Functionalized gel microspheres as well as preparation method and application thereof

Abstract

The functional gel microspheres are provided by taking the angle of tissue engineering biomechanics as a starting point, the functional gel microspheres comprise GM (at) mTG porous microspheres and JTG, synthetic tiger bone powder JTG with anti-inflammatory and anti-oxidation characteristics is utilized, multifunctional methacrylic acid gelatin GelMA microspheres are developed, and the JTG is locally delivered to be used for IVDD treatment. In vitro, the functional microspheres not only improve the release kinetics of JTG, but also effectively inhibit the inflammatory response and oxidative stress of lipopolysaccharide-induced nucleus pulposus NP cells, and promote cell proliferation. In vivo, by adjusting the biomechanical matrix rigidity of the functionalized microspheres, the functions of promoting secretion of extracellular matrix ECM, relieving inflammatory response, maintaining the water content of NP and the height of the intervertebral disc, maintaining the complete intervertebral disc tissue structure and the like are achieved, and finally regeneration of IVD is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering biology, takes tissue engineering biomechanics as a starting point, and specifically relates to a functionalized gel microsphere and a preparation method and application thereof. Background Art

[0002] Intervertebral disc degeneration (IVDD) is the most important cause of LBP. IVDD is a complex process caused by the combined action of multiple factors, among which nucleus pulposus degeneration is the key pathological link of intervertebral disc degeneration, but its degeneration mechanism is still unclear and is related to multiple factors.

[0003] Inflammatory response plays an important role in the development of IVDD. The elevated levels of inflammatory factors such as tumor necrosis factor (TNF-α) and interleukin family (such as IL-1 and IL-6) secreted by the nucleus pulposus (NP) and annulus fibrosus (AF) cells themselves are the initiating factors and important links of disc degeneration, and are also important markers for clinical diagnosis of inflammation. In a long-term inflammatory microenvironment, the synthesis and catabolism balance of nucleus pulposus tissue is disturbed, and the normal tissue repair process is broken. Under inflammatory stimulation, the phenotype of nucleus pulposus cells changes, promoting the secretion of proteases such as metalloproteinases (MMPs) and aggrecanases (ADAMTS), thereby inhibiting the synthesis of proteins (GAG) such as type II collagen (Col-II) and proteoglycans, affecting the accumulation of extracellular matrix (ECM), resulting in the gel-like appearance of nucleus pulposus tissue and loss of biomechanical properties. In addition, under the action of excessive inflammation, immune cells such as neutrophils, macrophages, and T cells are further enriched in the disc environment, activating downstream inflammatory cascade reactions and inducing more severe disc degeneration. We attempted to construct a functionalized microsphere therapeutic strategy with tunable biomechanical properties and anti-inflammatory effects to reduce the inflammatory response of NP cells, promote the synthesis of Col-II and ECM, and thus repair the degenerative intervertebral disc.

[0004] Disruption of the mechanical environment of the intervertebral disc will aggravate disc degeneration. The nucleus pulposus is located in the center of the intervertebral disc and is surrounded by the cartilaginous endplate (CEP) and the annulus fibrosus. It is essential to maintain the biomechanical function of the intervertebral disc. The NP and AF are mainly composed of water, glycosaminoglycans (GAGs), and collagen fibers. Collagen fibers form a mesh structure in the intervertebral disc, providing high tensile and stiffness strength to the tissue. Under typical conditions, the AF and NP cooperate to ensure that the intervertebral disc has the mechanical properties required to withstand vertebral stress. When degeneration occurs, the significant loss of GAGs in these structures reduces the hydration capacity of the intervertebral disc. At the same time, the reduction of type II collagen fibers further reduces mechanical integrity and elasticity, resulting in a decrease in structural support capacity. The CEP is a layer of hyaline cartilage that connects the intervertebral disc to the adjacent vertebral body. It acts as a transition layer to relieve stress at the disc-bone interface and effectively balance the flexibility of the intervertebral disc and the rigidity of the vertebral bone. CEP degeneration is believed to play a role in the early stages of IVDD. When CEP undergoes calcification, dehydration and other degeneration, its biomechanical properties may be changed, and the reduction of GAG content may lead to reduced permeability, which hinders the material exchange of the intervertebral disc and aggravates IVDD. A large number of studies have confirmed that a certain number of functional cells, a stable mechanical environment and sufficient nutrient supply are necessary conditions to ensure the regeneration of intervertebral disc IVD, and a stable mechanical microenvironment is a prerequisite for ensuring the survival of functional cells and nutrient supply. For example, dynamic factors such as matrix hardness and cell elasticity in the IVD microenvironment play an important role in the aggravation of IVDD. The biomechanical environment is the environment that cells truly sense and regulate. Whether it is muscle movement training, gene targeted intervention, tissue engineering material research and development or surgical intervention, the biomechanical source of IVDD cannot be avoided. In recent years, with the continuous deepening of tissue engineering technology research, more and more biomaterials have shown superior performance in maintaining a healthy mechanical environment of the intervertebral disc and restoring the physical and chemical properties of the intervertebral disc. Among them, hydrogel is one of the most commonly used materials.

[0005] Hydrogel has a unique three-dimensional network structure, and its water absorption and swelling properties are significantly superior to other materials. At the same time, as a photosensitive biohydrogel material, hydrogel has good biocompatibility, photocuring properties, and adjustable mechanical properties. However, large pieces of hydrogel materials usually have poor mechanical strength and are usually unable to maintain their integrity in the high-pressure environment of the intervertebral disc. The hydrogel fragments formed by the crushing after being compressed cannot maintain their mechanical properties, and also affect the sustained release of the loaded drugs. Therefore, there is an urgent need for a carrier that has both biocompatibility and certain stable mechanical properties, which can achieve stable drug release and provide a good biomechanical environment for degenerative intervertebral discs.

[0006] According to traditional Chinese medicine theory, tiger bones have anti-inflammatory, analgesic, immunomodulatory and fracture healing effects. However, according to the requirements of the Convention on International Trade in Endangered Species, tigers should be protected as endangered animals. China banned the use of tiger bones as medicine in 1993. Jintiange Capsule (JTG) uses bionic medicine research, uses bones from specific parts of non-protected animals, and uses the characteristic spectrum of natural tiger bones as a standard to develop a new generation of tiger bone substitute preparations. It is a new type of Chinese medicine with a national invention patent. Studies have shown that JTG can increase the positive expression of IL-2 and IL-4, reduce the positive expression of IL-1 and IL-6, and promote the mechanism of osteoporotic healing. JTG has been approved by the China Food and Drug Administration for the prevention and treatment of osteoporosis and osteoarthritis (CFDA China Z20030080). The main components of JTG are bone powder and collagen. Considering its anti-inflammatory and analgesic pharmacological effects, it is close to the clinical treatment of intervertebral disc degeneration. Moreover, its anti-osteoporosis effect is likely to have a synergistic effect with inhibiting disc degeneration. Therefore, we tried to use JTG to inhibit disc degeneration. Summary of the invention

[0007] The present invention takes the perspective of tissue engineering biomechanics as a starting point, and provides a method for preparing functionalized microspheres with adjustable matrix stiffness, and its application in the process of intervertebral disc degeneration. The microspheres are successfully loaded with JTG, effectively inhibiting the inflammatory response and oxidative stress of NP cells induced by lipopolysaccharide, and promoting cell proliferation. In vivo, it can stimulate ECM secretion, relieve inflammatory response, and maintain the intact intervertebral disc tissue structure.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides a functionalized gel microsphere, including GM@mTG porous microspheres and JTG.

[0009] The functionalized gel microspheres were cross-linked by glutaminase mTG to achieve dynamic regulation of their matrix stiffness, and JTG was successfully loaded using the porous structure of GelMA to obtain GM@mTG-J microspheres.

[0010] At the same time, a method for preparing the functionalized gel microspheres is also provided, comprising the following steps:

[0011] S1. The GelMA hydrogel precursor aqueous solution and the mTG aqueous solution are uniformly mixed in the LAP solution to obtain the GM@mTG hydrogel precursor aqueous solution, which is enzymatically cross-linked and then photo-cross-linked to achieve dynamic regulation of the mechanical stiffness of the GM@mTG porous microspheres, so that the stiffness of the GM@mTG porous microspheres is gradually increased;

[0012] S2. Incubate the GM@mTG porous microspheres with the JTG to obtain GM@mTG-J microspheres.

[0013] In step 1), the volume ratio of the GelMA hydrogel precursor aqueous solution and the mTG aqueous solution is 1:1. After being fully mixed and cross-linked by enzyme, 405 nm ultraviolet light is used for irradiation, and the mechanical stiffness of the GM@mTG porous microspheres is regulated by controlling the irradiation time of the 405 nm ultraviolet light.

[0014] The GelMA hydrogel precursor aqueous solution is an 8% GelMA hydrogel precursor aqueous solution; and the mTG aqueous solution is a 5 mg / mL mTG aqueous solution.

[0015] In step 2), the GM@mTG-J microspheres are prepared as follows:

[0016] Span 80 and 5% w / w mineral oil were used as the continuous phase;

[0017] JTG with a concentration of 32 μg / mL was mixed with 8% GM@mTG hydrogel precursor aqueous solution in equal volume ratio as the dispersed phase;

[0018] Monodisperse spherical droplets were prepared by oil-in-water microfluidics. Subsequently, the spherical droplets were photocrosslinked under ultraviolet irradiation, and the uniform spherical droplets downstream were collected. The collected spherical droplets were repeatedly washed with 75% ethanol to remove the continuous phase, and then thoroughly washed with phosphate-buffered saline (PBS). The washed monodisperse spherical droplets were frozen and dried at -80°C for 48 h to obtain GM@mTG-J microspheres.

[0019] The functionalized gel microspheres are used in preparing medicines for treating intervertebral disc degeneration or materials for repairing intervertebral disc degeneration.

[0020] The functionalized gel microspheres prepared by the above preparation method are used in preparing drugs for treating intervertebral disc degeneration, or in preparing materials for repairing intervertebral disc degeneration.

[0021] The present invention grafts JTG with modified GM@mTG porous microspheres through covalent bonds to construct functionalized gel microspheres. The microspheres achieve successful loading of drugs and realize local injection of IVDD through a microinjector. Experiments show that in vitro, GM@mTG-J microspheres not only improve the release kinetics of JTG, but also effectively inhibit the inflammatory response and oxidative stress of nucleus pulposus cells induced by lipopolysaccharide, and promote cell proliferation. In vivo, by regulating the biomechanical matrix stiffness of the functionalized microspheres, the synthesis and catabolism balance in the intervertebral disc environment is improved, the synthesis of ECM is promoted, the inflammatory response is relieved, the water content of NP and the height of the intervertebral disc are maintained, and the intact intervertebral disc tissue structure is maintained, etc., which can be used to treat inflammation and regenerate IVDD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 -Synthesis and characterization of microspheres. (A) and (B) Monodisperse GM@mTG microspheres at different magnifications, (C) Particle size distribution of synthesized microspheres (n=50), (D) SEM images of modified GM@mTG microspheres, (E) FTIR spectra of GM and GM@mTG, (F) Frequency scanning rheological analysis of GM and GM@mTG.

[0023] Figure 2 -Effects of microspheres with different matrix stiffness on cell expression. (A) Changes in storage modulus of GM@mTG gel microspheres, (B) Changes in proliferation rate of NP cells in microspheres with different stiffness, (C) and (D) qRT-PCR detection of the expression of ECM-related genes (Col-II and Aggrecan) in cells (n=3).

[0024] Figure 3 -GM@mTG-J microsphere characterization and JTG controlled release performance. (A) Frequency scanning rheological analysis of GM@mTG and GM@mTG-J, (B) FTIR spectrum characterization of GM@mTG and GM@mTG-J, (C) Degradation of synthesized microspheres in PBS, (D) Degradation in Col-II, (E) Release curve of JTG.

[0025] Figure 4 -GM@mTG-J microspheres in vitro safety and cell proliferation evaluation. (A) NP cell survival diagram observed by calcein staining (green represents live cells), (B) NP cell proliferation rate change diagram in functionalized microspheres.

[0026] Figure 5 -Imaging evaluation of animal experiments. (A) Diagram of the construction of rat tail IVDD model, (B) Representative CT scans and MRI images (local images) of different treatment groups in the IVDD model, (C) and (D) Line and bar graphs analyzing the changes in rat tail vertebrae DHI at different times, (E) MRI grading of intervertebral discs at 4 and 8 weeks. DETAILED DESCRIPTION

[0027] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific implementation methods, structural features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and embodiments.

[0028] In the present invention, based on the biomechanical theory, functionalized methacrylate gelatin (GM@mTG) microspheres were developed using JTG with anti-inflammatory and antioxidant properties. Local injection at the lesion site efficiently delivers and releases JTG for IVDD treatment. On the other hand, by adjusting the mechanical matrix stiffness of the gel microspheres, a mechanical microenvironment that is more suitable for IVD regeneration was explored. In vitro, the functionalized microspheres not only improved the release kinetics of JTG, but also promoted NP cell adhesion and proliferation, and inhibited LPS-induced cellular inflammatory response and oxidative stress. In addition, RNA monitoring revealed the effect of biomechanical properties on ECM anabolism. In vivo, a more stable biomechanical microenvironment is provided for IVD regeneration and repair, and the role of the functionalized microspheres in inhibiting inflammatory response, maintaining intervertebral disc height, water content and fibrous tissue structure, promoting ECM deposition and IVD regeneration, etc. was verified in the rat IVDD model.

[0029] In a specific implementation, the method for preparing functionalized gel microspheres includes:

[0030] (1) Preparation of methacrylic anhydride modified gelatin GelMA

[0031] Dissolve 10g gelatin in 100mL PBS solution, then add 1mL methacrylic anhydride (MA) solution (mass ratio 8%), stir thoroughly for 2h, dialyze in deionized water for one week, and freeze-dry to obtain GelMA precursor. Mix 0.5g GelMA with 9mL 0.25% photoinitiator (LAP) solution, heat in a 40-50℃ water bath in the dark for 30min, shake several times during the period, and GelMA hydrogel can be prepared. Immediately filter with a sterile syringe filter with a specification of 0.22μm to obtain a sterile photocurable GelMA hydrogel precursor solution.

[0032] (2) Modification of GelMA gel

[0033] The GM@mTG hydrogel precursor solution was made by uniformly mixing an 8% GelMA aqueous solution and a 5 mg / mL mTG aqueous solution in a LAP (0.05% w / w) solution as the dispersed phase. After photocrosslinking under ultraviolet irradiation, the GM@mTG porous microspheres were obtained by washing and freeze-drying.

[0034] (3) Preparation of GM@mTG-J gel microspheres

[0035] Microspheres were prepared using a coaxial needle with inner and outer dimensions of 30G and 28G, respectively. The outer needle syringe was connected to the microfluidic device, and Span 80 and mineral oil (5% w / w) were used as the continuous oil phase. JTG at a concentration of 32μg / mL and 8% GM@mTG hydrogel precursor liquid were mixed in equal proportions in LAP (0.05% w / w) solution as the dispersed phase. By adjusting the flow rate of the two phases, the ideal jet flow pattern and particle size were obtained. Under ultraviolet (λ=405nm) irradiation, uniform droplets were collected downstream to obtain fresh GM@mTG-J microspheres as functionalized gel microspheres. The collected microspheres were repeatedly washed with 75% ethanol to remove the continuous phase. Then they were thoroughly washed with phosphate buffered saline (PBS). The washed microspheres were frozen at -80℃ and finally freeze-dried for 48h.

[0036] Functionalized gel microspheres, namely GM@mTG-J microspheres, have the following specific applications in the treatment of intervertebral disc degeneration:

[0037] (1) By adjusting the matrix stiffness of the gel microspheres, a mechanical microenvironment suitable for the growth and stability of the intervertebral disc is constructed, which can promote the anabolism of ECM;

[0038] (2) GelMA gel can promote cell proliferation and adhesion, as well as the expression of proteoglycans and type II collagenase

[0039] (3) Long-term release of JTG can improve the proliferation of NP cells

[0040] (4) Sustained release of JTG inflammatory response inhibitors to regulate local inflammatory and oxidative microenvironment;

[0041] The present invention will be further described below in conjunction with embodiments:

[0042] The experimental materials used are all common commercial products and can be purchased on the market. Among them, JTG was purchased from Xi'an Jinhua Pharmaceutical Co., Ltd.

[0043] Example 1 Preparation and characterization of modified GM@mTG microspheres and functionalized GM@mTG-J microspheres

[0044] 1. Experimental methods

[0045] The preparation process of GM@mTG-J microspheres with adjustable biomechanical properties is as follows:

[0046] 1. Synthesis of modified GM@mTG gel microspheres

[0047] Microspheres were prepared using a coaxial needle with inner and outer dimensions of 30G and 28G, respectively. The outer needle syringe was connected to the microfluidic device, with Span 80 and mineral oil (5% w / w) as the continuous oil phase, and the hydrogel precursor liquid was composed of 8% GelMA (substitution degree 60%) and 5mg / mL mTG, mixed in an equal volume ratio in LAP (0.05% w / w) solution as the dispersed phase. By adjusting the flow rate of the two phases, the ideal jet flow pattern and particle size were obtained. Under ultraviolet (λ=405nm) irradiation, the uniform droplets downstream were collected to obtain fresh GelMA@mTG (GM@mTG) microspheres. The collected microspheres were repeatedly washed with 75% ethanol to remove the continuous phase, and then thoroughly washed with phosphate buffered saline (PBS). The washed microspheres were frozen at -80°C and finally freeze-dried for 48h.

[0048] 2. Bonding of JTG and GM@mTG microspheres

[0049] When preparing JTG-grafted hydrogel microspheres (GM@mTG-J), JTG with a concentration of 32 μg / mL was mixed with 8% GM@mTG hydrogel precursor solution in an equal volume ratio as a dispersed phase, and then the above steps were repeated to obtain GM@mTG-J microspheres.

[0050] 3. Characterization of Functionalized Microspheres

[0051] like Figure 1 As shown in the figure, the appearance and particle size of the synthesized gel microspheres were first analyzed using an optical microscope (Optical Microscope, STM7, UK). The completely freeze-dried GM, GM@mTG and GM@mTG-J were then placed in a Fourier transform infrared spectrometer (Thermo Scientific, Nicolet 6700, USA) to analyze their chemical composition. Thin slices were prepared by grinding with potassium bromide and irradiated at 400-4000 cm -1 Scanning within a range, a scanning electron microscope (Scanning ElectronMicroscope, ZEM20, Germany) was used to characterize the surface microstructure of the microspheres. Finally, a mechanical rheometer (Discovery HR2Hybrid Rheometer, USA) was used to test the rheological properties of the microspheres. The fully cured GM, GM@mTG and GM@mTG-V were carefully removed from the mold and loaded on the mechanical rheometer. Then a frequency scan (frequency 0.1-10 Hz, strain 1%, temperature 37°C) was performed to obtain the storage modulus (G′) and loss modulus (G″). Microspheres with different matrix stiffness, namely GM@mTG microspheres photocrosslinked for 30s, 60s, 90s, 120s and 150s, were prepared respectively, and the changes in the mechanical modulus were tested using a rheometer.

[0052] 4. Optimization of Microsphere Matrix Stiffness

[0053] Rat intervertebral disc NP cells were cultured in DMEM / F12 medium supplemented with 1% FBS and 0.1% penicillin / streptomycin. After every 3 days of proliferation, the cells were digested and subcultured and the culture medium was replaced. The cells of the 4th generation were used for subsequent experiments. The CCK-8 experiment was used to detect the changes in the proliferation activity of NP cells. First, GM@mTG microspheres of different stiffness were spread in the well plate, and then the centrifuged cell suspension was added. Then, the morphological changes of the cells on the surface of the microspheres were observed on the 3rd day, and then 50μl of CCK-8 reagent was added to each well. The reaction was protected from light for 3h, and the absorbance value of each well was detected by an ELISA instrument (with a detection wavelength of 450nm and a reference wavelength of 600nm), and the proliferation rate of NP cells in gel microspheres of different stiffness was calculated.

[0054] In order to investigate the effect of mechanical stiffness of microspheres on cell ECM expression, we photocrosslinked the microspheres for 30s, 60s, 90s, 120s and 150s to obtain GM@mTG microspheres with different stiffness, and then co-cultured with NP cells for 3 days. RT-qPCR was used to detect the relative expression levels of mRNA of COL-Ⅱ, aggrecan and GAPDH genes in each group. The primer sequences were designed by Sangon Biotech (Shanghai, China), and total RNA was extracted using TRIzol Reagent (Vazyme, Nanjing, China). 0.5 μg of isolated RNA was taken and reverse transcribed into cDNA using a cDNA synthesis kit (Vazyme, Nanjing, China). qPCR amplification was performed according to standard procedures. All samples were standardized with GAPDH. All primer sequences are shown in Table 1.

[0055] Table 1 - Primer sequences for RT-qPCR

[0056]

[0057] 5. Degradation performance of microspheres and release efficiency of JTG

[0058] In rat intervertebral disc tissue, GM@mTG-J microspheres are degraded under the action of collagenase, so that JTG is released and exerts pharmacological effects. Therefore, we evaluated the degradation rate of GM@mTG-J microspheres and the sustained release efficiency of JTG. At 37°C, incubated in PBS or PBS containing type ⅠⅠ collagenase (1U / mL), the in vitro degradation performance of GM@mTG-J microspheres was evaluated, and 3 parallel samples were set up. In order to maintain the activity of the enzyme, fresh collagenase solution was replaced every 2 days. The microspheres were first weighed before the experiment (W 0 Then, the microspheres were taken out and weighed at different time points (W 1 ), using the formula The degradation rate at different time points was calculated. In order to evaluate the in vitro release characteristics of JTG, the GM@mTG-J microspheres were placed in 0.01, 0.002 and 0.0004u / ml type II collagenase after complete gelation and incubated at 37°C for 10 days. The supernatant was aspirated at the planned time points (1st, 2nd, 5th, 7th and 10th days) and stored at 4°C. The JTG in the supernatant at different time points was quantified using the collagen ELISA kit, and its release efficiency was calculated.

[0059] (II) Results Analysis

[0060] 1. Preparation and Characterization of Hydrogel Microspheres

[0061] Based on the co-flow shear between oil and water, droplets are formed downstream of the coaxial needle in the microfluidic device. GM@mTG microspheres are obtained by further cross-linking and freeze-drying. The size of the microspheres can be adjusted by adjusting the flow rate of oil and water. Under the optical microscope, it was observed that the fresh GM@mTG microspheres of different diameters prepared by microfluidics all have the characteristics of complete morphology, uniform size, and good dispersion ( Figure 1 A and B). When the oil and water flow rates are 800 μL / h and 80 μL / h respectively, the diameter of the microspheres is about 150 μm ( Figure 1 C). Previous studies have shown that smaller gel microspheres (diameter <300 μm) have a larger specific surface area, which can enhance intercellular material transport and achieve efficient nutrient exchange. Therefore, in order to ensure smooth in vivo injection, we selected microspheres with a diameter of 150 μm for subsequent experiments. Subsequently, the surface structure, chemical bonds, and mechanical properties of the microspheres were analyzed to verify that mTG was successfully bonded to the microspheres. First, the porous structure ( Figure 1 D) FTIR spectrum showed that compared with the GM group, the GM@mTG group had a -1 The absorption peaks in the range of 1087 cm-1 and 1097 cm-2 were all enhanced, which may be due to the introduction of mTG to increase the cross-linking strength of GM and make its structure more stable. -1 and 1027cm -1 These measurements indicate that the synthesis of GM@mTG microspheres was successful ( Figure 1 E). In the range of 1% strain and 0.1-10 Hz frequency, the rheological properties of the two groups showed similar G″ and different G′ ( Figure 1F), with the introduction of mTG, the G′ of the GM@mTG group was significantly enhanced, the rheological properties were improved, and higher viscoelasticity and stiffness properties were obtained. This may be due to the fact that the unmodified lysine groups in GM combined with the glutamine in mTG through amide bonds, resulting in an enzymatic cross-linking reaction, which improved the three-dimensional strength of the hydrogel.

[0062] 2. Effect of mechanical stiffness of GM@mTG microspheres on cell ECM metabolism

[0063] The imbalance of ECM synthesis / decomposition metabolism in NP cells is closely related to the mechanical properties of the matrix, so it is necessary to optimize the matrix stiffness of the gel microspheres. GM@mTG microspheres adopt a two-step crosslinking method, namely enzymatic crosslinking first and then photocrosslinking, which can achieve dynamic regulation of stiffness. After enzymatic crosslinking, the stiffness of the hydrogel gradually increases by extending the photocrosslinking time. When the photocrosslinking time is extended to 30s, the stiffness of the microspheres is only 301.59±12.58Pa. As the photocrosslinking time is extended to 150s, the stiffness of the microspheres increases to 1825.49±31.39Pa ( Figure 2 A). This indicates that it is feasible to dynamically regulate the stiffness of GM@mTG microspheres by extending the photocrosslinking time. The proliferation ability of NP cells in GM@mTG microspheres with different stiffness was then observed. In the microspheres photocrosslinked for 90 seconds, the cell proliferation rate was the highest, reaching 79.71%±1.38%. However, when the photocrosslinking exceeded 90 seconds, the cell proliferation rate gradually decreased. When the crosslinking was performed for 150 seconds, the cell proliferation rate was only 39.35±0.63% ( Figure 2 B), indicating that NP cells are not suitable for growth in a harder matrix.

[0064] On this basis, we further explored the effect of the mechanical stiffness of GM@mTG microspheres on the expression of ECM in LPS-induced cells. Col-II and Agg are the main components of ECM and are involved in the normal metabolism of intervertebral discs. Compared with the Control group (not treated with LPS), the gene expression levels of Col-II and Agg in LPS-induced NPs were significantly reduced, proving that LPS successfully induced inflammation. The study also found that after co-culture with GM@mTG microspheres, the expression levels of these genes in NP cells gradually increased with the increase in the photocross-linking time of the microspheres. However, when the photocross-linking of the microspheres exceeded 90s, the expression of the relevant genes decreased significantly again. Compared with the LPS group, there was no statistical difference in the expression levels of Col-II and Agg when the photocross-linking was 150s ( Figure 2C and D). These results indicate that the inflammatory microenvironment caused by LPS reduces the synthesis of ECM to a certain extent, but microspheres with a mechanical stiffness in the range of 424.41 to 1291.19 Pa can promote the synthesis of ECM. Among them, the microspheres prepared by photocrosslinking for 90 s (799.78 ± 43.58 Pa) have the most obvious promoting effect. It is worth noting that when the GM@mTG microspheres were photocrosslinked for 150 s and the mechanical stiffness reached 1589.37 ± 67.39 Pa, a similar effect was not achieved. Therefore, in subsequent experiments, photocrosslinking for 90 s was selected as one of the important conditions for preparing microspheres.

[0065] 3. Bonding and release of JTG and GM@mTG microspheres

[0066] In this study, based on biomechanics, a mechanical microenvironment that can reverse the imbalance of ECM metabolism was constructed as one of the important mechanisms to promote disc regeneration. In addition, we introduced JTG to solve the severe inflammatory response and excessive oxidative stress associated with IVDD. With the loading of JTG, the mechanical modulus of GM@mTG microspheres did not fluctuate significantly, and the mechanical stiffness did not change significantly ( Figure 3 A), indicating that the microspheres have good mechanical stability. FTIR spectrum shows that compared with the GM@mTG group, the GM@mTG-J group has a -1 The absorption intensity at 1080 cm increased, which may be due to the introduction of JTG, which increased the CC structure and the CO structure caused the absorption intensity at 1080 cm -1 The relative intensity change of the CO stretching vibration peak at 3284 cm -1 The slight blue shift of the absorption peak near , which may be attributed to the breaking of hydrogen bonds in the hydrogel microspheres caused by small molecules, indicates that the JTG grafting is successful ( Figure 3 B).

[0067] GM@mTG-J microspheres hardly degraded in PBS solution, with a degradation rate of only 23.25% ± 0.02% after 11 days of incubation. However, they could only maintain for about 72 h in PBS containing 1 U / mL type II collagenase ( Figure 3 C and D), indicating that it has enzymatic degradation. In different concentrations of type II collagenase, the release of JTG is a sustained release, and the release rate is more than 50% ( Figure 3 E), where the release of JTG in 0.002 U / ml type II collagenase best reflects the in vivo release process of the intervertebral disc, and can also indirectly indicate the chemical cross-linking effect between JTG and GM@mTG gel.

[0068] Example 2 Detection of NP cells in the functionalized GM@mTG-J microsphere co-culture system

[0069] 1. Experimental methods

[0070] 1. Biocompatibility study of GM@mTG-J

[0071] First, the GM@mTG and GM@mTG-J microspheres with optimized matrix stiffness were spread in the well plate, and then the centrifuged cell suspension and 50μl of 10μg / ml lipopolysaccharide (LPS) were added in sequence, and the GM@mTG group, LPS group, GM@mTG-J (8μg / ml) group, GM@mTG-J (16μg / ml) group and GM@mTG-J (32μg / ml) group were set up. Then, the morphological changes of cells on the surface of the microspheres were observed on the 3rd and 5th days, and then 50μl of CCK-8 reagent was added to each well, and the reaction was protected from light for 3h. The absorbance value of each well was detected by an enzyme marker to observe the effect of different concentrations of JTG on the proliferation of NP cells. Then, the cell survival state was observed by live cell fluorescence staining (Calcein-AM). After culturing in the above five groups of hydrogels for 5 days, the NP cells were stained with Calcein-AM, and the cell survival state was observed under a fluorescence microscope after preparation.

[0072] (II) Results Analysis

[0073] 1. First, NP cells were cultured in different concentrations of GM@mTG-J for different periods of time, and then CCK-8 and Calcein-AM cell staining experiments were performed. It can be found that NP cells can grow well, and compared with the LPS group, the cell survival rate in the GM@mTG-J group can be significantly increased ( Figure 4 A), indicating that the functionalized microspheres have good biocompatibility. In addition, compared with the LPS group, in the GM@mTG-J group, the proliferation rate of NP cells increased significantly with the extension of culture time and the increase of JTG concentration. On the 5th day of culture, the proliferation rate can reach 87.65% ± 4.18%, with obvious changes ( Figure 4 B), indicating that JTG can promote NP cell proliferation and accelerate cell growth.

[0074] Example 3 In vivo evaluation of the effect of functionalized GM@mTG-J microspheres

[0075] 1. Animal Model and Surgical Methods

[0076] Clean-grade male SD rats, with an average body weight of 350 g, were purchased from the Experimental Animal Center of the School of Medicine of Xi'an Jiaotong University and were kept in the laboratory of the Institute of Bionic Engineering and Mechanics, School of Life Sciences of Xi'an Jiaotong University. The animal experiment protocol was reviewed and approved by the Experimental Animal Welfare Ethics Committee of the School of Medicine of Xi'an Jiaotong University (No. Lundongkepizi 2019 No. 100). The experimental rats were fed in cages, with three or four rats in each cage, free access to food and water, daily feeding, water change every other day, alternating light and dark time for 12 hours each, room temperature controlled at 24-26°C, humidity around 50%, and all experimental mice were in good condition.

[0077] Rats were randomly divided into 5 groups. After the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg), the puncture sites were routinely disinfected and covered with a drape. A 20G puncture needle was used to enter the middle of the NP tissue through the coccygeal 7-8 (Co7-8) and 8-9 (Co8-9) segments (the depth was controlled at 5 mm). After passing through the annulus fibrosus, the puncture needle was rotated 360° and maintained for 30 seconds to ensure the degeneration effect ( Figure 5 A). There were 6 groups in total: (i) no needle puncture (healthy group); (ii) needle puncture and injection of PBS (defective group); (iii) needle puncture and injection of GM@mTG-J (32μg / ml) microspheres photocrosslinked for 30s (30s group); (iv) needle puncture and injection of GM@mTG-J (32μg / ml) microspheres photocrosslinked for 60s (60s group); (v) needle puncture and injection of GM@mTG-J (32μg / ml) microspheres photocrosslinked for 90s (90s group) and (vi) needle puncture and injection of GM@mTG-J (32μg / ml) microspheres photocrosslinked for 120s (120s group). All microsphere injections were disinfected with alcohol immediately after acupuncture, and the injection volume was 10μL. After surgery, the rats were transferred to a warm, ventilated environment, and all rats were housed in a clean, ventilated environment at 25℃ and 50% humidity.

[0078] 2. Radiological evaluation

[0079] X-ray and MRI imaging of the rats' coccygeal vertebrae were performed 4 and 8 weeks after surgery. All rats were placed in the supine position after anesthesia with their tails straight. The disc height index (DHI) was measured on the X-ray images, and the intergroup comparison was performed using Image J software (DHI = the sum of the anterior, middle, and posterior edge heights of the two disc spaces / the sum of the anterior, middle, and posterior edge heights of the adjacent vertebrae, DHI% = DHI measured at each time point / DHI of the sham operation group × 100%). T2-weighted images were collected on the coronal plane using a 1.5T magnetic resonance scanner. Finally, three radiologists who were blinded to the grouping were invited to evaluate the MRI images and score the MRI according to the Pfirrmann classification.

[0080] (II) Results Analysis

[0081] 1. Efficacy of GM@mTG-J microspheres against IVDD in vivo

[0082] In this study, we improved the excessive inflammatory environment in the intervertebral disc by constructing a stable mechanical microenvironment and loading anti-inflammatory drugs, and promoted the reconstruction of ECM in vivo to achieve the purpose of repairing IVDD. Therefore, we took the biomechanical properties of hydrogels as the starting point, compared the repair ability of matrix stiffness on early and late IVDD, and established a rat caudal IVDD model ( Figure 5 A). Imaging and histology are effective methods for evaluating disc degeneration and regeneration. In the IVDD group, DHI decreased rapidly, the vertebral structure was significantly damaged, and the disc tissue structure was significantly destroyed. MRI showed that the water content of the disc tissue in the modeling segment decreased significantly, and there was almost no residual nucleus pulposus tissue ( Figure 5 B), indicating that the intervertebral disc has undergone severe degeneration, and the IVDD model was successfully established. Based on the successful construction of the IVDD model, functionalized microspheres with different matrix stiffness were injected for 4 weeks and 8 weeks of treatment.

[0083] CT scan images showed that DHI showed a recovery trend after 4 weeks of treatment. The DHI of the GM@mTG-J (60s) group and the GM@mTG-J (90s) group increased significantly, but was lower than that of the healthy group. After 8 weeks of treatment, the DHI signal increased further, and the vertebrae were obviously repaired ( Figure 5 C and D). This indicates that the biomechanical microenvironment constructed by GM@mTG-J (90s) is more suitable for disc regeneration. MRI T2-weighted imaging (T2WI) is a simple and intuitive visual evaluation method for a variety of diseases that can assess changes in disc water content and has been widely used in the diagnosis and grading of IVDD. The degree of hydration in the nucleus pulposus tissue of healthy intervertebral discs is high. In the IVDD group, the intervertebral disc morphology was significantly destroyed, collagen and aggrecan were reduced, the number of nucleus pulposus cells decreased, and the hydration of the nucleus pulposus tissue was significantly weakened, indicating that irreversible damage occurred after disc puncture. With the introduction of functionalized gel microspheres, the MRI grading of the GM@mTG-J (30s) group and the GM@mTG-J (120s) group was similar to that of the IVDD group. However, the morphology of the intervertebral disc tissue in the GM@mTG-J (60s) and GM@mTG-J (90s) groups gradually became complete, the water content of the intervertebral disc tissue increased significantly, and the MRI score decreased significantly. The MRI score of the GM@mTG-J (90s) group decreased most significantly ( Figure 5 B and E). The matrix stiffness of the microspheres in the GM@mTG-J (90s) group was 799.78±43.58Pa, and the biomechanical environment constructed was more suitable for the repair and regeneration of nucleus pulposus tissue.

[0084] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A functionalized gel microsphere, characterized in that: Including GM@mTG porous microspheres and JTG.

2. The functionalized gel microsphere according to claim 1, characterized in that: Through the cross-linking effect of glutaminase mTG, dynamic regulation of its matrix stiffness was achieved, and JTG was successfully loaded using the porous structure of GelMA to obtain GM@mTG-J microspheres.

3. The method for preparing functionalized gel microspheres according to claim 1 or 2, characterized in that: The steps include: S1. The GelMA hydrogel precursor aqueous solution and the mTG aqueous solution are uniformly mixed in the LAP solution to obtain the GM@mTG hydrogel precursor aqueous solution, which is enzymatically cross-linked and then photo-cross-linked to achieve dynamic regulation of the mechanical stiffness of the GM@mTG porous microspheres, so that the stiffness of the GM@mTG porous microspheres is gradually increased; S2. Incubate the GM@mTG porous microspheres with the JTG to obtain GM@mTG-J microspheres.

4. The preparation method according to claim 3, characterized in that: In step S1, the volume ratio of the GelMA hydrogel precursor aqueous solution and the mTG aqueous solution is 1:

1. After being fully mixed and cross-linked by enzyme, 405 nm ultraviolet light is used for irradiation, and the mechanical stiffness of the GM@mTG porous microspheres is regulated by controlling the irradiation time of the 405 nm ultraviolet light.

5. The preparation method according to claim 4, characterized in that: The GelMA hydrogel precursor aqueous solution is an 8% GelMA hydrogel precursor aqueous solution; the mTG aqueous solution is a 5 mg / mL mTG aqueous solution.

6. The preparation method according to claim 3, 4 or 5, characterized in that: In step S2, the GM@mTG-J microspheres are prepared according to the following method: Span 80 and 5% w / w mineral oil were used as the continuous phase; JTG with a concentration of 32 μg / mL was mixed with 8% GM@mTG hydrogel precursor aqueous solution in equal volume ratio as the dispersed phase; Monodisperse spherical droplets were prepared by oil-in-water microfluidics. Subsequently, the spherical droplets were photocrosslinked under ultraviolet irradiation, and the uniform spherical droplets downstream were collected. The collected spherical droplets were repeatedly washed with 75% ethanol to remove the continuous phase, and then thoroughly washed with phosphate-buffered saline (PBS). The washed monodisperse spherical droplets were frozen and dried at -80°C for 48 h to obtain GM@mTG-J microspheres.

7. Use of the functionalized gel microspheres according to claim 1 in preparing a drug for treating intervertebral disc degeneration, or in preparing a material for repairing intervertebral disc degeneration.

8. Use of the functionalized gel microspheres prepared according to the preparation method of claim 3, 4, 5 or 6 in preparing a drug for treating intervertebral disc degeneration, or in preparing a material for repairing intervertebral disc degeneration.

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