Injectable antibacterial hydrogel for irregular wound repair and its preparation method and application

Abstract

The present invention provides an injectable antibacterial hydrogel that can be used for irregular wound repair. The antibacterial hydrogel is composed of modified recombinant humanized type III collagen and chitosan grafted with dihydrocaffeic acid. The modified recombinant type III humanized collagen hydrogel has a three-dimensional network structure, and the chitosan grafted with dihydrocaffeic acid is evenly distributed within the three-dimensional network structure of the modified recombinant type III humanized collagen hydrogel. The modified recombinant type III humanized collagen is formed by photocuring recombinant type III humanized collagen through photoinduced cross-linking. The present invention also provides a method for preparing the antibacterial hydrogel and its use in wound repair. This invention improves the material's ability to promote wound healing while meeting its antibacterial properties, while also enhancing its suitability for irregular wounds.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to an injectable antibacterial hydrogel that can be used for repairing irregular wounds, and a preparation method and application thereof. Background Art

[0002] Skin, the largest tissue in the human body, serves as a barrier that maintains a stable internal environment and prevents microbial invasion. It performs crucial functions, including regulating body temperature, maintaining fluid balance, and monitoring the immune system. However, due to various factors, such as trauma, disease, and aging, full-thickness skin defects are difficult to heal quickly. Therefore, the development of materials that promote skin healing and inhibit bacterial infection is crucial for clinical treatment.

[0003] Type III collagen is the primary collagen in human skin, present between the epidermis and dermis. Recombinant humanized type III collagen is produced by fermenting human genes into yeast. It shares the same repair and hydration functions as human type III collagen and boasts a stable source and high yield, making it an ideal skin repair material. However, its application is limited by its poor mechanical properties, rapid degradation both in vitro and in vivo, and insufficient antimicrobial properties. Chitosan is a natural biopolymer primarily derived from the deacetylation reaction of chitin. Chitosan exhibits excellent cytocompatibility, biodegradability, non-toxicity, wound healing, and antibacterial properties. Therefore, introducing chitosan into a recombinant humanized type III collagen system can improve its antimicrobial and degradation properties.

[0004] Prior art involves blending recombinant humanized type III collagen with carboxylated chitosan, then chemically crosslinking them using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). This allows the amino groups of the recombinant humanized type III collagen to react with the carboxyl groups of the carboxylated chitosan to form amide bonds, transforming the gel into a porous scaffold material. This material is freeze-dried to produce a porous scaffold material. However, this porous scaffold material is solid, making it difficult to perfectly conform to the wound site when used for skin repair, especially when applied to complex wounds. This makes it difficult to fully cover and conform to complex wounds, thus limiting its use in complex wound repair. Furthermore, the porous scaffold material's ability to promote cell proliferation and spreading, as well as its wound healing performance, needs to be improved. Therefore, how to provide a wound dressing that has better wound healing ability, is non-biotoxic, and is more suitable for irregular wound repair based on the advantages of recombinant humanized type III collagen in skin repair and chitosan in antibacterial properties, while meeting the requirements of biodegradation and antibacterial functions, remains a challenge in the field of skin repair materials and is one of the problems that need to be urgently solved in this field. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an injectable antibacterial hydrogel that can be used for irregular wound repair, as well as its preparation method and application, so as to improve the material's ability to promote wound healing while meeting the antibacterial properties and improve the material's applicability to irregular wounds.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0007] Disclosed is an injectable antibacterial hydrogel that can be used for repairing irregular wounds. The antibacterial hydrogel is composed of modified recombinant humanized type III collagen and chitosan grafted with dihydrocaffeic acid. The modified recombinant type III humanized collagen hydrogel has a three-dimensional network structure, and the chitosan grafted with dihydrocaffeic acid is uniformly distributed in the three-dimensional network structure of the modified recombinant type III humanized collagen hydrogel. In the antibacterial hydrogel, the mass ratio of the modified recombinant type III humanized collagen to the chitosan grafted with dihydrocaffeic acid is (8-11):1. The modified recombinant type III humanized collagen is formed by light-curing recombinant type III humanized collagen through photoinitiated cross-linking.

[0008] In the above-mentioned technical solution of the injectable antibacterial hydrogel that can be used for irregular wound repair, the antibacterial hydrogel is formed by photo-initiated cross-linking of a gel precursor solution containing recombinant humanized type III collagen with photocuring properties, chitosan grafted with dihydrocaffeic acid, and a photoinitiator.

[0009] In the above-mentioned technical solution for the injectable antimicrobial hydrogel for irregular wound repair, the light-curable recombinant humanized type III collagen is preferably methacrylic anhydride-modified recombinant humanized type III collagen. The methacrylic anhydride-modified recombinant humanized type III collagen is formed by an acylation reaction between methacrylic anhydride and the amino groups of the recombinant humanized type III collagen. After the reaction, the methacrylic anhydride occupies the majority of the amino functional groups on the recombinant humanized type III collagen.

[0010] A feasible method for preparing recombinant humanized type III collagen modified with methacrylic anhydride is as follows:

[0011] (1) Dissolve recombinant humanized type III collagen (rhCol III) in Na2CO3-NaHCO3 buffer to obtain rhCol III solution. Add methacrylic anhydride to the rhCol III solution and stir at 35-45°C for 2-4 hours.

[0012] (2) The reaction solution obtained in step (1) is transferred into a dialysis bag, dialyzed with ultrapure water, and freeze-dried to obtain recombinant humanized type III collagen modified with methacrylic anhydride.

[0013] During the preparation process, the grafting rate of methacrylic anhydride in the methacrylic anhydride-modified recombinant humanized type III collagen can be adjusted by adjusting the ratio of methacrylic anhydride to recombinant humanized type III collagen.

[0014] Furthermore, in the above-mentioned technical solution of the injectable antibacterial hydrogel that can be used for repairing irregular wounds, the grafting rate of methacrylic anhydride in the recombinant humanized type III collagen with light-curing properties is 65% to 100%.

[0015] In the aforementioned injectable antibacterial hydrogel for irregular wound repair, the chitosan grafted with dihydrocaffeic acid is formed by an amidation reaction between the carboxyl groups of dihydrocaffeic acid and the amino groups of chitosan. The grafting ratio of dihydrocaffeic acid in the chitosan grafted with dihydrocaffeic acid is preferably 10% to 30%. Dihydrocaffeic acid is a metabolite of caffeic acid and has potent antioxidant properties. Modifying chitosan with dihydrocaffeic acid can enhance its antioxidant and antibacterial properties.

[0016] In the above-mentioned technical solution of the injectable antibacterial hydrogel that can be used for irregular wound repair, a feasible method for preparing chitosan grafted with dihydrocaffeic acid is as follows:

[0017] (1) dissolving chitosan in a hydrochloric acid aqueous solution to form a chitosan solution; dissolving dihydrocaffeic acid (HCA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in an ethanol-water solution to obtain an HCA-EDC mixed solution;

[0018] (2) adjusting the pH value of the chitosan solution to 5.4-5.6, adding the HCA-EDC mixture dropwise to the chitosan solution under stirring, adjusting the pH value of the resulting mixture to 5.4-5.6, and stirring at room temperature for 10-15 hours;

[0019] (3) The reaction solution obtained in step (2) is placed in a dialysis bag, dialyzed with deionized water having a pH value of 3.9 to 4.1, and freeze-dried to obtain chitosan grafted with dihydrocaffeic acid.

[0020] During the preparation process, the concentration and ratio of the chitosan solution to the HCA-EDC mixed solution are determined according to the grafting rate of dihydrocaffeic acid in the chitosan grafted with dihydrocaffeic acid to be prepared.

[0021] The present invention also provides a method for preparing the above-mentioned injectable antibacterial hydrogel that can be used for irregular wound repair, comprising the following steps:

[0022] Recombinant humanized type III collagen with photocuring properties, chitosan grafted with dihydrocaffeic acid, and a photoinitiator are dissolved in water, and the bubbles in the resulting solution are removed to obtain a gel precursor solution. Under blue light irradiation, the recombinant humanized type III collagen with photocuring properties undergoes a cross-linking reaction to obtain an injectable antibacterial hydrogel that can be used for irregular wound repair.

[0023] In the gel precursor solution described in the technical solution of the above preparation method, the mass ratio of the recombinant humanized type III collagen with photocuring properties to the chitosan grafted with dihydrocaffeic acid is (8-11):1.

[0024] In the gel precursor solution described in the technical solution of the above preparation method, the total concentration of the recombinant humanized type III collagen with photocuring properties and the chitosan grafted with dihydrocaffeic acid is 80-120 mg / mL.

[0025] In the technical solution of the above preparation method, the recombinant humanized type III collagen with light-curing properties is preferably recombinant humanized type III collagen modified with methacrylic anhydride.

[0026] In the gel precursor solution of the technical solution of the preparation method, the content of the photoinitiator is 0.5wt% to 2wt%. Furthermore, a feasible photoinitiator is a photoinitiator that can trigger a crosslinking reaction using blue light, such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0027] The injectable antibacterial hydrogel provided by the present invention, which can be used for irregular wound repair, has excellent skin healing, antibacterial and antioxidant properties, and biocompatibility, and is biodegradable in vivo. The present invention conducted physical and chemical characterization of the antibacterial hydrogel, in vitro cell proliferation and migration experiments, antibacterial experiments, and animal skin repair experiments. The results showed that:

[0028] (1) Physical and chemical experiments show that the antibacterial hydrogel provided by the present invention has good mechanical properties, and the storage modulus can reach 12KPa; it has good water absorption capacity, can complete rapid water absorption within 3 minutes, and reach more than 85% of the maximum swelling, and has good balanced swelling performance, which is conducive to the rapid absorption of blood, exudate, etc., and is conducive to maintaining a stable shape at the wound site; it also has excellent degradation performance, and can be degraded by about 70% in 6 days under a PBS buffer environment, and can fully adapt to the changes of irregular wounds during deformation and recovery.

[0029] (2) In vitro cell proliferation and migration experiments showed that the antibacterial hydrogel provided by the present invention has excellent biocompatibility. Compared with the first day of co-culture, the cell migration degree can reach 35% and the cell proliferation degree can be tripled on the third day of co-culture.

[0030] (3) Antibacterial experiments show that the antibacterial hydrogel provided by the present invention has excellent antibacterial properties and has a good inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus.

[0031] (4) Animal skin repair experiments showed that the antibacterial hydrogel provided by the present invention has an excellent ability to promote skin healing. When repairing full-thickness skin defects in mice, a healing rate of 92% can be achieved on the 12th day of repair, which is higher than the healing rate of 70% in the blank control group and the healing rate of about 82% in the prior art.

[0032] Based on the above experimental results, the injectable antibacterial hydrogel provided by the present invention can be used in the preparation of wound repair materials, antibacterial materials, and medical materials loaded with cells, drugs or other bioactive substances.

[0033] The present invention also provides the use of the above-mentioned injectable antibacterial hydrogel that can be used for irregular wound repair in wound repair. During the use, a gel precursor containing recombinant humanized type III collagen with photocuring properties, chitosan grafted with dihydrocaffeic acid and a photoinitiator is injected into the wound to be repaired, and blue light irradiation is applied to induce a cross-linking reaction to convert the gel precursor into a gel state to completely cover the area to be repaired, thereby repairing the wound to be repaired.

[0034] Compared with the prior art, the technical solution provided by the present invention produces the following beneficial technical effects:

[0035] 1. The present invention provides an injectable antibacterial hydrogel that can be used to repair irregular wounds. The hydrogel is composed of modified recombinant humanized type III collagen and chitosan grafted with dihydrocaffeic acid. The modified recombinant humanized type III collagen hydrogel has a three-dimensional network structure, and the chitosan grafted with dihydrocaffeic acid is evenly distributed in the three-dimensional network structure of the modified recombinant humanized type III collagen hydrogel. The injectable antibacterial hydrogel is formed by photoinitiated cross-linking of a gel precursor solution containing recombinant humanized type III collagen with photocurable properties, chitosan grafted with dihydrocaffeic acid, and a photoinitiator. By introducing chitosan grafted with dihydrocaffeic acid, the present invention enables the skin repair material based on recombinant humanized type III collagen to have excellent antibacterial properties, thereby improving the problem that the existing recombinant collagen-based skin repair materials have poor antibacterial properties and are not conducive to wound repair. Furthermore, by modifying recombinant humanized type III collagen to impart light-curing properties, the hydrogel can be injected into the wound to be repaired, followed by application of appropriate light conditions to achieve gelation. This broadens the applicability of the antibacterial hydrogel to wounds of varying morphologies, making it particularly suitable for repairing and protecting complex, irregular wounds. This effectively addresses the problem of existing recombinant collagen-chitosan composite skin repair materials, which require molds for shaping and are in a solid state, making them difficult to apply to irregular wounds.

[0036] 2. In the injectable antibacterial hydrogel that can be used for irregular wound repair according to the present invention, the chitosan grafted with dihydrocaffeic acid is distributed in the three-dimensional network structure of the modified recombinant humanized type III collagen hydrogel. On the one hand, the three-dimensional network structure of the modified recombinant humanized type III collagen hydrogel is conducive to the adhesion, proliferation and diffusion of cells. On the other hand, the chitosan grafted with dihydrocaffeic acid and the modified recombinant humanized type III collagen are mainly physically blended, and no obvious chemical bonding occurs between the two. Therefore, the chitosan grafted with dihydrocaffeic acid is more easily released from the hydrogel, which is conducive to the chitosan grafted with dihydrocaffeic acid to better play its role, for example, it can better promote the growth of cells related to wound healing, promote macrophages to produce active factors that contribute to wound healing, promote microvascular regeneration of skin wound tissue, etc., and provide a favorable environment for skin cell growth. These two factors make the hydrogel provided by the present invention have excellent wound repair ability.

[0037] 3. In vitro cell proliferation and migration experiments demonstrate that the antimicrobial hydrogel provided by the present invention exhibits excellent biocompatibility. Compared to the first day of co-culture, cell migration reached 35% and cell proliferation tripled by the third day of co-culture. Antibacterial experiments also demonstrated that the antimicrobial hydrogel exhibited excellent antimicrobial properties, effectively inhibiting the growth of both Escherichia coli and Staphylococcus aureus. Animal skin repair experiments also demonstrated that the antimicrobial hydrogel provided by the present invention exhibited excellent skin healing properties. When repairing full-thickness skin defects in mice, a 92% healing rate was achieved by the 12th day of repair. Compared to the prior art, at the same 90% healing rate, the antimicrobial hydrogel of the present invention achieved this within 12 days, while similar prior art failed to achieve this level of healing after 14 days. Furthermore, the healing rate of the present invention was approximately 10% higher over the same period. Compared to the blank control group, at the same 70% healing rate, the antimicrobial hydrogel of the present invention achieved this level of healing within 8 days, while the blank control group required 12 days of repair. Furthermore, the healing rate of the present invention was 10% to 30% higher over the same period. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 These are Fourier transform infrared spectra of the modified rhCol III and modified CS prepared in Examples 1 and 2, as well as the raw materials rhCol III and CS used.

[0039] Figure 2 1 is the hydrogen nuclear magnetic resonance spectrum of the modified rhCol III prepared in Example 1 and the raw material rhCol III used.

[0040] Figure 3 1 is the hydrogen nuclear magnetic resonance spectrum of the modified CS prepared in Example 1 and the raw material CS used.

[0041] Figure 4 These are the scanning electron microscopy test results of the internal pore structure of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3 after freeze-drying.

[0042] Figure 5 These are the swelling curves of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhColIII-CS4 after freeze-drying.

[0043] Figure 6 These are the storage modulus curves of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhColIII-CS4.

[0044] Figure 7 These are the degradation curves of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhColIII-CS4.

[0045] Figure 8 These are the antibacterial performance test results of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhColIII-CS4 against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

[0046] Figure 9 (a) shows the bacterial distribution on the bacteria / hydrogel sample, (b) shows the bacterial counting results of each hydrogel, where A, B, C, D, E, and F below the two groups of bar graphs represent the control group, rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4, respectively; (c) shows the SLSM test results of the live-dead staining results; and (d) shows the bacterial morphology under SEM.

[0047] Figure 10 This is a graph showing the cell proliferation of L929 cells in rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4.

[0048] Figure 11 These are laser confocal scanning electron microscopy images of L929 cells after culture in rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 for different periods of time.

[0049] Figure 12 These are the results of cell scratch assays for rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhColIII-CS4. Figures (A) and (B) are the results of inverted microscope observation and wound healing rate calculation, respectively.

[0050] Figure 13Figure 3. Repair effects of rhCol III-CS0 and rhCol III-CS2 mice full-thickness skin defect experiments. Figures (A), (B), and (C) show the gross appearance of the wound, the wound healing rate, and the changes in morphology and area during wound healing, respectively. DETAILED DESCRIPTION

[0051] The following examples further illustrate the injectable antibacterial hydrogel for irregular wound repair and its preparation method provided by the present invention. It should be noted that the following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by persons skilled in the art based on the above disclosure and implemented in accordance with the present invention remain within the scope of protection of the present invention.

[0052] Example 1

[0053] In this example, recombinant humanized type III collagen modified with methacrylic anhydride was prepared according to the following steps:

[0054] (1) Recombinant humanized type III collagen (rhCol III) was added to a buffer solution and stirred at room temperature until the solution became clear and transparent to obtain a rhCol III solution. Methacrylic anhydride (MA) was added to the rhCol III solution and reacted at 40°C for 3 h under stirring.

[0055] In this step, the buffer is a Na2CO3-NaHCO3 buffer, wherein the concentration of Na2CO3 is 8 mg / mL and the concentration of NaHCO3 is 14.7 mg / mL; the concentration of the rhCol III solution is 130 mg / mL, and the volume ratio of the rhCol III solution to MA is 30:1.

[0056] (2) The reaction solution obtained in step (1) was transferred to a 2000Da dialysis bag and dialyzed in ultrapure water for 72 hours. The ultrapure water was frequently replaced during the dialysis process. After the dialysis, the obtained product was freeze-dried to obtain recombinant humanized type III collagen modified with methacrylic anhydride, which was recorded as modified rhCol III.

[0057] The Fourier transform infrared spectra of the modified rhCol III prepared in this example and the raw material rhCol III used are shown in FIG. Figure 1 As shown by Figure 1 It can be seen that compared with the raw material rhCol III, the modified rhCol III has a -1 The peak of modified rhColIII was significantly weakened, forming a weak double peak, which indicates that MA mainly reacted with the amino group of lysine in collagen; the peak of modified rhColIII was at 925cm -1There is a new peak at 1629cm, which is judged to be the out-of-plane bending (deformation) vibration peak of carbon and hydrogen of RC=CH2 structure; -1 、1540cm -1 The peak intensity increased significantly, which is caused by the vibration of the two hydrogen-bonded C=O groups. This shows that MA reacts with the primary amine of the lysine residue in rhCol III, and C=C and C=O are successfully modified on the rhCol III molecular chain.

[0058] The H NMR spectra of the modified rhCol III prepared in this example and the raw material rhCol III used are shown in FIG. Figure 2 As shown, Figures (A) and (B) are the H NMR spectra of the raw material rhCol III and the modified rhCol III, respectively. Figure 2 It can be seen that a new characteristic peak appears at 5-6 ppm in the spectrum after MA modification, which is judged to be a double bond characteristic peak, indicating that MA has been successfully grafted onto rhCol III. The grafting rate of MA in the modified rhCol III prepared in this example is calculated based on the nuclear magnetic resonance hydrogen spectrum and is approximately 66.7%. By adjusting the ratio of rhCol III to MA during preparation, the grafting rate of MA in the modified rhCol III can be adjusted between 65% and 100%. The structure of the modified rhCol III is shown below:

[0059]

[0060] Example 2

[0061] In this embodiment, chitosan grafted with dihydrocaffeic acid was prepared by the following steps:

[0062] (1) Chitosan (CS) was added to deionized water, and the pH value was adjusted to 2.5 with 0.1 mol / L HCl solution. The solution was stirred at room temperature until CS was completely dissolved to form a yellow transparent solution, thereby obtaining a CS solution with a CS concentration of 15 mg / mL.

[0063] Dihydrocaffeic acid (HCA) and EDC were dissolved in 45 mL of an ethanol-water solution to obtain an HCA-EDC mixture. The HCA-EDC mixture had an HCA concentration of 21 mg / mL and an EDC concentration of 66.67 mg / mL. The ethanol-water solution had a volume ratio of ethanol to water of 1:1.

[0064] (2) The pH of the CS solution was adjusted to 5.5 with a 0.1 mol / L aqueous NaOH solution. The HCA-EDC mixture was added dropwise to the CS solution with stirring. The pH of the resulting mixture was adjusted to 5.5 with a 0.1 mol / L aqueous NaOH solution, and stirred at room temperature for 12 h. In this step, the volume ratio of the HCA-EDC mixture to the CS solution was controlled to be 0.45:1.

[0065] (3) The reaction solution obtained in step (2) was placed in an 8000-12000Da dialysis bag and dialyzed with deionized water with a pH value of 4.0 for 72 hours. After the dialysis, the obtained product was freeze-dried to obtain chitosan grafted with dihydrocaffeic acid, which was recorded as modified CS.

[0066] The Fourier transform infrared spectra of the modified CS prepared in this example and the raw material CS used are shown in Figure 2. Figure 1 As shown by Figure 1 It can be seen that the modified CS has a peak at 1520 cm -1 A new absorption peak appeared nearby, which was judged to be caused by the stretching vibration of C=C of dihydrocaffeic acid, proving that dihydrocaffeic acid was successfully grafted onto chitosan.

[0067] The H NMR spectra of the modified CS prepared in this example and the raw material CS used are as follows: Figure 3 As shown, Figures (A) and (B) are the H NMR spectra of the raw material CS and the modified CS, respectively. Figure 3 It can be seen that the modified CS has a new characteristic peak at 6-7ppm, which is judged to be the NH characteristic peak, indicating that dihydrocaffeic acid has been successfully grafted onto chitosan. The grafting rate of dihydrocaffeic acid in the modified CS prepared in this example is calculated based on the nuclear magnetic resonance hydrogen spectrum and is approximately 14.5%. By adjusting the ratio of chitosan to dihydrocaffeic acid during preparation and the molecular weight of chitosan, the grafting rate of dihydrocaffeic acid in the modified CS can be adjusted between 10% and 30%. The structure of the modified CS is shown below:

[0068]

[0069] Example 3

[0070] In this embodiment, an injectable antibacterial hydrogel that can be used for irregular wound repair is prepared in the following steps:

[0071] (1) The modified rhCol III prepared in Example 1 and the modified CS prepared in Example 2 were dissolved in ultrapure water and mixed thoroughly to obtain a mixed solution of modified rhCol III and modified CS. In the mixed solution, the total concentration of modified rhCol III and modified CS was 100 mg / mL, and the mass ratio of modified rhCol III to modified CS was 10.4:1.

[0072] (2) Adding a photoinitiator LAP to the mixed solution of the modified rhCol III and the modified CS obtained in step (1) and mixing thoroughly to remove bubbles therein to obtain a gel precursor solution, wherein the concentration of the photoinitiator in the gel precursor solution is 0.5 wt %.

[0073] (3) The gel precursor obtained in step (2) was cured by irradiation with blue light (365 nm) to obtain an injectable antibacterial hydrogel that can be used for irregular wound repair, which was recorded as rhCol III-CS2.

[0074] (4) Following the same procedures as steps (1) to (3), the mass ratio of modified rhCol III to modified CS in step (1) was changed to 1:0, the concentration of the mixed solution remained unchanged, and the prepared hydrogel was designated as rhCol III-CS0.

[0075] (5) Following the same procedures as steps (1) to (3), the mass ratio of modified rhCol III to modified CS in step (1) was changed to 21.9:1, the concentration of the mixed solution remained unchanged, and the prepared hydrogel was designated as rhCol III-CS1.

[0076] (6) Following the same procedures as steps (1) to (3), the mass ratio of modified rhCol III to modified CS in step (1) was changed to 6.6:1, the concentration of the mixed solution remained unchanged, and the prepared hydrogel was designated as rhCol III-CS3.

[0077] (7) Following the same procedures as steps (1) to (3), the mass ratio of modified rhCol III to modified CS in step (1) was changed to 4.7:1, the concentration of the mixed solution remained unchanged, and the prepared hydrogel was designated as rhCol III-CS4.

[0078] The rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in this example were freeze-dried, then subjected to gold spraying treatment and scanned under a scanning electron microscope. The results are shown in FIG. Figure 4 As shown by Figure 4 It can be seen that cross-linked hydrogels with a three-dimensional network structure have an interconnected pore structure, which is conducive to the rapid penetration of nutrients and is also beneficial to cell growth.

[0079] Example 4

[0080] In this example, the swelling rates of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3 were tested.

[0081] rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 were freeze-dried and weighed. They were then immersed in ultrapure water and allowed to swell at room temperature. The samples were taken out and weighed at regular intervals to calculate the swelling ratio: swelling ratio = (Wt-Wo) / Wo, where Wo is the weight before being placed in ultrapure water (after freeze-drying) and Wt is the weight when taken out and weighed after being immersed in ultrapure water for a period of time.

[0082] According to the swelling rate at different time points, the swelling curve is drawn. The results are as follows Figure 5 As shown in the figure, freeze-dried rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 rapidly absorbed water within 3 minutes, reaching over 85% of their maximum swelling. These materials also exhibited well-balanced swelling properties, facilitating the rapid absorption of blood and exudate, while maintaining a stable morphology at the wound site. As the modified CS content increased, the maximum swelling rate of the materials gradually increased, demonstrating that the modified CS enhanced their water absorption.

[0083] Example 5

[0084] In this example, the storage modulus of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3 was tested.

[0085] The storage modulus of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 were tested by dynamic mechanical analysis (DMA). Figure 6 As shown. Figure 6 It can be seen that with the increase of the modified CS content in the hydrogel, the storage modulus of the hydrogel will decrease, but overall, the mechanical properties of these hydrogels can meet the application requirements for wound repair.

[0086] Example 6

[0087] In this example, the biodegradability of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3 was tested.

[0088] rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 were freeze-dried, weighed, immersed in PBS buffer, and placed at a constant temperature of 20°C for degradation. The samples were taken out at regular intervals, freeze-dried, and weighed. The degradation rate was calculated as follows: degradation rate = (Wo - Wt) / Wo, where Wo is the weight before exposure to PBS buffer and Wt is the weight after freeze-drying after a period of degradation.

[0089] According to the degradation rate at different time points, the degradation curve is drawn. The results are as follows Figure 7 As shown. Figure 7 It can be seen that rhColIII-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 all have biodegradability and can be biodegraded under the action of PBS buffer, which is beneficial to tissue regeneration and repair.

[0090] Example 7

[0091] The antibacterial activities of the hydrogels (rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4) prepared in Example 3 were evaluated using Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923).

[0092] A single colony was inoculated into 10 mL of tryptic soy broth (TSB) and cultured at 37°C for 12 h. 4 CFUs were mixed evenly with each sterilized hydrogel in 1 mL of bacterial suspension and cultured statically. After 12 h, the mixed culture was diluted 10 5 The cells were evenly plated on trypticase soy agar (TSA) at 100 μg / mL and incubated for 24 hours. The number of colonies in each group was recorded. The control group received only the bacterial suspension without gel. The bacterial survival rate and inhibition rate were calculated.

[0093] Bacterial survival rate (%) = N1 / N0×100%, where N1 is the number of bacteria in each hydrogel, N0 is the number of bacteria in the control group using only bacterial suspension, and inhibition rate (%) = 1-bacterial survival rate (%).

[0094] Bacterial survival rate Figure 8 As shown, Figure 8 A, B, C, D, and E below the two groups of bar graphs represent rhColIII-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4, respectively. Figure 8 It can be seen that the antibacterial rates of rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 against Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923) are all above 96%, and the antibacterial rate of rhCol III-CS3 against Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923) is above 99%, showing good antibacterial effect.

[0095] In addition, the obtained bacteria / hydrogel samples were stained with the Live / Dead backlit bacterial activity kit and visualized by confocal laser scanning microscopy (CLSM, Leica TCS SP2, Leica Microsystems, Germany). In the CLSM detection results, green fluorescence represents live bacteria and red fluorescence represents dead bacteria. After dehydration with gradient ethanol, the microscopic morphology of bacteria in each hydrogel was observed using a scanning electron microscope (SEM, HITACHI S-800, Japan). The results are shown in Figure 2. Figure 9 As shown, Figure 9 (a) shows the bacterial distribution on the bacteria / hydrogel sample, (b) shows the bacterial counting results of each hydrogel, where A, B, C, D, E, and F below the two groups of bar graphs represent the control group, rhCol III-CS0, rhCol III-CS1, rhColIII-CS2, rhCol III-CS3, and rhCol III-CS4, respectively; (c) shows the SLSM test results of the live-dead staining results; and (d) shows the bacterial morphology under SEM.

[0096] Depend on Figure 9 It can be seen that rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 have obvious inhibitory effects on Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923).

[0097] Example 8

[0098] In this example, the biocompatibility of rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3 was tested.

[0099] rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 of the same size were sterilized by UV light and immersed in sterile PBS buffer overnight. L929 cells were digested with trypsin and 5×10 cells were added to each hydrogel sample. 3 The cell suspension was added dropwise onto each hydrogel and allowed to stand in an incubator for 30 min. The culture medium was then added and the cells were cultured in an incubator at 37°C and 5% CO2. The culture medium was replaced every other day during the culture period.

[0100] The culture medium is obtained by adding a mixture of penicillin and streptomycin and fetal bovine serum to DMEM basal culture medium. The mass concentration of the penicillin and streptomycin mixture in the DMEM culture medium is 1%, and the mass concentration of the fetal bovine serum is 10%. The penicillin and streptomycin mixture is provided by HyClone.

[0101] After culturing for 1 day and 3 days, the hydrogels were taken out and the cell proliferation was tested using CCK-8. The results were as follows: Figure 10 As shown, Figure 10 The A, B, C, D, and E columns below the two groups of histograms represent rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4, respectively. After culturing for one and three days, the hydrogels were removed and washed twice with PBS buffer. The washed hydrogels were then immersed in a PBS solution containing FDA and PI for approximately one minute. The growth and distribution of the cells were observed using a confocal laser scanning microscope (CLSM). The results are shown in Figure 2. Figure 11 shown.

[0102] Depend on Figure 10 、 11 As shown, L929 cells grew well in rhCol III-CS2, with significantly better proliferation than in the other groups, indicating that rhCol III-CS2 has the best cytocompatibility. In contrast, L929 cells in rhCol III-CS0 proliferated more slowly and even aggregated. This suggests that an appropriate content of modified CS in the hydrogel can promote cell spreading and proliferation on the hydrogel.

[0103] Example 9

[0104] In this example, a cell scratch test was performed on rhCol III-CS0, rhCol III-CS1, rhCol III-CS2, rhCol III-CS3, and rhCol III-CS4 prepared in Example 3.

[0105] Cultured in serum-free medium, using 48-well plates, seeded with 1 × 10 4 L929 cells were cultured for 12 hours, and the wound surface was treated with a sterile pipette tip, rinsed 3 times with PBS, and then incubated with hydrogel. The cells were photographed under an inverted microscope after 0 hours and 24 hours of incubation, and the wound healing rate (wound closure (%)) was calculated. Wound closure (%) = (A0-A24) / A0×100%, where A0 is the wound area at 0 hours of incubation and A24 is the wound area after 24 hours of incubation. The results are shown in Figure 2. Figure 12 As shown in Figure 1, (A) and (B) are the results of observation under an inverted microscope and the calculation results of wound healing rate respectively. Figure 12 It can be seen that the wound recovery effect of rhCol III-CS2 is better than that of other groups.

[0106] Example 10

[0107] In this example, the application effects of rhCol III-CS0 and rhCol III-CS2 prepared in Example 3 as skin repair materials were tested.

[0108] Male BALB / c mice (weighing 25 g ± 5 g, 6-8 weeks old) were used. Mice were housed for one week prior to surgery to acclimate to the environment, and all surgical instruments were sterilized by high temperature and high pressure. The mice were randomly divided into three groups: rhCol III-CS0, rhCol III-CS2, and a blank control group.

[0109] During surgery, mice were anesthetized with an intraperitoneal injection of 5% chloral hydrate (7.5 mL / 1 kg) and fixed on the operating table with their backs facing up. The backs of the mice were then cleaned and circular full-thickness skin defects with a diameter of 10 mm were created on both sides of the backs of the mice using a perforator. The rhCol III-CS0 group prepared in Example 3 was dripped into the wounds of the rhCol III-CS0 group mice, and the rhCol III-CS2 group prepared in Example 3 was dripped into the wounds of the rhCol III-CS2 group mice. The blank control group did not drip any material into the wounds, but was irradiated with a blue light flashlight for 30 seconds and secured with an elastic bandage around the waist and abdomen. After surgery, the mice were raised in a suitable environment and sacrificed on days 4, 8, and 12. The gross appearance and area of ​​the skin wounds were recorded, and the wound repair rate was calculated. Three defect experiments were performed simultaneously at each time point in the rhCol III-CS0 group, rhCol III-CS2 group, and blank control group.

[0110] The test results of this embodiment are as follows Figure 13 As shown, (A) (B) (C) are the general view of the wound, the wound healing rate, and the changes in morphology and area during the wound healing process. Figure 13 (B) Figure 3 A, B, and C below the group bar graph represent the blank control group, rhCol III-CS0 group, and rhCol III-CS2 group, respectively. Figure 13 As shown, compared with the blank control group, the wound healing rates in the rhCol III-CS0 and rhCol III-CS2 groups were faster, indicating that rhCol III-CS0 and rhCol III-CS2 can accelerate wound healing, reduce scarring, and that rhCol III-CS2 is more effective in promoting wound healing. Compared with the blank control group, the wound healing rate in the rhCol III-CS2 group was at least 20% higher at both 8 and 14 days. Compared with the rhCol III-CS0 group, the wound healing rate in the rhCol III-CS2 group was at least 10% higher at both 8 and 14 days. After 12 days of repair, the wound healing rate in the rhCol III-CS2 group reached over 90%, demonstrating a significant effect in promoting skin repair.

Claims

1. An injectable antibacterial hydrogel that can be used for irregular wound repair, characterized in that: The antibacterial hydrogel is formed by photoinitiated cross-linking of a gel precursor solution containing modified recombinant type III humanized collagen with photocuring properties, chitosan grafted with dihydrocaffeic acid, and a photoinitiator. The modified recombinant humanized type III collagen has a three-dimensional network structure, and the chitosan grafted with dihydrocaffeic acid is evenly distributed in the three-dimensional network structure of the modified recombinant humanized type III collagen. In the antibacterial hydrogel, the mass ratio of the modified recombinant humanized type III collagen to the chitosan grafted with dihydrocaffeic acid is (8-11):

1.

2. The injectable antibacterial hydrogel for irregular wound repair according to claim 1, characterized in that: The modified recombinant humanized type III collagen with light-curing properties is recombinant humanized type III collagen modified with methacrylic anhydride.

3. The injectable antibacterial hydrogel for irregular wound repair according to claim 2, characterized in that: In the modified recombinant humanized type III collagen with light-curing properties, the grafting rate of methacrylic anhydride is 65% to 100%.

4. The injectable antibacterial hydrogel for irregular wound repair according to any one of claims 1 to 3, characterized in that: The chitosan grafted with dihydrocaffeic acid is formed by amidation reaction between the carboxyl group of dihydrocaffeic acid and the amino group of chitosan. In the chitosan grafted with dihydrocaffeic acid, the grafting rate of dihydrocaffeic acid is 10% to 30%.

5. The method for preparing the injectable antibacterial hydrogel for repairing irregular wounds according to any one of claims 1 to 3, characterized in that: The following steps are involved: Modified recombinant humanized type III collagen with photocuring properties, chitosan grafted with dihydrocaffeic acid, and a photoinitiator are dissolved in water, and the bubbles in the resulting solution are removed to obtain a gel precursor solution. Under blue light irradiation, the modified recombinant humanized type III collagen with photocuring properties undergoes a cross-linking reaction to obtain an injectable antibacterial hydrogel that can be used for irregular wound repair.

6. The method for preparing the injectable antibacterial hydrogel for irregular wound repair according to claim 5, characterized in that: In the gel precursor solution, the total concentration of modified recombinant humanized type III collagen with photocurable properties and chitosan grafted with dihydrocaffeic acid is 80~120 mg / mL.

7. The method for preparing the injectable antibacterial hydrogel for repairing irregular wounds according to claim 5, characterized in that: In the gel precursor solution, the content of the photoinitiator is 0.5 wt%~2 wt%.

8. Use of the injectable antibacterial hydrogel for irregular wound repair according to any one of claims 1 to 3 in the preparation of wound repair materials.

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