Nanozyme-loaded nucleus pulposus matrix hydrogel microspheres

Abstract

The present invention discloses a nanzyme-loaded nucleus pulposus matrix hydrogel microsphere, belonging to the technical field of biomaterials. The present invention provides a simply prepared LOX-MnO2 nanzyme, loads it on GDNP, and forms a glucose-rich nucleus pulposus acellular matrix hydrogel microsphere loaded with lactate oxidase-manganese dioxide nanzyme through a constructed novel two-stage temperature-controlled microfluidic system. The preparation of the present invention is simple, convenient for storage, injectable, easy for large-scale production, and has potential for clinical application. The system aims to regulate the nutritional imbalance and harsh microenvironment after stem cells are implanted into the intervertebral disc, thereby improving the efficiency of stem cell therapy and providing a new strategy for the treatment of IDD.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a nanoenzyme-carrying nucleus pulposus matrix hydrogel microsphere. Background Art

[0002] Intervertebral disc degeneration (IDD) is a common degenerative disease that can lead to intervertebral disc (IVD) herniation and decreased spinal mobility, and induce radicular symptoms and chronic low back pain (LBP). LBP may greatly affect the quality of life of patients and increase social and economic burdens. However, due to insufficient understanding of the pathogenesis of the disease and the mechanism of postoperative adverse reactions, commonly used methods such as nucleotomy, vertebral fusion, local blockade and conservative drug treatment have not yet achieved satisfactory results. Developing new treatment strategies for IDD is an urgent task for clinical researchers. Stem cell-based tissue engineering science and biomaterial technology are the basic pillars of regenerative medicine. Mesenchymal stem cells (MSCs) have great potential in the treatment of IDD due to their characteristics of division and proliferation, multilineage differentiation potential, easy isolation and low immunogenicity. Currently, MSCs have been used in many clinical trials for IDD and have been shown to be safe and well tolerated.

[0003] Due to the hypovascular nature of normal IVD, nutrients are provided by the blood vessels around it and transported to the center of IVD through osmosis. Because the concentration of nutrients decreases with the distance from the blood vessels, the IVD is relatively nutrient-deficient and can only support a limited number of cells. When IDD occurs, bone marrow space occlusion, cartilage endplate calcification, and lumbar atherosclerosis will lead to a further reduction in nutrient supply. In stem cell therapy, the implantation of exogenous MSCs will lead to a sharp increase in the number of cells in the IVD. The implanted MSCs compete with the remaining living in situ IVD cells for nutrients, aggravating the nutrient deficiency of the IVD, which further causes cell death. Although MSCs can effectively regulate inflammation and stimulate extracellular matrix production, they must survive to perform their functions. Therefore, sufficient energy, especially glucose, can be provided to maintain the activity of implanted MSCs. The imbalance between nutrient demand and supply after MSC implantation, especially in the case of IDD, will reduce the efficiency of stem cell therapy.

[0004] The hypoxic microenvironment in IVD determines that anaerobic glycolysis is the main energy metabolism mode of nucleus pulposus cells (NPCs). This mode causes the accumulation of glycolytic metabolites in the intervertebral disc. It is reported that lactic acid is the main metabolite of anaerobic glycolysis, and its content in the intervertebral disc tissue of IDD is higher than that in blood and other tissues. High concentrations of lactic acid can impair the activity of MSCs and IVD cells. Therefore, when using stem cells for treatment, it is also necessary to consider eliminating the adverse effects of metabolites to improve the energy metabolism environment of cells and maintain cell survival and function.

[0005] Lactate oxidase (LOX) catalyzes the oxidation of lactate to produce pyruvate and hydrogen peroxide (H 2 O 2 ), and H 2 O 2 It is a key component of reactive oxygen species that induces oxidative stress and leads to cell death in IDD. Shen et al. designed a set of MnO-based 2 ) nanozymes. Using MnO 2 Catalytic H 2 O 2 decomposed into oxygen and water, this system can metabolize the H produced by lactic acid consumption 2 O 2 , and provide additional O 2 However, the report based on manganese dioxide (MnO 2 ) The synthesis of nanozymes is relatively complicated, requiring four steps and an additional heat source to reach a reaction temperature of 60°C, which is not conducive to large-scale industrial production and clinical application. Therefore, it is necessary to improve the synthesis method of nanozymes. However, even so, nanozymes are faced with the disadvantages of small size, easy diffusion, easy removal by cells, and inability to be locally enriched for a long time. Therefore, a suitable delivery system is urgently needed to maintain its local effect. Summary of the invention

[0006] In view of the above problems existing in the prior art, the present invention proposes a nanozyme-loaded nucleus pulposus matrix hydrogel microsphere.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A nanozyme-loaded nucleus pulposus matrix hydrogel microsphere, which simultaneously loads lactate oxidase and manganese dioxide nanozyme, wherein the glucose content in the nucleus pulposus matrix hydrogel microsphere is greater than or equal to 2.5-5mM.

[0009] A method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres, comprising: 2Lactate oxidase was added to the nanodispersion and LOX-MnO was obtained after dispersion and centrifugation. 2 Nanozyme particles; adding genipin to the digestion solution of decellularized nucleus pulposus tissue, and obtaining a pre-gel solution by adjusting the pH and osmotic pressure; adding genipin, glucose solution and the LOX-MnO 2 Nanozyme particles are pumped into a microfluidic chip and sheared into droplets, which are then gelled to obtain nanozyme-loaded nucleus pulposus matrix hydrogel microspheres.

[0010] Furthermore, the method for preparing the nanozyme-loaded nucleus pulposus matrix hydrogel microspheres specifically comprises the following steps:

[0011] (1)LOX-MnO 2 Synthesis of nanozyme particles

[0012] Potassium permanganate was dissolved in double distilled water, and then polyallylamine hydrochloride solution was added dropwise, and stirred at 25°C for 30 min to obtain a MnO 2 The mixture of nanoparticles was placed in a dialysis bag for dialysis and freeze-dried to obtain freeze-dried powder. The freeze-dried powder was added to double distilled water to obtain MnO 2 The nanodispersion was added with lactate oxidase, stirred at 25 °C and 200 rpm for 30 min, and finally centrifuged at 20,000 rpm for 30 min and washed to obtain LOX-MnO 2 Nanozyme particles;

[0013] (2) Nanozyme-loaded nucleus pulposus matrix hydrogel microspheres

[0014] Pepsin containing 1% w / v HCl is added to the pretreated decellularized nucleus pulposus tissue powder for digestion, the obtained digestion solution is centrifuged, genipin is added, the pH is adjusted to 7.3-7.5, and the osmotic pressure is adjusted to an isotonic state to form a pregel solution, which is recorded as a DNP pregel solution;

[0015] Glucose and genipin are added to the DNP pregel solution to obtain a GDNP pregel solution, and then the LOX-MnO in step (1) is added to the GDNP pregel solution. 2 The LMGDNP pre-gel solution obtained by nanozyme particles and genipin is pumped into a microfluidic chip, and droplets are obtained by shearing. The droplets are gelled to obtain glucose-rich nucleus pulposus acellular matrix hydrogel microspheres loaded with lactate oxidase-manganese dioxide nanozyme, that is, nucleus pulposus matrix hydrogel microspheres loaded with nanozyme.

[0016] Furthermore, in step (2), the pretreatment refers to: repeatedly freezing and thawing the fresh nucleus pulposus block in liquid nitrogen and a 37°C water bath for 5 times, and then treating it with 2wt% TritonX-100 at room temperature and shaking for 48h; centrifuging and discarding the supernatant, treating it with 1wt% SDS at room temperature and shaking for 24h, and finally treating it with 200U / mL DNase at 37°C and shaking for 12h to form a decellularized nucleus pulposus tissue block, washing it with sterile water, and crushing it to obtain a decellularized nucleus pulposus tissue powder.

[0017] Furthermore, in step (2), the digestion treatment refers to digestion at 25°C for 48 hours.

[0018] Further, in step (2), the GDNP and LOX-MnO 2 The mass ratio of nanozyme particles is 100:1.

[0019] The present invention also provides a two-stage temperature-controlled microfluidic system, which is used to prepare the nanozyme-loaded nucleus pulposus matrix hydrogel microspheres, and specifically consists of a polydimethylsiloxane chip, a microinjection pump, a heated magnetic stirrer, a polytetrafluoroethylene tube and a silicone resin tube.

[0020] The present invention also provides an application of the nanozyme-loaded nucleus pulposus matrix hydrogel microspheres in biomaterials.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The present invention uses microfluidic technology to prepare glucose-rich NP matrix hydrogel microspheres, and the glucose content in the prepared gel microspheres is as high as 2.5-5mM, providing nutrition supply and differentiation-promoting clues for MSCs. In addition, the present invention also constructs a simple preparation method of LOX-MnO 2 Nanozyme particles are loaded on GDNP to form glucose-rich nucleus pulposus acellular matrix hydrogel microspheres loaded with lactate oxidase-manganese dioxide nanozymes to remove excess lactic acid in the intervertebral disc and reduce damage to cells by metabolic byproducts. The system is simple to prepare, easy to store, injectable, easy to mass produce, and has potential for clinical application. The system is designed to regulate the nutritional imbalance and harsh microenvironment of stem cells after implantation into the intervertebral disc, thereby improving the efficiency of stem cell therapy and providing a new strategy for the treatment of IDD. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0024] Figure 1 LOX-MnO 2Schematic diagram of the construction of nanozyme particles (LM);

[0025] Figure 2 Schematic diagram of the construction of glucose-rich nucleus pulposus acellular matrix hydrogel microspheres loaded with lactate oxidase-manganese dioxide nanozyme;

[0026] Figure 3 is the relationship between glucose content and cell viability;

[0027] Figure 4 It is a two-stage temperature-controlled microfluidic system (TSTC-MS);

[0028] Figure 5 LOX-MnO 2 The morphology and size of nanozyme particles; A is PAH-coated MnO 2 TEM images of nanoparticles and LM; B is the size distribution of nanoparticles; C is MnO 2 Nanoparticles and LOX-MnO 2 Size distribution of nanozymes, data are presented as mean ± SD, n = 3 (referring to 3 repeated experiments);

[0029] Figure 6 LOX-MnO 2 Composition and characteristics of nanozymes; A is LOX loaded on PAH-MnO 2 The influence of the nanoparticles on the zeta potential; B is the SEM image and elemental analysis of the nanoparticles; C is the silver staining of SDS-PAGE to evaluate the LOX, MnO 2 and LM nanoparticles; D is LOX, MnO 2 and UV-Vis spectra of LM nanoparticles;

[0030] Figure 7 The results are as follows: A is HE staining to evaluate the decellularization efficiency, scale bar: 100 μm; B is DAPI staining to evaluate the decellularization efficiency, scale bar: 100 μm; C is the quantitative analysis of DNA content in fresh nucleus pulposus tissue blocks (FNP-B) and decellularized nucleus pulposus tissue slices (DNP-B); D is the GAG ​​analysis of FNP-B and DNP-B to evaluate the content of glycosaminoglycans in the tissue. Data are expressed as mean ± standard deviation, n = 3, *p < 0.05, **p < 0.01;

[0031] Figure 8 Macroscopic and microscopic morphology of microspheres; A is the microspheres collected and dispersed in liquid paraffin, B is the size distribution and microstructure of the microspheres, scale bars: 100 μm and 2.5 μm;

[0032] Fig. 9Figure 2 is the graph of LOX release from LMGDNP at different times; A is the cumulative release curve of LOX in LMGDNP based on BCA determination; B is the LOX-MnO 2 Lactate removal efficiency of nanozymes and microspheres;

[0033] Fig.10 LOX-MnO 2 Nanozymes and microspheres promote H 2 O 2 Decomposed efficiency evaluation;

[0034] Fig.11 Calcein-AM / PI fluorescence images of BMSCs loaded on different microspheres or cultured as cell spheres for 14 days, scale bar, 100 μm;

[0035] Fig.12 The biocompatibility of LMGDNP; A is the CCK8 assay to detect the cell viability of BMSCs loaded on different microspheres or cultured as cell spheres; B is the CCK8 assay to evaluate the cell viability of BMSCs cultured in a microsphere feeding system containing or not containing lactic acid for 24 hours; C is Calcein-AM / PI fluorescence staining to evaluate the survival of BMSCs cultured in a microsphere feeding system containing or not containing lactic acid for 24 hours, scale bar: 100μm;

[0036] Fig.13 To evaluate the differentiation-promoting ability of dECM microspheres; A is the expression of NPCs markers (Krt19, CD24, Col2 and Acan) in BMSCs cultured on microspheres for 21 days as shown by immunofluorescence, green is the target indicator, blue is DAPI, and orange is genipin autofluorescence, scale bar: 100 μm; B is the relative mRNA expression of Krt19, CD24, Col2 and Acan in BMSCs cultured on microspheres for 14 days and 21 days, data are expressed as mean ± SD, n = 3, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The "room temperature" mentioned in the present invention is 25±2°C unless otherwise specified.

[0043] The raw materials used in the following examples of the present invention are all commercially available.

[0044] In recent years, decellularized extracellular matrix (dECM) has attracted more and more attention in regenerative medicine due to its good biocompatibility, biomimetic fiber structure and bioactivity. In the early stage of the study, a dECM hydrogel derived from porcine sciatic nerve was prepared, and microspheres were prepared using a microfluidic system, which showed good cell compatibility and sustained release of small molecule drugs. In addition, dECM hydrogel derived from nucleus pulposus (NP) matrix effectively promoted the tissue-specific differentiation of stem cells into NPCs. Therefore, the present invention will prepare hydrogel microspheres based on NP matrix as a delivery system for stem cells and nanozymes.

[0045] The present invention uses microfluidic technology to prepare glucose-enriched decellularized nucleus pulposus hydrogel microspheres (GDNP) to provide nutrient supply and differentiation-promoting clues for MSCs. In addition, the present invention also constructs a simple preparation method of LOX-MnO 2 Nanozymes were loaded on GDNP to form glucose-rich nucleus pulposus acellular matrix hydrogel microspheres loaded with lactate oxidase-manganese dioxide nanozymes (LOX-MnO 2 -loaded & glucose-enriched decellularized nucleus pulposus hydrogelmicrospheres, LMGDNP pregel solution) to remove excess lactic acid in the intervertebral disc and reduce damage to cells by metabolic byproducts. The system is simple to prepare, easy to store, injectable, easy to mass produce, and has potential for clinical application. The system is designed to regulate the nutritional imbalance and harsh microenvironment of stem cells after implantation into the intervertebral disc, thereby improving the efficiency of stem cell therapy and providing a new strategy for the treatment of IDD.

[0046] The main preparation process is: the NP tissue collected from the bovine tail is decellularized to remove the cellular components, and then the pre-gel solution is formed by pepsin-mediated enzymatic digestion, namely the DNP pre-gel solution. 2 and glucose were added to the DNP pregel solution to obtain the LMGDNP pregel solution, which was pumped into the microfluidic chip to form LMGDNP droplets by shear force when encountering the oil phase. The droplets were gelled by a silicone tube immersed in a 37°C water bath and collected to obtain LMGDNP microspheres (reference Figure 2 ).

[0047] Nucleus pulposus cells were treated with lactic acid (0, 10, 15, 20 mM) and glucose (0.5, 1.25, 2.5, 3.75, 5 mM). As the lactic acid concentration increased, the cell viability gradually decreased. When the glucose concentration reached 2.5 mM, the cell viability was at a high level (reference Figure 3 ). Therefore, the glucose concentration in the microspheres in subsequent experiments was selected to be no less than 2.5 mM, and 5 mM was preferred in the following embodiments of the present invention.

[0048] Example 1

[0049] (1) Preparation of LOX-MnO 2 Nanozyme particles (LOX-MnO 2 Schematic diagram of nanozyme construction Figure 1 )

[0050] 64 mg of KMnO 4 Dissolve in 18 mL of double distilled water, then drop 2 mL of 36 mg / mL poly(allylamine hydrochloride, PAH) solution into it, react at 25 ° C for 30 minutes, and then obtain the MnO 2 The mixture of nanoparticles was placed in a dialysis bag (molecular weight 3 kDa), dialyzed against double distilled water for one week, and then freeze-dried to obtain lyophilized powder for calculation of yield. 2 Nanoparticles, freeze-dried powder was added to double distilled water to obtain 3.6 mg / mL MnO 2 Nanodispersion, 50 U LOX was added to 1 mL of MnO 2 The nanodispersion was stirred (200 rpm) at 25 °C for 30 min, then centrifuged at 20,000 rpm for 30 min, and washed three times with PBS to obtain LOX-MnO 2 Nanoparticles (denoted as LM nanoparticles).

[0051] (2) Preparation of glucose-rich nucleus pulposus acellular matrix hydrogel microspheres (LMGDNP) loaded with lactate oxidase-manganese dioxide nanozyme

[0052] Step 1: Within 6 hours after slaughter at the slaughterhouse, harvest the nucleus pulposus tissue from the tail section of the cattle and cut it into small pieces, called fresh nucleus pulposus blocks (FNP-B). FNP-B is repeatedly frozen and thawed in liquid nitrogen and a 37°C water bath for 5 times; treated with 2wt% TritonX-100 under shaking conditions for 48 hours; centrifuged and discarded the supernatant, treated with 1wt% SDS under shaking conditions for 24 hours, and treated with 200U / mL DNase under shaking conditions at 37°C for 12 hours to remove residual DNA, forming acellular nucleus pulposus tissue blocks (DNP-B). After washing with sterile water, crushing, the powder is digested in pepsin (0.1% w / v) containing 1% w / v HCl (0.01M) at 25°C for 48 hours to obtain a digestion solution, and the digestion solution is centrifuged to remove undissolved particles. The pH of the digestion solution was adjusted to 7.4 using 0.1 M NaOH, genipin (0.02% w / v) was added and the osmotic pressure was adjusted to an isotonic state to form a pre-gel solution. Glucose solution and genipin (0.02% w / v) were added to the DNP pre-gel solution to obtain a GDNP pre-gel solution with a glucose concentration of 5 mM. LM nanoparticles and genipin (0.02% w / v) were added to the GDNP pre-gel solution (the mass ratio of LM nanoparticles to the GDNP pre-gel solution was 1:100) to obtain a LMGDNP pre-gel solution, which was stored at 4°C until use.

[0053] Step 2: Design using AutoCAD software and use photolithography to make a silicon wafer mold. Mix the polydimethylsiloxane (PDMS) substrate with the curing agent (the mass ratio of PDMS to curing agent is 10:1, curing agent product number: Sylgard184) and degas for 15 minutes, then pour it onto the silicon wafer mold and degas for 2 hours, and then peel off the PDMS from the silicon wafer mold after curing at 70°C for 4 hours. Two holes with a diameter of 1.0 mm for the inflow channel and a hole with a diameter of 2.0 mm for the outflow channel are dug out on the obtained PDMS to obtain a chip. At the same time, a flat PDMS cover plate is prepared on the silicon wafer. Finally, the PDMS surface is activated using an oxygen plasma surface treatment instrument, the PDMS cover plate is carefully placed on the chip, and placed at 70°C for 30 minutes to obtain a microfluidic chip.

[0054] Step 3: Prepare microspheres (such as Figure 4 ). TSTC-MS consists of a PDMS chip, a microsyringe pump with two channels, a heatable magnetic stirrer, two polytetrafluoroethylene tubes (inner diameter = 1.0 mm) and a silicone tube (inner diameter = 2.0 mm). The aqueous phase (LMGDNP pregel solution) and the oil phase (liquid paraffin containing 20% ​​v / v Span80) were mixed in the microfluidic chip at 4°C, and an emulsion of oil-in-water droplets was formed using shear force. The formation of droplets was observed using a small inverted laboratory microscope. The flow rate ratio of the water / oil phase was 1:10. The emulsion flowed into the silicone tube in a 37°C water bath and gelled to generate microspheres. The microspheres were dropped into a double-layer liquid phase consisting of ether and PBS solution to remove mineral oil. The microspheres were concentrated in the PBS solution, and after centrifugation and washing three times with PBS, the microspheres were stored at 37°C for further use. For sterilization, the microspheres were centrifuged at 2000 rpm for 5 min to remove PBS and resuspended in 75% ethanol at 25°C for 24 h. Finally, the microspheres were washed three times with sterile PBS to remove residual ethanol to obtain glucose-rich nucleus pulposus decellularized matrix hydrogel microspheres (LMGDNP) loaded with lactate oxidase-manganese dioxide nanozyme.

[0055] The aqueous LMGDNP pregel solution in the above step (2) was replaced with DNP pregel solution sol and GDNP pregel solution sol respectively, and other microsphere preparation conditions remained unchanged to obtain DNP microspheres and GDNP microspheres respectively. The subsequent performance tests were carried out with these as comparisons.

[0056] Figure 5 LOX-MnO2 The morphology and size of nanozymes. Figure 5 As can be seen from A, PAH-coated MnO 2 The nanoparticles behaved as stable colloidal dispersions, while LOX loading changed the morphological structure. At the same time, after LOX loading, the diameter of LM nanozyme particles increased significantly ( Figure 5 B and C in ).

[0057] Figure 6 LOX-MnO 2 Composition and properties of nanozymes. After loading LOX, MnO 2 The zeta potential of the nanoparticles dropped from about 20 mV to about 0 mV, forming a stable non-covalent complex ( Figure 6 A). BCA determination of MnO 2 The LOX loading capacity of the nanoparticles was 54.18 ± 6.97%. EDS was used to verify the element distribution and showed that manganese (Mn) and oxygen (O) were uniformly distributed in LM and MnO. 2 Nanoparticles ( Figure 6 B) LOX, MnO 2 The LOX and LM nanoparticles were delivered to the MnO nanoparticles in SDS-PAGE (polyacrylamide gel electrophoresis) and silver-stained. Similar bands appeared at 40 kDa in the LOX and LM groups, indicating that LOX was successfully loaded on the MnO nanoparticles. 2 Nanoparticles ( Figure 6 C) UV-Vis spectrum shows that MnO 2 The spectra of MnO and LM are similar, indicating that loading LOX hardly changes the 2 Ultraviolet absorption properties of nanoparticles ( Figure 6 D).

[0058] Figure 7 To evaluate the effectiveness of decellularization. HE staining showed that after decellularization, the cellular components were removed, while the ECM was retained during the decellularization process ( Figure 7 Similarly, DAPI staining showed that the cell nuclei in the tissue were removed, indicating that the decellularization treatment was effective ( Figure 7 The DNA content decreased from 281.17±62.69ng / mg in FNP-B to 37.80±5.44ng / mg in DNP-B ( Figure 7 C), while GAG ​​(the main component of the extracellular matrix) decreased from 13.41±1.63μg / mg in FNP-B to 9.73±1.06μg / mg in DNP-B ( Figure 7 D), indicating that the decellularization treatment significantly removed cellular components while retaining most of the extracellular matrix.

[0059] Figure 8 The macroscopic and microscopic morphologies of the microspheres are shown in Figure 2. The obtained microspheres were immersed in the oil phase and precipitated at the bottom. Due to the presence of genipin in the microspheres, GDNP appears light blue, while LMGDNP appears light blue due to the presence of MnO 2 The presence of brown ( Figure 8 In real-time observation, the diameters of the droplets were consistent (DNP: 197.63 ± 7.34 μm; GDNP: 195.49 ± 9.10 μm; LMGDNP: 187.94 ± 14.36 μm), and the nanofibrous structure of the collected microspheres was similar to the self-assembled nanostructure of the DNP hydrogel ( Figure 8 B), which can simulate the extracellular matrix microenvironment of nucleus pulposus cells to support cell attachment, proliferation and directed differentiation.

[0060] Fig. 9 Figure 2 is a graph showing the release of LOX from LMGDNP at different times. As shown in the BCA assay, LMGDNP exhibits a sustained release of LOX ( Fig. 9 The lactate concentrations measured at different time points showed that the lactate content in both the LMGDNP and LM groups gradually decreased, but LMGDNP did not seem to be as effective as LM, which may be attributed to the delayed release of LOX into the enzyme reaction system in the LMGDNP group ( Fig. 9 B).

[0061] Fig.10 LOX-MnO 2 Nanozymes and microspheres promote H 2 O 2 Evaluation of the efficiency of decomposition. LOX scavenging lactate will cause H 2 O 2 The accumulation of H in the system can lead to oxidative stress. 2 O 2 The scavenging efficiency of MnO was found to be significantly improved by LMGDNP and LM, and the delayed oxygen production in the LMGDNP group indicated that LM was slowly released in the system, resulting in the 2 The concentration of nanoparticles was lower than that of LM group. LMGDNP and LM had significant effects on the elimination of H 2 O 2 It has a catalase-like effect in generating oxygen to improve the hypoxic microenvironment in the IVD.

[0062] Fig.11Calcein-AM / PI fluorescence images of BMSCs loaded on microspheres DNP, GDNP, LMGDNP, and GelMA (methacrylated hydrogel, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) (BMSCs loading was obtained by co-culturing with microspheres on low-adhesion well plates for 24 hours) or as cell spheres after 14 days of culture. Calcein / propidium iodide staining showed that bone marrow mesenchymal stem cells (BMSCs) cultured on GelMA, DNP, GDNP, and LMGDNP did not show obvious cell death on day 14, while under cell sphere culture conditions, PI positive staining increased significantly in the center of the cell sphere, indicating that significant cell death occurred in the center of the cell sphere. Compared with cell spheres, cells cultured on the surface of microspheres may have better nutrition and oxygen supply, resulting in higher cell survival rate.

[0063] Fig.12 The CCK8 assay showed that the cell viability of the DNP group increased more significantly compared with GelMA and cell spheres ( Fig.12 A in the figure indicates that dECM microspheres are more suitable for cell survival than GelMA. In addition, the GDNP and LMGDNP groups showed better cell viability than the DNP group ( Fig.12 A in the figure), which may be related to the ability of glucose to enhance cell viability ( Fig.12 B in the figure). The lactate concentration in the nucleus pulposus of LBP patients is between 2 and 6 mM. In order to evaluate the ability of microspheres to consume lactate, lactate (6 mM) was added to the microsphere culture system. CCK8 assay showed that the cell viability of the lactate + GDNP group was significantly decreased compared with the lactate + LMGDNP group ( Fig.12 C). Calcein-AM / PI staining also showed that lactate treatment led to increased cell death, while LMGDNP could significantly reduce cell death caused by lactate ( Fig.12 D).

[0064] To evaluate the differentiation-promoting ability of dECM microspheres, BMSCs were cultured on GelMA, GDNP, and LMGDNP for 14 and 21 days, and then the cell surface markers (Krt19, CD24, Col2, and Acan) of NPCs were detected. On day 21, immunofluorescence staining of Krt19, CD24, Col2, and Acan showed obvious positive staining in the GDNP and LMGDNP groups, while the above indicators in the GelMA group were not significantly positive ( Fig.13 PCR assay showed that the mRNA expressions of Krt19, CD24, Col2, and Acan were also significantly increased in the GDNP and LMGDNP groups ( Fig.13 B).

[0065] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres, characterized in that: Lactate oxidase is added to the MnO2 nanodispersion, and LOX-MnO2 nanozyme particles are obtained after dispersion and centrifugation; Genipin is added to the digestion fluid of decellularized nucleus pulposus tissue, and a pre-gel solution is obtained by adjusting pH and osmotic pressure; Adding genipin, glucose solution and the LOX-MnO2 nanozyme particles to the pre-gel solution again, pumping it into a microfluidic chip to shear it into droplets, and then gelling it to obtain nanozyme-loaded nucleus pulposus matrix hydrogel microspheres; The nanozyme-loaded nucleus pulposus matrix hydrogel microspheres simultaneously load lactate oxidase and manganese dioxide nanozyme, and the glucose content in the nucleus pulposus matrix hydrogel microspheres is equal to 2.5-5mM.

2. The method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres according to claim 1, characterized in that: The specific steps include: (1) Synthesis of LOX-MnO2 nanozymes Potassium permanganate was dissolved in double distilled water, and then a polyallylamine hydrochloride solution was added dropwise, and the mixture was stirred at 25°C for 30 minutes. The obtained mixture containing MnO2 nanoparticles was placed in a dialysis bag for dialysis, and freeze-dried to obtain a lyophilized powder; the lyophilized powder was added to double distilled water to obtain a MnO2 nanodispersion, lactate oxidase was added, and the mixture was stirred at 25°C and 200 rpm for 30 minutes, and finally centrifuged at 20,000 rpm for 30 minutes, and washed to obtain LOX-MnO2 nanoenzyme particles; (2) Nanozyme-loaded nucleus pulposus matrix hydrogel microspheres Pepsin containing 1% w / v HCl is added to the pretreated decellularized nucleus pulposus tissue powder for digestion, the obtained digestion solution is centrifuged, genipin is added, the pH is adjusted to 7.3-7.5, and the osmotic pressure is adjusted to an isotonic state to form a pregel solution, which is recorded as a DNP pregel solution; Glucose and genipin are added to the DNP pregel solution to obtain a GDNP pregel solution, and then the LOX-MnO2 nanozyme particles in step (1) are added to the GDNP pregel solution. The obtained LMGDNP pregel solution is pumped into a microfluidic chip, and droplets are obtained by shearing. The droplets are gelled to obtain glucose-rich nucleus pulposus decellularized matrix hydrogel microspheres loaded with lactate oxidase-manganese dioxide nanozyme, i.e., nucleus pulposus matrix hydrogel microspheres loaded with nanozyme.

3. The method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres according to claim 2, characterized in that: In step (2), the pretreatment refers to: repeatedly freezing and thawing the fresh nucleus pulposus block in liquid nitrogen and a 37°C water bath for 5 times, and then treating it with 2wt% TritonX-100 at room temperature and shaking for 48h; centrifuging and discarding the supernatant, treating it with 1wt% SDS at room temperature and shaking for 24h, and finally treating it with 200U / mL DNAse at 37°C and shaking for 12h to form a decellularized nucleus pulposus tissue block, washing it with sterile water, and crushing it to obtain a decellularized nucleus pulposus tissue powder.

4. The method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres according to claim 2, characterized in that: In step (2), the digestion treatment refers to digestion at 25°C for 48 hours.

5. The method for preparing nanozyme-loaded nucleus pulposus matrix hydrogel microspheres according to claim 2, characterized in that: In step (2), the mass ratio of the GDNP to the LOX-MnO2 nanozyme particles is 100:1.

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