Preparation method of multifunctional modified bone adhesion gel and application of multifunctional modified bone adhesion gel in promotion of diabetic fracture repair

Abstract

The invention discloses a preparation method of multifunctional modified bone adhesion gel and application of the multifunctional modified bone adhesion gel in promotion of diabetic fracture repair. The multifunctional modified bone adhesion gel is prepared by mixing and reacting bioactive glass nanoparticles (GA-MBGNs) modified by gallic acid and sodium alginate (AG-BA) modified by phenylboronic acid in a PBS (Phosphate Buffer Solution); the gel material disclosed by the invention can effectively play a bone adhesion role in an acid environment, so that the hydrogel still has relatively high strength when being applied to diabetic fracture repair. Meanwhile, the slow release of gallic acid molecules in the AGM system can improve the diabetic microenvironment and assist in promoting the healing of diabetic fractures, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing a multifunctional modified bone adhesion gel and its application in promoting the repair of diabetic fractures, belonging to the field of biomedical technology. Background Technology

[0002] Type 2 diabetes is prevalent worldwide, with a prevalence rate of approximately 11% in my country. Type 2 diabetes can lead to various chronic complications, such as diabetic macrovascular disease, retinopathy, nephropathy, and neuropathy. In recent years, the risk of fractures and bone defects in patients with type 2 diabetes has received increasing attention. Numerous epidemiological studies have shown that the fracture risk in diabetic patients is significantly higher than in the general population. Traditional methods for treating fractures primarily utilize internal fixation devices to precisely reduce the fracture ends; however, this method has inherent limitations. Besides poor fracture fixation, it often requires a secondary surgery to remove the internal fixation device, causing additional trauma. Therefore, more and more doctors and researchers have turned to researching alternative fracture treatment methods, among which bone adhesives have emerged as a promising solution. Bone adhesives are expected to exhibit rapid and strong adhesion to bone tissue while promoting bone repair, offering advantages in convenience, safety, efficiency, and cost-effectiveness compared to internal fixation methods.

[0003] Hydrogels are polymeric materials obtained by physical or chemical crosslinking of hydrophilic polymers. Hydrogels composed of different polymer matrices have different functions. Sodium alginate (AG), a commonly used polymer matrix in the biomedical field, has a large number of carboxyl and hydroxyl groups distributed in its molecular backbone, making it biocompatible and easily modified. Phenylboronic acid (BA) molecules can coordinate with the ortho-hydroxyl structure of the sodium alginate backbone to form dynamic borate ester bonds, giving the obtained BA-modified sodium alginate hydrogel (AG-BA) self-healing properties. In addition, the unique cis-diol structure of BA molecules can also adhere to the surface of biological tissues through hydrogen bonding and hydrophobic attraction under weakly alkaline pH conditions, giving the BA-modified sodium alginate hydrogel tissue adhesion properties, which is expected to be used as a bone adhesive for the repair of diabetic fracture sites.

[0004] However, the microenvironment of diabetic bone defects exhibits upregulated expression of inflammatory factors and high release of reactive oxygen species. This low-pH, slightly acidic environment reduces the stability of phenylboronic acid-modified sodium alginate hydrogels. Mesoactive glass nanoparticles (MBGNs) are widely reported biomaterials with osteogenic effects. Immersion in physiological fluids can form an apatite mineral layer on the surface of MBGNs, creating a slightly alkaline microenvironment that promotes bone mineralization and accelerates bone tissue repair and regeneration. Furthermore, the surface active layer of MBGNs can be further grafted with various bioactive molecules to exert multifaceted bioregulatory effects. Grafting gallic acid (GA), which has antioxidant and anti-inflammatory functions, onto the surface of bioactive glass nanoparticles (gallic acid modified mesoactive glass nanoparticles, GA-MBGNs) can enhance the inflammatory regulatory effects of the material without affecting the original osteogenic effect of MBGNs, potentially improving the diabetic microenvironment.

[0005] However, there are currently no reports on the preparation of a multifunctional hydrogel AGM with self-healing, adhesive, anti-inflammatory, antioxidant and osteogenic functions based on gallic acid-modified bioactive glass nanoparticles (GA-MBGN) and phenylboronic acid-modified sodium alginate system (AG-BA). Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing a multifunctional modified bone adhesion gel and its application in promoting the repair of diabetic fractures.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a multifunctional modified bone adhesion gel, which is prepared by mixing and reacting gallic acid-modified bioactive glass nanoparticles (GA-MBGNs) and phenylboronic acid-modified sodium alginate (AG-BA) in PBS buffer.

[0009] The present invention also provides a method for preparing the above-mentioned multifunctional modified bone adhesion gel, characterized by comprising the following steps: preparing gallic acid-modified bioactive glass nanoparticles and phenylboronic acid-modified sodium alginate respectively, and then mixing the gallic acid-modified bioactive glass nanoparticles and phenylboronic acid-modified sodium alginate and reacting them in PBS buffer to prepare the multifunctional modified bone adhesion gel.

[0010] Preferably, the preparation method of the gallic acid modified bioactive glass nanoparticles includes: dissolving gallic acid powder in phosphate buffer, stirring and mixing, adding MBGNs, dispersing and stirring evenly; then, centrifuging the resulting suspension, collecting the solid particles and drying them to obtain GA-loaded MBGNs (GA-MBGNs).

[0011] Preferably, the bioactive glass nanoparticles (MBGNs) are prepared by template-assisted sol-gel method using block copolymers (such as Pluronic F127) or surfactants (CTAB) as templates and silicon sources as raw materials.

[0012] In some embodiments of the present invention, the preparation method of the MBGNs includes: dissolving an organic template agent in deionized water, adding ethyl acetate and mixing evenly, then adding ammonia and stirring, then adding a silicon source and a calcium source and stirring to react, and after the reaction is completed, collecting the nanoparticles generated by centrifugation, and then washing, drying and calcining them in sequence to obtain MBGNs.

[0013] Preferably, the organic template agent is hexadecyltrimethylammonium bromide;

[0014] Preferably, the silicon source is at least one of tetraethyl orthosilicate, methyl orthosilicate, and butyl orthosilicate.

[0015] Preferably, the preparation method of the phenylboronic acid modified sodium alginate includes: dissolving sodium alginate in 2-morpholine ethanesulfonic acid buffer and adjusting the pH value to 5-6; then, adding a coupling activator to activate the carboxylate ions, and then adding phenylboronic acid to react; after the reaction is completed, purification and separation are performed to obtain phenylboronic acid modified sodium alginate (AG-BA).

[0016] Preferably, the mass ratio of gallic acid to MBGNs is 0.1–1 mg:1 g.

[0017] Preferably, the coupling activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0018] Preferably, the mass ratio of sodium alginate to phenylboronic acid is 1:0.1 to 0.5.

[0019] Preferably, the preparation method of sodium alginate modified with phenylboronic acid uses dialysis to purify and separate the product, the molecular weight cutoff of the dialysis is 3500 Da, and the pH of the solution is maintained at 5.5 during the dialysis process.

[0020] The present invention also provides the application of the above-mentioned multifunctional modified bone adhesion gel in the preparation of products for treating diabetic fractures or for repairing diabetic fractures.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention first synthesizes gallic acid-modified bioactive glass nanoparticles (GA-MBGN), and then incorporates them into a phenylboronic acid-modified sodium alginate system (AG-BA) to obtain a multifunctional hydrogel, AGM, with self-healing, adhesive, anti-inflammatory, antioxidant, and osteogenic functions. This material can effectively exert bone adhesion in an acidic environment, allowing the hydrogel to maintain high strength when applied to the repair of diabetic fractures. Simultaneously, the slow release of gallic acid molecules in the AGM system can improve the diabetic microenvironment and aid in the healing of diabetic fractures, showing promising clinical application prospects. Attached Figure Description

[0023] Figure 1 SEM scan results of AG-BA gel;

[0024] Figure 2 SEM scan results of AGM gel.

[0025] Figure 3 Schematic diagram of a three-point bending test.

[0026] Figure 4 : Maximum bending strength results for different groups.

[0027] Figure 5 Results of elastic modulus tests for different groups.

[0028] Figure 6 : Maximum load intensity results for different groups. Detailed Implementation

[0029] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0030] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Example

[0032] A method for preparing a multifunctional modified bone adhesion gel includes the following steps:

[0033] 1. Synthesis of MBGN

[0034] 1.36 g of CTAB (hexadecyltrimethylammonium bromide) was dissolved in 65 mL of ultrapure water and stirred at 37 °C for 30 min. Once the solution became clear, heating was stopped, and 20 mL of ethyl acetate (EA) was added dropwise. After stirring for another 30 min, 27 mL of ammonia hydroxide was added until the pH reached 9.5. Subsequently, 8 mL of TEOS (tetraethoxysilane) was slowly added. The mixture was stirred continuously for 4 hours, and the precipitate was collected by centrifugation (3000 rpm, 5 min) and washed twice each with ultrapure water and ethanol. Finally, the collected particles were dried overnight at 60 °C and then calcined at 600 °C for 3 hours.

[0035] 2. Synthesis of GA-MBGN

[0036] GA powder was dissolved in phosphate buffer (pH 7.4) at a concentration of 50 μg / ml and stirred in the dark for 30 minutes. Then, 0.2 g MBGNs were dispersed in 20 ml of GA solution and stirred for 5 hours. Subsequently, the suspension was centrifuged at 3000 rpm for 5 minutes to obtain GA-loaded MBGNs (GA-MBGNs), and the particles were dried in a fume hood for 48 hours.

[0037] 3. Synthesis of AG-BA molecules

[0038] 2 g of sodium alginate was dissolved in 0.1 M 2-morpholine ethanesulfonic acid buffer, and the pH of the solution was adjusted to 5.5 using 0.1 M hydrochloric acid. Subsequently, 1.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.2 g of N-hydroxysuccinimide (NHS) were added dropwise to the above solution. Then, 0.6 g of phenylboronic acid was added to the mixture, and the reaction was carried out at room temperature for 12 hours. After the reaction was complete, the product solution was purified by dialysis in pure water using a dialysis bag (MWCO: 3500 Da), with the pH of the solution maintained at 5.5 during dialysis.

[0039] 4. Synthesis of AG-BA hydrogel

[0040] AG-BA was dissolved in deionized water to prepare a 3 wt% solution. Then, 100 mM PBS buffer (pH 7.4) was added to the above solution to bring the final solution concentration to 2.3 wt%.

[0041] 5. Synthesis of AGM hydrogel

[0042] AG-BA was dissolved in deionized water to prepare a 3 wt% solution. GA-MBGN was then incorporated into the solution at a concentration of 1 wt%. Finally, 100 mM PBS buffer (pH 7.4) was added to the solution to bring the final AG-BA solution concentration to 2.3 wt%, thus obtaining AGM hydrogel.

[0043] Performance results:

[0044] Scanning electron microscopy revealed that the incorporation of GA-MBGN did not affect the three-dimensional porous structure of AG-BA gel, and could induce good osteoblast ingrowth, promoting bone regeneration. Figures 1-2 As shown.

[0045] A diabetic mouse fracture model was established, and the repair effect of AGM hydrogel on promoting fracture healing was observed. The specific procedures were as follows: A longitudinal incision was made on the lateral thigh of the diabetic mouse, the muscles were bluntly dissected, and the distal femur was exposed. The mid-shaft of the femur was transversely cut using a wire saw or manual bone saw; a hole was made above the patella, and a stainless steel needle was inserted into the femoral medullary cavity for fixation; AGM gel was implanted into the fracture ends as a fracture repair material (AGM group), while the control group did not have any bone adhesion material placed. Fresh femurs from diabetic rats were collected 6 weeks after fracture repair. The three-point bending test was used to investigate the repair effect. Figure 3 The study observed the differences in femoral elastic modulus and maximum load-bearing capacity between the AGM and control groups of diabetic mice to investigate whether AGM had better bone adhesion and bone regeneration effects. Results showed that AGM exhibited higher flexural strength compared to the control group. Figure 4 ), elastic modulus ( Figure 5 ) and compressive strength ( Figure 6 It can promote significant bone regeneration.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A multifunctional modified bone adhesion gel, characterized in that, The multifunctional modified bone adhesion gel is prepared by mixing and reacting gallic acid-modified bioactive glass nanoparticles (GA-MBGNs) and phenylboronic acid-modified sodium alginate (AG-BA) in PBS buffer.

2. The method for preparing the multifunctional modified bone adhesion gel according to claim 1, characterized in that, Includes the following steps: Gallic acid-modified bioactive glass nanoparticles and phenylboronic acid-modified sodium alginate were prepared separately. Then, the gallic acid-modified bioactive glass nanoparticles and phenylboronic acid-modified sodium alginate were mixed and reacted in PBS buffer to prepare the multifunctional modified bone adhesion gel.

3. The preparation method according to claim 2, characterized in that, The preparation method of the gallic acid modified bioactive glass nanoparticles includes: dissolving gallic acid powder in phosphate buffer, stirring and mixing, adding MBGNs, dispersing and stirring evenly; then, centrifuging the resulting suspension, collecting the solid particles and drying them to obtain GA-loaded MBGNs (GA-MBGNs).

4. The preparation method according to claim 2, characterized in that, The preparation method of the phenylboronic acid modified sodium alginate includes: dissolving sodium alginate in 2-morpholine ethanesulfonic acid buffer and adjusting the pH value to 5-6; then, adding a coupling activator to activate the carboxylate ions, and then adding phenylboronic acid to react; after the reaction is completed, purification and separation are performed to obtain phenylboronic acid modified sodium alginate (AG-BA).

5. The preparation method according to claim 3, characterized in that, The mass ratio of gallic acid to MBGNs is 0.1–1 mg:1 g.

6. The preparation method according to claim 4, characterized in that, The coupling activators are 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

7. The preparation method according to claim 4, characterized in that, The mass ratio of sodium alginate to phenylboronic acid is 1:0.1 to 0.

5.

8. The preparation method according to claim 4, characterized in that, The preparation method of sodium alginate modified with phenylboronic acid uses dialysis to purify and separate the product. The molecular weight cutoff for dialysis is 3500 Da, and the pH of the solution is maintained at 5.5 during the dialysis process.

9. The use of the multifunctional modified bone adhesion gel according to claim 1 in the preparation of products for treating diabetic fractures or for repairing diabetic fractures.

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