Use of an injectable hydrogel in the preparation of a medicament for treating early ONFH based on regulating VAO

Abstract

The application provides application of an injectable hydrogel in preparation of a drug for treating early ONFH based on regulation of VAO, and specifically includes application in a drug for inhibiting differentiation and fusion of VAO into BAO, application in a drug for resisting bone resorption of BAO, application in a drug for promoting or reserving VAO, application in a drug for mediating VAO to promote bone formation, application in a drug for mediating VAO to promote angiogenesis, and application in a drug for mediating VAO to promote H-type angiogenesis and coupling bone formation to treat early ONFH. The injectable hydrogel is a SA / HA / GDL injectable hydrogel loaded with a GO-PEI-miR7b overexpression gene vector, has suitable gelation time, certain mechanical properties and gene vector slow-release function, can specifically inhibit generation of BAO and bone resorption, protect and increase the number of VAO, and then promote bone-specific H-type angiogenesis and coupling bone formation, and has excellent effect in regeneration and repair treatment of early ONFH. The application can provide theoretical support for development of a new drug for treating early ONFH.

Description

TECHNICAL FIELD

[0001] The application relates to the medical technical field, in particular to application of an injectable hydrogel in preparation of a drug for treating early ONFH based on VAO regulation. BACKGROUND

[0002] Osteonecrosis of the femoral head (ONFH) is also known as avascular necrosis of the femoral head. ONFH is caused by blood supply damage or interruption, which leads to partial death of bone cells and bone marrow components, bone tissue necrosis and subsequent repair, and changes in the structure of the femoral head and even collapse, resulting in hip pain and dysfunction. ONFH is prone to occur in young adults, and the disease develops rapidly and has a high disability rate. If not treated effectively, about 94% of patients will have femoral head collapse within 5 years, and will lose hip function. Due to the increasing incidence of ONFH in recent years, it is gradually becoming a serious global health problem, and therefore is attracting more and more attention and attention from the medical community.

[0003] Among various early ONFH hip joint preservation treatment methods, femoral head core decompression combined with various bone repair biomaterial filling and / or structural bone support can effectively enhance bone formation, promote angiogenesis, reduce the risk of proximal femoral fracture, improve the efficacy of ONFH, and is recommended as a conventional treatment method for early ONFH. However, studies have found that even in the case of implanting a biomaterial to provide support, the destruction of the bone structure after core decompression, the reduction of macroscopic mechanical properties and the subsequent collapse are considered to be caused by the destruction of the microenvironment in the femoral head during the repair process. Therefore, through artificial intervention to delay or change the process of ONFH collapse, a balanced osteoblast, osteoclast and blood vessel related cell system is reconstructed, and sufficient mechanical support is provided, which has become a hot spot in the research of biomaterials for early ONFH repair in recent years.

[0004] In early ONFH, the microstructure of the femoral head has no obvious change, the shape of the femoral head remains round, and there is only a small amount of bone resorption inside the femoral head. However, the changes in osteoblast (OB) and osteoclast (OC) activity are similar to those in advanced ONFH, the number of OC per unit area in the necrotic area is significantly increased, and the OC activity is higher than the OB activity, showing a destructive repair process. In addition, the OC activity in the necrotic area is significantly increased, while the OB activity in the sclerotic area is significantly increased, and due to the excessive activity of bone resorption, microcavities appear in the bone remodeling process in the early stage of ONFH, which also leads to the change of the mechanical properties and structure of the trabecular bone of the femoral head. Therefore, in addition to providing sufficient mechanical support, it is crucial in the early treatment of ONFH to delay or change the process of femoral head necrosis and collapse by artificial intervention, to rebuild the bone metabolic balance system of OB and OC action, to promote new angiogenesis and ingrowth, and to maintain the stability and functional state of the bone microenvironment.

[0005] The ability of the human skeletal system to precisely regulate various cells to maintain the stability and functional state of the bone microenvironment depends on the interaction between OB, OC, endothelial cells (EC) and endothelial progenitor cells (EPC) and other cells in the bone microenvironment. In recent years, a number of breakthrough studies have pointed out that the maintenance of the stability and functional state of the bone microenvironment is not an isolated process, but the result of the coupling of bone resorption-bone formation mediated by OC and OB and angiogenesis mediated by EC and EPC. HIF-1α is a key molecule associated with angiogenesis and bone formation, and HIF-1α can continuously increase neovascularization to induce more precursor OB generation, and the mature precursor OB can form a large number of independent bone formation units. Activation of Notch signal in endothelial cells will increase H-type endothelial cells and promote bone growth. PreOC is a key cell for regulating H-type angiogenesis and coupling angiogenesis and bone formation, and preOC secretes PDGF-BB to induce H-type angiogenesis, and then H-type vessels promote bone formation by secreting S1P. The applicant's previous research results also found that preOC can promote the proliferation, migration, differentiation and lumen formation of vascular endothelial progenitor cells by secreting VEGFa. Recent research has established a new non-absorbing osteoclast subpopulation - vessel-associated osteoclast (VAO). Unlike bone-associated osteoclasts (BAO) located on the surface of trabecular bone (distal end) with larger volume (60-100 μm) and multinucleated (4-6 cell nuclei), VAO is distributed at the bone / cartilage interface (proximal end) with relatively small volume (15-20 μm) and 1-2 cell nuclei. Further studies have confirmed that VAO specifically co-expresses with H-type capillaries, and VAO can regulate the growth of H-type vessels in bone, thereby coupling bone formation. In view of the similarity of the anatomical structure of the femoral head, the applicant's previous research on human femoral head necrosis specimens found that VAO also exists at the bone / cartilage interface of the femoral head, and BAO exists on the surface of trabecular bone. The discovery of the above new mechanisms in the bone microenvironment suggests that VAO and BAO can be used as targets for biomaterial research, providing a new idea for the treatment of early ONFH.

[0006] Hydrogel is a polymer network that is insoluble in water, can swell in water and hold a large amount of water without being dissolved, has good tissue compatibility, not only allows nutrient transport and metabolic waste diffusion and discharge, but also provides biological signals for cells. The hydrogel network filled with a large amount of water has a fluid-like property, which is very similar to the extracellular matrix of the body tissue, enhances its affinity with the tissue, reduces the stimulation of the implanted material to the surrounding normal tissue, has good biological safety and tissue compatibility, and can be applied to tissue repair and regeneration as a large class of materials. In addition, drugs, biologically active particles and composite growth factors can be easily added to the hydrogel to prepare a scaffold material with special functions.

[0007] Hydrogel materials can be divided into preformed hydrogels and injectable hydrogels according to their preparation principles. The preformed hydrogel is generally a gel-forming process that is relatively fast (such as sodium alginate beads formed by cross-linking with CaCl2 solution) or requires special external stimuli such as light cross-linking, resulting in the whole system not having injectability. Although the preformed hydrogel has good biocompatibility, it also needs surgical implantation and is difficult to meet the irregular defect shape of the pre-repaired tissue or organ, and also difficult to avoid surgical trauma and possible complications at the implant site, causing pain to the patient. The injectable hydrogel refers to a sol-gel precursor sol that can be injected through a syringe needle to a designated site before gelation occurs, and the sol-gel phase transition occurs in a short time to form a non-flowable hydrogel, avoiding traumatic surgery, and also meeting the complex shape of different wounds, especially suitable for minimally invasive repair, and is one of the leading directions of the development of tissue engineering scaffolds.

[0008] Therefore, in view of the good biological safety and tissue compatibility of hydrogel, the injectable hydrogel is used as a carrier to load drugs, seed cells, active molecules and gene carriers, etc., and the VAO is regulated by using the slow-release method to mediate bone-specific angiogenesis, thereby promoting bone formation, inhibiting BAO and bone resorption, rebuilding the balanced osteoblast, osteoclast and blood vessel-related cell action system, promoting angiogenesis and ingrowth, maintaining the stability of the bone microenvironment and the functional state, or providing a new idea for minimally invasive surgical treatment of early ONFH. According to the data retrieval conducted by the applicant, there is no related report on the use of injectable hydrogel based on the regulation of VAO for minimally invasive treatment of early ONFH. SUMMARY

[0009] Therefore, in order to overcome the deficiencies of the prior art, the present application provides an application of an injectable hydrogel in the preparation of a drug for treating early ONFH based on the regulation of VAO. The injectable hydrogel of the present application has a suitable gelation time, certain mechanical properties and gene carrier slow-release function, promotes H-type angiogenesis mediated by VAO and couples bone formation, and has excellent effect in minimally invasive treatment of early ONFH.

[0010] The injectable hydrogel provided by the present application is used in the preparation of a drug for treating early ONFH based on regulating VAO, and the injectable hydrogel is a SA / HA / GDL injectable hydrogel loaded with a GO-PEI-miR7b overexpression gene vector.

[0011] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for inhibiting the differentiation and fusion of VAO into BAO.

[0012] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for resisting the bone resorption ability of BAO.

[0013] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for promoting or retaining VAO.

[0014] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for mediating VAO to promote bone formation.

[0015] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for mediating VAO to promote angiogenesis.

[0016] The present application also provides the use of the above-mentioned injectable hydrogel in the preparation of a drug for mediating VAO to promote H-type angiogenesis and coupling bone formation to treat early ONFH.

[0017] Further, the preparation method of the injectable hydrogel comprises the following steps:

[0018] A. preparing a GO-PEI complex;

[0019] B. mixing the GO-PEI complex with a miR7b plasmid, incubating at room temperature to obtain a GO-PEI-miR7b overexpression gene vector;

[0020] C. adding HA powder to a SA aqueous solution, and after ultrasonic treatment, magnetically stirring to obtain a SA / HA mixed solution; adding the GO-PEI-miR7b overexpression gene vector loaded with the miR7b plasmid into the SA / HA mixed solution, and magnetically stirring to obtain a SA / HA / GO-PEI-miR7b mixed solution;

[0021] D. mixing the SA / HA / GO-PEI-miR7b mixed solution and a GDL aqueous solution, and then injecting into a mold, and after gelation at room temperature, demolding to obtain a SA / HA / GDL injectable hydrogel loaded with a GO-PEI-miR7b overexpression gene vector.

[0022] Further, the step A. slowly add the PEI aqueous solution into the GO aqueous solution within 10 minutes, after ultrasonic treatment for 10 min, stir the mixture overnight, centrifugal wash 3-5 times with deionized water, and then re-disperse in deionized water to obtain the GO-PEI composite; the concentration of the GO aqueous solution is 0.1 mg / mL, the concentration of the PEI aqueous solution is 1 mg / mL, the molecular weight of the PEI is 10 kDa, the mass ratio of the GO aqueous solution to the PEI aqueous solution is 1:10, the centrifugal speed is 8000 rpm, and the volume of the deionized water used for the re-dispersion is 1 mL; measure the absorbance value of the GO-PEI composite aqueous solution at 230 nm, and then calculate the concentration of the GO-PEI composite by conversion through the extinction coefficient, and adjust the concentration of the GO-PEI composite to 0.5 mg / mL;

[0023] The GO-PEI is determined to be linked by zeta potential measurement and particle size detection.

[0024] Further, the extinction coefficient of the GO aqueous solution is 39 mg / ml / cm.

[0025] Further, the step B. mix the GO-PEI composite with the miR7b plasmid at an N / P ratio of 80, and incubate at room temperature for 1 h to obtain a GO-PEI-miR7b overexpression gene vector.

[0026] The GO-PEI-miR7b overexpression gene vector is added into RAW264.7 cells containing complete culture medium, and co-incubated for 48 h, and then fluorescence photographing and qRT-PCR detection are performed to determine that the miR7b has been expressed in the cells.

[0027] Further, the step C. add the HA with a mass percentage concentration of 3% into the SA aqueous solution with a mass percentage concentration of 2.8%, and then perform ultrasonic treatment at 40 kHz and 200 w for 20 min, and then perform magnetic stirring for 6 h to obtain a SA / HA mixed solution; the GO-PEI-miR7b gene vector loaded with the miR7b plasmid is added into the SA / HA mixed solution, and then magnetic stirring is performed for 1 h to obtain a SA / HA / GO-PEI-miR7b mixed solution, and then the SA / HA / GO-PEI-miR7b mixed solution is placed in an ice water bath at 4 ℃.

[0028] Further, the GO-PEI-miR7b gene vector loaded with 2 μg of the miR7b plasmid is contained in every 200 μl of the SA / HA mixed solution.

[0029] Further, the step D. configure the GDL aqueous solution with a mass percentage of 10% and place in an ice water bath at 4 ℃; and then mix the SA / HA / GO-PEI-miR7b mixed solution and the GDL aqueous solution according to a volume ratio of 5:1 to perform gelation.

[0030] The application has the following beneficial effects:

[0031] The SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector constructed by the application is suitable for early ONFH minimally invasive surgical treatment, and can not only avoid traumatic surgery, but also meet the complex shape of different individual necrotic femoral heads; the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector constructed by the application can control the gelation time, water content and equilibrium swelling rate, mechanical properties, degradation performance and GO-PEI-miR7b overexpression gene vector release performance by adjusting the SA / HA and HA / GDL ratios in the hydrogel composition, so as to realize effective control of the ONFH repair process; the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector constructed by the application has a honeycomb structure, good biodegradability, biosafety and tissue compatibility, and is suitable for cell adhesion growth and proliferation; the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector constructed by the application has no toxic effect in vivo and in vitro, can significantly inhibit VAO differentiation and fusion into BAO, and inhibit the generation of BAO and bone resorption; the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector constructed by the application can significantly protect and increase the number of VAOs in vivo and in vitro, and then promote bone-specific H angiogenesis and coupling bone formation, and has excellent effect in early ONFH regeneration and repair treatment. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Preparation of GO-PEI complex and GO-PEI-miR7b overexpression gene vector and screening of optimal parameter conditions

[0033] A, zeta potential analysis of GO-PEI complex; B, particle size analysis of GO-PEI complex; C, gel retardation electrophoresis of GO-PEI-miR7b overexpression gene vector; D, fluorescence photograph after GO-PEI-miR7b overexpression gene vector treated cells (bar = 100 μm); E, real-time fluorescence quantitative PCR after GO-PEI-miR7b overexpression gene vector treated cells;

[0034] Figure 2 Component and structure analysis of the injectable hydrogel of the application

[0035] A, Fourier transform infrared spectroscopy (FITR) detection; B, scanning electron microscope (SEM) observation;

[0036] Figure 3Gelation and gelation time of SA / HA / GDL hydrogel with different proportions

[0037] A&B, gelation; C, gelation time;

[0038] Figure 4 Water content and swelling properties of SA / HA / GDL hydrogel with different proportions

[0039] A, water content; B, swelling properties;

[0040] Figure 5 Degradation and DNA release properties of SA / HA / GDL hydrogel with different proportions

[0041] A, degradation; B, DNA release properties;

[0042] Figure 6 Mechanical properties of SA / HA / GDL hydrogel with different proportions

[0043] A, universal testing machine-stress / strain diagram; B, universal testing machine-compressive modulus; C, rheometer-elastic modulus G' / viscosity modulus G";

[0044] Figure 7 Transfection effect and expression of GO-PEI-miR7b overexpression gene vector released by SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on RAW264.7 cells

[0045] A, transmission electron microscopy (TEM) observation (120 hours) (bar = 1 μm); B, fluorescence microscope imaging observation (120 hours) (bar = 100 μm); C, qRT-PCR detection of mRNA expression of miR7b (72, 168 hours); *P < 0.05, **P < 0.01;

[0046] Figure 8 Effect of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on VAO and BAO osteoclast-related functions

[0047] A, CCK-8 cell proliferation detection (24, 72, 120, 168 hours); B, qRT-PCR detection of mRNA expression of osteoclast differentiation specific genes c-fos, RANK, NFATc1, DC-STAMP (168 hours); C, qRT-PCR detection of mRNA expression of VAO and BAO specific genes Ctsk, TNF-α, IL-6; D, bone resorption pit detection and percentage of osteoclast area analysis (168 hours) (bar = 100 μm); *P <0.05, **P <0.01;

[0048] Figure 9 Effect of GO-PEI-miR7b overexpression gene vector loaded SA / HA / GDL injectable hydrogel on VAO and BAO osteogenic differentiation in vitro

[0049] A, ALP activity detection (168 hours); B, qRT-PCR detection of mRNA expression of osteoblast differentiation specific genes ALP, Runx2, COL1a1 (168 hours); C, ALP staining (168 hours) (bar = 50 μm); *P <0.05, **P <0.01;

[0050] Figure 10 Effect of GO-PEI-miR7b overexpression gene vector loaded SA / HA / GDL injectable hydrogel on VAO and BAO angiogenesis in vitro

[0051] A, Transwell migration and tube formation detection (168 hours) (bar = 100 μm); B, statistical analysis of the number of migrated cells per well, main links and total length of tube; C, qRT-PCR detection of mRNA expression of angiogenesis specific genes VEGFR2, CD31, vWF (168 hours); *P <0.05, **P <0.01;

[0052] Figure 11 Evaluation of the repair effect of GO-PEI-miR7b overexpression gene vector loaded SA / HA / GDL injectable hydrogel on early ONFH in rats

[0053] A, X-ray, μCT, μCT three-dimensional reconstruction imaging, μCT angiography reconstruction imaging and H&E staining results of femoral head (bar = 100 μm); B, μCT result analysis, bone mineral density (BMD), trabecular bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th); C, μCT angiography result analysis, blood vessel volume and blood vessel surface area; *P <0.05, **P <0.01;

[0054] Figure 12 Effect of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on H-type angiogenesis in early ONFH rat femoral head

[0055] A, H-type vessel Emcn and CD31 immunofluorescence double-labeling staining in different regions of femoral head (bar = 100 μm); B, Emcn cell number per unit length hi CD31 hi Positive cell number; *P < 0.05, **P < 0.01;

[0056] Figure 13 Effect of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on VAO and BAO in early ONFH rat femoral head

[0057] A, osteoclast subtype Emcn and VPP3 immunofluorescence double-labeling staining in different regions of femoral head (bar = 100 μm); B, VAO and BAO cell number per unit length; *P < 0.05, **P < 0.01; DETAILED DESCRIPTION

[0058] The present application will be described in detail below by specific embodiments, it is understood that the following examples are only as explanation and illustration, not in any form limit the scope of the present application. In the following examples, the biochemical reagents not specifically described are conventional reagents in the art, which can be prepared according to conventional methods in the art or commercially available, specifications for laboratory grade.

[0059] Example 1, GO-PEI-miR7b gene vector construction and verification

[0060] (1) To prepare GO-PEI complex, 10 kDa PEI solution (1 mg / mL) was slowly added to GO solution (0.1 mg / mL) within 10 min, after ultrasonic treatment for about 10 min, the mixture was stirred overnight, washed with deionized water (DI) by centrifugation 3-5 times, centrifugal speed 8000 rpm, and then re-dispersed in deionized water. The absorbance value of GO-PEI complex aqueous solution at 230 nm was measured, and the GO-PEI complex concentration was calculated by the extinction coefficient (the extinction coefficient of GO aqueous solution used was 39 mg / mL / cm). The GO-PEI complex concentration was adjusted to 0.5 mg / mL. In this experiment, a series of GO-PEI complexes were obtained by mixing GO: PEI solutions with weight ratios of 1:1, 1:2, 1:5, 1:10, 1:20 and 1:40. And the zeta potential measurement and particle size detection were used to determine the GO-PEI linkage. (SeeFigure 1 A, 2B)

[0061] (2) Appropriate amounts of GO-PEI were mixed with miR7b plasmid at different N / P ratios of 0, 20, 40, 80, 100, 150, 200, 300, and 400, and gel retardation electrophoresis was performed to observe the miR7b plasmid linkage. GO-PEI ratios of 1:5 and 1:10 were selected for subsequent validation. (See...) Figure 1 C)

[0062] (3) Mix appropriate amounts of GO-PEI with miR7b plasmid at different N / P ratios of 80 and 300 (using PEI alone mixed with miR7b plasmid at the N / P ratio as a control), incubate at room temperature for 1 h, then add to RAW264.7 cells containing complete culture medium, incubate for a total of 48 h, and perform fluorescence imaging and qRT-PCR to detect the expression of miR7b in the cells. (See...) Figure 1 D)

[0063] Through the above screening, the optimal construction conditions for the GO-PEI-miR7b overexpression gene vector of the present invention were obtained: GO:PEI = 1:10, N / P = 80.

[0064] Example 2: Construction and characterization of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector

[0065] (1) Construction of GO-PEI-miR7b hydrogel

[0066] Construct hydrogels with different SA / HA / GDL ratios loaded with GO-PEI-miR7b overexpression gene vectors:

[0067] ①HA fixation and GDL fixation: 3% HA powder was added to completely dissolved 2%, 2.5%, 2.8%, 3%, and 3.5% SA solutions, respectively. The mixture was ultrasonicated (40kHz, 200w) for 20 min and then magnetically stirred for 6 h to obtain an SA / HA mixture. A GO-PEI-miR7b gene vector loaded with miR7b plasmid was constructed according to GO:PEI = 1:10 and N / P = 80. The vector was added to the SA / HA mixture (each 200 μl of SA / HA mixture contained 2 μg of miR7b plasmid loaded in the GO-PEI-miR7b gene vector). The mixture was magnetically stirred for 1 h to obtain an SA / HA / GO-PEI-miR7b mixture, which was then placed in an ice-water bath at 4℃. A 10% GDL solution was prepared, fully dissolved, and placed in an ice-water bath at 4℃. The SA / HA / GO-PEI-miR7b mixture and the 10% GDL solution were mixed at a volume ratio of 5:1 and then gelled.

[0068] ② HA fixation, SA fixation: add 3% HA powder to the completely dissolved 2.8% SA solution, ultrasonic (40 kHz, 200 w) treatment for 20 min, then magnetic stirring for 6 h to obtain the SA / HA mixed solution; according to GO: PEI = 1: 10, N / P = 80, construct the GO-PEI-miR7b gene carrier loaded with 2 μg of miR7b plasmid, add the SA / HA mixed solution (each 200 μl of the SA / HA mixed solution contains the GO-PEI-miR7b gene carrier loaded with 2 μg of miR7b plasmid), magnetic stirring for 1 h to obtain the SA / HA / GO-PEI-miR7b mixed solution, then place in the 4℃ ice water bath; respectively configure 6%, 8%, 10%, 12%, 14% GDL solution, after fully dissolving, place in the 4℃ ice water bath; mix the SA / HA / GO-PEI-miR7b mixed solution and the GDL solution with different concentrations according to the volume ratio of 5:1, then carry out gelation.

[0069] Among them, the total volume of 480 μl is injected into a 5 mL centrifuge tube for gelation time detection, the total volume of 600 μl is injected into a 5 mL glass bottle for gelation observation, the total volume of 600 μl is injected into a polytetrafluoroethylene mold with an inner diameter of 1 cm and a height of 1 cm for 24 hours demolding for gelation observation and compression performance detection, the total volume of 240 μl is injected into a polytetrafluoroethylene mold with an inner diameter of 0.6 cm and a height of 1 cm for 24 hours demolding for FTIR, SEM, water content, swelling performance and DNA release performance detection, and the total volume of 480 μl is injected into a polytetrafluoroethylene mold with an inner diameter of 0.6 cm and a height of 1 cm for 24 hours demolding for degradation performance detection.

[0070] (2) GO-PEI-miR7b loaded hydrogel structure characterization (see Figure 2 )

[0071] The SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b is successfully constructed, and the internal structure is a porous honeycomb structure.

[0072] (3) GO-PEI-miR7b loaded hydrogel performance characterization

[0073] ① The gelation time is shortened with the increase of the concentration of GDL or SA. (see Figure 3 )

[0074] ② The water content is reduced with the increase of the concentration of GDL or SA; the swelling rate is reduced with the increase of the concentration of GDL or SA. (see Figure 4 )

[0075] ③ The degradation performance is reduced with the increase of the concentration of GDL or SA; the DNA release rate is reduced with the increase of the concentration of GDL or SA. (see Figure 5 )

[0076] IV. The compressive strength increases with the increase of GDL or SA concentration, the compressive modulus increases with the increase of GDL or SA concentration; the elastic modulus increases with the increase of GDL or SA concentration, and the viscous modulus has no significant change. (See Figure 6 )

[0077] The optimal construction condition of the hydrogel of the application obtained by the above screening for subsequent cell and zoology experiments is 2.8% SA-3% HA-10% GDL.

[0078] Example 3, application of the injectable hydrogel of the application in preparation of a medicament for treating early ONFH based on regulating VAO

[0079] Application of the injectable hydrogel of the application in preparation of a medicament for inhibiting differentiation and fusion of VAO into BAO. Figure 8 B)

[0080] Application of the injectable hydrogel of the application in preparation of a medicament for resisting bone resorption ability of BAO. Figure 8 D)

[0081] Application of the injectable hydrogel of the application in preparation of a medicament for promoting or retaining VAO. Figure 8 C)

[0082] Application of the injectable hydrogel of the application in preparation of a medicament for mediating VAO to promote bone formation. Figure 9 )

[0083] Application of the injectable hydrogel of the application in preparation of a medicament for mediating VAO to promote angiogenesis. Figure 10 )

[0084] Application of the injectable hydrogel of the application in preparation of a medicament for mediating VAO to promote H-type angiogenesis and coupling bone formation to treat early ONFH. Figure 11 、 13 )

[0085] Test Example 1: cell biology evaluation of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector for early ONFH regeneration and repair

[0086] 1. Constructing of GO-PEI-miR7b hydrogel: 3% HA powder was added into the completely dissolved 2.8% SA solution, and then the mixture was treated by ultrasonic (40 kHz, 200 w) for 20 min and stirred by magnetic force for 6 h to obtain SA / HA mixture. GO-PEI-miR7b gene vector loaded with miR7b plasmid was constructed according to GO: PEI = 1: 10 and N / P = 80, and then was added into the SA / HA mixture (2 μg or 4 ug of miR7b plasmid was loaded into 200 μl of SA / HA mixture, in which 2 μg was used for 24-well plate related experiment and 4 ug was used for 6-well plate related experiment), and stirred by magnetic force for 1 h to obtain SA / HA / GO-PEI-miR7b mixture, which was then placed in 4 ℃ ice water bath. 10% GDL solution was prepared and dissolved completely, and then was placed in 4 ℃ ice water bath. The SA / HA / GO-PEI-miR7b mixture and 10% GDL solution were injected into polytetrafluoroethylene mold with inner diameter of 0.6 cm and height of 1 cm according to the volume ratio of 5: 1 and total volume of 240 μl (GO-PEI-miR7b negative control gene vector was used as negative control, and simple SA / HA / GDL injectable hydrogel without gene vector was used as blank control), and then was demolded after 24 h for cell biology evaluation.

[0087] 2. Verification of the effect and expression of GO-PEI-miR7b overexpression gene vector transfected RAW264.7 cells released by SA / HA / GDL

[0088] (1) Cell grouping and treatment

[0089] ① Cell grouping: RAW264.7 cells, RAW264.7 cells + GO-PEI-miR7b overexpression gene vector loaded SA / HA / GDL hydrogel.

[0090] ② Cell treatment: RAW264.7 cells were inoculated in 24-well plate at 1x10 4 6 cells / ml, and the culture medium was discarded the next day. The culture medium was composed of DMEM high glucose culture medium + 10% fetal bovine serum + 1% double antibiotic (penicillin-streptomycin). GO-PEI-miR7b overexpression gene vector loaded with 2 ug of miR7b plasmid was added into each well containing fresh culture medium, and the well without hydrogel material was used as control. The culture medium was replaced every 48 h, and related detection was performed after 24, 72, 120 and 168 h of culture observation.

[0091] (2) Transmission electron microscope: At the observation point 120 hours, the culture medium and hydrogel were discarded, and the cells were scraped and washed with PBS twice (800 rpm) and centrifuged, fixed with glutaraldehyde, and the GO-PEI-miR7b into the RAW264.7 cells was observed by transmission electron microscope.

[0092] (3) Fluorescence imaging: At the observation point 120 hours, the culture medium and hydrogel were discarded, and the expression of GO-PEI-miR7b overexpression gene vector in RAW264.7 cells was observed by inverted fluorescence microscope.

[0093] (4) qRT-PCR detection of miR7b expression: At the observation points 72 and 168 hours, the culture medium and hydrogel were discarded, the cells were lysed with Trizol, the total RNA in the cells was extracted, and the target gene miR7b was quantitatively detected by real-time quantitative RT-PCR.

[0094] Conclusion: The GO-PEI-miR7b overexpression gene vector released by the SA / HA / GDL hydrogel can enter the cytoplasm of RAW264.7 cells, the black layered fibers ( Figure 7 A), and express the green fluorescent protein carried by the gene vector in the cells ( Figure 7 B), and qRT-PCR detection further confirms that the gene vector can enter RAW264.7 cells and highly express miR-7b gene ( Figure 7 C).

[0095] 3, The effect of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on VAO and BAO osteoclast-related functions

[0096] (1) Cell grouping and treatment:

[0097] ① Cell grouping: RAW264.7, RAW264.7+osteoclast induction, RAW264.7+osteoclast induction+simple SA / HA / GDL hydrogel, RAW264.7+osteoclast induction+SA / HA / GDL hydrogel loaded with negative control gene vector, RAW264.7+osteoclast induction+SA / HA / GDL hydrogel loaded with GO-PEI-miR7b overexpression gene vector.

[0098] ② Cell treatment: RAW264.7 cells were treated with 1x10 4RAW264.7 cells were seeded into 24-well plates, and the culture medium was discarded the next day. The culture medium consisted of DMEM high-glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin. RANKL (final concentration 100 ng / mL) and M-CSF (final concentration 50 ng / mL) were used to induce osteoclast differentiation. Simultaneously, SA / HA / GDL hydrogels loaded with the GO-PEI-miR7b overexpression gene vector containing 2 μg miR7b plasmid were added to each well containing fresh culture medium. The GO-PEI-miR7b negative control gene vector served as a negative control, and the unloaded SA / HA / GDL injectable hydrogel served as a blank control. The culture medium was changed every 48 hours. After 24, 72, 120, and 168 hours of culture, relevant assays were performed. For the bone resorption pit assay, 0.5 mm calf bone fragments were pre-coated into the 24-well culture plates.

[0099] (2) Cell proliferation activity assay: At observation points of 24, 72, 120, and 168 hours, the culture medium and hydrogel were discarded, washed three times with PBS, and 500 μl of freshly prepared culture medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 2 hours. After 2 hours, the culture medium to be tested was aspirated into a 96-well plate, and the absorbance value at 450 nm was measured immediately.

[0100] (3) Detection of bone fragment absorption depressions: After 168 hours at the observation point, discard the culture medium and hydrogel, and wash three times with PBS. Bleach with 10% sodium hypochlorite (bleach) at room temperature for 10 min. Wash three times with double-distilled water, 5 min each time. Let dry at room temperature (3-5 hours). Stain with toluidine blue solution for 3 min. Rinse 3-5 times with double-distilled water, 5 min each time. Add 200 μl of double-distilled water or PBS to each well, observe and photograph under an inverted optical microscope.

[0101] (4) qRT-PCR detection of osteoclast differentiation-specific genes and VAO and BAO-specific gene expression: At the observation point, after 168h, the culture medium and hydrogel were discarded, cells were lysed with Trizol, total RNA was extracted from the cells, and real-time quantitative RT-PCR was used to quantitatively detect the target genes c-fos, RANK, NFATc1, DC-STAMP, Ctsk, TNF-α and IL-6.

[0102] Conclusion: During the differentiation of RAW264.7 cells into mature osteoclasts (BAO) induced by RANKL and M-CSF, the SA / HA / GDL hydrogel loaded with the GO-PEI-miR7b overexpression gene vector had no effect on cell proliferation activity. Figure 8 A), but significantly inhibited the expression of the osteoclast fusion gene DC-STAMP ( Figure 8B) c-fos, NFATc1, RANK expression related to osteoclast differentiation Figure 8 B), and further inhibit osteoclast bone resorption activity Figure 8 D). In addition, the GO-PEI-miR7b overexpression gene vector released by the SA / HA / GDL hydrogel can make the VAO positively correlated marker TNF-α and IL-6 highly expressed, and the negatively correlated marker Ctsk lowly expressed Figure 8 C). It is confirmed that during the differentiation of RAW264.7 cells into mature osteoclasts induced by RANKL and M-CSF, VAO and BAO exist at the same time, and GO-PEI-miR7b overexpression gene vector can prevent VAO fusion and transformation to BAO by inhibiting the expression of target gene DC-STAMP, that is, specifically inhibit BAO, and protect and increase the number of VAO.

[0103] 4. The effect of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector on the regulation of VAO and BAO on MC3T3-E1 osteogenic differentiation in vitro

[0104] (1) Cell grouping and treatment:

[0105] ① Cell grouping: Group 1, no upper chamber, lower culture hole MC3T3-E1 + osteogenic induction; Group 2, upper chamber RAW264.7, lower culture hole MC3T3-E1 + osteogenic induction; Group 3, upper chamber RAW264.7 + osteoclast induction, lower culture hole MC3T3-E1 + osteogenic induction; Group 4, upper chamber RAW264.7 + osteoclast induction + simple SA / HA / GDL hydrogel, lower culture hole MC3T3-E1 + osteogenic induction; Group 5, upper chamber RAW264.7 + osteoclast induction + SA / HA / GDL hydrogel loaded with negative control gene vector, lower culture hole MC3T3-E1 + osteogenic induction; Group 6, upper chamber RAW264.7 + osteoclast induction + SA / HA / GDL hydrogel loaded with GO-PEI-miR7b overexpression gene vector, lower culture hole MC3T3-E1 + osteogenic induction.

[0106] ② Cell treatment: MC3T3-E1 cells were inoculated in the lower culture hole of the 6-well plate at 2×10 5 per hole, and the next day the culture medium was discarded. The culture medium composition was: MEMα culture medium + 10% fetal bovine serum + 1% double antibiotic (penicillin-streptomycin), and β-glycerophosphate (final concentration 10 mM), ascorbic acid (final concentration 50 μg / ml) were used to induce MC3T3-E1 cells to differentiate into osteoblasts. The induction medium was changed every 2 days, and the culture was observed for 7 days. At 6 days, RAW264.7 cells were inoculated in the upper chamber at 5×10 4The RAW264.7 cells were inoculated in the upper chamber of a 6-well plate, and the medium was discarded the next day. The medium composition was: DMEM high-sugar culture solution + 10% fetal bovine serum + 1% double-antibiotic (penicillin-streptomycin), and RAW264.7 cells were induced to differentiate into osteoclasts by RANKL (final concentration 100 ng / mL) and M-CSF (final concentration 50 ng / mL). The upper chamber inoculated with RAW264.7 cells was placed in the lower culture well inoculated with MC3T3-E1 cells, and co-culture was started. At the same time, GO-PEI-miR7b overexpression gene vector loaded with 4 ug of miR7b plasmid was added to each well containing fresh medium, and GO-PEI-miR7b negative control gene vector was used as a negative control. Simple SA / HA / GDL injectable hydrogel without gene vector was used as a blank control. The cell induction medium was replaced every 48 h, and the co-culture was observed for 168 h, followed by relevant detection.

[0107] (2) Alkaline phosphatase (ALP) activity detection: At the observation point of 168 h, the medium and hydrogel were discarded, the MC3T3-E1 cells in the lower culture well were washed with PBS for 3 times, the cells were lysed with RIPA buffer, and the supernatant was obtained after homogenization and centrifugation. The total protein content in the cell cavity was determined by Pierce BCA protein detection kit (Thermo Scientific), and the protein concentration was adjusted to 5 ug / ul. After dilution by 10 times, the ALP activity was determined by p-nitrophenyl phosphate (p-NPP) based colorimetry. The standard curve was drawn with p-nitrophenol, and the ALP activity was normalized to the total protein, and finally expressed as arbitrary units per gram of protein.

[0108] (3) Alkaline phosphatase (ALP) staining: At the observation point of 168 h, the medium and hydrogel were discarded, the MC3T3-E1 cells in the lower culture well were washed with PBS for 3 times, and the cells were fixed with ALP fixing solution for 3 min and washed with distilled water for 5-10 s. Freshly prepared ALP incubation solution (AS-BI staining solution and FBB staining solution mixed at a volume ratio of 1:1) was added dropwise and incubated in the dark for 20 min, and then washed with distilled water for 5-10 s. Nucleus fast red staining solution was added for 3 min, and then washed with PBS for 3 times. The observation and photography were performed under an inverted optical microscope.

[0109] (4) qRT-PCR detection of osteogenic differentiation specific gene expression: At the observation point of 168 h, the medium and hydrogel were discarded, the MC3T3-E1 cells in the lower culture well were lysed with Trizol, and the total RNA in the cells was extracted. Real-time quantitative RT-PCR was used to quantitatively detect the target genes ALP, Runx2 and COL-I.

[0110] Conclusion: GO-PEI-miR7b overexpression gene vector released from SA / HA / GDL hydrogel can promote ALP activity Figure 9 A, 9C), and promote osteogenic related gene expression Figure 9 B), that is, GO-PEI-miR7b overexpression gene vector protects and increases the number of VAO by specifically inhibiting BAO, and the retained VAO eventually promotes osteogenic differentiation of MC3T3-E1 cells.

[0111] 5. In vitro effect of GO-PEI-miR7b overexpression gene vector loaded SA / HA / GDL injectable hydrogel on regulating angiogenesis of VAO and BAO

[0112] (1) Cell grouping and treatment:

[0113] ① Cell grouping: group 1, no upper chamber, bEnd.3 in lower culture well; group 2, RAW264.7 in upper chamber, bEnd.3 in lower culture well; group 3, RAW264.7 cells + osteoclast induction in upper chamber, bEnd.3 in lower culture well; group 4, RAW264.7 + osteoclast induction + simple SA / HA / GDL hydrogel in upper chamber, bEnd.3 in lower culture well; group 5, RAW264.7 + osteoclast induction + SA / HA / GDL hydrogel loaded with negative control gene vector in upper chamber, bEnd.3 in lower culture well; group 6, RAW264.7 + osteoclast induction + SA / HA / GDL hydrogel loaded with GO-PEI-miR7b overexpression gene vector in upper chamber, bEnd.3 in lower culture well.

[0114] ② Cell treatment: bend.3 cells were inoculated in the lower culture well of 6-well plate at 2×10 5 cells / well, and the culture medium was discarded the next day. The composition of the culture medium was: DMEM high glucose culture medium + 10% fetal bovine serum + 1% double antibiotic (penicillin-streptomycin), and the new culture medium was replaced. At the same time, RAW264.7 cells were inoculated in the upper chamber at 5×10 4The RAW264.7 cells were inoculated in the upper chamber of a 6-well plate, and the medium was discarded the next day. The medium composition was: DMEM high-sugar culture solution + 10% fetal bovine serum + 1% double-antibiotic (penicillin-streptomycin), and RAW264.7 cells were induced to differentiate into osteoclasts by RANKL (final concentration 100 ng / mL) and M-CSF (final concentration 50 ng / mL). The upper chamber inoculated with RAW264.7 cells was placed in the lower culture well inoculated with bend.3 cells, and the co-culture was started. At the same time, the GO-PEI-miR7b overexpression gene vector loaded with 2 ug of miR7b plasmid was added to each well containing fresh medium, and the GO-PEI-miR7b negative control gene vector was used as a negative control. The simple SA / HA / GDL injectable hydrogel without gene vector was used as a blank control. The cell culture medium was replaced every 48 h, and the co-culture was observed for 168 h before the relevant detection.

[0115] (2) Transwell migration detection: at the observation point of 168 hours, the medium and hydrogel were discarded, the bEnd.3 cells in the lower culture well were washed with PBS for 3 times, trypsinized for 3 min, and then centrifuged with PBS for 3 times (800 rpm) after the digestion was terminated by the medium. The bEnd.3 cells were resuspended in serum-free medium, and the cell density was adjusted to 1×10 5 cells / ml. 200 ul (2×10 4 cells) of the cell suspension was added to the Transwell chamber of a 24-well plate, 500 ul of serum-containing medium was added to the lower chamber of the 24-well plate, and the plate was incubated in an incubator for 24 hours. After 24 hours, the Transwell chamber was taken out, the medium in the well was discarded, and the cells were washed with PBS for 2 times. The cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS for 2 times, and dyed with 0.1% crystal violet for 2 min, and then washed with PBS for 2 times. The upper layer of non-migrated cells was gently wiped off with a cotton swab, the membrane of the chamber was removed with a surgical blade, and the cells were mounted with glycerol for observation and photography under an inverted optical microscope.

[0116] (3) Tube formation detection: at the observation point of 168 hours, the medium and hydrogel were discarded, the bEnd.3 cells in the lower culture well were washed with PBS for 3 times, trypsinized for 3 min, and then centrifuged with PBS for 3 times (800 rpm) after the digestion was terminated by the medium. The bEnd.3 cells were resuspended in fresh medium, and the cell density was adjusted to 6×10 5 cells / ml. 200 ul of liquid Matrigel matrix glue was evenly added to the wells of a 24-well plate using a pre-cooled gun head, and the plate was incubated at 37℃ for 30 min. 500 ul (3×10 5 cells) of the cell suspension was added to the wells of the 24-well plate pre-coated with the matrix glue, and the plate was incubated at 37℃ for 6 hours. After 6 hours, the cells were observed and photographed under an inverted microscope.

[0117] (4) qRT-PCR detection of angiogenesis-specific gene expression: At the observation point, after 168 hours, the culture medium and hydrogel were discarded, and the bEnd.3 cells in the lower culture wells were lysed with Trizol. Total RNA was extracted from the cells, and the target genes VEGFR, CD31 and vWF were quantitatively detected by real-time quantitative RT-PCR.

[0118] Conclusion: The GO-PEI-miR7b overexpression gene vector released by SA / HA / GDL hydrogel can promote the expression of angiogenesis-related genes. Figure 10 C), while promoting vascular endothelial cell migration and lumen formation ( Figure 10 A, 10B), namely, the GO-PEI-miR7b overexpression gene vector protects and increases the number of VAOs by specifically inhibiting BAOs, and the preserved VAOs ultimately promote angiogenesis.

[0119] Experimental Example 2: Evaluation of an animal model of early ONFH regeneration and repair using SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector.

[0120] 1. Early ONFH rat model construction:

[0121] (1) Experimental groups: normal control group (n=10) and early ONFH group (n=40).

[0122] (2) Model Construction: Fifty adult male SD rats (approximately 12 weeks old, weighing approximately 250 grams) were acclimatized for one week, accurately weighed, and randomly divided into groups using a random number table. Rats in the OFHN group received two intraperitoneal injections of *E. coli* endotoxin (LPS) at a dose of 10 μg / kg (0.8 ml solvent volume per injection), 24 hours apart. Rats in the control group received the same dose of saline intraperitoneally. Twenty-four hours after the last LPS injection, rats in the control group received three consecutive intramuscular injections of methylprednisolone (MPS) at a dose of 40 mg / kg (0.2 mL solvent volume per injection), 24 hours apart. Rats in the control group received the same dose of saline intramuscularly. Four weeks after the last MPS injection, repair was achieved using an SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector.

[0123] 2. Construction and injection administration of SA / HA / GDL injectable hydrogels loaded with GO-PEI-miR7b overexpression gene vector:

[0124] (1) Experimental grouping: early ONFH group (n=10), early ONFH+CD group (n=10), early ONFH+CD+simple SA / HA / GDL hydrogel group (n=10), early ONFH+CD+SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector group (n=10)

[0125] (2) Construction of SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector: 3% HA powder was added to the completely dissolved 2.8% SA solution, and after ultrasonic treatment (40 kHz, 200 w) for 20 min, magnetic stirring was performed for 6 h to obtain a SA / HA mixture; according to GO:PEI=1:10, N / P=80, a GO-PEI-miR7b gene vector loaded with miR7b plasmid was constructed and added to the SA / HA mixture (200 μl of the SA / HA mixture contained 50 μg of the GO-PEI-miR7b gene vector loaded with miR7b plasmid), and magnetic stirring was performed for 1 h to obtain a SA / HA / GO-PEI-miR7b mixture, which was then placed in a 4°C ice water bath; a 10% GDL solution was prepared and placed in a 4°C ice water bath after complete dissolution; the SA / HA / GO-PEI-miR7b mixture and the 10% GDL solution were mixed in a ratio of 5:1 (total volume 240 μl) using a three-way injection needle connector valve for standby use (with simple SA / HA / GDL injectable hydrogel without gene vector as a control).

[0126] (3) Core decompression (CD) and hydrogel injection: CD rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (0.3 ml / 100 g). The rats were placed in a prone position, and the skin and subcutaneous tissue were incised longitudinally over the greater trochanter. The gluteus maximus muscle was split along the direction of the muscle bundle, and the anterior 2 / 3 of the gluteus medius muscle was separated from the bone attachment. The anterior-lateral capsule of the hip joint was transected along the edge of the trochanter, and the femoral head was dislocated. The femoral neck periosteum and fibrous fold at the base were stripped. The marrow nucleus of the femoral head was decompressed from the round ligament stop point to the head and neck. A 1-mm-diameter drill bit parallel to the neck axis was used to drill, and the drill hole was flushed with normal saline. After drilling, 1 ml of a syringe was used to suck the residual normal saline and tissue fluid. The SA / HA / GO-PEI-miR7b mixture and 10% GDL solution were mixed at a volume ratio of 5:1, with a total volume of 240 μl. Then, 20 μl of the SA / HA / GDL hydrogel (containing 5 μg of miR7b plasmid-loaded GO-PEI-miR7b gene vector) was injected into the hole. After the hydrogel was gelled for 8 minutes, medical biological glue was applied to the hole for 3 minutes to seal the hole. The femoral head was reduced, and the hip joint capsule and gluteal muscle, skin were sutured layer by layer. The early ONFH group of rats did not undergo femoral head dislocation, and the rest were the same as the CD rats. Within 3 days after the operation, the rats were given intramuscular injection of 40,000 units of penicillin to prevent infection. At 8 weeks after the operation, the rats were sampled for follow-up experiments.

[0127] 3. Animal model detection and evaluation:

[0128] (1) Angiography: After 8 weeks, the rats were anesthetized, and the abdominal aorta and abdominal vein were dissected. The proximal aorta was ligated, and the abdominal vein was cut. Heparinized normal saline, 4% paraformaldehyde, and angiographic agent (MV-122, Flow Tech, Inc., Carver, MA, USA) were sequentially injected through the distal abdominal aorta until continuous outflow from the abdominal vein was observed. Then, the rats were placed at 4°C overnight to ensure complete polymerization, and the bilateral femoral heads and femurs were excised and the soft tissue was removed. After 72 hours of fixation with 4% paraformaldehyde, the samples were decalcified with 10% EDTA solution at 4°C for 20 days, with the decalcification solution being replaced every 3 days. Then, the bone microstructure was analyzed by small animal microCT detection, with a tube voltage of 50 kV, a tube current of 0.1 mA, and a resolution of 10 mm. Two-dimensional images were collected using DataReview, and three-dimensional images of the blood vessels were reconstructed using CTVOX.

[0129] (2) μCT imaging observation: After 8 weeks, the rats were anesthetized to death, and the bilateral femoral heads and femurs were excised and the soft tissue was removed. After 72 hours of fixation with 4% paraformaldehyde, the bone microstructure was analyzed by small animal microCT detection, with a tube voltage of 50 kV, a tube current of 0.1 mA, and a resolution of 10 mm. Two-dimensional images were collected using DataReview, and three-dimensional images of the bone were reconstructed using CTVOX.

[0130] (3) Histomorphology observation: After μCT detection, the samples were placed in 10% EDTA solution at 4℃ for 20 days, and the decalcification solution was replaced every 3 days. After 20 days, dehydration, paraffin embedding and sectioning were performed, and H&E staining was used to observe the histopathological changes, and immunofluorescence staining was used to observe the expression and distribution of H-type blood vessels (Emcn and CD31), VAO and BAO (Emcn and VPP3), and miR7b targeted molecule DC-STAMP (VPP3 and DC-STAMP).

[0131] Conclusion: SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector can inhibit BAO specifically, protect and increase the number of VAO, and thus promote bone-specific H-type angiogenesis and coupling bone formation, with excellent effect in early ONFH minimally invasive treatment.

[0132] ① X-ray ( Figure 11 AX-Ray), μCT and three-dimensional imaging ( Figure 11 A2D, 3D-bone), μCT angiography imaging ( Figure 11 A3D-bloodvessel) and H&E imaging ( Figure 11 AH&E): In the ONFH group, most of the subchondral trabecular bone disappeared, the subchondral bone marrow was sparse, the bone density was reduced ( Figure 11 B), and the blood vessels were significantly reduced ( Figure 11 C), but no collapse was found, indicating that the early ONFH model was successfully constructed; CD and CD+SA / HA / GDL can promote the neovascularization of the necrotic femoral head ( Figure 11 A, 11C), but have no significant effect on bone formation ( Figure 11 A, 11B), which may be due to the opening of the femoral head after CD, the reduction of the pressure in the femoral head, and the growth of the new blood vessels; SA / HA / GDL injectable hydrogel loaded with GO-PEI-miR7b overexpression gene vector can significantly promote the bone and vascular neovascularization of the necrotic femoral head ( Figure 11 A, 11B, 11C), and basically restore the bone and vascular structure of the femoral head.

[0133] ② H-type blood vessel distribution (Emcn and CD31 immunofluorescence double staining): H-type blood vessels exist in the subchondral trabecular bone, the junction of subchondral bone and cancellous bone, and the cancellous bone trabeculae, mainly distributed in the edge of the cancellous bone space ( Figure 12 A). Compared with the control group, the number of H blood vessels and Emcn hi CD31 hi positive cells in the ONFH group was significantly reduced ( Figure 12A, 12B); CD and CD+SA / HA / GDL can slightly increase H vessels and Emcn hi CD31 hi Number of positive cells ( Figure 12 A, 12B), the reasons are the same as analyzed above; while the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector can significantly increase H vessels and Emcn hi CD31 hi Number of positive cells ( Figure 12 A, 12B).

[0134] ③ Distribution of VAO and BAO (Emcn and VPP3 immunofluorescence double staining): VAO and BAO are present in subchondral bone trabeculae, the junction of subchondral bone and cancellous bone, and cancellous bone trabeculae. VAO is mainly distributed alongside H-type vessels, measuring 15-20 μm in size and containing 1-2 nuclei, while BAO is not distributed alongside H-type vessels, measuring 60-100 μm in size and containing more than 4 nuclei. Figure 13 A). Compared with the control group, the number of VAO-positive cells was significantly reduced in the ONFH group, while the number of BAO-positive cells was significantly increased. Figure 13 A, 13B); CD and CD+SA / HA / GDL had no significant effect on the number of VAO and BAO positive cells. Figure 13 A, 13B); while the SA / HA / GDL injectable hydrogel loaded with the GO-PEI-miR7b overexpression gene vector significantly increased the number of VAO-positive cells and decreased the number of BAO-positive cells (A, 13B); Figure 13 A, 13B).

[0135] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Use of an injectable hydrogel in the preparation of a medicament for the treatment of early ONFH based on the modulation of VAO, characterized in that, The injectable hydrogel is a SA / HA / GDL injectable hydrogel loaded with a GO-PEI-miR7b overexpression gene vector, The preparation method of the injectable hydrogel comprises the following steps: A. preparing a GO-PEI complex; B. mixing the GO-PEI complex with a miR7b plasmid, incubating at room temperature to obtain a GO-PEI-miR7b overexpression gene vector; C. adding 3% HA powder to a 2.8% SA aqueous solution, magnetically stirring after ultrasonic treatment to obtain a SA / HA mixture; adding the GO-PEI-miR7b overexpression gene vector loaded with the miR7b plasmid into the SA / HA mixture, the mass ratio of GO to PEI being 1:10, the nitrogen / phosphorus ratio of the GO-PEI complex to the miR7b plasmid being 80, magnetically stirring to obtain a SA / HA / GO-PEI-miR7b mixture; D. mixing the SA / HA / GO-PEI-miR7b mixture and a 10% GDL aqueous solution, then injecting into a mold, the SA / HA / GO-PEI-miR7b mixture and the 10% GDL solution being in a volume ratio of 5:1, demolding after gelation at room temperature to obtain a SA / HA / GDL injectable hydrogel loaded with a GO-PEI-miR7b overexpression gene vector.

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