber. S3. Apply a standing wave to pattern and orient the microfibers in the pre-assembly liquid, and then solidify the pre-assembly liquid. The aspect ratio of the microfiber is greater than 2.

2. The microfiber arrangement and assembly method according to claim 1, characterized in that, The length of the microfibers is in the hundreds of nanometers, micrometers, or millimeters.

3. The microfiber arrangement and assembly method according to claim 1 or 2, characterized in that, The microfibers include at least one of natural fibers, naturally derived fibers, synthetic organic fibers, carbon-based fibers, and ceramic fibers.

4. The microfiber arrangement and assembly method according to claim 3, characterized in that, The microfibers are selected from at least one of collagen fibers, silk fibroin fibers, cellulose fibers, potassium titanate whiskers, polylactic acid fibers, and polycaprolactone fibers.

5. The microfiber arrangement and assembly method according to claim 1, characterized in that, The curable driving fluid is selected from one of the following: photocurable liquid, thermosensitive hydrogel, fibrinogen, and thrombin system.

6. The microfiber arrangement and assembly method according to claim 5, characterized in that, The components of the photocurable liquid include a photocurable substrate, a photoinitiator, and a solvent; And / or, the curing conditions for the photocurable liquid are irradiation with 400-410nm blue light for 10-150s.

7. The microfiber arrangement and assembly method according to claim 6, characterized in that, The photocurable substrate includes methacrylic anhydride-modified biomaterials; And / or, the photoinitiator includes at least one of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

8. The microfiber arrangement and assembly method according to claim 1, characterized in that, The depth of the assembly chamber is 0.8-5 mm.

9. The microfiber arrangement and assembly method according to claim 1, characterized in that, The frequency of the standing wave is 15-300Hz; the amplitude is 50-1500mV.

10. The application of the microfiber arrangement and assembly method according to any one of claims 1-9 in simulating the construction of three-dimensional matrix fiber topology of tissues and organs.

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