Light-operated antibacterial injectable multifunctional hydrogel adhesive capable of promoting cell growth as well as preparation method and application of light-operated antibacterial injectable multifunctional hydrogel adhesive

Abstract

The invention discloses a light-operated antibacterial injectable multifunctional hydrogel adhesive capable of promoting cell growth and a preparation method and application of the light-operated antibacterial injectable multifunctional hydrogel adhesive capable of promoting cell growth, the adhesive is mainly composed of alkenyl tannic acid nanoparticles, photosensitizer chlorin e6, Cu < 2 + >, transglutaminase and gelatin, the concentration of the alkenyl tannic acid nanoparticles is 0.35-1.43 mg / mL, and the concentration of the gelatin is 0.35-0.45 mg / mL. The concentration of the photosensitizer chlorin e6 is 25-150 [mu] g / mL, the concentration of Cu < 2 + > is 17-18 ng / mL, and the concentration of transglutaminase is 1.79-7.14 mg / mL. The invention further discloses application of the hydrogel adhesive in preparation of products for repairing wounds, especially skin and various mucous membrane wounds or soft tissues around oral implants.

Description

Technical Field

[0001] The present invention belongs to the technical field of injectable hydrogels, and particularly relates to an injectable multifunctional hydrogel adhesive capable of light-controlled antibacterial and promoting cell growth, and a preparation method and application thereof. Background Art

[0002] Peri-implantitis is one of the main complications in the field of oral implantology. Its pathogenesis is complex and involves bacterial infection, excessive accumulation of reactive oxygen species (ROS), and persistent inflammatory response. Existing measures mainly include two aspects: antibacterial and promoting cell growth. However, the oral cavity is a bacteria-containing environment, and bacteria are likely to enter the gap between the implant and soft tissue after implantation, coexist with cells, and have similar biological response characteristics. Therefore, means for promoting cell growth (such as implant shape design, material selection, and surface modification) are also beneficial to bacteria, while means for inhibiting bacteria may cause damage to cells. In addition, the problem of antibiotic resistance is becoming increasingly serious, and it has become an urgent task to develop an antibacterial means that is harmless to cells and highly efficient. Summary of the Invention

[0003] The purpose of the present invention is to provide an injectable multifunctional hydrogel adhesive capable of light-controlled antibacterial and promoting cell growth.

[0004] The present invention also aims to provide a preparation method of the above-mentioned injectable multifunctional hydrogel adhesive capable of light-controlled antibacterial and promoting cell growth.

[0005] The last purpose of the present invention is to provide the application of the above hydrogel adhesive in the preparation of wound or soft tissue repair products around oral implants.

[0006] The above first object of the present invention can be achieved by the following technical solution: an injectable multifunctional hydrogel adhesive capable of light-controlled antibacterial and promoting cell growth, mainly composed of alkenylated tannic acid nanoparticles, photosensitizer chlorin e6, Cu 2+ , transglutaminase, and gelatin, wherein the concentration of the alkenylated tannic acid nanoparticles is 0.35 - 1.43 mg / mL, the concentration of the photosensitizer chlorin e6 is 25 - 150 μg / mL, the concentration of Cu 2+ is 17 - 18 ng / mL, and the concentration of the transglutaminase is 1.79 - 7.14 mg / mL.

[0007] Preferably, the concentration of the alkenylated tannic acid nanoparticles is 0.35 - 0.70 mg / mL, the concentration of the photosensitizer chlorin e6 is 80 - 120 μg / mL, the concentration of Cu 2+ is 17.5 - 18 ng / mL, and the concentration of the transglutaminase is 1.79 - 3.58 mg / mL.

[0008] More preferably, the concentration of the vinylated tannic acid nanoparticles is 0.35 mg / mL, the concentration of the photosensitizer chlorin e6 is 100 μg / mL, the concentration of Cu 2+ is 17.86 ng / mL, and the concentration of transglutaminase is 1.79 mg / mL.

[0009] Preferably, the Cu 2+ is selected from but not limited to CuSO4·5H2O solution.

[0010] The present invention provides a transglutaminase (TG)-crosslinked gelatin-based multifunctional hydrogel (Gelatin-based TG enzyme crosslinked functional hydrogel, GT-F hydrogel) loaded with vinylated tannic acid nanoparticles (vTA NP), photosensitizer chlorin e6 (Ce6), and Cu 2+ . Both gelatin and TG enzyme are of biological origin, and after crosslinking, they have good biosafety. The singlet oxygen generated by the photosensitizer Ce6 through light irradiation can quickly kill bacteria in the hydrogel without damaging surrounding cells. The inventors of the present application have successfully constructed vinylated modified tannic acid nanoparticles (application number 202210702829.0) in the early stage, which can be uniformly dispersed in the hydrogel, provide strong wet adhesion ability, and slowly release during the degradation of the hydrogel to scavenge excessive ROS. Cu 2+ , as an inorganic signaling factor, can activate angiogenesis-related factors and promote soft tissue regeneration.

[0011] The TG enzyme-crosslinked gelatin-based hydrogel of the present invention loaded with vTA NP, Ce6, and Cu 2+ is named GT-F hydrogel (Gelatin-based TG enzyme crosslinked functional hydrogel), and its physical and chemical properties are optimized by adjusting the proportions of each component to meet the needs of soft tissue regeneration.

[0012] The injectable multifunctional hydrogel adhesive of the present invention provides a sterile and cell-growth-friendly microenvironment. On the one hand, it can immediately kill bacteria during implant surgery and establish a physical barrier to prevent the invasion of oral bacteria; on the other hand, it can provide biological signals to promote soft tissue regeneration, scavenge adverse factors (such as bacteria and excessive ROS), and form a healthy and stable soft tissue integration. The injectable hydrogel is suitable for application in small and irregular soft tissue spaces around implants due to its property of being moldable as needed.

[0013] Therefore, the injectable multifunctional hydrogel of the present invention has the following functions: 1) Adhering to and sealing the wound to construct an immediate barrier; 2) Rapidly killing bacteria in the hydrogel to prevent their colonization and biofilm formation; 3) Loading bioactive molecules to promote soft tissue regeneration; 4) Having strong adhesion ability in oral environments such as wetness, chewing, and swallowing; 5) Having appropriate mechanical properties and slowly degrading to match the ingrowth of soft tissues.

[0014] The second object of the present invention can be achieved by the following technical solution: The preparation method of the above-mentioned light-controlled antibacterial and cell growth-promoting injectable multifunctional hydrogel adhesive includes the following steps: Take a solution of vinylated tannic acid nanoparticles, a solution of photosensitizer chlorin e6, a solution containing Cu 2+ solution, a solution of transglutaminase, and a gelatin solution. After mixing, place them in a mold and let it stand in an environment of 45-55 °C to obtain the light-controlled antibacterial and cell growth-promoting injectable multifunctional hydrogel adhesive.

[0015] The last object of the present invention can be achieved by the following technical solution: The application of the above-mentioned hydrogel adhesive in the preparation of products for soft tissue repair around wounds or oral implants.

[0016] More preferably, the application of the above-mentioned hydrogel adhesive in the preparation of products for soft tissue repair around skin and various mucosal wounds or oral implants.

[0017] The present invention has the following advantages: The light-controlled antibacterial and cell growth-promoting injectable multifunctional hydrogel adhesive (GT-F hydrogel) constructed by the present invention can adhere the metal implant to the soft tissue, form a barrier to block the downward movement of bacteria in the oral cavity, rapidly sterilize under light control, and release bioactive molecules in a long-term controlled manner to exert anti-inflammatory and angiogenesis-promoting effects, thereby promoting the integration of soft tissues around the implant and ensuring the success of implantation. Description of the Drawings

[0018] Figure 1 shows the gelation situation and gelation time of hydrogels with different TGase contents in Example 1;

[0019] Figure 2 shows the influence of TGase content and vTA NP on the tensile fracture strength of the hydrogel in Example 1 (*P < 0.05), Content of TG is the TG content, and Stress refers to the tensile fracture strength;

[0020] Figure 3 shows the storage modulus (G’) and loss modulus (G”) of hydrogels with different TGase contents in Example 1. Note: 6%, 3%, 1.5%, and 0.75% are the mass ratios of TGase in the total system. Note: Ang.frenquency is the angular frequency, G’ is the storage modulus, and G” is the loss modulus;

[0021] Figure 4 It is the cytocompatibility of hydrogels with different vTA NP contents in Example 1. Figure A shows the proliferation of L929 cells on the hydrogel surface detected by CCK8. Figure B shows the fluorescence staining image after co-culturing the hydrogel with L929 cells for 4 days. Blue represents the cell nucleus, and green represents the cytoskeleton. Note: OD 450 is the OD value at 450 nm; Day 1, Day 4, and Day 7 are the 1st, 4th, and 7th days; DAPI is 4',6-diamidino-2-phenylindole, a fluorescent probe for nuclear staining, and Actin-Tracker is a fluorescent probe for labeling actin in cells;

[0022] Figure 5 It is the bacterial survival rate of Ce6 before and after light irradiation in Example 1. Figure A shows S.a, Figure B shows MRSA, Figure C shows E.c, and Figure D shows P.g (*P < 0.05). Note: Bacterial Viability is the relative viability of bacteria; Concentration is the concentration of Ce6; S.a is Staphylococcus aureus, E.c is Escherichia coli, MRSA is Methicillin-resistant Staphylococcus aureus, and P.g is Porphyromonas gingivalis;

[0023] Figure 6 It is the singlet oxygen generated by Ce6 under different light irradiation times in Example 1. Figure A shows the color change of the mixture of DPBF probe and Ce6 after different irradiation times. Figure B shows the UV-vis spectrum of the mixture of DPBF probe and Ce6 after different irradiation times. Note: Wavelength is the wavelength, and Abs is the absorbance;

[0024] Figure 7 It is the SEM observation of the morphology of GT and GT-F hydrogels in Example 1;

[0025] Figure 8 It is the injectability of GT-F hydrogel in Example 1. Figure A shows the injectability of GT hydrogel. Figure B shows that the GT hydrogel maintains a stable shape at 50 °C after gelation. Figure C shows the injectability of GT-F hydrogel. Figure D shows that the GT-F hydrogel maintains a stable shape at 50 °C after gelation. Figure E shows the injectability of gelatin at 50 °C. Figure F shows that the gelatin dissolves after incubation at 50 °C. Note: For easy observation, gentian violet dye stains GT and Gel;

[0026] Figure 9 It is the tensile property diagram of GT-F hydrogel detected by a universal mechanical machine in Example 1 (Figure A) and the relationship curve between the standing time and the tensile fracture strength after gelation of GT and GT-F hydrogels (Figure B). Note: Time is the time, and Stress is the tensile fracture strength;

[0027] Figure 10The storage modulus (G’) and loss modulus (G”) of GT and GT-F hydrogels 72 h after gelation in Example 1. Note: Ang.frenquency is the angular frequency, G’ is the storage modulus, and G” is the loss modulus;

[0028] Figure 11 The bonding strength between GT and GT-F hydrogels in porcine skin tissue and between porcine skin tissue and titanium sheet in Example 1 (*P < 0.05) (Figure A) and the schematic diagram of the good bonding stability of GT and GT-F hydrogels (Figure B). Note: pigskin is porcine skin, Ti sheet is titanium sheet, and Stress is the bonding strength;

[0029] Figure 12 The schematic diagram of the swelling experiment of GT-F hydrogel in Example 1;

[0030] Figure 13 The swelling curves of GT and GT-F hydrogels in the examples. Note: Time is time, and Swelling Rate is the swelling rate;

[0031] Figure 14 The in vitro degradation curves of GT and GT-F hydrogels in Example 1. Note: Time is time, and RemainedWeight is the remaining mass;

[0032] Figure 15 Cu in GT-F hydrogel in Example 1 2+ and vTA NP release curves. Note: Time is time, and CumulativeRelease Rate is the cumulative release rate;

[0033] Figure 16 The cell proliferation of the cytotoxicity of the hydrogel cultured with the leaching solution in Example 1 (Figures A - C) and cell viability (Figures D - F). Note: Time is time, OD 450 is the OD value at 450 nm, Cell Viability is the relative cell viability, L929 is a mouse fibroblast cell line, HGF is a human gingival fibroblast, and HGE is a human gingival epithelial cell;

[0034] Figure 17 The fluorescence images of cells cultured with the hydrogel leaching solution in Example 1. Blue represents the cell nucleus, and green represents the cytoskeleton. Note: L929 is a mouse fibroblast cell line, HGF is a human gingival fibroblast, and HGE is a human gingival epithelial cell;

[0035] Figure 18It is the cell cytotoxicity cell proliferation of the surface cell culture evaluation hydrogel in Example 1 (Figure A - Figure C) and the cell viability (Figure D - Figure F). Note: Time is time, OD 450 is the OD value at 450 nm, Cell Viability is the relative cell viability, L929 is a mouse fibroblast cell line, HGF is a human gingival fibroblast, HGE is a human gingival epithelial cell, and Well is for directly culturing cells on the surface of the well plate;

[0036] Figure 19 It is the fluorescence image of cells cultured on the surface of the GT-F hydrogel in Example 1. The blue represents the cell nucleus, and the green represents the cytoskeleton. Note: L929 is a mouse fibroblast cell line, HGF is a human gingival fibroblast, HGE is a human gingival epithelial cell, and Well is for directly culturing cells on the surface of the well plate;

[0037] Figure 20 It is a schematic diagram of cell inoculation on one side of the bottom of the well plate in Example 1. Note: cell suspension is the cell suspension, well is the well plate, culture12 h is for culturing for 12 hours, and inject hydrogel means injecting the hydrogel precursor solution into the empty part of the well plate;

[0038] Figure 21 It is an experimental diagram of L929 cells growing into the GT-F hydrogel in Example 1. Among them, Figure A is a schematic diagram of inoculating cells on one side of the well plate, Figure B is the cell growth situation observed under the microscope 12 h after seeding the cells, Figure C is a schematic diagram of the GT-F hydrogel gelling beside the cells, and Figure D is the cell growth situation observed under the microscope 24 h after the hydrogel gels. Note: cell suspension is the cell suspension, well is the well plate, and hydrogel is the hydrogel;

[0039] Figure 22 In Example 1, an optical microscope and a laser confocal microscope were used to observe the situation of L929 cells growing into the GT-F hydrogel. Among them, Figure A and Figure B are the situations of cells growing into the hydrogel under the optical microscope, and Figure C is the fluorescence image of cells growing into the hydrogel under the laser confocal microscope. The blue represents the cell nucleus, and the green represents the cytoskeleton. Note: hydrogel is the hydrogel;

[0040] Figure 23 It is a schematic diagram of the Transwell model of the GT-F hydrogel physically blocking bacteria (Figure A) and a schematic diagram of the Transwell model of the GT-F hydrogel blocking bacteria around the implant (Figure B);

[0041] Figure 24It is the ability of the GT-F hydrogel in Example 1 to physically block bacteria. In Figure A, it shows the colony formation results of BHI medium in the lower chamber of Transwell. In Figure B, it shows the physical barrier rates of the GT-F hydrogel against S.a and E.c. Note: S.a is Staphylococcus aureus, E.c is Escherichia coli, and Control is the blank control group; Barrier Rate refers to the physical barrier rate of the hydrogel against bacteria.

[0042] Figure 25 It is the fluorescence image of simulating the GT-F hydrogel to block bacteria around the implant in Example 1. Blue represents the cell nucleus, and green represents the cytoskeleton. Note: No S.a is the group without adding Staphylococcus aureus, S.a is the group adding Staphylococcus aureus, HGE(upper) refers to human gingival epithelial cells cultured in the upper layer of the Transwell chamber, HGF(lower) refers to human gingival fibroblasts cultured in the lower layer of the Transwell chamber, and Control is the blank control group.

[0043] Figure 26 It is the influence of vTA NP on the ability of Ce6 to generate singlet oxygen in Example 1. Note: Decrease of Absat 410nm is the decrease value of absorbance at 410nm.

[0044] Figure 27 It is the detection of the ability of the GT-F hydrogel to generate singlet oxygen by the DPBF probe in Example 1. In Figure A, it shows the fluorescence images of the hydrogel before and after light irradiation, with green being the DPBF probe. In Figure B, it shows the statistical results of fluorescence intensity (*P < 0.05). The GT-F hydrogel has the ability to generate singlet oxygen under light irradiation. Note: hydrogel is the hydrogel, and Fluorescence Intensity is the fluorescence intensity.

[0045] Figure 28 It is the fluorescence image of live and dead bacteria staining of bacteria in the GT-F hydrogel in Example 1. Green represents live bacteria, and red represents dead bacteria. Note: LIVE refers to the staining result of live bacteria, DEAD refers to the staining result of dead bacteria, and MERGE is the merged image of live and dead staining results.

[0046] Figure 29It is the fluorescence image of the live and dead bacteria staining in the hydrogel in Example 1. The green color represents live bacteria and the red color represents dead bacteria. "without irradiation" refers to the group without 660 nm light irradiation, "5 mint irradiation" refers to the group irradiated with 660 nm light for 5 minutes, and "without irradiation cultured 24h" refers to the group of the bacteria-containing hydrogel without 660 nm light irradiation after being cultured for 24 hours; Live / Dead 3D is the three-dimensional image of the live and dead bacteria staining results, and Live / Dead SCAN is the sectional scan image after the live and dead bacteria staining;

[0047] Figure 30 It is the DPPH scavenging ability of vTA NP solution and GT + vTA hydrogel in Example 1 (*P < 0.05). Note: DPPH Scavenging Rate refers to the DPPH scavenging rate, vTA NPa.q. is the vTA nanoparticle solution, and GT + vTA is the gelatin-based hydrogel system crosslinked by TG enzyme containing vTA nanoparticles;

[0048] Figure 31 It is the DPPH scavenging rate of vTA NP after mixing vTA NP and Ce6 in solution and irradiating with 660 nm light for 5 min in Example 1 (*P < 0.05). Note: DPPH Scavenging Rate refers to the DPPH scavenging rate, and 0 min and 5 min refer to the group without 660 nm light irradiation (i.e., the non-irradiation group) and the group irradiated with 660 nm light for 5 minutes respectively;

[0049] Figure 32 It is the DPPH scavenging rate of GT + vTA + Ce6 and GT + vTA hydrogels after light irradiation (*P < 0.05). Note: DPPH Scavenging Rate refers to the DPPH scavenging rate, vTA NPa.q. is the vTA nanoparticle solution, and GT + vTA is the gelatin-based hydrogel system crosslinked by TG enzyme containing vTA nanoparticles;

[0050] Figure 33 It is the L929 cells cultured with the leaching solution of hydrogels with different components in Example 1. After being treated with 100 μM H2O2, the intracellular ROS level was detected by DCFH-DA probe. (A) DCFH fluorescence intensity, (B) Increase in intensity after H2O2 treatment (*P < 0.05). Note: Intensity refers to the fluorescence intensity of the DCFH probe, and Increase of Intensity is the increase in fluorescence intensity, representing the increase in intracellular ROS level;

[0051] Figure 34L929 cells cultured in the leaching solutions of hydrogels with different components in Example 1, DCFH staining images after treatment with 100 μM H2O2. Green represents the DCFH probe, and blue represents the cell nucleus. Note: DAPI is the fluorescent probe for nuclear staining, and DCFH is the probe for intracellular ROS staining;

[0052] Figure 35 Intracellular ROS levels in L929 cells cultured on the surfaces of hydrogels with different components in Example 1 after treatment with 100 μM H2O2. Figure A shows the DCFH fluorescence intensity, and Figure B shows the increase in fluorescence intensity after adding H2O2 (*P < 0.05). Note: Intensity refers to the fluorescence intensity of the DCFH probe, and Increase of Intensity represents the increase in intracellular ROS levels;

[0053] Figure 36 DCFH staining images of L929 cells cultured on the surfaces of hydrogels with different components in Example 1 after treatment with 100 μM H2O2. Green represents the DCFH probe, and blue represents the cell nucleus. Note: DAPI is the fluorescent probe for nuclear staining, DCFH is the probe for intracellular ROS staining, and MERGE is the merged image of the two stains;

[0054] Figure 37 Effect of hydrogel leaching solutions with different components in Example 1 on the angiogenic ability of HUVECs;

[0055] Figure 38 Statistical chart of the pro-angiogenesis experiment in Example 1. Figure A shows the number of junctions, and Figure B shows the number of branches (*P < 0.05). Note: Nb Junction is the number of junctions of newly formed blood vessels, and Nb branches is the number of branches of newly formed blood vessels. Detailed implementation mode

[0056] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only for illustrative purposes and should not be construed as limiting the present invention. Unless otherwise specified, the raw materials and equipment used in the following embodiments are obtained through conventional commercial channels.

[0057] Example 1

[0058] I. Synthesis and characterization of TGase-crosslinked gelatin-based multifunctional hydrogels loaded with vinylated tannic acid nanoparticles

[0059] Loaded with vTA NP, Ce6, Cu 2+The TG enzyme-crosslinked gelatin-based hydrogel is named GT-F hydrogel (Gelatin-based TG enzyme crosslinked functional hydrogel). By adjusting the proportion of each component, its physical and chemical properties are optimized to meet the needs of soft tissue regeneration.

[0060] 1.1 Optimization of the amount of TG enzyme

[0061] Hydrogels with different TG enzyme contents (0.75%, 1.5%, 3%, 6%) were prepared, and their gelation time and mechanical properties were detected. The specific operation was as follows: 1 mL of 150 mg / mL gelatin (Gelatin, Gel) solution was mixed with 100 μL of 12.5, 25, 50, 100 mg / mL TG enzyme solution, 1.4 mg / mL Ce6 solution, 10 mg / mL vTA NP solution, and 250 ng / mL CuSO4·5H2O solution respectively, and then injected into a mold and left to stand in an oven at 50 °C, and the gelation time was recorded. The tensile fracture strength of the GT-F hydrogel was detected using a universal testing machine at a tensile rate of 10 mm / min, and vTA NP was not added as a control. Hydrogel rheological mechanics experiment: At a constant temperature of 37 °C, a rotational rheometer was used to detect, the shear rate was 1 - 100 rad / s, the frequency was 1 Hz, and the fixed strain was 1%, and its storage modulus (Storage Modulus, G') and loss modulus (Loss Modulus, G") were detected. The experimental results showed that when the TG enzyme content was 0.75%, the gelation time exceeded 30 min, and when the TG enzyme content was 12%, the mixture had fluidity and could not form a gel. When the TG enzyme content was 1.5%, the gelation time of the hydrogel was 13 min ( Figure 1 ), the tensile fracture strength was 106.7 ± 1.8 kPa ( Figure 2 ), and the storage modulus was 3483.99 Pa ( Figure 3 ), with the best performance and meeting clinical requirements. Because of its optimal performance in gelation time and mechanical properties, 1.5% TG enzyme was finally selected for subsequent experiments.

[0062] Under the condition of 50 °C, the gelation time of the 1.5% TG enzyme hydrogel was 13 min. In practical applications, the 50 °C hydrogel precursor solution is injected around the wound, and it can be solidified in about 1 - 2 minutes (because the wound temperature is lower than 37 °C, and the gelatin rapidly physically solidifies). This characteristic makes clinical operation more convenient and does not affect the overall mechanical properties of the hydrogel. After physical solidification, the TG enzyme continues to react until complete gelation.

[0063] 1.2 Optimization of the content of vTA NP

[0064] Mix 1 mL of 150 mg / mL gelatin solution with 100 μL each of 25 mg / mL TGase solution, vTA NP solutions at different concentrations (20, 10, 5 mg / mL), 1 mg / mL Ce6 solution, and 250 ng / mL CuSO4·5H2O solution. The vTA NP solution is irradiated with ultraviolet light for 30 min, and the other solutions are filtered and sterilized through a 0.22 μm filter, then left to gel in an oven at 50 °C. The vTA NP contents in the prepared hydrogels are 1.3%, 0.65%, and 0.3% respectively. Seed L929 cells on the surface of the hydrogels and culture them for 1, 4, and 7 days under the conditions of 5% CO2 and 37 °C. Detect cell viability using the CCK8 method, and fix the cells with 4% paraformaldehyde on the 4th day of culture. Stain with Actin-Tracker and DAPI, and observe cell morphology through a laser confocal microscope.

[0065] Figure 4 The results show that L929 cells showed a continuous growth trend on the surfaces of hydrogels with different vTA NP contents within 1 - 7 days, and the lower the vTA NP content, the higher the cell viability. After 4 days of culture, the number of cells on the surface of the hydrogel with a vTA NP content of 1.3% was small, and the cell morphology was round; the cell morphology on the surface of the 0.65% hydrogel was good, spindle-shaped, and the fusion rate was about 80%; the cell growth on the surface of the 0.3% hydrogel was the best, with the largest number of cells and a fusion rate of about 90%. Therefore, the hydrogel with a vTA NP content of 0.3% was selected for subsequent research.

[0066] 1.3 Optimization of Ce6 dosage

[0067] Mix 100 μL of Ce6 solutions at different concentrations (12.5, 25, 50, 100 μg / mL) with 100 μL each of Staphylococcus aureus (S.a), Escherichia coli (E.c), Methicillin Resistant Staphylococcus Aureus (MRSA), and Porphyromonas gingivalis (P.g) bacterial suspensions at 1×10 8 CFU / mL respectively. Irradiate with a 660 nm laser with a power of 300 mW / cm 2 for 5 min, and set a non-irradiated group as a control. After culturing in the dark at 37 °C for 2 h, centrifuge at 4000 rpm for 5 min, remove the supernatant, resuspend the precipitate in 100 μL of medium, and read the OD value at 600 nm using an enzyme-linked immunosorbent assay reader. The calculation formula for cell survival rate is as follows:

[0068] Bacterial survival rate (%) = OD 处理组 / OD 对照组 × 100%.

[0069] Figure 5 As shown, for S.a, the bacterial survival rates before and after light irradiation at 12.5 μg / mL were 99.1 ± 0.8% and 93.0 ± 0.3%; at 25 μg / mL were 94.8 ± 2.6% and 84.2 ± 0.2%; at 50 μg / mL were 69.9 ± 0.2% and 48.9 ± 0.2%; at 100 μg / mL were 25.1 ± 9.5% and 18.0 ± 0.6%. For MRSA, the bacterial survival rates before and after light irradiation at 12.5 μg / mL were 99.0 ± 0.4% and 95.7 ± 0.1%; at 25 μg / mL were 93.5 ± 10.4% and 74.5 ± 3.4%; at 50 μg / mL were 70.5 ± 10.7% and 68.2 ± 0.3%; at 100 μg / mL were 46.2 ± 4.1% and 10.6 ± 7.4%. For E.c, the bacterial survival rates before and after light irradiation at 12.5 μg / mL were 99.6 ± 0.6% and 76.4 ± 0.3%; at 25 μg / mL were 95.1 ± 1.5% and 71.4 ± 0.5%; at 50 μg / mL were 80.4 ± 2.4% and 25.2 ± 0.2%; at 100 μg / mL were 58.3 ± 0.2% and 25.4 ± 0.6%. For P.g, the bacterial survival rates before and after light irradiation at 12.5 μg / mL were 97.8 ± 2.3% and 24.1 ± 0.4%; at 25 μg / mL were 96.2 ± 0.5% and 19.2 ± 0.7%; at 50 μg / mL were 97.3 ± 1.5% and 13.9 ± 0.2%; at 100 μg / mL were 97.6 ± 1.5% and 11.4 ± 0.7%. After light irradiation, the bacterial survival rates of the treatment groups at other concentrations all decreased significantly (P < 0.05), except for MRSA at 12.5 μg / mL. For the above four bacteria, more than 70% of the bacteria could be killed after 100 μg / mL Ce6 was irradiated by light. Therefore, 100 μg / mL was selected as the loading concentration of Ce6 in the hydrogel.

[0070] 1.4 Optimization of light irradiation time

[0071] Using 1,3 - Diphenylisobenzofuran (DPBF) singlet oxygen probe to detect the generation of singlet oxygen by Ce6 under light control. Singlet oxygen can cause the absorbance of DPBF at 410 nm to decrease, and there is a linear relationship between the amount of singlet oxygen and the decrease value of absorbance. Mix 1 mL of 100 μg / mL Ce6 solution with 1 mL of 10 μg / mL DPBF solution, irradiate with 660 nm light for 5, 10, 15, 30 min, and use UV - vis to detect the absorbance of the mixture at 410 nm. Use the DPBF solution as the negative control and the mixture of Ce6 and DPBF without light irradiation as the positive control.

[0072] The results showed that the solution of DPBF probe and Ce6 was fluorescent yellow after mixing, and turned into light brown after illumination ( Figure 6 The absorption peak of the blend at 410nm is significantly higher than that of the DPBF working solution. After 5 minutes of illumination, the absorption peak at 410nm decreases significantly. However, the UV-vis spectra after 10, 15, and 30 minutes of illumination have no significant difference from that after 5 minutes ( Figure 6 Therefore, 5 min was selected as the illumination time for subsequent experiments.

[0073] Observation of 1.5GT-F hydrogel morphology

[0074] According to the literature Cu 2+ The concentration of Cu in hydrogels that promotes angiogenesis but is not cytotoxic was determined. 2+ The concentration was 250ng / mL. The composition ratio of GT-F hydrogel was: 1mL gelatin solution (150mg / mL) was mixed with 100μL each of TG enzyme solution (25mg / mL), vTANP solution (10mg / mL), Ce6 solution (1.4mg / mL), and CuSO4·5H2O solution (250ng / mL). As a control group, 1mL gelatin solution was mixed with 100μL TG enzyme solution and 300μL ultrapure water to prepare GT hydrogel (Gelatin-based TG enzymes crosslinked hydrogel, GT). The above two hydrogels were freeze-dried after gelation in the mold, cut in liquid nitrogen to expose the cross section, and observed by scanning electron microscopy (SEM) after gold spraying. The results showed that the cross-sections of GT and GT-F hydrogels were loose and porous network structures, the porous skeletons were smooth and continuous, and no vTA NP agglomeration was observed. There was no significant difference in the pore size of the two groups of hydrogels, both of which were 80-100μm ( Figure 7 ).

[0075] 1.6GT-F hydrogel injectability

[0076] 1 mL of 50°C GT-F hydrogel, GT hydrogel and uncrosslinked gelatin solution (Gel) were placed in syringes, pushed onto the surface of the culture dish and English letters were written, and then incubated in a 50°C oven for 13 minutes for observation. The results showed that at 50°C, both GT-F and GT hydrogel precursor solutions had good fluidity and could be pushed out of the syringe smoothly. After incubation for 13 minutes, both hydrogels successfully gelled and maintained a stable shape and did not dissolve at 50°C. In contrast, gelatin solutions that were not cross-linked by TG enzyme could not gel, indicating that TG enzyme cross-linking is a key step in achieving stable gelation of hydrogels ( Figure 8 ).

[0077] 1.7 Mechanical properties of GT-F hydrogel

[0078] After the GT-F and GT hydrogels were completely gelled, they were allowed to stand for 12, 24, 48, 72, and 96 h respectively, and their mechanical properties were detected using a universal testing machine and a rotational rheometer.

[0079] The results showed that the GT-F hydrogel exhibited large deformations during the stretching process without fracture, indicating good extensibility ( Figure 9 Figure A in ). The mechanical properties of the GT and GT-F hydrogels continuously increased within 0 - 72 h after gelation. After 12 h, the tensile fracture strengths of the GT and GT-F hydrogels were 23.3 ± 1.2 kPa and 24.5 ± 0.3 kPa respectively; after 72 h, they increased to 37.3 ± 1.2 kPa and 91.4 ± 2.9 kPa respectively, and tended to be stable after 72 h, showing no further increase trend until 96 h ( Figure 9 Figure B in ). The rheological mechanical experiments showed that the storage modulus (G') of the GT-F hydrogel reached 3484.0 Pa, much higher than that of the GT hydrogel at 413.1 Pa; the loss moduli (G”) were 1471.06 Pa and 94.52 Pa respectively ( Figure 10 ). These results indicate that the mechanical properties of the GT-F hydrogel were significantly enhanced over time after gelation, and tended to be stable after 72 h, showing good mechanical properties and stability.

[0080] 1.8 Adhesive properties of GT-F hydrogel

[0081] The adhesive properties of the GT-F hydrogel between tissues and between tissue and metal were detected. The hydrogel precursor solution was evenly applied to the surface of titanium sheets or treated pig skins. After gelation at 50 °C, the adhesive properties were detected using a universal testing machine. The adhesive strength of the GT hydrogel between pig skin tissues was 131.0 ± 25.7 kPa, and that of the GT-F hydrogel was 287.9 ± 39.3 kPa; the adhesive strength of the GT hydrogel between pig skin tissue and titanium sheet was 273.1 ± 11.0 kPa, and that of the GT-F hydrogel was 885.7 ± 47.9 kPa. The adhesive strength of the GT-F hydrogel at both interfaces was significantly higher than that of the GT hydrogel (P < 0.05), indicating that the loaded vTA NPs significantly improved the adhesive properties of the hydrogel ( Figure 11 Figure A in ). Both the GT and GT-F hydrogels could closely adhere to the surface of pig skin tissues and withstand large deformations without detachment or peeling under repeated torsion and bending ( Figure 11 Figure B in ).

[0082] Due to the loading of vTA in the hydrogel, its catechol and pyrogallol structures endow the GT-F hydrogel with excellent adhesion properties, enabling it to firmly adhere metal to soft tissue and having recognized wet adhesion ability. The experimental results show that the addition of vTA significantly enhances the adhesion ability of the hydrogel. The adhesion stability of the GT-F hydrogel enables it to meet the requirements of closing wounds by adhesion around the implant abutment. In addition, the GT-F hydrogel exhibits good adhesion stability in a complex oral environment for the following reasons: 1) Gelatin itself has good ability to adhere to soft tissue, and its hydrophilic groups such as amino, carboxyl, and hydroxyl groups undergo chemical and physical reactions with functional groups in the skin and mucosa. 2) The catechol and pyrogallol groups in vTA form stable adhesion with the metal surface through chelation. 3) The injectability of the GT-F hydrogel enables it to fill irregular gaps, reducing stress concentration, thus ensuring the adhesion performance. In summary, the GT-F hydrogel has good adhesion ability to both tissues and metal implants, and can meet the requirements of closing wounds by adhesion around the implant.

[0083] 1.9 Swelling performance of GT-F hydrogel

[0084] Place the GT-F hydrogel in a 25 mL acid burette ( Figure 12 ), add 15 mL of ultrapure water, record the initial total volume (V0), seal it, and let it stand at room temperature. Read the total volume (V t ) at 1, 2, 3, 4, 6, 8, 10, and 12 h respectively, and plot the swelling curve. The calculation formula for the volume swelling ratio is as follows:

[0085] Volume swelling ratio (%) = (V t - V0) / V0 × 100%.

[0086] The results show that the GT hydrogel reaches swelling equilibrium at 8 h with a swelling ratio of 8.4 ± 0.1%; the GT-F hydrogel reaches swelling equilibrium at 6 h with a swelling ratio of 6.8 ± 0.1%. The GT-F hydrogel has a faster swelling rate and a slightly lower swelling ratio than the GT hydrogel ( Figure 13 ).

[0087] 1.10 Degradation performance of GT-F hydrogel

[0088] Weigh the formed GT-F hydrogel (W0), soak it in 30 mL of artificial saliva, and place it in a shaker at 37 °C. Take out the hydrogel samples at 1, 3, 5, 7, 9, 11, 13, and 15 days respectively, dry the surface and then weigh (W t ), and plot the degradation curve. The degradation calculation formula is as follows:

[0089] Remaining mass ratio (%) = W t / W0 × 100%.

[0090] The results showed that the weights of the GT and GT-F hydrogels increased during the first 1 - 3 days of immersion and then started to decrease after 3 days. On the 13th day, the remaining mass of the GT hydrogel was 30.8 ± 26.8%, while that of the GT-F hydrogel was 7.5 ± 2.6%( Figure 14 ). The GT-F hydrogel completely disintegrated on the 14th day and could not be taken out for weighing. The degradation rate of the GT-F hydrogel matched the healing rate of the soft tissues around oral implants (the healing time was about 14 days).

[0091] 1.11 Drug release performance of the GT-F hydrogel

[0092] The GT-F hydrogel was immersed in 10 mL of ultrapure water. At the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days, 2 mL of the leaching solution was taken out and an equal volume of ultrapure water was added. Inductively Coupled Plasma atomic emission spectrometry (ICP) was used to measure the concentration of Cu 2+ in the leaching solution. The Folin-Ciocalteu method was used to detect the concentration of vTA NP in the leaching solution. The hydrogel leaching solution was mixed with 50 μL of Folin-Ciocalteu reagent, allowed to stand for 6 min, then 100 μL of 70 mg / mL sodium carbonate solution and 750 μL of ultrapure water were added, and it was incubated in the dark for 30 min. The OD value at 725 nm was read with an enzyme-linked immunosorbent assay reader. Based on the linear relationship between the vTA NP concentration and the OD value, the concentration of vTA NP in the leaching solution was calculated to analyze the release of vTA NP from the hydrogel. The ion concentration in the leaching solution samples taken at each time point was marked as Ct (unit: mg / mL), t represents the time point, and the cumulative release amount formula is as follows: Cumulative release amount = Ct×10 + Ct-1×2 + Ct-2×2 + …… + C1×2. The cumulative release rate calculation formula is as follows: Cumulative release rate (%) = Cumulative release amount / Total input amount × 100%.

[0093] The results showed that on the 13th day, the degradation degree of the hydrogel was high and the leaching solution was too turbid to be detected. When immersed in ultrapure water for 11 days, the cumulative release amount of Cu 2+ was 1.67 ± 0.04 ng, and the cumulative release rate was 26.2 ± 0.4%; the cumulative release amount of vTA NP was 0.52 ± 0.01 mg, and the cumulative release rate was 52.0 ± 1.2%( Figure 15 ).

[0094] The physicochemical characterization results of the GT-F hydrogel indicate that it has the following characteristics: 1) It has injectability and can easily fill the irregular wounds between the implant and the surrounding soft tissues; 2) Its mechanical properties can be optimized by adjusting the components and ratios, providing a suitable scaffold for soft tissue regeneration; 3) Under wet conditions, the GT-F hydrogel exhibits strong adhesive ability to soft tissues and implants, can quickly seal the wounds and has good adhesive stability; 4) The degradation rate of the GT-F hydrogel matches the healing time of the soft tissues around the implant (about 14 days); 5) It can be used as a drug reservoir to load and control the release of various functional molecules (such as Ce6, vTA and Cu 2+ ). In summary, the GT-F hydrogel meets the requirements for enhancing soft tissue integration around oral implants in terms of physicochemical properties and shows good application prospects.

[0095] II. Biological properties of the hydrogel

[0096] 2.1 Cytotoxicity of the GT-F hydrogel

[0097] Culturing cells with the extract: Prepare the GT-F hydrogel, GT + vTA + Ce6 hydrogel (1 mL Gel solution + 100 μL TG solution + 100 μL Ce6 solution + 100 μL vTA NP solution + 100 μL ultrapure water) and GT + Ce6 hydrogel (1 mL Gel solution + 100 μL TG solution + 100 μL Ce6 solution + 200 μL ultrapure water), and co-culture them with L929 cells, human gingival fibroblasts (HGF) and human gingival epithelial cells (HGE) under the conditions of 5% CO2 and 37 °C, with PBS as the control. The CCK8 method and Actin-Tracker and DAPI staining methods are used to evaluate cell viability. The results show that L929 cells have good cell activity when cultured in the extract of each component hydrogel for 1 - 7 days, and the cell viability is greater than 80%; the cell viability of HGF cells is slightly lower on the second day (77%), and greater than 80% at the other time points; the cell viability of HGE cells is greater than 80% at each time point ( Figure 16 ). The results of laser confocal microscopy show that the cells in each group grow well, the fusion degree reaches about 70%, and the cell morphology extends ( Figure 17 ).

[0098] Culturing cells on the surface of hydrogels: Inject 200 μL of GT-F, GT+vTA+Ce6, and GT+Ce6 hydrogel precursor solutions into 48-well plates, and add L929, HGF, and HGE cell suspensions for co-culture. The CCK8 method was used to measure cell viability, and cells directly growing on the bottom of the well plate served as the control group. The results showed that cells directly cultured on the surface of hydrogels with different components had good cell compatibility for 1-7 days, and the cell growth trend was good. For L929 cells, except that the cell compatibility of the GT+Ce6 and GT+vTA+Ce6 groups was slightly lower than 80% on the second day, other components and time points were greater than 80%; for HGF cells, except that the cell compatibility of the GT+Ce6 group was slightly lower than 80% on the first and second days, other components and time points were greater than 80%; for HGE cells, except that the cell compatibility of the GT+Ce6 group was slightly lower than 80% on the first and second days and the GT+vTA+Ce6 group was slightly lower than 80% on the second day, other components and time points were greater than 80%( Figure 18 ). Three days after cells were seeded on the surface of various component hydrogels, the cell growth state was good, the confluence reached about 80%, and the cell morphology was extended( Figure 19 ).

[0099] Detection of cells growing into the hydrogel: Drop 50 μL of L929 cell suspension (containing 5×10 4 cells) on one side of the bottom of a 12-well plate. After the cells adhered to the wall, inject the hydrogel precursor solution beside the cells( Figure 20 ). After 24 h, the cells grew to cover the bottom of the well plate, and a small number of cells grew into the hydrogel area. After 4 days, the cells grew to cover the bottom of the well plate and the hydrogel after the hydrogel gelled( Figure 21 ). Observation under a microscope and a confocal laser scanning microscope after cell staining showed that the cells grew well on both the bottom of the well plate and the surface of the hydrogel, and the confluence was more than 90%( Figure 22 ).

[0100] The experimental results showed that the GT-F hydrogel had good biocompatibility, and cells could grow healthily on the surface and inside of the hydrogel, and the cells could grow from the well plate into the hydrogel. This meets the requirement of the growth of soft tissue cells around implants into the hydrogel in clinical applications. It is speculated that injecting GT-F into the gap between the implant abutment and the surrounding soft tissue can achieve the transition from hydrogel-mediated physical closure to soft tissue-mediated biological closure. As a natural polymer with excellent biocompatibility and biodegradability, gelatin can simulate the natural environment of mammalian cells, provide a suitable growth space for cells, and promote cell adhesion and proliferation. The reticular structure inside the GT-F hydrogel meets the pore diameter required for the growth of fibroblasts (about 80-100 μm), which can better promote cell migration and diffusion.

[0101] 2.2 Detection of the ability of GT-F hydrogel to physically block bacteria

[0102] The oral cavity is a bacterium-containing environment. To establish a sterile environment for the soft tissues around the implant, it is necessary to isolate the bacteria in the oral cavity. In this application, a Transwell in vitro model was used to evaluate the physical barrier ability of GT-F hydrogel against bacteria, simulating the situation before the complete healing of the gingival epithelium.

[0103] Detection of bacterial barrier rate: Add 1 mL of GT-F hydrogel to the bottom of the upper chamber of the Transwell. After the hydrogel gels, add 1 mL of a bacterial solution of 5×10 5 CFU / mL of S.a or E.c, and add sterile BHI medium to the lower chamber ( Figure 23 Figure A in the middle). After 24 h, the penetration of bacteria through the hydrogel was detected by the colony formation experiment of the BHI medium in the lower chamber. The Transwell without hydrogel was used as a blank control. The calculation formula for the barrier rate is:

[0104] Bacterial barrier rate (%) = number of colonies in the control group / number of colonies in the GT-F group × 100%.

[0105] The results showed that after 24 h, there were no bacteria in the lower chamber of the Transwell coated with the hydrogel, while there were a large number of bacteria in the lower chamber of the control group ( Figure 24 Figure A in the middle), and the barrier rates of GT-F hydrogel against both bacteria were 100% ( Figure 24 Figure B in the middle).

[0106] Simulation of the bacterial barrier experiment around the implant: Add hydrogel to the bottom of the upper chamber of the Transwell. After the hydrogel gels, inoculate HGE (1×10 5 cells) on the surface, and inoculate HGF (1×10 5 cells) in the lower layer. After culturing for 72 h, add 1 mL of a bacterial solution of 1×10 8 CFU / mL of S.a to the upper chamber. After 12 h, stain with Actin-Tracker and DAPI, and observe the cell morphology with a laser confocal microscope ( Figure 23 Figure B in the middle). The results showed that after 72 h, the HGE cells in the upper chamber did not form an epithelial seal. After adding S.a, the HGE cells were damaged and had an irregular shape. The HGF in the lower chamber of the GT-F group was not affected, while the HGF in the group without hydrogel was damaged and the cells floated ( Figure 25 ). This indicates that GT-F hydrogel can block the damage of bacteria to the underlying HGF cells.

[0107] Although the pore size of GT-F is larger than that of bacteria, its barrier effect on bacteria can be attributed to: 1) the internal porous network and the superposition of pore layers in the hydrogel; 2) the interaction between vTA NPs and bacteria to "stick" the bacteria; 3) the antibacterial function of vTA NPs. These factors work together to effectively isolate bacteria at the early stage of healing, cut off the invasion route of bacteria, and prevent bacterial colonization.

[0108] 2.3 Singlet oxygen generation ability of GT-F hydrogel

[0109] In the GT-F hydrogel constructed in this application, there is both Ce6 that generates singlet oxygen and vTA NPs that scavenge ROS. It is very important to explore whether the singlet oxygen generated by Ce6 will be scavenged by vTA NPs.

[0110] Effect of vTA NPs on the singlet oxygen generation ability of Ce6 in solution state: Prepare Gel + vTA + Ce6 and Gel + Ce6 solutions, add the DPBF probe and then irradiate with light, and detect the absorbance change at 410 nm by a microplate reader. After irradiation, there is no significant difference in the decrease in absorbance between the Gel + vTA + Ce6 and Gel + Ce6 solutions, indicating that vTA NPs have no significant effect on the singlet oxygen generation ability of Ce6 ( Figure 26 )

[0111] Effect of vTA NPs in GT-F hydrogel on the singlet oxygen generation ability of Ce6: Add the DPBF singlet oxygen probe to the GT-F hydrogel precursor solution, irradiate with light after gelation, and detect the fluorescence intensity change at 410 nm by a laser confocal microscope. The results show that the fluorescence intensity of the GT-F hydrogel decreases significantly after irradiation, indicating that the hydrogel can generate singlet oxygen ( Figure 27 )

[0112] vTA NPs in the hydrogel can scavenge a small amount of singlet oxygen, but will not completely scavenge it. The reasons may be: 1) The action distance of singlet oxygen is short. In GT-F, the concentrations of Ce6 and vTA NPs are not high and are evenly distributed in the hydrogel, and the number of vTA NPs within the action distance of singlet oxygen is limited; 2) vTA exists in the form of nanoparticles, and the number of phenolic hydroxyl groups on the surface is small; the molecular weight also has an important impact on the free radical scavenging ability of polyphenols. Oligomers have a faster free radical scavenging rate and a higher scavenging ability, while macromolecules and polymers may hinder their free radical scavenging rate due to steric hindrance; 3) The same polyphenol has different scavenging abilities for different types of reactive oxygen species. The phenolic hydroxyl groups in the vTA NP structure are more likely to scavenge ·O2 - , and have a weak scavenging ability for singlet oxygen. In summary, in the GT-F system, although Ce6 and vTA NPs coexist, the singlet oxygen generated by Ce6 will not be completely scavenged by the phenolic hydroxyl groups in vTA NPs.

[0113] 2.4 Light-controlled bactericidal ability of GT-F hydrogel

[0114] Construct GT-F hydrogel containing bacteria: Mix 1 mL of GT-F hydrogel precursor solution with 400 μL of 5×10 5 CFU / mL S. a bacterial solution at 37 °C and then gelate, and verify that there are live bacteria inside the hydrogel by live / dead bacteria staining. Figure 28)。

[0115] GT-F hydrogel's photocontrolled bactericidal ability: After mixing the GT-F hydrogel precursor solution with bacteria to form a gel, 660 nm light irradiation and non-irradiation treatments were carried out respectively. After 24 hours, live / dead bacteria staining was performed. The results showed that all the bacteria in the hydrogel died after 5 minutes of light irradiation, while a large number of live bacteria still remained in the non-irradiated hydrogel after 24 hours, indicating that the rapid bactericidal ability of the GT-F hydrogel depends on the singlet oxygen generated by Ce6 under light irradiation ( Figure 29 )。

[0116] In addition to preventing the colonization of pathogenic bacteria, it is equally important to kill the bacteria invading during the operation as early as possible. With the increase in drug-resistant bacteria, there is an urgent need to find alternative antibacterial therapies to traditional antibiotics. Photodynamic therapy has significant efficacy against drug-resistant bacteria and reduces the emergence of new drug-resistant mechanisms. In this application, the rapid bactericidal function of the GT-F hydrogel stems from the singlet oxygen generated by the photosensitizer Ce6. Loading Ce6 into the hydrogel can generate sufficient amounts of singlet oxygen for bactericidal action after 5 minutes of light irradiation at a wavelength of 660 nm. And the generated singlet oxygen has an action distance of only 10 -10 m and a lifetime of only 10 -6 s. It can only kill bacteria within a certain distance range and will not damage the surrounding tissues. Wrapping the bacteria in the hydrogel, the bacteria and Ce6 are evenly distributed, and the generated singlet oxygen can kill the adjacent bacteria. And due to the short lifetime of singlet oxygen, there will be no subsequent cytotoxicity after stopping light irradiation. For the non-irradiated hydrogel, a large number of live bacteria still remained after 24 hours, indicating that the vTA NPs and Cu 2+ in the hydrogel did not reach the bactericidal concentration, and the contact between vTA NPs and bacteria was insufficient, so the bactericidal effect was limited.

[0117] 2.5 Antioxidant stress ability of GT-F hydrogel

[0118] To investigate whether the singlet oxygen generated by Ce6 under light irradiation would oxidize vTA NPs and affect their DPPH scavenging ability, the following experiments were carried out.

[0119] DPPH scavenging ability of vTA NPs loaded in the hydrogel: Prepare a vTA NP solution with a concentration of 0.7 mg / mL and a GT + vTA hydrogel with equal vTA NP concentrations. Mash the hydrogel and place it in a 1000 Da dialysis bag, and also place the vTA NP solution in the dialysis bag. Immerse them in 10 mL of a DPPH working solution with a concentration of 2 mg / mL and react on a shaker at room temperature in the dark for 30 minutes. Read the OD value at 517 nm with an enzyme-linked immunosorbent assay reader. The results showed that the DPPH scavenging ability of vTA NPs loaded in the hydrogel was slightly weaker than that in the solution state, but still retained the DPPH scavenging ability ( Figure 30 )。This indicates that the vTA NPs in the hydrogel can still effectively scavenge DPPH free radicals, although their efficiency is slightly lower than that of vTA NPs in the solution.

[0120] The DPPH scavenging ability was detected after vTA NPs and Ce6 were blended in solution and irradiated with light: 50 μL of 1 mg / mL vTA NP solution and 50 μL of 10 mg / mL Ce6 solution were blended, irradiated with a 660 nm wavelength light source for 5 min or without light irradiation, reacted with 200 μL of 2 mg / mL DPPH working solution, and the DPPH scavenging ability was detected. The results showed that when vTA NPs and Ce6 were blended in solution and irradiated with light, they retained the DPPH scavenging ability, which was 93.0 ± 8.1% of that without light irradiation ( Figure 31 ). This indicates that the singlet oxygen generated by Ce6 has little effect on the DPPH scavenging ability of vTA NPs, and vTA NPs can still maintain a high scavenging efficiency under light irradiation.

[0121] The DPPH scavenging ability of vTA NPs and Ce6 loaded in hydrogels: 0.5 mL of each of the GT + vTA + Ce6 and GT + vTA hydrogel precursor solutions was taken and placed in a 1.5 mL EP tube, irradiated with 660 nm light for 5 min, and the DPPH scavenging ability was detected. The results showed that when vTA NPs and Ce6 were blended in the hydrogel state, although Ce6 generated singlet oxygen under light irradiation, vTA NPs retained the DPPH scavenging ability, and the DPPH scavenging rate of the GT + vTA + Ce6 hydrogel after light irradiation was 40.6 ± 3.7% ( Figure 32 ). This indicates that in the hydrogel, the DPPH scavenging ability of vTA NPs is partially inhibited, but there is still a significant scavenging effect.

[0122] When vTA and Ce6 were mixed in solution state and irradiated with light, the singlet oxygen generated by Ce6 would oxidize a part of the phenolic hydroxyl groups in vTA into quinones, resulting in the loss of a part of its DPPH scavenging ability. However, because vTA is in the form of nanoparticles and the phenolic hydroxyl groups encapsulated inside are not easily oxidized, and as the nanoparticles degrade, the interface is continuously updated, and the unoxidized phenolic hydroxyl groups are exposed. Therefore, vTA in the hydrogel has a long-term ability to scavenge ROS. When vTA and Ce6 were loaded in the GT-F hydrogel, the experimental results showed that after the hydrogel was irradiated with light, the singlet oxygen generated by Ce6 only oxidized a small part of the phenolic hydroxyl groups in vTA in the hydrogel. Considering GT-F as a whole, it has the DPPH scavenging ability and can be used to scavenge excessive ROS around oral implants, provide a suitable environment for soft tissue regeneration, and thus promote the integration of soft tissues around implants.

[0123] The antioxidant stress ability of GT-F hydrogel: H2O2 is a common and stable ROS and is often used to construct a cell oxidative stress model. The ability of GT-F hydrogel to resist oxidative stress was evaluated by detecting the changes in intracellular ROS levels after H2O2 treatment of the hydrogel extract and cells directly cultured on the surface.

[0124] Antioxidant stress capacity of hydrogel extracts: L929 cells were co-cultured with hydrogel extracts of different components (GT+Ce6, GT+vTA+Ce6, GT-F), and PBS was used as a control. After treatment with 100 μM H2O2 for 24 h, the intracellular ROS level was measured by the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method. The increase in DCFH fluorescence intensity represents the increase in intracellular ROS level. The cells were stained with DAPI and observed under a laser confocal microscope. The results showed that the intracellular ROS in each group increased after H2O2 treatment ( Figure 33 Figure A in the middle), but the increase in intracellular ROS in the hydrogel group was significantly lower than that in the blank control group (P < 0.05, Figure 33 Figure B in the middle). The fluorescence staining results showed that there was no obvious green fluorescence of DPBF in the cells before H2O2 treatment, and the green fluorescence intensity increased after treatment. The fluorescence intensity in the hydrogel group was lower than that in the blank control group ( Figure 34 ). This indicates that the hydrogel extract can effectively reduce the accumulation of intracellular ROS and protect cells from oxidative stress damage.

[0125] Antioxidant stress capacity of cells directly cultured on the hydrogel surface: L929 cells were directly cultured on the surfaces of hydrogels with different components, and cells directly growing on the bottom of the well plate were used as a control group. The remaining operations were the same as above. The results showed that after H2O2 treatment, the DCFH fluorescence intensity of the cells cultured on the surfaces of each group of hydrogels increased. Among them, the GT group and the blank control group had a larger increase, while the GT+vTA group had the least increase in fluorescence intensity ( Figure 35 Figure A in the middle). The increase in fluorescence intensity in each hydrogel group was significantly lower than that in the blank control group (P < 0.05, Figure 35 Figure B in the middle). Fluorescence staining showed that there was no obvious green fluorescence in the cells before H2O2 treatment, and the green fluorescence increased after treatment. Among them, the GT-F group had the least increase, and the blank control group had the most increase ( Figure 36 ). This indicates that the GT-F hydrogel can effectively reduce the accumulation of intracellular ROS and protect cells from oxidative stress damage.

[0126] 2.6 Angiogenesis-promoting ability of GT-F hydrogel

[0127] After the GT-F hydrogel was extracted with PBS at a volume ratio of 1:1 for 24 h, 100 μL of the extract was taken and mixed with 100 μL of the solution containing 1×10 4Mix the suspension of human umbilical vein endothelial cells (HUVEC) and inoculate it into a 48-well plate containing Matrigel matrix gel. After 4 h, observe the angiogenesis by microscope, and use Image J software to count the number of blood vessels in 6 fields of view in each group. The results show that the GT-F group has the most vascular growth, while the GT group has less angiogenesis than the other three groups( Figure 37 ). Further analysis of the number of crosslinks and branches found that the number of crosslinks in the GT+Ce6, GT+vTA+Ce6, GT-F and GT groups were 32.9±8.2, 30.3±5.2, 45.6±4.4 and 32.8±6.9 respectively, and the number of branches were 43.9±5.7, 42.2±11.6, 52.6±9.8 and 35.0±8.3 respectively. The GT-F group was significantly higher than the GT group in both indicators (P < 0.05, Figure 38 ), indicating that the GT-F hydrogel has stronger angiogenesis-promoting ability. Angiogenesis is crucial for the health and stability of the soft tissue around the implant. Good angiogenesis can provide sufficient nutrition and oxygen for the soft tissue, enhancing its ability to resist external infections. On the contrary, the ischemic state will weaken the antibacterial ability of the regenerated soft tissue around the implant, making it more vulnerable to infection, and at the same time accelerating the progression of inflammation, resulting in more serious tissue damage and complications. Choose to load Cu 2+ as an inorganic signaling factor to promote the migration, growth and lumen formation of endothelial cells, thus effectively promoting angiogenesis in the soft tissue around the implant, which not only helps to improve the nutritional supply and structural stability of the soft tissue, but also enhances its antibacterial ability and reduces the risk of infection, thus creating favorable conditions for the long-term stability and success of the implant.

[0128] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments only. Those of ordinary skill in the art can achieve the purpose of the present invention based on the content disclosed above. Any improvements and deformations made on the basis of the concept of the present invention fall within the protection scope of the present invention. The specific protection scope shall be subject to the content recorded in the claims.

Claims

1. An injectable multifunctional hydrogel adhesive for light-controlled antibacterial and cell growth promotion, characterized in that, Mainly composed of alkenylated tannic acid nanoparticles, photosensitizer chlorin e6, Cu 2+ , transglutaminase and gelatin, wherein the concentration of the alkenylated tannic acid nanoparticles is 0.35 - 1.43 mg / mL, the concentration of the photosensitizer chlorin e6 is 25 - 150 μg / mL, the concentration of the Cu 2+ is 17 - 18 ng / mL, and the concentration of the transglutaminase is 1.79 - 7.14 mg / mL.

2. The photocontrolled antibacterial and cell growth-promoting injectable multifunctional hydrogel adhesive according to claim 1, characterized in that The concentration of the alkenylated tannic acid nanoparticles is 0.35 - 0.70 mg / mL, the concentration of the photosensitizer chlorin e6 is 80 - 120 μg / mL, the concentration of Cu 2+ is 17.5 - 18 ng / mL, and the concentration of transglutaminase is 1.79 - 3.58 mg / mL.

3. The injectable multifunctional hydrogel adhesive for light-controlled antibacterial and cell growth promotion according to claim 2, wherein The concentration of the alkenylated tannic acid nanoparticles is 0.35 mg / mL, the concentration of the photosensitizer chlorin e6 is 100 μg / mL, the concentration of Cu 2+ is 17.86 ng / mL, and the concentration of transglutaminase is 1.79 mg / mL.

4. The preparation method of the light-controlled antibacterial and cell growth-promoting injectable multifunctional hydrogel adhesive according to any one of claims 1 to 3, characterized in that, It includes the following steps: taking a solution of vinylated tannic acid nanoparticles, a solution of photosensitizer chlorin e6, a solution containing Cu 2+ , a solution of transglutaminase, and a gelatin solution, mixing them and placing the mixture in a mold, and then standing still in an environment of 45-55°C to obtain an injectable multifunctional hydrogel adhesive with light-controlled antibacterial and cell growth promoting properties.

5. Use of the hydrogel adhesive according to any one of claims 1 to 3 in the preparation of a product for repairing soft tissues around a wound or an oral implant.

6. Use of the hydrogel adhesive according to any one of claims 1 to 3 in the preparation of a product for repairing soft tissues around a wound or an oral implant in the skin and various mucous membranes.

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