Targeted pathological B cell temperature-sensitive hydrogel bone repair material as well as preparation method and application thereof

Abstract

The invention provides a targeted pathological B cell temperature-sensitive hydrogel bone repair material as well as a preparation method and application thereof, and belongs to the technical field of bone repair biomaterials.The targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel provided by the invention is prepared by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs, the mass ratio of the hydroxypropyl chitin HPCH to the B3-Cu-Zn-MBGs is (5: 1) to (1: 1); according to the HPCH / B3-Cu-Zn-MBGs provided by the invention, the joint swelling is remarkably improved through the comprehensive action of the HPCH / B3-Cu-Zn-MBGs, the arthritis index is reduced, and the clinical symptoms of RA (rheumatoid arthritis) are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone repair biomaterials, and particularly relates to a temperature-sensitive hydrogel bone repair material targeting pathological B cells, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that mainly causes damage to tissues such as joint synovium, cartilage, and bone, is characterized by symmetrical and polyarticular inflammation, and may involve extra-articular tissues (such as skin, cardiovascular system, lungs, etc.), seriously damaging the quality of life of patients and bringing a heavy social and economic burden. In untreated patients with rheumatoid arthritis, destructive bone erosion and joint deformity will occur, and eventually joint dysfunction may result. The disability rate is 50% two years after the onset and 70% three years after the onset (Sokka T. Work disability in early rheumatoid arthritis [J]. Clin Exp Rheumatol, 2003, 21(5 Suppl 31): S71-4.). Therefore, conducting research on the pathogenesis and repair and regeneration of rheumatoid arthritis is of great significance for more effectively preventing and treating bone loss and improving the health level of the body.

[0003] Rheumatoid arthritis belongs to the category of osteoimmune diseases, and there are physiological changes such as reduced bone mineral density, decreased bone quality, and damaged bone microarchitecture. Previous studies have found that there is a large accumulation of immune B cells in the local lesions of rheumatoid arthritis (Sun W, Meednu N, Rosenberg A, et al. B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation[J]. Nat Commun, 2018, 9(1): 5127.). B cells play a role in the humoral immunity of the adaptive immune system by secreting antibodies, and they can also present antigens and secrete various cytokines, that is, they have non-antibody-secreting functions. The B cells aggregated in the rheumatoid joints can not only secrete pro-inflammatory cytokines such as TNF-α, IL-6, IL-17, and IL-23 (Fillatreau S. B cells and their cytokine activities implications in human diseases[J]. Clin Immunol, 2018, 186: 26-31.), directly regulating bone destruction; but also promote osteoclast activation by secreting RANKL or activators of RANKL, leading to bone erosion (Yeo L, Lom H, Juarez M, et al. Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis[J]. Ann Rheum Dis, 2015, 74(5): 928-35.). Previous studies have also found that a large number of B cells aggregate in the joint area of rheumatoid arthritis, adjacent to osteoblasts, and secrete CCL3 and TNF-α to inhibit osteoblast differentiation and bone formation (Sun W, Meednu N, Rosenberg A, et al. B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation[J]. Nat Commun, 2018, 9(1): 5127.).Rheumatoid arthritis is driven by immune memory cells including memory B cells and memory plasma cells (Komatsu N, Takayanagi H. Mechanisms of joint destruction in rheumatoid arthritis - immune cell - fibroblast - bone interactions[J]. Nat Rev Rheumatol, 2022, 18(7):415 - 429.).

[0004] In recent years, with the development of osteoimmunology, the design concept of orthopedic biomaterials has gradually shifted from "immune - friendly" to "immune - regulatory" (Sadowska J M, Wei F, Guo J, et al. The effect of biomimetic calcium - deficient hydroxyapatite and sintered β - tricalcium phosphate on osteoimmune reaction and osteogenesis[J]. Acta Biomaterialia, 2019, 96:605 - 618.). When developing new bone repair materials, it is necessary to consider both promoting bone formation and immune regulation of the local microenvironment at the same time. Biomaterials with good osteoimmune regulation ability can induce the formation of an immune environment conducive to the regeneration and repair of bone tissue. Injectable hydrogels can form solids at the bone defect site through minimally invasive injection, filling complex defect sites in the body, and are potential platforms for drug delivery in bone tissue engineering. Currently, there is no report on bone repair materials regulated by B cells (especially immune B cells locally aggregated in rheumatoid arthritis lesions). Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a thermosensitive hydrogel of a bone repair material HPCH / B3 - Cu - Zn - MBGs targeting pathological B cells, its preparation method and application. The present invention uses B3 - Cu - Zn - MBGs as a carrier, which can eliminate aggregated pathological B cells in rheumatoid arthritis (RA) and inhibit their abnormal activation of the immune system. By promoting the apoptosis of B cells, it reduces the negative impact of B cells on bone homeostasis and improves the immune microenvironment in the joint.

[0006] Furthermore, the present invention prepares a thermosensitive hydrogel using hydroxypropyl chitosan and B3 - Cu - Zn - MBGs, which can transform into a gel state at body temperature, effectively fill the bone defect site and promote the differentiation of local osteoblasts.

[0007] The HPCH / B3-Cu-Zn-MBGs provided by the present invention can regulate the local bone immune microenvironment through its thermosensitive properties and pharmacological activities. By reducing the aggregation of B cells, HPCH / B3-Cu-Zn-MBGs promotes the differentiation of osteoblasts, enhances the ability of bone regeneration, improves the cytokine balance in the bone immune system, and further alleviates the immune damage of RA.

[0008] The present invention provides a thermosensitive hydrogel of a bone repair material HPCH / B3-Cu-Zn-MBGs targeting pathological B cells, which is obtained by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs.

[0009] Preferably, the mass ratio of the hydroxypropyl chitin HPCH to B3-Cu-Zn-MBGs is 5:1 to 1:1.

[0010] The present invention provides a preparation method of the thermosensitive hydrogel of the bone repair material HPCH / B3-Cu-Zn-MBGs targeting pathological B cells, comprising the following steps:

[0011] 1) Mix hydroxypropyl chitin HPCH with water and stir to obtain a first material;

[0012] 2) Mix B3-Cu-Zn-MBGs with water to obtain a second material;

[0013] 3) Mix the first material and the second material to obtain a thermosensitive hydrogel;

[0014] There is no limitation on the order between step 1) and step 2);

[0015] During the mixing process of step 3), stirring is accompanied, the rotation speed of the stirring is 300 - 400 rpm, and the stirring time is 10 - 20 min.

[0016] Preferably, the stirring time in step 1) is 2 - 4 h.

[0017] Preferably, the mixing ratio of the hydroxypropyl chitin HPCH to water in step 1) is 15 mg:(0.5 - 1.0) ml.

[0018] Preferably, the ratio of the B3-Cu-Zn-MBGs to water in step 2) is (3 - 15) mg:(0.2 - 0.3) ml.

[0019] The present invention provides the application of the thermosensitive hydrogel of the bone repair material HPCH / B3-Cu-Zn-MBGs targeting pathological B cells in the preparation of a drug for treating rheumatoid arthritis.

[0020] Preferably, the dosage form of the drug is an injectable hydrogel.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel provided by the present invention is prepared by mixing hydroxypropyl chitosan HPCH and B3-Cu-Zn-MBGs. Using B3-Cu-Zn-MBGs as a carrier, it can eliminate the aggregated pathological B cells in rheumatoid arthritis (RA) and inhibit their abnormal activation of the immune system. By promoting the apoptosis of B cells, it reduces the negative impact of B cells on bone homeostasis and improves the immune microenvironment in the joint. Further, the thermosensitive hydrogel prepared by the present invention using hydroxypropyl chitosan and B3-Cu-Zn-MBGs also has thermoresponsive properties, can rapidly transform into a gel state in vivo, and form a solid at the bone defect site, effectively filling complex defect areas, and has strong bone regeneration potential and application value.

[0022] The present invention uses TNF transgenic mice (TNF-Tg) as an animal model of rheumatoid arthritis, and in vivo proves that HPCH / B3-Cu-Zn-MBGs can effectively inhibit the aggregation of B cells in damaged joints, promote the differentiation of local osteoblasts, significantly improve joint swelling and reduce the arthritis index. By using an appropriate ratio of HPCH / B3-Cu-Zn-MBGs (3:1), while promoting bone formation at the damaged site, it effectively eliminates the aggregated pathological B cells. Flow cytometry analysis shows that the number of local MSCs in the joint increases, the osteogenic effect is significantly enhanced, and the number of OCP osteoclasts decreases, thereby regulating the bone immune microenvironment and significantly improving the clinical symptoms of RA, showing a significant therapeutic effect and having the potential to become a candidate drug for the treatment of rheumatoid arthritis. Brief Description of the Drawings

[0023] Figure 1 For the characterization and performance detection of the HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel material, wherein (A) is the scanning electron microscope (SEM) image of HPCH / B3-Cu-Zn-MBGs, showing the microscopic morphological structure of HPCH / B3-Cu-Zn-MBGs; (B) is in a liquid state at room temperature and transforms into a gel state at 37 °C, illustrating the thermosensitive transition characteristics; (C) is the EDX energy spectrum analysis chart, indicating the elemental composition and distribution of B3-Cu-Zn-MBGs.

[0024] Figure 2Effect of HPCH / B3-Cu-Zn-MBGs at different concentrations on the proliferation of MPCs cells and B cells. (A) shows the CCK-8 assay results for the proliferation of MPCs cells on days 0, 1, 2, and 3; (B) shows the CCK-8 assay results for the proliferation of B cells at 0, 2, 6, 12, and 24 h. Data are presented as mean ± SEM (n = 6), *p < 0.05, **p < 0.01.

[0025] Figure 3 Effect of HPCH / B3-Cu-Zn-MBGs at different concentrations on B cells and MPCs after treatment: (A) Double staining with calcein (live cells, green fluorescence) and propidium iodide (dead cells, red fluorescence) shows the ratio of live cells to dead cells in B cells at 0, 2, 6, 12, and 24 h; (B) ALP staining of MPCs after osteogenic induction; (C) Live / dead double staining images (upper panel) of B cells and MPCs (mesenchymal progenitor cells) after 3 days of co-culture, and ALP staining results (lower panel) of MPCs after osteogenic induction.

[0026] Figure 4 Arthritis symptoms of TNF transgenic mice in each treatment group after ankle injection: (A) Representative photographs of the hind paws of TNF-tg mice on day 28 after administration; (B) Arthritis index score; Data are presented as mean ± SEM (n = 6), *p < 0.05, **p < 0.01.

[0027] Figure 5 μ-CT 3D reconstruction images and scanned sectional images of the ankle joints of TNF-tg mice in each treatment group 28 days after ankle injection. (A) is the control group (veh), and (B) is the treatment group (3:1); (C) shows the morphometric data of the bone volume of the ankle joint relative to the tissue volume (%); (D) shows the morphometric data of the bone surface area to bone volume ratio (%). Data are presented as mean ± SEM (n = 6), **p < 0.01 compared with the control group.

[0028] Figure 6 Flow cytometry analysis results of the ankle joints of TNF-tg mice in each treatment group 28 days after ankle injection: (A, B) show the proportion of B cells; (C, D) show the proportion of MPC cells; (E, F) show the proportion of OCP (osteoclast precursor cells). Data are presented as mean ± SEM (n = 5), *p < 0.05 compared with the control group. Detailed implementation

[0029] The present invention provides a targeted pathological B-cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel, which is prepared by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs.

[0030] The hydroxypropyl chitin (Hydroxypropyl Chitin, HPCH) used in the present invention has both excellent biocompatibility and adjustable thermal responsiveness. That is, under the low-temperature conditions of the in vitro environment, it usually exists in the form of a solution, and once it enters the body temperature (about 37 °C), it will quickly turn into a solid. Through its biomimetic extracellular matrix microenvironment and dynamic phase change characteristics, it exhibits significant bone induction activity.

[0031] Cu induces macrophages to polarize into an anti-inflammatory phenotype, and MSCs highly express BMP-2 and collagen I under Cu 2+ stimulation, coordinating bone formation and immune tolerance. Mesoporous Bioactive Glasses Nanoparticles (MBGN) have become an intelligent platform for local drug / ion delivery due to their unique nano-mesoporous structure. In the present invention, MBGN is used to load Cu, endowing it with the biological function of coordinating bone formation and immune tolerance.

[0032] In the present invention, the mass ratio of the hydroxypropyl chitin HPCH to B3-Cu-Zn-MBGs is 5:1 to 1:1, and can be optionally 5:1, 3:1, 2:1, 1:1, preferably 3:1.

[0033] The present invention provides a preparation method of the targeted pathological B-cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel, including the following steps: 1) Mix and stir hydroxypropyl chitin HPCH with water to obtain a first material; 2) Mix B3-Cu-Zn-MBGs with water to obtain a second material; 3) Mix the first material and the second material to obtain a thermosensitive hydrogel.

[0034] In the present invention, hydroxypropyl chitin HPCH is mixed and stirred with water to obtain a first material. The mixing ratio of the hydroxypropyl chitin HPCH to water is 15 mg:(0.5 - 1.0) ml, preferably 15 mg:(0.6 - 0.8) ml, and further preferably 15 mg:0.75 ml. The stirring time is preferably 2 - 4 h, and further preferably 3 h; in the present invention, the temperature of the mixing and stirring is preferably room temperature, and further preferably 15 - 25 °C. In the present invention, the stirring is magnetic stirring, and the stirring speed is preferably 300 - 400 rpm.

[0035] In the present invention, B3-Cu-Zn-MBGs are mixed with water to obtain a second material, and the ratio of B3-Cu-Zn-MBGs to water is (3-15) mg:(0.2-0.3) ml, preferably (3-15) mg:0.25 ml. In the present invention, the B3-Cu-Zn-MBGs are preferably prepared by the following steps: 2.1) Synthesize B3-MBGs by a microemulsion-assisted sol-gel method; 2.2) Prepare B3-Cu-Zn-MBGs by a modified post-impregnation method.

[0036] After obtaining the first material and the second material in the present invention, the two are mixed, and the mixing process is accompanied by stirring, and the stirring is preferably magnetic stirring; the rotation speed of the stirring is 300-400 rpm, and the stirring time is preferably 10-20 min, more preferably 12-18 min, and even more preferably 15 min.

[0037] The present invention also provides the application of the targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel in the preparation of drugs for treating rheumatoid arthritis. In the present invention, the dosage form of the drug is preferably an injectable hydrogel. The HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel provided by the present invention can effectively kill locally aggregated pathological B cells, promote bone formation, and improve the bone immune microenvironment, thereby providing a new solution for the treatment of rheumatoid arthritis.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] 1. Preparation of HPCH / B3-Cu-Zn-MBGs

[0041] Step 1: Synthesize B3-MBGs by a microemulsion-assisted sol-gel method;

[0042] Dissolve 1.2 g of cetyltrimethylammonium bromide in 40 mL of an ethanol / water mixed solvent (ethanol / water volume ratio 3:1), and magnetically stir (600 rpm) to form a micelle system. Dropwise add triethyl borate (5.82 mL) and triethyl phosphate (0.89 mL) in sequence, control the dropping rate (0.5 mL / min), maintain pH = 9.0 (adjusted with ammonia water), and stir for 2 hours to complete the pre-polymerization of the boron-phosphorus network. Add the metal nitrate solution (Na + 、K + 、Mg 2+ 、Ca 2(+), Stir for 4 hours to form a homogeneous sol. Collect the formed bioactive glass particles by centrifugation (4000 rpm, 15 min), and wash them 3 times with deionized water and ethanol. The collected particles are dried overnight in air at 60 °C, and then calcined in a furnace at 700 °C in air at a heating rate of 2 °C / min for 3 h to finally obtain B3-MBGs with a spherical mesoporous structure.

[0043] Step 2: Prepare B3-Cu-Zn-MBGs by a modified post-impregnation method;

[0044] Prepare an ethanol / water mixture (ethanol / water volume ratio 2:1), add 0.83 g of copper nitrate (Cu(NO3)2·3H2O) and 0.49 g of zinc nitrate (Zn(NO3)2·6H2O), and adjust the total metal ion concentration to 1.2 M. Disperse B3-MBGs (10 g / L) in the above impregnation solution and impregnate with ultrasonic assistance (40 kHz, 150 W) for 12 hours to ensure the 2+ / Zn 2+ gradient distribution of Cu / Zn within the mesopores. The loaded particles are vacuum filtered and washed successively with 0.1 M nitric acid and deionized water until neutral. In a reducing atmosphere (95% N2 + 5% H2), heat to 550 °C at a rate of 2 °C / min and calcine for 3 hours to promote the stable embedding of Cu / Zn in the borosilicate network in the form of oxides.

[0045] Step 3: Prepare thermosensitive hydrogels with different ratios of HPCH / B3-Cu-Zn-MBGs according to the mass ratio;

[0046] At room temperature, add 15 mg of HPCH to 0.75 mL of ultrapure water and stir magnetically for 3 h. Add 3 mg of B3-Cu-Zn-MBGs to 0.25 mL of ultrapure water, mix and stir magnetically for 15 min to prepare 1 mL of (5:1) HPCH / B3-Cu-Zn-MBGs;

[0047] Add 15 mg of HPCH to 0.75 mL of ultrapure water and stir magnetically for 3 h. Add 5 mg of B3-Cu-Zn-MBGs to 0.25 mL of ultrapure water, mix and stir magnetically for 15 min to prepare 1 mL of (3:1) HPCH / B3-Cu-Zn-MBGs;

[0048] Add 15 mg of HPCH to 0.75 mL of ultrapure water and stir magnetically for 3 h. Add 10 mg of B3-Cu-Zn-MBGs to 0.25 mL of ultrapure water, mix and stir magnetically for 15 min to prepare 1 mL of (3:2) HPCH / B3-Cu-Zn-MBGs;

[0049] 15 mg of HPCH was added to 0.75 mL of ultrapure water and stirred magnetically for 3 h. 15 mg of B3-Cu-Zn-MBGs was added to 0.25 mL of ultrapure water, and after mixing, it was stirred magnetically for 15 min to prepare 1 mL (1:1) of HPCH / B3-Cu-Zn-MBGs.

[0050] 2. Material Characterization and Performance Detection

[0051] The microstructure of the surface of 1:1 HPCH / B3-Cu-Zn-MBGs was shown by scanning electron microscope (SEM) as shown in (A) in Figure 1 , and the elemental composition was shown by EDX spectrum, and the results were as shown in (C) in Figure 1 .

[0052] HPCH / B3-Cu-Zn-MBGs was in a liquid state at room temperature and in a gel state at 37 °C, with thermosensitive transition characteristics, and the results were as shown in (B) in Figure 1 .

[0053] Example 2

[0054] 1. Extraction and Culture of B Cells and MPC Cells

[0055] B cells were isolated from the spleens of mice using a B cell isolation kit (#130-121-031, Miltenyi Biotec) according to the manufacturer's instructions. Purified B cells (5×10 5 cells / well in a 96-well plate) were further stimulated with 2.5 μg / mL anti-CD40 (#553722, BD Biosciences) plus 20 ng / mL IL4 (#AF-214-14, PeproTech) and 10 μg / mL LPS (#L4391, Sigma) for subsequent experiments.

[0056] Three-week-old wild-type (WT) mice were taken, the tibias and femurs were dissected, cut into bone pieces with scissors, cultured in a culture dish for 3 days, and the bone pieces were transferred to a clean culture dish as the first generation and cultured for another 7 days until the cells grew confluently. MPCs from the second generation were used subsequently.

[0057] All mice were housed and maintained in the SPF-class experimental animal center of Nanjing Medical University. The use of animals in this study has been approved by the Animal Experiment Ethics Committee of Nanjing Medical University (approval number: 1906018).

[0058] HPCH and thermosensitive hydrogels of HPCH / B3-Cu-Zn-MBGs with different concentrations (5:1, 3:1, 3:2, 1:1) were used for co-culturing B cells with MPCs and B cells with MSCs.

[0059] For MPCs, blank group, HPCH, thermosensitive hydrogel of HPCH / B3-Cu-Zn-MBGs 5:1 (denoted as 5:1 in the figure), thermosensitive hydrogel of HPCH / B3-Cu-Zn-MBGs 3:1 (denoted as 3:1 in the figure), thermosensitive hydrogel of HPCH / B3-Cu-Zn-MBGs 3:2 (denoted as 3:2 in the figure), and thermosensitive hydrogel of HPCH / B3-Cu-Zn-MBGs 1:1 (denoted as 1:1 in the figure) were respectively set up.

[0060] For B cells, the above-mentioned treatment groups were also set up.

[0061] 2. CCK-8 cell viability assay

[0062] According to the kit instructions, B cells and MPCs were seeded in 96-well plates at a certain number. According to the cell proliferation situation, at the predetermined time points, 100 μL of fresh medium and 10 μL of CCK-8 reagent (#K1018, Apex bio) were used to replace the conditioned medium. After incubation in the dark for 2 h, the optical density (OD) at a wavelength of 450 nm (OD 450) was measured according to the manufacturer's instructions. The results are as Figure 2 shown that the killing effect of the thermosensitive hydrogel of HPCH / B3-Cu-Zn-MBGs on B cells gradually increased with the increase of time and concentration, while the effect on MPCs was not significant.

[0063] 3. Cell ALP staining

[0064] For MPCs treated with the thermosensitive hydrogel and MPCs after co-culturing B cells-MPCs, they were co-cultured in osteoblast induction medium for 3 days. ALP staining was performed using BCIP / NBT alkaline phosphatase color development kit (#C3206, Beyotime), and the staining was analyzed. The results are as Figure 3 shown that compared with the control group, the expression of osteogenic differentiation marker ALP in MPCs of the thermosensitive hydrogel treatment group was significantly increased. In the B cell-MPC co-culture system, the osteogenic differentiation ability of MPCs was significantly inhibited, and the ALP activity was lower than that of the control group. When the thermosensitive hydrogel was added to the co-culture system for intervention, the osteogenic inhibition effect mediated by B cells could be partially reversed, and the ALP activity showed a recovery trend.

[0065] Example 3

[0066] 1. Experimental mice and drug administration

[0067] TNF transgenic (TNF-Tg) mouse strain (strain 3647), carrying a modified human TNF transgene in which the 3′ end of the TNF gene is replaced by the 3′ end of the human α-globin gene, has been successfully raised and propagated in the laboratory. Starting from 2 months of age, TNF-Tg transgenic mice develop arthritis in the ankle joints, which progresses with age, and systemic bone loss and osteoporosis appear at 4 months of age. All mice were housed and maintained in the SPF-class experimental animal center of Nanjing Medical University. The use of animals in this study has been approved by the Animal Experiment Ethics Committee of Nanjing Medical University (approval number: 1906018).

[0068] When the condition of 2-month-old TNF-Tg transgenic mice had progressed partially, 12 mice were randomly divided into a treatment group (3:1) and a control group (Veh.). 5 μL of HPCH / B3-Cu-Zn-MBGs (3:1) was injected into the bilateral ankle joint cavities of the mice in the treatment group, and the control group was given an equal volume of normal saline simultaneously.

[0069] 2. Clinical evaluation of arthritis

[0070] The arthritis index is one of the two important indicators for judging the anti-RA effect of drugs and reflects the inflammatory reaction occurring in the joints. The experiment was carried out on days 0, 7, 14, 21, and 28, and the joint swelling and deformity were observed every 7 days. The severity of arthritis was measured using the arthritis scoring method, and the clinical arthritis score was evaluated from 0 to 4 according to the report: 0 points, normal; 1 point, only mild swelling of the toes; 2, 3, and 4 points indicate mild, moderate, and severe swelling of the toes and ankles, respectively. The results are as Figure 4 shown, and the treatment group significantly improved joint swelling and reduced the arthritis index.

[0071] 3. Imaging and histomorphological evaluation

[0072] After 28 days of drug administration, tissue samples were taken from all mice, and the ankle joints were collected and fixed in 4% paraformaldehyde. The ankle joints were subjected to μ-CT scanning and three-dimensional reconstruction to observe the changes in bone mass. The results are as Figure 5 shown.

[0073] 4. Flow cytometry labeling, detection, and sorting

[0074] After 28 days of drug administration intervention, the ankle joint tissues of mice in each group were taken for single-cell analysis. The specific procedure is as follows: The tissue samples were prepared into single-cell suspensions by enzymatic digestion combined with mechanical grinding, and the cell concentration was adjusted to 1×10 6cells / 100 μL, incubated with a multi-color fluorescently labeled antibody in the dark at 4 °C for 30 minutes. After washing with PBS (containing 2% FBS), the cell subset ratios of B cells, mesenchymal progenitor cells (MPCs), and osteoclast precursors (OCPs) were detected by flow cytometry. The results are as Figure 6 shown. After local injection intervention, the number of local B cells in the experimental group was significantly reduced compared with the control group; the population ratio of MPCs increased significantly; the population ratio of OCPs showed a downward trend. These results suggest that the thermosensitive hydrogel can affect the bone metabolism balance by regulating the significantly downregulated B cells to change the local immune microenvironment.

[0075] As can be seen from the above embodiments, the HPCH / B3-Cu-Zn-MBGs provided by the present invention can regulate the local bone immune microenvironment through its thermosensitive properties and pharmacological activities. By reducing the aggregation of B cells, HPCH / B3-Cu-Zn-MBGs promoted the differentiation of osteoblasts, enhanced the ability of bone regeneration, and improved the cytokine balance in the bone immune system, further reducing the immune damage of RA.

[0076] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A target pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel, characterized in that, It is obtained by mixing hydroxypropyl chitosan HPCH and B3-Cu-Zn-MBGs.

2. The temperature-sensitive hydrogel of the targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs according to claim 1, wherein The mass ratio of the hydroxypropyl chitosan HPCH to the B3-Cu-Zn-MBGs is 5:1 to 1:

1.

3. The preparation method of the temperature-sensitive hydrogel of the targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs according to claim 1 or 2, characterized in that, It includes the following steps: 1) Mix hydroxypropyl chitosan HPCH with water and stir to obtain a first material; 2) Mix B3-Cu-Zn-MBGs with water to obtain a second material; 3) Mix the first material and the second material to obtain a thermosensitive hydrogel; There is no limitation on the sequence between step 1) and step 2); During the mixing process in step 3), stirring is accompanied. The rotation speed of the stirring is 300 - 400 rpm, and the stirring time is 10 - 20 min.

4. The preparation method according to claim 3, wherein The stirring time in step 1) is 2 - 4 h.

5. The preparation method according to claim 4, characterized in that, The mixing ratio of the hydroxypropyl chitosan HPCH to water in step 1) is 15 mg:(0.5 - 1.0) mL.

6. The preparation method according to claim 3, characterized in that, The ratio of the B3-Cu-Zn-MBGs to water in step 2) is (3 - 15) mg:(0.2 - 0.3) mL.

7. Application of the targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel described in claim 1 or 2, and the targeted pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel prepared by the preparation method described in any one of claims 3 - 6 in the preparation of a drug for treating rheumatoid arthritis.

8. The application according to claim 7, wherein The dosage form of the drug is an injectable hydrogel.

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