Preparation method of anisotropic microgel for regulating and controlling cell mechanics

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of anisotropic microgel for regulating and controlling cell mechanics. According to the anisotropic microgel and the preparation method thereof, mechanical and biological signal crosstalk can be formed through long-axis polarity and cell membrane-intracellular skeleton-cell nucleus, the adhesion, proliferation and anti-apoptosis capabilities of nucleus pulposus cells are improved, the stability of the nuclear membrane is improved, DNA damage and leakage are reduced, cGAS-STING expression is down-regulated, the young state level of the loaded nucleus pulposus cells is maintained, and the anisotropic microgel can be used for preparing the medicine for treating the nucleus pulposus cells. Extracellular matrix metabolism unbalance in intervertebral disc degeneration is corrected, secretion of various inflammatory factors and protease is reduced, and the pathological process of intervertebral disc degeneration is delayed.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the preparation of anisotropic microgel for regulating cell mechanical mechanics. Background Art

[0002] Extracellular matrix (ECM) biological signal transduction refers to the dynamic process by which cells and adhesion tissues convert physical, biochemical and other information in the external environment into biological signals inside cells through various pathways. Compared with biochemical information transmission such as ligand-receptor, cells have a stronger perception and response sensitivity to the regulation and adaptive changes of peripheral physical and mechanical factors, which is crucial for the overall maintenance of various physiological functions of the tissue and the delay of aging and decline. The intervertebral disc (IVD) is a stress-transmitting tissue that bears the weight and buffers the body's trunk. The long-term ischemic and hypoxic environment caused by the lack of blood vessel growth makes the intervertebral disc considered to be the first organ in the human body to age. With the loss of ECM water content and the reduction of proteoglycan synthesis, nucleus pulposus cells (NPs) highly express the senescence-associated secretory phenotype (SASP), and the secretion of inflammatory factors and matrix-degrading enzymes such as tumor necrosis factor (TNF-α), interleukin (IL) 1β, matrix metalloproteinases (MMPs), and adipose-like metalloproteinases (ADAMTS) increases, promoting aseptic chronic inflammation, positive feedback inducing NPs aging or apoptosis, and accelerating the pathological progression of intervertebral disc degeneration (IVDD). At present, in the research of tissue engineering, the local microenvironment is mainly regulated by functionalized biological information delivery carriers to promote the structural and functional recovery of IVD, but the diffusion and inactivation of biological information are inevitable. Directly editing the anti-aging behavior of NPs through the inherent mechanical and physical factors of biomaterials, and in-depth exploration of the biological mechanism of related external ECM physical signals and internal cell information transduction will undoubtedly bring new hope for delaying the aging process of NPs and even reversing IVDD.

[0003] The physical and mechanical factors of ECM include stiffness, viscoelasticity, dimension, pressure, etc. However, the research on mechanical signal transduction has long faced many challenges, such as: the exploration of the mechanical properties of viscoelasticity is not intuitive enough; compression and stretching may cause forced pathological changes in cells; directly interfering with or enhancing the signal transduction pathway cannot fully verify the impact of the physical and chemical properties of ECM. Recently, the research field focusing on the main physical characteristics of external ECM - three-dimensional geometric morphology has been widely recognized and valued by the academic community. Zahler et al. found that micropatterns with different geometric shapes may significantly regulate the proliferation activity of Human Umbilical Vein Endothelial Cells (HUVEC) through the Myocardin-related transcription factor (MRTF)-Serum response factor (SRF) and Hippo-Yes-associated protein (YAP) signaling axis; Zhang et al. further found that the focal adhesion (FA) at the edge of the long axis of the cuboid is denser than that of the cube, proving that the three-dimensional geometric morphology of cells with anisotropy and polar spreading may have stronger mechanical signal transduction ability and proliferation activity; In addition, some scholars have found that whether it is a single cell or a cell group, tissue or organism responds to geometric properties such as ECM curvature by adjusting its shape and migration behavior, which may be through the Integrin β1-Focal adhesion kinase (FAK) axis to regulate cell adhesion to perceive changes in external geometric information. However, restricting cells to the three-dimensional space of the overall biomaterial lacks the exploration of the active adaptation process of cells, and the existing biomaterials for the study of three-dimensional geometric morphology are also difficult to achieve the exploration of the overall behavior of multi-cell groups. Therefore, exploring the potential NPs morphology-micromechanics-biological activity transduction effects and mechanisms based on biomaterials with strong independence and controllable ECM geometric structures will be more helpful in developing therapeutic strategies to combat IVD aging by utilizing ECM geometric properties.

[0004] The intervertebral disc is mainly a closed body composed of upper and lower cartilage endplates, external annulus fibrosus and internal nucleus pulposus tissue. The narrow working space makes injectable nanoparticles (Nano-particles) and hydrogel perfusion the preferred choice for the treatment of IVDD. However, the small particle size of Nano-particles and the amorphous morphology of hydrogels make it difficult to study the geometric properties of ECM by regulating NPs. The injectable micron-sized gel particles prepared by microfluidic technology have more intuitive and controllable three-dimensional morphology and mechanical properties, and are more suitable for exploring the advantages of NPs morphological changes. It has become another excellent choice for improving the microenvironment of tissue repair, and is also considered to be an ideal process for exploring the interaction between multiple cells and ECM. However, the anisotropic microgels currently developed are mostly directly cross-linked on the chip surface. The extremely high cross-linking dependence and viscosity are likely to face the challenges of chip flow channel blockage and difficulty in batch preparation; and the main components of the gel are mostly synthetic organic matter, lacking the simulation of natural ECM. Therefore, how to improve the current microfluidic process for preparing microgel particles is crucial to further explore how anisotropic microgels promote IVDD regeneration by maintaining the "youthful state" of NPs through mechanical transduction pathways. Summary of the invention

[0005] In view of this, the present invention provides a method for preparing anisotropic microgels for regulating cell mechanics, and the prepared anisotropic microgels can delay the pathological process of IVDD.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention also provides a method for preparing microgel based on a microfluidic chip, wherein the dispersed phase is not photocured before flowing out of the chip, and is photocured in a silicone tube after flowing out of the chip.

[0008] In some specific embodiments of the present invention, the above method comprises the following steps:

[0009] S1, passing the continuous phase and the dispersed phase through a dual-channel PDMS microfluidic chip with an inner diameter of 100 μm, and not photocuring the dispersed phase before it flows out of the dual-channel PDMS microfluidic chip;

[0010] S2. Connect a silicone tube to the outlet of the dual-channel PDMS microfluidic chip, and photo-cure the dispersed phase flowing through the silicone tube to obtain a microgel.

[0011] In some specific embodiments of the present invention, the flow rate ratio of the dispersed phase to the continuous phase in the above method is 2: (2-24) μL / min.

[0012] In some specific embodiments of the present invention, the dispersed phase in the above method comprises 7% (w / v) GelMA solution, 0.5% (w / v) photoinitiator and PBS.

[0013] In some specific embodiments of the present invention, the continuous phase in the above method is an isopropyl myristate solution containing 10% (w / w) Span80.

[0014] The present invention provides anisotropic microgel, which is a rod-shaped microgel with a length of 360-420 μm, which can be 370 μm, 375 μm, 380 μm, 385 μm, 390 μm, 395 μm, 400 μm, 405 μm, 410 μm or 415 μm; and a diameter of 90-110 μm, which can be 92 μm, 94 μm, 96 μm, 98 μm, 100 μm, 102 μm, 104 μm, 106 μm or 108 μm.

[0015] In some specific embodiments of the present invention, the anisotropic microgel is mainly composed of methacryloyl gelatin (GelMA).

[0016] In some specific embodiments of the present invention, the anisotropic microgel is prepared by the above method.

[0017] The present invention also provides the use of anisotropic microgel in any of the following items:

[0018] (1) Increase the expression of Integrinβ1, β-tubulin, Vinculin and / or Myosin;

[0019] (2) increasing the expression of YAP, TAZ, Lamin A / C and / or Lamin B1;

[0020] (3) enlarge nuclear pores and / or promote the nuclear expression of YAP;

[0021] (4) Increase the proportion of Ki-67+ cells;

[0022] (5) Enhanced expression of p-PI3K and / or p-AKT;

[0023] (6) Reduce Caspase3 expression;

[0024] (7) Increase the expression of BCL-2;

[0025] (8) Increase the relative fluorescence intensity of JC-1;

[0026] (9) Improve the tolerance of nucleus pulposus cells to oxidative stress damage;

[0027] (10) Improving the glycolytic function and / or glycolytic capacity of nucleus pulposus cells;

[0028] (11) Reduce the expression of MMP13;

[0029] (12) increasing the expression of CTGF, ACAN and / or COL-II;

[0030] (13) Reduce the expression of SA-β-Gal, P16 INK4a, STING, IL-6, IL-1β, P53, P21, P16, cGAMP, γ-H2AX and / or TNF-α.

[0031] The present invention also provides the use of anisotropic microgel in any of the following items:

[0032] (i) promoting the proliferation of nucleus pulposus cells and / or inhibiting the aging of nucleus pulposus cells;

[0033] (ii) improve disc collagen remodeling and inhibit disc extracellular matrix degradation;

[0034] (iii) preventing the decrease of disc water content and / or preventing disc collapse;

[0035] (iv) Delaying intervertebral disc degeneration.

[0036] The present invention also provides the use of anisotropic microgel in any of the following items:

[0037] (I) preparing products that promote proliferation of nucleus pulposus cells and / or inhibit aging of nucleus pulposus cells;

[0038] (II) preparing products that improve intervertebral disc collagen remodeling and inhibit intervertebral disc extracellular matrix degradation;

[0039] (III) preparing a product for preventing the intervertebral disc from decreasing in water content and / or preventing the intervertebral disc from collapsing;

[0040] (IV) Preparation of products for delaying intervertebral disc degeneration.

[0041] In some specific embodiments of the present invention, the anisotropic microgel used in the above application is a rod-shaped microgel with a length of 360-420 μm, which can be 370 μm, 375 μm, 380 μm, 385 μm, 390 μm, 395 μm, 400 μm, 405 μm, 410 μm or 415 μm; a diameter of 90-110 μm, which can be 92 μm, 94 μm, 96 μm, 98 μm, 100 μm, 102 μm, 104 μm, 106 μm or 108 μm.

[0042] The present invention also provides a medicine comprising the anisotropic microgel and acceptable adjuvants or auxiliary agents.

[0043] The present invention also provides a culture medium comprising the anisotropic microgel.

[0044] The present invention also provides a method for treating a disease, which is based on the above-mentioned anisotropic microgel or drug treatment;

[0045] The disease includes at least one of intervertebral disc herniation, intervertebral disc degeneration, intervertebral disc inflammation, and intervertebral disc-induced low back pain.

[0046] The present invention has the following effects:

[0047] This application constructs traditional MicroSphere and anisotropic Rod-Shaped-Microgels (MicroRod) through improved microfluidic technology, and finds that MicroRod forms mechanical and biological signal crosstalk with cell membrane-intracellular skeleton-nucleus through long-axis polarity, thereby improving NPs adhesion, proliferation and anti-apoptosis ability. Anisotropic microgels trigger YAP / TAZ (Yes-associated protein / Tafazzin) to improve nuclear membrane stability, reduce DNA damage leakage, downregulate cGAS-STING expression, maintain the "youthful" level of loaded NPs, correct ECM metabolic imbalance in IVDD, reduce the secretion of multiple inflammatory factors and proteases, and delay the pathological process of IVDD.

[0048] This application not only provides a new biological material platform for exploring the impact of ECM three-dimensional spatial geometry on mechanical signal transduction in cell groups, but also provides new theoretical support for mechanical mechanics to improve cell activity, delay cell aging, and promote regeneration of aged tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0050] Figure 1 Schematic diagram showing how anisotropic microgels maintain the "youth" of cells through mechanical transduction pathways to promote the regeneration of aged tissues, wherein A shows the preparation process of anisotropic microgels, B shows the general morphology of cell-laden microspheres and microrods, and C shows the interaction mechanism between mechanical signal transduction and cytoskeleton and nuclear mechanics;

[0051] Figure 2: shows the preparation and characterization of anisotropic microgels, wherein A shows a schematic diagram of an improved microfluidic chip and an overall schematic diagram of a local flow channel, B shows the adjustment of the long axis diameter of microrods by stabilizing the flow rate of the aqueous phase and adjusting the flow rate of the oil phase, C shows a scanning electron microscope (SEM) image, D shows the diameter of the microspheres, E shows the length of the microrods, F shows infrared analysis, G shows energy dispersive spectrum (EDS) analysis, H shows Fourier transform infrared (FTIR) spectrum, I shows atomic force microscope (AFM) images of microspheres and microrods, J shows the statistics of Young's modulus of two microgels, and K shows the overall morphology of two microgels under an optical microscope;

[0052] Figure 3 Figure 2 shows the cell adhesion and skeleton spreading of anisotropic microgels, where A shows F-actin immunofluorescence staining at 3 and 7 days (the direction of the yellow arrow indicates that the anisotropic microgel-loaded NPs spread along the long axis of the rod), B shows Integrin immunofluorescence, C shows F-actin immunofluorescence quantification, D-E shows WB detection and semi-quantitative analysis of adhesion-related protein molecules, F shows Integrin semi-quantitative analysis, G-H shows β-catenin immunofluorescence and quantification, I-J shows Vinculin immunofluorescence and quantification, K-L shows cytoskeleton-related protein expression and semi-quantitative analysis, and M-N shows Myosin immunofluorescence and semi-quantitative analysis;

[0053] Figure 4 Figure 2 shows that anisotropic microgels regulate nuclear mechanics and nuclear shape remodeling, where A shows YAP immunofluorescence staining, B shows semi-quantitative analysis of YAP nucleocytoplasmic ratio, C-D shows Lamin A / C fluorescence staining and semi-quantitative analysis (yellow arrows mark F-actin indentation), E shows the degree of nuclear pore opening observed by biological transmission electron microscopy (red arrows mark nuclear pores), F shows quantification of nuclear pore size, G-H shows the expression of proteins related to nuclear mechanics, I-J shows TAZ immunofluorescence staining and semi-quantitative analysis, and K shows a schematic diagram of the interaction mechanism between ECM and nuclear mechanics;

[0054] Figure 5Figure 2 shows RNA sequencing analysis of H2O2 treated MicroRod and MicroSphere co-cultured with nucleus pulposus cells, where A shows a heat map of differentially expressed genes, B shows the 15 major KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of DEGs in Cellular Processes and Environmental Information Processing, C shows a representative GO (Gene Ontology) analysis related to cell adhesion, skeletal regulation, signal transduction, and important physiological functions, and D shows the possible biological function changes of nucleus pulposus cells on anisotropic microgels suggested by RNA sequencing analysis;

[0055] Figure 6 Figure 2 shows the investigation of the proliferation ability of anisotropic microgel-loaded cells, where A shows a schematic diagram of the co-culture system of MicroSphere, MicroRod and NPs oxidative stress, B-C show the flow cytometry detection and semi-quantitative analysis of Ki-67 protein, D shows the CCK-8 detection at 3 and 7 days, E-F show live-dead staining, G-H show the expression of PI3K-AKT pathway proteins and semi-quantitative analysis, I shows the CCK-8 detection after cytochalasin D intervention, and J-K show the expression of PI3K-AKT pathway proteins after cytochalasin D intervention;

[0056] Figure 7 Figure 2 shows the anti-apoptosis ability of anisotropic microgel-loaded NPs, where A-B show WB of important apoptosis-related molecules, C-D show JC-1 staining and quantification, E show MitoSox flow cytometry, F-G show MitoTracker-Red staining and semi-quantitative analysis, H show mitochondrial morphology, I show cell energy metabolism analysis, J-K show COL-II and MMP13 staining, L-M show COL-II and MMP13 staining, N-O show WB detection of ECM synthesis and degradation-related protein expression, P-Q show the expression of important apoptosis-related molecule proteins after cytochalasin D blocked mechanical sensing;

[0057] Figure 8Figure 2 shows the study of anisotropic microgels maintaining nuclear membrane integrity and delaying aging, where A-B show SA-β-Gal (Senescence-Associated β-Galactosidase) staining and semi-quantitative analysis after H2O2 intervention, C-H show P16 INK4a, STING, TNF-α immunofluorescence staining and semi-quantitative analysis, I-J show Lamin A / C immunofluorescence staining and semi-quantitative analysis (yellow arrows represent the filamentous indentations produced by F-actin compressing the nuclear membrane), K shows the protein expression of molecules related to aging, nuclear membrane integrity, and DNA damage, L-M show IL-6 immunofluorescence staining and semi-quantitative analysis, N shows WB protein expression semi-quantitative analysis of molecules related to aging, nuclear membrane integrity, and DNA damage, O shows ELISA detection of cGAMP content, P shows cGAMP content after cytochalasin D intervention, Q-R show Lamin B1 and STING protein expression and semi-quantitative analysis after cytochalasin intervention, S-T show IL-6 and MMP13 protein expression and semi-quantitative analysis after C-176 inhibition;

[0058] Fig. 9 Figure 2 shows the imaging and histological analysis of cell-laden anisotropic microgel injection therapy, wherein A shows the schematic diagram of IVDD modeling and MicroRod and MicroSphere injection therapy, B shows DHI analysis, C shows X-ray analysis, D shows in vivo imaging of small animals after Cy5 labeling of two types of GelMA microgels, E to F show MRI analysis, G shows H&E staining, H shows histological grading, I shows red / fast green staining, J shows TUNEL fluorescence staining, and K shows TUNEL fluorescence semi-quantitative analysis;

[0059] Fig.10 Figure 2 shows the in vivo ECM metabolism and aging after cell-loaded anisotropic microgel injection therapy, where A shows COL-II fluorescence staining, B shows MMP13 fluorescence staining, C shows SA-β-gal tissue staining, D shows IL-6 fluorescence staining, E shows semi-quantitative analysis of COL-II fluorescence staining, F shows semi-quantitative analysis of MMP13 fluorescence staining, G shows the percentage of SA-β-gal+ cells, and H shows semi-quantitative analysis of IL-6 fluorescence staining. DETAILED DESCRIPTION

[0060] The present invention discloses a preparation of anisotropic microgels for regulating cell mechanical mechanics. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] Specifically, one implementation of the present application is:

[0062] 1. Preparation and physicochemical characterization of anisotropic microgels

[0063] (1) Preparation of MicroRod and MicroSphere: Based on previous research, GelMA hydrogel microrods MicroRod were prepared by microfluidic chip extrusion technology and precise photocrosslinking. After photocrosslinking was cancelled, the microgel droplets rebounded into a spherical shape due to surface tension, and crosslinking was performed again to obtain GelMA hydrogel microspheres MicroSphere with the same volume of droplets.

[0064] (2) Characterization of the physical and chemical properties of MicroRod and MicroSphere gels: ① Scanning electron microscopy (SEM) and optical microscopy were used to observe the surface morphology and internal structure of the microgels, and to evaluate the distribution, diameter size, and aspect ratio of MicroRod and MicroSphere; ② Dynamic light scattering (DLS) was used to evaluate the surface charge of MicroRod and MicroSphere; ③ X-ray energy dispersive spectrometry (EDS) was used to detect the elemental differences between MicroRod and MicroSphere; ④ Atomic mechanical microscopy (AFM) was used to evaluate the elastic modulus and surface roughness of the microgels, and to draw force curves; ⑤ Fourier transform infrared absorption spectroscopy (FTIR) was used to detect the chemical groups of the microgels; ⑥ Water contact angle (WCA) was used to evaluate the hydrophilicity of MicroRod and MicroSphere; ⑦ The degradation rate of MicroRod and MicroSphere was calculated by PBS immersion method; ⑧ Optical microscopy was used to observe and compare the general flow diagram of microgels in the microfluidic chip under different flow rate ratios.

[0065] 2. In vitro study on the inhibition of nucleus pulposus cell senescence by mechanical reprogramming of anisotropic microgels

[0066] (1) Cytocompatibility: MicroRod and MicroSphere were co-cultured with nucleus pulposus cells for 3 and 7 days, respectively. The survival rate was evaluated by live-dead staining, cell proliferation was detected by CCK-8, and cell geometry and adhesion were observed by SEM. Ki67 flow cytometry was used to detect the proliferation ability of nucleus pulposus cells, and the state of nucleus pulposus cells after co-culture with MicroRod and MicroSphere microgels was photographed under a light microscope.

[0067] (2) Investigation of cytoskeleton spreading: MicroRod and MicroSphere were co-cultured with nucleus pulposus cells, and the morphology of F-actin, MYH, VCL, and Integrin was observed by immunofluorescence (IF), and semi-quantitative analysis was performed.

[0068] (3) Investigation of mechanical signal transduction and YAP nuclear entry: ① MicroRod and MicroSphere were co-cultured with nucleus pulposus cells, and the distribution of YAP and TAZ on the surface cells of the two microgels was observed by immunofluorescence (IF), the nuclear-cytoplasmic ratio of YAP and TAZ was calculated, and semi-quantitative analysis was performed; ② The morphology and strength of Lamin (laminin) A / C were observed by immunofluorescence staining to evaluate the state of the nuclear membrane complex after the mechanical tension of the skeleton was transmitted to the cell nucleus; ③ The nuclear morphology, the overlap ratio of cytoskeleton F-actin and the cell nucleus were observed by confocal microscopy, the F-actin orientation order parameters, and the nuclear shape factor were calculated to evaluate the association between skeleton stretching and nuclear morphological remodeling; ④ The degree of nuclear pore opening and size after co-culture of nucleus pulposus cells with Sphere and Rod microgels were evaluated by biological projection electron microscopy.

[0069] (4) Investigation of the inhibition of nucleus pulposus cell aging by anisotropic microgel particles: ① SA-β-gal staining was used to investigate the aging of MicroRod and MicroSphere scaffold-loaded cells; 200 μM H2O2 was added to the co-culture system and SA-β-gal staining was performed again for control; ② WB was used to detect the protein expression of aging-related markers such as P53, P21, and P16; PCR was used to detect the mRNA expression of aging-related markers such as P53, P21, and P16; co-culture immunofluorescence was used to detect the distribution and intensity of aging-related markers such as P53, P21, and P16 on microgel co-cultured cells; ③ WB was used to detect the expression of proteins related to extracellular matrix synthesis and degradation such as MMP13, ADAMTS-5, COLII, and AGGRECAN; co-culture immunofluorescence was used to detect the distribution and intensity of proteins related to extracellular matrix synthesis and degradation such as MMP13, ADAMTS-5, COLII, and AGGRECAN on microgel co-cultured cells; ④ Lamin B1 immunofluorescence staining was used to evaluate the integrity of the nuclear membrane of nucleus pulposus cells and observe the changes in nuclear mechanics and the interaction between the actin filaments of the cytoskeleton; the changes in the depression, wrinkling, lobulation, and protrusion of the MicroRod and MicroSphere cell nuclei under oxidative stress were evaluated; ⑤ the cGAMP content was evaluated by ELISA to analyze DNA leakage damage and STING activation.

[0070] 3. In vivo study of anisotropic microgels delaying IVDD

[0071] A rat model of intervertebral disc degeneration induced by acupuncture in the coccyx was established, divided into MicroRod, MicroSphere, sham operation (Sham) and blank control (BC). During the period, the animal's mental state, incision, activity and diet were closely observed, and blood routine, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and liver and kidney function were closely monitored. The animal ethics review regulations of the First Affiliated Hospital of Soochow University were strictly followed.

[0072] (1) Biosafety assessment: 7 days after surgery, heart, liver, spleen, lung, kidney, and brain tissue specimens were collected from SD rats and stained with hematoxylin-eosin (H&E) to systematically evaluate the safety of the composite fiber.

[0073] (2) IVDD microenvironment assessment: ① H&E staining was used to preliminarily evaluate the damage and repair of the nucleus pulposus; ② Red-fast green staining was used to evaluate the degradation of proteoglycans; ③ SA-β-gal staining was used to explore the aging of the nucleus pulposus; ④ Immunofluorescence detection of the expression of aging-related markers such as P53, P21, and P16; ⑤ Immunohistochemistry was used to detect the expression of inflammatory factors such as IL-6, TNF-α, and IL-1β; ⑥ Tissue IF (TUNEL) and FCM (Annexin-V / PI double staining) were used to detect cell apoptosis; ⑦ Immunofluorescence was used to detect the expression of proteins related to extracellular matrix synthesis and decomposition, such as MMP3, ADAMTS5, COLII, and AGGRECAN.

[0074] (3) Study on delaying NPs degeneration: ① Imaging: Magnetic resonance imaging (MRI) was performed on animals 7 days, 4 weeks, and 8 weeks after surgery to evaluate the nucleus pulposus grade, and X-ray was used to evaluate the disc height change (DHI); ② Histomorphology: Disc samples were collected 7 days, 4 weeks, and 8 weeks after surgery, and H&E staining was used to evaluate the gross morphology and nucleus pulposus regeneration; the nucleus pulposus histological score was performed by red-fixing green; ③ In vivo imaging of small animals was used to investigate the distribution, implantation, and degradation of MicroRod and MicroSphere microgels.

[0075] This application is based on methacryloyl gelatin (Gelma) with excellent biosafety and close to IVD ECM components as raw materials. By designing narrow flow channels to produce physical extrusion and combining external "on-off" precise alternating cross-linking methods, the preparation process of microfluidic gel particles is improved and simplified, and the "oil-cut-water" droplets of the same volume are processed into two microgel particles (MicroSphere and MicroRod) with completely identical physical and chemical properties except for the geometric shape. By comparing and analyzing the two geometric shapes of microgel particles to regulate the biological activities of NPs at in vitro and in vivo levels, it was found that the NPs cytoskeleton acts as a relay station that responds to ECM anisotropy, enhances the linkage between cell membrane and cell nucleus, activates the expression of adaptive young nuclear transcriptional network, and reverses the progression of IVDD. This application not only provides a new and effective research method for exploring the field of how the three-dimensional geometric morphological characteristics of ECM affect the mechanical signal transduction of cell groups, but also provides an experimental basis for the development of therapeutic strategies for mechanical stress to delay cell / tissue aging.

[0076] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0077] The present invention will be further described below in conjunction with the embodiments.

[0078] Example 1

[0079] 1. Preparation of GelMA

[0080] Add about 20g of gelatin to 200mL PBS solution, then stir in a 60℃ water bath for 1h until the gelatin is completely dissolved to obtain a 10% (w / v) gelatin solution. After that, use a micropump syringe to extract 16mL of methacrylic anhydride under light-proof conditions, add it dropwise to the gelatin solution at a rate of 0.25mL / min, and stir continuously for 2h. Then add 800mL of preheated PBS and continue stirring for 15min. Then, divide the solution into 12-14kDa dialysis bags and dialyze for 2 weeks, replacing PBS every 2 days to finally obtain the GelMA solution, which is freeze-dried and stored in a -80℃ refrigerator.

[0081] 2. Preparation of MicroRod Gel Particles

[0082] Prepared by microfluidic technology in the form of oil-in-water. First, a dual-channel PDMS microfluidic chip with a uniform inner diameter and depth of 100 μm was synthesized by melt casting of PDMS (dimethylsiloxane). The prepared 10% (w / w) Span80 isopropyl myristate solution was used as the continuous phase (oil phase) and connected to the outer needle of the coaxial electrospinning nozzle. The prepared 7% (w / v) GelMA solution and 0.5% (w / v) photoinitiator were dissolved in PBS, and the inner needle of the nozzle was connected after sufficient stirring and mixing as the dispersed phase (water phase). The two phases were connected to the EFL integrated microfluidic preparation device, and the flow rate ratio of the water phase (i.e., GelMA phase) to the oil phase was adjusted to 2:2, 2:4, 2:8, and 2:16 μL / min, respectively. The switch was turned on and the exhaust was respectively performed until continuous monodisperse spherical droplets were generated at the shearing position. The droplet morphology was observed using an optical microscope in the integrated microfluidic preparation device until a rod-shaped gel with an aspect ratio of about 4:1 was formed. A 0.1*0.2mm steel needle channel is connected at the outlet. A 100μm silicone tube is inserted outside to allow the rod-shaped gel to fully stabilize its shape in the silicone tube. Then, the disc-shaped cold light source emitter in the integrated microfluidic preparation device is used to photocure the gel microrods in the silicone tube. After the droplet size is uniform, place it in a beaker pre-filled with an oily solution. Take it out every 10 to 20 minutes, irradiate it with ultraviolet light, and replace the beaker (maintain a certain temperature of the needle channel system in the entire environment to prevent cross-linking and blockage in the needle channel). After that, the collected cured hydrogel microrods are repeatedly washed with 75% ethanol and PBS (75% ethanol 3 times, PBS 5 times), and then PBS is replaced every 3 hours to remove the photoinitiator and oil. Finally, the purified microrods are frozen in a -80℃ refrigerator overnight, and immediately freeze-dried for 48 hours after being taken out.

[0083] 3. Preparation of MicroSphere Gel Particles

[0084] Same as the above method, a 0.1*0.2mm steel needle channel is connected at the outlet. A 100μm silicone tube is nested outside, but the cold light source is not turned on in the silicone tube for curing. Instead, the microdroplets are collected into a beaker pre-filled with an oily solution after the size is uniform. The microgel rebounds into a spherical shape due to surface tension. Take it out every 10 to 20 minutes, irradiate it with ultraviolet light, and replace the beaker (maintain a certain temperature of the needle channel system in the whole environment to prevent cross-linking and blockage in the needle channel). After that, the collected cured hydrogel microspheres are repeatedly washed with 75% ethanol and PBS (75% ethanol 3 times, PBS 5 times), and then PBS is replaced every 3 hours to remove the photoinitiator and oil. Finally, the purified microspheres are frozen in a -80℃ refrigerator overnight, and freeze-dried for 48 hours immediately after taking them out.

[0085] Example 2

[0086] Physical and chemical characterization of two geometric microgel particles: MicroSphere and MicroRod:

[0087] 1. Scanning electron microscopy (SEM)

[0088] The freeze-dried MicroRod and MicroSphere gels were evenly pasted on the surface of the SEM sample metal stage with conductive glue pre-pasted, and then gold was sprayed on the fiber surface using a vacuum ion sputtering coater. The sample stage was then placed in a scanning electron microscope, and the morphology of each group of microgels was observed using an accelerating voltage of 10kV. At the same time, an optical microscope was used to observe the distribution and homogeneity of the unfreeze-dried microgels, and the pore size distribution, diameter size, and aspect ratio changes of MicroRod and MicroSphere prepared under different flow rate ratios were evaluated.

[0089] 2. X-ray spectrum analysis

[0090] The MicroRod and MicroSphere gels were freeze-dried, sprayed with gold, and then EDS was used to analyze the surface elements and chemical bond content of the microgels to evaluate whether the surface elements of the two were consistent. The acceleration voltage was 10 kV and the working distance WD = 8.5.

[0091] 3.AFM

[0092] MicroRod and MicroSphere microgels were freeze-dried and observed using AFM. The surface roughness of different microgels was analyzed based on the change in the interaction force between the needle tip and the sample surface, and the corresponding changes in the surface topology of the microgel scaffold were evaluated. At the same time, the mechanical curves were detected to evaluate the elastic modulus of the two microgels.

[0093] 4. FTIR

[0094] The prepared MicroRod and MicroSphere gel freeze-dried samples were added to potassium bromide (BrK) powder at a mass ratio of 1:50, and then fully ground with an agate mortar. The ground powder was then pressed into a disc. Then, the pressed sample discs of each group were placed on the stage, and various samples were scanned by FITR with a resolution of 4 cm -1 , scanning times 128 times, scanning range 400~4000cm -1 Finally, the curve data were saved, various curve data were plotted into graphs using Origin software, and the differences in functional groups between different groups were analyzed.

[0095] 5. Water contact angle

[0096] The static water contact angles of the surfaces of the above-mentioned microgel scaffolds were measured by a water contact angle analyzer to evaluate the differences in their surface hydrophilic and hydrophobic properties.

[0097] 6. Microgel flow in the chip

[0098] During the preparation of MicroRod and MicroSphere microgels, an optical microscope equipped with the EFL integrated microfluidic preparation device was used to observe the droplet shear in the flow channel of the microfluidic chip, and different flow rate ratios were used to take general pictures of the microgel flow.

[0099] Example 3

[0100] Study on the in vitro effects of anisotropic microgels

[0101] 1. Establishment of the co-culture system of anisotropic microgels and nucleus pulposus cells

[0102] Establish a co-culture system of MicroRod and MicroSphere microgels and nucleus pulposus cell NPs: Dissolve MicroSphere and MicroRod microgels in 75% alcohol and sterilize them in a 50mL centrifuge tube under UV for 30 minutes. Pour out the alcohol, rinse three times with DMEM / F12 medium and resuspend. Then use EFL cell sphere culture 24-well flat-bottom plate (EFL-SP201) to construct an anti-adhesion well plate system, seed each well with about 200 Sphere and Rod microgels, and then inoculate nucleus pulposus cells at a rate of about 5×10 4 Each well was seeded with 100 mL of DMEM / F12 medium, and 15% FBSDMEM / F12 medium was replaced every day for 1, 4, and 7 days.

[0103] 2. Cytocompatibility

[0104] ① Live-dead staining: After the two groups of microgels were co-cultured with nucleus pulposus cells for 1, 4, and 7 days, the working solution was prepared according to the instructions of the live / dead kit, with 1 μL of A solution and 2 μL of B solution added to 2 mL of PBS solution. Each group of microgels planted in the well plate was stained with the working solution at room temperature for 30 minutes. The cell morphology and growth orientation were observed using a confocal fluorescence microscope (ZIESS). The survival rate was analyzed and evaluated.

[0105] ②CCK-8: After the two groups of microgels were co-cultured with nucleus pulposus cells for 1, 4, and 7 days, CCK-8 was used to detect the proliferation ability of each group of cells. According to the ratio of 1 mL of CCK-8 solution to 10 mL of complete culture medium, 100 μL of the prepared CCK-8 working solution was added to the 96-well plate. After incubation in the incubator in the dark, the absorbance of the cells in each group of wells at a wavelength of 450 nm was measured by an ELISA instrument to analyze and evaluate the proliferation of each group of cells.

[0106] ③Cell morphology and adhesion status: After the two groups of microgels were co-cultured with nucleus pulposus cells for 1, 4, and 7 days, they were fixed with 4% paraformaldehyde. After alcohol gradient dehydration and critical point drying, they were observed and photographed under SEM to observe and analyze the morphology, growth direction, and adhesion status of the nucleus pulposus cells loaded on the two microgels.

[0107] ④Ki67 flow cytometry: After removing the supernatant of the co-culture system, add 100 μL PBS to each tube, then add Ki67 flow cytometry antibodies in turn and incubate at 4°C for 30 minutes. After gently mixing, incubate at 37°C in the dark for 20 minutes, and then analyze on a flow cytometer. The results were analyzed using FlowJo 10.0 software.

[0108] 3. Study on cytoskeleton spreading and mechanical signal transduction

[0109] ① Immunofluorescence: The co-culture system was constructed according to the above method. The two groups of microgels were co-cultured with nucleus pulposus cells for 1, 4, and 7 days, respectively. After fixation, permeation, and blocking, they were incubated with F-actin, MYH, VCL, and Integrin primary antibodies overnight, washed, and then incubated with specific secondary antibodies at room temperature for 2 hours. They were stained with phalloidin and DAPI in sequence, and then the morphology and spreading state of each skeleton protein were observed with a confocal microscope. Semi-quantitative fluorescence analysis was performed using ImageJ software. The distribution of YAP and TAZ on the surface cells of the two microgels was observed by immunofluorescence, and the nuclear-cytoplasmic ratio of YAP and TAZ was calculated, and semi-quantitative fluorescence analysis was performed using ImageJ software. At the same time, the morphology and intensity of Lamin A / C were observed by immunofluorescence staining to evaluate the state of the nuclear membrane complex after the mechanical tension of the skeleton was transmitted to the cell nucleus. The nuclear morphology, the overlap ratio of cytoskeleton F-actin and the cell nucleus were observed by the confocal microscope layer scanning overlay function, and the F-actin orientation order parameter and the nuclear shape factor were calculated.

[0110] 4. Mitochondrial and nuclear pore morphology

[0111] After adding GelMA lysis solution to the co-culture system, collect the nucleus pulposus cells. Centrifuge at 1500-3000 rpm for 5-10 minutes, collect the cells at the bottom of the tube until 2-3 rice-like samples are visible to the naked eye, discard the supernatant, slowly add 4°C pre-cooled fixative along the tube wall for 2 hours, then pour out the fixative, rinse the sample three times with 0.1M, pH 7.0 phosphate buffer, 15 minutes each time; fix the sample with 1% osmium acid solution for 1-2 hours; remove the osmium acid waste liquid, rinse the sample three times with 0.1M, pH 7.0 phosphate buffer, 15 minutes each time; dehydrate the sample with gradient concentrations (including 30%, 50%, 70%, 80%, 90% and 95% five concentrations) of ethanol solution, each concentration is treated for 15 minutes, and then treated with 100% ethanol for 20 minutes; finally, transition to pure acetone for 20 minutes. Treat the sample with a mixture of embedding agent and acetone (V / V=1 / 1) for 1 hour; treat the sample with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 hours; treat the sample with pure embedding agent overnight; embed the sample after infiltration treatment and heat it at 70°C overnight to obtain the embedded sample. The sample is sliced ​​in an ultra-thin slicer to obtain 70-90nm slices, which are stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 minutes each. After drying, the openness, number, and size of the cell nuclear pores and mitochondria are observed under a transmission electron microscope, and the nuclear pore diameter is quantified.

[0112] 5. Interaction between Mechanics and Oxygen Metabolism

[0113] ① Obtain the microsphere-cell co-culture system by the above method. Prepare JC-1 staining working solution according to the instructions of the JC-1 kit (Solarbio, China). Add an appropriate amount of working solution, incubate in a 37°C cell culture incubator for 20 minutes, wash twice with JC-1 staining buffer 1X, wash twice with culture medium, observe under an inverted fluorescence microscope, and perform semi-quantitative fluorescence analysis using ImageJ software.

[0114] ② After adding GelMA lysis solution to the co-culture system, the nucleus pulposus cells were collected. The total protein extraction kit was used to extract each group of proteins and the protein concentration was quantified by the BCA protein quantification kit. Then, the protein samples were separated by electrophoresis, and after rapid transfer at 400mA constant current for 20min, the target proteins of each group were transferred to the PVDF membrane, and then blocked with WB blocking solution, and the membranes were cut according to the molecular weight of the required target protein. Then, they were incubated with the primary antibodies of apoptosis-related indicators Calpain, Caspase-9, Caspase-3, Bcl-2, and Bax proteins and incubated at 4℃ overnight for 16h. Then, the bands were washed 3 times with TBST, incubated with horseradish peroxide-labeled secondary antibodies at room temperature for 2h, washed 3 times with TBST again, developed with ECL luminescence kit, and scanned and observed by gel imaging system. The obtained pictures were quantitatively analyzed by ImageJ grayscale. The grayscale value of each target protein and the corresponding β-actin grayscale value ratio were measured to evaluate the differences in protein expression between the groups.

[0115] ③ The mitochondrial oxygen consumption (OCRs) and extracellular acidification rates (ECARs) were studied using the Seahorse Extracellular Flux XFe24 Analyzer. Nucleus pulposus cells were collected after adding GelMA lysis buffer to the co-culture system. 1×10 5 Cells were seeded in Seahorse XF-24 plates and incubated in a CO2-free incubator for 1 hour before use in the assay. To measure ECAR, glucose (10 × 10 -3 m), Oligo(1×10 -6 m) and 2-DG (50×10 -3 m) were injected into the cells sequentially. Glycolysis was evaluated by subtracting the last ECAR value before glucose injection from the maximum ECAR value before oligo treatment; glycolytic capacity was evaluated by subtracting the last ECAR value before glucose injection from the maximum ECAR value after oligo injection; glycolytic reserve was expressed as glycolytic capacity minus glycolytic amount. For OCR analysis, Oligo (1×10 -6 m), FCCP (1.5×10 -6 m) and rotenone / antimycin (R / A) (2×10 -6m) were injected into cells, and basal respiration was assessed as the last OCR before oligo injection minus the non-mitochondrial respiration; maximal respiration was calculated as the maximum OCR after FCCP injection minus the minimum OCR after R / A injection; ATP production was assessed as the final OCR before oligo injection minus the minimum OCR after oligo injection.

[0116] 6. Research on inhibiting aging of nucleus pulposus cells

[0117] Follow the instructions of the SA-β-gal assay kit, fix the cells at room temperature for 15 minutes, add the prepared SA-β-gal staining working solution and incubate at 37°C overnight. Then observe under a light microscope. Use WB and PCR to verify the distribution and morphological expression of aging-related marker proteins such as P53, P21, and P16, DNA damage-related marker proteins such as γH2AX, aging-related inflammatory factors such as IL-6, TNF-α, IL-1β, and Lamin B1 protein.

[0118] Example 4

[0119] In vivo study of anisotropic microgels delaying IVDD

[0120] 1. In vivo experiments and animal screening

[0121] Male SD rats, with an average weight of 300-350 g, were purchased from the Experimental Animal Center of Soochow University. The surgical and treatment effects were good. The study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.

[0122] 2. Establishment of the rat coccygeal disc degeneration model

[0123] Rats were intraperitoneally injected with 10% (wt%) chloral hydrate. After complete anesthesia, the rat tail was disinfected, and then the rat 7-9 IVD was punctured continuously with a needle to induce degeneration. To ensure the induction of degeneration, the internal tissue of the intervertebral disc was damaged by suction after acupuncture. Then, about 15 μL of GM and GM@CS-BP microsphere solution was injected using a micropump syringe, and PBS was injected into the negative control group. After surgery, the rats were placed in a warm and ventilated place.

[0124] 3. X-ray and MR imaging evaluation

[0125] At 4 and 8 weeks after surgery, 3 rats were randomly selected from each group and underwent X-ray and MRI examinations before sacrifice. Each rat was placed in the supine position and its tail was placed on a molybdenum target radiography device. The acquired X-ray images were measured and the disc height index (DHI%) was calculated using ImageJ software. MRI examinations were performed using a 1.5T system (GE). T2-weighted images were obtained in the coronal plane. Imaging clinicians who were unaware of the grouping evaluated the T2-weighted signal intensity and scored MRI as grades I to IV according to the modified Thomson classification.

[0126] 4. Histological Evaluation and Immunofluorescence and Immunohistochemistry

[0127] After 4 and 8 weeks, the rats were killed, and the IVD was removed and immersed in formalin. The rats were decalcified in 10% EDTA for 30 days and embedded in paraffin. The specimens were sliced ​​into sections of about 5 μm. The changes in the tissue structure and collagen in the intervertebral disc were then observed by hematoxylin-eosin staining (H&E staining) and safranin O-fast green staining (SO-FastGreen staining). The histological grading used the scale established by Masuda, which was scored from 4 points (normal) to 12 points (severe degeneration). The sections were also stained with fluorescent stains such as IL-6, TNF-α, and IL-1β.

[0128] Effect Example 1: Characterization and co-culture experimental results of anisotropic microgels regulating cell mechanomechanics

[0129] 1. Preparation and Characterization of Anisotropic Microgels

[0130] This application designs a narrow channel to produce physical extrusion, and combines precise external "on-off" alternating cross-linking technology to successfully mass-produce GelMA with excellent biosafety and close to ECM components into MicroRods with high aspect ratio and anisotropy. A microgel cell loading system that fits the physiological components of ECM and simulates the dimensions of cell growth has been constructed. Specifically, combined with the "oil-water" principle of the microfluidic chip, the depth and width of the chip flow channel are limited to 100μm, and the tangent GelMA droplets are physically compressed in the narrow channel and deformed into rods. Its height and width are consistent with the flow channel, that is, 100μm. After that, the droplets are photocured and cross-linked in a rod-shaped form to obtain MicroRod. Since the direct cross-linking of the traditional MicroRod preparation on the chip is prone to blockage of the flow channel, this application simultaneously connects a 100μm diameter silicone tube at the end of the microfluidic chip flow channel, so that the rod-shaped droplets running in the chip continue to enter the silicone tube part. After that, the external silicone tube was fully cross-linked by ultraviolet light (UV1) to obtain MicroRod, avoiding the problem of clogging. At the same time, when the droplet flows out of the silicone tube, it naturally rebounds into a spherical shape due to the release of flow channel pressure and the effect of surface tension. At this time, the external ultraviolet light (UV2) was turned on to cross-link and obtain MicroSphere ( Figure 2 A in the figure). The volume, composition, and physicochemical properties of MicroSphere and MicroRod were unified during the distributed cross-linking process, and an excellent platform was constructed to explore the effect of ECM geometry on signal transduction in multiple cell populations.

[0131] Afterwards, the morphology of anisotropic MicroRod and isotropic MicroSphere was observed by SEM ( Figure 2 C), the diameter of MicroSphere was found to be about 183.15±30.16μm ( Figure 2 D in the figure), while the long diameter of MicroRod is about 393.14±25.16μm ( Figure 2 In order to explore whether the physical and chemical properties of the two are similar, mapping and EDS scanning analysis were also carried out ( Figure 2 F, G in the figure), it was found that both MicroSphere and MicroRod only showed concentrated expression of C, N, and O elements, which was related to the main components of GelMA. In order to explore whether the chemical groups of the two groups have changed, Fourier transform infrared spectroscopy analysis was performed ( Figure 2 Both of them can detect the characteristic 1620cm of GelMA -1 The stretching vibration peak of the peptide bond at 3270 cm -1 The stretching vibration peak of NH was then measured using AFM. Figure 2I) and calculated the Young's modulus. It was found that although the photocrosslinking steps were different, there was no significant difference in the stiffness of the two, that is, 26.46±4.69 kPa for the ball and 26.63±6.0 kPa for the rod ( Figure 2 There is no significant difference in the RA values ​​between the two, which proves that the surface morphology is similar.

[0132] 2. Cell adhesion and skeleton spreading of anisotropic microgels

[0133] Adhesion and cytoskeleton-related staining were performed on the co-culture system ( Figure 3 A in the figure). After 3 days of co-culture, although there was no significant difference in the fluorescence intensity of F-actin, the cell F-actin morphology on the anisotropic MicroRod surface showed a high orientation, full spreading, and orderly growth along the long axis, while the spreading of NPs on the isotropic MicroSphere was disordered and scattered. After 7 days of co-culture, the cells on the rod gel were more densely arranged and orderly, and the trend of stretching along the long axis was more obvious. Even the fluorescence intensity of F-actin was enhanced compared with the MicroSphere group ( Figure 3 C). This provides favorable biological evidence that ECM geometry induces the directional spreading of adherent cells.

[0134] In order to further explore the relationship between ECM geometry and cell adhesion, cell adhesion-related substances were investigated. Integrin is a class of molecules that are commonly found on the surface of vertebrate cells and mediate mutual recognition and adhesion between cells and between cells and extracellular matrix. Integrins, as transmembrane connectors, play a bidirectional role in communication between the extracellular matrix and the intracellular actin skeleton, connecting the extracellular matrix with the intracellular skeleton network into a whole, and play an important role in the process of cell adhesion. Integrinβ1 was immunofluorescently stained ( Figure 3 The expression distribution of Integrinβ1 in MicroRod increased by 1.67 times compared with that in MicroSphere ( Figure 3 F). WB detection of Integrinβ1 protein expression also showed differences ( Figure 3D~E in the figure). The β1 subunit of integrin has been shown to affect FAK activity and the phosphorylation of PI3K and AKT, leading to enhanced adhesion and reduced apoptosis of NPs. β-catenin is an intercellular adhesion molecule that participates in intercellular adhesion and cell-to-cell signaling. β-catenin is also an important component of the Wnt signaling pathway, regulating processes such as cell proliferation, differentiation, and growth. Both WB protein expression and immunofluorescence of MicroRod showed high expression of β-catenin ( Figure 3 These results confirm that the anisotropy of the MicroRod group significantly affects the adhesion-related processes of NPs.

[0135] In addition, integrins also have the function of transmitting extracellular signals into cells. When the extracellular domain of integrin binds to extracellular ligands (such as fibronectin or laminin), it will induce a configurational change at the end of the intracellular domain. The configurational change in turn changes the interaction between the end of the cytoplasmic domain of integrin and adjacent proteins. This physical connection between the extracellular matrix and actin is called "Focal Adhesion". The mechanical properties of the extracellular matrix can be sensed by FA and further regulate downstream signaling pathways. Therefore, FA plays an important role in mediating cell attachment and anchoring to the ECM, and the size of FA can directly reflect the tension level of the cell. FA formed by actin filaments and integrins requires the joint participation of talin, FAK and vinculin. Among them, FAK is a tyrosine kinase and may be the first signal molecule in the cell to receive external physical signals. When cells spread and move, FAK participates in the formation and regulation of focal adhesions. The expression of FAK was explored by WB, and the results showed that the protein expression of FAK in MicroRod was significantly increased ( Figure 3 E in Figure 1). Focal adhesion protein is a cytoskeletal protein and focal adhesion component protein. It is enriched in cell-cell and cell-matrix adhesion sites and is involved in a variety of physiological processes such as cell adhesion, extension, migration, cell morphology maintenance, and force-chemical signal transduction. Figure 3 I, J) and WB( Figure 3K) in the MicroRod group also observed an increase in Vinculin expression. These represent a significant increase in FA, which may indicate that the MicroRod co-culture environment provides stronger adhesion and support between cells and the matrix. Nicolas and Safran et al. first proposed a linear spring chain model for force-induced anisotropic growth of focal adhesions. That is, the local contraction force from stress fibers will deform FA, and these force-induced deformations will change the local density of proteins around the adhesion site, and the non-uniform protein density distribution will lead to different molecular dynamics (conformational changes or molecular reorganization) in the front and back regions of the edge, thereby triggering differential new protein aggregation and adhesion towards anisotropic growth of the biochemical cascade reaction. This may explain the phenomenon that the anisotropy of MicroRod causes the upregulation of FA-related protein molecules.

[0136] The response of cells to mechanical signals depends on the viscoelastic properties of cells, which largely rely on the highly dynamic cytoskeleton. The viscoelastic properties of the cytoskeleton originate from the properties and dynamic interactions of three cytoskeletal filaments, actin, intermediate filaments, and microtubules. The ability of cells to maintain and respond to mechanical stress does not depend on the properties of individual filaments, but on the properties of a complex cytoskeletal network that constantly adapts to chemical and mechanical signals in the cellular environment and transmits them in the form of cytoskeletal spreading. In WB, high expression of β-tubulin and Myosin, representative of the cytoskeleton, was detected ( Figure 3 K, L in the figure). Immunofluorescence experiment ( Figure 3 M, N in the figure show the directional arrangement of Myosin and the growth along the longitudinal axis. This may represent the comprehensive response and adaptive changes of cells to anisotropic microgels through a series of adhesion and mechanical perception-induced cytoskeleton spreading, and also suggests the strong correlation between cell adhesion, mechanical signal transduction, growth, and differentiation signals mediated by the cytoskeleton.

[0137] Effect Example 2: Experimental results of oxygen metabolism balance functionalized hydrogel microspheres in vitro and in vivo

[0138] 1. Anisotropic microgels regulate nuclear mechanics and nuclear shape remodeling

[0139] Cells sense the mechanical tension of the external environment, which is a necessary micro-condition for maintaining cell survival. External physical and chemical factors are important factors affecting the physiological functions of cells. Among them, YAP / TAZ regulates various cell functions by integrating different cell signaling pathways. When the cytoskeleton tension increases, F-actin highly overlaps with the cell nucleus, mobilizing the skeleton mechanical force to transmit into the nucleus, opening the nuclear pore complex on the nuclear membrane, inducing YAP and other transcription activation-related factors to enter the nucleus, and increasing the expression of cell proliferation-related genes. When phosphorylated, YAP is retained in the cytoplasm and degraded. The enhancement of F-actin stress weakens the phosphorylation of YAP and induces the nuclear translocation of YAP. It is worth noting that on NPs loaded on the anisotropic MicroRod, high nuclear expression of YAP was observed, and its nuclear-to-cytoplasmic ratio was significantly higher than that of cells on MicroSphere ( Figure 4 A and B in Figure 2 confirmed the strong nuclear translocation of YAP. At the same time, the protein expression of YAP / TAZ also increased significantly.

[0140] The nuclear entry of YAP is related to the transmission and concentration of mechanical force along the cytoskeleton to the nucleus. There is a high degree of tacit understanding between the nucleus and the cytoskeleton. The cytoplasmic actin skeleton is connected to the inner nuclear envelope arranged on the nucleus through the LINC complex composed of Nesprin and SUN proteins. This network connects the cytoskeleton, the Lamin-based nuclear membrane and the nuclear pore complex that controls transport in and out of the nucleus. Lamin then senses the tension of the cytoskeleton and further interacts with the chromatin in the nucleus to cause stretching changes in the nucleus and reduce the condensation of chromatin. Immunofluorescence staining ( Figure 4 C and D in Figure 3 show that the expression of Lamin A / C around the nucleus of cells loaded on the anisotropic MicroRod is more obvious, and obvious "indentation" of the actin skeleton can be observed (marked by yellow arrows), confirming the interaction between the cytoskeleton and the nuclear skeleton. Figure 4 E) shows that the nuclear pores (marked by red arrows) in the MicroRod group are significantly larger than those in the MicroSphere group ( Figure 4 F in the figure indicates an increase in nuclear membrane permeability. Then, WB was used to detect the expression of molecules related to mechanical signal transduction ( Figure 4 G in the figure). A significant increase in YAP was observed in the MicroRod group. TAZ, as a YAP paralog, regulates gene expression by binding to TEAD transcription factors, thereby affecting cell proliferation and differentiation. In addition, an increase in TAZ and Lamin A / C protein expression was observed ( Figure 4 H in the figure). Immunofluorescence showed that the fluorescence intensity of TAZ in the MicroRod group was significantly higher than that in the MicroSphere group ( Figure 4 I~J).

[0141] In summary, the effect of mechanical stress on the growth and differentiation fate of cells is complex, and its role in the survival and resistance of nucleus pulposus cells in harsh environments is seriously underestimated. In order to explore the potential mechanism of anisotropic microgel-induced enhanced mechanical signal transduction and possible biological effects, microgel-NPs were co-cultured for 7 days, then intervened with H2O2 and total cell RNA was extracted for transcriptome sequencing. Compared with cells loaded on MicroSphere, 2131 genes were upregulated and 2279 genes were downregulated in the MicroRod group. Heat map analysis of differentially expressed genes (DEGs) showed ( Figure 5 A), there was a significant difference between the two groups, demonstrating that anisotropy has an important influence on the tolerance of NPs in pathological environments.

[0142] The potential signaling pathways were explored using KEGG pathway, and the top fifteen enriched pathways of Cellular Processes and Environmental Information Processing were listed ( Figure 5 B in the figure). MicroRod significantly enhanced the cytoskeleton and adhesion-related signaling pathways, such as focal adhesion and regulation of actin cytoskeleton. Similarly, DEGs were collected for analysis of the gene ontology GO database, which is usually divided into three categories: biological process (BP), molecular function (MF), and cellular component (CC), and ten representative enriched pathways in each category were selected ( Figure 5 C) also showed significant enrichment in adhesion and cytoskeleton regulation. This suggests that MicroRod induces changes in the adhesion-related processes of surface-loaded cells through its own anisotropy, as well as subsequent adjustments to the cytoskeleton. GSEA was then performed based on the marker gene set, and the results showed a significant upregulation of focal adhesion and integrin mediated cell adhesion.

[0143] The results of transcriptional sequencing showed that the obvious changes in the biological effects of NPs may be caused by mechanical signal transduction. For example, KEGG analysis found changes related to the PI3K-AKT pathway and Apoptosis, confirming the possible improvement of cell proliferation and anti-apoptosis ability. The GO analysis suggested the possible delay of cell aging. GSEA further showed the downregulation of the P53 signaling pathway closely related to cell aging and the obvious increase of the cell cycle.

[0144] 2. Study on the ability of anisotropic microgels to promote nucleus pulposus proliferation, resist apoptosis, and delay aging

[0145] Since the intervertebral disc is in an ischemic and hypoxic microenvironment for a long time, the dynamic balance of reactive oxygen production and removal is broken, causing a large amount of reactive oxygen species to accumulate inside the intervertebral disc. The accumulation of these products will cause an increase in the level of tissue lipid peroxidation, trigger changes in mitochondrial membrane potential, increase mitochondrial outer membrane permeability, and a large amount of apoptotic factor outflow, ultimately leading to cell apoptosis. It is very important whether anisotropic microgels can continue to maintain the "young state" of loaded NPs under oxidative stress ( Figure 6 A in Figure 1). GSEA analysis revealed that the PI3K-AKT pathway was upregulated in the MicroRod group, suggesting that this signaling pathway may be involved in the corresponding proliferation and anti-apoptotic effects. The intracellular signal transduction network is complex. As one of the important signaling molecules, PI3K has gradually been understood in recent years in its relationship with ECM-integrin-mediated signal transduction. Studies have found that the p85 subunit of PI3K is tyrosine phosphorylated during cell adhesion, and FAK can phosphorylate this subunit. After PI3K is activated, the second messenger PIP3 is produced on the plasma membrane. PIP3 binds to the intracellular signaling protein AKT and PDK1 (phosphoinositide dependent kinase-1) containing the PH domain, prompting PDK1 to phosphorylate Ser308 of the AKT protein, leading to the activation of AKT. At the same time, the activated AKT activates or inhibits its downstream target proteins Bad, Caspase3, NF-κB, etc. through phosphorylation, thereby regulating cell proliferation, differentiation, apoptosis, and migration. The FAK / PI3K / AKT axis may be the intermediate link in the cascade of signal transduction between multiple cytokines and integrins, representing the comprehensive response of cells to the external environment.

[0146] In order to further confirm these possible biological effects, the proliferation ability of cells and the activity of oxidative stress state were investigated. First, Ki-67 protein flow cytometry was used to detect the proliferation of NPs in the co-culture system ( Figure 6 B). Ki-67 positive ratio analysis showed that the proportion of Ki-67+ cells in the MicroRod group increased significantly compared with that in the MicroSphere group ( Figure 6 The same phenomenon was observed in the cell viability test using the Cell Counting Kit-8 (CCK-8) to determine the biocompatibility and proliferation ( Figure 6 D in the figure). In addition, since the intervertebral disc is in a hypoxic environment for a long time, the proliferation ability of NPs in a hypoxic environment was also explored using H2O2. By continuously increasing the intervention concentration of H2O2 in the co-culture system, the proliferation ability of NPs on MicroRod was always higher than that of the MicroShpere group. Afterwards, WB was used to analyze the protein expression of the PI3K-AKT pathway, and the results showed that the expression of p-PI3K and p-AKT was enhanced ( Figure 6G, H in Figure 3). Live-dead staining further illustrates that the MicroRod group maintains the proliferation ability of NPs in a hypoxic environment ( Figure 6 E, F in it).

[0147] To further verify the association between cell mechanical signal transduction and NPs activity, cytochalasin D (a F-actin stress fiber polymerization inhibitor) was used to intervene in the co-culture system to block the effect of the anisotropy of MicroRod on the cytoskeleton reconstruction. Figure 6 The results of the positive ratio analysis of I) and Ki-67 showed that the proliferation activity of NPs in the MicroRod group was no different from that in the MicroSphere group. Finally, WB analysis of the expression of PI3K-AKT pathway proteins was performed, and it was found that there was no significant difference in the phosphorylation degree between the two groups ( Figure 6 J, K in Figure 3). These results confirm the possible activation of the PI3K-AKT pathway caused by the cytoskeletal stress regulation and adaptive process, as well as the enhancement of NPs proliferation activity.

[0148] In order to explore the effect of the anisotropy of MicroRod on the apoptotic function of loaded NPs, WB detection of important apoptotic markers was performed under the intervention of H2O2. The results showed that the protein expression of the main terminal cleavage enzyme Caspase3 decreased and the anti-apoptotic protein BCL-2 increased in the MicroRod group ( Figure 7 A and B in Figure 2 confirm the possible apoptosis inhibition. The decrease of mitochondrial membrane potential is a hallmark event of early cell apoptosis, and the change of JC-1 from red fluorescence to green fluorescence can reflect the decrease of cell membrane potential. The relative fluorescence intensity (RFI) of J-aggregates and Monomer in the MicroRod group increased significantly compared with that in the MicroSphere group, indicating the improvement of mitochondrial depolarization in early apoptosis ( Figure 7 C and D in the figure). MitoSox flow cytometry produces red fluorescence under conditions of mitochondrial oxidative damage, reflecting the generation and increase of superoxide anion reactive oxygen species in mitochondria. A significant increase in MitoSox intensity was observed in the MicroSphere group, demonstrating the tolerance of cells on the MicroRod to oxidative stress damage ( Figure 7 E). However, in MitoTracker-Red fluorescence staining, the overall distribution and fluorescence intensity of mitochondria did not change significantly ( Figure 6 F, G in Figure). The mitochondrial morphology was observed using biological cryo-transmission electron microscopy ( Figure 7H in the figure). In the MicroSphere group, the mitochondria of NPs were swollen, the cristae disappeared, and even vacuoles appeared, indicating mitochondrial damage caused by oxidative stress. In the MicroRod group, although there was partial swelling of mitochondria, the mitochondria were still able to maintain the long oval shape and cristae. At the same time, because NPs reside in the avascular microenvironment of the intervertebral disc, their metabolic pathways have been shown to be dominated by glycolysis, and their glycolysis capacity can reflect the metabolic flux and the overall biosynthetic capacity of NPs. The ECAR of NPs on the two microgels was investigated using the Seahorse Cell Energy Metabolism Analyzer ( Figure 7 I in the figure). The cells on both microgels initially showed a mild glycolytic metabolic state. When glucose was added, the acidification of the MicroRod group increased significantly, indicating an improvement in glycolytic function. After the addition of oligomycin to completely inhibit oxidative phosphorylation, the glycolytic capacity of the MicroRod group increased relative to the MicroSphere group, confirming that anisotropic microgels improved the metabolic function and energy storage of NPs. This may be due to the improvement in the overall biosynthetic capacity of NPs caused by the activity of cells in the MicroRod group and the demand for ECM synthesis.

[0149] The effects of various adverse factors in the IVD environment lead to SASP in NPs. MMP13 can specifically degrade the expression of the main components of the intervertebral disc ECM, ultimately leading to metabolic imbalance of the ECM. Immunofluorescence staining ( Figure 7 J and K in Figure 3 show that the intensity of the main proteins secreted and degraded by ECM in the MicroRod group was higher than that in the MicroSphere group ( Figure 7 L, M in the figure). The WB results also showed that the protein expression of ACAN and COL-II increased and the protein expression of MMP13 decreased ( Figure 7 N in the figure). As a target gene of YAP, CTGF has also been shown to improve the ECM synthesis ability of NPs under pathological conditions. WB showed that the expression of CTGF protein in the MicroRod group was significantly enhanced, suggesting the possible association between mechanical stress and ECM metabolic function ( Figure 7 O in the figure). After blocking F-actin function with cytochalasin D, apoptosis-related studies were conducted. WB and qRT-PCR ( Figure 7 The results showed that there was no significant difference in the mRNA and protein expression of Caspase3 and BCL-2 between MicroSpere and MicroRod groups ( Figure 7 Q in ), suggesting that mechanical stress induces an enhancement in the ability of NPs to inhibit apoptosis.

[0150] The researchers tested whether mechanical stress in NPs would have a similar effect on delaying aging. SA-β-Gal is a β-galactosidase that exhibits increased activity during cell aging. After using H2O2 to intervene in the co-culture system of two microgels and NPs, the activity level of SA-β-Gal in the cells was detected to assess the degree of cell aging ( Figure 8 The results showed that the number of SA-β-Gal+NPs in the MicroRod group was significantly lower than that in the MicroSphere group ( Figure 8 B in the figure). P16INK4a is a protein closely related to cell cycle regulation and cell senescence. Its expression is upregulated when cells are senescent or damaged by DNA. Immunofluorescence showed that the intensity of P16 INK4a in the MicroRod group decreased, confirming the delay of senescence process ( Figure 8 C, F).

[0151] Afterwards, STING and TNF-α immunofluorescence staining ( Figure 8 D, E, G, and H in the figure show that the strength of both cells in the MicroRod group decreased significantly. This suggests the possible association between mechanical stress and the cGAS-STING pathway and downstream inflammatory signals, and further indicates that the geometric changes in the cells loaded on the anisotropic MicroRod may delay the STING-mediated immune response. In addition, in the Lamin A / C staining after H2O2 intervention ( Figure 8 I), the cell nuclei of the MicroRod group have obvious Lamin A / C distribution around the nuclei. The tensile stress of F-actin leads to the formation of filamentous indentations (indicated by yellow arrows), which may be the reason why the nuclei of the MicroRod group are elliptical and extended. On the contrary, the nuclei of the MicroSphere group show changes such as shrinkage, depression, protrusion, and lobulation, which may be due to the loose cytoskeleton structure of the MicroSphere group and the lack of nuclear membrane stress, which leads to the weakening of the support of the nuclear lamina to the nuclear membrane ( Figure 8 In addition, changes such as nuclear membrane indentation may also lead to abnormal integrity and function of the nuclear membrane, and the internal chromatin gradually becomes tight, which ultimately makes it difficult for genes on the chromatin to be transcribed and expressed.

[0152] IL-6 is an important SASP molecule associated with aging and can significantly inhibit NP proliferation, migration and extracellular matrix synthesis. Figure 8 The protein expression of IL-6 was detected to be decreased, and at the same time, the protein expression of classic aging-related molecules P53, P21, and P16 ( Figure 8 The decrease in cGAS and STING protein expression suggests that cGAS-STING mediated inhibition of aging process ( Figure 8Immunofluorescence staining ( Figure 8 L, M) showed that the fluorescence intensity of IL-6 and IL-1β in the MicroRod group was significantly inhibited, and ELISA test showed that the content of cGAMP in the MicroRod group decreased ( Figure 8 O in the figure). This indicates that the inflammatory signaling cascade associated with STING was blocked in the MicroRod group. In addition, the protein expression of Lamin B1, an important molecule that maintains the nuclear structure and mediates the integrity of the nuclear membrane, increased significantly ( Figure 8 K in ), and decreased expression of γ-H2AX, a key regulator of DNA damage ( Figure 8 These phenomena may be due to the anisotropy of MicroRod inducing cell geometry to regulate mechanical perception, play a role in maintaining nuclear membrane integrity and reducing DNA damage, and then delay the aging process of NPs.

[0153] To further confirm the association between mechanical stress, cytoskeleton, nuclear membrane integrity, and aging, cytochalasin D was used to block the cytoskeleton's perception of mechanical signals, and cGAMP detection was performed ( Figure 8 The results showed that there was no significant difference in the cGAMP content between the MicroSphere and MicroRod groups. At the same time, WB and qRT-PCR showed that there was no significant difference in the mRNA and protein expression of Lamin B1 and STING ( Figure 8 Q, R in the figure). This suggests that the maintenance of nuclear membrane integrity on anisotropic microgels is inhibited after blocking F-actin function, and these phenomena may trigger the subsequent activation of the immune signal transduction linker STING. Finally, in order to confirm the association between the SASP of NPs and the STING pathway, STING-IN-2 (C-176) was used to covalently inhibit STING activity. The results showed that there was no significant difference in the protein and mRNA expression of IL-6 and MMP13 between the MicroSphere group and the MicroRod group ( Figure 8 These phenomena suggest that the molecular mechanism of regulating cell senescence centered on the anisotropic-YAP / TAZ-cGAS-STING signaling pathway may also exist in NPs, revealing the potential connection between mechanical sensing and innate immunity, and providing a theoretical basis for the therapeutic strategy of anisotropic microgels to maintain YAP / TAZ mechanical signals and further inhibit STING to limit aging-related inflammation.

[0154] 3. In vivo effects of anisotropic microgels regulating cell mechanomechanics

[0155] After constructing the rat IVD degeneration model ( Fig. 9A in the figure, MicroSphere and MicroRod loaded with NPS were injected and their biological effects were investigated. X-ray examination was performed on rats implanted with the two microgels for 4 and 8 weeks ( Fig. 9 C). There were significant differences in the intervertebral disc height between the blank control group and the simple puncture group NC at 4 and 8 weeks (p<0.01), and the height at 8 weeks decreased significantly compared with that at 4 weeks, proving the successful construction of the intervertebral disc degeneration model ( Fig. 9 B). The intervertebral disc height of the MicroRod group at 4 and 8 weeks was significantly restored compared with that of the MicroSphere group (p<0.01). This demonstrates the ability of anisotropic microgels to improve the intervertebral space caused by the restoration of intervertebral disc degeneration in vivo. The higher T2-weighted signal in magnetic resonance imaging indicates a higher water content in the nucleus pulposus. As intervertebral disc degeneration progresses, the massive loss of proteoglycans will damage the water content in the IVD, and the intervertebral disc will gradually "blacken", which is consistent with MRI analysis ( Fig. 9 The nucleus pulposus of the NC group showed a significant low T2-weighted signal at 4 and 8 weeks, while the nucleus pulposus of the Control group still maintained a high water content (p<0.01), demonstrating irreversible damage after disc puncture. MRI grading (Pfirrmann classification) showed that the intradiscal grading of the MicroRod group was significantly improved compared with the MicroSphere group (p<0.01), and it was more obvious at 8 weeks ( Fig. 9 E). It is proved that anisotropy can regulate the local pathological environment to repair the water content and collapse degree of the intervertebral disc and delay the degeneration of the intervertebral disc.

[0156] In addition, in vivo imaging observations were performed to evaluate the retention efficiency of the two microgels within the intervertebral disc environment ( Fig. 9 D in the figure). Fluorescence signals were clearly visible 1 day after injection, proving the success of the two microgel injections. At the same time, due to the strong degradation ability of GelMA, the radiation efficiency signals of the microgels were almost undetectable at 14 days. However, cell-loaded microgel injection therapy still has the potential to provide good ECM support and timely cell replenishment in the puncture environment. Hematoxylin-eosin (H&E) staining can be used to observe the morphology of the nucleus pulposus ( Fig. 9 G in the figure). The area of ​​the nucleus pulposus in both microgel groups was reduced compared with that in the control group, but a more obvious tissue edge was observed. Although a certain area of ​​NP could be observed in the MicroSphere group, the NP was atrophied compared with that in the MicroSphere group, which was not enough to represent the regeneration of NP. Safranin / fast green staining was used to evaluate the proteoglycan (orange) and collagen (blue) content in IVD ( Fig. 9The I in the figure). The MicroRod group showed obvious orange aggregation compared with the NC group, representing the remodeling of proteoglycan. Although the MicroSphere group expressed a certain amount of proteoglycan, it did not form a complete structure. The Masuda scale showed that the scores of the MicroRod group after 4 and 8 weeks were significantly lower than those of the MicroSphere group (p<0.01), indicating the occurrence of the regeneration process ( Fig. 9 H in the figure). Tissue Tunel staining ( Fig. 9 J) found that at 4 weeks, the intensity of the Tunel in the MicroRod group was significantly lower than that in the MicroSphere group, proving the inhibition of apoptosis, but there was no significant difference between the two. At 8 weeks, the intensity of the Tunel in the MicroRod group was significantly lower than that in the MicroSphere group, proving the attenuation of apoptosis ( Fig. 9 in K).

[0157] Finally, the ECM metabolism and aging in the IVDD environment were investigated. After targeted injection of two cell-loaded microgels, the tissue fluorescence intensity of CoL-II ​​was significantly increased compared with the NC group ( Fig.10 The MicroRod group showed a more significant enhancement than the MicroSphere group ( Fig.10 The opposite situation was observed in the fluorescence staining of MMP13 ( Fig.10 B, F in Figure 3). This demonstrates that the anisotropic microgels inhibited the degradation of local ECM and improved collagen remodeling. Fig.10 C) found that the NC group after puncture had more SA-β-gal+ cells, proving the presence of a large number of senescent cells in the IVDD environment ( Fig.10 G in the figure). Both cell-loaded microgel treatments were able to reduce the number of SA-β-gal+ cells, indicating the feasibility of injection therapy. The number of senescent cells in the MicroRod group decreased more significantly. The fluorescence intensity of the senescence-related molecule P53 and the important molecules of SASP, IL-6 and TNF-α in the injection environment ( Fig.10 The same trend is also shown in D Fig.10 H in.

[0158] In summary, the results of in vivo experiments show that although the DHI% and MRI signals of the MicroSphere group are better than those of the NC group, they are still weaker than those of the MicroRod group, and more obvious after 8 weeks. Histological staining showed that the remnants of nucleus pulposus tissue and the deposition of proteoglycans in the MicroRod group were the most obvious, its apoptosis and aging were relatively improved, and the ECM metabolic disorder was restored to a certain extent. This may be because the anisotropy of MicroRod inhibits the aging process by affecting the response of YAP / TAZ to mechanical stress. These highly active "rejuvenated" cells injected by targeted injection may affect the expression of various inflammatory factors and proteases by paracrine improvement of SASP, block the activation of downstream inflammatory pathways, cut off the vicious cycle between aging and inflammation, improve the overall environment of IVDD and promote nucleus pulposus regeneration.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing microgel based on a microfluidic chip, characterized in that: The dispersed phase is not light-cured before flowing out of the chip, but is light-cured in the silicone tube after flowing out of the chip.

2. The method according to claim 1, characterized in that The following steps are involved: S1, passing the continuous phase and the dispersed phase through a dual-channel PDMS microfluidic chip with an inner diameter of 100 μm, and not photocuring the dispersed phase before it flows out of the dual-channel PDMS microfluidic chip; S2. Connect a silicone tube to the outlet of the dual-channel PDMS microfluidic chip, and photo-cure the dispersed phase flowing through the silicone tube to obtain a microgel.

3. The method according to claim 2, characterized in that The flow rate ratio of the dispersed phase to the continuous phase is 2:(2-24) μL / min.

4. The method according to claim 2 or 3, characterized in that The dispersed phase includes 7% (w / v) GelMA solution, 0.5% (w / v) photoinitiator and PBS.

5. The method according to any one of claims 2 to 4, characterized in that: The continuous phase is an isopropyl myristate solution containing 10% (w / w) Span80.

6. Application of anisotropic microgels in any of the following: (1) Increase the expression of Integrinβ1, β-tubulin, Vinculin and / or Myosin; (2) increasing the expression of YAP, TAZ, Lamin A / C and / or Lamin B1; (3) enlarge nuclear pores and / or promote the nuclear expression of YAP; (4) Increase the proportion of Ki-67+ cells; (5) Enhanced expression of p-PI3K and / or p-AKT; (6) Reduce Caspase3 expression; (7) Increase the expression of BCL-2; (8) Increase the relative fluorescence intensity of JC-1; (9) Improve the tolerance of nucleus pulposus cells to oxidative stress damage; (10) Improving the glycolytic function and / or glycolytic capacity of nucleus pulposus cells; (11) Reduce the expression of MMP13; (12) increasing the expression of CTGF, ACAN and / or COL-II; (13) Reduce the expression of SA-β-Gal, P16 INK4a, STING, IL-6, IL-1β, P53, P21, P16, cGAMP, γ-H2AX and / or TNF-α.

7. Application of anisotropic microgels in any of the following: (I) preparing products that promote proliferation of nucleus pulposus cells and / or inhibit aging of nucleus pulposus cells; (II) preparing products that improve intervertebral disc collagen remodeling and inhibit intervertebral disc extracellular matrix degradation; (III) preparing a product for preventing the intervertebral disc from decreasing in water content and / or preventing the intervertebral disc from collapsing; (IV) Preparation of products for delaying intervertebral disc degeneration.

8. The use according to claim 6 or 7, characterized in that The anisotropic microgel is a rod-shaped microgel with a length of 360 to 420 μm and a diameter of 90 to 110 μm.

9. A drug, characterized in that Contains anisotropic microgel and acceptable excipients or adjuvants; The anisotropic microgel is a rod-shaped microgel with a length of 360 to 420 μm and a diameter of 90 to 110 μm.

10. A culture medium, characterized in that Contains anisotropic microgels; The anisotropic microgel is a rod-shaped microgel with a length of 360 to 420 μm and a diameter of 90 to 110 μm.

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