Urethral injury repair gel and its preparation method and application

Abstract

The invention discloses a urethral injury repair gel and a preparation method and application thereof. The repair gel is formed by chemical cross-linking of an APF copolymer and a procoagulant polypeptide, wherein the APF copolymer is composed of terminally aminoated PF127. The repair gel has collagen peptides for antibacterial and anti-inflammatory effects as inhibitors, and preferably also has an adipose stem cell membrane extracellular matrix to promote angiogenesis and matrix secretion in the later stage of wound healing, thereby achieving the purpose of promoting wound healing and preventing scar tissue formation after injury, and providing a multifunctional all-in-one full-process repair gel for the adverse microenvironment of urine.

Description

Technical Field

[0001] The invention belongs to the technical field of urethral injury repair, and specifically relates to a multifunctional all-in-one repair gel for the whole process of repair, so as to promote scar-free repair of urethral wounds. Background Art

[0002] After urethral injury, local urine and blood become natural culture media for bacteria, leading to bacterial infection and obvious inflammatory response, which will greatly affect the normal repair of the urethra and even cause abnormal proliferation of local urethral cells, remodeling of extracellular matrix, abnormal increase of myofibroblasts, significant changes in collagen composition and its proportion, and collagen type, ultimately forming scar tissue and even leading to urethral stenosis.

[0003] Although there are many clinical treatment methods, such as urethral anastomosis and urethral replacement, for the treatment of urethral stricture, which have achieved excellent results, there is still a gap in promoting urethral wound healing and preventing or reducing the formation of stricture after urethral injury. Therefore, in clinical practice, most patients who have experienced urethral injury will develop urethral stricture at different times.

[0004] Most existing urethral repair materials focus on hydrogel dressings or patches to replace suture treatment at the site of injury, and there is almost no research on in situ local injection of hydrogel treatment. Currently, most urethral repair materials only focus on a single stage in the healing process (such as early inflammation or late proliferation stage). Although certain results have been achieved using these materials, their general efficacy and applicability remain to be studied due to the lack of understanding of the multi-factor characteristics of the unfavorable local urine microenvironment after urethral trauma.

[0005] However, most of the existing tissue engineering repair materials are used for animal skin repair, and lack attention to the difficulty of healing in the poor urethral microenvironment. In the current basic research of tissue engineering, the urethral injury repair modeling method is mostly a urethral mucosal defect model, so most of them are patch-type or dressing-type hydrogels for alternative suture treatment. This type of modeling and treatment method mainly simulates the existing clinical urethral replacement plastic surgery for urethral injuries and scar tissue formation, and there is almost no research on the in situ injection of hydrogels at the injury site to prevent the formation of urethral scar tissue. In addition, the existing urethral repair materials are mostly arrangements and combinations of different materials, and have not been specifically developed and designed based on the local poor urine prognosis microenvironment. Summary of the invention

[0006] The purpose of the present invention is to provide a multifunctional urethra injury repair gel suitable for full-process repair, as well as a preparation method and application of the repair gel.

[0007] As a first aspect of the present invention, a method for preparing a urethral injury repair gel is provided, comprising:

[0008] 1) subjecting the PF127 hydrogel to terminal amination reaction to obtain an APF copolymer;

[0009] 2) chemically cross-linking the APF copolymer and the procoagulant polypeptide to obtain a chemically cross-linked repair gel;

[0010] Wherein, an inhibitor is added to the cross-linked gel, and the inhibitor includes an antibacterial agent and / or an anti-inflammatory agent.

[0011] Preferably, the synthesis conditions of step 1 are as follows:

[0012] Fully dissolve PF127 in dichloromethane, add triethylamine and stir in an ice-water bath until fully mixed, inject methanesulfonyl chloride into the liquid twice with a syringe, react at room temperature for 2-24 hours, and after the reaction is completed, filter the reaction solution to remove solids, rotary distill until there is almost no sol, pour in petroleum ether to obtain a solid, dry the solid and dissolve it in ammonia water, stir vigorously for 0.5-5 days, let stand until the foam disappears, extract with dichloromethane, and then use anhydrous magnesium sulfate to remove water from the dichloromethane phase to finally obtain APF polymer.

[0013] Preferably, the synthesis conditions of step 2 are as follows:

[0014] Procoagulant polypeptide powder, 2% wt 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride powder and 2% wt N-hydroxysuccinimide powder were added to the APF solution for cross-linking reaction, and stirred overnight at 4°C to obtain a chemically cross-linked repair gel.

[0015] Preferably, adipose stem cell membrane decellularized matrix is ​​added to the repair gel to promote angiogenesis and matrix secretion in the later stage of healing, thereby avoiding the final formation of scar tissue.

[0016] Preferably, the procoagulant polypeptide is fish collagen peptide.

[0017] Preferably, the inhibitor is LL-37 antimicrobial peptide.

[0018] Preferably, the repair gel is in a gel state at room temperature and in a sol state at 35-37°C.

[0019] Preferably, the APF copolymer accounts for 15-25wt%.

[0020] Preferably, the procoagulant polypeptide accounts for 15-25 wt%.

[0021] Preferably, the fish collagen peptide accounts for 5-15wt%.

[0022] As a second aspect of the present invention, a urethral injury repair gel is provided, comprising: an APF copolymer, a procoagulant polypeptide and an inhibitor, wherein the AFP copolymer is a terminally amino-containing PF127 hydrogel, and the gel is formed by chemically cross-linking the APF copolymer and the procoagulant polypeptide, and the APF copolymer is a terminally amino-containing PF127 copolymer; the inhibitor comprises an antibacterial agent and / or an anti-inflammatory agent.

[0023] Preferably, the repair gel is further added with a decellularized matrix that can promote angiogenesis and matrix secretion in the later stage of healing to avoid the final formation of scar tissue.

[0024] Preferably, the decellularized matrix is ​​an adipose stem cell membrane decellularized matrix, the procoagulant polypeptide is a fish collagen peptide, and the inhibitor is an LL-37 antimicrobial peptide.

[0025] Preferably, the procoagulant polypeptide accounts for 5-10 wt % in the repair gel.

[0026] Preferably, in the repair gel, the inhibitor concentration is 0.5 wt %.

[0027] Preferably, in the repair gel: the concentration of the decellularized matrix is ​​80 mg / ml.

[0028] As other aspects of the present invention, there is also provided:

[0029] Use of any of the above-mentioned urethra injury repair gels in the preparation of materials for promoting urethra injury repair.

[0030] Use of any of the above-mentioned urethral injury repair gels in the preparation of materials for promoting urethral angiogenesis.

[0031] Use of any of the above-mentioned urethral injury repair gels in the preparation of materials for promoting urethral epithelial regeneration.

[0032] Use of any of the above-mentioned urethral injury repair gels in the preparation of materials for promoting urethral smooth muscle regeneration.

[0033] The present invention designs a urethral injury repair gel that can be multifunctional and repair the whole process in view of the adverse microenvironment of urine. It has the ability to promote blood coagulation, antibacterial, inhibit inflammatory response, and promote cell proliferation and migration. It plays a role in the coagulation stage, inflammation stage and proliferation stage of urethral wound healing, and can promote the healing of urethral wounds, and can also prevent the formation of scar tissue after urethral injury. The invention has a high potential for clinical application and can provide new means and ideas for the treatment of urethral injuries in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the PF127 and APF in the embodiment1 H NMR spectrum;

[0035] Figure 2 : is the storage modulus and elastic modulus of APF10, APF20 and APF30 in the embodiment changing with temperature;

[0036] Figure 3 : is the storage modulus and elastic modulus of APF20 / C5, APF20 / C10 and APF20 / C20 in the embodiment as a function of temperature;

[0037] Figure 4 It is a chart showing the results of measuring the contents of various substances before and after decellularization in the embodiment;

[0038] Figure 5 1 is a test chart for evaluating the biosafety and hemostatic performance of the materials in the embodiment, wherein A is measured by the coagulation experiment, B is measured by the BCI method, and C is measured by the dynamic coagulation time;

[0039] Figure 6 1 is a test diagram of the antibacterial properties of different materials evaluated by various methods in the embodiments, wherein A is a plate coating method, B and C are bacterial live and dead staining methods, D is an inhibition ring method, and E is a biological scanning electron microscopy method;

[0040] Figure 7 This is a graph showing the ability of the hydrogel in the embodiment to promote the proliferation and migration of adipose stem cells;

[0041] Figure 8 The chart for verifying the anti-inflammatory effect of the hydrogel in the embodiment; A is the flow cytometry determination, B is the semi-quantitative analysis of the flow cytometry results, C is the WB determination, D is the immunofluorescence staining of rabbit urethra sections, and E is the semi-quantitative analysis of the immunofluorescence staining results;

[0042] Fig. 9 This is a chart comparing the effects of different materials injected in the examples to prevent scar formation after urethral injury. DETAILED DESCRIPTION

[0043] A urethral injury repair gel comprises: an APF copolymer, a procoagulant polypeptide and an inhibitor, wherein the AFP copolymer is a terminally amino PF127 hydrogel, the APF copolymer is a gel formed by chemically cross-linking with the procoagulant polypeptide, and the APF copolymer is a terminally amino PF127 copolymer; the inhibitor comprises an antibacterial agent and / or an anti-inflammatory agent.

[0044] The preparation method of the above-mentioned repair gel comprises:

[0045] 1) subjecting the PF127 hydrogel to terminal amination reaction to obtain an APF copolymer;

[0046] 2) chemically cross-linking the APF copolymer and the procoagulant polypeptide to obtain a chemically cross-linked repair gel;

[0047] Wherein, an inhibitor is added to the cross-linked gel, and the inhibitor includes an antibacterial agent and / or an anti-inflammatory agent.

[0048] The repair gel can be used in the repair of urethral injuries, in the preparation of materials for promoting the repair of urethral injuries, in the preparation of materials for promoting the regeneration of urethral blood vessels, in the preparation of materials for promoting the regeneration of urethral epithelium, and in the preparation of materials for promoting the regeneration of urethral smooth muscle.

[0049] Example

[0050] 1. Synthesis of multifunctional all-in-one repair hydrogel

[0051] In the research design of the present invention, we use the thermosensitive Pluronic F127 (i.e., PF127) hydrogel with high biocompatibility as a carrier, wherein Pluronic F127 is a type of polyoxyethylene-polyoxypropylene ether non-ionic triblock copolymer (PEO-PPO-PEO) composed of polyoxyethylene (PEO) and polyoxypropylene (PPO). However, PF127 hydrogel is unstable and has a high degradation rate. In order to extend the time for the hydrogel to function, we amino-terminated Pluronic F127 (i.e., APF) copolymers by terminal amination, and chemically cross-linked it with fish collagen peptides with procoagulant function, thereby obtaining a multifunctional all-in-one repair hydrogel.

[0052] 1.1 Synthesis of amino-terminated PF127

[0053] Take 20g PF127 and fully dissolve it in 300mL dichloromethane, add 15mL triethylamine, and stir in an ice-water bath until fully mixed. Use a syringe to inject a total of 7.5mL of methanesulfonyl chloride into the liquid surface twice to avoid excessive heat release and white smoke. The reaction is carried out at room temperature for 12 hours. Filter the reaction solution to remove solids, and after rotary distillation until there is almost no sol, pour petroleum ether to obtain a solid.

[0054] After the solid was dried, it was dissolved in 200 mL of ammonia water and stirred vigorously for 3 days. After standing until the foam disappeared, it was extracted three times with dichloromethane, and then the dichloromethane phase was dehydrated with anhydrous magnesium sulfate to finally obtain the terminal amino-terminated APF copolymer.

[0055] like Figure 1 As shown, compared with PF127 copolymer, APF 1New peaks appeared at 1.8 ppm and 2.8 ppm in the H NMR spectrum, indicating that the terminal of PF127 was modified by amino group, that is, its terminal was aminated to form APF copolymer.

[0056] 1.2 Exploration of PF127 concentration in hydrogel system

[0057] Since the normal rabbit urethra temperature is about 35°C, a system with room temperature as Sol and 35°C as Gel was selected to construct the hydrogel system.

[0058] Through rheological testing, we found that 10% wt APF copolymer could not gel in rabbits, while 30% wt APF hydrogel was in a gel state at room temperature, which would make injection difficult.

[0059] Therefore, we selected 20%wt APF for subsequent experiments. The temperature-modulus curves of APF10, APF20 and APF30 are shown in Figure 2 As shown, A, B, and C correspond to the three in turn, and the phase transition temperature is marked in Figures B and C. Figure 2 It can be seen that the storage modulus and elastic modulus of the three change with increasing temperature.

[0060] 1.3 Exploration of fish collagen peptide concentration in hydrogel system

[0061] In 100 ml of APF solution containing 20% ​​wt, 5g / 10g / 20g fish collagen peptide (Collagen) powder and 2% wt 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (1-Ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide, i.e. EDC) powder and 2% wt N-hydroxysuccinimide (NHS) powder were added for cross-linking, and stirred overnight at 4°C to obtain 5% wt fish collagen peptide + APF20 (APF / C5) hydrogel, 10% wt fish collagen peptide + APF20 (APF / C10) hydrogel and 20% wt fish collagen peptide + APF20 (APF / C20) hydrogel.

[0062] Through rheological testing, it was found that as the concentration of fish collagen peptide in the system increased, the phase transition temperature of the hydrogel continued to decrease. Figure 3 The storage modulus and elastic modulus of APF20 / C5, APF20 / C10 and APF20 / C20 change with increasing temperature. However, when the fish collagen peptide concentration is 20%wt, the system is in a gel state at room temperature. This may be due to the excessively high concentration of collagen, which gels prematurely under EDC / NHS activation, making it difficult to inject and thus eliminated.

[0063] Therefore, we selected APF / C5 and APF / C10 hydrogels for subsequent synthesis and verification experiments.

[0064] 1.4 Synthesis of LL-37 antimicrobial peptide

[0065] Take an appropriate amount of modified resin (Trityl Chloride resin) for the synthesis of antibacterial peptides, first add 20% pip / DMF to the reactor, shake for 20 minutes, filter to remove the solvent, add DMF to the system, shake the reactor for 1 minute, and filter to remove the liquid; this operation is performed three times.

[0066] Take an appropriate amount of ninhydrin A reagent, ninhydrin B reagent and a small amount of resin and add them to the detection tube. Place the detection tube at 100°C for 30-60 seconds and check whether the resin changes color. If it does, it means that Fmoc (9-fluorenylmethyloxycarbonyl protecting group, 9-fluorenylmethyloxycarbonyl, abbreviated as Fmoc) has been successfully removed.

[0067] Add the prepared amino acid solution to the reactor, then add the DIC solution, shake the reactor for 1 hour, and then add the condensation reagent for cyclization modification. After the resin is cleaved, the crude analysis is performed, and finally it is purified by RP-HPLC, and the fractions are collected and freeze-dried.

[0068] 1.5 Obtaining adipose-derived stem cell membrane decellularized matrix powder

[0069] Take the subcutaneous fat tissue of New Zealand rabbits, cut it into pieces, add it to the serum-free DMEM medium containing 1mg / ml collagenase, shake it at 37℃ for 2h, and culture the digested adipose stem cells on the plate. When cultured to P2, add 5mg / ml vitamin C to the culture medium and culture it into a cell membrane sheet. The obtained cell membrane sheet is immersed in the decellularization solution A and shaken at a frequency of 100-150r / min for 48 hours on a shaker at 37℃. The decellularization solution A is a PBS buffer containing 10mM Tris-HCl, 10mM EDTA, 1% (volume concentration) TritonX-100 and 1% SDS (volume concentration). The tissue is then placed in the decellularization solution B and shaken at a frequency of 100-150r / min for 24 hours. The decellularization solution B is a PBS buffer containing 400μg / mL deoxyribonuclease I. The decellularized sample is freeze-dried and ground into microparticles with a grinder, and then sterilized by C060γ irradiation.

[0070] DNA quantification before and after decellularization ( Figure 4 A), collagen quantification ( Figure 4 B) and sGAG quantification ( Figure 4In center C), we found that the decellularization step removed genetic material while retaining extracellular matrix components.

[0071] In addition, ELISA experiments also confirmed that, except for bFGF, the content of other growth factors did not decrease significantly after decellularization ( Figure 4 (middle D).

[0072] 2. Verification of the procoagulant function of the hydrogel

[0073] Collagen peptides have been shown to have a procoagulant effect. We have verified the biosafety and procoagulant properties of APF / C5 and APF / C10 through hemolysis experiments, blood coagulation index (BCI method) and dynamic coagulation time.

[0074] 2.1 Hemolysis experiment

[0075] Fresh anticoagulated blood was drawn from the ear vein of healthy New Zealand rabbits.

[0076] ①APF group: 1 g APF hydrogel was placed in a test tube and 10 ml of normal saline was added.

[0077] ②APF / C5 group: 1 g of APF / C5 hydrogel was placed in a test tube and 10 ml of normal saline was added.

[0078] ③APF / C10 group: 1 g of APF / C10 hydrogel was placed in a test tube and 10 ml of normal saline was added.

[0079] ④Negative control group: Add 10 ml of normal saline into the test tube.

[0080] ⑤Positive control group: Add 10 ml of distilled water into the test tube.

[0081] Then, 0.2 ml of SD rat blood was added to each of the four test tubes, mixed thoroughly, and incubated at 37°C for 60 minutes. The liquid in each test tube was taken out and centrifuged at 2500 rpm for 5 minutes. The supernatant was then taken and the absorbance was measured using an ultraviolet spectrophotometer at a wavelength of 545 nm. The results were repeated three times and the average value was taken.

[0082] The hemolysis rate was defined as: (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%, and the experiment was repeated three times.

[0083] 2.2 Blood coagulation index (BCI method)

[0084] We took 0.2 ml of APF hydrogel, 0.2 ml of APF / C5 hydrogel, and 0.2 ml of APF / C10 hydrogel into a clean centrifuge tube, and then moved the centrifuge tube containing the material into a water bath and let it stand at 37°C for 5 minutes. Then, 0.1 ml of anticoagulated whole blood was dropped on the material, and 200 ul of 0.2 M calcium chloride solution was immediately added. Five minutes later, we added 25 ml of deionized water to each centrifuge tube, and then transferred it to a shaking water bath in a pot, with a water bath temperature of 37°C and a water flow rate of 50 rpm for 5 minutes. After that, we took the solution out of the centrifuge tube and measured the absorbance value of the solution in each centrifuge tube at a wavelength of 545 nm using a UV-visible spectrophotometer (A1). We took 0.1 ml of blood, added 25 ml of deionized water, and measured the absorbance value at the same wavelength as the reference value (A2). Repeat this process three times, and calculate the coagulation index BCI using the formula BCI = A1 / A2.

[0085] 2.3 Dynamic coagulation time

[0086] Take 6 portions of 0.2 ml APF hydrogel, 0.2 ml APF / C5 hydrogel and 0.2 ml APF / C10 hydrogel and place them in clean centrifuge tubes. We set seven time points, with a time interval of 5 minutes between each two adjacent points. Take 200 ml of anticoagulated whole blood and drop it on the surface of each material. Repeat the same operation for each material in each culture dish.

[0087] After the operation was completed, we started timing and added 50 ml of distilled water to the centrifuge tube every 5 minutes. After standing for 5 minutes, the leaching solution was recovered and the optical density of free hemoglobin in the leaching solution at different time points was measured using a spectrophotometer with a wavelength of 540 nm, and then a dynamic coagulation curve was drawn.

[0088] Through the BCI method and dynamic coagulation time measurement, we found that the addition of fish collagen peptides gave the hydrogel hemostatic ability, which increased with the increase of fish collagen peptide concentration (see Figure 5 ), so we finally chose APF / C10 (hereinafter referred to as AFP / C) for subsequent experiments.

[0089] 3. Verification of the antibacterial properties of hydrogels

[0090] Due to the harsh local microenvironment of urethral injury, it is necessary for the hydrogel to have antibacterial and anti-inflammatory effects. We introduced LL-37 antimicrobial peptide into APF / C10 hydrogel. According to the different concentrations of LL-37 antimicrobial peptide, we named the hydrogels APF / C10 / L-0.02, APF / C10 / L-0.1 and APF / C10 / L-0.5, corresponding to LL-37 concentrations of 0.02%wt, 0.1%wt and 0.5%wt, respectively.

[0091] We evaluated the antibacterial effects of different concentrations of LL-37 antimicrobial peptide by using the inhibition ring method, plate count method, live / dead staining method, and biological scanning electron microscopy to observe bacterial morphology.

[0092] ① Inhibition ring method: Place paper pieces soaked with different groups of solutions on a solid nutrient culture medium evenly coated with bacterial suspension, culture at 37°C for 12-24 hours, and finally measure the size of the inhibition ring.

[0093] ② Plate coating method: a certain bacterial solution concentration (about 10 6 CFU / ml) of bacterial suspension was incubated with different gels for 12 hours. After the incubation, the bacterial suspension was inoculated onto the surface of the solid nutrient medium and cultured at 37°C for 12-24 hours, and then the culture dish was taken out and photographed for evaluation.

[0094] ③ Live / dead staining method: After the bacteria are spread on the plate, the live bacteria and dead bacteria are dyed green and red respectively using the live and dead bacteria double staining kit, and then the live and dead bacteria are observed under a microscope to semi-quantitatively evaluate the bacterial death ratio.

[0095] ④ The bacteria treated with different hydrogels were fixed with 2.5% glutaraldehyde solution at 4°C for 40 minutes, then dehydrated with ethanol aqueous solution and dried for SEM observation.

[0096] We used S. aureus and E. coli as bacterial models and evenly spread the two bacterial cultures pretreated with different hydrogels on the bacterial culture dishes. After 24 hours, the number of colonies was reduced as seen with the naked eye (see Figure 6 The results of bacterial live and dead staining also showed the same trend. Figure 6 As can be seen in B and C, S. aureus and E. coli treated with PBS, APF and APF / C10 all showed obvious green fluorescence, and as the concentration of LL-37 increased, the red fluorescence became stronger and stronger, which reflects the common antibacterial effect of LL-37 on S. aureus and E. coli.

[0097] Through the inhibition ring method, we found that as the concentration of LL-37 antimicrobial peptide in the gel increased, the inhibition zone gradually increased, and the APF / C10 / L-0.5 group had the largest inhibition zone (see Figure 6 In addition to the inhibition zone, plate coating method, and live / dead staining method, we also used scanning electron microscopy to observe the morphological changes of S. aureus and E. coli after different treatments to determine the bactericidal effect of the gel.

[0098] like Figure 5As shown in Figure E, the morphology of S. aureus and E. coli treated with PBS, APF, and APF / C10 was normal, and the cell membrane was smooth and intact, indicating that their damage to the bacteria was negligible. However, with the addition of LL-37, the bacterial cell membrane became wrinkled and shrunk, especially when the concentration of LL-37 increased to 0.5%wt, the bacterial cell membrane was completely destroyed, and the bacteria were deformed or even ruptured.

[0099] These results all confirmed that APF / C10 / L-0.5 hydrogel has a significant bactericidal effect, so we selected APF / C10 / L-0.5 (hereinafter abbreviated as APF / C / L) hydrogel for subsequent experiments.

[0100] 4. Verification of the ability of hydrogels to promote cell proliferation and migration

[0101] Although the currently synthesized APF / C / L hydrogel has the effects of promoting blood coagulation, antibacterial and anti-inflammatory, it can play a huge role in the early stage of wound healing. However, due to its lack of biological activity, it cannot play a good role in the proliferation stage of wound repair. Therefore, we introduced adipose stem cell sheet extracellular matrix (ADSCs sheet dECM). It is rich in a variety of growth factors and nutrients, and can promote angiogenesis and matrix secretion in the later stage of wound healing.

[0102] We freeze-dried the decellularized ADSCs sheets and ground them into powder, and added them into hydrogels at different concentrations (5, 10, 20, 40, 80, 160 mg / ml) to detect the optimal concentration for promoting the replication and proliferation of adipose stem cells.

[0103] We named the different hydrogels APF / C / L and APF / C / L@dECM5-80, corresponding to APF / C10 / L-0.5 and APF / C10 / L-0.5@dECM5-80 mg / ml, respectively. Figure 7 (B) The ability of dECM powder to promote cell proliferation was evaluated, and the optimal dECM powder concentration was selected.

[0104] The results showed that when the concentration of ADSCs sheets dECM powder increased to 80 mg / ml (APF / C / L@dECM80), the ability of the hydrogel to promote cell proliferation tended to stabilize. The scratch test also confirmed that APF / C / L@dECM80 hydrogel can promote cell migration (see Figure 7 (A, C).

[0105] Therefore, we finally selected a hydrogel with a dECM powder concentration of 80 mg / ml (hereinafter abbreviated as APF / C / L@dECM) for subsequent experiments.

[0106] 5. Verification of the anti-inflammatory properties of hydrogels

[0107] 5.1 In vitro anti-inflammatory validation

[0108] We first cultured RAW264.7 cells in vitro to verify the anti-inflammatory properties of the hydrogel. We used 100 ng / ml LPS and 20 ng / ml IFN-γ to induce macrophage polarization. Flow cytometry results showed that after co-culturing the cells with different hydrogels, the proportion of CD86+M1 macrophages in the APF / C / L group and the APF / C / L@dECM group was significantly reduced, and the proportion of CD206+M2 macrophages was slightly increased (see Figure 8 (A, B).

[0109] WB results showed the same trend. In RAW264.7 cells co-cultured with APF / C / L and APF / C / L@dECM hydrogels, validation indicators such as CD86, IL-6, and IL-1β were significantly decreased (see Figure 8 (C), which indicates that APF / C / L and APF / C / L@dECM hydrogels can effectively inhibit the polarization of M0 macrophages to M1 and suppress the release of inflammatory factors.

[0110] 5.2 In vivo anti-inflammatory validation

[0111] Subsequently, we injected different groups of hydrogels into the area around the rabbit urethral injury, and immunofluorescence staining of urethral sections revealed that the urethral verification indexes in the APF / C / L and APF / C / L@dECM hydrogel treatment groups were significantly reduced (see Figure 8 (E, F), indicating that these two hydrogels also have excellent anti-inflammatory effects in rabbits due to the presence of LL-37 antimicrobial peptide.

[0112] 6. The effect of multifunctional all-in-one full-process repair hydrogel in preventing scar formation after urethral injury

[0113] Through the above experiments, we verified that APF / C / L@dECM hydrogel can exert antibacterial and hemostatic functions, as well as anti-inflammatory and cell proliferation and migration promoting effects.

[0114] Next, in vivo experiments will be conducted to verify the role of APF / C / L@dECM hydrogel in promoting wound repair and preventing scar tissue formation after urethral injury.

[0115] We used hemostats to clamp the urethra to injure the entire urethral tissue. This was used to simulate the urethra condition of patients with urethral injury in clinical settings. We then injected different groups of hydrogels into the injured urethral tissues around the rabbits, and performed histological examinations, immunofluorescence staining, and urethrography 4 weeks (4W) and 8 weeks (8W) after treatment to evaluate the therapeutic effects of each group of hydrogels.

[0116] As Fig. 9 As shown in A, gross morphology showed that obvious scar tissue was formed in the control group, APF group and APF / C group, leading to urethral stenosis and poor repair effect. The scar area in the APF / C / L group was relatively small, which may be due to the early anti-inflammatory effect of the LL-37 antimicrobial peptide contained in it. The urethral repair effect of the APF / C / L@dECM group was significantly better than that of the other groups. The repaired urethra was smooth without any hypertrophic scar formation.

[0117] We then performed urethrography on the rabbits. The results showed that the lumen of the control, APF, and APF / C groups was very narrow due to scar hyperplasia, and the lumen of the APF / C / L group was relatively wide, but still had a certain degree of obstruction rate (see Fig. 9 It is worth noting that compared with the other four groups, the APF / C / L@dECM group had the lowest obstruction rate and the highest maximum flow rate (Qmax), even close to the normal group, which further indicated that the use of this APF / C / L@dECM group to treat urethral trauma did not result in urethral scarring or urethral fistula formation (see Fig. 9 C and D). More importantly, the generated tissue was completely integrated with the adjacent normal tissue without obvious sense of boundary. No obvious implantation reaction or infection was observed during the sampling process, and the rabbits remained in good health.

[0118] Histological staining showed that the urethra healing in the control group, APF group and APF / C group was poor, the local tissue structure of the trauma was disordered, a large number of fibroblasts and collagen fibers were deposited, and thick hypertrophic scar tissue was formed (see Fig. 9 E and F). This indicates that APF polymer and fish collagen peptide alone do not promote wound healing and may even cause a certain rejection reaction. In contrast, although the APF / C / L group had less scar formation after 4 weeks of repair, showing a certain repair function, it still formed obvious scar tissue after 8 weeks, and Masson staining indicated obvious collagen deposition in the local area.

[0119] These results confirm that the procoagulant, antibacterial and anti-inflammatory effects of APF / C / L@dECM hydrogel in the early stage of wound repair and its role in promoting cell proliferation, migration and vascularization in the later stage of wound repair can promote the healing of rabbit urethral trauma and avoid the ultimate formation of scar tissue.

Claims

1. A method for preparing a urethral injury repair gel, characterized in that: include: 1) subjecting the PF127 hydrogel to terminal amination reaction to obtain an APF copolymer; 2) chemically cross-linking the APF copolymer and the procoagulant polypeptide to obtain a chemically cross-linked repair gel; Wherein, an inhibitor is added to the cross-linked gel, and the inhibitor includes an antibacterial agent and / or an anti-inflammatory agent.

2. The method according to claim 1, characterized in that: Synthesis conditions of step 1: fully dissolve PF127 in dichloromethane, add triethylamine and stir in an ice-water bath until fully mixed, inject methanesulfonyl chloride into the liquid twice with a syringe, react at room temperature for 2-24 hours, and after the reaction is completed, filter the reaction solution to remove solids, rotary distill until there is almost no sol, pour petroleum ether to obtain a solid, dry the solid and dissolve it in ammonia water, stir vigorously for 0.5-5 days, let stand until the foam disappears, extract with dichloromethane, and then use anhydrous magnesium sulfate to remove water from the dichloromethane phase to finally obtain APF polymer.

3. The method according to claim 1 or 2, characterized in that: Synthesis conditions of step 2: Add procoagulant polypeptide powder, 2% wt 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride powder and 2% wt N-hydroxysuccinimide powder to the APF solution for cross-linking reaction, and stir overnight at 4°C to obtain a chemically cross-linked repair gel.

4. The method according to claim 3, characterized in that The repair gel is further added with adipose stem cell membrane decellularized matrix, the procoagulant polypeptide is fish collagen peptide, and the inhibitor is LL-37 antimicrobial peptide; The repair gel is in a gel state at room temperature and in a sol state at 35-37°C.

5. A urethral injury repair gel, characterized in that: include: APF copolymer, procoagulant polypeptide and inhibitor, the AFP copolymer is a terminal amino PF127 hydrogel, the APF copolymer and the procoagulant polypeptide are chemically cross-linked to form a gel, the APF copolymer is a terminal amino PF127 copolymer; the inhibitor includes an antibacterial agent and / or an anti-inflammatory agent.

6. The urethra injury repair gel according to claim 6, characterized in that: The repair gel is also added with a decellularized matrix which can promote angiogenesis and matrix secretion in the later stage of healing.

7. The urethra injury repair gel according to claim 6, characterized in that: The decellularized matrix is ​​adipose stem cell membrane decellularized matrix, the procoagulant polypeptide is fish collagen peptide, and the inhibitor is LL-37 antibacterial peptide.

8. The urethra injury repair gel according to claim 6 or 7, characterized in that: In the repair gel: the procoagulant polypeptide accounts for 5-10wt%, the inhibitor concentration is 0.5wt%, and the decellularized matrix concentration is 80mg / ml.

9. Use of the urethra injury repair gel according to claim 5 in the preparation of materials for promoting urethra injury repair.

10. Use of the urethral injury repair gel according to claim 5 in the preparation of materials for promoting urethral angiogenesis, materials for promoting urethral epithelium regeneration, or materials for promoting urethral smooth muscle regeneration.

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