Method and system for printing silk fibroin scaffold by using ultrasound

Abstract

The invention discloses a method and a system for printing a silk fibroin scaffold by using ultrasound, and belongs to the technical field of biological material manufacturing and tissue engineering. The method comprises the following steps: preparing silk fibroin bio-ink, polydopamine-coated barium titanate composite powder and lipid microbubbles; mixing the three components to obtain composite printing ink; and sequentially applying ultrasonic waves to predetermined three-dimensional point positions in a tool loaded with the ink by using a focused ultrasonic transducer, triggering a local cavitation effect under the assistance of microbubbles, inducing silk fibroin to solidify at a fixed point, and accumulating point by point to form a three-dimensional bracket. The invention further provides an ultrasonic printing device for implementing the method. The preparation method avoids the use of a chemical cross-linking agent, and is green and safe; the cavitation threshold value is reduced through microbubbles, and low-energy and high-efficiency solidification is achieved; fine printing of a complex structure is achieved by combining focused ultrasound and three-dimensional motion control. And a piezoelectric function is integrated, so that the prepared stent has good biocompatibility and bioelectricity activity.

Description

[0001] A method and system for ultrasonic printing of silk fibroin scaffolds Technical Field

[0002] This invention relates to the interdisciplinary fields of biomaterials manufacturing, additive manufacturing, and tissue engineering, and in particular to a method and system for ultrasonically printing silk fibroin scaffolds. Background Technology

[0003] Silk fibroin, as a natural biopolymer, has broad application prospects in tissue engineering, drug delivery systems, and biomedical devices due to its excellent biocompatibility, biodegradability, and controllable mechanical properties. Traditional methods for preparing silk fibroin materials can be divided into two categories: chemical cross-linking and physical curing. Chemical cross-linking typically uses cross-linking agents such as glutaraldehyde and genipin, or alcohol solvents to induce coagulation. While this allows for rapid material molding, it carries the risk of chemical residue toxicity, potentially adversely affecting cell viability and tissue compatibility. Physical curing methods, such as salting out and freeze-drying, avoid chemical toxicity, but their long coagulation times and poor process controllability make it difficult to achieve complex structures. These technological bottlenecks limit its in-depth application in high-end biomedical fields.

[0004] Currently, to enhance the functionality of silk fibroin in the biomedical field, silk fibroin can be combined with functional materials to improve its mechanical strength, piezoelectric properties, etc. Piezoelectric biomaterials, due to their unique force-electric conversion characteristics, have shown unique advantages in promoting tissue regeneration and constructing biosensors. Barium titanate, as a classic piezoelectric ceramic material, has good piezoelectric properties and biocompatibility. It is usually combined with silk fibroin through electrospinning, in-situ synthesis, etc., but there are the following challenges: (1) Traditional composite methods mostly use physical blending followed by chemical cross-linking, which makes it difficult to achieve uniform dispersion at the nanoscale; (2) The natural conformation of silk fibroin and the piezoelectric crystal phase of barium titanate are easily destroyed during the composite process; (3) Existing technologies have not yet achieved spatially controllable molding of piezoelectric composite materials.

[0005] In recent years, ultrasound-induced biomaterial molding technology has gradually attracted attention. This method utilizes the mechanical and thermal effects generated by ultrasonic cavitation to induce the self-assembly of protein molecules in an aqueous environment. It is characterized by being green and rapid, and can improve the problem of uniform dispersion of functional composite materials. However, existing ultrasonic solidification technology still has the following limitations: (1) To achieve effective cavitation, a high ultrasonic energy input is often required, which can easily cause local overheating and damage temperature-sensitive bioactive components. (2) Conventional ultrasonic treatment lacks functional design, and the resulting material has limited properties, making it difficult to meet the growing demand for materials with high electroactivity and intelligent responsiveness in biomedical applications. (3) The ultrasonic solidification process has insufficient control precision, making it difficult to achieve spatial gradient regulation of material structure and properties.

[0006] Therefore, there is a need to develop an innovative method and system for preparing silk fibroin materials and their composites. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a method and system for ultrasonic printing of silk fibroin scaffolds. This method introduces microbubbles to synergistically reduce the cavitation threshold and combines dual-stage focused ultrasound (first, steady-state cavitation to uniformly disperse piezoelectric materials, and then transient cavitation to solidify silk fibroin) to achieve low-energy, green, and precise integrated molding of piezoelectric functional scaffolds.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The method for ultrasonically printing silk fibroin scaffolds provided by this invention includes the following steps: S1. Preparation of microbubbles containing lipids and perfluorocarbon gas; S2. Prepare silk fibroin bio-ink and polydopamine-coated barium titanate composite powder; mix and disperse the silk fibroin bio-ink and the polydopamine-coated barium titanate composite powder, and then add the microbubbles to obtain composite printing ink. S3. Place the composite printing ink in the printing fixture, and control the focus of the focused ultrasonic transducer to align with a predetermined position in the composite printing ink according to a preset three-dimensional path; start the focused ultrasonic transducer to emit ultrasonic waves, and use the microbubbles as cavitation nucleating agents to induce cavitation effect in the composite printing ink at the focus, thereby causing the silk fibroin to coagulate. The emission of the ultrasonic waves includes a first processing stage and a second processing stage performed sequentially. The ultrasonic parameters used in the first processing stage are those that induce steady-state cavitation in the microbubbles, which are used to ensure that the barium titanate composite powder coated with polydopamine is uniformly dispersed under the action of microbubble oscillation. The ultrasonic parameters used in the second processing stage are those that cause transient cavitation in the microbubbles, which are used to rapidly coagulate the silk fibroin and lock the arrangement structure of the polydopamine-coated barium titanate composite powder. S4. Repeat step S3 to gradually accumulate and form a silk fibroin scaffold with a three-dimensional structure.

[0009] Furthermore, in step S1, the lipid is a mixture of dipalmitoylphosphatidylcholine (DPPC) and distearate phosphatidylethanolamine (DSPE); the perfluorocarbon gas is perfluoropropane.

[0010] Furthermore, in step S3, the ultrasonic frequency used in the first processing stage is 0.8 to 1 MHz, and the spatial peak time average power density is no greater than 0.3 W / cm². 2 The processing time is 60 to 120 seconds.

[0011] In step S3, the ultrasonic frequency used in the second processing stage is 0.8-1.2MHz, and the spatial peak time average power density is 0.8 to 1.2W / cm². 2 The processing time is 5 to 15 seconds.

[0012] Furthermore, in step S3, the temperature of the composite printing ink is maintained at 25-37°C by a temperature control module.

[0013] Furthermore, in step S3, the printing fixture is equipped with a temperature control module to maintain the temperature of the composite printing ink at 31℃±6℃.

[0014] The ambient water temperature of the composite printing ink is controlled at 25-37℃ during ultrasonic emission.

[0015] Furthermore, in step S3, the timing of switching from the first processing stage to the second processing stage is controlled by real-time monitoring of the acoustic impedance or backscattering signal of the composite ink at the focal point.

[0016] Furthermore, in step S2, the mass ratio of the polydopamine-coated barium titanate composite powder to the silk fibroin in the silk fibroin bio-ink is 1%-20%.

[0017] In step S2, the barium titanate composite powder coated with polydopamine is mixed with the silk fibroin bio-ink and then ultrasonically treated for 30 minutes to achieve uniform dispersion.

[0018] The system for ultrasonically printing silk fibroin scaffolds provided by the present invention is used to implement the above method, including a three-dimensional displacement platform, and further including a printing fixture, a focused ultrasonic transducer, a temperature control module, and a control system disposed on the three-dimensional displacement platform; The printing fixture is used to hold composite printing ink and is mounted on the three-dimensional displacement platform; The focused ultrasonic transducer emits ultrasonic waves whose focal point can be aligned with a predetermined spatial position within the printing fixture. The temperature control module is used to maintain the temperature inside the printing fixture at 25-37℃; The control system is electrically connected to the three-dimensional displacement platform and the focused ultrasonic transducer, respectively, and is used to control the motion path of the three-dimensional displacement platform and the start / stop and emission parameters of the focused ultrasonic transducer, so as to solidify the composite printing ink in the printing fixture point by point according to the preset three-dimensional model.

[0019] Furthermore, the focused ultrasound transducer is a traveling wave focused transducer, a spherical focused transducer, or a ring focused transducer.

[0020] Furthermore, the number of focused ultrasound transducers is multiple, forming a transducer array.

[0021] The beneficial effects of this invention are as follows: This invention provides a method and system for ultrasonically printing silk fibroin scaffolds, belonging to the field of biomaterials and additive manufacturing technology. The method includes: preparing a composite ink containing silk fibroin, barium titanate coated with polydopamine, and lipid microbubbles; using a focused ultrasonic transducer to control its focal point to scan the ink along a preset three-dimensional path; and through the cavitation effect assisted by microbubbles, inducing uniform dispersion of barium titanate in two stages, followed by point-by-point solidification of silk fibroin, thus gradually accumulating to form a three-dimensional scaffold. The system includes a three-dimensional displacement platform, printing fixture, focused ultrasonic transducer, and control system. This invention avoids chemical cross-linking, making it green and safe; microbubbles lower the cavitation threshold, achieving low-energy solidification; and it integrates piezoelectric functional composites with the precise molding of complex structures, resulting in a scaffold with both good biocompatibility and bioelectric activity.

[0022] The method provided by this invention has the following beneficial effects: Green and safe with high biocompatibility: It completely abandons chemical cross-linking agents and achieves silk fibroin coagulation based on the pure physical ultrasonic cavitation effect, eliminating harmful chemical residues from the source and ensuring the biosafety of the material.

[0023] Low energy consumption and bio-friendly: By introducing microbubbles as cavitation nucleating agents, the ultrasonic energy threshold required to initiate effective cavitation is significantly reduced, thereby enabling efficient coagulation under mild conditions of low power and low dosage, greatly reducing the damage of ultrasonic thermal effects to potentially loaded active biological components such as cells and protein drugs.

[0024] Integrated Functional Composites and Precision Molding: Simultaneously with ultrasonic-induced solidification, staged acoustic field control is used to first uniformly disperse the piezoelectric filler (barium titanate), then lock its structure. This one-step method can produce intelligent biocomposite materials that possess both excellent biocompatibility and a clear piezoelectric response. Combining focused ultrasound with precise three-dimensional motion control enables the fine printing of complex three-dimensional microstructures.

[0025] The manufacturing process is highly controllable: by programmatically controlling ultrasonic parameters (such as power, mode, time) and scanning path, the local properties of the stent (such as mechanical strength, porosity, and piezoelectricity) can be precisely controlled, enabling customized manufacturing with designable performance.

[0026] The above and other objects, advantages, and features of the present invention will be more fully set forth and demonstrated through the following detailed description of specific embodiments in conjunction with the accompanying drawings. Those skilled in the art, upon referring to the following detailed description and the accompanying drawings, will be able to better understand and realize the above advantages of the present invention. Other objects, features, and advantages of the present invention will become clearer after being described in detail in the detailed description section in conjunction with the accompanying drawings. Attached Figure Description

[0027] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration.

[0028] Figure 1 This is a flowchart from raw materials to printed product; Figure 2 Diagram of the acoustic printing platform; Figure 3 This is a diagram of piezoelectric microbubble silk fibroin coagulation.

[0029] In the diagram, 1 represents the ink fixture; 2 represents the robotic arm; 3 represents the heating rod; 4 represents the thermometer; 5 represents the temperature control; 6 represents focused ultrasound; and 7 represents the drive motor. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] Example 1 like Figure 1 As shown, Figure 1 The flowchart from raw materials to printed form is provided in this embodiment. The method and system for ultrasonic printing of silk fibroin scaffolds include the following steps: S1. Prepare microbubbles containing lipids and perfluorinated carbon gas; wherein the lipids contain 5-30 mol% of the cationic lipid dioleoyltrimethylpropane ammonium (DOTAP). S2. Prepare silk fibroin bio-ink and polydopamine-coated barium titanate composite powder; mix and disperse the silk fibroin bio-ink and the polydopamine-coated barium titanate composite powder, and then add the microbubbles to obtain composite printing ink. S3. Place the composite printing ink in the printing fixture, and control the focus of the focused ultrasonic transducer to align with a predetermined position in the composite printing ink according to a preset three-dimensional path; start the focused ultrasonic transducer to emit ultrasonic waves, and use the microbubbles as cavitation nucleating agents to induce cavitation effect in the composite printing ink at the focus, thereby causing the silk fibroin to coagulate. The ultrasonic wave emission includes a first processing stage (steady-state cavitation arrangement of filler) and a second processing stage (transient cavitation coagulation of protein), as detailed below: The ultrasonic parameters used in the first processing stage are those that induce steady-state cavitation in the microbubbles, which are used to ensure that the barium titanate composite powder coated with polydopamine is uniformly dispersed under the action of microbubble oscillation. The ultrasonic parameters used in the second processing stage are those that cause transient cavitation in the microbubbles, which are used to rapidly coagulate the silk fibroin and lock the arrangement structure of the polydopamine-coated barium titanate composite powder. The trigger condition for switching from the first processing stage to the second processing stage is: real-time monitoring of the acoustic impedance or backscattering signal of the composite ink at the focal point. When the signal reaches a predetermined threshold, indicating that the barium titanate filler has achieved a predetermined degree of orientation, the system automatically switches to the second processing stage. This embodiment utilizes the additional acoustic effect generated by charged microbubbles or controls the processing stages based on real-time material status feedback to achieve a two-stage ultrasonic process, enabling more efficient and precise controllable orientation of functional fillers in a three-dimensional gel network.

[0032] This embodiment can also switch from the first processing stage to the second processing stage through time control. After 40 seconds have elapsed in the first processing stage, the second processing stage begins, and the processing time in the second processing stage is 20 seconds.

[0033] S4. Repeat step S3 to gradually accumulate and form a silk fibroin scaffold with a three-dimensional structure.

[0034] In step S1 of this embodiment, the lipid is a mixture of dipalmitoylphosphatidylcholine (DPPC) and distearate phosphatidylethanolamine (DSPE); the perfluorocarbon gas is perfluoropropane.

[0035] In step S1 of this embodiment, the concentration of the microbubbles in the composite printing ink is 10. 6 -10 8 per mL.

[0036] In step S3 of this embodiment, the ultrasonic frequency used in the first processing stage is 0.8-1MHz, and the spatial peak time average power density is no greater than 0.3W / cm². 2 The processing time is 60 to 120 seconds.

[0037] In step S3 of this embodiment, the ultrasonic frequency used in the second processing stage is 0.8-1.2MHz, and the spatial peak time average power density is 0.8 to 1.2W / cm². 2 The processing time is 5 to 15 seconds.

[0038] In step S3 of this embodiment, the acoustic power density of the ultrasonic waves emitted by the focused ultrasonic transducer is no higher than 1.5 W / cm². 2 .

[0039] In step S3 of this embodiment, the temperature of the composite printing ink is maintained at 25-37°C by a temperature control module.

[0040] In step S3 of this embodiment, the printing fixture is equipped with a temperature control module to maintain the temperature of the composite printing ink at 25-37℃. In this embodiment, the ambient water temperature of the composite printing ink during ultrasonic emission is controlled at 28℃±2℃.

[0041] In step S2 of this embodiment, the mass ratio of the polydopamine-coated barium titanate composite powder to the silk fibroin in the silk fibroin bio-ink is 1%-20%.

[0042] In step S2 of this embodiment, the polydopamine-coated barium titanate composite powder is mixed with the silk fibroin bio-ink and then ultrasonically treated for 30 minutes to achieve uniform dispersion. After the treated composite ink mixture is left to stand in a transparent container at a predetermined temperature, the time it takes for obvious stratification or floating to occur is used to determine whether uniform dispersion has been achieved. In this embodiment, the determination is made within the planned printing operation time window (e.g., 30-60 minutes), when the microbubble concentration ratio at the bottom and top of the ink is not less than 85%.

[0043] The focused ultrasound transducer described in this embodiment is a traveling wave focused transducer, a spherical focused transducer, or a ring focused transducer.

[0044] In this embodiment, the ultrasonic driving frequency is 0.8-1.2MHz, the waveform is a continuous wave, and the processing time for a single point is 10 seconds to 180 seconds.

[0045] The system for ultrasonically printing silk fibroin scaffolds provided in this embodiment is used to implement the above method. It includes a three-dimensional displacement platform, and also includes a printing fixture, a focused ultrasonic transducer, a temperature control module, and a control system disposed on the three-dimensional displacement platform. The printing fixture is used to hold composite printing ink and is mounted on the three-dimensional displacement platform; The focused ultrasonic transducer emits ultrasonic waves whose focal point can be aligned with a predetermined spatial position within the printing fixture. The temperature control module is used to maintain the temperature inside the printing fixture at 25-37℃; The control system is electrically connected to the three-dimensional displacement platform and the focused ultrasonic transducer, respectively, and is used to control the motion path of the three-dimensional displacement platform and the start / stop and emission parameters of the focused ultrasonic transducer, so as to solidify the composite printing ink in the printing fixture point by point according to the preset three-dimensional model, with an XYZ three-axis accuracy of 1mm.

[0046] The focused ultrasound transducer described in this embodiment is a traveling wave focused transducer, a spherical focused transducer, or a ring focused transducer.

[0047] In this embodiment, there are multiple focused ultrasound transducers, forming a transducer array.

[0048] This embodiment first prepares silk fibroin bio-ink, polydopamine-coated barium titanate composite powder, and lipid microbubbles; then, the three are mixed to obtain a composite printing ink; finally, a focused ultrasonic transducer is used to sequentially apply ultrasonic waves to predetermined three-dimensional points within a tooling containing the ink, inducing local cavitation effects with the assistance of microbubbles, thereby inducing the localized coagulation of silk fibroin and accumulating it point by point to form a three-dimensional scaffold. The ultrasonic printing system provided in this embodiment avoids the use of chemical cross-linking agents, making it green and safe; by lowering the cavitation threshold through microbubbles, low-energy, high-efficiency coagulation is achieved; combining focused ultrasound with three-dimensional motion control enables the fine printing of complex structures; and it integrates piezoelectric functionality, resulting in a scaffold with both good biocompatibility and bioelectric activity.

[0049] Example 2 This embodiment details the method and system implementation process of ultrasonic printing of silk fibroin scaffolds. This method overcomes the problems of existing silk fibroin coagulation technologies, such as slow coagulation speed, uncontrollable process, potential introduction of harmful chemicals, and poor uniformity and mechanical properties of the prepared materials. It achieves a rapid, controllable, green, low-dose, and piezoelectric material preparation process. The specific implementation process is as follows: I. Preparation of silk fibroin bio-ink: Add 2.12g sodium carbonate to 1000ml water, boil the silk fibroin in the sodium carbonate solution for 40min to degumme, wash and dry at 50℃ for 500min, dissolve 26.9g lithium bromide in 25ml water at 60℃ for 5h; dialyze with 3500kDa dialysis belt for 5 days; finally concentrate the silk fibroin to the required concentration of 4%-20% using PEG6000 solution.

[0050] II. Preparation of PDA (polydopamine)-encapsulated barium titanate piezoelectric materials: Take an appropriate amount of tetragonal barium titanate powder, disperse it in anhydrous ethanol, and sonicate it for 30 minutes to remove surface impurities.

[0051] Then centrifuge at 8000 rpm for 10 minutes, collect the solid precipitate, and wash it 2-3 times with deionized water.

[0052] The washed solid was placed under vacuum at 60°C for 6 hours to obtain barium titanate powder with a clean surface for later use.

[0053] Prepare 10 mmol·L⁻ 1 The Tris buffer solution, with an initial pH of 10, was adjusted to pH 8.5 by adding dilute hydrochloric acid.

[0054] 1.0 g of the pretreated barium titanate powder was dispersed in 20 mL of Tris-HCl buffer solution at pH 8.5 and sonicated for 30 minutes under ice bath conditions.

[0055] Then add 0.1g of dopamine hydrochloride and continue sonication for 10 minutes.

[0056] The mixture was placed at room temperature and magnetically stirred for 24 hours.

[0057] After the reaction is complete, the mixture is driven at 9000 r·min⁻ 1 Centrifuge at a speed of 1000 rpm for 10 minutes and collect the solid.

[0058] To fully remove unreacted impurities and byproducts, the obtained solid was redispersed in deionized water and washed by centrifugation three times.

[0059] Finally, the washed precipitate was placed in a vacuum freeze dryer and dried for 48 hours to obtain a gray-black barium titanate@polydopamine composite powder.

[0060] III. Preparation of microbubbles: Weigh 50 mg of DPPC and 30 mg of DSPE lipid powder for later use.

[0061] Subsequently, 9.0 mL of PBS buffer was transferred, 1.0 mL of glycerol was added, and the mixture was stirred to prepare a PBS-glycerol mixed solution.

[0062] Add the weighed lipid powder to the mixed solution and place it in an ultrasonic cleaner at 50°C for 1 hour to allow the lipids to be fully dispersed.

[0063] Next, a small amount of the above solution was placed at the bottom of a small vial, and the air inside the vial was drawn out with a syringe and perfluorinated carbon gas was injected to replace the atmosphere.

[0064] Finally, fix the vial in the clamp of the dental amalgam mixer and vibrate for 1 minute to complete the foam preparation.

[0065] IV. Preparation of piezoelectric microbubble silk fibroin: The barium titanate@polydopamine composite powder prepared above is mixed with silk fibroin in different mass ratios, and an appropriate amount of deionized water or buffer solution is added. The mixture is ultrasonically treated for 30 minutes to make the composite powder uniformly dispersed in the silk fibroin solution. Before printing, an appropriate amount of microbubbles are injected to obtain piezoelectric silk fibroin composite materials with different piezoelectric properties.

[0066] V. Sound Printing: The prepared piezoelectric microbubble silk fibroin aqueous solution is injected into the silk fibroin ink fixture. The fixture is moved by a three-dimensional displacement platform so that the silk fibroin solution to be treated is placed at the focal point of the focused ultrasound. The ultrasound system is started and ultrasound with specific parameters is applied to solidify the silk fibroin solution.

[0067] Some parameters are as follows: Drive frequency: 0.8-1.2MHz; Waveform: Continuous wave; Processing time: 10s to 180s; Water temperature: 25-37℃.

[0068] like Figure 1 As shown, Figure 1 This is a flowchart illustrating the preparation process of silk fibroin hydrogels. The flowchart shows the process from raw materials to the final molded scaffold via a pre-defined three-dimensional path. The key steps include: preparation of silk fibroin bio-ink, preparation of polydopamine-coated barium titanate powder, microbubble preparation, obtaining composite printing ink (mixing and dispersing), and finally, molding via focused ultrasound. In this embodiment, the pre-defined three-dimensional path is achieved by controlling the three-axis motion mode of a robotic arm; alternatively, the pre-defined path can be completed by manually controlling the X, Y, and Z-axis motion trajectories of the robotic arm.

[0069] Preparation of silk fibroin solution: Raw materials include silkworm silk (i.e., cicada cocoons), deionized water, and lithium bromide; Degumming: Boil silk in a solution such as sodium carbonate to remove the sericin protein that wraps around the fibroin fibers, thus obtaining pure fibroin fibers. Dissolution: The degummed silk fibroin fibers are dissolved in a lithium bromide solution, and a silk fibroin lithium bromide solution is formed under heating conditions (such as 60°C); Concentration: The above solution is dialyzed to remove lithium bromide, and then concentrated using polyethylene glycol (PEG) and other concentration stages to finally obtain a silk fibroin solution of the required concentration, which is used as the matrix material for bio-ink.

[0070] Preparation of polydopamine-coated barium titanate (PDA@barium titanate): a surface modification process for piezoelectric functional fillers; Raw materials: include tetragonal barium titanate powder, which is a piezoelectric ceramic material; Pretreatment: The barium titanate powder is cleaned (e.g., ultrasonically cleaned in ethanol), centrifuged and dried to remove surface impurities, thus obtaining pretreated barium titanate. Hydrophilization: The pretreated barium titanate powder is dispersed in Tris buffer solution, and dopamine hydrochloride is added. In an alkaline environment, dopamine undergoes oxidative self-polymerization, forming a highly adhesive polydopamine (PDA) coating on the surface of barium titanate particles. This was used to achieve hydrophilic modification of barium titanate, which improved its dispersion stability in subsequent aqueous inks and its interfacial compatibility with silk fibroin, resulting in PDA@barium titanate composite powder. Preparation of lipid microbubbles: The process of preparing microbubbles as cavitation nucleating agents; Raw materials include lipid powder and glycerol, wherein the lipid powder is a mixture of dipalmitoylphosphatidylcholine (DPPC) and distearate (DSPE). The glycerol is used as a cosolvent and stabilizer; Ultrasonic dispersion: Lipid powder, glycerol and PBS buffer are mixed and dispersed by ultrasonication to form a uniform lipid powder-glycerol mixture solution, so that the lipids are fully hydrated; Atmosphere replacement and shaping: The above solution is placed in a sealed vial (such as a vial), and air is drawn out with a syringe before perfluorocarbon gas (such as perfluoropropane) is injected to complete the atmosphere replacement. Subsequently, the gas is sheared and dispersed in the lipid solution by high-speed mechanical oscillation (such as using a dental amalgam mixer) to form a stable microbubble suspension with lipid as the shell and perfluorocarbon gas as the core; Formation of piezoelectric silk fibroin microbubble bio-ink: Piezoelectric silk fibroin microbubble bio-ink was prepared by mixing silk fibroin solution, PDA@barium titanate composite powder and microbubbles. The bio-ink prepared in this embodiment integrates the biocompatibility of silk fibroin, the piezoelectric properties of barium titanate, and the ultrasonic response properties of microbubbles, and can be used as a direct material for subsequent focused ultrasound printing.

[0071] like Figure 2 As shown, Figure 2 The diagram shows the overall structure and layout of the ultrasonic printing system used in this method. It includes a three-dimensional displacement platform, a printing fixture (containing composite printing ink) mounted on it, and a focused ultrasonic transducer. The ultrasonic waves emitted by the focused ultrasonic transducer are concentrated at a single focal point, which can be positioned at any three-dimensional coordinate point of the ink within the printing fixture by controlling the three-dimensional displacement platform or the transducer itself. Figure 2 The ultrasonic printing system on display operates through a dynamic cycle of precise coordination among multiple components. Utilizing the cavitation effect of focused ultrasound, it induces the physical coagulation of silk fibroin ink containing microbubbles and functional fillers point by point, thereby accumulating and forming a three-dimensional scaffold.

[0072] The entire process begins with the initialization and preparation phase. An ink fixture containing composite printing ink is fixed to the end of a robotic arm. After the system starts up, heating rods integrated into the water bath surrounding the fixture begin to work, while thermometers monitor the water temperature in real time and feed the data back to the control system, forming a closed-loop temperature control module. Together, they precisely maintain the ink environment at a physiologically compatible temperature of 25-37°C, laying the foundation for subsequent bio-friendly printing.

[0073] Subsequently, the system enters a point-to-point printing cycle. The first step is precise positioning: the central control system sends instructions to the drive motor according to the preset 3D model path. The motor drives the robotic arm to carry the ink fixture in 3D motion, precisely moving the first target point of the ink in the fixture to the acoustic focus area of ​​the focused ultrasonic transducer.

[0074] Once positioning is complete, the ultrasonic processing stage begins immediately. This process consists of two key sub-stages, both achieved through precise control of the focused ultrasonic transducer by the control system. The first is the alignment stage: the transducer emits low-power, steady-state cavitation parameters of ultrasound. Microbubbles located at the focal point oscillate stably in the acoustic field, and the resulting microfluidics and radiative forces drive the polydopamine-coated barium titanate powder to disperse evenly and orderly along the acoustic field direction. Next comes the solidification and locking stage: the control system instantly switches the ultrasonic parameters to a high-power, transient cavitation mode. The microbubbles violently collapse, generating a strong local mechanical effect that instantly triggers the formation and solidification of the silk fibroin molecular network, thus permanently "locking" the previously aligned barium titanate filler structure within the gel network. At this point, a tiny three-dimensional voxel is printed.

[0075] Subsequently, the system repeats the "positioning-arranging-solidification" cycle. Driven by a motor, the robotic arm continuously delivers the next predetermined point in the ink fixture to the ultrasonic focal point, where it is solidified by a focused ultrasonic transducer. By scanning point by point and accumulating layer by layer, a solid scaffold with a complex three-dimensional structure and uniformly dispersed internal functional fillers is finally "grown" directly from the liquid ink within the temperature-controlled ink fixture.

[0076] The system provided in this embodiment achieves spatial positioning through a drive motor and robotic arm, environmental control maintained by a thermometer and heating rod, and targeted energy stimulation provided by a focused ultrasound transducer through seamless collaboration. This constitutes a green, precise, and integrated advanced biomanufacturing platform that can realize multiple functions.

[0077] Example 3 This embodiment further illustrates the method with specific illustrations and implementation processes, and the preparation of an auricle-shaped cartilage regeneration scaffold with piezoelectric properties is as follows: 1. Prepare an 8% silk fibroin ink according to the aforementioned method. Prepare BaTiO3@PDA powder (BaTiO3 core particle size 200nm, PDA layer thickness approximately 10nm).

[0078] 2. Add BaTiO3@PDA powder at a ratio of 5wt% to silk fibroin ink and ultrasonically disperse it for 30 minutes at 40W power in an ice bath.

[0079] 3. Take the above-mentioned mixed ink and gently mix it with freshly prepared C3F8 lipid microbubble suspension (average diameter 2.0 μm) to achieve a final microbubble concentration of 5 × 10⁻⁶. 7 The composite ink was obtained by using 1 / mL of 1 / mL.

[0080] 4. Inject the composite ink into the transparent PDMS printing cavity. Use a spherical focusing transducer with a center frequency of 1.0MHz and a focal length of 15mm, and set it to pulsed ultrasonic mode (pulse width 1ms, repetition frequency 100Hz, spatial peak time average power density 0.8W / cm²). 2 ).

[0081] 5. Import the 3D digital model of the auricle from the computer and slice it. Control the movement of the 3D platform so that the transducer focus scans the ink along the slicing path, with each point irradiated for 45 seconds.

[0082] 6. After scanning, the fully formed auricle-shaped scaffold was removed from the chamber, washed with PBS, and placed in cell culture medium for later use. This scaffold has a fine structure, good mechanical support, and piezoelectric response (measured d). 33 (Approximately 15 pC / N), which can be used to inoculate chondrocytes for auricular regeneration research.

[0083] like Figure 3 As shown, Figure 3 This is a diagram of piezoelectric microbubble silk fibroin coagulation. Figure 3 The dynamic changes in material state under two-stage ultrasonic treatment are demonstrated. To quantitatively demonstrate the technical effectiveness of this strategy, the following comparative experiment was designed, and the key data are shown in the table below: Comparative experimental data on the effects of two-stage ultrasound strategies Groups and Status Ultrasonic treatment strategy Filler orientation degree (XRD orientation factor, 0-1) <![CDATA[Piezoelectric properties (d 33 , pC / N)]]> Single-point gelation time (seconds) Cell compatibility (24h survival rate after inoculation, %) Key Explanation initial state No ultrasonic treatment, liquid ink 0.05 (completely random) Unformed and cannot be measured - - In the baseline state, the ink is liquid and the filler is randomly dispersed. Control group 1 Single-stage transient cavitation (direct high power) 0.22±0.04 4.5±1.0 25±5 75.3±5.2 Although high-energy cavitation can rapidly induce solidification, the violent disturbance disrupts the cell arrangement, and the thermal effect damages the cells. Control group 2 Reverse the order (transient state first, then steady state) 0.25±0.05 5.2±1.2 105±10 82.1±4.0 The protein network solidified first, severely hindering the subsequent arrangement of filler material, proving that the order is crucial. After the first phase Steady-state cavitation only (low power) 0.78±0.06 Not fully solidified, weak >300 (not solidified) 95.0±2.5 (estimated) The filler is highly uniformly dispersed, but the matrix is ​​not cured, and the structure cannot be fixed. The arrangement effect is visually demonstrated. After the second phase Two-stage strategy (steady-state first, then transient) 0.80±0.05 (Structure locked) 17.5±2.5 100±5 (Total Time) 94.8±2.0 <![CDATA[Achieve rapid solidification while maintaining a high degree of orientation. d 33 It is about 300% higher than that in the single-stage process, and the cell activity is comparable to that of pure steady-state treatment, proving that the locking process is mild.]]> Figure 3 Before printing, A in the image is in its initial liquid state. Figure 3 In the first stage of B, polydopamine is uniformly dispersed. Figure 3 The second stage of C in the process was successfully completed, and the silk fibroin coagulation printing was completed. Figure 3 The dynamic process and final effect of the two-stage ultrasonic coagulation method are demonstrated. Figure 3 In the figure, A represents the initial state before printing. The composite printing ink is in liquid state, and the components are evenly dispersed. Figure 3B in the text corresponds to the first processing stage (steady-state cavitation). Under the action of gentle ultrasound, the barium titanate powder coated with polydopamine in the ink is significantly and uniformly dispersed along the direction of the sound field, but the silk fibroin matrix has not yet solidified. Figure 3 In the diagram, C represents the second processing stage (transient cavitation). After completion, high-energy ultrasound induces a rapid cavitation effect, causing the silk fibroin to instantly solidify and form a stable printing point by locking the uniformly dispersed barium titanate structure within it. This sequence of figures clearly demonstrates that the present invention can achieve integrated molding of filler arrangement and matrix solidification in a step-by-step and controllable manner. Figure 3 By comparing the experimental data, the following conclusions can be drawn: Visual process verification: Figure 3 The sequence A→B→C in the table directly corresponds to the material state and performance transitions from "initial state" to "after the first stage" to "after the second stage" in the table above, proving the feasibility of implementing the strategy step by step.

[0084] Synergistic effect quantification: Data shows that the strategy of this method ( Figure 3 The final state C in the equation is not a simple summation of A→B→C. Its core effect lies in: Efficient arrangement: Under mild conditions (B state), an orientation degree close to the theoretical limit (0.78) is obtained.

[0085] Non-destructive locking: By using short-term, precise transient cavitation, the highly oriented structure in the B state is "frozen" in the C state, achieving piezoelectric performance (17.5 pC / N) that is 3-4 times that of single-stage treatment, without sacrificing biocompatibility (survival rate >94%).

[0086] Proof of non-obviousness: The inferiority of control group 2 (inverted order) shows that the specific order of "arrangement followed by locking" is a necessary condition for obtaining ultra-high voltage electrical performance, and the technical effects (high orientation + high voltage + high activity) produced by this order are not suggested by the prior art.

[0087] In summary, the experimental data and the attached... Figure 3 The findings confirm that the dual-stage ultrasonic strategy of this invention, through the temporal control of "first steady-state arrangement, then transient locking," successfully solves the problem of balancing "high orientation of filler," "efficient matrix curing," and "good biocompatibility" in a mild integrated process, resulting in a significant synergistic effect.

[0088] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for printing silk fibroin scaffolds using ultrasound, characterized in that, Includes the following steps: S1. Preparation of microbubbles containing lipids and perfluorocarbon gas; S2. Prepare silk fibroin bio-ink and polydopamine-coated barium titanate composite powder; mix and disperse the silk fibroin bio-ink and the polydopamine-coated barium titanate composite powder, and then add the microbubbles to obtain composite printing ink. S3. Place the composite printing ink in the printing fixture, and control the focus of the focused ultrasonic transducer to align with a predetermined position in the composite printing ink according to a preset three-dimensional path; start the focused ultrasonic transducer to emit ultrasonic waves, and use the microbubbles as cavitation nucleating agents to induce cavitation effect in the composite printing ink at the focus, thereby causing the silk fibroin to coagulate. The emission of the ultrasonic wave includes a first processing stage and a second processing stage performed sequentially. The ultrasonic parameters used in the first processing stage are those that induce steady-state cavitation in the microbubbles, which are used to uniformly disperse the barium titanate composite powder coated with polydopamine under the action of microbubble oscillation. The ultrasonic parameters used in the second processing stage are those that cause transient cavitation in the microbubbles, which are used to rapidly coagulate the silk fibroin and lock the arrangement structure of the polydopamine-coated barium titanate composite powder. S4. Repeat step S3 to gradually accumulate and form a silk fibroin scaffold with a three-dimensional structure.

2. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1, characterized in that, In step S1, the lipid is a mixture of dipalmitoylphosphatidylcholine (DPPC) and distearate phosphatidylethanolamine (DSPE); the perfluorocarbon gas is perfluoropropane.

3. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1, characterized in that, In step S3, the first treatment stage employs ultrasonic frequencies of 0.8 to 1 MHz, with a spatial peak time-averaged power density of no more than 0.3 W / cm 2 , for a treatment time of 30 to 120 seconds. In step S3, the second treatment stage employs ultrasonic frequencies of 0.8 to 1.2 MHz, spatial peak time-averaged power densities of 0.8 to 1.2 W / cm 2 , and treatment times of 5 to 30 seconds.

4. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1, characterized in that, In step S3, the temperature of the composite printing ink is maintained at 25-37°C by the temperature control module.

5. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1, characterized in that, In step S3, the printing fixture is equipped with a temperature control module to maintain the temperature of the composite printing ink at 25-37°C; the ambient water temperature of the composite printing ink during ultrasonic emission is controlled at 25-37°C.

6. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1 or 4, characterized in that, In step S3, the timing of switching from the first processing stage to the second processing stage is controlled by real-time monitoring of the acoustic impedance or backscattering signal of the composite ink at the focal point.

7. The method for ultrasonically printing silk fibroin scaffolds as described in claim 1, characterized in that, In step S2, the mass ratio of the polydopamine-coated barium titanate composite powder to the silk fibroin in the silk fibroin bio-ink is 1%-20%. In step S2, the barium titanate composite powder coated with polydopamine is mixed with the silk fibroin bio-ink and then ultrasonically treated at a temperature below 37°C to achieve uniform dispersion.

8. A system for ultrasonically printing silk fibroin scaffolds, comprising a three-dimensional displacement platform, characterized in that, It also includes a printing fixture, a focused ultrasonic transducer, a power source, a temperature control module, and a control system, all mounted on a three-dimensional displacement platform. The printing fixture is used to hold composite printing ink and is mounted on the three-dimensional displacement platform; The focused ultrasonic transducer emits ultrasonic waves that are focused at a predetermined spatial position within the printing fixture. The power source is used to provide power to the transducer; The temperature control module is used to maintain the temperature inside the printing fixture at 25-37℃; The control system is electrically connected to the three-dimensional displacement platform and the focused ultrasonic transducer, respectively, and is used to control the motion path of the three-dimensional displacement platform and the start / stop and emission parameters of the focused ultrasonic transducer, so as to solidify the composite printing ink in the printing fixture point by point according to the preset three-dimensional model.

9. The system for ultrasonically printing silk fibroin scaffolds as described in claim 8, characterized in that, The focused ultrasonic transducer is a traveling wave focused transducer, a spherical focused transducer, or a ring focused transducer, used to achieve high-precision curing.

10. The system for ultrasonically printing silk fibroin scaffolds as described in claim 8, characterized in that, The number of focused ultrasonic transducer transmitting units is multiple, forming a transducer array.

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