Injectable antibacterial hydrogel for treating osteomyelitis and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and relates to injectable antibacterial hydrogel as well as a preparation method and application thereof. The method comprises the following steps: (1) preparing a sodium alginate antibacterial peptide solution, and adding sodium alginate powder into an antibacterial peptide aqueous solution with a certain concentration for ultrasonic dissolution; (2) preparing a chitosan, sodium alginate and antibacterial peptide mixed solution: adding chitosan powder into the sodium alginate and antibacterial peptide aqueous solution, performing ultrasonic treatment for a period of time, adding acetic acid, and stirring until the acetic acid is completely dissolved; (3) preparing a beta-GP solution: putting beta-GP particles into an alkaline solution with a certain concentration, and carrying out ultrasonic stirring until the beta-GP particles are completely dissolved; and (4) dropwise adding the beta-GP solution into the chitosan / sodium alginate / antibacterial peptide mixed solution under an ice bath condition, uniformly stirring, sucking the liquid by using an injector, and injecting the liquid to an injured part, thereby obtaining the multifunctional antibacterial hydrogel material under a body temperature condition. The multifunctional injectable antibacterial hydrogel material prepared by the invention has a rapid gelling effect, also shows long-term and effective antibacterial performance, effectively cures chronic osteomyelitis infection, and has good biocompatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to an injectable antibacterial hydrogel for treating osteomyelitis and a preparation method thereof. Background Art

[0002] Bone tissue infection is a common orthopedic complication, which is a bone tissue disease caused by microbial infection. Common causes include surgical wound infection, trauma, osteoclast infection, etc. Osteomyelitis is a common disease in bone tissue infection, characterized by progressive inflammatory bone destruction accompanied by reactive bone formation, and can involve a small part or several areas of any bone. The initial infection that leads to osteomyelitis can be inoculated from the blood of the pathogen to another part of the body, or directly inoculated through trauma or surgical wounds. This leads to an acute infection that usually lasts for days or weeks and may require antibiotics and surgical intervention. When the pathogenic microorganism persists for more than 10 days and causes further bone destruction, the infection is considered to be chronic osteomyelitis. Conventional systemic antimicrobial therapy is usually sufficient to clear acute bone infection; and chronic osteomyelitis is very difficult to treat, requiring radical surgical debridement of necrotic and infected tissues, followed by extensive antibiotic treatment. However, long-term and extensive antibiotic treatment is very likely to cause bacterial resistance and has low therapeutic effect. At present, the new antibacterial injectable hydrogel has a good development prospect and application value as a new generation of antibacterial therapeutic materials.

[0003] At present, various natural and synthetic materials have been widely used in the manufacture of antibacterial hydrogels, such as polysaccharides, inorganic salts, biological products, and nano-self-assembled peptides. However, single-component hydrogels cannot meet the irregular shapes of infected wounds during antibacterial treatment and maintain long-term release of antibacterial agents. Therefore, the ideal antibacterial hydrogel should have multiple functions, such as fitting irregular infection environments, isolating bacterial invasion, and releasing antibacterial substances for a long time to maintain long-term antibacterial effects.

[0004] Antimicrobial peptides are short-sequence peptides, most of which are cationic peptides, and have a wide range of inhibitory effects on bacteria, fungi, and viruses. Antimicrobial peptides have broad-spectrum antimicrobial activity even at low concentrations, can kill bacteria quickly, and have a low resistance trend, which helps to solve public health problems such as antibiotic shortages and antibiotic resistance. By interacting with the bacterial cell membrane, destroying the structural integrity of the cell membrane, leading to the excretion of bacterial contents, a rapid antimicrobial effect is achieved, which may be completed within a few seconds of contact with the microorganism. Importantly, this special mechanism of action is not prone to drug resistance. However, high toxicity and low stability limit their application. In order to expand the scope of application of antimicrobial peptides, rational design is needed to maintain or enhance the antimicrobial activity of antimicrobial peptides and reduce their hemolytic activity and cytotoxicity. In addition, suitable carriers provide the possibility of combining antimicrobial peptides with other materials, which can also enhance their function and stability.

[0005] Chitosan is a linear polysaccharide obtained by partial N-deacetylation of chitin, which has been identified as a non-toxic polymer with good antibacterial properties, biodegradability, and good biocompatibility. So far, it has been used in the preparation of various biomedical materials, such as gels, sponges, micro- or nanoparticles, and membranes. It can be further compounded with other materials to adjust its physical and biochemical properties to meet the application conditions of infected wounds. β-Glycerol phosphate (GP) is a natural body component used as a source of phosphate and also used to treat phosphate metabolism disorders. Mixing CS with GP in an aqueous medium results in a stimulus response in the formation of gels that respond to both in situ pH and temperature changes. The increase in pH leads to the neutralization of the amino groups in the CS chains, while the increase in temperature leads to the loss of hydration water, resulting in precipitation, increased physical cross-linking of the polymer chains, and subsequent formation of a hydrogel. Sodium alginate is a natural polysaccharide isolated from the cell wall of brown algae, which has good biocompatibility and biodegradability. As an anionic polysaccharide, sodium alginate can be effectively combined with a variety of cationic antimicrobial agents to control the sustained release of drugs, making it easy for antimicrobial materials to play a long-term role. At the same time, the addition of sodium alginate cross-links with chitosan molecules to form a reticular porous structure, which improves the mechanical properties of the hydrogel. In orthopedic diseases, bacterial infection is a potential serious complication. Therefore, advanced antimicrobial materials are urgently needed to remove microbial contamination. Therefore, hydrogel materials should support the loading of antimicrobial agents to achieve long-term antibacterial, bone repair and other functions.

[0006] Based on the above content, the present invention proposes an injectable antibacterial hydrogel for the treatment of osteomyelitis and a preparation method thereof, and further protects the application of the prepared injectable antibacterial hydrogel in the preparation of drugs or articles for the treatment of osteomyelitis and antibacterial treatment. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide an injectable antibacterial hydrogel for the treatment of osteomyelitis and a preparation method thereof to solve the problems raised in the background technology. The present invention uses chitosan, β-GP, sodium alginate and antibacterial polypeptides as raw materials, and mixes the antibacterial polypeptides with the natural composite matrix chitosan / sodium alginate to prepare an injectable antibacterial hydrogel having a highly cross-linked porous structure, excellent biocompatibility, excellent biodegradability, excellent antibacterial performance, and the ability to effectively promote the treatment of osteomyelitis.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] The first aspect of the present invention provides a method for preparing an injectable antibacterial hydrogel for treating osteomyelitis, comprising the following contents:

[0010] S1. preparing sodium alginate antimicrobial peptide solution: adding sodium alginate powder to an antimicrobial peptide aqueous solution of a certain concentration and dissolving by ultrasonication to obtain a sodium alginate antimicrobial peptide solution;

[0011] S2, preparing a chitosan sodium alginate antimicrobial peptide mixed solution: adding chitosan powder to the sodium alginate antimicrobial peptide aqueous solution prepared in step 1), adding acetic acid and stirring until completely dissolved after ultrasonic treatment for a period of time, to prepare a chitosan sodium alginate antimicrobial peptide mixed solution;

[0012] S3, preparing β-GP solution: placing β-GP particles in an alkaline solution of a certain concentration, stirring by ultrasonic until completely dissolved, to prepare β-GP solution;

[0013] S4. Preparation of antibacterial hydrogel material: under ice bath condition, add the β-GP solution prepared in S3 drop by drop into the chitosan sodium alginate antibacterial peptide mixture prepared in S2, stir evenly, use a syringe to absorb the mixed liquid and inject it into the injured area to form a multifunctional antibacterial hydrogel material under body temperature condition.

[0014] Preferably, the ultrasonic power of the ultrasonic dissolution in S1 is 30% to 90%; and the ultrasonic time is 5 to 60 minutes.

[0015] Preferably, the power of the ultrasonic treatment in S2 is 30% to 90%; and the ultrasonic treatment time is 5 to 60 minutes.

[0016] Preferably, the temperature of the ice bath in S2 is 0-5° C.; the speed of the stirring treatment is 100-150 rpm; and the stirring time is 5-60 minutes.

[0017] Preferably, the concentration of the sodium alginate is 0.01-0.5% by mass; more preferably, 0.05-3%.

[0018] Preferably, the power of the ultrasonic treatment in S3 is 30% to 90%; and the ultrasonic treatment time is 5 to 60 minutes.

[0019] Preferably, the alkaline solution in S3 includes but is not limited to one or more of CaCO3, Ca(OH)2, Na2CO3, NaHCO3, NaOH, K2CO3, KHCO3 and KOH solutions.

[0020] Preferably, the peptide in the aqueous antimicrobial peptide solution is a cationic polypeptide; in terms of mass fraction, the amount of the cationic polypeptide added is 10 to 50% of the amount of sodium alginate added; more preferably, it is 20 to 40%.

[0021] Preferably, the molecular weight of the chitosan is 1 to 500 KDa; more preferably, it is 10 to 300 KDa.

[0022] Preferably, the concentration of chitosan is 0.5-5% by mass; more preferably, 0.1%-3%.

[0023] Preferably, the mass percentage of the acetic acid is 0.1-5%.

[0024] Preferably, the amount of alkali added to the alkaline solution is 1 to 20% based on the mass fraction of β-GP particles; more preferably, it is 5 to 15%.

[0025] Preferably, in terms of mass fraction, the concentration of β-GP in the β-GP solution is 1 to 20%, and more preferably 5 to 15%.

[0026] Preferably, the mixing volume ratio of the β-GP solution to the chitosan sodium alginate antimicrobial peptide solution is 1-18:18-1; more preferably, it is 1-10:10-1.

[0027] The second aspect of the present invention is to provide an injectable antibacterial hydrogel prepared by the above preparation method.

[0028] The third aspect of the present invention is to provide the use of the above-mentioned injectable antibacterial hydrogel in the preparation of drugs or articles for the treatment of osteomyelitis and antibacterial treatment.

[0029] Compared with the prior art, the present invention provides an injectable antibacterial hydrogel for treating osteomyelitis and a preparation method thereof, which has the following beneficial effects:

[0030] The present invention proposes a method for preparing an injectable antibacterial hydrogel for treating osteomyelitis, wherein an antibacterial polypeptide is loaded into a hydrogel matrix formed by cross-linking chitosan / sodium alginate, and finally an injectable antibacterial hydrogel with excellent comprehensive performance is obtained. The injectable antibacterial hydrogel prepared by the preparation method proposed by the present invention has excellent and long-lasting antibacterial performance, can meet the treatment of irregular infected wounds, and has excellent biocompatibility and biodegradability. The preparation method is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of Gram staining of the injectable antibacterial hydrogel mentioned in the experimental example of the present invention;

[0032] Figure 2 Schematic diagram of H&E staining of the injectable antibacterial hydrogel mentioned in the experimental example of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, but the described embodiments are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] Embodiment 1:

[0035] A method for preparing an injectable antibacterial hydrogel for treating osteomyelitis comprises the following steps:

[0036] S1. Preparation of sodium alginate antimicrobial peptide solution: adding sodium alginate powder to an antimicrobial peptide aqueous solution of a certain concentration and dissolving by ultrasonication to obtain a sodium alginate antimicrobial peptide solution; the details are as follows:

[0037] S11. Prepare an antimicrobial peptide aqueous solution: weigh 3 mg of antimicrobial peptide powder and add it to a beaker, slowly add an appropriate amount of ultrapure water to the beaker, and gently stir with a stirring rod until it is completely dissolved to obtain an antimicrobial peptide aqueous solution;

[0038] S12, preparing sodium alginate antimicrobial peptide solution: adding 10 mg of sodium alginate powder to the antimicrobial peptide aqueous solution and dissolving it by ultrasonication at 60% power to obtain a sodium alginate antimicrobial peptide solution; S2, preparing a chitosan sodium alginate antimicrobial peptide mixed solution: adding chitosan powder to the sodium alginate antimicrobial peptide aqueous solution prepared in S1, ultrasonicating for a period of time, adding acetic acid and stirring until completely dissolved, to obtain a chitosan sodium alginate antimicrobial peptide mixed solution; the details are as follows:

[0039] S21, preparing chitosan powder: weighing chitosan solid and crushing it into powder using a grinder, and sieving it according to needs to obtain chitosan powder of different particle sizes;

[0040] S22, weigh 200 mg of chitosan powder prepared in S21 and add it to 10 mL of sodium alginate antimicrobial peptide aqueous solution prepared in S12, treat with ultrasonic power of 30% for 15 min, then add 100 μL of acetic acid and stir until completely dissolved to prepare chitosan sodium alginate antimicrobial peptide mixed solution;

[0041] S3. Preparation of β-GP solution: placing β-GP particles in an alkaline solution of a certain concentration, stirring by ultrasonic until completely dissolved, to obtain a β-GP solution; the details are as follows:

[0042] S31. Prepare an alkaline solution: weigh 0.8 g of alkaline substance powder and transfer it to a beaker, slowly add 10 mL of distilled water to the beaker, and gently stir with a glass rod until dissolved, transfer the dissolved solution to a volumetric flask, continue to add distilled water to the volumetric flask to complete the fixed volume, and mix the solution to obtain an 8% alkaline solution;

[0043] S32, preparing β-GP solution: weighing 8 g of β-GP particles, and adding them to the alkaline solution prepared in S31, ultrasonically treating at a power of 30% for 15 min until completely dissolved, to prepare a β-GP solution;

[0044] S4, preparation of antibacterial hydrogel material: under ice bath condition, add the β-GP solution prepared in step 3) drop by drop into the chitosan sodium alginate antibacterial peptide mixed solution prepared in step 2) and stir evenly, then use a syringe to draw the mixed liquid and inject it into the wound, and form a multifunctional antibacterial hydrogel material under body temperature condition. The details are as follows:

[0045] S41, preparing a mixed liquid: adding 1 mL of the β-GP solution prepared in S32 dropwise to 9 mL of the chitosan sodium alginate antimicrobial peptide mixed liquid prepared in S22 under ice bath conditions, and stirring evenly;

[0046] S42, preparing an antibacterial hydrogel material: using a syringe to absorb the mixed liquid prepared in S41 and injecting it into the wound, so as to form a multifunctional antibacterial hydrogel material under body temperature conditions.

[0047] Embodiment 2:

[0048] The steps are basically the same as those in Example 1, except that "3 mg antimicrobial peptide powder (sequence FFRKVLKLIRKIWR)" in S11 is replaced by "1 mg antimicrobial peptide powder (sequence FFRKVLKLIRKIWR)".

[0049] Embodiment 3:

[0050] The steps are basically the same as those in Example 1, except that "3 mg antimicrobial peptide powder (sequence FFRKVLKLIRKIWR)" in S11 is replaced by "5 mg antimicrobial peptide powder (sequence FFRKVLKLIRKIWR)".

[0051] Embodiment 4:

[0052] The steps are basically the same as those in Example 1, except that "1 mg antimicrobial peptide powder (sequence FFRKVLKLIRKIWR)" in S11 is replaced by "2 mg antimicrobial peptide (sequence FFRKVLKLIRKIWR)".

[0053] Embodiment 5:

[0054] The steps are basically the same as those in Example 1, except that, when preparing the sodium alginate antimicrobial peptide solution in S11, "adding sodium alginate powder to the antimicrobial peptide aqueous solution" is replaced by "adding the antimicrobial peptide aqueous solution to sodium alginate powder".

[0055] Embodiment 6:

[0056] The steps are basically the same as those in Example 1, except that when the sodium alginate powder is added in S12, "10 mg sodium alginate powder" is replaced by "20 mg sodium alginate powder".

[0057] Embodiment 7:

[0058] The steps are basically the same as those in Example 1, except that when weighing the chitosan powder in S22, "200 mg chitosan powder" is replaced by "300 mg chitosan powder".

[0059] Embodiment 8:

[0060] The steps are basically the same as those in Example 1, except that when weighing the alkaline substance powder in S31, "800 mg of alkaline substance powder" is replaced by "1200 mg of alkaline substance powder".

[0061] Embodiment 9:

[0062] The steps are basically the same as those in Example 1, except that when weighing β-GP particles in S32, "8 g β-GP particles" is replaced with "15 g β-GP particles".

[0063] Embodiment 10:

[0064] The steps are basically the same as those in Example 1, except that: when preparing the mixed liquid in S41, the "ice bath condition" is replaced by "normal temperature condition".

[0065] Embodiment 11:

[0066] The steps are basically the same as those in Example 1, except that when preparing the mixed liquid in S41, "1 mL of β-GP solution" is replaced by "9 mL of β-GP solution".

[0067] Comparative Example 1:

[0068] The steps are basically the same as those in Example 1, except that no antimicrobial peptide is added to S1.

[0069] Comparative Example 2:

[0070] The steps are basically the same as those in Example 1, except that chitosan powder is not added to S2.

[0071] Comparative Example 3:

[0072] The steps are basically the same as those in Example 1, except that no acetic acid solution is added to S2.

[0073] Comparative Example 4:

[0074] The steps are basically the same as those in Example 1, except that no β-GP solution is added to S3.

[0075] Experimental example:

[0076] 1. Performance research

[0077] (a) Appearance and morphology analysis of hydrogels

[0078] Observe whether the appearance of the hydrogel is uniform, transparent or translucent, and whether there are impurities, bubbles, etc. Determine its morphological changes before and after injection. When the amount of antimicrobial peptide added to the hydrogel is too small, the hydrogel will form slowly. When the amount added is too large, the hydrogel is prone to bubbles and uneven formation. Similarly, changes in the content of sodium alginate, chitosan, β-GP, and alkaline solution in the hydrogel system also affect the difficulty of hydrogel formation and the gelation state. In the comparative example, when the antimicrobial peptide was not added, the hydrogel formed extremely slowly, and in other comparative examples, the hydrogel formation was prone to uneven.

[0079] Table 1 Test results of hydrogel gel state in different embodiments

[0080]

[0081]

[0082] (b) Analysis of hydrogel swelling

[0083] The swelling properties of the injectable antibacterial hydrogels for treating osteomyelitis prepared in Examples 1-11 and the hydrogels prepared in Comparative Examples 1-4 were tested. The results can be seen in Table 2.

[0084] Table 2 Test results of hydrogel swelling properties in different embodiments

[0085]

[0086] It can be seen from the results in Table 2 that under the preferred formula, the swelling ratio of the injectable hydrogel is about 4, and the swelling property is good. When the amount of antimicrobial peptide added to the hydrogel is too small, the hydrogel is slowly formed and the swelling ratio is high. When the amount added is too large, the hydrogel is prone to bubbles and uneven formation, and the swelling ratio is further increased. When the content of sodium alginate, chitosan, β-GP, and alkaline solution in the hydrogel system changes, the degree of structural bonding between the hydrogels is affected, and the swelling ratio of the hydrogel also begins to increase. In the comparative example, when the antimicrobial peptide is not added, the hydrogel is difficult to form and the swelling ratio is high; in other comparative examples, the hydrogel gelation is more difficult than that in the embodiment and has a higher swelling ratio.

[0087] (c) Evaluation of hydrogel gel time

[0088] The gel time performance of the injectable antibacterial hydrogels for treating osteomyelitis prepared in Examples 1-11 and the hydrogels prepared in Comparative Examples 1-4 was tested by a timer. The results are shown in Table 3.

[0089] Table 3 Test results of hydrogel gel time in different embodiments

[0090]

[0091] It can be seen from the results in Table 3 that the gel time of the injectable antibacterial hydrogel for the treatment of osteomyelitis under the preferred formula is maintained at about 60s, and it has excellent injectability. When the amount of antimicrobial peptide added to the hydrogel is too small, the hydrogel is slowly formed and the gel time is greatly prolonged; when the amount added is too large, the hydrogel is prone to bubbles and uneven formation, and the gel time is further prolonged. Similarly, when the content of sodium alginate and chitosan in the hydrogel system changes, the structural bonding between the hydrogels is not tight enough, and the gel time of the hydrogel is prolonged. When the content of β-GP and alkaline solution in the hydrogel system increases, the bonding between the hydrogel molecules is too fast, and the gel time is too short, affecting the operation of osteomyelitis treatment. In the comparative example, when the antimicrobial peptide is not added, the hydrogel is extremely difficult to form and the gel time is extremely long; in other comparative examples, the hydrogel gelation is difficult and has an extremely long gel time.

[0092] (d) Gel temperature of hydrogel

[0093] For hydrogel therapeutic materials with excellent injectable properties, a suitable gelling temperature is essential, which enables the hydrogel to gel quickly in the human body and exert its effectiveness. The temperature of the sol-gel state transition of the hydrogel was tested by a rheometer. The results are shown in Table 4.

[0094] Table 4 Hydrogel gel temperature test results in different embodiments

[0095]

[0096] It can be seen from the results in Table 4 that under the preferred formula, the gel temperature of the injectable antibacterial hydrogel for the treatment of osteomyelitis is around 37°C, and it can transform from a sol to a gel state under body temperature conditions, which plays an important role in the treatment of osteomyelitis with antibacterial injectable hydrogels. When the hydrogel components of the embodiment change, the hydrogel is difficult to form under body temperature conditions, and the gelation time is greatly improved. When the antimicrobial peptide is not added in the comparative example, the cross-linking structure of the hydrogel is destroyed, and it is difficult to form under body temperature conditions; in other comparative examples, the hydrogel is difficult to gel and requires a higher temperature to gel, and cannot be used in the treatment of osteomyelitis. In summary, the results show that the addition amount of AMP (antimicrobial peptide), sodium alginate, chitosan powder, acetic acid and β-GP solution has a significant effect on the swelling, gelation time and gelation temperature of the hydrogel.

[0097] (e) Biocompatibility of hydrogels

[0098] Biomaterials must undergo safety testing before being used in the human body, and hemolysis is one of the testing methods. Generally speaking, the lower the hemolysis rate, the less impact on red blood cells, and the safer the material. After culturing the hydrogel with red blood cells for a period of time, the absorbance of the supernatant is measured to determine whether the material is hemolytic. The results are shown in Table 5.

[0099] Table 5 Biocompatibility test results of hydrogels in different embodiments

[0100]

[0101] As can be seen from the results in Table 5, the hemolysis rate of the injectable antibacterial hydrogel for the treatment of osteomyelitis prepared by the present invention is less than 5%, which meets the national safety regulations on the blood compatibility of biomaterials. Among them, the addition of antimicrobial peptides within the preferred range can promote cell proliferation and contribute to the treatment and recovery of osteomyelitis. Cationic polypeptides have certain hemolytic activity and cytotoxicity, and their stability in vivo is poor. How to control the release of polypeptides is a key scientific issue in the application of polypeptides. In the injectable hydrogel prepared by the present invention, the addition amount of AMP (antimicrobial peptide), sodium alginate, chitosan powder, acetic acid and β-GP solution has a significant effect on the release of polypeptides, that is, it also has a greater impact on the safety of the hydrogel. The injectable antibacterial hydrogel for the treatment of osteomyelitis has good biocompatibility.

[0102] (f) Antibacterial properties of hydrogels

[0103] The important indicator of biomaterials used in the treatment of osteomyelitis is antibacterial properties. Generally speaking, the better the antibacterial effect of the hydrogel material, the better the therapeutic effect on osteomyelitis. The results are shown in Table 6.

[0104] Table 6 Biocompatibility test results of hydrogels in different embodiments

[0105]

[0106]

[0107] As shown in Table 6, the hydrogels prepared by the present invention all have certain antibacterial properties, which are mainly due to the addition of antimicrobial peptides. Therefore, the amount of antimicrobial peptides added and the release behavior from the hydrogel are the main factors determining its antimicrobial properties. Generally speaking, the more antimicrobial peptides are added, the faster the release is, and the better the antimicrobial properties are, but safety issues will also be faced. Therefore, how to add antimicrobial peptides and control the release of antimicrobial peptides so that they have excellent antimicrobial properties without causing safety issues is a key problem to be solved by the present invention. Through optimization and selection, the injectable antimicrobial hydrogel for the treatment of osteomyelitis prepared by the present invention has excellent antimicrobial effect and good biocompatibility.

[0108] From the above results, it can be seen that the addition amount of AMP (antimicrobial peptide), sodium alginate, chitosan powder, acetic acid and β-GP solution has a great influence on the biocompatibility and antibacterial properties of the hydrogel.

[0109] 2. Application of injectable antibacterial hydrogel in wound repair for osteomyelitis treatment

[0110] 2.1 Construction of rat osteomyelitis model

[0111] Eighteen male SD rats weighing about 220 g (purchased from Guangzhou Ruige Biotechnology Co., Ltd.) were selected as experimental animals. The entire experimental process complies with the ethical standards for animal experiments.

[0112] Osteomyelitis model establishment and grouping: SD rats were randomly divided into three groups: osteomyelitis blank control group, osteomyelitis + CA hydrogel group (Comparative Example 1), and osteomyelitis + CAH hydrogel group (Example 1). The rats were anesthetized, and the skin of the surgical area was shaved and disinfected. The rats were anesthetized by intraperitoneal injection of pentobarbital, and the femoral condyles of both legs were surgically exposed. The Kirschner wire with a diameter of 1.5 mm was perforated, and the MRSA solution (100 μL, 10 8 CFU / mL) was injected into the leg bone marrow cavity through the bone defect to establish an osteomyelitis model. The control group was not given any other treatment after being rinsed with saline. Then 0.3mL of CA and CAH hydrogel solutions were injected into the defect respectively. After the solution gelled, the incision was closed layer by layer. The rats were killed for observation 4 weeks after the operation.

[0113] 2.2 Application Performance Evaluation

[0114] (a) Evaluation of the therapeutic effect of osteomyelitis

[0115] The osteomyelitis treatment ability of the injectable antibacterial hydrogel for osteomyelitis treatment was evaluated by the osteomyelitis treatment effect in rats. The results are shown in Table 7.

[0116] Table 7 Treatment effect of hydrogel osteomyelitis in different embodiments

[0117]

[0118] From the results in Table 7, it can be seen that under the preferred formula, the injectable antibacterial hydrogel for treating osteomyelitis has a BV / TV value of about 20% after 30 days of treatment of osteomyelitis, which can effectively treat osteomyelitis in vivo and achieve bone repair effect; while when the components of the system 1 in Example 1 are changed, the BV / TV value of osteomyelitis treatment decreases. The BV / TV value of the hydrogel in Comparative Example 1 is about 13%, and the hydrogel has a poor osteomyelitis treatment effect.

[0119] (b) Evaluation of antibacterial activity in vivo

[0120] Bacterial infection can affect the treatment of osteomyelitis, so the in vivo antibacterial properties were tested.

[0121] After the rat osteomyelitis model was successfully established, 0.3 mL of CA (Comparative Example 1) and CAH (Example 1) hydrogel solutions were injected into the defect, respectively. After the hydrogel solutions were allowed to gel, the incisions were closed layer by layer.

[0122] Gram staining was used to evaluate the antibacterial ability of the hydrogel material in vivo after 30 days of treatment. Figure 1 .

[0123] Depend on Figure 1 The results show that, under the preferred formula, after 30 days of treatment of osteomyelitis, there is basically no stained bacteria in the osteomyelitis site, indicating that the bacteria are basically killed; while there is obvious bacterial staining in the blank control and comparative example 1, and the comparative example has a poor antibacterial effect on osteomyelitis. At the same time, it is shown that the injectable antibacterial hydrogel prepared by the present invention can slowly release antimicrobial peptides in the body, thereby exerting excellent antibacterial properties.

[0124] (c) Histological evaluation

[0125] Femurs of rats in different experimental groups were collected for histological staining. The staining steps were as follows:

[0126] For histological analysis, rat femurs from both legs were buffered with 4% paraformaldehyde and PBS (pH 7.4) overnight, decalcified with 10% EDTA, and embedded in paraffin. 4 mm thick sections were made and stained with hematoxylin and eosin (H&E) to observe tissue morphology. Figure 2 shown.

[0127] Depend on Figure 2 The results show that under the preferred formula, the inflammatory factors in the osteomyelitis site were significantly reduced after 30 days of treatment of osteomyelitis with the injectable antibacterial hydrogel, indicating that the injectable antibacterial hydrogel has a good anti-inflammatory effect in the treatment of osteomyelitis; while the inflammatory factors in the blank control group and the comparison example were basically not reduced, and the anti-inflammatory treatment effect of the comparison example on osteomyelitis was poor.

[0128] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A method for preparing an injectable antibacterial hydrogel for treating osteomyelitis, characterized in that: The steps include: S1. preparing a sodium alginate antimicrobial peptide solution: adding sodium alginate powder to an antimicrobial peptide aqueous solution of a certain concentration and dissolving by ultrasonication to obtain a sodium alginate antimicrobial peptide solution; S2, preparing a chitosan sodium alginate antimicrobial peptide mixed solution: adding chitosan powder to the sodium alginate antimicrobial peptide aqueous solution prepared in step 1), adding acetic acid and stirring until completely dissolved after ultrasonic treatment for a period of time, to prepare a chitosan sodium alginate antimicrobial peptide mixed solution; S3, preparing β-GP solution: placing β-GP particles in an alkaline solution of a certain concentration, stirring by ultrasonic until completely dissolved, to prepare a β-GP solution; S4. Preparation of antibacterial hydrogel material: under ice bath condition, add the β-GP solution prepared in S3 drop by drop into the chitosan sodium alginate antibacterial peptide mixture prepared in S2, stir evenly, use a syringe to absorb the mixed liquid and inject it into the injured area to form a multifunctional antibacterial hydrogel material under body temperature condition.

2. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: The alkaline solution in S3 includes, but is not limited to, one or more of CaCO3, Ca(OH)2, Na2CO3, NaHCO3, NaOH, K2CO3, KHCO3 and KOH solutions.

3. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: Calculated by mass fraction, the concentration of the sodium alginate solution is 0.01-0.5%.

4. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: The peptide in the antimicrobial peptide aqueous solution is a cationic polypeptide; in terms of mass fraction, the added amount of the cationic polypeptide is 10-50% of the added amount of the sodium alginate.

5. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: The molecular weight of the chitosan is 1-500 KDa; in terms of mass fraction, the concentration of the chitosan is 0.5-5%.

6. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: The amount of alkali added to the alkaline solution is 1-20% based on the mass fraction of β-GP particles.

7. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: In terms of mass fraction, the concentration of β-GP in the β-GP solution is 10 to 100%.

8. The method for preparing the injectable antibacterial hydrogel for treating osteomyelitis according to claim 1, characterized in that: The mixing volume ratio of the β-GP solution to the chitosan sodium alginate antimicrobial peptide solution is 1-18:18-1.

9. An injectable antibacterial hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable antibacterial hydrogel according to claim 9 in preparing drugs or articles for treating osteomyelitis or antibacterial treatment.

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