Hydrogel microspheres with both mechanical properties and differentiation-promoting functions and preparation method and application thereof

Abstract

A hydrogel microsphere having both mechanical properties and differentiation-promoting functions and its preparation method and application, the preparation method of the hydrogel microsphere comprising: (1) using gelatin and methacrylic anhydride to synthesize a photo-crosslinkable gelatin methacrylate prepolymer; (2) preparing a GelMA hydrogel by a microfluidic device; (3) connecting fibronectin to the GelMA hydrogel obtained in step (2) to obtain a fibronectin-modified GelMA hydrogel; (4) binding platelet-derived growth factor-BB to the fibronectin-modified GelMA hydrogel obtained in step (3) to obtain a hydrogel microsphere having both mechanical properties and differentiation-promoting functions. The hydrogel prepared by the present invention solves the contradiction between the existing hydrogel material that a lower modulus is required to better promote the differentiation of nucleus pulposus stem cells, but a higher mechanical strength is required to ensure its use performance when implanted in vivo, not only achieving a good differentiation effect, but also improving the modulus of the hydrogel microsphere.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a hydrogel microsphere having both mechanical properties and differentiation-promoting functions and a preparation method and application thereof, which can be used for endogenous degenerative tissue regeneration, especially endogenous regeneration of intervertebral disc degenerative tissue. Background Art

[0002] Under the regulation of special induced differentiation, stem cells have the potential to proliferate and differentiate into a variety of cells, thus making it possible to compensate for or replace the functions of degenerated cells or tissues. However, the abnormal mechanical microenvironment in degenerated tissues induces the misdirection of cell fate, making it difficult for stem cells cultured in vitro to achieve efficient endogenous regeneration in vivo.

[0003] Endogenous regeneration strategies generally use functional biomaterials to activate endogenous progenitor / stem cells of tissues to migrate to damaged areas. Endogenous cells replace apoptotic cells by differentiation and secrete anti-inflammatory cytokines to achieve tissue reconstruction. However, in the process of tissue repair, even if the biomaterials successfully recruit enough endogenous cells, it is difficult to fully restore normal tissue function, which often leads to the failure of endogenous repair.

[0004] The mechanism of endogenous repair failure is due to the harsh microenvironment of damaged tissues, which affects the survival and differentiation of endogenous cells, while untrained cells lack the ability to differentiate into target lineages and regulate inflammation. Therefore, it is often necessary to properly train endogenous progenitor / stem cells in order to guide their differentiation into therapeutic cells to achieve precise endogenous repair.

[0005] Cell training strategies based on biomaterials have been applied to the directed differentiation of progenitor / stem cells. These training strategies can be mainly divided into two categories: biochemical induction and physical stimulation. Biochemical induction includes the use of inductive biochemical factors and genetic engineering methods. Inductive biochemical factors, such as BMP-2 and TGF-β3, can promote the bone and cartilage differentiation of stem cells. However, the dosage of these inductive biochemical factors has always been controversial. In order to ensure the effective differentiation of stem cells, high doses of inductive biochemical factors are usually required, and high doses of inductive biochemical factors may activate inflammation and additional osteophyte formation in vivo. Genetic engineering methods precisely train stem cell behavior by regulating specific genes of interest, but it is difficult to strike a balance between gene transfection efficiency and cytotoxicity. Physical stimulation has higher biosafety than biochemical induction. Physical signal training such as electromagnetic stimulation has been shown to promote neural and cardiac differentiation of stem cells in vitro, but as an exogenous physical stimulus, unstable signal input and energy supply problems limit its application in vivo.

[0006] Mechanical signals transmitted by the extracellular matrix (ECM) have been shown to be a huge factor in regulating stem cell behavior and fate. The cell's integrin receptors bind to ECM ligands to achieve stable adhesion, the cell's actin produces a skeleton to feedback the ECM's mechanical signals, and transcribes the mechanical signals to the nucleus through Yes-associated protein (YAP), thereby activating downstream signaling pathways to regulate its own behavior. During the regeneration process, damaged tissues undergo a series of matrix remodeling and ECM modulus changes to promote the proliferation and migration of endogenous stem cells and achieve early self-repair. This phenomenon suggests that the elastic modulus of the ECM is an important mechanical cue for training stem cell behavior patterns.

[0007] However, in severe tissue damage and long-term degenerative diseases, abnormal changes in matrix modulus can lead to exhaustion of the endogenous stem cell pool and uncontrolled differentiation, resulting in repair failure. For example, cartilage from patients with osteoarthritis exhibits a much higher elastic modulus than healthy cartilage; and hardening of ocular tissue is closely related to the progression of ocular neurodegenerative diseases.

[0008] Many experiments have demonstrated that substrates that mimic tissue-specific moduli can promote stem cell differentiation into specific tissue lineages. [1] Studies have shown that soft matrix (<5Kpa) inhibits the formation of actin filaments and promotes the differentiation of stem cells into fat and endothelial cells; while hard matrix (>10Kpa) promotes the diffusion of stem cells and actin polymerization, thereby activating the nuclear localization of YAP, which in turn reflects a higher differentiation trend into osteoblasts and myocardium. [2] et al. found that on hydrogels with lower elastic modulus (about 1 KPa), pluripotent stem cells differentiated into neural cells; on hydrogels with medium elastic modulus (about 10 KPa), cells differentiated into muscle cells because the elastic modulus was similar to that of muscle tissue; on hydrogels with higher hardness (about 100 KPa), it was more suitable for differentiation into osteoblasts. [3] It was found that the increase in the elastic modulus of the material itself is more conducive to the differentiation of intestinal cells. It can be seen that the elastic modulus of the material can significantly affect cell adhesion, migration, proliferation and differentiation, and the specific modulus of different tissues is closely related to cell differentiation.

[0009] Intervertebral disc degeneration can be attributed to the aging and excessive apoptosis of nucleus pulposus (NP) cells, which leads to an imbalance in the synthesis / metabolism of the extracellular matrix (ECM) and ultimately to ECM degradation. Studies have shown that NP-derived mesenchymal stem cells isolated from degenerated intervertebral discs have strong proliferation and differentiation potential.

[0010] In recent years, many studies have shown that there are native progenitor / stem cell niches in the cartilage endplate, annulus fibrosus and nucleus pulposus of the intervertebral disc, and these cells also have the omnipotence of osteogenic, chondrogenic and adipogenic differentiation. Therefore, activating these endogenous cells to migrate from the niche area to the nucleus pulposus is a new method that is expected to treat intervertebral disc degeneration.

[0011] However, since the nucleus pulposus of the degenerated intervertebral disc has the characteristic of local hardening, the higher elastic modulus will transmit bad mechanical signals, leading to the incorrect differentiation of endogenous cells, aggravating the fibrosis and degeneration of the intervertebral disc. Therefore, for the intervertebral disc, the existing implants with mechanical training often sacrifice the corresponding elastic modulus to ensure the differentiation of intervertebral disc progenitors / stem cells into nucleus pulposus cells, but this has an adverse effect on the performance of the implant material. Since the intervertebral disc is a stress tissue of joint activity, the intervertebral disc implant material needs to have sufficient mechanical properties and biological requirements. The modulus and strength of some scaffolds even reach tens of MPa. [4] Therefore, on the one hand, people want the intervertebral disc implant materials to meet their high mechanical performance requirements, and on the other hand, they hope that the materials have a relatively low elastic modulus to promote the differentiation of intervertebral disc progenitors / stem cells into nucleus pulposus cells, which has become a major contradiction in the development of intervertebral disc implant materials at this stage.

[0012] For example, Navaro et al. [5] A hydrogel was synthesized to facilitate the adhesion of nucleus pulposus stem cells and promote the expression of nucleus pulposus related genes Aggrecan and Sox9. The results showed that the hydrogel with a lower shear storage modulus (1KPa) was better than the hydrogel with a higher shear storage modulus (2KPa). Therefore, the conclusion drawn from the existing research is that in order to achieve better directional differentiation of nucleus pulposus stem cells and promote the expression of related differentiation genes, it is more inclined to prepare a hydrogel with a shear storage modulus of 1KPa. Obviously, people hope that the modulus of the hydrogel material is higher to meet its performance in the body.

[0013] At present, there are very few literature reports on how to solve the contradiction that endogenous stem cells in the intervertebral disc require a lower modulus during directed differentiation, but require a higher strength when used in vivo. This issue has become a major problem in the research of intervertebral disc implant materials.

[0014] Therefore, in the process of achieving endogenous treatment of intervertebral disc regeneration, how to provide an in vivo implant material that has a relatively high elastic modulus, can better meet the application of the intervertebral disc in vivo, and can also train stem cells to differentiate into nucleus pulposus cells has become a technical problem that needs to be solved urgently.

[0015] The references cited are as follows:

[0016] [1]K.-K. Tian, ​​S.-C. Huang, X.-X. Xia, Z.-G. Qian, Biomacromolecules2022, 23, 1777.

[0017] [2]Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directstem cell lineage specification [J]. Cell, 2006, 126(4): 677-89.

[0018] [3] Li Yue, Master's thesis of Zhejiang Gongshang University, Effect of elastic modulus of gelatin / nanofiber composites on 3D intestinal epithelial cell model and evaluation of probiotic immune efficacy, published on June 2022.

[0019] [4] Han Xiao, PhD thesis, Chinese Academy of Medical Sciences and Peking Union Medical College, Design and performance evaluation of polyurethane porous intervertebral disc scaffold under stress based on the concept of three-periodic minimal surfaces, 2022.4.

[0020] [5]Y. Navaro, N. Bleich-Kimelman, L. Hazanov, I. Mironi-Harpaz, Y.Shachaf, S. Garty, Y. Smith, G. Pelled, D. Gazit, D. Seliktar, Z. Gazit,Biomaterials 2015, 49, 68. Summary of the invention

[0021] The present invention is to solve the above technical problems, thereby providing a hydrogel microsphere with both mechanical properties and differentiation-promoting function and its preparation method and application. The technical purpose of the present invention is to provide a hydrogel microsphere and a preparation method to solve the contradiction between the existing hydrogel materials that require a lower modulus to better promote the differentiation of nucleus pulposus stem cells when treating endogenous regeneration of intervertebral discs, but require a higher mechanical strength to ensure its performance when implanted in vivo.

[0022] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0023] The present invention first provides a method for preparing hydrogel microspheres having both mechanical properties and differentiation-promoting functions, comprising the following steps:

[0024] (1) Photo-crosslinkable gelatin methacrylate (GelMA) prepolymer was synthesized using gelatin and methacrylic anhydride;

[0025] (2) Preparation of gelatin methacrylate hydrogel microspheres by microfluidic method;

[0026] (3) shaking and incubating the fibronectin solution and the gelatin methacrylate hydrogel microspheres obtained in step (2) in a constant temperature water bath to obtain fibronectin-modified gelatin methacrylate hydrogel microspheres;

[0027] (4) Binding platelet-derived growth factor-BB to the fibronectin-modified gelatin methacrylate hydrogel microspheres obtained in step (3) to obtain hydrogel microspheres having both mechanical properties and differentiation-promoting functions.

[0028] Gelatin methacrylate (GelMA) is a photocrosslinked hydrogel synthesized by replacing lysine and hydroxylysine of gelatin with methacrylic anhydride (MA). The mechanical properties of GelMA hydrogel can be adjusted by changing the degree of substitution of methacrylic anhydride and the exposure time of ultraviolet light. In the present invention, type A gelatin obtained by acid treatment is used for MA modification because fibronectin has a higher affinity for type A gelatin than type B gelatin obtained by alkali treatment.

[0029] Existing studies have shown (such as reference [5] mentioned above) that lower modulus is more conducive to the adhesion of nucleus pulposus stem cells and promotes the expression of nucleus pulposus related genes Aggrecan and Sox9. However, too low modulus will affect the application of implant materials in vivo. Existing methods cannot well resolve the contradiction that low modulus can promote differentiation while high modulus has better effect. The hydrogel microspheres prepared by the method of the present invention can well resolve this contradiction. It not only provides a hydrogel microsphere that can achieve the same differentiation effect, but also improves the modulus of the hydrogel microspheres, which can be better used in vivo.

[0030] The inventors combined microfluidics and photopolymerization technology to construct gelatin methacrylate (GelMA) microspheres as a mechanical training platform, and achieved precise regulation of the elastic modulus (from 1Kpa to 30Kpa) by changing the degree of substitution of the photogroup and the cross-linking conditions. Furthermore, fibronectin (Fn) as an additional ECM ligand is tightly anchored to the GelMA microspheres through its natural gelatin / collagen binding domain to promote the adhesion and mechanical transduction of endogenous cells. At the same time, platelet-derived growth factor-BB (PDGF-BB) is bound to Fn-modified GelMA (Fn@GelMA) through the heparin binding domain of Fn to construct microspheres that can recruit endogenous stem cells. Through the above method, the present invention constructs hydrogel microspheres with relatively improved elastic modulus and without affecting the differentiation effect, which has great application prospects in the field of intervertebral disc implant material regeneration.

[0031] The hydrogel material provided by the present invention can support the sustained release of PDGF-BB for more than 28 days based on the strong growth factor binding ability of Fn, and can promote the proliferation and migration of nucleus pulposus stem cells in vitro. In vivo, mechanical training microspheres can form a temporary stem cell niche, and the successful recruitment of intervertebral disc progenitor / stem cells and up-regulated ECM synthesis were also observed in the rat acupuncture disc degeneration model.

[0032] Furthermore, the steps of the microfluidic method described in step (2) are as follows: a mixed solution of gelatin methacrylate prepolymer and a photoinitiator is injected into a microfluidic channel to form monodisperse hydrogel droplets under the shear of the oil phase, and then the droplets are cross-linked using 365nm blue light to form solidified hydrogel microspheres.

[0033] Furthermore, the concentration of gelatin methacrylate prepolymer in the gelatin methacrylate hydrogel microspheres is 7.5 wt %, and the degree of substitution of photogroup methacrylate in the gelatin methacrylate material is 20 %.

[0034] Furthermore, the concentration of the fibronectin solution in step (3) is 2 μg / ml.

[0035] Furthermore, in step (3), the weight ratio of fibronectin to gelatin methacrylate hydrogel microspheres is 1:3.

[0036] Furthermore, in step (3), the mixture is incubated with shaking at 37° C. for 1 hour.

[0037] Furthermore, the mass ratio of platelet-derived growth factor-BB to fibronectin-modified gelatin methacrylate hydrogel microspheres in step (4) is 1:200,000-250,000.

[0038] Furthermore, in step (4), the diluted platelet-derived growth factor-BB solution and the fibronectin-modified gelatin methacrylate hydrogel microspheres are incubated with shaking at 4° C. for 12 hours.

[0039] The second object of the present invention is to provide a hydrogel microsphere prepared by the method as described above, which has both mechanical properties and promotes differentiation. The elastic modulus of the hydrogel microsphere is 2KPa, and the average particle size of the hydrogel microsphere is 80-200μm.

[0040] The third object of the present invention is to provide the use of the hydrogel microspheres prepared by the method as described above in the preparation of implant materials for treating intervertebral disc degeneration.

[0041] Furthermore, the present invention also provides the use of the hydrogel microspheres prepared by the above method in the preparation of a drug for treating endogenous regeneration of intervertebral disc.

[0042] The beneficial effects of the present invention are as follows:

[0043] The present invention constructs a hydrogel microsphere with both mechanical properties and differentiation-promoting functions, which combines the characteristics of endogenous cell recruitment and instructing cell differentiation to repair intervertebral disc degeneration. The hydrogel microspheres provided by the present invention can independently and flexibly adjust the elastic modulus and ECM ligand density, and can mobilize the migration of endogenous stem cells and train cells and guide their differentiation fate. In vitro and in vivo experiments show that the hydrogel microspheres of the present invention can effectively train stem cells to differentiate into nucleus pulposus-like cells through mechanical signal transduction, achieving maximum nucleus pulposus repair ability. At the same time, the elastic modulus of the hydrogel material has been improved compared with the prior art, and can be better used for in vivo applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Middle: a) 1H NMR spectra of gelatin and GelMA with 20% and 100% MA substitution; b) Schematic diagram of mechanical properties of GelMA and changes in ECM density; c) Morphological characterization of microspheres: i) Microscope image of microspheres; ii) Particle size distribution; d) Compression properties of hydrogels: i) Stress-strain curve of hydrogels; ii) Elastic modulus of hydrogels; (n=3); iii) Rheological analysis of hydrogels; (n=3); e) Characterization of Fn-GelMA microspheres: i) Schematic diagram of the interaction between GelMA and Fn; ii) Immunofluorescence image of Fn-GelMA microspheres; iii) Naming principle of microspheres; f) Quantification of the degree of ECM ligand modification at different densities; (n=3); g) SEM image of freeze-dried microspheres; ∗∗∗∗p<0.0001.

[0045] Figure 2 Middle: a) Schematic illustration of the effect of microsphere mechanical cues on cytoskeletal polymerization; b) Confocal immunofluorescence images of F-actin polymerization and actin arrangement; c) Quantification of F-actin localization in NPSCs; (n=5); d) Quantification of the average diameter of actin filaments; (n=10); ∗p<0.05; ∗∗p<0.01 and ∗∗∗∗p<0.0001.

[0046] Figure 3Characterization of NPSCs’ NP-like differentiation; a) Schematic diagram of mechanotransduction directing cell fate; b) Schematic diagram of the procedure for detecting cell gene expression; c) Immunofluorescence images of YAP and Col II; d) Quantification of YAP nuclear localization; (n=5) compared with the Ultrasoft-L group; e) Semi-quantitative fluorescence intensity of Col II; (n=5) compared with the Ultrasoft-L group; f) Expression of differentiation genes; compared with the control group; (n=3); g) Expression of catabolic genes; compared with the control group; (n=3); ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001.

[0047] Figure 4 Middle: a) Schematic diagram of Fn binding to PDGF-BB; b) Release curves of GelMA microspheres and Fn-GelMA microspheres; (n=3) c) Evaluation of cell migration; i) Schematic diagram of cell scratch healing assay; ii) Microscopic images of scratches at 0, 12, and 24 h; iii) Quantification of the percentage of scratch healing; (n=3) d) Evaluation of cell chemotaxis; i) Schematic diagram of chemotaxis assay; ii) Microscopic images of NPSCs migrating from the upper chamber; iii) Quantification of the number of migrated cells; (n=3); e) Evaluation of the ability of microspheres to capture NPSCs in a 3D culture model; i) Microspheres were cultured with NPSCs on non-adherent plates; ii-iv) Immunofluorescence images of cells captured and adhered to GelMA, Fn-GelMA, and PDGF@Fn-GelMA microspheres after 24 h; ∗p<0.05 and ∗∗∗∗p<0.0001; f) Schematic diagram of microspheres recruiting endogenous progenitor / stem cells in the intervertebral disc.

[0048] Figure 5 Radiological evaluation of the rat IVD degeneration model; a) Schematic diagram of the establishment of the rat coccygeal puncture model and radiological investigation; b) Schematic diagram of the calculation of intervertebral disc height change; c) X-ray images of IVD at 4 and 8 weeks after surgery; d) MRI images and heat maps of IVD water content at 4 and 8 weeks after surgery; e) DHI changes of IVD at 4 and 8 weeks after surgery; (n=6); f) Signal density changes of water content at 4 and 8 weeks after surgery; (n=6); ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; and ∗∗∗∗p<0.0001.

[0049] Figure 6Histological analysis of animal experiments; ab) H&E and safranin fast green staining images of IVDs at 4W and 8W after surgery; histological individual scores evaluated according to four categories at 4W (c) and 8W (d) after surgery; histological total scores at 4W (e) and 8W (f) after surgery; (n=6); ∗∗p<0.01 and ∗∗∗∗p<0.0001. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention. Example

[0051] 1. Gelatin Methacrylate (GelMA) Synthesis

[0052] 20 g of type A gelatin (derived from pig skin) was dissolved in 200 ml of carbonate-bicarbonate buffer (0.1 M) at 60 °C with stirring. 1 ml or 5 ml of methacrylic anhydride was added dropwise to the gelatin solution and reacted at 50 °C for 3 hours. Excessive PBS was added to stop the reaction. The diluted mixed solution was dialyzed in deionized water at 37 °C for 3 days, and then the purified sample was freeze-dried for 3 days to form a white GelMA foam. The GelMA foam was dissolved in deuterium oxide and hydrated with 1 H NMR measures the degree of substitution of methacrylate groups.

[0053] 2. Fabrication of Microfluidic Device

[0054] A simple microfluidic device was constructed by coaxially inserting a 32G medical needle into a 27G needle. The aqueous phase was injected from the 32G needle, and a hole was drilled in the cavity of the 27G needle to allow the oil phase to flow in. All other connecting parts were sealed with resin glue.

[0055] 3. Generation of Microfluidic Hydrogel Microspheres

[0056] Freeze-dried GelMA foam was dissolved in deionized water at 37 °C at a concentration of 7.5 wt%. GelMA solution containing 0.25% lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) photoinitiator was injected into the dispersed phase channel. Paraffin oil containing 5 wt% Span 80 surfactant was introduced into the continuous phase channel. The flow rate ratio of the inner and outer phases was controlled to vary from 5:1 to 8:1 to adjust the microsphere size from 80 to 200 μm. The GelMA fluid was squeezed by the oil phase and broke at the junction of the coaxial needles to form droplets. The GelMA droplets were then collected in a low-temperature bath (1-6 °C) and cross-linked under ultraviolet irradiation (365 nm). The cross-linked microspheres were repeatedly washed with ether and deionized water to remove residual paraffin oil and surfactant.

[0057] 4. Preparation of Fibronectin-GelMA Microspheres

[0058] The microspheres were sterilized by soaking in 75% ethanol overnight. The sterilized microspheres were then soaked in anhydrous ethanol and blown dry on a clean bench to remove residual surface moisture. Fibronectin solution (2 μg / ml) was then added and the microspheres were incubated with fibronectin at 37°C for 1 hour with shaking. The microspheres were washed with PBS to remove unbound free fibronectin.

[0059] 5. Physical Characterization of Fibronectin-GelMA Microspheres

[0060] The elastic modulus of the microspheres was measured by atomic force microscopy nanoindentation to obtain the stress-strain curve of the microspheres. The elastic modulus of the hydrogel microspheres was obtained by calculating the slope of the linear strain region from 0% to 30%. The storage modulus of the hydrogel microspheres was measured using a rheometer. Whether the elastic modulus of the microspheres changed before and after fibronectin modification was measured. The microspheres were freeze-dried and the microstructure was observed under a scanning electron microscope.

[0061] 6. Isolation and identification of nucleus pulposus stem cells

[0062] The nucleus pulposus was isolated from the rat coccygeal intervertebral disc, cut into pieces, and digested in DMEM / F12 medium containing 0.1% type II collagenase for 2 hours to isolate the nucleus pulposus cells. The isolated cells were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum. The cells were reseeded in a 6-well plate (density of 500 cells / ml) and the serum concentration was adjusted to 2% until stem cell clones were formed. The phenotype of the stem cells, including CD90, CD29, CD44 and CD45, was identified using flow cytometry.

[0063] 7. Fibronectin-GelMA microspheres regulate the skeleton and differentiation of nucleus pulposus stem cells

[0064] The microspheres were mixed with cell suspension (1×106 / ml) were mixed and co-cultured on low-adhesion culture plates. After 3 days of culture, phalloidin stain and YAP antibody were used to examine the cell skeleton formation and YAP expression level. Co-culture continued for 14 days, and qPCR and immunofluorescence staining were used to examine the level of cell differentiation, with Col II, ACAN, SOX9 and COL I as the indicators.

[0065] 8. Loading and release of platelet-derived growth factor-BB

[0066] 1 ml of microspheres were incubated with 1 ml of platelet-derived growth factor-BB (recombinant human PDGF-BB, molecular weight 24.8 KDa, Sigma-Aldrich) at a concentration of 300 ng / ml at 4°C overnight. The microspheres were then centrifuged at low speed (500 rpm) and the supernatant was collected. The concentration of unbound growth factor in the supernatant was measured using an ELISA kit for PDGF-BB to determine the loading efficiency. For release experiments, PBS containing 0.1% BSA was used as the release medium and released in a shaker at 37°C. The release medium supernatant was collected at the specified time points and the concentration of PDGF-BB released in the supernatant was measured.

[0067] 9. Biocompatibility and in vitro recruitment

[0068] The microspheres were placed in the upper chamber of a 24-well plate (pore size 0.4 µm), and nucleus pulposus stem cells (1×10 6 On days 3 and 7, cell viability was examined using live-dead cell stain. To assess cell proliferation, cells were seeded in 96-well plates (1×10 4 / ml), and microspheres were added for co-culture. CCK8 solution was added on days 1, 2, and 5 for 1 hour, and the absorbance at 450 nm was measured to examine cell proliferation.

[0069] To evaluate cell migration in vitro, microspheres were placed in the upper chamber, while NPs were seeded in the lower chamber (0.4 µm pores). When cells reached 70-80% confluence, a straight scratch was made with a 200 µL pipette tip, and the medium was replaced with serum-free medium for continued culture. The wound healing was observed at 0, 12, and 24 hours. For chemotaxis assays, microspheres were placed in the lower chamber, while cells were seeded in the upper chamber (8 µm pores). The upper chamber was serum-free medium, while the lower chamber contained 10% serum medium. After 24 hours of culture, the chambers were collected and the cells were fixed with 4% paraformaldehyde. They were then stained with 0.5% crystal violet, eluted with acetic acid, and the absorbance of the eluate at 590 nm was measured to quantify the number of cells that migrated. To evaluate the three-dimensional recruitment ability of microspheres, microspheres and cells were seeded on low-adhesion culture plates. After 24 hours, live cell stain was added to observe cells on the surface of microspheres.

[0070] 10. In vivo testing

[0071] 10-week-old rats were selected and anesthetized with intraperitoneal injection of pentobarbital (50 mg / kg). The intervertebral discs of the 5-6, 6-7, 7-8, and 8-9 segments of the rat's coccygeal vertebrae were punctured with an 18G needle. The needle punctured the center of the intervertebral disc and then rotated 360° and stayed for 30 seconds. Subsequently, 10 μL of materials (PDGF@Fn Soft-L MS, PDGF@Fn Stiff-H MS, free PDGF-BB, and PBS) were injected into the intervertebral disc using a 1 ml syringe.

[0072] At 4 and 8 weeks after surgery, the disc height was measured using X-rays, and the water content of the disc was measured using 0.5T MRI. The rats were then killed, and the disc tissues were collected and fixed with 4% paraformaldehyde at 4°C and decalcified. The samples were embedded in paraffin, cut into 5 μm sections, and stained with H&E, Safranin O / fast green, and aggrecan.

[0073] 1. Preparation and characterization of microspheres with adjustable mechanical properties

[0074] The GelMA microspheres with adjustable mechanical modulus prepared by coaxial microfluidics were characterized. The 1H NMR spectrum results are shown in Figure 1 In part a, the new signals appearing at 5.4 ppm and 5.6 ppm indicate the successful modification of MA. The degree of methacrylation is calculated by comparing the decrease of the peak of GelMA at 2.9 ppm compared to gelatin. Finally, it is determined that the substitution degree of the synthesized GelMA is 20% and 100%, respectively.

[0075] To prepare microfluidic microspheres, the aqueous and oil phases consisting of 7.5% GelMA precursor solutions with different methacrylate degrees were injected into a coaxial microfluidic device to form hydrogel droplets of oil-in-water emulsions, which were subsequently cured by UV cross-linking. The prepared microspheres showed good size distribution (coefficient variation <5%), with an average diameter of less than 150 μm, and were able to be injected for administration ( Figure 1 Part c, i-ii).

[0076] The results of atomic force microscopy showed that by regulating the cross-linking conditions, microspheres with elastic moduli of 1Kpa (named Ultrasoft), 2Kpa (named Soft) and 10Kpa (named Stiff) were successfully produced. Figure 1 (Part d in the figure).

[0077] Next, fibronectin was modified on the microspheres in low-density and high-density forms, with the concentrations of fibronectin set to 2 μg / ml (named L) and 10 μg / ml (named H), respectively. By binding to different concentrations of fibronectin, the ligand density of the microspheres can be easily adjusted ( Figure 1 (Fig. e, i). The storage modulus of fibronectin-bound GelMA was not affected by fibronectin modification.

[0078] The Fn coating was visualized using FITC-labeled fibronectin antibody, demonstrating the tight binding between GelMA and fibronectin, whereas no fluorescence signal was observed on unmodified GelMA microspheres ( Figure 1 The detailed naming scheme for each group is in Figure 1 At the same time, the fluorescence intensity increased with the increase of fibrin density, and at the same density, there was no significant difference in the binding of GelMA microspheres with different elastic moduli to fibrin ( Figure 1 (f) SEM images of six types of Fn-GelMA microspheres are shown in Figure 1 Part g. It can be observed that the microspheres of all groups exhibited smooth surfaces and porous microstructures, and the microspheres with lower modulus also exhibited larger pore sizes. In addition, when the microspheres were combined with fibronectin, salt crystals were seen to form on the surface of the microspheres.

[0079] 2. Isolation and characterization of nucleus pulposus-derived stem cells

[0080] Nucleus pulposus stem cells (NPSCs) were used as representative IVD progenitor / stem cells for subsequent experiments. NPSCs were extracted from the nucleus pulposus tissue of the rat coccygeal intervertebral disc using a monoclonal method. Nucleus pulposus-derived cells were seeded at a very low density, and a portion of the cells were able to attach to the culture plate and then divide rapidly. After a week of culture, single cells were able to form cell clones.

[0081] It was observed that the extracted cells exhibited the spindle shape characteristic of mesenchymal stem cells and grew in parallel or vortex patterns. In contrast, nucleus pulposus cells showed polygonal morphology and random growth patterns. Cell clones were collected and flow cytometry was used to examine the expression of surface markers. The results showed that NPSCs were highly positive for typical MSC markers CD29, CD44, and CD90, but negative for hematopoietic stem cell marker CD45. In contrast, nucleus pulposus cells expressed low levels of MSC markers (<50%). These results indicate that highly purified NPSCs were successfully isolated from nucleus pulposus-derived cells.

[0082] 3. Evaluation of microsphere training-guided stem cell differentiation

[0083] First, we focus on the effect of mechanical training of microspheres on the cytoskeleton formation of NPSCs ( Figure 2 NPSCs were cultured on the microspheres for 3 days, and all material groups supported cell adhesion and spreading, as well as actin filament aggregation ( Figure 2 (part b). In addition, the formation of actin stress fibers was shown to be regulated by the elastic modulus and ligand density of the microspheres. NPSCs cultured on Ultrasoft-L microspheres exhibited a significantly wider actin leading edge compared to other groups. It was also observed that actin assembly was mainly produced at the leading edge of the cells, indicating that the fiber network was mainly polymerized along the plasma membrane of stem cells. By calculating the ratio of actin immunofluorescence density in the intracellular / cortical layer, it was found that the relatively soft matrix and low ECM ligand density could maintain low expression of F-actin along the cell membrane boundary. There was no statistical difference in the intracellular / cortical fluorescence ratio between the Ultrasoft group and Soft-L ( Figure 2 (Part c).

[0084] Normally, if the actin inner / cortical ratio is low, it indicates that mesenchymal stem cells differentiate into adipocytes and cartilage, while the polymerization and fiber extension of actin throughout the cytoplasm are highly correlated with the differentiation of osteoblasts and annulus fibrosus cells. Similar to chondrocytes, actin tends to accumulate at the periphery of nucleus pulposus cells. Therefore, NPSCs cultured on the Ultrasoft group and Soft-L group showed the potential to differentiate into nucleus pulposus-like cells.

[0085] In addition, the orientation of actin filaments in cells was investigated. When cultured on Ultrasoft-L microspheres, NPSCs showed random orientation of actin filaments, while as the density of microsphere ligands increased, an orderly orientation of F-actin was observed. Similar phenomena were also observed in Soft microspheres.

[0086] The diffuse distribution of F-actin was associated with reduced cytoskeletal integrity, indicating that less cytoskeletal tension was generated on Ultrasoft-L and Soft-L microspheres. In addition, the average diameter of actin fibers increased significantly with increasing elastic modulus and ligand density ( Figure 2 This also proves that the cells generate greater cytoskeletal tension.

[0087] YAP activity depends on actin-generated cytoskeletal tension to regulate stem cell fate. Figure 3 Next, we investigated the effect of cytoskeletal tension generated by actin fibers on YAP nuclear translocation. Figure 3As shown in part c, the cytoplasmic localization of YAP is manipulated by the elastic modulus and ligand density of the microspheres. Retention of YAP in the cytoplasm was observed in Ultrasoft microspheres regardless of the ECM density. Interestingly, a significant translocation of YAP occurred when the ligand density of Soft-L microspheres was changed. In addition, the enhanced YAP nuclear localization trend was highly correlated with the continuous increase in the microsphere elastic modulus and ECM ligand density ( Figure 3 (Part d in the figure).

[0088] In this experiment, the Soft-H group and the Stiff group were shown to be sufficient to support the nuclear localization of YAP in nucleus pulposus stem cells, which may hinder the differentiation of stem cells into nucleus pulposus-like cells. This result is similar to the results of existing studies.

[0089] To further verify that appropriate mechanical cues can guide NPSCs to nucleus pulposus-like differentiation, the expression of nucleus pulposus-specific genes was evaluated when NPSCs were cultured on different groups of microspheres. All groups were cultured in complete medium without the addition of chondrogenic differentiation agents. The expression level of Col II was visualized by immunofluorescence staining ( Figure 3 The highest expression of Col II was observed in the Soft-L group, and the fluorescence intensity decreased slightly with the increase of ligand density ( Figure 3 In particular, only very low fluorescence signals were observed in the Ultrasoft and Stiff microsphere groups.

[0090] Next, the mRNA levels of Sox9, ACAN, NCAM-1, and Col I were examined by qPCR. Sox9, ACAN, and NCAM-1 were selected as nucleus pulposus-specific genes, while Col I is considered a marker of fibrosis. NPSCs cultured on a plate were used as a control group. It should be noted that the elastic modulus of traditional polystyrene culture plates is about 1 GPa, which is much higher than that of most natural tissues. Figure 3As shown in part f, no obvious upregulation of NP genes was observed in the Ultrasoft microsphere group, regardless of how the density of fibronectin was changed. This is consistent with the results of Col II staining. Therefore, NPSCs cultured on Ultrasoft microspheres always produced fewer NP genes, a phenomenon that may be attributed to its preference for the adipogenic differentiation lineage. Among all microsphere groups, the Soft-L group presented the highest NP-like differentiation potential. Interestingly, for both the Soft and Stiff microsphere groups, a significant decrease in NP gene expression could be observed with increasing ligand density. This is consistent with the results of cytoskeleton formation and YAP localization in previous experiments, indicating that excessive cytoskeletal tension will likely hinder NPSC differentiation into NP-like. The expression level of Col I also demonstrated that the fibrotic tendency is highly correlated with the matrix modulus. All microsphere groups showed a downregulation of Col I levels compared with the control group. In addition, the expression of Col I can be enhanced by increasing the elastic modulus and ECM density.

[0091] The process of ECM remodeling involves the degradation and synthesis of ECM components, so we next investigated the expression levels of catabolic genes, including Mmps, Adamts, and Timps, in the early stages of cell differentiation. Figure 3 (g). Upregulation of ECM remodeling enzymes was observed in most groups except the Ultrasoft group. Upregulation of Adamts 5 was observed in all experimental groups except the Ultrasoft group. In the Ultrasoft group, there was no significant increase in the expression of Mmp 13 and Adamts 4, indicating that this modulus range is more conducive to adipogenesis. In short, the expression of essential catabolic genes in NPSCs validates the ability of microspheres to regulate cell differentiation.

[0092] Based on the purpose of the present invention to repair the nucleus pulposus, it is concluded that Soft-L microspheres are a suitable candidate for intervertebral disc stem cell training. On the other hand, other microsphere groups can be used to treat various other diseases according to the characteristics of the tissue. For example, Stiff microspheres are expected to be used as a training platform for repairing annulus fibrosus injuries.

[0093] 4. Construction of cell recruitment microspheres

[0094] Based on the previous results, Soft-L microspheres were used for subsequent experiments. Cell recruitment microspheres were prepared by loading PDGF-BB on Fn-GelMA microspheres ( Figure 4 In the middle part (a), the concentration of PDGF-BB was optimized, and it was expected that the microspheres would form a synthetic niche in vivo to recruit endogenous progenitor / stem cells for colonization. The loading efficiency of PDGF on Fn-GelMA microspheres and unmodified GelMA microspheres was approximately 97.50% and 95.39%.

[0095] In the release experiment, Fn-GelMA microspheres supported a slower release of PDGF than GelMA microspheres, with 8.67% of PDGF-BB released from the microspheres after 28 days. In contrast, GelMA microspheres released more PDGF at any time point, with a total release of 14.06% ( Figure 4 The higher loading capacity and longer release period indicate that Fn enhanced the binding of GelMA microspheres to PDGF-BB, because the heparin binding sites provided by the 12FnIII-13FnIII-14FnIII fragment of Fn can efficiently bind to various growth factors.

[0096] In vitro biocompatibility experiments showed that PDGF@Fn-GelMA microspheres could maintain high viability of NPSCs, with very few dead cells observed in live / dead staining images. In addition, CCK8 results showed that the combination of PDGF-BB could promote cell proliferation. Therefore, given the limited number of progenitor cells, endogenous regeneration has always been challenging, and the synthetic niche proposed in this invention can promote the expansion of endogenous cells before cell differentiation.

[0097] To evaluate the recruitment capacity of microspheres, a co-culture system was constructed using the Transwell system to simulate cell migration within the stem cell niche. In the scratch assay, microspheres were placed in the upper chamber, while NPSCs were seeded in the lower chamber ( Figure 4 Part c, i). Figure 4 As shown in part c, ii, the migration area increased in all groups after 12 and 48 hours. The highest scratch healing rate was observed in the PDGF@Fn-GelMA group, reaching 81.5% after 48 hours ( Figure 4 In the Transwell migration assay, microspheres were placed in the lower chamber and NPSCs were seeded in the upper chamber ( Figure 4 Different from the results of the scratch test, there was no significant difference between the Fn-GelMA microsphere group and the control group after 24 h. However, significantly enhanced cell migration was observed in the PDFD@ Fn-GelMA microsphere group ( Figure 4 (d) Part ii-iii).

[0098] To further investigate the ability of microspheres to recruit stem cells, microspheres and NPSCs were plated on non-adherent culture plates and cultured for 24 h, followed by staining with Calcein AM ( Figure 4 Although some cells were observed adhering to the microsphere surface in the GelMA and Fn-GelMA groups, a large number of cells were detected on the PDGF@Fn-GelMA microspheres and formed cell aggregates ( Figure 4The above results indicate that the controlled release of PDGF-BB from microspheres promoted the migration of NPSCs.

[0099] Therefore, the PDFD@Fn-GelMA microspheres prepared in the present invention can be used to recruit progenitor / stem cells residing in the nucleus pulposus, annulus fibrosus and cartilage endplate to the damaged area ( Figure 4 (see Part f in the figure).

[0100] 5. Microspheres train IVDs regeneration in vivo

[0101] To further investigate the therapeutic effect of microsphere mechanical biotraining on nucleus pulposus regeneration in vivo, a rat acupuncture disc degeneration model was established using an 18G needle, and radiological evaluations were recorded at 4 and 8 weeks after surgery ( Figure 5 The disc height index (DHI) was measured by X-ray images of the rat tail and reflects the changes in the disc space ( Figure 5 Part b). Rats were treated with microspheres and free PDGF-BB, respectively. To emphasize the importance of mechanotransduction in in vivo regeneration, Stiff-H microspheres (abbreviated as Stiff MS) ​​were also loaded with the same dose of PDGF-BB for treatment in order to compare with Soft-L microspheres (abbreviated as Soft MS) because they showed the greatest difference in previous in vitro differentiation experiments. Four weeks after surgery, X-ray images showed that there was no statistical difference in DHI changes in the soft microsphere group compared with the control group, but IDH in the other groups decreased statistically significantly ( Figure 5 Subsequently, at 8 weeks, the soft microsphere group maintained the DHI level to the greatest extent, while the other treatment groups experienced rapid collapse of the disc space. The disc contains 70-80% water, so magnetic resonance imaging (MRI) is used to measure changes in water content to reflect the degenerative process of the IVD. The signal intensity of the IVD decreased in all groups after 4 and 8 weeks, and it can be seen that the water content of the disc can be best maintained with soft microsphere treatment ( Figure 5 These results suggest that cell training through appropriate mechanical signals can further promote nucleus pulposus regeneration while maintaining the structural integrity of the intervertebral disc and reducing the loss of water content.

[0102] Histological analysis was performed 4 and 8 weeks after surgery. HE staining showed that the intervertebral disc structure of the soft microsphere group remained intact, and there was a clear boundary between the nucleus pulposus and the annulus fibrosus at 4 and 8 weeks after surgery ( Figure 6Figure 5 (a and b). In addition, the largest nucleus pulposus area was observed in the soft microsphere group. However, in the other treatment groups, it was observed that the boundary between the annulus fibrosus and the nucleus pulposus was severely interrupted. Safranin O / fast green and immunohistochemical staining were used to reflect the expression of proteoglycans. Although the staining intensity of all groups at week 8 was slightly lower than that at week 4, the soft microsphere group had the highest proteoglycan expression among the treatment groups at any time point.

[0103] Histologic scoring is based on four categories of degenerative changes. A detailed grading table is available at Figure 6 Parts c and d are visualized, and higher scores represent more severe degeneration. Figure 6 Parts e and f show the overall histological scores at 4 and 8 weeks after surgery, indicating that the histological scores of the soft microsphere group were always significantly lower than those of the other treatment groups. In addition, treatment with hard microspheres also delayed disc degeneration compared with the PDGF group, which may be due to the controlled release of growth factors in the microspheres. However, due to the lack of appropriate mechanical training, the hard gel could not achieve endogenous regeneration.

[0104] The above results indicate that the mechanical biosignaling in the Soft MS group has an enhanced regenerative effect in vivo.

Claims

1. A method for preparing hydrogel microspheres having both mechanical properties and differentiation-promoting functions, characterized in that: The following steps are involved: (1) Synthesis of photo-crosslinkable gelatin methacrylate prepolymer using gelatin and methacrylic anhydride; (2) preparing gelatin methacrylate hydrogel microspheres by a microfluidic method; specifically, a mixed solution of gelatin methacrylate prepolymer and a photoinitiator is injected into a microfluidic channel to form monodisperse hydrogel droplets under the shear of the oil phase, and then the droplets are cross-linked using 365 nm blue light to form solidified hydrogel microspheres to obtain gelatin methacrylate hydrogel microspheres; (3) shaking and incubating the fibronectin solution and the gelatin methacrylate hydrogel microspheres obtained in step (2) in a constant temperature water bath to obtain fibronectin-modified gelatin methacrylate hydrogel microspheres; (4) Binding platelet-derived growth factor-BB to the fibronectin-modified gelatin methacrylate hydrogel microspheres obtained in step (3) to obtain hydrogel microspheres having both mechanical properties and differentiation-promoting functions.

2. The preparation method according to claim 1, characterized in that: The concentration of gelatin methacrylate prepolymer in the gelatin methacrylate hydrogel microspheres is 7.5 wt %, and the degree of substitution of photogroup methacrylate in the gelatin methacrylate material is 20 %.

3. The preparation method according to claim 1, characterized in that: The concentration of the fibronectin solution in step (3) is 2 μg / ml, and the weight ratio of the fibronectin to the gelatin methacrylate hydrogel microspheres is 1:

3.

4. The preparation method according to claim 1, characterized in that: In step (3), the mixture was incubated with shaking at 37°C for 1 hour.

5. The preparation method according to claim 1, characterized in that: The mass ratio of platelet-derived growth factor-BB to fibronectin-modified gelatin methacrylate hydrogel microspheres in step (4) is 1:200,000-250,000.

6. The preparation method according to claim 1 or 5, characterized in that: In step (4), the diluted platelet-derived growth factor-BB solution and the fibronectin-modified gelatin methacrylate hydrogel microspheres are incubated with shaking at 4°C for 12 hours.

7. A hydrogel microsphere having both mechanical properties and differentiation-promoting function prepared by the method according to any one of claims 1 to 6, characterized in that: The elastic modulus of the hydrogel microspheres is 2 KPa; and the average particle size of the hydrogel microspheres is 80-200 μm.

8. Use of the hydrogel microspheres according to claim 7 in preparing implant materials for treating intervertebral disc degeneration.

9. Use of the hydrogel microspheres according to claim 7 in preparing a drug for treating endogenous regeneration of intervertebral disc.

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