Hydrogel dressing for inhibiting wound scar formation and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a hydrogel dressing for inhibiting wound scar formation and a preparation method and application thereof. The invention firstly provides a composition for preparing the anti-scar hydrogel dressing, wherein the composition is prepared from arsenic trioxide and auxiliary materials; the auxiliary materials comprise chitosan and sodium alginate. The preparation method of the hydrogel dressing is provided based on the composition, and comprises the following steps: mixing a chitosan solution and a sodium alginate solution to obtain CS / SA pre-gel; the preparation method comprises the following steps: dispersing arsenic trioxide into a sodium alginate solution, and adding chitosan to obtain an ATO-coated CS / SA pregel solution; and respectively soaking the CS / SA pre-gel and the ATO coated CS / SA pre-gel solution in a CaCl2 solution to obtain the ATO coated CS / SA hydrogel. The ATO coated CS / SA hydrogel can effectively inhibit formation of wound scars, overcomes the toxicity problem of ATO, and has great clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a hydrogel dressing for inhibiting scar formation on a wound surface, and a preparation method and application thereof. Background Art

[0002] As the first barrier of the human body, the skin plays an important role in defending against external damage. When the skin is injured by trauma, cuts, burns, scalds, skin infections, surgical operations, etc., if no intervention is performed, the natural healing process of the wound may lead to scar formation. When scar growth exceeds a certain limit, various complications will occur, such as destruction of appearance, loss of tissue function, limited movement, severe psychological pain, etc., which will bring great physical and mental pain to the patient. Therefore, it is necessary to intervene in the wound healing stage to prevent the formation of scars or reduce their effects. Known drugs with the function of inhibiting scar formation include silicone drugs, hormone drugs, and Chinese medicine external preparations. Silicone drugs are a class of drugs that are widely used in clinical practice. They can effectively improve the appearance of scars and reduce the incidence of hypertrophic scars, but this type of drug is prone to cause complications such as skin maceration, skin itching, and dermatitis. Chinese medicine preparations such as Centella asiatica cream ointment have the effects of detumescence, detoxification, dehumidification and itching, and have an effect on both new and old scars, but Chinese medicine preparations are slow to take effect. Although hormonal drugs such as glucocorticoids have the function of reducing scars, they may cause side effects such as osteoporosis. Therefore, it is urgent to develop new strategies for wound-free scar healing to improve the current situation.

[0003] Hydrogels are soft and wet polymers with three-dimensional microstructures. They are ideal for tissue repair due to their excellent biocompatibility, biodegradability, moisture retention and drug loading capacity. Chitosan (CS) and sodium alginate (SA) are widely used in the development of wound dressings because of their antibacterial, hemostatic and cell proliferation-promoting functions, which can accelerate wound healing and prevent infection. Dual-base hydrogels can also be used as a drug delivery system, and their structural properties allow precise control of drug release to achieve sustained and effective therapeutic effects.

[0004] Arsenic trioxide (ATO) is the active ingredient of the traditional Chinese medicine arsenic. As one of the oldest poisons, ATO has attracted much attention due to its significant therapeutic effects on malignant diseases such as malaria, the Black Death, ulcers, cancer, and leukemia. Arsenic has a long history of application in traditional Chinese medicine. In the Compendium of Materia Medica by Li Shizhen of the Ming Dynasty, it was recorded that arsenic was used to treat various suppurative diseases and tuberculosis diseases. In addition, ATO is also a specific drug invented independently by my country and the world's first for the treatment of acute promyelocytic leukemia (APL). It was not until 1995 that ATO was approved by the US Food and Drug Administration (FDA) for the treatment of APL patients, and it has gradually been used and promoted worldwide. Existing studies have shown that ATO can regulate the transition of vascular smooth muscle cells from a synthetic phenotype to a contractile phenotype, thereby affecting the formation of the new intima; and the YAP1 signaling pathway is involved in the mechanism of this process. YAP and PDZ binding motif (TAZ) play an important role in regulating various biological functions such as organ size and tissue regeneration, and respond to various internal and external signals through the Hippo signaling pathway. ATO can effectively inhibit YAP / TAZ activity, thus endowing ATO with the potential to promote scarless wound healing as an anti-fibrotic agent. It is worth noting that although inhibition of YAP / TAZ signaling can reduce tissue fibrosis and scar formation, extensive inhibition of YAP / TAZ may lead to significant side effects and hinder wound re-epithelialization. Therefore, it is very necessary to explore suitable methods for topical application of ATO to treat scarless wound repair. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide a composition for preparing an anti-scar hydrogel dressing. The present invention uses a combination of arsenic trioxide, chitosan and sodium alginate for wound treatment, which can effectively inhibit the formation of scars on the wound surface.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention discloses a composition for preparing an anti-scar hydrogel dressing, which is composed of arsenic trioxide and auxiliary materials; the auxiliary materials include chitosan and sodium alginate; and the volume ratio of the arsenic trioxide to the auxiliary materials is 3:10000-20000.

[0008] Furthermore, the volume ratio of the arsenic trioxide, the chitosan and the sodium alginate is 3:7000-13000:3000-7000, preferably 3:10000:5000. The volume ratio is the total usage volume ratio.

[0009] Preferably, the concentration of arsenic trioxide is 10 mM.

[0010] More preferably, the arsenic trioxide is in the state of an arsenate solution dissolved in 5M NaOH (aq), and the As2O3 concentration in the solution is 10mM.

[0011] Furthermore, the auxiliary materials also include acetic acid solution, distilled water and / or CaCl2 solution.

[0012] The acetic acid solution is used as a dispersant for chitosan; the distilled water is a solvent for sodium alginate; and the CaCl2 solution is a cross-linking agent.

[0013] Preferably, the concentration of the chitosan solution is 1%-5%, more preferably 2%.

[0014] Preferably, the concentration of the sodium alginate solution is 3%-10%, more preferably 5%.

[0015] Preferably, the concentration of the acetic acid solution is 2%; the concentration of the CaCl2 solution is 0.03M.

[0016] The second object of the present invention is to provide a method for preparing the above composition into a hydrogel dressing.

[0017] To achieve the above object, the present invention adopts the following technical solutions:

[0018] The method for preparing the above composition into a hydrogel dressing comprises the following steps:

[0019] (1) mixing chitosan solution and sodium alginate solution to obtain CS / SA pregel;

[0020] (2) dispersing arsenic trioxide into a sodium alginate solution and adding chitosan to obtain an ATO@CS / SA pregel solution;

[0021] (3) The CS / SA pregel obtained in step (1) and the ATO@CS / SA pregel solution obtained in step (2) are respectively immersed in CaCl2 to obtain ATO@CS / SA hydrogel.

[0022] The present invention adopts a one-pot method to prepare ATO@CS / SA hydrogel, and the -COO - Group and Ca 2+ and CS-NH3 + The groups combine to form a hydrogel, that is, it is transformed from a liquid to a semi-solid hydrogel state through calcium ion cross-linking. At the same time, CS / SA acts as a biocompatible carrier, effectively alleviating the potential toxicity induced by ATO.

[0023] Furthermore, in step (2), SA is used as a dispersant for ATO.

[0024] Furthermore, in step (1), the volume ratio of the chitosan solution to the sodium alginate solution is 1.5-3:1, preferably 2:1.

[0025] Furthermore, in step (2), the volume ratio of the arsenic trioxide to the sodium alginate solution is 1-10:3000-10000, preferably 3:5000; the volume ratio of the chitosan to the sodium alginate is 1.5-3:1, preferably 2:1.

[0026] Furthermore, the concentration of the chitosan solution is 1%-5%; the concentration of the sodium alginate solution is 3%-10%; and the concentration of arsenic trioxide is 10 mM.

[0027] Preferably, the concentration of the chitosan solution is 2%; the concentration of the sodium alginate solution is 5%; and the concentration of arsenic trioxide is 10 mM.

[0028] The third object of the present invention is to provide a hydrogel dressing prepared by the above method.

[0029] To achieve the above object, the present invention adopts the following technical solutions:

[0030] The hydrogel dressing is prepared by the above method.

[0031] Furthermore, the hydrogel dressing has a porous internal structure.

[0032] The hydrogel prepared by the present invention has the ability to promote the repair of full-thickness skin defects, inhibit excessive fibrosis of fibroblasts, inhibit excessive proliferation of vascular endothelial cells, inhibit the expression of YAP, inhibit the expression of TAZ, and inhibit excessive proliferation of collagen at the wound surface.

[0033] A fourth object of the present invention is to provide a use of the aforementioned composition and / or the aforementioned gel dressing in the preparation of a product for promoting the repair of full-thickness skin defects.

[0034] A fifth object of the present invention is to provide a use of the aforementioned composition and / or the aforementioned gel dressing in the preparation of a product for inhibiting scar formation on a wound surface and / or promoting scar repair.

[0035] The sixth object of the present invention is to provide a use of the aforementioned composition and / or the aforementioned gel dressing in the preparation of any one or more products in the following 1)-10), including:

[0036] 1) Fibroblast migration promoter;

[0037] 2) Inhibitors of abnormal cell proliferation and tissue fibrosis;

[0038] 3) Inhibitors of abnormal vascular proliferation;

[0039] 4) Inhibitors of excessive collagen proliferation on wound surfaces;

[0040] 5) YAP expression inhibitors;

[0041] 6) TAZ expression inhibitors;

[0042] 7) α-SMA expression inhibitors;

[0043] 8) TGF-β3 expression promoter;

[0044] 9) TGF-β1 expression inhibitor;

[0045] 10) CD31 expression inhibitors.

[0046] The beneficial effects of the present invention are:

[0047] 1. The present invention has developed a new type of ATO@CS / SA hydrogel for wound treatment, which can effectively inhibit the formation of scars on the wound surface. Studies have found that ATO@CS / SA hydrogel can inhibit angiogenesis, reduce abnormal cell proliferation and tissue fibrosis by inhibiting the YAP / TAZ signaling pathway, thereby inhibiting the formation of scars and promoting scarless wound healing in vivo. The results of the study showed that after treatment with ATO@CS / SA hydrogel, the expression of YAP / TAZ in the wound surface was at a low level, CD31 was moderately expressed, the expression of fibrosis factor TGF-β1 was reduced, and the expression of anti-scar factor TGF-β3 was increased. It shows that ATO@CS / SA hydrogel plays a key role in promoting scarless wound healing in vivo.

[0048] 2. The ATO@CS / SA hydrogel of the present invention not only effectively inhibits scar formation when promoting normal wound healing, but also plays an active role in inhibiting scar formation after the wound is completely healed. In the 90-day in vivo scar observation, ATO@CS / SA showed a long-term effect on scar formation. The ATO@CS / SA hydrogel can effectively promote scar-free wound healing in vivo.

[0049] 3. The ATO@CS / SA hydrogel of the present invention has good injectability, biocompatibility and blood compatibility.

[0050] 4. The ATO@CS / SA hydrogel of the present invention has good hemostatic properties. The amino groups of CS are protonated in the blood to form -NH3 + , attracting negatively charged platelets, aggregating red blood cells and accelerating thrombus formation; at the same time, the hydrogel combines with the tissues around the wound to effectively block the bleeding site, thereby achieving a hemostatic effect and keeping the wound relatively stable during the critical bleeding period.

[0051] 5. The ATO@CS / SA hydrogel of the present invention can effectively accelerate fibroblast migration, promote dermal tissue regeneration, and promote wound healing.

[0052] 6. The present invention found that the combination of ATO, CS and SA can not only effectively inhibit the formation of wound scars, but also solve the problem of ATO's own toxicity. After treatment with ATO@CS / SA hydrogel, the structures of the rats' heart, liver, spleen, lung, kidney, testicle and brain remained intact, without obvious pathological changes caused by the deposition of ATO or ATO@CS / SA hydrogel. The present invention uses CS / SA as a biocompatible carrier to effectively reduce the potential toxicity induced by ATO. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Diagram of the design strategy of ATO@CS / SA hydrogel for scarless wound repair;

[0054] Figure 2 Figure 2 shows the ATO@CS / SA gel network before and after immersion in 0.03 M CaCl2 solution;

[0055] Figure 3 This is the result of the injectability test of ATO@CS / SA hydrogel;

[0056] Figure 4 SEM image of ATO / CS / SA hydrogel;

[0057] Figure 5 SEM-EDS elemental image of freeze-dried ATO@CS / SA hydrogel;

[0058] Figure 6 FT-IR spectra of freeze-dried CS, SA, ATO / SA, CS / SA and ATO@CS / SA;

[0059] Figure 7 is a graph of cell viability of L929 cells after drug action;

[0060] Figure 8 This is a graph showing the proliferation of L929 cells;

[0061] Fig. 9 is a graph of the hemolysis ratio of the hydrogel (n=3);

[0062] Fig.10 The figure is a dynamic whole blood coagulation test of the liver and the corresponding statistical analysis, wherein: Fig.10 -A is the result of the dynamic whole blood coagulation test of the liver. Fig.10 -B is Fig.10 -A is a graph showing the statistical analysis results;

[0063] Fig.11 The figure is the result of the dynamic whole blood coagulation test at the tail, where: Fig.11 -A is the result diagram of the dynamic whole blood coagulation test at the tail, Fig.11 -B is Fig.11 -A is a graph showing the statistical analysis results;

[0064] Fig.12 The figure is the result of L929 cell migration analysis;

[0065] Fig.13 for Fig.12 Statistical analysis results diagram;

[0066] Fig.14 Diagram of the experimental steps of the treatment and evaluation protocol for full-thickness skin excision of Sprague-Dawley rats in the in vivo wound healing assay;

[0067] Fig.15 Schematic diagram of wound healing sites on days 0, 3, 7, 10, and 14;

[0068] Fig.16 The figure is a statistical diagram of wound closure rate (n=5) on days 0, 3, 7, 10 and 14;

[0069] Fig.17 -A is the HE staining image on the 7th and 14th day; Fig.17 -B is the Masson staining image on day 14;

[0070] Fig.18 HE staining of the heart, liver, spleen, lung, kidney, testis and brain of SD rats on the 14th day of drug treatment;

[0071] Fig.19 This is a diagram of the experimental steps of the treatment and evaluation scheme for SD rats with full-thickness skin excision in Example 7;

[0072] Fig. 20 Schematic diagram of wound healing sites on day 0, day 30, and day 90;

[0073] Fig.21 The statistical diagrams of wound healing on day 0, day 30 and day 90;

[0074] Fig. 22 Masson staining images at day 30 and day 90;

[0075] Fig.23 Schematic diagram of promoting scarless wound healing by inhibiting the YAP / TAZ signaling pathway;

[0076] Fig.24The results of immunofluorescence experiments of YAP in L929 cells treated with drugs for 1 day, where: Fig.24 -A is the YAP immunofluorescence image of L929 cells; Fig.24 -B is Fig.24 -A is a graph showing the statistical analysis results;

[0077] Fig.25 This is the result of immunofluorescence experiment of YAP in skin wound tissue after treatment on the 14th day. Fig.25 -A is the immunofluorescence image of YAP in skin wounds; Fig.25 -B is Fig.25 -A is a graph showing the statistical analysis results;

[0078] Fig.26 The results of immunofluorescence experiments of TAZ in L929 cells treated with drugs for 1 day, Fig.26 -A is the TAZ immunofluorescence image of L929 cells; Fig.26 -B is Fig.26 -A is a graph showing the statistical analysis results;

[0079] Fig. 27 This is the result of immunofluorescence experiment of TAZ in skin wound tissue after treatment on the 14th day, where: Fig. 27 -A is the immunofluorescence image of TAZ in skin wounds; Fig. 27 -B is Fig. 27 -A is a graph showing the statistical analysis results;

[0080] Fig.28 The results of the immunofluorescence experiment of α-SMA in L929 cells treated with drugs for 1 day are shown in the figure. Fig.28 -A is the immunofluorescence image of α-SMA of L929 cells; Fig.28 -B is Fig.28 -A is a graph showing the statistical analysis results;

[0081] Fig.29 This is the result of the immunofluorescence experiment of α-SMA in the skin wound tissue after treatment on the 14th day, where: Fig.29 -A is the immunofluorescence image of α-SMA in the skin wound area; Fig.29 -B is Fig.29 -A is a graph showing the statistical analysis results;

[0082] Fig.30 This is the immunohistochemical staining of TGF-β1 and TGF-β3 in the skin wound tissue after treatment on the 14th day;

[0083] Fig.31 for Fig.30 Statistical analysis results of Fig.31-A is a diagram showing the statistical analysis results of TGF-β1; Fig.31 -B is a diagram showing the statistical analysis results of TGF-β1;

[0084] Fig.32 The results of blood perfusion test, HE staining and CD31 IHC are shown;

[0085] Fig.33 for Fig.32 Statistical data graph of blood perfusion test;

[0086] Fig.34 The immunofluorescence image of YAP in HUVECs 1 day after endothelial cell administration;

[0087] Fig.35 The immunofluorescence image of TAZ in HUVECs 1 day after endothelial cell administration;

[0088] Fig.36 -A is a graph showing the expression level of CD31; Fig.36 -B is a graph showing the nuclear entry rate of YAP in HUVECs; Fig.36 -C is a graph showing the nuclear entry rate of TAZ in HUVECs. DETAILED DESCRIPTION

[0089] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0090] In order to overcome scar formation, the present invention has developed a new type of ATO-loaded CS / SA hydrogel (referred to as ATO@CS / SA hydrogel) for wound treatment. The design strategy of ATO@CS / SA hydrogel for scarless wound repair is as follows Figure 1As shown. ATO controls the expression of YAP / TAZ at a low level, reduces excessive fibrosis of fibroblasts and excessive proliferation of vascular endothelial cells, while CS / SA, as a biocompatible carrier, reduces the potential toxicity induced by ATO. In vitro experiments showed that ATO@CS / SA has good biocompatibility and blood compatibility. After co-culturing the hydrogel with fibroblasts and HUVECs cells, the expression of α-SMA and YAP / TAZ in the cells was not completely inhibited, but maintained at a low level. This reasonable regulation will not affect normal wound healing and avoid scar formation. In the in vivo scar observation for up to 90 days, ATO@CS / SA showed a long-term effect on scar formation. Even after the wound was completely healed, ATO still played an active role in inhibiting scar formation. In histological observations, the expression of YAP / TAZ in the wound surface was also at a low level after treatment with ATO@CS / SA. In addition, moderate expression of CD31, decreased expression of fibrosis factor TGF-β1, and increased expression of anti-scar factor TGF-β3 indicated that ATO inhibited excessive vascular proliferation and excessive fibrosis. These results show that the ATO@CS / SA hydrogel developed by the present invention challenges the traditional understanding of ATO toxicity, explores its potential in scar inhibition, and the appropriate application of the hydrogel also gives it clinical application value.

[0091] In the embodiment of the present invention, 10 mM ATO (aq) is an arsenate solution state in which ATO is dissolved in 5 M NaOH (aq), and the concentration of As2O3 in the solution is 10 M.

[0092] Example 1. Preparation of ATO@CS / SA hydrogel

[0093] ATO@CS / SA hydrogel was prepared by a one-pot method and transformed from a liquid to a semisolid hydrogel state by calcium ion crosslinking. Figure 2 The specific preparation steps of ATO@CS / SA hydrogel are as follows:

[0094] (1) Dispersing 0.8 g of chitosan into 40 mL of 2% acetic acid solution to obtain a 2% chitosan solution;

[0095] (2) Dissolve 1.2 g of sodium alginate in 24 mL of distilled water to obtain a 5% sodium alginate solution;

[0096] (3) 2% chitosan solution and 5% sodium alginate solution were mixed in a volume ratio of 2:1 to prepare CS / SA pregel;

[0097] (4) 10 mM ATO (aq) was dispersed into a sodium alginate solution at a ratio of 3:5000 to form a solution containing 6 μM ATO, and then the solution was mixed with chitosan at a ratio of chitosan: sodium alginate solution = 1:2 (v / v) to form an ATO@CS / SA pregel solution;

[0098] (5) The above two pregels were fully immersed in 0.03 M CaCl2(aq) respectively to finally form a semisolid hydrogel, namely ATO@CS / SA hydrogel.

[0099] Example 2. Characterization of ATO@CS / SA hydrogel

[0100] (1) Structural characterization of hydrogels

[0101] Scanning electron microscopy-energy dispersive X-ray spectrometer (SEM-EDS, Hitachi SU8010) and Fourier transform infrared spectrometer (FTIR, Thermo Scientific Nicolet FTIR 6700spectrometer) were used to verify the preparation of ATO@CS / SA hydrogel. In addition, the infrared spectra of freeze-dried CS, SA, ATO / SA, and CS / SA were recorded in the range of 4000 to 600 cm -1 ..

[0102] Results: The morphological properties of ATO@CS / SA hydrogels are shown in Figure 4 As shown, the hydrogel has a porous internal structure. Porosity is an important feature of hemostatic dressings related to blood absorption rate. Previous studies have shown that high porosity and large internal pore structure can effectively provide the necessary space for cell growth. SEM-EDS results are shown in Figure 5 As shown in Figure 2, As element is evenly distributed in the hydrogel. The CN bond stretching vibration corresponds to 1350-1100 cm -1 1150-800cm corresponding to the stretching vibration of the C-O-C bond -1 The peak intensity of chitosan decreased after gelation, which proved that the introduction of ATO and sodium alginate reduced the polarity of the above chemical bonds, indicating that the synthesis was successful.

[0103] 3263cm in FTIR spectrum -1 The absorption peak at 2925 cm is attributed to the OH stretching vibration, while the absorption peak at 2925 cm -1 The absorption peak at 1555 cm is attributed to the CH stretching vibration. -1 The peak intensity at 27° decreases, which corresponds to the NH bond, indicating that the -COO - Group and Ca 2+ and CS-NH3+ At the same time, comparing the infrared spectra of ATO / SA and SA, the addition of ATO did not significantly change the spectrum of SA, indicating that SA is an effective dispersant for ATO. Figure 6 .

[0104] (2) Injectability test of ATO@CS / SA hydrogel

[0105] Injectable hydrogels have attracted widespread attention due to their painless and minimally invasive properties. To demonstrate the injectability of hydrogels, a syringe filled with hydrogel was used to inject the letters "ATO" on a glass plate.

[0106] The results are as follows Figure 3 As shown, the ATO@CS / SA hydrogel prepared in the present invention has good injectability.

[0107] Example 3. Cell biocompatibility of ATO@CS / SA hydrogel

[0108] Biocompatibility is essential for wound dressings because they are in direct contact with the skin. Therefore, the cell biocompatibility of ATO@CS / SA hydrogels was evaluated. Figure 3 As shown, in Figure 7 The live / dead cell staining results of L929 cells showed that no obvious cell death was observed after 24 hours of drug action, indicating that ATO@CS / SA hydrogel has low lethality to cells. Figure 8 The results show that both CS / SA hydrogel and ATO@CS / SA hydrogel groups have a more obvious tendency to promote cell proliferation. In order to evaluate the blood compatibility of ATO@CS / SA hydrogel, the present invention conducted an in vitro hemolysis test. The results are as follows Fig. 9 As shown, a clear color distinction can be seen between the two hydrogel groups, the negative control group (PBS) and the positive control group (Triton X-100); quantitative data showed that the hemolysis rate was significantly reduced after ATO was added to the hydrogel.

[0109] Example 4. Hemostatic properties of hydrogels

[0110] Hemostasis initiates the initial stage of the natural wound healing process after injury. In addition to promoting platelet aggregation and blood clot formation at the wound site, suitable wound dressings can also adhere to surrounding tissues and quickly coagulate to help stop bleeding quickly. Therefore, timely hemostasis after injury is crucial to reducing blood loss and improving survival rate. The present invention uses two bleeding models to evaluate the hemostatic ability of ATO@CS / SA hydrogel: rat liver bleeding and tail amputation model. In the liver bleeding model, the blood loss of the control group (untreated) was about 200 mg, while after treatment with CS / SA and ATO@CS / SA hydrogels, the blood loss was reduced to about 100 mg, respectively, see for details. Fig.10 Similarly, in the tail docking model, the blood loss in the CS / SA and ATO@CS / SA treatment groups was reduced to about 50 mg, while that in the control group was about 100 mg. Fig.11 This is because the amino group of CS can be protonated in the blood to form -NH3 + , which can attract negatively charged platelets, aggregate red blood cells, and accelerate thrombosis. At the same time, the hydrogel can combine with the tissues around the wound to effectively block the bleeding site, thereby achieving a hemostatic effect and keeping the wound relatively stable during the critical bleeding period.

[0111] Example 5. In vitro cell migration assay

[0112] To evaluate the effectiveness of the hydrogel in accelerating dermal tissue regeneration, a cell scratch assay was used to simulate the wound migration process. Effective promotion of wound healing requires rapid migration of fibroblasts, which plays an important role in re-epithelialization, the third stage of wound healing. Significant changes in migration levels were observed after different treatments of the cell scratch. Fig.12 and Fig.13 As shown, within the first 12 hours, the wound area (%) was reduced most significantly when co-cultured with CS / SA hydrogel and ATO@CS / SA hydrogel compared with the other groups. Within 24 hours, ATO and ATO@CS / SA hydrogels showed a greater ability to promote fibroblast migration, while the enhancement effect of CS / SA hydrogel in reducing wound area was slightly less obvious.

[0113] Example 6. In vivo wound healing assay

[0114] A rat full-thickness defect model was established to comprehensively evaluate the wound healing performance of ATO@CS / SA hydrogel. The morphology of all wounds on different days was recorded. Fig.14 .like Fig.15 and Fig.16As shown in Figure 3, application of ATO@CS / SA hydrogel achieved satisfactory wound closure on day 7. The ATO@CS / SA group showed better wound healing than the control group on each recorded day, with the most significant improvement shown on day 10. On day 14, the ATO@CS / SA treated group was close to complete healing.

[0115] The present invention further conducted histological analysis to evaluate the deeper repair effect of ATO@CS / SA hydrogel on regenerated skin. Fig.17 -A, HE staining showed that the wound bed was significantly smaller, the granulation tissue was significantly narrowed, the epithelial tissue was widely formed, the dermal tissue was abundant, and the hair follicles and sebaceous glands were clearly visible. In contrast, there were no appendages such as hair follicles or sebaceous glands in the scar, and hypertrophic features were observed in the control wound. The application of ATO and ATO hydrogel resulted in a thinner and flatter epidermis, demonstrating the scar inhibitory effect of ATO@CS / SA. In addition, Masson staining showed that compared with the other three groups, the skin treated with ATO@CS / SA had significantly fewer collagen fibers, similar to the normal collagen structure of rat skin, and the blood vessels were more organized, see for details. Fig.17 -B. The accumulation of ATO mainly leads to its cytotoxic effects, which damages tissues and organs. HE staining results showed that the structures of the heart, liver, spleen, lung, kidney, testis, and brain remained intact, with no obvious pathological changes caused by the deposition of ATO or ATO@CS / SA hydrogel, see for details. Fig.18 .

[0116] Example 7. Promoting scarless wound healing in vivo

[0117] The therapeutic effect of ATO@CS / SA hydrogel on promoting scarless wound healing in a rat skin defect model was further explored. Using a full-thickness skin defect animal model, photographs and tissue samples were collected from the wound site at two time points: 30 days and 90 days after injury. Fig.19 The scar areas of the ATO@CS / SA group and the ATO group were lower than those of the control group and the CS / SA group, indicating that the key to scar-free healing lies in the introduction of ATO. Fig. 20 and Fig.21 In addition, some hair follicles and sebaceous glands were observed, making the tissue very similar to healthy skin. By Masson staining, it was observed that on the 30th day, ATO@CS / SA hydrogels initially formed hair follicles in the epithelial layer, and by the 90th day, the hair follicles and sebaceous glands had been fully formed and restored, see Fig. 22 The remodeling phase can last from months to years, during which time the newly repaired tissue structure continues to mature and gradually approaches its pre-injury state in appearance and flexibility.

[0118] Example 8. Inhibition of YAP / TAZ in fibroblasts reduces adverse remodeling and improves scarless healing

[0119] YAP / TAZ is a key effector of the Hippo signaling pathway, affecting cell growth and response to mechanical stimulation. After injury, YAP and TAZ are activated by mechanical strain and translocate to the nuclei of differentiated epidermal cells to promote regeneration, but the continued activation of YAP / TAZ can lead to abnormal cell proliferation and tissue fibrosis. The inhibitory effect of ATO on YAP has been confirmed by our team's previous studies. Fig.23 As shown, in order to confirm the role of the YAP / TAZ signaling pathway in the effect of ATO on scarless wound healing, the present invention used immunofluorescence staining to evaluate the expression of YAP and TAZ in wound tissue. Fig.24 As shown in Figure 2, both ATO and ATO@CS / SA significantly reduced the nuclear expression of YAP and TAZ in L929. The inhibition of YAP can also be easily found in the wound area on day 14, as detailed in Fig.25 At the same time, the expression of TAZ in L929 and wounds treated with ATO and ATO@CS / SA also decreased, see Fig.26 and Fig. 27 .

[0120] The above experimental results indicate that ATO treatment inhibits the YAP / TAZ signaling pathway and plays a key role in promoting scarless wound healing by reducing abnormal cell proliferation and tissue fibrosis.

[0121] Example 9. Inhibiting fibrosis and promoting scarless wound healing

[0122] Myofibroblasts (MF) are the main scar-forming cell type in the skin. Myofibroblasts expressing α-smooth muscle actin (α-SMA) are key cells that drive tissue fibrosis and scar formation. After ATO or ATO@CS / SA treatment, the expression of α-SMA in L929 immunofluorescence and tissue immunofluorescence on day 14 showed a downward trend, see Fig.28 and Fig.29 , which may be due to the inhibition of YAP, avoiding the excessive proliferation of fibroblasts, allowing normal healing and regeneration of the skin and restoring structural function. Transforming growth factor-β is an important pathological feature and a key factor in the progression of scarring. Unlike TGF-β1, which induces extensive fibrosis, TGF-β3 antagonizes tissue fibrosis and induces scarless wound healing. In this study, after treatment with ATO@CS / SA, on day 14, the wounds showed a decrease in TGF-β1 expression compared with other groups, while TGF-β3 expression increased, see for details. Fig.30 and Fig.31 .

[0123] Example 10. Inhibiting angiogenesis and promoting scarless wound healing

[0124] Angiogenesis refers to the process by which endothelial cells (ECs) generate new blood vessels from existing ones, and it is closely intertwined with the metabolic environment of the tissue. However, excessive angiogenesis can lead to scar formation. Blood perfusion imaging showed that ATO alone significantly reduced blood perfusion in rat wounds compared with the control group and CS / SA hydrogel group, while blood perfusion in the ATO@CS / SA hydrogel treatment group was not completely inhibited. Fig.32 and Fig.33 Immunohistochemical analysis showed that the expression of CD31 in the ATO@CS / SA hydrogel group was lower than that in the control group and the CS / SA hydrogel group, which was consistent with the blood perfusion results. Fig.32 and Fig.34 However, the introduction of ATO did not adversely affect the normal healing mechanism, and CD31 was still expressed at an appropriate level. To clarify the relationship between YAP signal transduction and angiogenesis inhibition, the present invention detected the nuclear entry rate of YAP in human umbilical vein endothelial cells (HUVECs). The results showed that in the ATO@CS / SA hydrogel group, the nuclear entry rates of YAP and TAZ were significantly reduced, see Fig.35 , Fig.36 -A. Fig.36 -B and Fig.36 -C. Therefore, it can be inferred that ATO@CS / SA hydrogel reduces abnormal vascular proliferation and promotes scarless wound healing by inhibiting the YAP / TAZ signaling pathway.

Claims

1. A composition for preparing an anti-scar hydrogel dressing, characterized in that: The composition consists of arsenic trioxide and auxiliary materials; the auxiliary materials include chitosan and sodium alginate; the volume ratio of the arsenic trioxide to the auxiliary materials is 3:10000-20000.

2. The composition according to claim 1, characterized in that The volume ratio of the arsenic trioxide, the chitosan and the sodium alginate is 3:7000-13000:3000-7000.

3. A method for preparing a hydrogel dressing from the composition according to any one of claims 1 to 2, characterized in that: The steps include: (1) mixing chitosan solution and sodium alginate solution to obtain CS / SA pregel; (2) dispersing arsenic trioxide into a sodium alginate solution and adding chitosan to obtain an ATO@CS / SA pregel solution; (3) The CS / SA pregel obtained in step (1) and the ATO@CS / SA pregel solution obtained in step (2) are respectively immersed in CaCl2 to obtain ATO@CS / SA hydrogel.

4. The method according to claim 3, characterized in that In step (1), the volume ratio of the chitosan solution to the sodium alginate solution is 1.5-3:

1.

5. The method according to claim 3, characterized in that: In step (2), the volume ratio of the arsenic trioxide to the sodium alginate solution is 1-10:3000-10000; the volume ratio of the chitosan to the sodium alginate is 1.5-3:

1.

6. The method according to claim 3, characterized in that The concentration of the chitosan solution is 1%-5%; the concentration of the sodium alginate solution is 3%-10%; and the concentration of arsenic trioxide is 10mM.

7. A hydrogel dressing prepared by the method according to any one of claims 3 to 6.

8. Use of the composition according to any one of claims 1 to 2 and / or the hydrogel dressing according to claim 7 in the preparation of a product for promoting the repair of full-thickness skin defects.

9. Use of the composition according to any one of claims 1 to 2 and / or the hydrogel dressing according to claim 7 in the preparation of a product for inhibiting scar formation on a wound surface and / or promoting scar repair.

10. Use of the composition according to any one of claims 1 to 2 and / or the hydrogel dressing according to claim 7 in the preparation of any one or more products in 1) to 10) below, characterized in that: include: 1) Fibroblast migration promoter; 2) Inhibitors of abnormal cell proliferation and tissue fibrosis; 3) Inhibitors of abnormal vascular proliferation; 4) Inhibitors of excessive collagen proliferation on wound surfaces; 5) YAP expression inhibitors; 6) TAZ expression inhibitors; 7) α-SMA expression inhibitors; 8) TGF-β3 expression promoter; 9) TGF-β1 expression inhibitor; 10) CD31 expression inhibitors.

Images