Controllable release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration

Abstract

The invention discloses a controllable release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration, and a preparation method comprises the following steps: adding horse radish peroxidase into a mixed solution containing modified chitosan, magnesium lithium silicate nanosheets and recombinant human type III collagen, uniformly mixing, adding H2O2, and adjusting the pH value of the solution to be neutral to obtain a hydrogel pre-solution; and injecting the hydrogel pre-liquid into a mold through an injector to obtain the hydrogel dressing. According to the multifunctional composite hydrogel dressing provided by the invention, components of hydrogel form a stable hydrogel network structure through interaction of chemical bonds, static electricity and hydrogen bonds, and controllable release of recombinant human III-type collagen is realized by controlling the input proportion of magnesium lithium silicate; the hydrogel also has multiple functions of resisting inflammation and oxidation, promoting cell migration and angiogenesis, inhibiting bacteria, promoting full-thickness skin defect repair, inhibiting scar generation, promoting skin appendage regeneration and the like, and provides a better treatment effect on clinical treatment of skin full-thickness defects.

Description

Technical Field

[0001] The present invention relates to the technical field of skin wound repair materials, in particular to a controllable-release type III collagen composite hydrogel dressing for promoting the repair and regeneration of full-thickness skin defects. Background Art

[0002] Skin injuries are common in daily life, primarily caused by mechanical trauma, burns, scalds, diabetic foot, and pressure ulcers. Large, full-thickness skin defects in clinical practice often have difficulty healing quickly. If infection or excessive inflammation occurs, they can easily transform into chronic wounds, significantly increasing the difficulty, time, and cost of treatment. Therefore, the development of antibacterial wound dressings that promote full-thickness skin repair and regeneration is of great clinical significance.

[0003] Traditional dressings, such as cotton wool and gauze, all absorb wound exudate to varying degrees and contribute to wound management. Their primary function is to absorb wound secretions and allow the evaporation of moisture, thereby keeping the wound dry and preventing harmful bacteria from entering the wound and causing infection. However, they can easily adhere to the wound, causing secondary damage during dressing changes, and their ability to absorb exudate is limited. Currently, novel dressings under development include alginate, hydrogels, films, hydrocolloids, foams, and some natural biomaterial dressings and antimicrobial dressings. These new wound dressings can absorb excess exudate or retain moisture, maintaining wound moisture within an optimal range and promoting wound healing. However, as research and development progress, these treatments have limitations, such as low wound neovascularization, skin contraction caused by the transformation of dermal fibroblasts, scarring, and a lack of attachments such as hair follicles and sweat glands in the newly formed skin. Furthermore, the addition of antibiotics, anti-inflammatory drugs, or growth factors to wound dressings is currently the primary method for promoting wound repair. However, the problems of these bioactive drugs being easily inactivated, prone to drug resistance and toxic side effects cannot be ignored.

[0004] The wound healing process generally involves hemostasis, inflammation, proliferation, and remodeling. Chronic wound healing is more challenging than standard wound healing. Vascular damage and impaired vascular regeneration are the pathological basis for most refractory wounds and are one of the main causes of wound regeneration dysfunction. Pathological changes in both macrovascular and microvascular vessels lead to insufficient blood flow to the wound surface, preventing it from delivering sufficient oxygen and nutrients for wound repair. This impairs the normal inflammatory response and increases the risk of infection, ultimately causing the wound to remain non-healing. Therefore, promoting wound vascular regeneration and restoring blood flow are important therapeutic targets for accelerating the healing of refractory wounds. Furthermore, chronic wounds are hampered by excessive inflammation, causing the healing process to remain in the inflammatory phase for a prolonged period. Disruptions in any of these wound healing processes can lead to excessive scarring. While there are reports of using dressing materials to modulate the wound's inflammatory microenvironment, these dressing materials often only address a single cause of wound non-healing, limiting overall healing effectiveness. Furthermore, complete repair of skin structure, such as hair follicle regeneration, is difficult to achieve, and scar tissue regeneration is often present within the wound. Therefore, it is necessary to develop multifunctional skin dressings that can simultaneously promote angiogenesis, control inflammation, reduce oxidative damage, promote wound repair and regeneration, and reduce scar formation. Summary of the Invention

[0005] In response to the clinical problems of poor vascular regeneration, chronic inflammation, susceptibility to infection, and scarring after healing in skin defects, which limit the wound healing and repair effect, the present invention provides a controlled-release type III collagen composite hydrogel dressing that promotes the repair and regeneration of full-thickness skin defects.

[0006] The controlled-release type III collagen composite hydrogel dressing provided by the present invention promotes the repair and regeneration of full-thickness skin defects. It is prepared by oxidative cross-linking in an aqueous solvent: modified chitosan (qCSc), lithium magnesium silicate (Laponite) nanosheets, and recombinant human type III collagen (rhCol III). The addition of Laponite nanosheets to the modified chitosan not only improves the mechanical and gel-forming properties of the hydrogel, but also allows for the controlled, slow release of rhCol III.

[0007] The molecular structural formula of the modified chitosan is as follows:

[0008]

[0009] The preparation method of the composite hydrogel dressing is as follows: horseradish peroxidase (HRP) is added to a mixed solution containing modified chitosan (qCSc), lithium magnesium silicate (Laponite) nanosheets and recombinant human type III collagen (rhCol III) and mixed evenly, and then H2O2 is added and the pH of the solution is adjusted to neutral to obtain a hydrogel pre-liquid, and the hydrogel pre-liquid is injected into a mold through a syringe to obtain a hydrogel dressing.

[0010] In the hydrogel pre-solution, the amount of modified chitosan accounts for 0.5%-5% of the total mass of the hydrogel pre-solution, the amount of lithium magnesium silicate nanosheets accounts for 0.2%-6% of the total mass of the hydrogel pre-solution, and the amount of recombinant human type III collagen accounts for 0.1-1% of the total mass of the hydrogel pre-solution. The concentration of horseradish peroxidase is 5-20 ppm, and the concentration of H2O2 is 1-2‰. The proportion of Laponite in the hydrogel pre-solution must be strictly controlled, as Laponite plays a major role in controlling the release of type III collagen.

[0011] Preferably, the modified chitosan is prepared by the following two steps:

[0012] (1) Chitosan (CS) was dissolved in a mixture of water and glacial acetic acid, 2,3-epoxypropyltrimethylammonium chloride was added, and the mixture was heated to 50-60°C and stirred for 20-25 hours. After the reaction, the mixture was dialyzed and freeze-dried to obtain quaternized chitosan, referred to as qCS.

[0013] (2) qCS was dissolved in water and the pH was adjusted to 4-5 to obtain solution A; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and caffeic acid (CA) were added to a mixed solvent of ethanol and water, stirred to dissolve, and then stirred and activated for 20-120 minutes. The activated solution was added dropwise to solution A, and the pH was adjusted to 4-5. The reaction was continued for 10-14 hours. After the reaction was completed, the modified chitosan was dialyzed and freeze-dried to obtain qCSc.

[0014] Compared with the prior art, the present invention is beneficial in that:

[0015] (1) The composite hydrogel dressing of the present invention is made of three components: modified chitosan, lithium magnesium silicate nanosheets, and recombinant human type III collagen. The hydrogel mainly forms a network through oxidative cross-linking of catechol groups; the free catechol groups on the qCSc molecular chain have strong physical adhesion and can interact with Laponite nanosheets and rhColⅢ; the Laponite nanosheets are negatively charged on the surface and positively charged on the end. Due to the charge effect, the Laponite nanosheets in the hydrogel can not only self-assemble, but also interact with qCSc and rhColⅢ through electrostatic interaction and silicate-mediated hydrogen bonding, thereby accelerating the gelation speed of the hydrogel, building a composite hydrogel with stable multi-network performance, and enhancing the mechanical properties of the hydrogel; in addition, the adsorption of rhColⅢ by catechol groups and Laponite nanosheets gives the hydrogel the ability to controllably release rhColⅢ.

[0016] (2) The present invention provides a multifunctional composite hydrogel dressing that integrates anti-inflammatory and antioxidant properties, promotes cell migration, angiogenesis, antibacterial properties, promotes full-thickness skin defect repair, and regenerates skin appendages such as hair follicles, providing a good therapeutic effect for the clinical treatment of full-thickness skin defects. This composite hydrogel promoted skin wound healing and repair, as well as subcutaneous hair follicle regeneration in a rat full-thickness skin defect model.

[0017] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the modification principle of chitosan.

[0019] Figure 2 This is a comparison chart of the anti-inflammatory effects of different modified chitosans.

[0020] Figure 3 This is a comparison chart of the antioxidant effects of different modified chitosans.

[0021] Figure 4 This is a diagram of the gelation mechanism of the composite hydrogel of the present invention.

[0022] Figure 5 These are scanning electron micrographs of qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel.

[0023] Figure 6 This is a diagram showing the effect of different amounts of Laponite nanosheets on hydrogel.

[0024] Figure 7 This is a graph showing the effect of Laponite nanosheet dosage on the release of rhColⅢ in the composite hydrogel.

[0025] Figure 8 This is a comparison chart of cell viability tested by different hydrogels in a cell proliferation experiment.

[0026] Figure 9 These are inverted fluorescence microscope photos of different hydrogels in cell live-death experiments.

[0027] Figure 10 This is a diagram showing the effects of different hydrogels in promoting cell migration.

[0028] Figure 11 This is a diagram showing the effect of different hydrogels in promoting HUVEC cell tube formation in vitro.

[0029] Figure 12 This is a graph showing the results of different hydrogels promoting HUVEC angiogenic gene expression.

[0030] Figure 13 This is a diagram showing the effect of different hydrogels on the scavenging ability of ROS in RAW264.7 cells.

[0031] Figure 14 Figure 2 shows the anti-inflammatory effects of different hydrogels.

[0032] Figure 15 This is a diagram showing the antibacterial effects of different hydrogels.

[0033] Figure 16 These are photos of skin healing in animal experiments using different hydrogels.

[0034] Figure 17 This is a comparison chart of skin healing area and healing rate in animal experiments using different hydrogels.

[0035] Figure 18 It is a comparison chart of the thickness of the epidermis of the new skin and the density of regenerated hair follicles in the new skin. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] Example 1

[0038] The modification principle of chitosan is as follows Figure 1 As shown in Figure 2, chitosan (CS) was modified with (2,3-epoxypropyltrimethylammonium chloride)-GTAC and caffeic acid (CA) in two steps to produce qCSc. In the first step, 1 g of CS was dissolved in 36 ml of water and 0.36 ml of glacial acetic acid. The mixture was stirred and heated to 55°C, followed by the addition of 0.94 g of GTAC. After 24 hours of reaction, the solution was transferred to a dialysis bag and dialyzed against RO water for three days before being freeze-dried in a freeze dryer to obtain quaternized chitosan (qCS). In the second step, 0.8 g of qCS was dissolved in 40 ml of water and an appropriate amount of dilute hydrochloric acid was added to adjust the pH to 4.5. 3.48 g of EDC, 2.09 g of NHS, and 1.63 g of CA were added to a mixed solvent of 48 ml of ethanol and 40 ml of water, stirred to dissolve, and then stirred for 2 hours for activation. The activated solution was added dropwise to the qCS solution, and the pH was adjusted to 4.5. After continuing the reaction for 12 hours, the solution was transferred to a dialysis bag and dialyzed for 3 days with a solvent of ethanol:RO water volume ratio = 2:1, and then dialyzed for 3 days with RO water. The modified chitosan qCSc was obtained by freeze-drying.

[0039] Figure 1The acetyl groups in the molecular structure of chitosan (CS) are inherent to the chitosan itself. Chitosan is produced by deacetylation of chitin, but this is generally not completely removed, leaving some residue. The first step of modification, modification with quaternary ammonium salts, improves the solubility of chitosan in water, thereby ensuring the smooth progress of the second step of caffeic acid modification. Furthermore, the residual amino group content is proportional to the degree of caffeic acid grafting in the second step of modification. The biofunctionality of chitosan is positively correlated with its amino group content. Therefore, to ensure the smooth progress of the second step of modification, a low degree of quaternary ammonium substitution is necessary. The present invention synthesizes qCS with two degrees of substitution (DSS) of 10% and 20%, namely qCS-10 and qCS-20. In the second step of caffeic acid synthesis, an activated caffeic acid solution is added to the qCS solution. The final mixed solution has a water:ethanol ratio of 2:1. At a pH of 4.6-4.8, the use of qCS-10 causes chitosan precipitation, hindering qCS synthesis. However, the use of qCS-20 eliminates this chitosan precipitation issue. Therefore, the present invention experimentally demonstrates that a DSS of approximately 20% is optimal, allowing the second step of modification to proceed smoothly.

[0040] The chitosan modification method of the present invention can effectively increase the degree of substitution of caffeic acid, making the bioactivity of chitosan more advantageous. At the same time, the use of glacial acetic acid is avoided in the caffeic acid grafting, which can avoid the side reactions caused by acetic acid. In addition, in the second step of modification, a mixed solution of water and ethanol is used as the dialysate to remove unreacted small molecule residues (especially CA, which is slightly soluble in cold water) as much as possible, making the product purer and avoiding the toxicity of small molecules. At the same time, the water solubility of qCSc is improved by the quaternary ammonium salt modification in the first step, and it has higher stability at physiological pH.

[0041] In addition, studies have found that the optimal reaction pH condition for EDC is 4.7-6.0. Too low a pH will greatly reduce the reaction efficiency and reduce the caffeic acid grafting rate. In the present invention, the water solubility of chitosan is improved by grafting modification with quaternary ammonium salts, so that it can be dissolved under pH neutral conditions, ensuring that the pH of the caffeic acid modification reaction is controlled in the range of 4.6-4.8. If the pH is too high, caffeic acid is easily oxidized, affecting the adhesion and antioxidant capacity of the product qCSc and related biological activities, and may even cause oxidative cross-linking of the product, leading to synthesis failure. In addition, the present invention uses EDC+NHS to activate caffeic acid, which can generate more stable activated esters, thereby improving the reaction efficiency, that is, increasing the caffeic acid grafting rate. The prepared qCSc has obvious anti-inflammatory and antioxidant properties, and a higher caffeic acid grafting rate is the guarantee of this function. Related experimental test data can be found in Figure 2 and Figure 3 . Figure 2The results of different material treatment groups were obtained by stimulating Raw246.7 cells with LPS. qCSc50 and qCSc500 refer to the final concentrations of modified chitosan in the culture medium of 50 and 500 μg / ml, respectively. The figure shows that modified chitosan qCSc has significant anti-inflammatory properties. The anti-inflammatory ability of qCSc is much stronger than that of qCS, and the anti-inflammatory performance is proportional to the material concentration. The antioxidant capacity of qCSc and qCS was determined in vitro by the DPPH method (24 hours). The results are shown in Figure 3 , it can be seen that the antioxidant capacity of qCSc is much stronger than that of qCS, and the antioxidant performance is proportional to the material concentration.

[0042] Example 2

[0043] A controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration is prepared by mixing a qCSc aqueous solution (prepared in Example 1) and a Laponite nanosheet dispersion, then adding an rhCol III solution and mixing, then adding HRP and mixing, adding H₂O₂, and adjusting the pH of the solution to neutral with NaOH. After rapid mixing, the dressing is injected into a mold via a syringe to obtain the composite hydrogel dressing, referred to as the qCSc / Lap / rhCol III composite hydrogel. The concentrations of the components of the hydrogel precursor solution are as follows: qCSc accounts for 1% of the total mass of the hydrogel precursor solution, lithium magnesium silicate nanosheets account for 0.25% of the total mass of the hydrogel precursor solution, recombinant human type III collagen accounts for 0.2% of the total mass of the hydrogel precursor solution, the HRP concentration is 10 ppm, and the H₂O₂ concentration is 1‰. This example uses recombinant humanized type III collagen from Jinbo Biotechnology.

[0044] The hydrogel formation mechanism is as follows Figure 4 As shown, the hydrogel network is primarily formed through oxidative cross-linking of catechol groups. The free catechol groups on the qCSc molecular chains exhibit strong physical adhesion, interacting with Laponite nanosheets and rhCol III. Due to the charge interaction, the Laponite nanosheets in the hydrogel both self-assemble and interact with the qCSc molecular chains and rhCol III, accelerating hydrogel formation and enhancing the hydrogel's mechanical properties. The adsorption of rhCol III by the catechol groups and Laponite nanosheets imparts the hydrogel with the ability to controllably release rhCol III.

[0045] Comparative Example 1

[0046] On the basis of Example 2, rhColⅢ was deleted during the preparation of the hydrogel to prepare a two-component qCSc / Lap hydrogel.

[0047] Comparative Example 2

[0048] On the basis of Example 2, Laponite nanosheets and rhColⅢ were deleted during the preparation of the hydrogel to prepare a single-component qCSc hydrogel.

[0049] The performance tests of the hydrogels prepared in Example 2 and Comparative Examples 1-2 are as follows:

[0050] (1) Figure 5 The following are scanning electron micrographs of qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhCol III composite hydrogel after freeze-drying. It can be seen that the qCSc / Lap / rhCol III composite hydrogel exhibits a porous structure after freeze-drying, similar to that of the qCSc and qCSc / Lap hydrogels.

[0051] (2) Based on Comparative Example 2, the content of Laponite nanosheets (abbreviated as Lap) in the qCSc / Lap hydrogel was changed to prepare the mechanical properties data of qCSc / Lap hydrogels with different Lap contents. Figure 6 . Figure 6 In the figure, (a) is the gelation time graph, (b) is the compressive strength graph, and (c) is the stress-strain curve. The percentage of Lap refers to the percentage of the mass of Laponite nanosheets to the total mass of the hydrogel. In Figure (c), Lap0, Lap0.5, Lap1.0, Lap1.5, and Lap2.0 refer to the percentage of the mass of Laponite nanosheets to the total mass of the hydrogel, which are 0, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. It can be seen that when Lap is mixed with modified chitosan, as the concentration of Lap added increases, the gelation time of the chitosan-based hydrogel shortens and the mechanical properties gradually enhance. This shows that there is an interaction between Lap and modified chitosan.

[0052] On the basis of Example 2, the amount of Laponite nanosheets added was changed to test the release of rhColⅢ in the composite hydrogel obtained. The method for determining the release of rhColⅢ was as follows: the hydrogel (volume of 200ul) was placed in a 2ml EP tube and 1ml PBS solution was added. The EP tube was placed in a 37°C constant temperature oven. All liquids were taken out at 2h, 6h, 12h and 24h, and 1ml of fresh PBS solution was added. The protein content of the sample was determined by the Coomassie Brilliant Blue method, which was the amount of rhColⅢ released during the time period. The cumulative release amount was obtained by adding the release amounts in different time periods. The ratio of the cumulative release amount to the initial content of rhColⅢ in the hydrogel (about 400ug) was the cumulative release rate. The test results are shown in Figure 7In the figure, LAP0, LAP2.5, LAP5, LAP7.5, and LAP10 refer to Laponite nanosheet concentrations in the hydrogel of 0, 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml, and 10 mg / ml, respectively. It can be seen that after the addition of type III collagen rhCol III, the release rate of rhCol III slows down with increasing Lap concentration. This demonstrates that the release rate of rhCol III can be regulated by controlling the amount of Lap added, achieving controlled release of rhCol III.

[0053] (3) In the cell proliferation experiment, four groups were set up for comparison: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. The control group was a blank group without hydrogel. DMEM culture medium with 10% serum was used in a 24-well plate, and 5x10 cells were added to each well. 4 L929 cells, after the cells were plated overnight, the corresponding hydrogel (volume 200ul) was added to the wells, and the cell viability was measured using CCK8 after 1 day and 3 days respectively. The test results are shown in Figure 8 The experimental results showed that compared with qCSc hydrogel and qCSc / Lap hydrogel, qCSc / Lap / rhColⅢ composite hydrogel had a significant pro-proliferation effect, which was mainly due to rhColⅢ.

[0054] (4) In the cell live-death experiment, four groups were set up for comparison: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. DMEM culture medium with 10% serum was used in a 24-well plate, and 5x10 cells were added to each well. 4 HUVEC cells were plated overnight and the corresponding hydrogel (volume 200ul) was added to the wells. FDA / PI staining was performed after 24h and 48h, respectively, and then photographed using an inverted fluorescence microscope. The experimental results are shown in Figure 9 The experimental results showed that there were basically no dead cells in the qCSc / Lap / rhColⅢ composite hydrogel group, indicating that the hydrogel material has good biocompatibility and is non-toxic.

[0055] (5) In the cell migration experiment, four groups were set up for comparison: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. A 24-well transwell plate was used, 1 ml of 10% serum DMEM medium was added to the well, 200 μl of cell suspension (resuspended in the DMEM medium extract of the hydrogel) was added to the chamber, and 4x10 cells were added to each well. 4After culturing HUVEC cells for 20 hours, they were fixed with 4% paraformaldehyde solution for 20 minutes and then stained with crystal violet for 30 minutes. The polycarbonate membrane at the bottom of the chamber was carefully removed and fixed on a glass slide. The sections were scanned and statistically analyzed using Image J. The experimental results are shown in Figure 2. Figure 10 As shown in the figure, compared with the Control group, all the hydrogel groups promoted cell migration, among which the qCSc / Lap / rhColⅢ composite hydrogel had the most significant promoting effect. This is because qCSc and rhColⅢ have a synergistic effect in promoting HUVEC migration.

[0056] (6) In the HUVEC cell tube formation experiment in vitro, four groups were set up for comparative experiments: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. A 96-well plate was used, and 100 μL of diluted Matrigel was added to each well (Matrigel: culture medium volume ratio = 2:1). After incubation in the incubator for 1 hour, the cell suspension (resuspended in DMEM culture medium extract of the hydrogel) was added, and 3x10 cells were added to each well. 4 After culturing HUVEC cells for 4 hours, bright field images were taken using a fluorescence microscope. The images were statistically analyzed using Image J. The experimental results are shown in Figure 2. Figure 11 As shown, compared with the Control group, the hydrogel group could promote the tube formation of HUVEC cells in vitro.

[0057] (7) In the HUVEC cell angiogenesis gene expression experiment, four groups were set up for comparison: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. DMEM culture medium with 10% serum was used, and 24-well plates were used. 5x10 4 HUVEC cells were plated overnight and the corresponding hydrogel (200 μl) was added to the wells. RNA was extracted using the Tiangen RNA extraction kit and reverse transcribed 3 days later. The expression of angiogenesis-related genes was determined by PCR. The experimental results are shown in Figure 12 It can be seen that compared with the Control group, the hydrogel group can promote the expression of HUVEC angiogenic genes.

[0058] (8) By adding 100 μM H2O2 to the culture medium to induce RAW264.7 cells to produce ROS, the ability of the material to scavenge ROS was investigated. In the experiment, four groups were set up: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. DMEM culture medium with 10% serum was used in a 24-well plate, and 2×10 cells were added to each well. 5RAW264.7 cells, 6 parallel samples, 2 well plates, after the cells were plated overnight, the corresponding hydrogel (volume 200ul) was added to the wells, and after 24 hours, it was stained with Hochest and DCFH-DA. Then one well plate was photographed with fluorescence, and the other well plate was analyzed using flow cytometry. The experimental results are shown in Figure 2. Figure 13 As shown in the figure, compared with the Control group, the hydrogel group could reduce the ROS level in RAW264.7 cells, and qCSc and rhColⅢ had a synergistic effect.

[0059] (9) The anti-inflammatory effect of the material was investigated by adding 250 ng / ml LPS to the culture medium to induce the expression of inflammatory factors in RAW264.7 cells. Four groups were set up in the experiment: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. DMEM culture medium with 10% serum was used in a 24-well plate, and 5x10 4 RAW264.7 cells were plated in 6 parallel samples. After the cells were plated overnight, the corresponding hydrogel (volume 200ul) was added to the wells. The supernatant was collected after 24h and 72h, and the IL-6 expression was measured by Elisa. The experimental results are shown in Figure 2. Figure 14 As shown in the results, compared with the Control group, the hydrogel group could reduce the expression of IL-6 in RAW264.7 cells, and qCSc and Laponite had synergistic anti-inflammatory effects.

[0060] (10) Take 100 μL of Staphylococcus aureus and Escherichia coli culture solutions respectively in 20 mL of LB medium, place them in a shaker at 37 °C for 12 h, and dilute the resulting culture solutions to OD 600 The value is 0.1, which means the bacterial concentration is 1×10 7 CFU / mL. Then dilute the bacterial solution 100 times, that is, the bacterial concentration is 1×10 5 CFU / mL, take two 24-well plates and add diluted Staphylococcus aureus and Escherichia coli solutions, 1 ml per well. Set up four groups: Control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. Add different hydrogels (volume 200ul) to each group, with 3 parallel samples in each group. After 12 hours, measure the OD on a microplate reader. 600 The antibacterial rate was calculated by taking the bacterial solution from the well plate and diluting it by the same multiple and then applying it to the culture dish with solid culture medium (the control group was diluted to 1×10 5 CFU / mL), and took photos and recorded them after 12 hours. Figure 15As shown, the results showed that the hydrogel had an inhibitory effect on Staphylococcus aureus and Escherichia coli, and the inhibitory ability came from qCSc.

[0061] (11) The animal experiment was modeled using SD male rats, weighing about 200g each. The experiment was divided into four groups, namely the gauze control group (Control), qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel. Two 10mm diameter full-thickness skin defects were symmetrically made on the back of each rat, and the wounds were fixed with silicone rings to prevent shrinkage. After the material was added, it was covered with 3M1624W transparent dressing and then covered with gauze and tape. Pictures were taken and samples were taken at 3d, 7d, and 12d, respectively. After sampling, the skin was fixed with paraformaldehyde. The results are shown in the figure below. Figure 16 and Figure 17 As shown in the figure, compared with the gauze control group (Control), the healing speed of the single-component qCSc hydrogel, two-component qCSc / Lap hydrogel and three-component qCSc / Lap / rhColⅢ hydrogel groups accelerated in turn. This shows that the three materials qCSc, Lap and rhColⅢ have a synergistic effect on wound repair. At 12 days, the healing rate of the qCSc / Lap / rhColⅢ hydrogel group reached 91.6%, which was much higher than the 43.9% of the control group. Image J was further used to perform statistics on the HE sections of the four groups of healed skin at 12 days, and the thickness of the new skin epidermis and the density of regenerated hair follicles in the new skin were statistically analyzed. The results are shown in Figure 2. Figure 18 The results showed that the epidermis of the newly formed skin was thicker than that of normal skin, but the thickness decreased in the control, qCSc hydrogel, qCSc / Lap hydrogel, and qCSc / Lap / rhColⅢ composite hydrogel groups. The qCSc / Lap / rhColⅢ group had an epidermal thickness closer to that of normal epidermis, with lower levels of collagen tissue proliferation (scarring) and better healing. The control group had almost no new hair follicles, while the density of new hair follicles in the hydrogel material groups increased. This indicates that the skin tissue achieved a certain degree of regeneration under the intervention of the materials, with the qCSc / Lap / rhColⅢ group showing the best regeneration.

[0062] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A controlled-release type III collagen composite hydrogel dressing for promoting the repair and regeneration of full-thickness skin defects, characterized in that: The hydrogel is prepared by oxidative cross-linking of three components: modified chitosan, lithium magnesium silicate nanosheets, and recombinant human type III collagen in an aqueous solvent. The components of the hydrogel interact through chemical bonds, electrostatic bonds, and hydrogen bonds to form a stable hydrogel network structure, which is injectable. By controlling the proportion of lithium magnesium silicate nanosheets, the controlled release of recombinant human type III collagen is achieved. The molecular structure of the modified chitosan is as follows:

2. The controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration according to claim 1, characterized in that: Horseradish peroxidase is added to a mixed solution containing modified chitosan, lithium magnesium silicate nanosheets and recombinant human type III collagen, and the mixture is evenly mixed. H2O2 is then added and the pH of the solution is adjusted to neutral to obtain a hydrogel pre-liquid. The hydrogel pre-liquid is injected into a mold through a syringe to obtain a hydrogel dressing.

3. The controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration according to claim 2, characterized in that: In the hydrogel pre-liquid, the amount of modified chitosan accounts for 0.5%-5% of the total mass of the hydrogel pre-liquid, the amount of lithium magnesium silicate nanosheets accounts for 0.2%-6% of the total mass of the hydrogel pre-liquid, and the amount of recombinant human type III collagen accounts for 0.1%-1% of the total mass of the hydrogel pre-liquid.

4. The controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration according to claim 3, characterized in that: The horseradish peroxidase concentration in the hydrogel pre-liquid is 5-20 ppm.

5. The controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration according to claim 3, characterized in that: In the hydrogel pre-liquid, the mass concentration of H2O2 is 1-2‰.

6. The controlled-release type III collagen composite hydrogel dressing for promoting full-thickness skin defect repair and regeneration according to claim 1, characterized in that: The modified chitosan is prepared by the following two steps: (1) Dissolving chitosan in a mixture of water and glacial acetic acid, adding 2,3-epoxypropyltrimethylammonium chloride, and heating to 50-60° C. with stirring for 20-25 hours. After the reaction, dialyzing and freeze-drying are performed to obtain quaternized chitosan; (2) Dissolve the quaternized chitosan in water and adjust the pH to 4-5 to obtain solution A; take 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and caffeic acid and add them to a mixed solvent of ethanol and water, stir and dissolve, and continue to stir and activate for 20-120 minutes, add the activated solution dropwise to solution A, adjust the pH to 4-5, continue the reaction for 10-14 hours, and after the reaction is completed, dialyze and freeze-dry to obtain modified chitosan.

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