An injectable hydrogel and its preparation method and application

Abstract

The present invention provides an injectable hydrogel and its preparation method and application. The method includes: oxidizing hyaluronic acid to obtain oxidized hyaluronic acid, and adding deionized water to prepare an oxidized hyaluronic acid solution; adding 4-aminophenylboronic acid to the oxidized hyaluronic acid solution, stirring evenly to obtain AOHA, and adding deionized water to prepare an AOHA solution; adding rosmarinic acid and lithium magnesium silicate to the solution containing AOHA, mixing and reacting to obtain an injectable hydrogel. This hydrogel has good immunomodulatory function, osteogenic induction, biocompatibility and antibacterial properties, and can be used for the treatment of infectious bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of orthopedic implant materials, and particularly relates to an injectable hydrogel, a preparation method thereof and an application thereof. Background Art

[0002] Infectious bone defect is a common orthopedic disease after open bone trauma and is an important problem faced by clinical surgeons. Due to the invasion of local pathogens and subsequent insufficient blood supply, the spread of necrotic tissue, apoptosis of osteoblasts, etc., it usually leads to a serious decline in the osteogenic ability of the infectious bone defect site, resulting in delayed bone healing. Therefore, targeting bacteria and bone marrow stromal cells (BMSCs) to eliminate in-situ pathogen infection and simultaneously construct an osteogenic microenvironment at the bone defect interface has always been considered the most reasonable strategy for treating infectious bone defects. With the progress of osteoimmunology, the key role of immune regulation in bacteria clearance and bone healing has been gradually elucidated. Among them, macrophages are one of the most important effector cells in immune responses and can secrete a variety of cytokines that directly affect initial inflammation, BMSC recruitment, and subsequent differentiation and mineralization. Therefore, biomaterials that simultaneously have antibacterial, promote macrophage M2 polarization, and induce osteogenic differentiation of BMSCs are expected to further accelerate the repair of infectious bone defects.

[0003] Rosmarinic acid (RA) is a natural phenolic compound present in various plants such as Boraginaceae and Lamiaceae, and has an ester structure of caffeic acid and 3,4-dihydroxyphenyl lactic acid. RA not only has broad-spectrum antibacterial activity, but also has the potential to induce macrophage M2 polarization. Lithium magnesium silicate (Lap) is a synthetic clay composed of disk-shaped crystals (about 25 nm in diameter and 0.92 nm in height). A 2% Lap aqueous solution can mediate the self-assembly of Lap crystals to form a thixotropic hydrogel. Studies have shown that even in the absence of exogenous growth factors such as BMP-2, Lap can up-regulate the expression of bone-related genes and promote type I collagen synthesis in vivo through its degradation products Mg 2+ 、Si(OH)4、Li + etc. to play an osteogenic promoting role.

[0004] In recent years, injectable hydrogels have been widely used in the field of tissue engineering. It can replace implantation surgery with minimally invasive injection methods and can form any desired shape to match irregular defects. Therefore, injectable hydrogels with characteristics such as high structural adaptability, minimally invasiveness, tissue similarity, drug delivery ability, etc. are considered ideal candidate materials for the repair of infectious bone defects. However, how to integrate the above components with antibacterial, immune regulation, and osteogenic differentiation into an injectable hydrogel remains a huge challenge. Summary of the Invention

[0005] The object of the present invention is to provide an injectable hydrogel, its preparation method and application. The hydrogel has good immunomodulatory function, osteogenic induction, biocompatibility and antibacterial property, and can be used for treating infectious bone defects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a preparation method of an injectable hydrogel is provided, and the method includes:

[0008] Oxidize hyaluronic acid to obtain oxidized hyaluronic acid, and add deionized water to prepare an oxidized hyaluronic acid solution;

[0009] Add 4-aminophenylboronic acid to the oxidized hyaluronic acid solution, stir evenly to obtain AOHA, and add deionized water to prepare an AOHA solution;

[0010] Add rosmarinic acid and lithium magnesium silicate to the solution containing AOHA, mix and react to obtain an injectable hydrogel.

[0011] Further, in the oxidized hyaluronic acid solution, the molar ratio of the oxidized hyaluronic acid to deionized water is 1.5:1 - 10:1.

[0012] Further, the molar ratio of the 4-aminophenylboronic acid to the oxidized hyaluronic acid is 1:5 - 4:5.

[0013] Further, in the AOHA solution, the molar ratio of the AOHA to deionized water is 1:50 - 1:20.

[0014] Further, the concentration of the rosmarinic acid added to the solution containing AOHA is 25 - 50 mg / ml.

[0015] Further, the molar ratio of the rosmarinic acid to the AOHA is 1:2 - 1:10, and the molar ratio of the lithium magnesium silicate to the AOHA is 3:1 - 5:1.

[0016] In the second aspect of the present invention, an injectable hydrogel prepared by the above method is provided.

[0017] In the third aspect of the present invention, the application of the injectable hydrogel in preparing a material for repairing infectious bone defects is provided.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] 1. The injectable hydrogel of the present invention is prepared by linking oxidized hyaluronic acid and 4-aminophenylboronic acid through a Schiff base reaction, loading rosmarinic acid as a drug into the system, and providing an alkaline environment by lithium magnesium silicate to form a first-level network, and then the "card-house" structure of lithium magnesium silicate itself forms a second-level network to enhance the first-level network. Due to the reversibility of Schiff base bonds, borate bonds and electrostatic interactions, the AOHA-RA / Lap hydrogel can not only be injected, but also exhibits self-strengthening characteristics after shear thinning.

[0020] After the injectable hydrogel of the present invention is implanted into the infected bone defect site of rats, it can continuously release rosmarinic acid and lithium magnesium silicate for a long time, and construct an osteogenic microenvironment with antibacterial, bone induction and immunomodulatory functions at the defect interface, so as to repair the infected bone defect.

[0021] 2. In the past, the treatment of infected bone defects was mainly aimed at bacteria and BMSCs. The present invention utilizes the immunomodulatory effect of rosmarinic acid, combined with its antibacterial effect and the osteogenic induction function of lithium magnesium silicate, so as to achieve a trinity treatment effect.

[0022] 3. In the present invention, the alkalinity of lithium magnesium silicate is used to provide conditions for the successful construction of the first-level network of the hydrogel. At the same time, the self-structure of lithium magnesium silicate is cleverly used to form a second-level network, which enhances the first-level network. The hydrogel exhibits self-strengthening characteristics after shear thinning, enabling it to adaptively fit the bone defect interface after in vivo injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 is a schematic diagram of the synthesis of AOHA-RA / Lap injectable hydrogel;

[0025] Figure 2 is a physical diagram of AOHA-RA / Lap injectable hydrogel and its effect diagram of filling bone defects in vitro; among them, Figure 2 A is a diagram of injecting AOHA-RA / Lap hydrogel, Figure 2 B is a physical diagram of AOHA-RA / Lap injectable hydrogel, Figure 2 C is a physical diagram of AOHA-RA / Lap hydrogel at the bone defect interface

[0026] Figure 3It is the rheological diagram of the AOHA-RA / Lap injectable hydrogel; among them, Figure 3 A is the Lap rheological diagram in the frequency mode, Figure 3 B is the AOHA-RA rheological diagram in the frequency mode, Figure 3 C is the AOHA-RA / Lap rheological diagram in the frequency mode, Figure 3 D is the AOHA-RA / Lap rheological diagram before applying shear force in the time mode, Figure 3 E is the AOHA-RA / Lap rheological diagram after applying shear force in the time mode

[0027] Figure 4 It is the electron microscopy morphology diagram of the AOHA-RA / Lap injectable hydrogel;

[0028] Figure 5 It is the biocompatibility result of the AOHA-RA / Lap injectable hydrogel; among them, Figure 5 A is the live-dead cell staining of RAW264.7 cells 3 days after receiving different treatments, Figure 5 B is the live-dead cell staining of BMSCs cells 3 days after receiving different treatments.

[0029] Figure 6 It is the result of the AOHA-RA / Lap injectable hydrogel regulating macrophage polarization;

[0030] Figure 7 It is the result of the AOHA-RA / Lap injectable hydrogel regulating the osteogenic differentiation of BMSCs;

[0031] Figure 8 It is the in vitro antibacterial result of the AOHA-RA / Lap injectable hydrogel;

[0032] Figure 9 It is the micro-CT result of the AOHA-RA / Lap injectable hydrogel repairing infectious bone defects in vivo. Detailed implementation manners

[0033] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0035] The general idea of the present invention is as follows:

[0036] The present invention discloses a multifunctional injectable hydrogel for the repair of infectious bone defects and its preparation method. Using a one-pot method, oxidized hyaluronic acid (OHA), 4-aminophenylboronic acid (4-APBA), and rosmarinic acid (RA) are mixed and then lithium magnesium silicate (Lap) is added to synthesize the AOHA-RA / Lap injectable hydrogel.

[0037] The AOHA-RA / Lap hydrogel exhibits self-strengthening characteristics after shear thinning and has antibacterial, macrophage M2 polarization-promoting, and bone marrow mesenchymal stem cell osteogenic differentiation-inducing functions.

[0038] After injecting the AOHA-RA / Lap hydrogel into the bone defect site, it can fit well with the bone defect interface; as the borate ester bonds gradually break, the AOHA-RA / Lap hydrogel can continuously release rosmarinic acid and lithium magnesium silicate for a long time.

[0039] Therefore, an osteogenic microenvironment with antibacterial, bone induction, and immunomodulatory functions is constructed at the defect-hydrogel interface to achieve the repair of infectious bone defects.

[0040] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0041] The present application will be described in detail below in conjunction with examples and experimental data.

[0042] Example 1. Preparation method of the AOHA-RA / Lap injectable hydrogel

[0043] 1. Preparation of OHA: Dissolve 2 g of sodium hyaluronate (HA) in 50 ml of deionized water, add it to a magnetic stirring water bath and stir at a speed of 600 r / min for 3 hours until dissolved. After cooling to room temperature, add 1.3 g of sodium periodate, stir in a 25°C water bath for 12 hours, then add ethylene glycol to quench the unreacted sodium periodate and continue stirring for 1 hour. Then put the product into a dialysis bag with a cut-off molecular weight of 3500 D and dialyze for 48 hours, changing the water every four hours on the first day and twice a day thereafter for a total of three days of dialysis, and then lyophilize to successfully prepare OHA ( Figure 1 ).

[0044] 2. Preparation of the AOHA solution: Weigh 0.2 g of OHA and dissolve it in 1 ml of deionized water, let it stand until completely dissolved, add 0.05 g of 4-APBA and stir at 25°C on a magnetic stirrer for 24 hours, then dialyze for 3 days, and lyophilize the obtained solution to obtain AHOA.

[0045] 3. Preparation of AOHA-RA / Lap injectable hydrogel: Weigh 0.2 g of AOHA and dissolve it in 1 ml of deionized water. Let it stand until it is completely dissolved. Add 0.025 g of RA and 0.1 g of Lap to the AOHA solution, stir evenly on a magnetic stirrer, and let it stand to obtain AOHA-RA / Lap hydrogel.

[0046] Example 2. Characterization of AOHA-RA / Lap injectable hydrogel

[0047] Place the hydrogel in a syringe, such as Figure 2 As shown in the figure, the newly synthesized composite hydrogel is in a viscous flow state; after standing, the viscous flow state changes into a gel state. The hydrogel is injected into the irregular bone defect with a needle. Due to its injectable properties, the hydrogel can gel in situ and can fit the complex shape of the bone defect. The rheological properties of the hydrogel are tested using a rheometer, such as Figure 3 As shown in the figure, in the frequency mode, the hydrogel storage modulus G' is greater than the corresponding loss modulus G", and in the time mode, the hydrogel can gel within 2 minutes, and the subsequent state is stable, which further proves the properties of the gel. Then, a JSM-7500F scanning electron microscope was used to photograph the surface of the hydrogel material, as shown in the figure. Figure 4 As shown, the material surface exhibits a loose and porous structure, which is conducive to drug loading and cell migration and proliferation.

[0048] Example 3. Biocompatibility of AOHA-RA / Lap injectable hydrogel

[0049] Calcein-AM / PI live-dead cell staining kit was used to evaluate the cytocompatibility of the hydrogel in BMSCs or RAW264.7. BMSCs or RAW264.7 cells (10 cells per well) were placed in the 5 ) were seeded in a 6-well plate, and the AOHA-RA / Lap hydrogel was placed in a transwell chamber. After 72 hours of culture, the dead cells and live cells were stained according to the instructions. Figure 5 As shown, the BMSCs and RAW264.7 cells in the blank control group and AOHA-RA / Lap hydrogel group survived well, and no obvious dead cells were observed.

[0050] Example 4: Effect of AOHA-RA / Lap injectable hydrogel on polarization of RAW264.7 cells in vitro

[0051] RAW264.7 cells were co - cultured with AOHA - RA / Lap hydrogel (placed in a transwell chamber) in complete medium for 3 days. During this process, LPS (promoting M1 polarization) was added, and the cells were subjected to immunofluorescence staining for iNOS (M1 surface marker) and CD206 (M2 surface marker). Cells without hydrogel addition were used as blank controls, and cells with only LPS addition were used as positive control groups. As Figure 6 shown, compared with the blank control group, the expression of iNOS on the surface of cells in the LPS group increased, but the expression of iNOS in the AOHA - RA / Lap hydrogel group was significantly lower than that in the LPS group. In addition, the expression of CD206 in the AOHA - RA / Lap hydrogel group was also significantly higher than that in the blank control and LPS groups.

[0052] Example 5. Effect of AOHA - RA / Lap injectable hydrogel on osteogenic differentiation of BMSCs in vitro

[0053] BMSCs were co - cultured with AOHA - RA / Lap hydrogel (placed in a transwell chamber) in osteogenic induction medium for 7 days, and ALP staining was performed using an alkaline phosphatase (ALP) staining kit. Cells without hydrogel addition were used as blank controls. On the 14th day of osteogenic induction, alizarin red (ARS) staining was used to evaluate the degree of calcium deposition in the extracellular matrix of BMSCs. As Figure 7 shown, both the ALP activity of BMSCs on the 7th day and the degree of calcium deposition in the extracellular matrix on the 21st day in the AOHA - RA / Lap hydrogel group were higher than those in the blank control group.

[0054] Example 6. In vitro antibacterial ability of AOHA - RA / Lap injectable hydrogel

[0055] AOHA - RA / Lap hydrogel (50 mg) was added to 1 mL of Escherichia coli or Staphylococcus aureus suspension (10 7 CFU / mL) respectively. The Escherichia coli or Staphylococcus aureus suspension co - cultured without hydrogel was used as a control, and the culture was carried out in a 24 - well plate at 37 °C for 12 h. Then, 10 μL of the bacterial solution was taken, diluted with 500 μL of PBS, and evenly spread on the surface of an agar plate. After incubation at 37 °C for 12 hours, the colonies were counted. As Figure 8 shown, on the agar plates of the AOHA - RA / Lap hydrogel group, the number of colonies of Escherichia coli or Staphylococcus aureus was extremely small, indicating that the AOHA - RA / Lap hydrogel has strong bactericidal activity against Escherichia coli or Staphylococcus aureus.

[0056] Example 7. Treatment of rat infectious skull defect model

[0057] A rat infectious skull defect model was made using Staphylococcus aureus, and the AOHA-RA / Lap hydrogel was implanted 2 weeks after the surgery. At 4 and 8 weeks after the hydrogel implantation, the rats were sacrificed and the skulls were taken and fixed in 4% paraformaldehyde solution. The bone defect area was scanned using a micro-CT system to evaluate the formation of new bone. As Figure 9 shown, at 4 weeks after the hydrogel implantation, almost no new bone formation was seen in the skulls of the blank control group due to Staphylococcus aureus infection, which led to bone destruction. In contrast, obvious new bone was visible in the AOHA-RA / Lap hydrogel group. At 8 weeks after the hydrogel implantation, bone regeneration in the skulls of the rats in the AOHA-RA / Lap hydrogel group was even more obvious, and the skull defect was almost completely filled with new bone, while only a small amount of new bone regeneration was visible in the blank control group.

[0058] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0059] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0060] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of an injectable hydrogel in the preparation of a material for repairing an infected bone defect, characterized in that: The preparation method of the injectable hydrogel comprises: Oxidizing hyaluronic acid to obtain oxidized hyaluronic acid, and adding deionized water to prepare an oxidized hyaluronic acid solution; Adding 4-aminophenylboronic acid to the oxidized hyaluronic acid solution, stirring evenly to obtain aminophenylboronic acid-oxidized hyaluronic acid, and adding deionized water to prepare an aminophenylboronic acid-oxidized hyaluronic acid solution; the molar ratio of the 4-aminophenylboronic acid to the oxidized hyaluronic acid is 1:5-4:5; Add rosmarinic acid and lithium magnesium silicate to a solution containing aminophenylboronic acid-oxidized hyaluronic acid, mix and react, and obtain an injectable hydrogel; the amount ratio of the rosmarinic acid to the aminophenylboronic acid-oxidized hyaluronic acid is 1:2-1:10, and the amount ratio of the lithium magnesium silicate to the aminophenylboronic acid-oxidized hyaluronic acid is 3:1-5:

1.

2. The use according to claim 1, characterized in that: In the oxidized hyaluronic acid solution, the molar ratio of the oxidized hyaluronic acid to deionized water is 1.5:1-10:

1.

3. The use according to claim 1, characterized in that: In the aminophenylboronic acid-oxidized hyaluronic acid solution, the amount of aminophenylboronic acid-oxidized hyaluronic acid to deionized water is 1:50-1:

20.

4. The use according to claim 1, characterized in that: The concentration of rosmarinic acid added to the solution containing aminophenylboronic acid-oxidized hyaluronic acid is 25-50 mg / ml.

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