Conductive hydrogel dressing, preparation method and application of conductive hydrogel dressing in electrically promoted healing

Abstract

The invention provides a conductive hydrogel dressing, a preparation method and application of the conductive hydrogel dressing in electrically promoted healing, and belongs to the technical field of biomedical materials in the chemical aspect of bandages, dressings, absorbent pads or surgical supplies. The preparation method of the conductive hydrogel dressing comprises the following steps: mixing a biocompatible high polymer material, borax, tannic acid and keratin for full reaction to obtain conductive hydrogel; the mass percentage content of the biocompatible high polymer material is 5-10%, the mass percentage content of the tannic acid is 0.25-2%, the mass percentage content of the keratin is 0.25-2%, and the mass percentage content of the borax is 0.5-2%. The biocompatible high polymer material is selected from one of polyvinyl alcohol, polyacrylic acid, a polyvinyl alcohol derivative and a polyacrylic acid derivative. The conductive hydrogel dressing has excellent conductivity, can be used for promoting cell migration and proliferation and accelerating wound healing, and also can monitor the electrical signal change of wound healing in real time through the conductivity of the conductive hydrogel dressing.

Description

Technical Field

[0001] The invention relates to a conductive hydrogel dressing, a preparation method and application thereof in electrical healing, and belongs to the technical field of biomedical materials in the chemical aspect of bandages, dressings, absorbent pads or surgical supplies. Background Art

[0002] Trauma has always been one of the most important and common catastrophic events in human history. Acute and chronic skin wounds have brought a heavy burden to global health. Trauma occupies an important position in the global death spectrum and is one of the leading causes of death among people aged 15-44 years worldwide. The number of deaths caused by trauma is estimated to reach millions each year. Therefore, promoting efficient and rapid repair of wounds has become an important public health and safety issue.

[0003] As the largest organ in the human body, the skin undertakes multiple physiological functions such as protection, temperature regulation and immune defense. Trauma can lead to the loss of these functions, and wound healing is a complex biological process involving the interaction of multiple cell types, growth factors and cytokines. From the perspective of wound bioelectric signals, normal (undamaged) skin epithelial cells form a resistance barrier through extracellular connections. The epithelial cell layer transports positively charged sodium ions, potassium ions and negatively charged chloride ions to the matrix side and outside the skin through ion channels and proton pumps on both sides of the cell membrane, forming a transepithelial potential with a potential of about 10~60 mV. When the skin is injured, the central potential drops to 0 mV, while the surrounding transepithelial potential remains unchanged, forming a lateral current pointing to the center of the wound. Epithelial cells migrate to the center of the wound under the stimulation of endogenous electric fields. Most cells (such as keratinocytes, macrophages, fibroblasts, endothelial cells, etc.) will move in a directional manner under the stimulation of a DC electric field within a certain intensity range, that is, "galvanotaxis". At the same time, electrical stimulation can reduce local edema, increase vascular permeability, help transport white blood cells and oxygen to the wound, restore the injury current, enhance antibacterial ability, stimulate blood perfusion, accelerate cell proliferation, and thus accelerate skin tissue repair.

[0004] When the skin is damaged, it can spontaneously repair itself, but improper treatment will lead to long-term non-healing of the wound. Therefore, wound care is necessary to prevent infection, dryness, relieve pain, accelerate healing and avoid scar formation. Traditional dressings such as gauze and films are mainly used to protect wounds, prevent infection, and maintain a moist environment to promote healing, but they lack active functions to promote healing, and cannot monitor the wound condition in real time, and cannot meet the dynamic monitoring and active promotion of healing requirements of modern wound management.

[0005] As an emerging research direction in the field of wound management, conductive hydrogel dressings combine the ability of hydrogels to maintain a moist environment and the electrical signal conduction properties of conductive materials. By simulating the electrical properties of skin tissue, they conduct electrical signals to promote wound healing. Its conductive properties are mainly derived from internal conductive materials, such as metal nanoparticles, carbon-based materials, and conductive polymers. These materials not only provide pathways for electrical signal conduction, but also respond to external electrical stimulation, playing a dual role in wound treatment. This dressing can sensitively sense weak currents at the wound site and actively participate in wound healing and tissue regeneration. Endogenous electrical stimulation (ES) has been proven to effectively guide biological cell movement and promote wound closure. When an external current, i.e., exogenous ES, is applied, the conductive hydrogel dressing can accurately transmit electrical signals to cells and tissues, play a similar function to "electronic skin", provide an ideal healing environment, significantly promote cell migration and proliferation, regulate growth factors, exert antibacterial and anti-inflammatory effects, and promote angiogenesis. More importantly, it can also convert physiological information at the wound site into readable electrical signals, providing a window for real-time monitoring of human health, breaking through the limitations of traditional dressings. Conductive hydrogel dressings are becoming a hot topic in wound management research due to their excellent wound healing promotion ability and additional functional potential, bringing faster and better healing effects, providing a new intelligent wound management method, and bringing unlimited possibilities and hope for future wound treatment. However, the conductivity of existing conductive hydrogel dressings still needs to be further improved. Summary of the invention

[0006] The present invention provides a conductive hydrogel dressing, a preparation method and application thereof in electrical healing, which can effectively solve the above problems.

[0007] The present invention is achieved in that: A method for preparing a conductive hydrogel dressing comprises mixing a biocompatible polymer material, borax, tannic acid and keratin for sufficient reaction to obtain a conductive hydrogel dressing; the mass percentage of the biocompatible polymer material is 5-10%, the mass percentage of the tannic acid is 0.25-2%, the mass percentage of the keratin is 0.25-2%, and the mass percentage of the borax is 0.5-2%. In some embodiments, the biocompatible polymer material is selected from polyvinyl alcohol, polyacrylic acid, a polyvinyl alcohol derivative, and a derivative of polyacrylic acid.

[0008] In some embodiments, the reaction temperature is 20-30°C.

[0009] In some embodiments, the mass percentage of the polyvinyl alcohol is 6.25%, the mass percentage of the tannic acid is 0.75%, the mass percentage of the keratin is 1.25%, and the mass percentage of the borax is 0.75%.

[0010] A conductive hydrogel dressing prepared by the method.

[0011] A device with electrical healing promotion and progress monitoring functions comprises the conductive hydrogel dressing.

[0012] A non-disease treatment method of electrical healing, wherein the conductive hydrogel dressing is connected to an electrical stimulation device, and electrical stimulation of a specific mode is applied for a certain period of time by controlling the intensity of the current at a certain interval.

[0013] In some embodiments, the specific mode of electrical stimulation includes one of constant current direct current stimulation, constant voltage direct current stimulation, alternating current stimulation, and pulse electrical stimulation.

[0014] In some embodiments, the parameters of the constant current DC stimulation are 0.3-1.0 mA; the parameters of the constant voltage DC stimulation are 1.5-6.0 V; the certain interval time is 24-48 h; and the certain time of electrical stimulation is 10-20 min.

[0015] A non-disease diagnostic method for monitoring the progress of wound healing, wherein the conductive hydrogel dressing is applied to the wound area, and the progress of wound healing is tracked in real time by monitoring the changes in electrical signals generated by the movement of joints near the wound area.

[0016] The beneficial effects of the present invention are: The conductive hydrogel dressing prepared by the present invention has excellent conductive properties. The dressing successfully integrates electrical stimulation therapy and can effectively accelerate cell migration and proliferation through constant current, constant voltage, AC electrical stimulation or pulse electrical stimulation, significantly optimizing the healing effect. In addition, its excellent conductive properties also support real-time monitoring of changes in the relative resistance signal of the wound area and accurate evaluation of the healing status. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The preparation flow chart of conductive hydrogel and its bonding.

[0019] Figure 2 Schematic diagram of electrically stimulated cells.

[0020] Figure 3 Schematic diagram of the electrical stimulation wound model.

[0021] Figure 4 Schematic diagram of wound healing model monitoring.

[0022] Figure 5 The electrical performance diagram of the conductive hydrogel. Among them: (A) conductivity test diagram, (B) conductivity diagram of the conductive hydrogel, (C) cut healing resistance response diagram of the conductive hydrogel.

[0023] Figure 6 The following are graphs showing the effect of different component ratios on electrical conductivity, including: (A) electrical conductivity of polyvinyl alcohol at different contents, (B) electrical conductivity of tannic acid at different contents, (C) electrical conductivity of borax at different contents, and (D) electrical conductivity of wool keratin at different contents.

[0024] Figure 7 The biocompatibility diagram of the conductive hydrogel. Among them: (A) hemolysis experiment diagram, (B) cell live-death staining diagram, (C) cell activity diagram, (D) bleeding volume recorded by weighing filter paper, (E) film-forming real-life photos of the rat liver bleeding model, (F) real-life photos of the bleeding volume recorded by filter paper at 30s, 30-60s, and 60-120s in the rat liver bleeding model, and (G) coagulation index determination.

[0025] Figure 8 The effect of electrical stimulation parameters on the migration of L929 cells, HUVEC cells and C2C12 cells. Among them: direct current stimulation with constant voltage (A) cell migration rate of L929 cells, (B) cell migration rate of HUVEC cells, (C) cell migration rate of C2C12 cells, direct current stimulation with constant current (D) cell migration rate of L929 cells, (E) cell migration rate of HUVEC cells, (F) cell migration rate of C2C12 cells.

[0026] Fig. 9 The cell scratch experiment pictures of different experimental groups. Among them: (A) microscope image, (B) quantitative in vitro scratch closure results.

[0027] Fig.10 Photograph of wound during wound healing process of conductive hydrogel.

[0028] Fig.11 Schematic diagram of the resistance response of the conductive hydrogel to large strain when attached to the skin, including: (A) finger bending, (B) rapid finger bending, (C) wrist bending, (D) fist clenching, (E) elbow bending and (F) knee bending.

[0029] Fig.12The conductive hydrogel is used to monitor the healing process of rat joint wounds. Among them: (A) relative resistance response of normal skin, (B) relative resistance response of damaged skin, (C) relative resistance response of healing skin, (D) relative resistance response of completely healed skin, and (E) relative resistance response of poorly healed skin. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] The embodiment of the present invention provides a method for preparing a conductive hydrogel dressing, which specifically includes fully mixing and reacting a plurality of components such as a polymer material with good biocompatibility, borax, tannic acid, and keratin, and finally preparing a hydrogel dressing with excellent conductive properties. In the preparation process, the borate ions and sodium ions in the borax play a key role. Among them, a part of the borate ions and the biocompatible polymer material, such as the hydroxyl group of polyvinyl alcohol, form a stable borate ester bond through a chemical reaction, thereby realizing the cross-linking structure of the material; while the other part of the borate ions remain in a free state. At the same time, a large number of sodium ions also exist freely in the colloid, and these free ions together give the hydrogel dressing good conductive properties.

[0032] The conductive hydrogel dressing not only has excellent conductive properties, but can also be effectively combined with electrical stimulation therapy to further promote the wound healing process through the action of electrical stimulation. In addition, the dressing can also sense and transmit electrophysiological signals in the wound area to achieve real-time monitoring of the wound healing process.

[0033] In some specific embodiments, the biocompatible polymer material is selected from a variety of materials, including but not limited to polyvinyl alcohol, polyacrylic acid, various derivatives of polyvinyl alcohol and one of various derivatives of polyacrylic acid. Through this selection, the safety and applicability of the material in a biological environment are ensured.

[0034] In other specific embodiments, the biocompatible polymer material is polyvinyl alcohol, which is an ideal choice due to its excellent biocompatibility, good mechanical properties and easy processing, ensuring high efficiency and reliability in practical applications.

[0035] In some specific embodiments, the selected keratin mainly comes from wool. As a natural polymer material, the keratin component of wool has unique biocompatibility and biodegradability. At the same time, the thiol (-SH) and negatively charged groups rich in its surface can specifically adsorb coagulation factor XII and platelets, accelerate the coagulation cascade reaction through electrostatic action, and improve the hemostatic efficacy and biocompatibility of the material. In addition, keratin is widely available, cheap and easy to obtain, and has a broad application prospect in the field of biomedical materials.

[0036] In some specific embodiments, the mass percentage of the biocompatible polymer material in the overall formula is set to 5% to 10%, and this ratio is intended to ensure the safety and stability of the material in the biological environment. At the same time, the mass percentage of the tannic acid in the formula is controlled between 0.25% and 2%, and this range is set to give full play to the antioxidant and antibacterial effects of tannic acid. In addition, the mass percentage of keratin in the formula is also limited to the range of 0.25% to 2%, and the addition of keratin helps to improve the mechanical strength and biocompatibility of the material. Furthermore, the mass percentage of the borax in the formula is set to 0.5% to 2%, and the presence of borax can effectively adjust the ionic conductivity of the material. Combining the ratios of these ingredients, the resulting material exhibits excellent electrical conductivity.

[0037] In other specific embodiments, the mass percentage of the polyvinyl alcohol in the overall formula is precisely set to 6.25%, and this proportion of polyvinyl alcohol can provide good matrix support and adhesion. At the same time, the mass percentage of the tannic acid in the formula is determined to be 0.75%, and this concentration of tannic acid can effectively exert its antioxidant and antibacterial properties. In addition, the mass percentage of keratin in the formula is set to 1.25%, and this proportion of keratin helps to further improve the mechanical properties and biocompatibility of the material. Furthermore, the mass percentage of the borax in the formula is precisely controlled to 0.75%, and this concentration of borax can effectively adjust the ionic conductivity of the material. Through these precise ratio designs, the resulting material also exhibits excellent electrical conductivity.

[0038] In some specific implementation cases, in order to ensure that the multiple components can achieve the best mixing effect and sufficient chemical reaction, the components need to be placed in a strictly temperature-controlled environment during the operation, and the temperature range of the environment is precisely set between 20 and 30 degrees Celsius. This temperature range not only helps the molecular movement between the components to reach an appropriate level of activity and promotes effective contact and interaction between them, but also effectively avoids adverse effects such as slow reaction due to too low temperature or decomposition of components due to too high temperature, thereby ensuring the smooth progress of the entire mixing and reaction process and the quality and stability of the final product.

[0039] The embodiment of the present invention aims to provide a conductive hydrogel dressing prepared by a specific method. The conductive hydrogel dressing adopts unique processes and technologies in the preparation process to ensure that it has good conductivity and biocompatibility and is suitable for application in a variety of scenarios.

[0040] The embodiment of the present invention also provides a device that integrates electrical healing and progress monitoring functions. The core component of the device is the aforementioned conductive hydrogel dressing. By combining the conductive hydrogel dressing with advanced electronic monitoring technology, the device can not only effectively promote the healing process of the wound, but also monitor the progress of the healing in real time.

[0041] The embodiment of the present invention provides a non-disease treatment method of electrical healing, wherein the conductive hydrogel dressing is connected to an electrical stimulation device, and a specific pattern of electrical stimulation is applied for a certain period of time by controlling the intensity of the current and at intervals of a certain length of time. This method can promote cell migration and accelerate wound healing. The dressing is designed to match the electrical properties of human skin to achieve the best electrical stimulation treatment effect.

[0042] In some specific embodiments, the specific mode of electrical stimulation includes multiple types, including but not limited to one of constant current direct current stimulation, constant voltage direct current stimulation, alternating current stimulation and pulse electrical stimulation. These different types of electrical stimulation each have unique application scenarios and effects.

[0043] In some embodiments, the specific parameter range of the constant current DC stimulation is set to 0.3 to 1.0 mA. The current intensity within this range can ensure safety and effectively promote cell migration and proliferation. Similarly, the parameter range of the constant voltage DC stimulation is set to 1.5 to 6.0 volts (V). This voltage range is also within a safe and controllable range and has been proven to have a certain effect of promoting cell migration and proliferation.

[0044] In addition, in some other embodiments, the parameters of the AC stimulation are set to a current intensity between 0.3 and 1.0 mA, and a frequency range between 5 and 20 Hz. This AC stimulation mode can play a positive role under different physiological and pathological conditions. The parameters of the pulsed electrical stimulation are set to a frequency range between 25 and 100 Hz. This high-frequency pulsed electrical stimulation shows significant biological effects in specific applications. By precisely controlling these electrical stimulation parameters, the regulatory effect on cell behavior can be maximized while ensuring safety.

[0045] In some specific embodiments, the target cells for a non-disease treatment method using electrical stimulation to promote healing include multiple cell types, including but not limited to mouse fibroblasts, mouse myoblasts, and human umbilical vein endothelial cells. These cells can be used alone or in combination to achieve the best healing-promoting effect.

[0046] Similarly, in some detailed embodiments, a target wound animal model applicable to a non-disease treatment method for promoting healing by electrical stimulation also includes a variety of different wound types. These models include, but are not limited to, skin wounds of mice and rats, diabetic ulcers of mice and rats, wounds at the locomotion joints of mice and rats, skin wounds of rabbit ears, infected wounds of mice and rats, and subcutaneous abscesses of mice and rats. These wound models can be of a single type or a combination of multiple types to more comprehensively evaluate and verify the effectiveness and safety of the electrical healing method.

[0047] In some specific embodiments, when the target cell type selected for the experiment is mouse fibroblasts (L929) or human umbilical vein endothelial cells (HUVEC), in order to achieve the best cell migration effect, the constant DC stimulation parameter used is set to 0.5 mA. This parameter has been verified by many experiments and is considered to be the most suitable electrical stimulation intensity for these two cell types. When the target cell type of the experiment is changed to mouse myoblasts (C2C12), in order to achieve the best cell migration effect, the constant DC stimulation parameter is adjusted to 0.8 mA accordingly. The setting of this parameter is also based on a large amount of experimental data, ensuring that mouse myoblasts can exhibit optimal migration ability at this electrical stimulation intensity.

[0048] In other embodiments, the certain interval time is strictly controlled between 24 and 48 hours; and the duration of electrical stimulation is set to 10 to 20 minutes. Specifically, under certain experimental conditions, the duration of electrical stimulation is precisely set to 15 minutes. The selection of this time range not only takes into account the effect of electrical stimulation on cell migration, but also takes into account the practical feasibility and safety of the experimental operation. After many experimental verifications, this parameter range can not only significantly improve the migration efficiency of cells, but also has a high safety, ensuring that no unnecessary damage to the cells will be caused during the experiment.

[0049] The embodiment of the present invention provides a non-disease diagnostic method for monitoring the wound healing process. The core of the method is to apply a specially made conductive hydrogel dressing to the wound area, and to accurately monitor the changes in electrical signals generated by the joints near the wound area during movement, thereby achieving real-time tracking of the wound healing process. In specific operations, the dressing is covered on the wound area. The dressing has adaptive characteristics and can adjust itself according to the specific shape of the wound to ensure that the dressing and the wound surface are in full contact, thereby improving the accuracy and effectiveness of monitoring.

[0050] During the monitoring process, the range of change of the wound resistance signal is set to be greater than 10%, which is significantly different from the range of change of the resistance signal of normal skin or healed wounds, which is usually between 5% and 10%. For skin that appears to be healed on the surface but still has damage inside, the range of change of the resistance signal is less than 5%. Based on the differences in these resistance signal changes, the scheme provided by the present invention can be used to accurately monitor the wound healing process and determine whether the wound is healed well.

[0051] In some specific embodiments, in order to further optimize the monitoring effect, the movement range of the joint near the wound area is controlled between 30 and 60 degrees. In other embodiments, the movement range of the joint is precisely set to 45 degrees. Through these detailed parameter settings, the changes in electrical signals can be more effectively captured, thereby providing more reliable data support for wound healing monitoring.

[0052] Example 1 A conductive hydrogel wound dressing integrating electrical healing promotion and process monitoring functions, and a preparation method thereof as follows Figure 1 shown.

[0053] (1) First, 40 g of wool was chopped, rinsed twice, and soaked in a mixture of 1 L of ethanol and acetone (volume ratio of 1:1). A homogenizer was used to stir at 200 rpm / min for 2 hours, then washed with deionized water 4 times, and then placed in a 60 ℃ oven to dry overnight to obtain defatted wool. Next, 5 g of defatted wool was dissolved in 50 mL of an aqueous solution containing 6 g of sodium sulfide, 24 g of urea, and 1.442 g of sodium dodecyl sulfate (SDS), heated to 60 ℃ in a water bath and stirred for 8 hours to obtain a crude keratin extract. After the crude keratin extract was balanced, it was placed in a centrifuge, set to a speed of 11000 rpm / min, and centrifuged for 30 minutes to remove the completely dissolved wool. The supernatant after centrifugation was poured into a 3500 D dialysis bag for dialysis purification, and a suitable length of dialysis membrane was cut and loaded with crude keratin solution, which was dialyzed in deionized water with a volume ratio of about 1:100 for 3 days, and the water was changed every 6 hours. After the dialysis was completed, the keratin solution was frozen overnight and then freeze-dried in a freeze dryer to obtain keratin powder.

[0054] (2) Next, weigh 0.5 g of polyvinyl alcohol (PVA, P), add 3 mL of ultrapure water, and stir at 95 °C for 1 hour to completely dissolve it to prepare a PVA solution. Weigh 0.1 g of keratin (W) and dissolve it in 2 mL of ultrapure water. After it is fully dissolved, add it to the PVA solution and stir slowly at room temperature for 1 hour. Then, dissolve 0.06 g of borax (B) and 0.06 g of tannic acid (T) in 3 mL of ultrapure water. After slowly stirring the mixture of PVA and keratin for 5 seconds, an ion conductive hydrogel dressing is formed.

[0055] Comparative Example 1 Polyvinyl alcohol (P) and borax (B) were mixed and stirred to prepare an ion conductive hydrogel dressing (PB). The remaining steps were the same as those in Example 1.

[0056] Comparative Example 2 Polyvinyl alcohol (P), keratin (W) and borax (B) were mixed and stirred to prepare an ion conductive hydrogel dressing (PWB). The remaining steps were the same as those in Example 1.

[0057] Comparative Example 3 Polyvinyl alcohol (P), tannic acid (T) and borax (B) were mixed and stirred to prepare an ion conductive hydrogel dressing (PTB). The remaining steps were the same as those in Example 1.

[0058] Comparative Example 4 In this Example 1, the mass percentages of other components are fixed, and the proportions of the single components are adjusted to form an ion conductive hydrogel dressing. The remaining operating steps are the same as those in Example 1.

[0059] Comparative Example 5 Polyvinyl alcohol (P), silk fibroin (S), tannic acid (T) and borax (B) were mixed and stirred to prepare an ion conductive hydrogel dressing (PSB). The remaining steps were the same as those in Example 1.

[0060] The electrical properties and biocompatibility of the conductive hydrogels prepared in Example 1 and Comparative Examples 1-5 were characterized. Figure 5-7 shown.

[0061] The electrical performance test was carried out by using a 3D printed mold (size: 30 mm×10 mm×10 mm) to standardize the sample morphology, and high-purity copper foil electrodes were attached to both ends of the mold to ensure that the electrode-gel interface contact area was constant. The conductive hydrogel prepared in Example 1 and Comparative Examples 1-5 was placed in the mold cavity and completely fitted with the mold. A precision source measurement unit (TH1991B, Tonghui Electronics, Jiangsu, China) was connected to measure the DC resistance value (R). The sampling interval was 60 seconds, and the average value of three measurements was taken. Based on Ohm's law, the conductivity (σ) was calculated by formula (1):

[0062] Wherein, L is the electrode distance and A is the effective conductive cross-sectional area.

[0063] The biocompatibility test is as follows: After the cytotoxicity test, the conductive hydrogels prepared in Example 1 and Comparative Examples 1-3 were extracted with complete culture medium at 37°C for 24 h and then used for later use. L929 cells in the logarithmic growth phase were taken and 1×10 4 The cells were inoculated at a density of 100 μL / well in a 96-well plate. After the cells adhered to the wall (about 6 h), the original culture medium was discarded and 100 μL of the hydrogel extracts (PB, PWB, PTB, PWTB, concentration 1 mg / mL) of Example 1 and Comparative Examples 1-3 were added to the control group culture medium (n=5 per group). After the cells were cultured for 24, 48 and 72 h, 10 μL of CCK-8 reagent was added to each well and incubated in the dark for 2 h. The absorbance (OD value) at a wavelength of 450 nm was measured using an ELISA instrument, where the blank group was a well containing only CCK-8 reagent and culture medium. The cell viability was calculated according to formula (2) to compare the biocompatibility of the materials.

[0064]

[0065] Figure 5 (A) shows that the hydrogel has conductive properties and is composed of Figure 5 In (B), it can be seen that the composite hydrogel doped with wool keratin and tannic acid has a higher conductivity than the single-component conductive hydrogel and is close to the conductivity of human skin. Figure 5In (C), it can be seen that its conductive properties also change with its strain. When the hydrogel is cut, the circuit is interrupted and the LED indicator turns off. Then the two sections are kept in contact for 24 hours to achieve the self-healing process, and the LED indicator on the circuit lights up again.

[0066] The mass percentages of other components in Example 1 are fixed, and the proportions of the individual components are adjusted to test the corresponding conductivity of the conductive hydrogel. Figure 6 It can be seen that by adjusting the ratio of polyvinyl alcohol, borax, tannic acid and keratin, the conductivity of the hydrogel will be different. The results show that when the mass percentage of polyvinyl alcohol is 6.25%, the mass percentage of tannic acid is 0.75%, the mass percentage of keratin is 1.25%, and the mass percentage of borax is 0.75%, the conductivity of the hydrogel is the best. This is because tannic acid can combine with the components in the hydrogel through hydrogen bonds or other chemical bonds, change the network structure, make it easier for ions to migrate in the network, and improve the conductivity. Tannic acid and keratin pass hydrogen bonds and ionic bonds, thereby improving the stability and solubility of keratin, and also improving the conductivity of the hydrogel. However, if the concentration of tannic acid is too high, keratin will denature to form flocculent precipitation, thereby hindering the migration of ions in the hydrogel system.

[0067] Take fresh anticoagulated mouse whole blood, centrifuge at 3000 rpm for 15 minutes, discard the upper plasma and light yellow layer, and retain the bottom red blood cell sediment; resuspend the red blood cell sediment with an equal volume of phosphate buffered saline (PBS, pH 7.4), repeat the above centrifugation operation until the supernatant is colorless and transparent, and obtain a purified red blood cell suspension. 20 mg of the hydrogel prepared in Example 1 and Comparative Examples 1-3 were placed in 1.5 mL centrifuge tubes respectively; 1 mL of 2% RBC suspension (PBS diluted) was added to each tube and incubated at 37°C for 4 hours; the blank group was a 2% RBC suspension diluted with PBS, and the positive group was a 2% RBC suspension diluted with deionized water. After the incubation, each tube was centrifuged at 3000 rpm for 10 minutes, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at a wavelength of 540 nm using an enzyme reader. Four replicate wells were set for each group (n=4). The hemolysis rate of each group was calculated according to formula (3):

[0068] Depend on Figure 7 As can be seen from the results in (A), the hemolysis rate of each component hydrogel is less than 5%, which meets the relevant safety standards. Figure 7From the results of cell compatibility of (BC), it can be seen that cell proliferation gradually increased within 24 hours, 48 ​​hours and 72 hours, and the proliferation level of the PWTB group was significantly higher at 72 hours compared with the control group. The live / dead staining results also showed that the green fluorescence intensity increased over time, indicating that cell viability gradually increased with the increase in culture time. The PWTB hydrogel group showed a significantly stronger green fluorescence signal than the control group, indicating that the hydrogel has a positive promoting effect on cell survival and proliferation.

[0069] The hydrogel of Example 1 was used to evaluate the in vivo hemostatic properties of the hydrogel in a rat liver bleeding model. After the animal was anesthetized, the abdomen was cut open to expose the liver. The liver was punctured on the liver using a syringe needle, and then a weighed filter paper was placed under the liver. The hydrogel was then quickly injected or adhered to the bleeding site, and the blood loss weight was recorded within a set time. A wound without any treatment was used as a control.

[0070] Depend on Figure 7 From the results of (DF), it can be seen that the data can intuitively show that compared with the untreated group, the group coated with the hydrogel of this embodiment can quickly stop bleeding in a short time, and the amount of bleeding is much lower than that of the blank group. The hydrogels of Example 1, Comparative Examples 1-3 and Comparative Example 5 were tested for blood coagulation index (BCI), and 0.1M CaCl2 was added to the heparinized mouse blood at a ratio of 10:1 to activate the blood. 50µL of activated blood was placed on the surface of the hydrogel, and 5 mL of deionized water was used to dissolve the uncoagulated blood at different time points. The OD value at 540 nm was tested by an enzyme marker. A solution of 50µL of blood in 5 mL of deionized water was used as the control group. BCI is calculated by the following formula 4:

[0071] The lower the BCI, the more effective it is in stimulating blood coagulation. Figure 7It can be seen from the results of (G) that compared with the comparative example 1 of the basic gelling component, this embodiment has a better hemostatic effect. The above hemostatic data may be due to the fact that keratin has abundant thiol groups (-SH) and disulfide bonds (-SS-), which can promote cell adhesion and tissue repair, and contribute to coagulation and wound healing. At the same time, keratin materials can promote platelet adhesion and aggregation by interacting with platelets under physiological conditions, thereby accelerating the hemostatic process. Single-component tannic acid mainly accelerates the formation of blood clots through protein precipitation and vasoconstriction, but when the concentration of tannic acid is too high, it may have cytotoxicity and cause platelet inactivation, affecting the hemostatic effect. Compared with the single-component comparative examples 2 and 3, keratin can form hydrogen bonds and covalent crosslinks with tannic acid, so that the PWTB hydrogel forms a denser and tougher network structure. At the same time, this embodiment cooperates with the protein adhesion, blood coagulation, vasoconstriction and hydrogel barrier of keratin and tannic acid, and effectively promotes hemostasis. Taking silk fibroin as a comparison, it can be observed that silk fibroin, as a natural polymer material similar to keratin, can promote tissue regeneration and cell adhesion, and its structure helps to form a stable barrier to assist hemostasis, but its pro-coagulant effect is not significant, which is similar to the results of basic gelling component comparative example 1.

[0072] Example 2 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate, ensuring that the spacing between the parallel lines is 0.5-1 cm. L929 cells in the logarithmic growth phase are digested with trypsin into a single-cell suspension and inoculated into a 12-well plate, with 4 x10^4 cells implanted in each well. After adding complete culture medium, place in a 37 ℃, 5% CO2 incubator for 24 hours until the cells completely cover the bottom of the well plate.

[0073] (2) Use a 10 μL pipette tip to make scratches along the ruler, with the scratch direction perpendicular to the marked line. Select a 0.2 mm platinum wire as the conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0074] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Apply constant current of 1.5 V, 3.0 V, 4.5 V, and 6.0 V for electrical stimulation. Each group has 2 replicates. Each group is electrically stimulated for 15 minutes per day, and DMEM medium is used for culture during this period. Observe cell migration at 0 h, 24 h, 48 h, and 72 h.

[0075] The cells stimulated by electricity in this example were placed under a microscope for observation and quantitative analysis. The cell scratch experiment showed that electrical stimulation had a significant effect on promoting the migration of L929 cells. Figure 8 Middle A shows that at 72 h, the cell migration effect under 4.5 V electrical stimulation was the best and the scratch area was the smallest.

[0076] Example 3 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate. The spacing between the parallel lines should be controlled between 0.5 and 1 cm. HUVEC cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and inoculated into a 12-well plate, with 4 x 10^4 cells per well. After adding complete culture medium, the plate was cultured in a 37°C, 5% CO2 incubator for 24 hours until the cells completely covered the bottom of the well plate.

[0077] (2) Use a 10 μL pipette tip to make scratches along the ruler, with the scratch direction perpendicular to the marked line. Take a 0.2 mm platinum wire as a conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0078] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Use constant currents of 1.5 V, 3.0 V, 4.5 V, and 6.0 V to electrically stimulate the two ends of the wire. Each group has two replicate wells. Each group is electrically stimulated for 15 minutes per day. During this period, DMEM culture medium is used to culture the cells. The cell migration is observed at 0 h, 24 h, and 48 h.

[0079] The cells stimulated by electricity in this example were placed under a microscope for observation and quantitative analysis. The cell scratch experiment showed that electrical stimulation had a certain effect on promoting HUVEC cell migration. Figure 8 Middle B shows that at 48 h, the cell migration effect of the 4.5 V group was the best and the scratch was basically healed.

[0080] Example 4 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate, with the line spacing controlled between 0.5-1 cm. Digest the C2C12 cells in the logarithmic growth phase with trypsin to form a single-cell suspension, then inoculate them into a 12-well plate, with 4 x 10^4 cells per well. After adding complete culture medium, place the plate in a 37 ℃, 5% CO2 incubator for 24 hours until the cells completely cover the bottom of the well plate.

[0081] (2) Use a 10 μL pipette tip and a ruler to make scratches perpendicular to the marking line. Take a 0.2 mm platinum wire as a conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0082] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Apply constant currents of 1.5 V, 3.0 V, 4.5 V, and 6.0 V for electrical stimulation. Each group has two replicate wells. Electrical stimulation lasts for 15 minutes every day. During this period, DMEM culture medium is used to maintain the culture. The cell migration is observed at 0 h, 24 h, and 48 h.

[0083] The cells treated with electrical stimulation were placed under a microscope for observation and quantitative analysis. The cell scratch test showed that electrical stimulation significantly promoted the migration of C2C12 cells. Figure 8 Middle C shows that at 48 h, the cell migration effect of the 4.5 V group was the most significant, and the scratch was basically healed.

[0084] Example 5 A scheme for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate, ensuring that the spacing between the parallel lines is between 0.5 and 1 cm. Digest the L929 cells in the logarithmic growth phase with trypsin to form a single-cell suspension, and inoculate them into a 12-well plate, with 4 x10^4 cells per well. After adding complete culture medium, place the plate in a 37°C, 5% CO2 incubator for 24 hours until the cells completely cover the bottom of the well plate.

[0085] (2) Use a 10 μL pipette tip and a ruler to make scratches perpendicular to the marked line. Take a 0.2 mm platinum wire as a conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0086] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Electrical stimulation was performed at constant currents of 0.3 mA, 0.5 mA, 0.8 mA, and 1.0 mA, respectively. Each group had 2 replicates and electrical stimulation lasted for 15 minutes every day. During this period, DMEM medium was used for culture, and cell migration was observed at 0 h, 24 h, 48 h, and 72 h.

[0087] The cells treated with electrical stimulation were placed under a microscope for observation and quantitative analysis. The cell scratch test showed that electrical stimulation had a significant effect on promoting the migration of L929 cells. Figure 8 Middle D shows that at 72 h, the cell migration effect of the 0.5 mA group was the most significant, and the scratch area was the smallest.

[0088] Example 6 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate, with the spacing between the lines controlled at 0.5-1 cm. HUVEC cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and inoculated into a 12-well plate, with 4 x 10^4 cells in each well. After adding complete culture medium, the plate was cultured in a 37 ℃, 5% CO2 incubator for 24 hours until the cells completely covered the bottom of the well plate.

[0089] (2) Using a ruler, use a 10 μL pipette tip to make a scratch perpendicular to the marked line. Take a 0.2 mm platinum wire as a conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0090] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Electrical stimulation was performed at constant currents of 0.3 mA, 0.5 mA, 0.8 mA, and 1.0 mA, respectively. Each group had two replicate wells and electrical stimulation was performed for 15 minutes every day. During this period, DMEM medium was used for culture, and cell migration was observed at 0 h, 24 h, and 48 h.

[0091] The electrically stimulated cells were placed under a microscope for observation and quantitative analysis. The cell scratch test showed that electrical stimulation has a significant effect on promoting HUVEC cell migration. Figure 8 Middle E shows that at 48 h, the cell migration effect of the 0.5 mA group was the best and the scratch was basically healed.

[0092] Example 7 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2As shown, the specific electrical stimulation program includes the following steps: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate. The spacing between the parallel lines should be between 0.5 and 1 cm. Digest the C2C12 cells in the logarithmic growth phase with trypsin to form a single-cell suspension, then inoculate them into a 12-well plate, with 4 x 10^4 cells per well. After adding complete culture medium, incubate in a 37°C, 5% CO2 incubator for 24 hours until the cells completely cover the bottom of the well plate.

[0093] (2) Use a 10 μL pipette tip to make scratches along the ruler, with the scratch direction perpendicular to the marked line. Take a 0.2 mm platinum wire as a conductor, place it in the culture medium, and fix it to the edge of the well plate with insulating tape.

[0094] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Use constant currents of 0.3 mA, 0.5 mA, 0.8 mA, and 1.0 mA for electrical stimulation. Set up 2 replicates in each group, and stimulate each group for 15 minutes per day. During this period, use DMEM culture medium to culture and observe cell migration at 0 h, 24 h, and 48 h.

[0095] The cells stimulated by electricity in this example were placed under a microscope for observation and quantitative analysis. The cell scratch test showed that electrical stimulation had a certain effect on promoting the migration of C2C12 cells. Figure 8 Middle F shows that at 48 h, the cell migration effect was best under the electrical stimulation condition of 0.8 mA, and the scratch was basically healed.

[0096] Example 8 A method for electrically stimulating cells using the conductive hydrogel prepared in Example 1 is as follows: Figure 2 The specific steps are as follows: (1) Use a marker to draw parallel lines on the bottom of a 12-well plate, ensuring that the distance between the parallel lines is maintained within the range of 0.5-1 cm. HUVEC cells in the logarithmic growth phase are digested with trypsin into a single-cell suspension and inoculated into a 12-well plate, with 4 x 10^4 cells implanted in each well. After adding complete culture medium, place in a 37 ℃, 5% CO2 incubator for 24 hours until the cells completely cover the bottom of the well plate.

[0097] (2) Use a 10 μL pipette tip and a ruler to make a scratch, making sure the scratch is perpendicular to the marked line. Use a 0.2 mm platinum wire as a conductor, place it in the culture medium, and secure it to the edge of the well plate with insulating tape.

[0098] (3) Use SS-L303SPD linear power supply to connect the two ends of the wire for electrical stimulation. Use the optimal constant current determined in Example 3 to electrically stimulate the two ends of the wire. Set up 2 replicates in each group and stimulate for 15 minutes every day. During this period, use DMEM culture medium or PWTB hydrogel extract for culture, and observe the cell migration at 0 h, 24 h, and 48 h respectively.

[0099] The cells treated with electrical stimulation in this example were placed under a microscope for observation and quantitative analysis. Fig. 9 The analysis results show that electrical stimulation combined with hydrogel can significantly enhance cell migration, thereby effectively promoting wound healing.

[0100] Example 9 A scheme of an electrical stimulation wound model using the conductive hydrogel prepared in Example 1 is as follows Figure 3 The specific scheme includes the following steps: (1) Female BALB / c mice, 6 weeks old and weighing an average of 18 g, were placed in an environment with a constant temperature of (23 ± 2) °C and a humidity of 50%-60%, following a circadian rhythm of 12 h light and 12 h dark, with free access to water and food.

[0101] (2) After anesthetizing the mice, a 9 mm full-thickness circular wound was made on their backs with a scalpel. The blank control group did not receive any treatment. For the electrical stimulation (ES) group, a linear power supply was placed near the edge of the wound, and ES treatment was performed every other day. After 15 minutes, the external power supply was removed. For the hydrogel + ES group, the conductive hydrogel prepared in Example 1 was covered on the wound, and treatment was performed every other day. Each time, the hydrogel was used for 15 minutes according to the electrical stimulation parameters of Example 5.

[0102] By observing the changes in the wound area in this implementation case (such as Fig.10 As shown), it can be seen that the wound contraction effect of the conductive hydrogel dressing group is better than that of the control group. Compared with the groups treated with hydrogel or ES alone, the hydrogel dressing combined with ES showed a more significant wound healing effect. This shows that ES treatment has a positive effect on wound healing, but there is still room for improvement. In contrast, the wound in the hydrogel + ES group was able to achieve complete closure, and its wound repair rate was significantly different from that of the conductive hydrogel group without ES, further confirming that the synergistic effect between conductive hydrogel and ES effectively accelerated wound healing.

[0103] Example 10 A conductive hydrogel prepared in Application Example 1 is used to monitor human body movement and prevent wound tearing. The specific monitoring scheme includes the following steps: (1) The conductive hydrogel of Example 1 is adhered to various joints of the human body and fixed at both ends of the hydrogel with wire clips.

[0104] (2) Connect the precision source measurement unit to the wire clamp to form a closed loop and measure the relative resistance change of the conductive hydrogel.

[0105] The resistance signal change of this embodiment is recorded by a precision source measurement unit. Fig.11 The conductive hydrogel sensor was connected to an integrated system to record the resistance change of the hydrogel caused by strain during human movement, thereby monitoring human movement and avoiding secondary tearing of wounds. In addition, due to its excellent adhesion properties, the hydrogel sensor can adhere tightly to human skin and accurately transmit electrical signals even in the case of intense exercise or repeated deformation.

[0106] Embodiment 11 A scheme for monitoring the wound healing process using the conductive hydrogel prepared in Example 1 is as follows: Figure 4 As shown, the specific monitoring program includes the following steps: (1) Male SD rats aged 8 weeks and weighing an average of 300 g were placed in an environment with a constant temperature of (23±2)°C and a humidity of 50%-60%, following a circadian rhythm of 12 hours of light and 12 hours of darkness, with free access to water and food. After the rats were anesthetized, an 8 mm circular wound was made on their left knee joints with a scalpel. The blank control group did not receive any treatment.

[0107] (2) The conductive hydrogel prepared in Example 1 was placed on the wound of the left knee joint of the rat and the undamaged skin surface of the right knee joint, and connected to form a closed loop according to the sensing test scheme of Example 8. The left knee joint of the rat was assisted to move with the amplitude constantly controlled at 45°, and the relative resistance change of the conductive hydrogel was recorded by a precision source measurement unit.

[0108] (3) One week later, when the wound on the left knee joint of the rat was about to heal, the test was performed again according to step (2) of Example 8.

[0109] (4) Two weeks later, when the wound on the left knee joint of the rat was completely healed, the test was performed again according to step (2) of Example 8.

[0110] (5) Prepare a piece of pig skin and silicone with a length of 50 mm, a width of 15 mm, and a thickness of 10 mm. Use a scalpel to make a shallow scratch on the back of the sample, making sure not to penetrate the material and keep the front of the sample intact. Form a deep wound on the back to simulate a poorly healed wound in which the skin surface is closed but the deep layer is not healed.

[0111] (6) Cover the surface of pig skin and silicone with the conductive hydrogel prepared in Example 1, fix the pig skin and silicone on a manipulator, and set the pulse width modulation of the manipulator to move from 1500 to 2500 with a time interval of 1200 ms.

[0112] The resistance signal change of this implementation case is recorded by a precision source measurement unit. The results are as follows: Fig.12 shown. Fig.12 (B) and (C) show that the resistance signal of the wound changes within the range of >10%. Fig.12 The resistance signal of normal skin or healed wound represented by (A) and (D) varies within the range of 5-10%. Fig.12 The middle (E) represents the skin that has healed on the surface but still has internal damage. The resistance signal change range is <5%. Therefore, this solution can be used to monitor the wound healing process and healing quality, assist doctors in diagnosis, and help patients better understand their own conditions.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a conductive hydrogel dressing, characterized in that: A biocompatible polymer material, borax, tannic acid and keratin are mixed and reacted fully to obtain a conductive hydrogel dressing; the mass percentage of the biocompatible polymer material is 5-10%, the mass percentage of the tannic acid is 0.25-2%, the mass percentage of the keratin is 0.25-2%, and the mass percentage of the borax is 0.5-2%.

2. The method for preparing a conductive hydrogel dressing according to claim 1, characterized in that: The biocompatible polymer material is selected from polyvinyl alcohol, polyacrylic acid, polyvinyl alcohol derivatives, and one of polyacrylic acid derivatives.

3. The method for preparing the conductive hydrogel dressing according to claim 1 or 2, characterized in that: The reaction temperature is 20-30°C.

4. The method for preparing a conductive hydrogel dressing according to claim 2, characterized in that: The mass percentage of the polyvinyl alcohol is 6.25%, the mass percentage of the tannic acid is 0.75%, the mass percentage of the keratin is 1.25%, and the mass percentage of the borax is 0.75%.

5. A conductive hydrogel dressing prepared by the method according to claim 1.

6. A device with electrical healing and progress monitoring functions, characterized in that: The conductive hydrogel dressing according to claim 5.

7. A non-disease treatment method of electrical healing, characterized in that: The conductive hydrogel dressing of claim 5 is connected to an electrical stimulation device, and a specific mode of electrical stimulation is applied for a certain period of time at certain intervals by controlling the intensity of the current.

8. The non-disease treatment method of electrical healing according to claim 7, characterized in that: The specific mode of electrical stimulation includes one of constant current direct current stimulation, constant voltage direct current stimulation, alternating current stimulation, and pulse electrical stimulation.

9. The non-disease treatment method of electrical healing according to claim 8, characterized in that: The parameters of the constant current direct current stimulation are 0.3-1.0 mA; the parameters of the constant voltage direct current stimulation are 1.5-6.0 V; the certain interval time is 24-48 h; and the certain time of electrical stimulation is 10-20 min.

10. A non-disease diagnostic method for monitoring wound healing progress, characterized in that: The conductive hydrogel dressing of claim 5 is applied to the wound area, and the progress of wound healing is tracked in real time by monitoring the changes in electrical signals generated by the movement of joints near the wound area.

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