A pathological b cell targeted thermosensitive hydrogel bone repair material and a preparation method and application thereof

Abstract

The application provides a targeted pathological B cell temperature-sensitive hydrogel bone repair material and 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 application is prepared by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs, and the mass ratio of the hydroxypropyl chitin HPCH and the B3-Cu-Zn-MBGs is 5:1-1:1. The HPCH / B3-Cu-Zn-MBGs provided by the application significantly improves joint swelling, reduces arthritis index, and reduces the clinical symptoms of RA through the comprehensive action thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bone repair biomaterials, and particularly relates to a pathological B cell targeted temperature-sensitive hydrogel bone repair material and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that mainly causes damage to synovial membrane, cartilage, bone and other tissues, has the characteristics of symmetrical and polyarthritis inflammation, and can involve extra-articular tissues (such as skin, cardiovascular system, lung, etc.), seriously damages the life quality of patients, and brings heavy social and economic burden. Untreated rheumatoid arthritis patients will have destructive bone erosion and joint deformation, and eventually lead to joint dysfunction, with a disability rate of 50% two years after the onset and a disability rate of 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, it is of great significance to carry out researches on the pathogenesis and repair regeneration of rheumatoid arthritis for more effectively preventing and treating bone loss and improving the health level of the body.

[0003] Rheumatoid arthritis is a bone immune disease, which is characterized by low bone density, decreased bone mass, and damaged bone microstructure. Previous studies have shown that immune B cells are aggregated in the local area 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 immune system of the adaptive immune system by secreting antibodies, and can also present antigens and secrete various cytokines, i.e. non-antibody secretion function. The aggregated B cells in the rheumatoid joint can not only secrete TNF-α, IL-6, IL-17, IL-23 and other pro-inflammatory cytokines (Fillatreau S. B cells and their cytokine activities implications in human diseases[J]. Clin Immunol, 2018, 186: 26-31.), but also promote osteoclast activation by secreting RANKL or RANKL activating factor, leading to bone erosion. Previous studies have also found that a large number of B cells are aggregated in the joint area of rheumatoid arthritis, close 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 regulation" (Sadowska J M, Wei F, Guo J, et al. The effect of biomimetic calcium deficient hydroxyapatite and sintered beta-tricalcium phosphate on osteoimmune reaction and osteogenesis [J]. Acta Biomaterialia, 2019, 96: 605-618.). The development of new bone repair materials needs to consider 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 bone tissue regeneration and repair. Injectable hydrogels can form solids by minimally invasive injection at bone defect sites, and fill complex defect sites in the body, which is a potential platform for drug delivery in bone tissue engineering. Currently, there is no report on bone repair materials regulated by B cells (especially immune B cells that accumulate locally in rheumatoid arthritis lesions). SUMMARY

[0005] Therefore, the purpose of the present application is to provide a pathological B cell targeted bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel, its preparation method and application. The present application uses B3-Cu-Zn-MBGs as a carrier, which can eliminate the aggregation of pathological B cells in rheumatoid arthritis (RA) and inhibit the abnormal activation of the immune system. By promoting the apoptosis of B cells, the negative impact of B cells on bone homeostasis is reduced, and the intra-articular immune microenvironment is improved.

[0006] Further, the present application uses hydroxypropyl chitin and B3-Cu-Zn-MBGs to prepare a temperature-sensitive hydrogel, which can be converted to a gel state at body temperature, effectively filling the bone defect site and promoting the differentiation of local osteoblasts.

[0007] The HPCH / B3-Cu-Zn-MBGs provided by the application can regulate local bone immune microenvironment through its temperature-sensitive characteristics and pharmacological activity, promote the differentiation of osteoblasts by reducing the aggregation of B cells, enhance the ability of bone regeneration, and improve the balance of cytokines in the bone immune system, further reducing the immune damage of RA.

[0008] The application provides a pathological B cell targeted bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel prepared by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs.

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

[0010] The application provides a preparation method of the pathological B cell targeted bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel.

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

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

[0013] 3) mixing the first material and the second material to obtain a temperature-sensitive hydrogel;

[0014] The order of steps 1) and 2) is not limited;

[0015] In the mixing process of step 3), stirring is accompanied, the stirring speed 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 and water in step 1) is 15 mg:(0.5-1.0) ml.

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

[0019] The application provides an application of the pathological B cell targeted bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel in the preparation of a drug for treating rheumatoid arthritis.

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

[0021] Compared with the prior art, the application has the following beneficial effects: the target pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel provided by the application is prepared by mixing hydroxypropyl chitin HPCH and B3-Cu-Zn-MBGs, and B3-Cu-Zn-MBGs is used as a carrier, so that the gathered pathological B cells in rheumatoid arthritis (RA) can be removed, and the abnormal activation of the immune system can be inhibited. By promoting the apoptosis of B cells, the negative influence of B cells on bone homeostasis is reduced, and the intra-articular immune microenvironment is improved. Further, the temperature-sensitive hydrogel prepared from hydroxypropyl chitin and B3-Cu-Zn-MBGs also has a thermal response characteristic, can rapidly change into a gel state in the body, and form a solid at a bone defect site, effectively fill a complex defect area, and has strong bone regeneration potential and application value.

[0022] The application uses TNF transgenic mice (TNF-Tg) as an animal model of rheumatoid arthritis to prove in vivo that HPCH / B3-Cu-Zn-MBGs can effectively inhibit the aggregation of B cells in the damaged joint, promote local osteoblast differentiation, and significantly improve joint swelling and reduce arthritis index. By using HPCH / B3-Cu-Zn-MBGs (3:1) in a proper ratio, the aggregation of pathological B cells is effectively removed while bone formation at the damaged site is promoted, flow cytometry analysis shows that the number of MSCs in the joint increases, osteogenesis is significantly enhanced, and OCP osteoclasts are reduced, so that the bone immune microenvironment is regulated, the clinical symptoms of RA are significantly improved, and a significant therapeutic effect is exhibited, and the application has the potential to become a candidate drug for treating rheumatoid arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For characterization and performance detection of the HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel material, (A) is a scanning electron microscope (SEM) image of HPCH / B3-Cu-Zn-MBGs, which shows the microstructure of HPCH / B3-Cu-Zn-MBGs; (B) is in a liquid state at room temperature and changes into a gel state at 37 DEG C, and the temperature-sensitive change characteristic is shown; (C) is an EDX energy spectrum analysis diagram, which shows the element composition and distribution of B3-Cu-Zn-MBGs.

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

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

[0026] Figure 4 Arthritis symptoms in TNF transgenic mice after ankle injection in each treatment group: (A) is a representative photograph of the hind paw of TNF-tg mice on day 28 after administration; (B) is the arthritis index score; data are expressed as mean ± SEM (n=6), *p<0.05, **p<0.01.

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

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

[0029] The application provides a pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel targeting pathology, which is prepared by mixing hydroxypropyl chitin (HPCH) and B3-Cu-Zn-MBGs.

[0030] The hydroxypropyl chitin (HPCH) used in the application has excellent biocompatibility and controllable thermal responsiveness, that is, in a low-temperature environment in vitro, the HPCH usually exists in the form of a solution, and once the temperature reaches the body temperature (about 37 DEG C), the HPCH rapidly changes into a solid, and the HPCH exhibits significant bone induction activity through the biomimetic extracellular matrix microenvironment and dynamic phase change characteristics.

[0031] Cu induces macrophages to polarize into an anti-inflammatory phenotype, and MSCs highly express BMP-2 and collagen I under Cu stimulation, and coordinate bone formation and immune tolerance. 2+ Mesoporous bioactive nanoparticles (MBGN) have become an intelligent platform for local drug / ion delivery due to the unique nano-mesoporous structure. In the application, the MBGN is loaded with Cu, and the MBGN is endowed with the biological function of coordinating bone formation and immune tolerance.

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

[0033] The application provides a preparation method of the pathological B cell bone repair material HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel targeting pathology, and the preparation method comprises the following steps: 1) mixing and stirring hydroxypropyl chitin (HPCH) and water to obtain a first material; 2) mixing B3-Cu-Zn-MBGs and water to obtain a second material; and 3) mixing the first material and the second material to obtain a temperature-sensitive hydrogel.

[0034] In the application, the 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 more preferably 15 mg:0.75 ml, and the stirring time is preferably 2-4 h, and more preferably 3 h. In the application, the mixing and stirring temperature is preferably room temperature, and more preferably 15-25 DEG C. In the application, the stirring is magnetic stirring, and the stirring speed is preferably 300-400 rpm.

[0035] In the present application, the B3-Cu-Zn-MBGs are mixed with water to obtain the second material, and the ratio of the 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 application, the B3-Cu-Zn-MBGs are preferably prepared by the following steps: 2.1) synthesizing B3-MBGs by using a microemulsion-assisted sol-gel method; and 2.2) preparing B3-Cu-Zn-MBGs by using a modified post-dipping method.

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

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

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

[0039] Example 1

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

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

[0042] 1.2 g of cetraxate bromide was dissolved in 40 mL of an ethanol / water mixed solvent (ethanol / water volume ratio 3:1), and a micellar system was formed by magnetic stirring (600 rpm). Triethyl borate (5.82 mL) and triethyl phosphate (0.89 mL) were added dropwise in sequence, the dropwise rate was controlled at 0.5 mL / min, the pH was maintained at 9.0 (adjusted by ammonia water), and the stirring was continued for 2 hours to complete the boron-phosphorus network pre-polymerization. Metal nitrate solutions (Na + , K + , Mg 2+ , Ca 2+) for 4 h to form a homogeneous sol. The bioactive glass particles formed were collected by centrifugation (4000 rpm, 15 min) and washed with deionized water and ethanol for 3 times. The collected particles were dried overnight in air at 60 °C and then calcined in a furnace at 700 °C for 3 h with a heating rate of 2 °C / min in air to obtain spherical mesoporous B3-MBGs.

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

[0044] An ethanol / water mixture (ethanol / water volume ratio 2:1) was prepared and 0.83 g of copper nitrate (Cu(NO3)2·3H2O) and 0.49 g of zinc nitrate (Zn(NO3)2·6H2O) were added to adjust the total metal ion concentration to 1.2 M. B3-MBGs (10 g / L) were dispersed in the above impregnation solution and immersed for 12 h with ultrasonic assistance (40 kHz, 150 W) to ensure the Cu 2+ / Zn 2+ Gradient distribution in mesopores. The particles after loading were vacuum filtered and washed with 0.1 M nitric acid and deionized water to neutral. Calcination was performed in a reducing atmosphere (95% N2+5% H2) at 550 °C for 3 h with a heating rate of 2 °C / min to promote the stable embedding of Cu / Zn in the borosilicate network in the form of oxides.

[0045] Step 3: Preparation of different proportions of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel according to the mass ratio;

[0046] At room temperature, 15 mg of HPCH was added to 0.75 mL of ultrapure water and magnetically stirred for 3 h, 3 mg of B3-Cu-Zn-MBGs was added to 0.25 mL of ultrapure water, and after mixing, magnetically stirred for 15 min to prepare 1 mL (5:1) of HPCH / B3-Cu-Zn-MBGs;

[0047] 15 mg of HPCH was added to 0.75 mL of ultrapure water and magnetically stirred for 3 h, 5 mg of B3-Cu-Zn-MBGs was added to 0.25 mL of ultrapure water, and after mixing, magnetically stirred for 15 min to prepare 1 mL (3:1) of HPCH / B3-Cu-Zn-MBGs;

[0048] 15 mg of HPCH was added to 0.75 mL of ultrapure water and magnetically stirred for 3 h, 10 mg of B3-Cu-Zn-MBGs was added to 0.25 mL of ultrapure water, and after mixing, magnetically stirred for 15 min to prepare 1 mL (3:2) of HPCH / B3-Cu-Zn-MBGs;

[0049] 15 mg HPCH was added into 0.75 mL ultrapure water and stirred magnetically for 3 h, 15 mg B3-Cu-Zn-MBGs was added into 0.25 mL ultrapure water, mixed and stirred magnetically for 15 min, configured into 1 mL (1:1) HPCH / B3-Cu-Zn-MBGs;

[0050] 2. Material characterization and performance detection

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

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

[0053] Example 2

[0054] 1. B cell and MPC cell extraction and cell culture

[0055] B cells were isolated from mouse spleen using B cell isolation kit (#130-121-031, Miltenyi Biotec) according to the manufacturer's instructions. Purified B cells (5 x 10 5 cells / well in 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] The tibia and femur of 3-week-old wild-type (WT) mice were stripped and cut into bone pieces with scissors, and the bone pieces were cultured in a culture dish for 3 days. The bone pieces were transferred to a clean culture dish as the first generation, and the cells were cultured for 7 days to grow to confluence. MPCs from the second generation were used subsequently.

[0057] All mice were bred and maintained in the SPF level 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 different concentrations (5:1, 3:1, 3:2, 1:1) of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel were used for B cell and MPCs co-culture and B cell and MSCs co-culture.

[0059] Blank group, HPCH, 5:1 of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel (indicated as 5:1 in the figure), 3:1 of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel (indicated as 3:1 in the figure), 3:2 of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel (indicated as 3:2 in the figure), and 1:1 of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel (indicated as 1:1 in the figure) were set for MPCs.

[0060] The above treatment groups were also set for B cells.

[0061] 2. CCK-8 cell viability assay

[0062] According to the kit instructions, B cells and MPCs were inoculated in a 96-well plate at a certain number, and according to the cell proliferation, at the predetermined time point, 100 μL of fresh culture medium and 10 μL of CCK-8 reagent (#K1018, Apex bio) were used instead of 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, and the results are shown in Figure 2 As shown in the figure, the killing effect of HPCH / B3-Cu-Zn-MBGs temperature-sensitive hydrogel on B cells gradually increased with time and concentration, while the effect on MPCs was not significant.

[0063] 3. Cell ALP staining

[0064] For MPCs treated with temperature-sensitive hydrogel and B cell-MPCs co-cultured MPCs, 3 days of co-culture in osteoblast induction medium. ALP staining was performed using BCIP / NBT alkaline phosphatase color reagent kit (#C3206, Beyotime), and the results of staining analysis are shown in Figure 3 As shown in the figure, compared with the control group, the expression of osteogenic differentiation marker ALP of MPCs in the temperature-sensitive hydrogel treatment group was significantly increased, and in the B cell-MPCs 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 co-culture system was intervened by adding temperature-sensitive hydrogel, the B cell-mediated osteogenic inhibition effect 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 with the 3' end of the human a-globin gene has been successfully bred in the laboratory. From 2 months of age, TNF-Tg transgenic mice develop arthritis in the ankle joint and progress with age, with systemic bone loss and osteoporosis at 4 months of age. All mice were housed and maintained at the SPF level experimental animal center of Nanjing Medical University, and the use of animals in this study has been approved by the Animal Ethics Committee of Nanjing Medical University (approval number: 1906018).

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

[0069] 2. Clinical evaluation of arthritis

[0070] Arthritis index is two important indicators to judge the effect of drugs against RA, reflecting the inflammatory response occurring in the joint. The test was observed every 7 days on the 0th, 7th, 14th, 21st, and 28th days for joint swelling and deformity. The severity of arthritis was measured by 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 toe mild swelling; 2, 3 and 4 points represent mild, moderate and severe swelling of the toes and ankles, respectively. As shown in Figure 4 , the treatment group significantly improved joint swelling and reduced the arthritis index.

[0071] 3. Imaging, histomorphological evaluation

[0072] All mice were subjected to tissue sampling after 28 days of drug administration, and the ankle joint was collected and fixed in 4% paraformaldehyde. The ankle joint was subjected to μ-CT scanning and three-dimensional reconstruction to observe the changes in bone mass, and the results are shown in Figure 5 .

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

[0074] After 28 days of drug intervention, the ankle joint tissue of mice in each group was taken for single cell analysis. The specific process is as follows: the tissue sample was prepared into a single cell suspension by enzyme digestion combined with mechanical grinding, and the cell concentration was adjusted to 1 × 10 6cells / 100 μL, combined with multi-color fluorescent labeled antibodies, and incubated at 4℃ for 30 minutes. After washing with PBS (containing 2% FBS), the proportions of B cell, mesenchymal progenitor cell (MPC) and osteoclast precursor (OCP) subpopulations were detected by flow cytometry. The results are shown in Figure 6 As shown in the results, after local injection intervention, the number of B cells in the joint of the experimental group was significantly reduced compared with the control group; the proportion of MPC population was significantly increased; and the proportion of OCP population showed a downward trend. The results suggest that the temperature-sensitive hydrogel can significantly down-regulate the local immune microenvironment by adjusting the B cell aggregation, HPCH / B3-Cu-Zn-MBGs promotes the differentiation of osteoblasts, enhances the ability of bone regeneration, and improves the balance of cytokines in the bone immune system, further reducing the immune damage of RA.

[0075] From the above examples, it can be seen that the HPCH / B3-Cu-Zn-MBGs provided by the present application can adjust the local bone immune microenvironment through its temperature-sensitive properties and pharmacological activity, reduce the aggregation of B cells, promote the differentiation of osteoblasts, enhance the ability of bone regeneration, and improve the balance of cytokines in the bone immune system, further reducing the immune damage of RA.

[0076] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A thermosensitive hydrogel, HPCH / B3-Cu-Zn-MBGs, used for targeted pathological B-cell bone repair, characterized in that... It was prepared by mixing hydroxypropyl chitosan (HPCH) and B3-Cu-Zn-MBGs; the mass ratio of hydroxypropyl chitosan (HPCH) to B3-Cu-Zn-MBGs was 5:1 to 1:

1. The B3-Cu-Zn-MBGs were prepared by the following steps: 1) B3-MBGs were synthesized by microemulsion-assisted sol-gel method; 2) B3-Cu-Zn-MBGs were prepared using a modified post-impregnation method, which enabled Cu / Zn to be stably embedded in the borosilicate network in oxide form.

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

3. The preparation method according to claim 2, characterized in that, The stirring time in step 1) is 2-4 hours.

4. The preparation method according to claim 3, characterized in that, In step 1), the ratio of hydroxypropyl chitosan (HPCH) to water is 15 mg: (0.5~1.0) mL.

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

6. The application of the HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel, a targeted pathological B-cell bone repair material according to claim 1, and the HPCH / B3-Cu-Zn-MBGs thermosensitive hydrogel prepared by the preparation method according to any one of claims 2 to 5, in the preparation of a drug for treating rheumatoid arthritis.

7. The application according to claim 6, characterized in that, The drug is in the form of an injectable hydrogel.

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