Magnetic driving assembly for enhancing osteogenic differentiation of stem cells in vitro as well as preparation method and application of magnetic driving assembly

Abstract

The invention belongs to the technical field of biomedical functional materials, and particularly relates to design, a preparation method and application of a magnetic driving assembly for enhancing stem cell osteogenic differentiation in vitro. The assembly comprises PDMS micro-column arrays at different intervals, black nanoscale iron powder and a programmable remote magnetic device, the magnetically-driven PDMS micro-column arrays at different intervals are doped with the same content of nanoscale iron powder to form MMP micro-column arrays, and the MMP and the magnetic assembly can form controllable magnetic induction to generate deformation bending. According to the invention, the external magnetic assembly is applied to generate a periodic magnetic field to bend the microcolumn, so that the three-dimensional space-time mechanical stimulation of stem cells inoculated on the surface of the microcolumn is accurately regulated and controlled; a new mechanism that heterogeneous topological constraint force generated by micro-column spacing regulation and control specifically enhances stem cell osteogenesis directional differentiation by activating YAP / TAZ mechanical transduction pathway is disclosed in a breakthrough manner. Compared with a traditional biochemical induction method, the mechanical and biological regulation and control strategy provided by the research not only gets rid of dependence on growth factors, but also establishes a relationship model of'mechanical force parameter-cell response threshold ', and provides a theoretical framework for constructing a dynamic interactive mechanical microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical functional materials, and specifically relates to the design, preparation method and application of a magnetic drive component for enhancing the osteogenic differentiation of stem cells in vitro. Background Art

[0002] Bone defect repair is a major challenge in clinical and regenerative medicine. Traditional treatments often face challenges such as low repair efficiency and insufficient microenvironmental regulation in complex scenarios, especially in infectious defects and those with decreased bone regeneration in aging. Research on stem cell fate regulation and microenvironmental osteogenic culture has provided new insights into bone regeneration. Although in vitro biomaterial micro-nanotopological structures (such as nanopatterns and micropillar arrays) play an important role in regulating stem cell behavior, their single design has significant limitations. Furthermore, traditional biomaterials often cannot dynamically respond to cell behavior due to their static structures, resulting in restricted cell migration and inefficient osteogenic differentiation.

[0003] Therefore, a technology that can dynamically simulate the extracellular matrix microenvironment is particularly important for regulating the osteogenic differentiation of stem cells. Based on this, magnetic drive technology activates the differentiation potential of stem cells by remotely controlling magnetic micropillars to apply dynamic mechanical force, providing an innovative solution for regulating the osteogenic differentiation of stem cells in vitro. Summary of the Invention

[0004] The present invention aims to address the aforementioned shortcomings of existing technologies, improve the efficiency of traditional in vitro stem cell osteogenic induction, and design a dynamic magnetically controlled micropillar that can be used to regulate stem cell osteogenic differentiation. The present invention fabricates static / dynamic micropillars using maskless laser direct-write lithography and two-step replica molding soft lithography. This method offers a simple preparation process, low cost, excellent morphological reproducibility, good biocompatibility, spatiotemporal programmability, and excellent osteogenic differentiation performance. The specific preparation steps are as follows:

[0005] 1) After the silicon wafer is cut, the surface organic solvent is removed using a cleaning solution. After removing the cleaning solution, the silicon wafer is dried and subjected to surface hydrophilic treatment. SU-8 photoresist is spin-coated using a spin coater to complete the pre-bake treatment.

[0006] 2) Using a maskless laser direct write lithography machine to expose the silicon wafer with spin-coated photoresist to complete the micropillar array manufacturing.

[0007] 3) After the micropillar array is hydrophobicized, PDMS is poured and baked and cured to obtain a PDMS micropore array.

[0008] 4) After the micropore array is hydrophobically treated, PDMS and PDMS mixed with nano-iron powder are cast respectively, baked and cured, and demolded to obtain PDMS static micropillars (MP) and PDMS dynamic magnetically controlled micropillars (MMP).

[0009] 5) Use a stepper motor drive controller to achieve spatiotemporal dynamic control of the magnetically controlled microcolumns, providing programmable mechanical stimulation for the inoculated stem cells.

[0010] In step 1), the silicon wafer cleaning solution is prepared using concentrated H2SO4 and H2O2 in a mass ratio of 7:3, and the volume used is 100 mL.

[0011] In step 1), the solution for removing the silicon wafer cleaning solution is 100 mL of deionized water and 100 mL of isopropyl alcohol.

[0012] In step 1), the power of the plasma cleaning apparatus used for the surface hydrophilic treatment is set to 90%, the oxygen flow is maintained at 3.5-4.0 mbar, and the treatment time is 10 minutes.

[0013] In step 1), the first speed of the glue spreader is 500 rpm, which lasts for 9 seconds, and then the second speed is set to 2000 rpm, which lasts for 30 seconds.

[0014] In step 2), the exposure dose of the photolithography machine is 145mJ / cm 2 ,The exposure size is 4mm*4mm, and the exposure dose correction is 8μm.

[0015] In step 3), the hydrophobic treatment is performed by dropping OTS solution (octadecyltrichlorosilane) around the silicon wafer, controlling the oven temperature to 150° C., and heating the wafer for 120 minutes.

[0016] In step 4), the hydrophobic treatment is performed by dropping 1H,1H,2H,2H-heptadecafluorodecyltrimethoxysilane solution around the PDMS micropores, placing the solution in a vacuum desiccator, and vacuum degassing for 8 hours.

[0017] In step 5), the magnetic control device is composed of a single-line rail single-slider lead screw slide, a stepper motor controller, a 28-stepper motor driver and a DC power supply.

[0018] The dynamic magnetically controlled micropillars obtained by the above method can be used as a new in vitro osteogenic culture platform to replace the original static cell osteogenic interface, and have better stem cell differentiation potential and osteogenic ability.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The preparation process of the present invention is simple and easy. Static / dynamic micro-column morphologies are prepared by using maskless laser direct writing lithography and two-step replication molding soft lithography. The morphologies have good reproducibility and can be prepared quickly and in large quantities.

[0021] (2) The novel dynamic magnetically controlled micropillars prepared in the present invention can effectively promote tissue adhesion and osteogenesis, thereby achieving better bone integration and soft tissue integration, providing new ideas and methods for the construction of a dynamic in vitro osteogenic culture platform, and achieving highly efficient osteogenic differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the preparation process of the static microcolumn / dynamic magnetically controlled microcolumn of the present invention.

[0023] Figure 2 This is the scanning electron microscope result of the static micropillar / dynamic magnetically controlled micropillar of the present invention.

[0024] Figure 3 This is the Young's modulus test result of the static microcolumn / dynamic magnetically controlled microcolumn of the present invention.

[0025] Figure 4 This is the contact angle test result of the static microcolumn / dynamic magnetically controlled microcolumn of the present invention.

[0026] Figure 5 This is the element surface scanning mapping result of the dynamic magnetic control microcolumn of the present invention.

[0027] Figure 6 It is the quantitative result of the iron powder doping content of the dynamic magnetic control microcolumn of the present invention.

[0028] Figure 7 This is the bending angle test result of the 5μm-pitch dynamic magnetically controlled microcolumn of the present invention.

[0029] Figure 8 This is the bending angle test result of the 10 μm pitch dynamic magnetically controlled microcolumn of the present invention.

[0030] Figure 9 This is the bending angle test result of the 15 μm pitch dynamic magnetically controlled microcolumn of the present invention.

[0031] Figure 10 This is the result of adhesion protein fluorescence staining of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of the static micropillars / dynamic magnetically controlled micropillars of the present invention.

[0032] Figure 11 The results of F-actin staining and heat map distribution of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of static microcolumns.

[0033] Figure 12 The results of F-actin staining and heat map distribution of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of dynamic magnetically controlled micropillars.

[0034] Figure 13These are the results of F-actin fluorescence quantitative analysis and cell area size test of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of static / dynamic magnetically controlled microcolumns.

[0035] Figure 14 This is the grayscale value distribution result of stress fibers of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of static / dynamic magnetically controlled micropillars.

[0036] Figure 15 These are the results of Alizarin Red S staining after culturing MC3T3-E1 (mouse osteoblast precursor cells) on the surface of static / dynamic magnetically controlled micropillars for 7 days.

[0037] Figure 16 These are the results of Alizarin Red S staining after 14 days of culture of MC3T3-E1 (mouse osteoblast precursor cells) on the surface of static / dynamic magnetically controlled micropillars.

[0038] Figure 17 These are the results of fluorescence staining and intensity quantitative analysis of the osteogenic marker Runx2 after 7 days of culture of MC3T3-E1 (mouse osteoblast precursor cells) on the surface of static / dynamic magnetically controlled micropillars.

[0039] Figure 18 These are the results of fluorescence staining and intensity quantitative analysis of the osteogenic marker Runx2 after 14 days of culture of MC3T3-E1 (mouse osteoblast precursor cells) on the surface of static / dynamic magnetically controlled micropillars.

[0040] Figure 19 These are the alkaline phosphatase staining results after culturing bMSCs (mouse bone marrow mesenchymal stem cells) on the surface of static / dynamic magnetically controlled microcolumns for 7 days.

[0041] Figure 20 These are the alkaline phosphatase staining results after culturing bMSCs (mouse bone marrow mesenchymal stem cells) on the surface of static / dynamic magnetically controlled microcolumns for 14 days.

[0042] Figure 21 These are the results of fluorescence staining and intensity quantitative analysis of the osteogenic marker Runx2 after culturing bMSC (mouse bone marrow mesenchymal stem cells) on the surface of static / dynamic magnetically controlled microcolumns for 7 days.

[0043] Figure 22 These are the results of fluorescence staining and intensity quantitative analysis of the osteogenic marker Runx2 after culturing bMSC (mouse bone marrow mesenchymal stem cells) on the surface of static / dynamic magnetically controlled microcolumns for 7 days. DETAILED DESCRIPTION

[0044] Several implementation cases are given below to further illustrate the present invention. The present invention can be implemented in a wider range and is not limited to these.

[0045] Example 1

[0046] Please refer to Figure 1 The following are the steps for preparing static micropillars / dynamic magnetically controlled micropillars:

[0047] 1. Silicon wafer processing

[0048] (1) Silicon wafer surface pretreatment: Take a 6-inch silicon wafer and use a diamond pen to cut the silicon wafer into 2cm*2cm square pieces. Place it in a polytetrafluoroethylene cleaning basket. Add 30mL of H2O2 and 70mL of concentrated H2SO4 to prepare Piranha solution. Soak in an oil bath at 90℃ for 2h to remove residual chemicals on the surface. After the solution cools down, discard it. First, add anhydrous ethanol and deionized water to rinse the silicon wafer twice. Then add isopropanol to cover the silicon wafer. Ultrasonicate at 100% power for 5min. Replace with new isopropanol solution. Repeat the ultrasonic step until the silicon wafer surface is clean (no Piranha solution residue). Blow dry the silicon wafer with nitrogen and place it in a vacuum desiccator for later use.

[0049] (2) Hydrophilic treatment of silicon wafer surface: Place the cut silicon wafer flatly into the O2-Plasma plasma cleaning instrument, turn on the vacuum pump to saturate the vacuum, then introduce oxygen for 10 minutes, maintain the vacuum degree at 0.3-0.4 mbar, press the "Generator" button to generate glow, and maintain this state for 5 minutes to complete the hydrophilic modification of the silicon wafer surface.

[0050] 2. Photolithography Preparation of Micropillar Silicon Wafer

[0051] (1) Spreading the photoresist: Use a dropper to draw 0.5-1 mL of SU-82015 photoresist and spread it evenly on the surface of the silicon wafer. Set the low speed of the spreader to 500 rpm for 9 seconds and the high speed to 2000 rpm for 30 seconds to obtain a photoresist with a predetermined thickness (about 16 μm).

[0052] (2) Baking before photolithography: Move the silicon wafer after photoresist coating to a heating plate. To avoid wrinkles caused by changes in the internal stress of the photoresist during baking, the silicon wafer needs to be heated in stages. Specifically: set the temperature of the heating plate to 65°C and maintain it for 3 minutes, then immediately raise the temperature to 95°C and maintain it for 4 minutes. After baking, keep the silicon wafer away from light and let it stand overnight to avoid uneven thickness due to evaporation of the solvent inside the photoresist.

[0053] (3) Photolithography: set the exposure dose to 145 mJ / cm 2 The exposure accuracy is 1μm. Clewin5 software is used to draw circles with a diameter of 6μm and pitches of 5μm, 10μm, and 15μm. The pre-drawn patterns are exposed using a DMO maskless lithography machine.

[0054] (4) Post-photolithography baking: Move the exposed silicon wafer to a hot plate, set the temperature to 95°C, and bake for 4 minutes.

[0055] (5) Development: Take a clean 100 mL beaker and add PGMEA (SU-8 photoresist developer) until the silicon wafer is covered. Due to the small size of the microcolumns, ultrasound is required to assist in the development process to reduce the influence of capillary forces on the microcolumn morphology during the development process. The ultrasound power is set to 40% for 30 seconds, and then the development is allowed to stand for 3 minutes until the unexposed photoresist is completely dissolved.

[0056] (6) Drying: After development, the surface of the silicon wafer is immediately rinsed with ultrapure water, and the silicon wafer is gently blown with an air gun until the surface is completely dry.

[0057] (7) Hardening: Place the dried silicon wafer on a heating plate, set the temperature to 150°C, and heat for 30 minutes to allow the photoresist to reach the maximum Young's modulus.

[0058] 3. Hydrophobic treatment of micro-pillar silicon wafer

[0059] Take a 10 cm glass dish, place the photolithographic microcolumn silicon wafer into the dish, add a small amount of octadecyltrichlorosilane (OTS) into the dish, put it into an oven and bake it at 150 ° C for 120 minutes. After cooling, put it into a vacuum desiccator for later use.

[0060] 4. Preparation of PDMS Microporous Positive Mold

[0061] (1) Preparation of PDMS: PDMS prepolymer and curing agent (SYLGARD 184) were mixed in a weight ratio of 10:1. Then, 10% of the total weight of PDMS in n-hexane was added and stirred thoroughly until dense, fine bubbles formed. A vacuum pump was then used to remove the bubbles. Vacuum degassing should be maintained in a sealed container for 30 min to avoid PDMS overflow due to vigorous vacuuming.

[0062] (2) PDMS pouring and curing: Pour the vacuum-degassed PDMS slowly onto the silicon wafer surface, use a vacuum pump to remove the tiny bubbles generated by pouring, and maintain for 30 minutes. Then, cure it in an oven at 90°C for 2 hours. After the PDMS cools down, use a scalpel to cut it into the desired size. Use pointed tweezers to gently grasp one side of the PDMS and peel it off from the silicon wafer surface to obtain the PDMS microporous negative mold.

[0063] 5. Hydrophobic Treatment of PDMS Microwell Array

[0064] Place the PDMS microporous negative mold into the O2-Plasma plasma cleaning instrument, turn on the vacuum pump and evacuate to saturation, then introduce oxygen for 10 minutes, maintain the vacuum degree at 0.3-0.4 mbar, press the "Generator" button to generate a glow, and maintain this state for 5 minutes to complete the hydrophilic modification of the PDMS microporous negative mold surface.

[0065] The PDMS microporous female mold was placed in a vacuum desiccator, and a small amount of 1H,1H,2H,2H-heptadecafluorodecyltrimethoxysilane was added. After vacuum degassing, the mixture was allowed to stand overnight to ensure that the decane gas in the desiccator reached a saturated vapor state.

[0066] 6. Preparation of PDMS Static Micropillars (MP)

[0067] After vacuum degassing, slowly pour the PDMS onto the surface of the PDMS microporous negative mold. Use a vacuum pump to remove small bubbles generated by pouring, and hold for 30 minutes. Then, oven-cure at 90°C for 2 hours. After the PDMS cools, use a scalpel to cut it to the appropriate size. Use pointed tweezers to gently grasp one side of the PDMS and peel it off from the surface to obtain the PDMS micropillar array.

[0068] 7. Preparation of PDMS Magnetic Micropillar Array (MMP)

[0069] Place the PDMS micropore negative mold horizontally on the bottom of a 3 cm plastic dish, add a small amount of nano-iron powder until it completely covers the top of the micropore negative mold, place a circular magnet with a diameter of 2 cm (N52 neodymium magnet, surface field of approximately 3000 Gs) on the bottom of the plastic dish and move it back and forth at least 5 times to ensure that the iron powder completely enters the micropores, and observe the distribution of the iron powder using a metallographic microscope. When it is observed that the iron powder has been completely and evenly incorporated into the pores, use a disposable cotton swab to wipe off excess iron powder on the surface.

[0070] The vacuum-degassed PDMS was slowly poured onto the surface of the PDMS microporous negative mold, which had been doped with nano-iron powder. A magnet was placed underneath to attract the nano-iron powder to rearrange itself and allowed to stand for 24 hours. This prevented the nano-iron powder from floating up due to secondary vacuum extraction and reduced the formation of cavities caused by tiny bubbles trapped in the micropores. After the standing time was complete, the mold was placed in an oven at 90°C for 2 hours to ensure complete curing of the PDMS. After the PDMS cooled, it was cut to the appropriate size using a scalpel. Using pointed tweezers, one side of the PDMS was gently grasped and peeled off from the surface to obtain a PDMS magnetically controlled micropillar array.

[0071] Example 2

[0072] Please refer to Figure 2 The present invention uses an environmental field emission scanning electron microscope to observe the surface morphology of static micropillars (MP) / dynamic magnetically controlled micropillars (MMP).

[0073] Environmental field scanning electron microscopy (SEM): PDMS micropillar and PDMS magnetron micropillar samples were placed on the sample stage and then sprayed with gold for 90 seconds. The sample images were magnified by 2000 and 4000 times using SEM, respectively, to observe the surface morphology of different samples.

[0074] The results show that the MP microcolumns and MMP microcolumns have high fidelity, and the only difference between them is the presence or absence of iron powder doping.

[0075] Example 3

[0076] Please refer to Figure 3 The present invention uses an atomic force microscope to measure the Young's modulus of static micropillars (MP) and dynamic magnetically controlled micropillars (MMP).

[0077] PeakForce QNM mode was used to scan the surface morphology and modulus of the micropillars (n=6) in different areas of the sample with a size of 500nm×500nm at the top. The scanning rate was 1Hz and the resolution was 256. The modulus vector diagram was drawn and normalized using NanoScopeAnalysis software. The corresponding average modulus size was calculated for different areas of the sample.

[0078] The results showed that there was no significant difference in the modulus between MP microcolumns and MMP microcolumns.

[0079] Example 4

[0080] Please refer to Figure 4 The present invention uses a contact angle meter to measure the contact angle of static micropillars (MP) / dynamic magnetically controlled micropillars (MMP).

[0081] This experiment uses a contact angle meter, recording the falling water droplets using continuous shooting. The continuous shooting frame rate is set to 10 fps, the continuous shooting time is set to 5 seconds, and the droplet volume is set to 1 μL per drop. The micropillar array is placed on the contact angle table with the front side facing up. The coarse knob is turned to raise the table to ensure that the water droplet lands precisely above the micropillars. The image is immediately frozen after the droplet is received. The contact angle is manually measured using the ring method. Each group of micropillars has at least three samples, and three areas of each sample are measured. The contact angle data is averaged from each group.

[0082] The results showed that there was no significant difference in the hydrophilicity and hydrophobicity between MP microcolumns and MMP microcolumns.

[0083] Example 5

[0084] Please refer to Figure 5 In order to determine the iron powder doping content inside the dynamic magnetic microcolumn (MMP), the present invention uses SEM combined with EDS to analyze the element content inside the sample. Static microcolumn / dynamic magnetic microcolumn

[0085] A PDMS magnetically controlled microcolumn sample was placed on a sample stage and then gold-sprayed for 90 seconds. An environmental field scanning electron microscope (SEM) coupled with EDS was used to magnify the sample image by 2000 and 4000 times, respectively, to observe the element mapping of different samples.

[0086] The results show that there is no significant difference in the iron powder doping content of MMP microcolumns with different spacing.

[0087] Example 6

[0088] Please refer to Figure 7 、 8 9. In order to determine the degree of bending angle of dynamic magnetically controlled micropillars (MMPs) driven by an external magnetic field, the present invention uses a metallographic microscope to measure the bending angle of the micropillars and verify their stability.

[0089] Using a metallographic microscope, we characterized the driving effect of the magnetically driven micropillars by photographing the bending of the micropillars under different magnetic fluxes.

[0090] The results show that the bending angle of the micro-pillars with a 5μm spacing is the largest at a distance of 1mm (magnetic field strength of 396mT), which is 26.4°. As the distance increases, the bending angle gradually decreases, and the bending angle at a distance of 10mm (magnetic field strength of 80mT) is 4.5°; the bending angle of the micro-pillars with a 10μm spacing is the largest at a distance of 1mm, which is 26.6°. As the distance increases, the bending angle gradually decreases, and the bending angle at a distance of 10mm is 4.2°; the bending angle of the micro-pillars with a 15μm spacing is the largest at a distance of 1mm, which is 26.2°. As the distance increases, the bending angle gradually decreases, and the bending angle at a distance of 10mm is 4.2°.

[0091] The results show that the pitch of the micropillar array does not affect the bending angle.

[0092] Example 7

[0093] Please refer to Figure 10 In order to determine the effects of static micropillars (MP) / dynamic magnetic micropillars (MMP) on focal adhesion of MC3T3-E1 cells, the present invention used a laser confocal microscope to perform immunofluorescence assay on the expression of focal adhesion.

[0094] 12 and 72 hours after seeding the microcolumns with MC3T3-E1 cells, remove the 96-well plate from the cell culture incubator to terminate the culture. Discard any remaining culture medium and rinse the wells two to three times with PBS buffer to remove unattached cells. Add 100 μL of 4% paraformaldehyde to each well and soak for 10 minutes to fix cell morphology. Rinse the samples three times with PBS buffer for 5 minutes each. Add 100 μL of 0.5% Triton X-100 to each well and soak for 10 minutes to enhance antibody permeability to the cell membrane. Rinse the samples three times with PBS buffer for 5 minutes each. Add 100 μL of 5% BSA solution to each well and soak for 1 hour to prevent nonspecific antibody binding. Dilute the vinculin antibody and phalloidin antibody at a 1:200 ratio in 5% BSA solution. Add 100 μL of the diluted vinculin antibody solution to each well and incubate overnight at 4°C in the dark. After 12 hours, remove the 96-well plate and rewarm for 45 minutes before recovering the focal adhesion antibody solution. Wash three times with PBS buffer for 5 minutes each. Prepare a 1:500 goat anti-rabbit solution, add 100 μL to each well, and incubate at room temperature in the dark for 2 hours. Aspirate the solution, recover the solution, and wash three times with PBS buffer for 5 minutes each. Prepare 5 μg / mL Hochest 33258 dye, add 100 μL to each well, and incubate at room temperature for 5 minutes. Wash three times with PBS buffer for 5 minutes each. Add 1-2 drops of anti-quenching agent to the coverslip. Use bent-tip tweezers to grasp the micropillars on both sides and remove them from the plate. Gently invert the micropillars onto the coverslip and photograph using a 100X confocal microscope.

[0095] The results showed that vinculin was specifically enriched at stress fiber anchoring points, and the spatial Pearson colocalization coefficient with F-actin was >0.84, confirming the activation of the mechanical force transduction pathway.

[0096] Example 8

[0097] Please refer to Figure 11 、 12 , 13, 14. In order to determine the effects of static micropillars (MP) / dynamic magnetic micropillars (MMP) on the cytoskeleton of MC3T3-E1 cells, the present invention used a laser confocal microscope to perform immunofluorescence assay on the expression of F-actin.

[0098] 12 and 72 hours after seeding the microcolumns with MC3T3-E1 cells, remove the 96-well plate from the cell culture incubator to terminate the culture. Discard any remaining culture medium and rinse the wells two to three times with PBS to remove unadhered cells. Add 100 μL of 4% paraformaldehyde to each well and soak for 10 minutes to fix cell morphology. Rinse the samples three times with PBS for 5 minutes each. Add 100 μL of 0.5% Triton X-100 to each well and soak for 10 minutes to enhance antibody permeability to the cell membrane. Rinse the samples three times with PBS for 5 minutes each. Add 100 μL of 5% BSA solution to each well and soak for 1 hour to prevent nonspecific antibody binding. Dilute phalloidin 1:200 in 5% BSA solution. Add 100 μL of this diluted phalloidin solution to each well and incubate overnight at 4°C in the dark. After 12 hours, remove the 96-well plate and rewarm for 45 minutes before recovering the phalloidin solution. Wash three times with PBS buffer for 5 minutes each. Prepare 5 μg / mL Hochest 33258 dye, add 100 μL to each well, and incubate at room temperature for 5 minutes. Wash three times with PBS buffer for 5 minutes each. Add 1 to 2 drops of anti-quenching agent to the coverslip. Use elbow-tipped tweezers to grasp the microcolumns on both sides and remove them from the plate. Gently invert the microcolumns onto the coverslip and photograph using a 100X confocal microscope.

[0099] The results showed that cytoskeleton reconstruction was stimulation-dependent: the MP group mainly guided cytoskeleton reorganization through topological morphology, while the MMP group synergistically regulated through dynamic magnetomechanical stimulation, inducing significant thickening of stress fibers and forming a strengthened cytoskeleton network. The stress fiber reorganization effect of the 10μm group cells was the most significant.

[0100] Example 9

[0101] Please refer to Figure 15 、 16 In order to determine the osteogenic differentiation ability of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of static micropillars (MP) / dynamic magnetic micropillars (MMP), the present invention used an inverted microscope to stain the cell mineralized calcium nodules on the inoculated micropillars, verifying the excellent osteogenic differentiation level of the dynamic magnetic micropillars.

[0102] MC3T3-E1 cells were seeded at a density of 10,000 cells / well on various substrates (flat, PDMS static micropillars, and PDMS magnetic micropillars) in 96-well plates. After 7 and 14 days of cell culture, the 96-well plates were removed from the cell culture incubator and the cultures were terminated. The old culture medium was aspirated, and the cells were washed three times with PBS buffer. Afterward, 100 μL of fixative solution was added to each well to fix the cell morphology for 10 minutes. Subsequently, 100 μL of Alizarin S staining solution (2%, pH 4.2) was added to each well and incubated at room temperature for 30 minutes. After three washes with deionized water buffer, the cells were photographed using an inverted microscope with a 20X lens. After imaging, 200 μL of 10% cetylpyridinium chloride was added to each well to dissolve the calcium nodules, and the OD values were measured at a wavelength of 590 nm using a microplate reader for semi-quantitative analysis.

[0103] The results showed that the microcolumn materials used in the experiment all had a certain effect in promoting cell osteogenic differentiation. The osteogenic differentiation effect of magnetically driven microcolumns with MMP was better than that of static microcolumns. Among them, the differentiation-promoting effect of microcolumns with a spacing of 10 μm was the best. The topological structure changes caused by the microcolumn spacing may affect the efficiency of mechanical signal transduction by regulating the cytoskeleton tension.

[0104] Example 10

[0105] Please refer to Figure 17 、 18 In order to determine the osteogenic differentiation ability of MC3T3-E1 (mouse osteoblast precursor cells) cultured on the surface of static micropillars (MP) / dynamic magnetically controlled micropillars (MMP), the expression of the osteogenic differentiation marker Runx2 was determined by immunofluorescence using a laser confocal microscope.

[0106] MC3T3-E1 cells were seeded at a density of 10,000 cells / well in 96-well plates (2D), flat substrates, MP micropillars, and MMP micropillars. After 7 and 14 days of culture, the cells were removed from the incubator to terminate the culture. Rabbit Anti-Runx2 was used as the primary antibody, and the staining method was the same as in Example 8.

[0107] The results are the same as those in Example 9.

[0108] Example 11

[0109] Please refer to Figure 19 、 20 In order to determine the osteogenic differentiation ability of bMSC (mouse bone marrow mesenchymal stem cells) cultured on the surface of static micropillars (MP) / dynamic magnetic control (MMP) micropillars, the present invention used an inverted microscope to stain the alkaline phosphatase of the cells inoculated on the micropillars, verifying the excellent osteogenic differentiation level of the dynamic magnetic control micropillars.

[0110] bMSC cells were seeded at a density of 15,000 cells / well on different substrates (flat, PDMS static microcolumns, PDMS magnetic microcolumns) and placed in a 96-well plate. After 7 and 14 days of cell culture, the 96-well plate was removed from the cell culture incubator and the culture was terminated. The old culture medium was aspirated, washed three times with PBS buffer, and 100 μL of 4% paraformaldehyde was added to each well to fix the cell morphology for 10 minutes. Then 100 μL of BCIP / NBT alkaline phosphatase detection solution was added to each well and incubated at room temperature for 30 minutes. Then it was washed three times with deionized water buffer and photographed using an inverted microscope with a 20X lens.

[0111] The results were similar to those of MC3T3-E1, confirming that this material system has the ability of mechanical biological regulation across cell lineages, providing theoretical support for the development of universal bone repair scaffolds.

[0112] Example 12

[0113] Please refer to Figure 20 、 21 In order to determine the osteogenic differentiation ability of bMSC (mouse bone marrow mesenchymal stem cells) cultured on the surface of static micropillars (MP) / dynamic magnetically controlled micropillars (MMP), the expression of osteogenic differentiation marker Runx2 was determined by immunofluorescence using a laser confocal microscope.

[0114] bMSCs were seeded at a density of 15,000 cells / well in 96-well plates (2D), flat substrates, MP microcolumns, and MMP microcolumns. After 7 and 14 days of culture, the cells were removed from the incubator to terminate the culture. Rabbit Anti-Runx2 was used as the primary antibody, and the staining method was the same as in Example 8.

[0115] The results are the same as in Example 11.

Claims

1. A magnetic drive component for enhancing the osteogenic differentiation of stem cells in vitro, its preparation method, and application patent. Its characteristics include the following steps: 1) Cut commercially available silicon wafers into 2cm*2cm sizes, place them in a silicon wafer cleaning solution and heat them in an oil bath, then remove the surface cleaning solution and dry them; 2) treating the silicon wafer with an oxygen-plasma cleaning apparatus to complete surface hydrophilic modification; 3) Place the hydrophilic silicon wafer on a heating plate to remove surface moisture; 4) Spin-coat SU-8 photoresist using a spin coater; 5) using a hot plate to bake the photoresist before exposure; 6) Exposure using a maskless laser direct writing lithography machine; 7) using a hot plate to perform post-exposure baking on the photoresist; 8) Soaking in a developer to remove unexposed photoresist; 9) Drying the photolithographic silicon wafer to obtain a micropillar array; 10) After the micropillar array is subjected to hydrophobic treatment, PDMS is poured and baked and cured to obtain a PDMS microwell array; 11) After the micropore array is hydrophobically treated, PDMS and PDMS mixed with nano-iron powder are cast respectively, baked and cured, and demolded to obtain PDMS static micropillars (MP) and PDMS dynamic magnetically controlled micropillars (MMP).

2. A magnetic drive component for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 1), the cleaning solution is prepared by using concentrated H2SO4 and H2O2 in a mass ratio of 7:3, and the volume used is 20 to 100 mL.

3. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 1), the oil bath heating temperature is 95° C., and the cleaning time is 30 to 60 minutes.

4. A magnetic drive component for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 1), the solution for removing the silicon wafer cleaning solution is 20-100 mL of deionized water and 20-100 mL of isopropyl alcohol.

5. A magnetic drive component for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 2), the power of the plasma cleaner was set to 90%, the oxygen flow was maintained at 3.5-4.0 mbar, and the treatment time was 10 minutes.

6. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 3), the heating device used is a digitally controlled heating plate, the temperature is controlled to 150° C., and the duration is 10 to 15 minutes.

7. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and its application patent as claimed in claim 1, characterized in that In step 4), the first speed of the spin coater is 500 rpm for 9 s, and then the second speed is set to 2900 rpm for 30 s to obtain the preset microcolumn height.

8. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 5), the heating plate temperature is set to 65° C. for 2 to 3 minutes, and then to 95° C. for 3 to 4 minutes.

9. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 6), the exposure dose of the lithography machine is 145mJ / cm 2 ,The exposure size is 4mm*4mm, and the exposure dose correction is 8μm.

10. A magnetic drive component for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 7), the temperature of the heating plate is set to 95° C., and the heating time is 3 to 4 minutes.

11. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 8), the developer is PGMEA, and the immersion time is 3 to 4 minutes.

12. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 9), the drying process includes cleaning with isopropyl alcohol and drying the silicon wafer with an ultrapure nitrogen gun.

13. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and application patent as claimed in claim 1, characterized in that In step 10), the hydrophobic treatment is performed by dropping OTS solution (octadecyltrichlorosilane) on the periphery of the silicon wafer, controlling the oven temperature at 150° C., and heating the wafer for 90 to 120 minutes.

14. A magnetic drive assembly for enhancing osteogenic differentiation of stem cells in vitro, its preparation method, and its application patent as claimed in claim 1, characterized in that In step 11), the hydrophobic treatment is performed by dropping 1H,1H,2H,2H-heptadecafluorodecyltrimethoxysilane solution around the PDMS micropores, placing the solution in a vacuum desiccator, and vacuum degassing for 8 to 12 hours.

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