Microsphere for promoting Ext1 expression and relieving osteoarthritis and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials. The invention provides microspheres for promoting Ext1 expression and relieving osteoarthritis and a preparation method of the microspheres. The microspheres comprise gelatin microspheres and bone marrow mesenchymal stem cells loaded on the microspheres. In the field, the effects of promoting Ext1 gene expression and relieving osteoarthritis are achieved for the first time under the condition that exogenous drugs are not adopted; meanwhile, the action time of the invention can be as long as 30 days. The invention provides a new direction for clinical treatment of osteoarthritis in the future, has huge potential in the aspects of personalized and non-surgical treatment, can reduce dependence on drugs and improve joint health, and provides an economical and practical solution.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a microsphere for promoting Ext1 expression and alleviating osteoarthritis and a preparation method thereof. Background Art

[0002] Osteoarthritis (OA) is a common chronic joint disease that mainly affects middle-aged and elderly people and seriously affects the quality of life of patients. For patients with severe osteoarthritis, surgery is an effective method. However, surgical treatment is invasive and prone to causing damage to the body. Conservative treatments other than surgery mainly include drug treatment and physical rehabilitation treatment. Generally, physical rehabilitation treatment usually requires a relatively complex treatment plan, and long-term use of drugs has the drawbacks of potential side effects and high treatment costs.

[0003] Considering that biomedical materials usually have good biocompatibility, if a biomedical material that can stimulate the body or cells to regulate genes related to osteoarthritis and alleviate osteoarthritis or a preparation using a biomedical material to load related cells can be discovered, the deficiencies of the above treatment methods can be solved.

[0004] As far as the inventors know, there is no biomedical material or preparation using a biomedical material to load related cells with the above effects in the prior art. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to provide a biomedical material preparation that can regulate genes related to osteoarthritis and alleviate osteoarthritis without relying on exogenous drugs.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A microsphere for promoting Ext1 expression and alleviating osteoarthritis, the microsphere comprising gelatin microspheres and bone marrow mesenchymal stem cells loaded on the microspheres.

[0008] Preferably, the addition amounts of the gelatin microspheres and the bone marrow mesenchymal stem cells are: 1×10 5 bone marrow mesenchymal stem cells are loaded per microgram of gelatin microspheres.

[0009] Preferably, the gelatin microspheres have a snowflake-like structure; the particle size of the gelatin microspheres does not exceed 1000 μm.

[0010] Preferably, the average particle size of the gelatin microspheres is 230.6 um.

[0011] Preferably, the gelatin microspheres are prepared by an emulsification method using an aqueous gelatin solution as the water phase and paraffin oil added with Span80 as the oil phase.

[0012] Preferably, the content of gelatin in the aqueous phase is 15% w / v.

[0013] Preferably, in the oil phase, the volume ratio of Span 80 to paraffin oil is 1:100.

[0014] Preferably, when preparing the gelatin microspheres, 1.5 g of gelatin powder is dissolved in 10 mL of deionized water and continuously stirred at 60 °C to prepare a 15% w / v gelatin solution as the aqueous phase; 0.2 mL of Span 80 is heated to 60 °C in 20 mL of paraffin oil to form the oil phase; the gelatin solution is slowly dropped into the oil phase solution for homogenization to form droplets in the W / O emulsion; after cooling to 4 °C, the droplets become gelatin microspheres, which are washed 3 times with acetone to remove the excess oil phase substances, washed three times with deionized water, and dried to obtain the gelatin microspheres.

[0015] Another object of the present invention is to provide a method for preparing microspheres that can promote Ext1 expression and relieve osteoarthritis, which is obtained by loading the aforementioned bone marrow mesenchymal stem cells on gelatin microspheres.

[0016] Another object of the present invention is to provide the application of the aforementioned microspheres in the preparation of preparations for relieving osteoarthritis.

[0017] As shown in the examples of the present invention, the inventors found that the expression of the Ext1 gene in the weight-bearing parts of the joints of osteoarthritis patients is weakened, while treatment with gelatin microspheres loaded with mesenchymal stem cells (MSCs) can promote the overexpression of the Ext1 gene in MSCs, thereby relieving the symptoms of osteoarthritis and regulating the immune response in the joint cavity in vivo.

[0018] As far as the inventors know, in this field, the present invention first realizes the effect of promoting the expression of the Ext1 gene to relieve osteoarthritis without using exogenous active substances, opening up a new research path for the research in this field.

[0019] During the operation on subjects with advanced osteoarthritis, the inventor obtained cartilage tissues from both weight-bearing and non-weight-bearing parts. Transcriptomic analysis showed that in the weight-bearing parts, the expression of the Ext1 gene was down-regulated, while the expression of immunomodulatory factors was up-regulated. Subsequently, the inventor constructed a gelatin microsphere-based mesenchymal stem cell (MSCs) system and confirmed in vitro experiments that the gelatin microspheres had good biocompatibility, which was beneficial to the three-dimensional culture of MSC cells and could simulate the growth state of cells in the in vivo three-dimensional environment. The porous structure of the gelatin microspheres was conducive to cell metabolism and secretion. Transcriptomic analysis of the cells showed that the three-dimensional culture state could promote the overexpression of Ext1 in mesenchymal stem cells (MSCs) and also regulate the immune response. Finally, the inventor conducted in vivo experiments to construct an OA (osteoarthritis) rat model and injected gelatin microspheres loaded with mesenchymal stem cells (MSCs) into the joint cavity. The experimental results showed that this method had biological safety and could act continuously in the joint cavity for 30 days. In summary, in the cartilage tissues of osteoarthritis patients, the expression level of the Ext1 gene was reduced. To solve this problem, the present invention constructed a gelatin microsphere system loaded with mesenchymal stem cells (MSCs) through biomaterial engineering. This system simulated the three-dimensional growth mode of in vivo cells, promoted the overexpression of the Ext1 gene, and regulated the immune response in the joint cavity, thereby achieving the effect of treating knee osteoarthritis.

[0020] Advantages of the present invention:

[0021] For the first time in the art, the present invention achieved the effect of promoting the expression of the Ext1 gene and alleviating osteoarthritis without using exogenous drugs; meanwhile, the action time of the present invention can be up to 30 days. The present invention provides a new direction for the future clinical treatment of osteoarthritis, has great potential in personalized and non-surgical treatment, can reduce the dependence on drugs, improve joint health, and provide an affordable solution. Description of the Drawings

[0022] Figure 1Experimental result figures reflecting the abnormal expression of the Ext1 gene and abnormal immune responses in osteoarthritis patients; among them, part A is a representative X-ray image of the knee joint of a patient with knee osteoarthritis; in part B, during the operation of a knee osteoarthritis patient, cartilage tissue samples were obtained from the medial (control group) and lateral (cartilage degeneration group) cartilage tissues of the knee joint, and RNA was extracted from them for transcriptome analysis; part C is a heat map showing the differential expression patterns of the knee joint cartilage tissue transcriptome; part D is a volcano plot depicting the significant changes in gene expression levels in the knee joint cartilage tissue transcriptome; part E summarizes the KEGG pathways enriched in the knee joint cartilage tissue transcriptome, including the relevant gene counts and significance levels; part F further selects immunomodulatory genes previously studied and considered related to the pathogenesis of osteoarthritis to create a correlation heat map.

[0023] Figure 2 Experimental result figures for the preparation of gelatin microspheres and three-dimensional culture of mesenchymal stem cells; among them, part A is a schematic diagram of the preparation process of collagen microspheres encapsulated with mesenchymal stem cells and collagen complexes; part B is the particle size detection of collagen microspheres; part C is the statistical analysis of the particle size distribution of collagen microspheres; part D is the scanning electron microscope image of collagen microspheres; part E shows that mesenchymal stem cells are cultured in collagen microspheres for 24 hours, and the collagen microspheres loaded with mesenchymal stem cells are stained with phalloidin to highlight the cytoskeleton; part F shows that the microspheres loaded with mesenchymal stem cells are stained with calcein AM and propidium iodide (PI) as a control group; part G is the two-dimensional fluorescence microscope image of the calcein AM and PI-stained microspheres loaded with mesenchymal stem cells; part H is the two-dimensional fluorescence overlay microscope image of the calcein AM and PI-stained microspheres loaded with mesenchymal stem cells; part I is the three-dimensional fluorescence microscope image of the calcein AM and PI-stained microspheres loaded with mesenchymal stem cells, showing the three-dimensional distribution of mesenchymal stem cells inside the microspheres.

[0024] Figures 3 - 7 Experimental result figures for enhancing the expression of the Ext1 gene in mesenchymal stem cells through three-dimensional culture using gelatin microspheres; among them, Figure 3 It shows that the control group consists of mesenchymal stem cells (MSCs) cultured in a traditional two-dimensional cell culture dish, while the experimental group involves attaching MSCs to gelatin microspheres for three-dimensional cell culture. After 48 hours of culture, RNA is extracted from both groups for transcriptome analysis, and a heat map showing the transcriptome analysis of MSCs cultured in three-dimensional gelatin microspheres is presented; Figure 4 It shows the KEGG (Kyoto Encyclopedia of Genes and Genomes) flux analysis diagram, presenting the results of the transcriptome data; Figure 5 It shows the distribution of differentially expressed genes identified in the transcriptome analysis; Figure 6It is a GO (Gene Ontology) enrichment map generated based on transcriptome data; Figure 7 It is a KEGG pathway map highlighting the biological pathways affected in the transcriptome of mesenchymal stem cells co-cultured with gelatin microspheres; where BP: Biological Process; MF: Molecular Function; CC: Cellular Component.

[0025] Figure 8 It shows that the addition of microspheres to the culture medium enhanced the immunomodulatory properties of MSCs (mesenchymal stem cells) by IFN-γ; among them, part A is a schematic diagram of the treatment of MSCs by IFN-γ; it shows that the control group consists of mesenchymal stem cells (MSCs), while the experimental group contains gelatin microspheres loaded with MSCs; after 24 hours of culture, IFN-γ was added to both groups; after 24 hours of intervention, the supernatant was collected from the cells and subjected to Luminex liquid suspension chip immunocytokine analysis; part B is a heat map depicting the cytokine expression analysis; part C is the principal component analysis (PCA) result of the cytokine expression analysis; part D shows the changes in the levels of inflammatory factors determined by cytokine analysis.

[0026] Figure 9 It is a figure showing the experimental results that cultured human bone marrow mesenchymal stem cells (MSCs) and gelatin microspheres (GM) help to alleviate the symptoms of osteoarthritis (OA) in vivo; among them, part A is a schematic timeline of the in vivo experiment; an osteoarthritis rat model was prepared by transecting the anterior cruciate ligament. The 8th week after the establishment of the rat osteoarthritis model was defined as day 1, and on day 1 (1d), gelatin microspheres loaded with mesenchymal stem cells were injected into the joint cavity. The Von Frey pain test measured the knee joint pain sensitivity by applying silk threads of different sizes to the knee joint to evaluate the mechanical pain threshold; part B shows in vivo imaging was performed on day 1 (1d), day 10 (10d), and day 21 (21d) after the intra-articular injection of gelatin microspheres loaded with mesenchymal stem cells. Live cells were labeled with DiR iodide. In this color scheme, the labeled cells are darker in color; part C shows that the Von Frey pain test of the rat knee joint was measured on day 1 (1d), day 6 (6d), day 12 (12d), day 18 (18d), and day 24 (24d). The Von Frey pain test evaluated joint pain by applying different forces to the skin around the joint to determine the pain threshold sensitivity. The lower the tactile abnormality, the more severe the pain; part D shows that the rats were sacrificed on day 24, and representative H&E staining images of the knee joint were used to show the control group, OA group, and OA+GM group. Detailed implementation methods

[0027] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0028] Example 1

[0029] 1.1 Materials

[0030] Gelatin was purchased from Sigma Company; Span 80 and acetone were purchased from Aladdin Company; iodine deep erythrocyte membrane fluorescence probe was purchased from Yeasen Biotechnology Company; DAPI staining solution was purchased from Beyotime Biotechnology Company; phalloidin-iFluor 488 was purchased from Yeasen Biotechnology Company; Calcein AM / PI was purchased from Beyotime Biotechnology Company; Pholloidin was purchased from Cytoskeleton Company; Mouse IFN-gamma recombinant protein was purchased from Thermo Fisher Scientific Company; type II collagenase was purchased from Gibco Company; α-MEM was purchased from Gibco Company; PFA and Triton X-100 were purchased from Yeasen Biotechnology Company.

[0031] 1.2 Methods

[0032] 1.2.1 Collection of human osteoarthritis samples

[0033] This study was approved by the Ethics Committee of Peking University People's Hospital (2019phb098-01). Discarded cartilage tissues were extracted from patients undergoing total knee arthroplasty. The inclusion criteria were as follows: age 68 years old, ±6.48 years old (mean ± SD), and the clinical diagnosis was based on the radiological diagnosis standard of Kellgren & Lawrence grade 3. The exclusion criteria included infectious arthritis or rheumatoid arthritis. According to the International Cartilage Repair Society (ICRS) standard, the remote area was defined as the central area of the tibial plateau, and the ICRS was grade I, which is a non-weight-bearing site. The lesion area was located on the lateral tibial plateau, and the ICRS grades were II and III respectively, both of which are weight-bearing sites.

[0034] 1.2.2 Transcriptome analysis

[0035] After obtaining human cartilage samples, chondrocytes were digested as follows: Cartilage samples were taken from both the distal and lesion areas. Each clinical specimen was digested continuously with 0.2% type II collagenase at 37 °C for 10 hours, washed with phosphate-buffered saline (PBS), filtered through a 100 mm nylon mesh, and RNA was collected using Trizol reagent for transcriptome analysis. RNA was collected using Trizol reagent for transcriptomics analysis. Data analysis and plotting were both performed in the R language environment. Differential gene screening was performed using the R package limma, and the screening criteria were a log2 fold change value greater than 1 or less than -1 and a p-value less than 0.05. A volcano plot of differential genes was created using the R package ggplot2, and a heatmap of differential genes was completed using the R package heatmap. Enrichment analysis was performed using the R package cluster Profiler, and GO and KEGG enrichment result plots were created using the R package ggplot2.

[0036] 1.2.3 Gelatin microspheres

[0037] 1.5 g of gelatin powder was dissolved in 10 mL of deionized water and continuously stirred at 60 °C to prepare a 15% (w / v) gelatin solution as the aqueous phase. Span 80 (0.2 mL) was heated to 60 °C in 20 mL of paraffin oil to form the oil phase. The gelatin solution was slowly dropped into the oil phase solution and homogenized to form emulsion droplets in a W / O emulsion. After cooling to 4 °C, the emulsion droplets became gelatin microspheres, which were washed 3 times with acetone to remove excess oil phase substances, washed 3 times with deionized water, and dried to obtain gelatin microspheres. Before use in cell culture, the gelatin was irradiated with ultraviolet light for 2 hours to ensure sterility.

[0038] 1.2.4 Scanning electron microscopy (SEM)

[0039] The gelatin microspheres were freeze-dried, surface-treated, and sputter-coated with gold. The prepared samples were placed on the SEM sample stage to ensure stability. In the scanning electron microscope, appropriate parameters such as acceleration voltage, working distance, and probe current were set. Then the samples were scanned, and secondary electron or backscattered electron images were collected. The acquired images were analyzed using image analysis software equipped with a scanning electron microscope to study the microstructure and morphology of the gelatin microspheres.

[0040] 1.2.5 Culturing bone marrow mesenchymal stem cells with gelatin microspheres

[0041] Bone marrow mesenchymal stem cells isolated from human embryonic stem cells, from CytoNiche Biotechnology Company. Bone marrow mesenchymal stem cells were grown in α-MEM containing 10% fetal bovine serum. Gelatin microspheres were sterilized by ultraviolet irradiation for 2 h. Bone marrow mesenchymal stem cells were washed, counted, centrifuged and resuspended. The cells were seeded into gelatin microspheres in 48-well plates at a final concentration of 5×10 5 cells / 5 mg gelatin microspheres and allowed to attach for 2 h at 37 °C. Finally, 500 μL of growth medium was added to each well and cultured for 9 days at 37 °C and 5% CO2, and the medium was changed every 3 days.

[0042] 1.2.6 Cell immunofluorescence

[0043] Cell samples were collected, fixed with 4% PFA, and then washed with PBS (phosphate buffered saline). Washed with Triton X-100, and bovine serum albumin (BSA) was used to block the antibody binding sites of non-specific interactions. Specific antibodies were added and incubated for a period of time at an appropriate temperature. The cells were washed with PBS to remove unbound antibodies. The cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) dye. The samples were observed with a confocal fluorescence microscope, and image acquisition and analysis were performed using imaging software.

[0044] 1.2.7 Cell culture after IFN-γ treatment

[0045] The above-mentioned mesenchymal stem cell source was passaged and transferred to 6-well plates. Bone marrow mesenchymal stem cells were seeded at a density of 3×10 5 cells / well. The control group was cultured with traditional two-dimensional culture of MSCs (2D culture), and the experimental group was cultured with gelatin microspheres for three-dimensional culture of MSCs (3D culture). Both groups were induced with IFN-γ to detect the regulatory effect of immune factors. After adhesion for 24 h, IFN-γ 50 ng / mL (Beyotime Biotechnology Company) was added to the medium and cultured for 24 h. Cells and conditioned medium were collected after IFN-γ stimulation for Luminex liquid suspension chip immunocytokine analysis.

[0046] 1.2.8 Animal osteoarthritis model

[0047] This study was approved by the Ethics Committee of Peking University People's Hospital (RDJP2023-21). Specific pathogen-free (SPF) grade Sprague-Dawley (SD) rats were randomly divided into a control group (Ctrl), an osteoarthritis group (OA), and an osteoarthritis treatment group (OA+GM), with 5 rats in each group. 8-week-old SD rats were anesthetized, and the surgical area of the knee joint was shaved and disinfected. An appropriate skin incision was made at the knee joint to expose the joint. Through blunt dissection, the anterior structures of the knee joint were observed, including the patella, patellar tendon, and the exposed transverse anterior cruciate ligament (ACL) of the joint capsule. The complete transection of the ligament was confirmed by moving the knee joint, and the joint stability was reduced. The joint capsule and skin incision were sutured with absorbable or non-absorbable sutures. After the operation, appropriate pain management and antibiotic prophylaxis were given to the rats. Independent movement was required after the anesthesia recovery. Cell therapy was injected at different time points after the operation, and relevant data such as joint stability, cartilage degeneration, and inflammatory response were collected and analyzed.

[0048] 1.2.9 Histological examination

[0049] The tissues were fixed in 10% formalin overnight, decalcified with 20% EDTA for 2 weeks, and paraffin sections were made. The tibiae were collected, fixed overnight at 4°C, and embedded in undecalcified methyl methacrylate. Sections with a thickness of 5 μm were stained.

[0050] 2. Results

[0051] 2.1 Downregulation of cartilage Ext1 gene expression in osteoarthritis patients

[0052] To explore the pathogenesis of osteoarthritis, cartilage samples from the weight-bearing and non-weight-bearing areas of the distal femoral condyles were collected during the operation of patients with advanced knee osteoarthritis ( Figure 1 Part A in it). Articular cartilage damage usually occurs in the weight-bearing area, where severe cartilage surface wear and cartilage degeneration can be seen. Chondrocytes were obtained from non-weight-bearing and weight-bearing areas, and cellular RNA was extracted for transcriptome analysis. KEGG analysis showed that the downregulation of genes such as Ext1 was related to the regulation of immune responses ( Figure 1 Parts B-C in it). Compared with the non-weight-bearing area, the expression of Ext1 and genes related to the regulation of extracellular matrix glycosylation synthesis was downregulated in chondrocytes from the weight-bearing area ( Figure 1 Part D in it). Functional enrichment analysis showed that in addition to the pathways related to the Ext1 gene, gene expression changes related to immune regulation also occurred ( Figure 1 Part E in it). Further screening was carried out on immunomodulatory genes that had been previously studied and were considered related to the pathogenesis of osteoarthritis, and a correlation heat map was thus drawn ( Figure 1 Part F in it). The above findings indicate that the degeneration of articular cartilage damage in osteoarthritis is related to the disorder of immune response regulation caused by the downregulation of Ext1 gene expression.

[0053] As far as the inventors know, in the prior art, the understanding of Ext1 mainly is that Ext1 is involved in the growth and development of chondrocytes. However, there is not enough understanding of how to regulate Ext1 to relieve osteoarthritis.

[0054] 2.2 Culturing mesenchymal stem cells in a three-dimensional gelatin microsphere culture medium

[0055] Based on the above research results, the present invention aims to treat osteoarthritis by co-culturing gelatin microspheres with mesenchymal stem cells (MSCs) to regulate Ext1 gene expression and regulate the immune response in the joint cavity. The present invention mixes a polymer modified with an RGD sequence with a gelling agent by a chemical cross-linking method to prepare three-dimensional gelatin microspheres ( Figure 2 part A in), and the average particle size of the microspheres is 230.6 μm ( Figure 2 parts B-C in). Through visual observation and scanning electron microscope (SEM) observation, it can be seen that the gelatin microspheres have a snowflake-like structure and can effectively adhere to MSCs ( Figure 2 part D in). This structure can protect MSCs from being affected by internal environmental factors. Immunofluorescence staining is used to observe the adhesion state of MSCs to gelatin. Gelatin microspheres help to maintain the three-dimensional culture state of cells, which is closer to the in vivo cell growth environment ( Figure 2 parts E-I in).

[0056] This study adopts the method of combining gelatin microspheres with mesenchymal stem cells (MSCs), and uses gelatin microspheres as a carrier to effectively deliver MSCs to the damaged area of osteoarthritis. Gelatin microspheres are like a protective shell to protect MSCs from the influence of the joint cavity environment, such as the fluid shear stress that may damage cells.

[0057] In the prior art, it is known that: (1) Gelatin microspheres provide a three-dimensional culture environment for cells and have been proven to promote cell adhesion in vitro without affecting cell proliferation and growth; (2) Gelatin microspheres are a unique carrier and are known for their biocompatibility, softness, and high water content among various biomaterials. They have been developed for different biomedical applications such as drug delivery and tissue engineering. For example, there has been a study using a hydrogel platform loaded with calcium boride nanosheets (CBN@GelDA hydrogel) as a high-loading and sustainable H2 precursor for the treatment of osteoarthritis.

[0058] However, as far as the inventors know, there is no report in the prior art that can relieve osteoarthritis only relying on gelatin microspheres and the loaded cells without relying on exogenous active substances (such as CBN). Therefore, the present invention provides a new idea for treating related diseases without relying on exogenous active substances in this field.

[0059] 2.3 Promotion of Ext1 gene expression and immune response by culturing mesenchymal stem cells with three-dimensional gelatin microspheres

[0060] The inventors found that co-culturing mesenchymal stem cells (MSCs) with gelatin microspheres is beneficial for their three-dimensional growth. The inventors further performed transcriptome sequencing on MSCs co-cultured with gelatin microspheres in a three-dimensional environment. The inventors screened and analyzed genes with an upregulation of more than 1.5-fold and a downregulation of more than 0.66-fold. First, we performed a heatmap analysis of the differentially expressed genes ( Figure 3 ). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed an increase in the expression of extracellular matrix (ECM) components, and the protein encoded by the Ext1 gene was expressed in the ECM pathway ( Figure 4 ). Functional analysis showed a change in the immune system ( Figure 5 ). GO (Gene Ontology) analysis showed an increase in cytokine-cytokine interactions and enhanced expression of immune system processes and responses to stimuli ( Figure 6 ). Pathway analysis showed an alteration in the ECM, which is related to the extracellular matrix of chondrocytes ( Figure 7 ). These results indicate that three-dimensional cultured MSCs co-cultured with gelatin microspheres promoted the expression of the Ext1 gene and simultaneously regulated the immune response.

[0061] Existing technologies have shown that intra-articular injection of mesenchymal stem cells (MSCs) is effective in treating osteoarthritis (OA) and promoting cartilage regeneration, but the repair efficiency of this method is limited. The present invention for the first time found that loading MSCs with the microsphere carrier of the present invention can promote the expression of the Ext1 gene, thereby achieving the therapeutic effect on osteoarthritis.

[0062] 2.4 IFN-γ-induced co-cultured mesenchymal stem cells with gelatin microspheres exhibit immunomodulatory ability

[0063] Transcriptomic results showed an increase in cytokine-cytokine interactions. To test the immunomodulatory ability of mesenchymal stem cells (MSCs) cultured with gelatin microspheres, MSCs adhered to gelatin microspheres were exposed to the pro-inflammatory cytokine IFN-γ. Both groups consisted of MSCs cultured with gelatin microspheres. The control group was not treated with IFN-γ, while the experimental group received IFN intervention ( Figure 8 Part A). The inventors detected 25 related inflammatory factors ( Figure 8 Part B) and found that the expression of five inflammatory factors changed after IFN-γ stimulation ( Figure 8 Part C), including IFN-γ, basic FGF, VEGF, IL-5, and IL-6 ( Figure 8Part D). The results of the liquid-phase suspension chip showed that mesenchymal stem cells loaded on gelatin microspheres enhanced their immunomodulatory effect.

[0064] 2.5 It was confirmed in a rat model of knee osteoarthritis that gelatin microspheres cultured with mesenchymal stem cells helped alleviate the symptoms of osteoarthritis in vivo

[0065] To evaluate the biosafety of gelatin in vivo, the inventors conducted an experiment in which gelatin microspheres were cultured with mesenchymal stem cells (MSCs) and then injected into the knee joint cavity of wild-type rats ( Figure 9 Part A). In vivo imaging detection showed that these cells could survive in the joint cavity for more than 21 days and did not migrate to other tissues outside the joint cavity ( Figure 9 Part B). In addition, no obvious abnormalities were observed in the peripheral blood of the rats. The von Frey pain test showed that increasing the amount of gelatin microspheres and MSCs injected into the osteoarthritic joint cavity could relieve knee pain ( Figure 9 Part C). Histological examination showed that the gelatin microspheres did not damage the normal cartilage tissue after entering the joint cavity ( Figure 9 Part D). These findings confirmed the safety of gelatin microspheres in in vivo treatment, which will provide a new strategy for tissue injury repair.

[0066] To verify the therapeutic effect of gelatin microspheres cultured with mesenchymal stem cells (MSCs) on osteoarthritis, the inventors constructed a rat knee osteoarthritis model by injuring the anterior cruciate ligament. The von Frey pain test showed that injecting gelatin microspheres cultured with MSCs could relieve the abnormal tactile pain associated with osteoarthritis. Histological examination showed that gelatin microspheres cultured with MSCs could alleviate the process of cartilage damage in osteoarthritis and protect the structural morphology of knee joint cartilage. The in vivo experimental results showed that gelatin microspheres cultured with MSCs could effectively treat knee osteoarthritis.

Claims

1. A microsphere that promotes Ext1 expression and alleviates osteoarthritis, characterized in that, The microspheres include gelatin microspheres and bone marrow mesenchymal stem cells loaded on the microspheres.

2. A microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 1, wherein The addition amounts of gelatin microspheres and bone marrow mesenchymal stem cells are: 1×10 5 bone marrow mesenchymal stem cells are loaded per microgram of gelatin microspheres.

3. A microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 1, characterized in that, The gelatin microspheres have a snowflake-like structure; the particle size of the gelatin microspheres does not exceed 1000 μm.

4. The microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 3, characterized in that, The average particle size of the gelatin microspheres is 230.6 μm.

5. The microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 1, wherein The gelatin microspheres are prepared by an emulsification method using an aqueous gelatin solution as the aqueous phase and paraffin oil added with Span80 as the oil phase.

6. The microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 5, characterized in that, In the aqueous phase, the content of gelatin is 15% w / v.

7. A microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 6, characterized in that In the oil phase, the volume ratio of Span 80 to paraffin oil is 1:

100.

8. A microsphere for promoting Ext1 expression and alleviating osteoarthritis according to claim 7, characterized in that, When preparing the gelatin microspheres, 1.5 g of gelatin powder is dissolved in 10 mL of deionized water and continuously stirred at 60 °C to prepare a 15% w / v gelatin solution as the aqueous phase; 0.2 mL of Span 80 is heated to 60 °C in 20 mL of paraffin oil to form the oil phase; the gelatin solution is slowly dropped into the oil phase solution for homogenization to form droplets in the W / O emulsion; after cooling to 4 °C, the droplets become gelatin microspheres, which are washed 3 times with acetone to remove the excess oil phase substances, washed three times with deionized water, and dried to obtain the gelatin microspheres.

9. A preparation method of microspheres for promoting Ext1 expression and alleviating osteoarthritis, characterized in that, The bone marrow mesenchymal stem cells in the microspheres according to any one of claims 1 to 8 are loaded on the gelatin microspheres to obtain the product.

10. Use of the microspheres according to any one of claims 1 to 8 in the preparation of a preparation for relieving osteoarthritis.

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