Hydrogel material and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a mixed hydrogel material with a rapid gelation function and application of the mixed hydrogel material. According to the invention, bioactive glass nanoparticles rich in magnesium and enoxacin are integrated into the design, and the hydrogel material is efficiently prepared. The manufacturing process of the material is only a few minutes, and meanwhile the material still keeps the optimal function. The material has good biocompatibility, remarkable antibacterial effect and strong immunoregulation characteristic, can successfully improve the recovery of intractable wounds, and also shows hemostatic performance at the same time. According to the invention, the healing of the infected wound can be safely and effectively accelerated, and a new solution is provided for the complex challenge of wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a hybrid hydrogel material with rapid gelation and its application. Background Art

[0002] With the continuous innovation of planting techniques, the problem of bone or soft tissue defects in the planting area has become increasingly prominent, making the hard and soft tissue augmentation surgery particularly important. Successful implant restoration not only requires the formation of a stable bone bond between the implant and the alveolar bone, but also ensures the stability of the soft tissue around the implant (DELAMO F S L, YU S-H, SAMMARTINO G, et al. Peri-implant soft tissue management: Cairo opinion consensus conference [Z]. MDPI. 2020.). Good tissue healing can establish an effective barrier to prevent the invasion of bacteria, thus creating a safe and healthy microenvironment for bone healing (WANG Y, ZHANG Y, MIRON R J. Health, maintenance, and recovery of soft tissues around implants [J]. Clinical implant dentistry and related research, 2016, 18(3): 618-34.). However, after implant surgery, there may be tissue edema, misalignment of sutures or wound dehiscence at the wound, which may then lead to infection. Once an infection occurs, it not only seriously hinders the healing process of the hard and soft tissues after implant surgery, but may also cause irreversible effects on the final aesthetic effect. Therefore, it is particularly important to accelerate the healing of infected wounds after implant surgery.

[0003] According to different participating cells and protein molecules, wound healing can be divided into four stages: hemostasis, inflammation, proliferation, and remodeling. After a wound is formed, the body quickly enters the hemostasis stage. Platelets first aggregate and are activated into fibrin clots, recruiting neutrophils and lymphocytes to participate in the inflammatory response while stopping bleeding. Then it enters the inflammation stage, where monocytes proliferate and differentiate into macrophages, releasing reactive oxygen species to remove foreign bodies, damaged autologous tissues, and bacterial proteases, and at the same time secreting cytokines that promote tissue proliferation and cell migration (RODRIGUES M, KOSARIC N, BONHAM CA, et al. Wound healing: a cellular perspective[J]. Physiological reviews, 2019, 99(1): 665 - 706.). Then in the proliferation stage, granulation tissue containing a large number of newly formed capillaries arranged disorderly proliferates in the provisional extracellular matrix (DIPIETRO LA. Angiogenesis and wound repair: when enough is enough[J]. Journal of Leucocyte Biology, 2016, 100(5): 979 - 84.), and finally the newly formed capillaries are remodeled to form a mature vascular network. In the remodeling stage, collagen fibers are also replaced and rearranged to complete the repair and remodeling of the tissue at the wound site (WILKINSON HN, HARDMAN MJ. Wound healing: Cellular mechanisms and pathological outcomes[J]. Open biology, 2020, 10(9): 200223.).

[0004] Wound healing is a complex and time-consuming biological process. However, this process does not always proceed smoothly. Especially when combined with bacterial infection after injury, the healing process will be severely affected (ROBSON M C. Wound infection: a failure of wound healing caused by an imbalance of bacteria[J]. Surgical Clinics of North America, 1997, 77(3): 637-50.). First of all, when the skin or other tissues are damaged, the exposure of the wound provides an opportunity for microorganisms such as bacteria. These microorganisms may come from the external environment or the patient's own flora. Once bacteria invade the wound, they will quickly multiply and cause infection. The infection will not only cause symptoms such as redness, pain, and fever of the wound, but may also damage the newly formed granulation tissue and blood vessels, delaying the healing speed (VACHHRAJANI V, KHAKHKHAR P. Science of wound healing and dressing materials[R]: Springer, 2020.). Secondly, bacterial infection will also trigger a series of immune responses. Although the activation of the immune system helps to eliminate bacteria, excessive immune responses will also lead to the aggravation of the inflammatory response, further damaging the wound tissue (GIAMARELLOS-BOURBOULIS E J, RAFTOGIANNIS M. The immune response to severe bacterial infections: consequences for therapy[J]. Expert review of anti-infective therapy, 2012, 10(3): 369-80.). This vicious cycle not only increases the patient's pain, but may also lead to the failure of the healing process. Therefore, anti-bacterial is particularly important in the process of wound healing.

[0005] The treatment plan for wounds has always aimed to reduce complications and achieve the remodeling of tissue structure and function. Clinically, for soft tissue wounds, a treatment mode mainly based on protective barriers is usually adopted. Gauze, bandages or medical absorbent cotton are still the most commonly used wound dressings. Due to their low price, guaranteed effectiveness and high breathability, gauze is used for most wounds. It can not only stop bleeding quickly but also act as a physical barrier to protect the wound from contact with the external environment and prevent infection, and at the same time play an auxiliary role in fixation and dressing (Bains D, Singh G, Kaur N, Singh N. Development of Biological Self-Cleaning Wound-Dressing Gauze for the Treatment of Bacterial Infection[J]. ACS sustainable chemistry & engineering. 2019;7:969-978.). However, there are still many drawbacks to this traditional treatment method. There are large pores in the gauze, and it is difficult for the incompletely closed wound surface to become a barrier against microorganisms. It not only has limited ability to absorb exudate but also easily dries out and hardens, adheres to the wound site, and granulation tissue is also likely to grow into the pores. This makes it more difficult to change the gauze, and even causes secondary trauma, destroys the self-healing process of the wound, and makes the patient more painful (Li S, Chen A, Chen Y, et al. Lotus leaf inspired antiadhesive and antibacterial gauze for enhanced infected dermal wound regeneration[J]. Chemical engineering journal. 2020;402:126202.). Therefore, this traditional dry treatment is more suitable for use as a dry wound or a secondary dressing, which also means that the treatment method for protecting wounds still needs to be optimized.

[0006] To improve the defects of traditional wound treatment, people apply ointments containing antibiotics to the wound and use gauze for fixation, or use artificial skin for protection. Although the moist environment of the wound is maintained, once the early protection is improper and the infection spreads, the short-term application of antibiotic ointments or oral medications is difficult to maintain long-term efficacy, but will instead prolong the healing process (Valacchi G, Zanardi I, Sticozzi C, Bocci V, Travagli V. Emerging topics in cutaneous wound repair[J]. Annals of the New York Academy of Sciences. 2012;1259:136-144.), and the overuse of antibiotics will also lead to the development of drug-resistant strains, resulting in irreversible side effects. Therefore, there is an urgent clinical need for a new treatment method that can exert therapeutic effects in the early stage of trauma and has the least side effects after use. Researchers have also turned their attention to natural active ingredients with therapeutic capabilities and related synthetic drugs. Many substances of natural origin have been successfully applied to modern dressings, including various types of collagen, marine-derived active substances such as chitosan, natural medicaments such as honey and Scutellaria baicalensis, or skin substitutes such as fish skin and pig skin have been developed and applied to clinical treatment (Rezvani Ghomi E, Khalili S, Nouri Khorasani S, Esmaeely Neisiany R, Ramakrishna S. Wound dressings: Current advances and future directions[J]. Journal of applied polymerscience. 2019;136:47738-n / a.).At the same time, researchers are constantly improving the way of delivering active substances, optimizing existing treatments, developing modern dressings that enhance healing capabilities, and providing more stable and efficient early treatments (Kolimi P, Narala S, Nyavanandi D, Youssef AAA, Dudhipala N. Innovative Treatment Strategies to Accelerate Wound Healing: Trajectory and Recent Advancements [J]. Cells. 2022; 11: 2439; Abdo J, Ortman H. Biologic and Synthetic Cellular and / or Tissue-Based Products and Smart Wound Dressings / Coverings [J]. The Surgical clinics of North America. 2020; 100: 741-756.). The demand for clinical treatment continues to promote researchers' research on materials. At present, there are various types of new dressings, including hydrogels, films, hydrocolloid foams and other composite materials, which can provide a good hydration environment for the wound, and have sealing and water retention properties. While protecting the wound, the active ingredients added to the material can promote wound healing. GD, Grumezescu AM. Natural and synthetic polymers for wounds and burns dressing [J]. Int J Pharm. 2014; 463 (2): 127-136.). These dressings and regenerative tissue engineering have developed a variety of active substances, providing more options for accelerating wound healing.

[0007] Among them, hydrogels, as a kind of material with a three-dimensional network structure, have various special effects such as wetness, flexibility, biodegradability, biocompatibility and softness (CALóE, KHUTORYANSKIY V V. Biomedical applications of hydrogels: A review of patents and commercial products [J]. European polymer journal, 2015, 65: 252-67.), and have received extensive attention in the field of wound dressings in recent years. The texture of hydrogels is similar to that of natural extracellular matrix, which not only provides a good microenvironment for wound healing, but also can serve as a physical barrier to block bacteria and prevent wound infection (JIANG F, CHI Z, DING Y, et al. Wound dressing hydrogel of enteromorpha prolifera polysaccharide–polyacrylamide composite: A facile transformation of marine blooming into biomedical material [J]. ACS Applied Materials & Interfaces, 2021, 13(12): 14530-42.). At the same time, hydrogels have good water absorption characteristics while being rich in water, can adsorb exudate in the wound area and maintain the wetness around the wound. In addition, hydrogel dressings are soft, adhere comfortably, and are easy to remove, and can effectively relieve pain to a certain extent. Therefore, a dressing that can quickly seal open wounds is developed, and this dressing can have the functions of hemostasis, antibacterial, and promoting wound healing. This broadens the treatment time window for subsequent treatment. Summary of the Invention

[0008] In the present invention, bioactive glass nanoparticles (MBG) rich in magnesium (Mg) and enoxacin (EN) are integrated into the design, and a hydrogel material is efficiently prepared. The manufacturing process of this material only takes a few minutes, has antibacterial and immunomodulatory capabilities, and at the same time maintains its integrity. Based on this, the present invention is completed.

[0009] In the first aspect, the present invention provides a hydrogel material, which is composed of mesoporous bioactive glass nanoparticles (MBG) rich in metal ions and antibiotics.

[0010] Further, the metal ions are selected from magnesium (Mg), zinc (Zn), copper (Cu) and / or silver (Ag).

[0011] Preferably, the metal ion is magnesium (Mg).

[0012] Further, the antibiotics include but are not limited to enoxacin (EN), levofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, sparfloxacin, etc.

[0013] Preferably, the antibiotic is enoxacin (EN).

[0014] Further, the mesoporous bioactive glass nanoparticles are composed of SiO 2 (silicon dioxide), Na 2 O (sodium oxide), CaO (calcium oxide), and P 2 O 5 (phosphorus pentoxide).

[0015] Further, the content ratio of the antibiotic and the magnesium-containing mesoporous bioactive glass nanospheres in the hydrogel material is 4:1.

[0016] In a second aspect, the present invention provides a preparation method of a hydrogel flexible electrode dressing, and the preparation method includes the following steps:

[0017] S1. Prepare mesoporous bioactive glass nanoparticles

[0018] S1.1 Mix the dissolved cetyltrimethylammonium bromide (CTAB) and ethyl acetate to obtain a mixed solution 1;

[0019] S1.2 Add an ammonia water solution to the mixed solution 1 to obtain a mixed solution 2;

[0020] S1.3 Add tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate to the mixed solution 2 to obtain mesoporous bioactive glass nanoparticles;

[0021] S2. Prepare mesoporous bioactive glass nanoparticles containing metal ions

[0022] S2.1 Add nitrate to the mesoporous bioactive glass nanoparticles prepared in S1, and centrifuge to collect the formed colloid;

[0023] S2.2 Collect the precipitate obtained in S2.1 to obtain mesoporous bioactive glass nanoparticles containing metal ions;

[0024] S3. Prepare mesoporous bioactive glass nanospheres containing metal ions and antibiotics

[0025] Mix the antibiotic with the mesoporous bioactive glass nanoparticles containing metal ions prepared in S2 to obtain mesoporous bioactive glass nanoparticles containing metal ions and antibiotics;

[0026] S4. Prepare the hydrogel

[0027] S4.1 Mix the monomers for preparing the hydrogel to prepare a prepolymer solution;

[0028] S4.2 Add the prepolymer solution into the mold, and add monomers into the solution.

[0029] S4.3 Extract the layered hydrogel from the mold in S4.2 to form a hydrogel containing metal ions, and a hydrogel containing metal ions and antibiotics.

[0030] Further, in step S2.1, the nitrate is selected from magnesium nitrate, zinc nitrate, copper nitrate, calcium nitrate, etc.

[0031] Furthermore, the nitrate is selected from magnesium nitrate or zinc nitrate.

[0032] Preferably, the nitrate is magnesium nitrate.

[0033] Further, in step S3, the antibiotics include but are not limited to enoxacin (EN), levofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, sparfloxacin, etc.

[0034] Preferably, the antibiotic is enoxacin (EN).

[0035] Further, in step S4.1, the monomers include acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and N,N'-methylenebisacrylamide.

[0036] Further, in step S4.2, the monomer is triethylborane-tetrahydrofuran complex (Et3B-THF).

[0037] Further, in step S4.2, a film needs to be placed under the mold to prevent the hydrogel material from sticking.

[0038] Furthermore, the film is a material that does not stick to the flexible electrode dressing.

[0039] Preferably, the film is polyimide.

[0040] In a third aspect, the present invention provides a hydrogel dressing, and the dressing contains the hydrogel material described in the first aspect; the material has at least one of the following functions:

[0041] 1) Promote wound healing;

[0042] 2) Inhibit wound infection;

[0043] 3) Promote wound hemostasis.

[0044] In a fourth aspect, the present invention provides an application of a hydrogel material in the preparation of a product for promoting wound healing; the material has at least one of the following functions:

[0045] 1) Promote wound healing;

[0046] 2) Inhibit wound infection;

[0047] 3) Promote wound hemostasis.

[0048] Furthermore, the product includes a hydrogel dressing and a hydrogel filler.

[0049] Even further, the hydrogel filler can be used for nose augmentation and breast augmentation.

[0050] Beneficial effects

[0051] 1. The present invention utilizes the unique initiation mechanism of the autoxidation reaction of triethylboron with oxygen to efficiently prepare a hydrogel material. The manufacturing process only takes a few minutes, and at the same time, the material still maintains its optimal functions.

[0052] 2. The present invention integrates innovative bioactive glass nanoparticles (MBG) rich in magnesium (Mg) and enoxacin (EN) into the design of the hydrogel material. The integration of these elements endows the hydrogel material (PAM-ME) with antibacterial and immunomodulatory capabilities while maintaining its integrity.

[0053] 3. In vitro studies of the present invention show that the PAM-ME hydrogel has good biocompatibility, significant antibacterial effects, and strong immunomodulatory properties.

[0054] 4. The PAM-ME hydrogel prepared by the present invention has been proven to successfully improve the recovery of intractable wounds and also exhibits hemostatic performance.

[0055] This invention creates a hybrid hydrogel that can safely and effectively accelerate the healing of infected wounds, providing a new solution to the complex challenges of wound healing. Description of the drawings

[0056] Figure 1 Is the gelation process of the hydrogel.

[0057] Figure 2 Is the appearance diagram of the hydrogel material.

[0058] Figure 3Characterization of Mg-MBG and PAM-based hydrogels. A. SEM images of Mg-MBG before and after loading enoxacin; scale bar: left panel: 500 nm, middle panel: 2 μm, right panel: 5 μm. B. TEM and EDS images of Mg-MBG after loading enoxacin; scale bar: 500 nm. C. Morphology of cross-sections of various hydrogels. D. Compressive stress-strain curve of PAM hydrogel. E. Compressive stress-strain curve of PAM-ME hydrogel. F. FTIR of EN, Mg-MBG, Mg-MBG@En, and various hydrogels. G. XRD of Mg-MBG, Mg-MBG@En, and various hydrogels. H. Swelling ratio of PAM and PAM-ME hydrogels. I. Concentration of Mg2 + released from PAM-M and PAM-ME hydrogels. J. Cumulative release amount of EN from PAM-ME hydrogel.

[0059] Figure 4 In vitro cell compatibility. A. Live / dead detection images of 3T3 cells (lower panel); scale bar: 200 μm. B. CCK-8 assay of bone marrow mesenchymal stem cells. C. Hemolysis test results of exposure to the hydrogels. D. CLSM analysis of the morphology of bone marrow mesenchymal stem cells co-cultured on the hydrogels for 24 h; scale bar: 100 μm. Results are expressed as mean ± SD (n = 3).

[0060] Figure 5 In vitro antibacterial activity. A-B. Live / dead staining of bacteria treated with various treatments and measurement of their survival rates; scale bar: 100 μm. C-D. Results of colony formation after treating bacteria with different hydrogels for 24 h. E. Bacterial morphology after treatment with different hydrogels detected by transmission electron microscopy. Results are expressed as mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01.

[0061] Figure 6 Study on the in vitro immunomodulatory effect of PAM-ME hydrogel. A. Expression of iNOS (M1 macrophage marker) and CD206 (M2 macrophage marker) after treatment with different hydrogels; scale bar: 20 μm. B. Statistical data of the fluorescence intensities of iNOS and CD206. C. Analysis of CD86 and CD206 in macrophages after treatment with different hydrogels by flow cytometry. D. Percentages of CD86 and CD206 cells. E. Expression of inflammation-related genes. Results are expressed as mean ± SD (n = 3). ****P < 0.0001, **P < 0.01.

[0062] Figure 7This is an in vivo hemostasis test. A. Representative photographs of the liver bleeding site at different times. B-C. Bleeding time and blood loss in the rat liver bleeding model. D. Typical photographs of the femoral artery bleeding site at different times. E-F. Bleeding time and blood loss in the rat femoral artery bleeding model. The results are expressed as mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

[0063] Figure 8 This is for the PAM-ME hydrogel to promote diabetic wound healing. A-B. Photographs of bacteria-related wounds: day 0 (orange), day 2 (yellow), day 4 (grey-green), day 6 (light green), day 8 (blue), day 10 (purple), and day 12 (pink), and the superimposed images of the wounds after treatment with various hydrogels. C. Unhealed wound area in each group at different time intervals. D-E. CLSM analysis of the morphology of bone marrow mesenchymal stem cells co-cultured on the hydrogel for 24 h; scale bar: 100 μm. The results are expressed as mean ± SD (n = 3). The results are expressed as mean ± SD (n = 3). ****P < 0.0001.

[0064] Figure 9 This is for the histopathological analysis of skin tissue. A. H&E staining; scale bar: 1 mm. B. Quantitative analysis of the corresponding granulation tissue width. C. Masson's trichrome staining; scale bar: 1 mm. D. Statistical analysis of collagen deposition. E. Immunofluorescence staining of CD86 and CD206 in wound tissue sections; scale bar 50 μm. F-G. Statistical analysis of the proportion of CD86 (F) and CD206 (G) positive macrophages. The results are expressed as mean ± SD (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01. Specific embodiments

[0065] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0066] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0067] In the embodiments of the present invention, PAM refers to a conventional hydrogel without adding other substances; PAM-M refers to a hydrogel containing Mg-MBG; PAM-ME refers to a hydrogel containing Mg-MBG@EN.

[0068] In the embodiment of the present invention, FG is fibrin glue, a commonly used clinical bioprotein product for hemostasis and promoting wound healing.

[0069] Synthesis of hydrogel in Example 1

[0070] 1.1 Preparation of mesoporous bioactive glass nanoparticles (Mg-MBG) containing magnesium and antibiotics

[0071] A modified microemulsion-assisted sol-gel method was adopted. Briefly, 0.6 g of cetyltrimethylammonium bromide (CTAB) was dissolved in 30 mL of deionized water and stirred. After the solution became clear, 8 mL of ethyl acetate was added. After stirring, 6 mL of ammonia aqueous solution (NH 3 H 2 O, 1 M) was added. After further stirring, 2.88 mL of tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate were added successively at intervals. Magnesium nitrate was added to the above suspension for further reaction. The formed colloid was collected by centrifugation and washed with deionized water and ethanol (99%). The collected precipitate was dried overnight and then calcined to obtain Mg-MBG. 0.5 mg of enoxacin was mixed with 50 mg of Mg-MBG in 5 mL of Tris-HCl (pH 8.8) and stirred to load enoxacin (EN) into the internal pores of Mg-MBG. Excess reagents were removed by centrifugation to obtain Mg-MBG@EN.

[0072] 1.2 Preparation of hydrogel

[0073] The monomers used to prepare the hydrogel, including acrylamide (AAm, 98%), 2-acrylamido-2-methylpropanesulfonic acid (AMPS, 98%), N,N'-methylenebisacrylamide (MBAA, 98%), and triethylborane-tetrahydrofuran complex (Et3B-THF, 1 M solution), were obtained and used from Energy Chemistry. A prepolymer solution was prepared by mixing 20 mL of AAm aqueous solution (24 wt%), 0.8 mL of MBAA aqueous solution (1 wt%), and 1.6 mL of AMPS aqueous solution (10 wt%). The prepolymer solution was added to a mold, and a polyimide film was placed under the mold. Then, 0.2 mL of Et3B-THF solution (1 M) was added to the solution. Polymerization occurred inside the mold, accompanied by heat release. After a few minutes, the layered hydrogel was extracted from the casting and placed in a container. Specifically, the hydrogel containing Mg-MBG formed during formation was denoted as PAM-M, and the hydrogel containing Mg-MBG@EN was denoted as PAM-ME. Subsequently, all hydrogels were stored for subsequent use.

[0074] As Figure 1 shown, the gel time of this material is relatively fast, and the gel process is basically completed in about 90 s. Figure 2Appearance diagrams of ordinary hydrogel and hydrogel containing Mg-MBG@EN.

[0075] Example 2: Hydrogel for regulating macrophage phenotype in vitro

[0076] First, the expressions of M1 and M2 surface markers (CD86, CD206) were detected by immunofluorescence staining and flow cytometry. That is, macrophages were co-cultured with the hydrogel described in Example 1. 50 ng / mL lipopolysaccharide (LPS) was used to create a systemic inflammatory microenvironment. For immunofluorescence, cells were fixed and blocked. Then the cells were incubated with anti-CD206 and anti-CD86 antibodies, and then the relevant secondary antibodies were used. The cells were stained with DAPI and observed under CLSM. ImageJ software was used to quantify CD206-positive and CD86-positive cells. According to the above steps, the cells after treatment were evaluated for changes in macrophage subtypes by flow cytometry using CD86 and CD206 antibodies. The supernatant was taken out, macrophages were collected, washed with PBS, and then stained with CD206 and CD86 antibodies. After staining, the cells were washed with 500 μL PBS and resuspended for flow cytometry analysis. RAW 264.7 cells followed the same culture protocol. Total RNA was extracted with TRIzol. The gene expression was normalized using the housekeeping gene ACTB. The primer sequences are shown in Supplementary Information (Table S1).

[0077] Table S1 Primer sequences

[0078]

[0079]

[0080] Example 3: Characterization of materials

[0081] The structural characteristics of Mg-MBG, Mg-MBG@EN and different hydrogels (PAM-M and PAM-ME) were detected by scanning electron microscopy (SEM). The internal structure and elemental composition of Mg-MBG@En were studied by transmission electron microscopy (TEM). The chemical composition of the hydrogel was analyzed by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Cylindrical hydrogel samples (10 mm high, 5 mm in diameter) were subjected to a compression test at 5 mm / min until the maximum strain. The hydrogel was incubated at 37 °C to evaluate the water retention rate, and the weight was recorded at regular intervals until equilibrium. Subsequently, the water retention rate was calculated using the following formula:

[0082] Water retention rate = W2 / W1 × 100%;

[0083] Here, W2 represents the weight of the hydrogel at different time intervals, and W1 represents its original weight.

[0084] The ion discharge ability of hydrogels containing nanoparticles was further evaluated. Equal amounts of hydrogels (PAM-M and PAM-ME) were immersed in phosphate buffer solution (PBS) for examination. Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to determine the level of Mg 2+ The release of EN from the hydrogels was studied by placing the PAM-ME hydrogel in a dialysis bag and ultrafiltrating it with an 8 kDa molecular weight membrane. Subsequently, the dialysis bag containing the hydrogel was immersed in phosphate buffered saline (PBS) and stirred. At specified time intervals, samples were taken and the concentration of the released EN was determined by ultraviolet spectrophotometry (λ = 286 nm).

[0085] Scanning electron microscopy examination showed that Mg-MBG consisted of uniform spherical particles with a diameter of approximately 100 nm ( Figure 3 A). Transmission electron microscopy imaging confirmed these findings, showing the uniform distribution of Mg-MBG and mesoporous channels suitable for drug encapsulation ( Figure 3 B). In addition, analysis using energy dispersive spectroscopy (EDS) confirmed the composition of Si, O, Ca, and Mg in the Mg-MBG structure.

[0086] The macroscopic morphologies of PAM, PAM-ME, and PAM-ME hydrogels are shown in the figure, indicating that although MBG nanoparticles and drugs were incorporated into the PAM-ME hydrogel, there was no obvious change in its overall morphology. The only obvious change was the color change from transparent to light yellow. This color change may be related to the loading of drug molecules and their interaction with MBG nanoparticles, rather than being attributed to MBG itself. It is worth noting that after the addition of MBG and drugs, the physical integrity of the hydrogel remained unchanged, indicating that the introduction of MBG nanoparticles did not significantly affect the basic network structure of the hydrogel. The internal microstructure of the hydrogel was studied by scanning electron microscopy. Examination of the freeze-dried hydrogel under scanning electron microscopy showed a porous structure. This porous arrangement supports cell growth and movement, facilitating the effective exchange of nutrients, oxygen, and waste necessary for tissue regeneration. In addition, the porosity of the hydrogel allows for the control of drug release. After nanoparticle incorporation, Mg-MBG and Mg-MBG@EN were uniformly dispersed in the double-crosslinked network, maintaining the integrity of the porous structure of the hydrogel ( Figure 3 C). The mechanical properties of the hydrogels were analyzed. Compared with the PAM hydrogel, the incorporation of nanoparticles in the PAM-ME hydrogel maintained its superior mechanical strength ( Figure 3 D-E).

[0087] Then various hydrogels were analyzed by FTIR, and unique Mg-mbg peaks were found at 812 cm -1 , attributed to the symmetric stretching vibration band of Si-Si, and at 1299 - 900 cm -1, associated with asymmetric silicon-silicon (bridging bond) and silicon-O- (non-bridging bond)( Figure 3 F). Figure 3 The XRD patterns shown in G indicate that the structural characteristics of PAM remain consistent before and after the introduction of nanoparticles. In addition, no substantial changes were observed in the structure or arrangement of the unique diffraction peaks of PAM. Analysis shows that integrating nanoparticles into the PAM hydrogel does not disrupt its inherent structure, thus maintaining the integrity of the biological properties of PAM. Over a period of time, the Mg 2+ concentration in PAM-M and PAM-ME hydrogels was continuously monitored( Figure 3 H-I). The Mg 2+ concentration gradually increased from 6 h to 7 days, indicating the sustained release of the drug. The drug release curve of the PAM-ME hydrogel after soaking in PBS for a specific time is shown in Figure 3 J. There was a rapid release within the first 48 h, followed by a slower release phase. Notably, more than 80% of the drug was released within 168 h.

[0088] Example 4 Biocompatibility Detection Method

[0089] 4.1 Cytotoxicity

[0090] Rat-derived bone marrow mesenchymal stem cells (RBMSCs) were maintained at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a 5% carbon dioxide incubator. The materials were divided into 4 groups (blank control, PAM, PAM-M, PAM-ME), and the proliferation of RBMSCs after different treatments was evaluated by the CCK-8 assay. The cells were initially seeded in Transwell inserts containing the hydrogel. After the hydrogel was soaked in the culture medium for 24 hours, 5 mg of the hydrogel material was placed in the culture medium. Cell suspensions were taken after 1, 3, and 5 days and placed in a well plate, and CCK-8 reagent was added for incubation. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader to calculate the cell viability.

[0091]

[0092] 4.2 Hemolytic Properties of Materials

[0093] The hemocompatibility of different hydrogels was evaluated using the hemolytic activity assay: Rat blood was obtained and centrifuged. The lower erythrocyte layer was separated. Centrifugation was repeated. The cells were resuspended in PBS. 100 mg of the hydrogel sample was mixed with 1 mL of erythrocytes and incubated at room temperature. PBS was used as the negative control and distilled water was used as the positive control to measure hemolysis. All samples were centrifuged, and the absorbance of the supernatant was measured at 545 nm.

[0094] 4.3 Cell viability and cytotoxicity staining

[0095] To evaluate the effect of the hydrogels on cell status, RBMSCs were co-cultured with different hydrogels (PAM-M and PAM-ME) for 24 h, and then fluorescein isothiocyanate-fluorescein actin staining was performed. The cell nuclei were labeled with DAPI, and the cell structure was observed using a confocal laser scanning microscope (CLSM). To stain the live cells, after thorough washing, the cells were incubated with calcein-am and PI, and finally imaged under a fluorescence microscope.

[0096] 4.4 Results

[0097] Cells were co-cultured on various hydrogel substrates. After a 24-h incubation period, viability and death staining were performed. Figure 4 A shows that in the CTRL, PAM, PAM-M, and PAM-ME hydrogel groups, most cells showed an active metabolic state, indicated by green fluorescence, while only a few cells showed a non-viable state. These observations suggest that the hydrogels tested promoted optimal cell adhesion and proliferation, thus supporting their candidacy for biomedical applications.

[0098] The results of the CCK8 assay showed that there was no change in cell viability after co-culture with the hydrogels for 1, 3, or 5 days, indicating no cytotoxic effect ( Figure 4 B). On days 3 and 5, all treatments showed an increase in cell permeability, indicating that the hydrogels supported cell proliferation. The results suggest that the hydrogel scaffolds provided a favorable environment for promoting cell proliferation.

[0099] The hemolysis test confirmed the blood compatibility of the hydrogels, with no significant hemolysis induced ( Figure 4 C).

[0100] Cell morphological analysis further confirmed that after treatment with the hydrogels, the cell structure showed effective spreading and robust growth ( Figure 4 D).

[0101] The results of the study highlighted the biocompatibility of the PAM-ME hybrid hydrogel. It demonstrated the ability to promote cell adhesion, facilitate cell spreading, and significantly enhance proliferation. These observations underscored the suitability of the material for biomedical applications, where cell support and compatibility are crucial. This evidence makes the hybrid hydrogel a promising candidate for tissue engineering and regenerative medicine efforts.

[0102] Example 5 In vitro antibacterial activity

[0103] Frozen-cultured Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were incubated overnight. After dilution 1:10000, the culture reached the logarithmic phase. Subsequently, bacteria with a concentration of 1×10 6 were co-cultured with the hydrogel to evaluate the antibacterial activity. The evaluation of the antibacterial properties of the hydrogel started with live / dead staining analysis. In this process, each sample was treated with a dye mixture containing Syto9 and propidium iodide (PI) and incubated. Then the bacteria were placed on a slide and observed under a fluorescence microscope. The bacteria interacted with various hydrogels and were then diluted by the spread plate method (SPM) and cultured on agar plates. After culturing, photos were taken and the colonies were counted. The ultrastructural changes of the bacterial cell membrane were detected by transmission electron microscopy. Specifically, bacteria (1×10 6 CFU / mL) were treated with different hydrogels for 24 h. Then they were fixed, exposed to osmium tetroxide, and dehydrated with an ethanol series. Then sections were made with a diamond knife. The sections were placed on copper grids, stained with lead citrate, and examined by transmission electron microscopy.

[0104] A significant number of bacteria (green fluorescence) survived in the CTRL and PAM, PAM-ME, or PAM-M hydrogel treatment groups (including S. aureus and E. coli). However, in the samples treated with the PAM-ME hydrogel, the green fluorescence intensity was significantly reduced, indicating a significant reduction in the number of bacteria, thus confirming the strong antibacterial effect of the PAM-ME hydrogel ( Figure 5 A). Statistical analysis of the bacterial survival rate ( Figure 5 B) further verified the previous results.

[0105] Subsequently, the colonies were quantitatively evaluated using SPM. Obviously, after the same treatment time, there were few colonies in the PAM-ME hydrogel treatment group, indicating that its antibacterial effect was significantly better than that of other groups compared with other groups ( Figure 5 C-D).

[0106] Transmission electron microscopy images showed that the bacterial cell membranes in the PAM-ME group (including S. aureus and E. coli) exhibited a contracted, blurred boundary, and wrinkled appearance. In contrast, the bacterial cell membranes in the CTRL group, PAM group, and PAM-M group were smooth, intact, and had obvious contours ( Figure 5 E).

[0107] Example 6 In vitro immunomodulatory properties

[0108] The expressions of M1 and M2 surface markers (CD86, CD206) were detected by immunofluorescence staining and flow cytometry. Briefly, macrophages were co-cultured with the hydrogels described. Lipopolysaccharide (LPS) at 50 ng / mL was used to create a systemic inflammatory microenvironment. For immunofluorescence, cells were fixed and blocked. Then we incubated the cells with anti-CD206 and anti-CD86 antibodies, followed by the use of relevant secondary antibodies. The cells were stained with DAPI and observed under CLSM. ImageJ software was used to quantify CD206-positive and CD86-positive cells. Following the above steps, the treated cells were evaluated for changes in macrophage subtypes using CD86 and CD206 antibodies by flow cytometry. They removed the supernatant, collected the macrophages, washed them with PBS, and then stained them with CD206 and CD86 antibodies. After staining, the cells were washed with PBS and resuspended for flow cytometry analysis. RAW 264.7 cells followed the same culture protocol. Total RNA was extracted using TRIzol.

[0109] As part of the experiment, immunofluorescence staining was performed. RAW264.7 cells were exposed to different hydrogels and simultaneously exposed to LPS for 24 h. Subsequently, the effects of these interventions on the expression levels of macrophage markers (iNOS for M1 macrophages; CD206 for M2 macrophages) were evaluated by immunofluorescence staining. Figure 6 The results in A - B showed that LPS treatment led to a significant increase in iNOS expression, with a significant increase in green fluorescence. In contrast, PAM-M and PAM-ME could reverse the expression of iNOS and significantly reduce the fluorescence intensity of iNOS, indicating their inhibitory effects on iNOS expression. Notably, the expression of CD206 showed an opposite pattern. Compared with the LPS group, the intensity of CD206 (red fluorescence) was significantly enhanced in the PAM-M and PAM-ME treatment groups. This indicates that PAM-M and PAM-ME hydrogels can inhibit the elevated iNOS expression induced by LPS while promoting the expression of CD206, the latter of which was inhibited by LPS. The results of flow cytometry verified the above results. Both the PAM-M and PAM-ME hydrogel treatment groups showed a significant decrease in CD86-positive (M1 macrophages) cells and a significant increase in CD206-positive (M2 macrophages) cells. As Figure 6 shown in C - D, the proportions of CD86-positive cells in the PAM-M group (14.5%) and PAM-ME group (14.2%) were significantly lower than those in the LPS group (33.2%) and PAM group (32.1%). In contrast, the proportions of CD206-positive cells in the PAM-M and PAM-ME treatment groups (11.9% and 10.6% respectively) were significantly higher than those in the LPS group (2.91%).

[0110] The qPCR method was used to detect the expression levels of inflammation-related genes in cells of different treatment groups to confirm the above results. Compared with the LPS-treated group, the gene expression levels of common M1 macrophage markers such as iNOS, IL-1β, and IL-6 were significantly decreased in the PAM and PAM-ME treatment groups, as shown in Figure 6 Figure E. The gene expression of M2 macrophage markers and CL-4 and CD206 was significantly increased, contrary to the above observations, indicating that PAM-M and PAM-ME have the ability to inhibit the expression of inflammation-related genes, thus leading to the reversal of the inflammatory environment.

[0111] The phenotypic properties of macrophages are formed by their inducers, quality, and surface markers. In this study, a pioneering composite hydrogel PAM-ME was synthesized by incorporating EN into Mg-MBG and then enveloping it with a hydrogel matrix. This section is dedicated to elucidating its regulatory effect on macrophages. The experimental results showed that the treatment with PAM-ME hydrogel has the ability to counteract the LPS-triggered M1 macrophage polarization and promote the enhancement of the M2 macrophage subtype. This result emphasizes the ability of PAM-ME hydrogel to regulate macrophage properties, especially by promoting the anti-inflammatory M2 phenotype while inhibiting the pro-inflammatory M1 phenotype. This indicates that PAM-ME hydrogel has great potential as a therapeutic method to improve wound healing outcomes. The ICP analysis results showed that the magnesium ion level in PAM-ME was 14.1. Extensive studies have shown that the strong anti-inflammatory properties associated with magnesium ions can effectively inhibit the activation of inflammatory genes and reduce the release of inflammatory factors. This emphasizes the immunomodulatory potential of PAM-ME hydrogel enhanced by integrating Mg-MBG.

[0112] Example 7 Hemostasis in Vivo

[0113] This study evaluated the hemostatic effect of hydrogels in a rat liver injury model. After anesthesia, the rats were disinfected, and the abdomen of the SD rats was opened to expose the largest left hepatic lobe. A biopsy puncture formed an incision (10 mm in diameter, 5 mm deep). Hemostasis was achieved with gauze for 5 s, and a pre-weighed filter paper was placed under the liver. Various hydrogels were applied (i.e., the hydrogel material was applied to the bleeding site and then hemostasis was achieved through adhesion and water absorption), with the untreated group as the control (CTRL). The blood loss was determined by weighing the filter paper before and after the bleeding event. The bleeding stop time was also recorded. The hemostatic properties of the hydrogels were further evaluated using a rat femoral artery injury model. Briefly, the femoral artery of the rat was first exposed and isolated, and then clamped with a hemostat. Then, the artery was punctured with a needle to cause trauma, followed by massive bleeding. The blood was blotted with gauze, and a pre-weighed filter paper was placed under the artery. Different hydrogels were used to treat the femoral artery injury to seal the bleeding site and control the bleeding. The untreated femoral artery was used as the negative control group. After releasing the vascular clamp, the bleeding situation was evaluated. The bleeding time and blood loss were recorded as described above.

[0114] The hemostatic effect of PAM-ME was initially evaluated using a rat liver bleeding model. Figure 7 A, The results shown in 7D revealed a significant difference between the CTRL group and the PAM-ME treatment group. As time passed, the bleeding gradually increased, and complete control of bleeding was effectively achieved within a short time at the wound site. There were significant differences in bleeding time and blood loss between the CTRL group and the PAM-ME group, as Figure 7 shown in B-C. In the liver bleeding model, the CTRL group showed an extended bleeding time of 400 s and a blood loss of 1500 mg. In contrast, in the group treated with PAM-ME, the bleeding time was significantly reduced to 100 s, and the blood loss was significantly reduced to 100 mg. This indicates that PAM-ME has a good hemostatic effect in vivo.

[0115] The hemostatic effect of BT-Gel was also evaluated in a rat femoral artery bleeding model, and the results were consistent with those of the rat liver bleeding model. Among them, the bleeding time of the CTRL group was 678 s, while the bleeding time of the PAM-ME treatment group was reduced by 325 s ( Figure 7 E). Correspondingly, the blood losses of the CTRL group and the PAM-ME group were 3324 mg and 2074 mg, respectively ( Figure 7 F). The in vivo study found that there was no significant difference in the hemostatic effect among PAM, PAM-M, and PAM-ME, indicating that the addition of Mg-MBG and EN does not affect the hemostatic ability of the gel.

[0116] Example 8 Wound healing in vivo

[0117] A mouse wound infection model was established using 4-5-week-old female ICR mice. A full-thickness skin wound was induced on the back using a punch machine. Subsequently, 10 μL of a S. aureus solution with a concentration of 2×10 9 CFU / mL was administered. Normal saline (CTRL), PAM, PAM-M, PAM-ME, and FG dressings (n = 8) were applied to the wounds. Wound images were collected at 0, 2, 4, 6, 8, 10, and 12 days after treatment, and the areas were measured using Image J software (n ≥ 3 per group). Then, wound tissues were collected for histological and immunofluorescence examinations. H&E staining was used to evaluate wound healing. Masson's trichrome staining was used to quantify collagen deposition. Immunohistochemical staining of iNOS and CD206 was used to observe macrophage infiltration and polarization at the wound site. Staining quantification was performed using ImageJ.

[0118] Throughout the study, all infected wounds in the experimental groups showed a trend of gradual healing, as Figure 8 shown in A - B. However, significant differences in infection intensity and recovery rate were observed among different treatment cohorts. Notably, long-term infections, tissue necrosis, and purulent secretions occurred in wounds treated with PBS (CTRL), PAM, PAM-M, and FG. In contrast, wounds treated with the PAM-ME hydrogel remained clean and dry throughout the 12-day treatment regimen, showing no obvious signs of infection or suppuration. The results indicated that the wound healing progress was fastest in the PAM-ME hydrogel treatment group. Specifically, at days 2, 4, 6, 8, 8, 10, and 12, the healing rates of the PAM-ME hydrogel group were 33.5%, 54.2%, 69.8%, 80.7%, 88.7%, and 94.8%, respectively, exceeding those of the control group, which only reached 66.7% by day 12, as Figure 8 shown in C. These results were further confirmed by the results of SPM analysis of infected skin tissues ( Figure 8 D - E). Therefore, these findings suggest that the improvement of the PAM-ME hydrogel may be related to its special antibacterial properties and Mg 2+ repair properties, inhibiting pathogen colonization and promoting a favorable healing environment.

[0119] Histological analysis using H&E staining verified the recorded wound healing rates ( Figure 9 A). Quantitative measurement of granulation tissue length showed that wound contraction in the PAM-ME group was significantly accelerated compared to other groups ( Figure 9 B). Studies have shown that appropriate collagen deposition is crucial for tissue strength and healing during skin remodeling. Therefore, Masson's trichrome staining was used to evaluate collagen production. By day 12 of observation, persistent crusting occurred in the CTRL group, indicating a delay in the wound recovery process, as Figure 9As shown in C. In contrast, the wounds treated with hydrogel showed an obvious red epidermal layer and an increase in collagen content. Notably, the PAM-ME hydrogel treatment cohort demonstrated the ability to promote chronic wound healing and the successful regeneration of dermal appendages, as Figure 9 shown in D. Collectively, these findings highlight that the PAM-ME hydrogel significantly enhanced wound healing properties compared to other treatment groups. The present invention also investigated the potential correlation between reduced inflammation and enhanced wound healing and macrophage polarization in bacterially infected wounds. The results of immunofluorescence staining of M1 (iNOS) and M2 (CD206) macrophages showed that the M2 / M1 ratio was most favorable in the PAM-ME hydrogel-treated group ( Figure 9 E). Notably, the application of the PAM-ME hydrogel resulted in a significant reduction in iNOS+ M1 macrophages ( Figure 9 F), while a significant increase in CD206+ M2 macrophages ( Figure 9 G). The CTRL group showed an obvious presence of iNOS+ macrophages, indicating a prolonged inflammation time compared to the treatment groups. These findings highlight the potential of the PAM-ME hydrogel to promote wound healing by regulating the modulation of macrophage subtypes within the wound site.

Claims

1. A hydrogel material, which is composed of mesoporous bioglass nanoparticles (MBG) rich in metal ions and antibiotics.

2. The hydrogel material as claimed in claim 1, wherein the metal ions are selected from magnesium (Mg), zinc (Zn), copper (Cu) and / or silver (Ag); the antibiotics include but are not limited to enoxacin (EN), levofloxacin, norfloxacin, ciprofloxacin, lomefloxacin and sparfloxacin.

3. The hydrogel material as claimed in claim 1, wherein the mesoporous bioglass nanoparticles are composed of SiO2 (silicon dioxide), Na2O (disodium oxide), CaO (calcium oxide) and P2O5 (phosphorus pentoxide).

4. The hydrogel material as claimed in claim 1, wherein the content ratio of the antibiotic to the mesoporous bioglass nanosphere particles containing magnesium in the hydrogel material is 4:

1.

5. A method for preparing a hydrogel flexible electrode dressing, the method comprising the following steps: S1. Preparation of Mesoporous Bioglass Nanoparticles S1.1 Mix dissolved hexadecyltrimethylammonium bromide (CTAB) and ethyl acetate to obtain a mixed solution 1; S1.2 adding an aqueous ammonia solution to the mixed solution 1 to obtain a mixed solution 2; S1.3 adding tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate to the mixed solution 2 to obtain mesoporous bioglass nanoparticles; S2. Preparation of mesoporous bioglass nanoparticles containing metal ions S2.1 adding nitrate to the mesoporous bioglass nanoparticles prepared in S1, and collecting the formed colloid by centrifugation; S2.2 collecting the precipitate obtained in S2.1 to obtain mesoporous bioglass nanoparticles containing metal ions; S3. Preparation of mesoporous bioglass nanospheres containing metal ions and antibiotics The antibiotics are mixed with the mesoporous bioglass nanoparticles containing metal ions prepared in S2 to obtain mesoporous bioglass nanoparticles containing metal ions and antibiotics; S4. Preparation of hydrogel S4.1 Mixing monomers for preparing hydrogel to prepare a prepolymer solution; S4.2 adding the prepolymer solution into the mold, and adding the monomer into the solution; S4.3 Layered hydrogels were extracted from the mold in S4.

2. The hydrogel containing metal ions during formation was denoted as PAM-M, while the hydrogel containing metal ions and antibiotics was denoted as PAM-ME.

6. The method according to claim 5, wherein in step S2.1, the nitrate is selected from magnesium nitrate, zinc nitrate, copper nitrate or silver nitrate; In step S3, the antibiotics include but are not limited to enoxacin (EN), levofloxacin, norfloxacin, ciprofloxacin, lomefloxacin and sparfloxacin; In step S4.1, the monomers include acrylamide, 2-acrylamide-2-methylpropanesulfonic acid and N,N'-methylenebisacrylamide; In step S4.2, the monomer is triethylborane-tetrahydrofuran complex (Et3B-THF).

7. A hydrogel dressing, comprising the hydrogel material according to the first aspect; the material has at least one of the following functions: 1) Promote wound healing; 2) Inhibit wound infection; 3) Promote wound hemostasis.

8. Use of a hydrogel material in the preparation of a product for promoting wound healing; the material has at least one of the following functions: 1) Promote wound healing; 2) Inhibit wound infection; 3) Promote wound hemostasis.

9. The use according to claim 8, wherein the product comprises a hydrogel dressing and a hydrogel filler.

10. The use as claimed in claim 8, wherein the hydrogel filler can be used for rhinoplasty and breast augmentation.

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