Photosensitive multifunctional tri-crosslinked hydrogel GM / OD-coated PRP, and preparation method and application thereof

Abstract

The invention discloses photosensitive multifunctional tri-crosslinked hydrogel GM / OD-coated PRP, a preparation method and application thereof, and relates to the technical field of hydrogel, and the preparation method comprises the following steps: (1) preparing methacrylated gelatin GM; (2) preparing oxidized dextran OD; (3) preparing platelet rich plasma (PRP); and (4) preparing the GM / OD-coated PRP tri-crosslinked hydrogel. In the invention, the GM can form an optical cross-linked network, the three-network composite hydrogel is formed by introducing OD, utilizing Schiff base reaction and combining amino groups in the GM and PRP, and the three-network composite hydrogel is used as a physical protection barrier to prevent digestive juice from overflowing and is used for preventing anastomotic fistula. According to the composite hydrogel, due to the addition of the PRP, the hydrogel is endowed with the capability of multiple growth factors at the same time, and the healing of an anastomotic stoma is promoted and the occurrence of anastomotic fistula is prevented by promoting cell proliferation, revascularization and vascularization. The GM / OD-coated PRP hydrogel has excellent bacteriostatic ability to staphylococcus aureus and escherichia coli, so that intestinal bacteria displacement can be prevented, and infection in the abdominal cavity is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a photosensitive multifunctional triple-crosslinked hydrogel GM / OD@PRP, a preparation method and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor, and surgical resection is the most common way to treat CRC. After removing the tumor part of the intestine, the intestinal structure and function are reconstructed by anastomosis of the two ends, but it is usually accompanied by the risk of various complications. Anastomotic leakage (AL) can cause digestive fluid to leak into the abdominal cavity, corrode surrounding organs, and cause secondary severe abdominal infection, pelvic abscess and bleeding, and even lead to death, seriously affecting the patient's long-term survival rate. After AL occurs, another surgery is required to prevent fistula formation at the end of the ileum, and a third surgery is performed to reconstruct the intestine after the anastomosis heals. Multiple surgeries can induce a systemic stress response activated by catecholamines and prostate hormone receptors, which may promote the growth of carcinogenic factors. Therefore, in order to improve the quality of life of patients, avoid multiple surgeries, and improve the cure rate, it is urgent to explore methods to prevent anastomotic leakage.

[0003] Hydrogel adhesives are non-traumatic, have fluid sealing capabilities, and have good wound healing abilities. Many studies have used innovative hydrogel properties for the treatment, repair, and detection of AL. However, due to the special environment of the intestine, biomaterials are not only required to have good healing abilities, but also to have certain antibacterial abilities. However, these two studies lack antibacterial experiments to prove their antibacterial properties. Barros et al. developed an injectable nano-silicon hydrochloric acid and gelatin-dopamine conjugate hydrogel for the treatment of AL. The material has significant mechanical stability, adhesion to host tissues, antibacterial properties, and high regenerative properties. It exhibits volume stability in a humid internal environment and maintains its strong adhesive strength. However, the prevention of AL requires not only hydrogels to have excellent antibacterial properties, biocompatibility, healing promotion, and certain mechanical properties, but also due to the particularity of the intestine, excessive adhesion is prone to adhesion, which leads to worsening of the abdominal cavity, which is not conducive to the prevention of AL.

[0004] Therefore, there is an urgent need to develop a hydrogel that can avoid both abdominal adhesion and abdominal infection and anastomotic fistula. Summary of the invention

[0005] In order to solve the above problems, the present invention discloses a photosensitive multifunctional three-cross-linked hydrogel GM / OD@PRP, a preparation method and an application thereof. Methacryloylated gelatin can form a photo-crosslinked network. By introducing oxidized dextran and utilizing Schiff base reaction, the amino groups in methacryloylated gelatin and platelet-rich plasma are combined to form a three-network composite hydrogel GM / OD@PRP, which serves as a physical protective barrier to prevent digestive fluid overflow and is used for the prevention of anastomotic fistula.

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

[0007] The first aspect of the present invention provides a method for preparing a photosensitive multifunctional triple-crosslinked hydrogel GM / OD@PRP, comprising the following steps:

[0008] (1) preparing methacryloyl gelatin GM;

[0009] (2) Preparation of oxidized dextran OD;

[0010] (3) Preparation of platelet-rich plasma (PRP);

[0011] (4) Preparation of GM / OD@PRP triple-crosslinked hydrogel.

[0012] Optionally, step (1) prepares methacrylic acid-treated gelatin GM, specifically comprising: adding gelatin to phosphate buffered saline at pH=5, stirring in a 50°C water bath until completely dissolved, to obtain a gelatin solution with a concentration of 10%; then slowly adding methacrylate to the gelatin solution, continuing to stir until the solution is completely mixed and turns milky white, diluting the resulting mixture with PBS, placing the diluted solution in a dialysis bag, and dialyzing it in deionized water at 50°C for 1 week, replacing the dialysis water every 6-8 hours to remove unreacted methacrylate; finally, freeze-drying the dialyzed solution and storing it at -20°C, adding a water-soluble photoinitiator, and gelling it under 365nm ultraviolet light to obtain methacrylic acid-treated gelatin GM.

[0013] Optionally, step (2) prepares oxidized dextran OD, specifically comprising: adding 5.0-10.0 g of dextran and 5.0-10.0 g of NaIO4 into 50-100 mL of deionized water, stirring continuously for 4 hours at room temperature in the dark, then adding 3 mL of ethylene glycol to the reaction solution and continuing stirring for 1 hour to quench unreacted NaIO4 in the solution; then, placing the reaction solution in a dialysis bag, and thoroughly dialyzing the reaction solution in deionized water for 5 days, replacing the dialysate every 4 hours; finally, freeze-drying the dialyzed solution and storing it at 4°C.

[0014] The substitution degree of oxidized dextran was determined by the hydroxylamine hydrochloride titration method. A 0.25 mol / L hydroxylamine hydrochloride solution containing 0.0002% methyl orange was mixed with 0.10 g OD, stirred at room temperature in the dark for 24 hours, and finally titrated with 0.10 mol / L NaOH. The OD substitution degree expression is:

[0015]

[0016] Wherein, V represents the volume of NaOH consumed (mL); c represents the concentration of NaOH (moL / L); and m represents the mass of OD (g).

[0017] Optionally, step (3) prepares platelet-rich plasma PRP, specifically comprising: anesthetizing SD rats (45 mg / kg) by intraperitoneal injection of sodium pentobarbital, and after the rats are completely anesthetized, extracting whole blood from the rat's abdominal aorta and transferring it to an anticoagulant reagent tube containing acid-citrate-glucose solution formula A (anticoagulant: blood = 1:4, v / v), then centrifuging the blood sample at 300 g for 7 minutes, transferring the supernatant to another tube, and then centrifuging at 1000 g for 5 minutes to obtain milky yellow platelet-rich plasma PRP from top to bottom, which is freeze-dried and stored at -80°C.

[0018] Optionally, step (4) prepares the GM / OD@PRP tri-crosslinked hydrogel, specifically comprising: dissolving GM in PBS and heating to dissolve at 50°C, dissolving OD and PRP directly in PBS, respectively, to obtain 15% (w / v) methacryloyl gelatin GM solution, 9% (w / v) OD solution and 25% (w / v) platelet-rich plasma PRP solution, respectively, mixing the three materials in an equal volume ratio, adding 0.5% (w / v) photoinitiator, and irradiating under 365nm ultraviolet light for 5-10 minutes to obtain the tri-crosslinked hydrogel GM / OD@PRP.

[0019] Optionally, the water-soluble photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0020] The second aspect of the present invention provides a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP, which is prepared by the above method.

[0021] The third aspect of the present invention proposes an application of the above-mentioned photosensitive multifunctional tri-crosslinked hydrogel GM / OD@PRP for preventing anastomotic fistula.

[0022] The beneficial effect of the present invention is that, in the present invention, GM can form a photo-coated network, and by introducing OD and utilizing the Schiff base reaction, the amino groups in GM and PRP are combined to form a three-network composite hydrogel, which serves as a physical protective barrier to prevent digestive fluid from overflowing and is used to prevent anastomotic fistula. Due to the addition of PRP, the composite hydrogel is endowed with the ability of multiple growth factors at the same time, which promotes the healing of the anastomosis by promoting cell proliferation, angiogenesis and angiogenesis, and prevents the occurrence of anastomotic fistula. The excellent antibacterial ability of GM / OD@PRP hydrogel against Staphylococcus aureus and Escherichia coli can prevent intestinal bacterial translocation and avoid the occurrence of intra-abdominal infection. In addition, the present invention verifies the effect of GM / OD@PRP hydrogel in preventing anastomotic fistula by establishing a rat colorectal anastomotic fistula model. The experimental results show that GM / OD@PRP hydrogel can avoid abdominal adhesion and abdominal infection, and successfully promotes anastomotic healing and avoids the occurrence of anastomotic fistula. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The preparation process and principle diagram of a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP;

[0024] Figure 2 The synthetic chemical structure and basic characterization performance test of the hydrogel of the present invention, wherein (A) is the synthesis step of the OD chemical structure; (B) is the synthesis step of the GM chemical structure; (C) is the Fourier transform infrared spectra (FTIR) of Dex and OD, gelatin and GM; (D) is the frequency scanning curve characterizing the rheological properties of different hydrogels; (E) is the swelling rate of GM, GM / OD and GM / OD@PRP; (F) is the degradation of GM, GM / OD and GM / OD@PRP hydrogels in simulated intestinal fluid;

[0025] Figure 3 Synthesis and self-healing test of GM / OD@PRP hydrogel, where (A) is the synthesis of GM / OD@PRP hydrogel; (B) is the gelation time of GM, GM / OD and GM / OD@PRP hydrogel; (C) is the pore size of GM, GM / OD and GM / OD@PRP hydrogel; (D) is the porosity of GM, GM / OD and GM / OD@PRP hydrogel; (E) is the self-healing test step of hydrogel; (F) is the adhesion strength of GM, GM / OD and GM / OD@PRP hydrogel;

[0026] Figure 4 In vitro antibacterial test of hydrogel, where (A) is a graph showing the inhibitory effect of hydrogel on S. aureus and E. coli; (B) is a bar graph showing the inhibitory effect of hydrogel on S. aureus and E. coli; (C) is a morphological graph of S. aureus treated with GM / OD@PRP hydrogel;

[0027] Figure 5 The biocompatibility test of the hydrogel, where (A) is the live / dead staining test result, (B) is the cell viability; (C) is the scratch test of the cell migration ability of the hydrogel; (D) is the cell migration rate; (E) is the cytotoxicity test of the hydrogel; (F) is the blood compatibility test;

[0028] Figure 6 Animal experiments and pathological tissue section analysis, among which (A) is the healing of colorectal anastomotic fistula in rats; (B) is the pathological section, (C) is the quantitative data analysis of Masson staining; (D) is the score result of abdominal adhesion in Beixiu glue group and GM / OD@PRP hydrogel group; (E) is the schematic diagram of intestinal burst test at the anastomotic site in Beixiu glue group and GM / OD@PRP hydrogel group; (F) is the burst pressure result of Beixiu glue group and GM / OD@PRP hydrogel group; Figure 7 Figure 2 is an immunohistochemical analysis, where (A) is an immunohistochemical analysis diagram, (B) is the relative value of VEGF in Beixiu glue group and GM / OD@PRP hydrogel group; (C) is a comparison of vascular area between Beixiu glue group and GM / OD@PRP hydrogel group; (D) is the relative value of B-FGF in Beixiu glue group and GM / OD@PRP hydrogel group; (E) is the expression of IL-6 in Beixiu glue group and GM / OD@PRP hydrogel group; (F) is the expression of TNF-α in Beixiu glue group and GM / OD@PRP hydrogel group. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Experimental Materials

[0032] Gelatine (type A, from pig skin) was provided by McLean Biochemical Co., Ltd., Shanghai, China; methacrylic anhydride and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; dextran (Mw=20 kDa) and sodium periodate (NaIO4) were provided by Shanghai Yuanye Biotechnology Co., Ltd.; and ethylene glycol was provided by Sinopharm Chemical Reagent Co., Ltd. Platelet-rich plasma (PRP) was collected from centrifuged whole blood drawn from the abdominal aorta of SD rats provided by Suzhou Xishan Biotechnology Co., Ltd., Jiangsu Province. Modified tryptone soy broth (mTSB broth), LB broth and agar powder were provided by Beijing Solebow Technology Co., Ltd. Live / dead cell staining kit was provided by Dalian Meilun Biotechnology Co., Ltd., and cell counting kit-8 (CCK-8) kit was provided by Shanghai McLean Biochemical Technology Co., Ltd. All chemical reagents were of analytical grade and used without further purification.

[0033] A method for preparing a photosensitive multifunctional triple cross-linked hydrogel GM / OD@PRP, such as Figure 1 As shown, the following steps are included:

[0034] (1) Preparation of methacryloyl gelatin GM

[0035] Specifically, the process includes: adding 10 g of gelatin to 100 mL of phosphate buffered saline (pH = 5), stirring in a 50°C water bath until completely dissolved, and obtaining a gelatin solution with a concentration of 10%; then slowly adding 6 mL of methyl methacrylate MA to the gelatin solution, continuing to stir until the solution is completely mixed and turns milky white, diluting the resulting mixture with 200 mL of PBS, placing the diluted solution in a dialysis bag (14 kDa, Jelupu), and dialyzing in deionized water at 50°C for 1 week, replacing the dialysis water every 6-8 hours to remove unreacted methacrylate; finally, freeze-drying the dialysis solution and storing it at -20°C.

[0036] (2) Preparation of oxidized dextran OD

[0037] Specifically, 5.0 g of dextran and 5.0 g of NaIO4 were added into 50 mL of deionized water, and the mixture was stirred continuously for 4 hours at room temperature in the dark. Then, 3 mL of ethylene glycol was added into the reaction solution and the stirring was continued for 1 hour to quench the unreacted NaIO4 in the solution. Subsequently, the reaction solution was placed into a dialysis bag (8 kDa, Jelupu), and the reaction solution was dialyzed thoroughly in deionized water for 5 days, and the dialysate was replaced every 4 hours. Finally, the dialyzed solution was freeze-dried and stored at 4°C.

[0038] The substitution degree of oxidized dextran was determined by the hydroxylamine hydrochloride titration method. A 0.25 mol / L hydroxylamine hydrochloride solution containing 0.0002% methyl orange was mixed with 0.10 g OD, stirred at room temperature in the dark for 24 hours, and finally titrated with 0.10 mol / L NaOH. The OD substitution degree expression is:

[0039]

[0040] Wherein, V represents the volume of NaOH consumed (mL); c represents the concentration of NaOH (moL / L); and m represents the mass of OD (g).

[0041] (3) Preparation of platelet-rich plasma (PRP)

[0042] Specifically, the method includes: taking blood from SD rats (weight ≥ 250 g), anesthetizing the SD rats (45 mg / kg) by intraperitoneal injection of sodium pentobarbital, and after the rats are fully anesthetized, drawing whole blood from the rat's abdominal aorta and transferring it to an anticoagulant reagent tube containing acid-citrate-glucose solution formula A (anticoagulant: blood = 1:4, v / v), and then centrifuging the blood sample at 300 g for 7 minutes, transferring the supernatant to another tube, and subsequently centrifuging at 1000 g for 5 minutes to obtain milky yellow platelet-rich plasma PRP from top to bottom, which is freeze-dried and stored at -80°C.

[0043] (4) Preparation of GM / OD@PRP triple cross-linked hydrogel

[0044] Specifically, GM was dissolved in PBS and heated at 50°C to dissolve, OD and PRP were directly dissolved in PBS to obtain 15% (w / v) methacryloyl gelatin GM solution, 9% (w / v) OD solution and 25% (w / v) platelet-rich plasma PRP solution, respectively, the three materials were mixed in an equal volume ratio, 0.5% (w / v) photoinitiator I2959 was added, and the mixture was irradiated under 365nm ultraviolet light for 5-10 minutes to obtain a triple-crosslinked hydrogel GM / OD@PRP.

[0045] Example 2

[0046] The photosensitive multifunctional tri-crosslinked hydrogel GM / OD@PRP prepared in Example 1 was used for the following tests.

[0047] (I) Surface chemistry and morphology characterization

[0048] 1. OD and GM infrared test

[0049] The FTIR spectra of Dex, OD, Gtn and GM were recorded by Fourier transform infrared spectrometer to determine whether the grafting was successful. The freeze-dried samples of Dex, OD, Gelatin and GM were placed in the FT-IR measurement point, and the spectrum scanning range was: 7800-380cm -1 Each sample was analyzed 32 times with a resolution of 2 cm -1 The scan completes the data collection.

[0050] In order to observe the microscopic characteristics of the material, the hydrogels GM, GM / OD and GM / OD@PRP were synthesized and freeze-dried for 12 hours. The hydrogels were cut open with a blade to expose the cross-section and fixed. The images were imaged using a field emission scanning electron microscope at an accelerating voltage of 15KV. Before imaging the hydrogels GM, GM / OD and GM / OD@PRP, a 10-nanometer gold-palladium alloy film was coated on the sample surface using a sputtering coating technique to enhance the conductivity. The images were continuously magnified from 25X and acquired in 8-bit grayscale mode.

[0051] 2. Antibacterial test

[0052] In this example, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used to verify the antibacterial properties of the hydrogel. The three groups of prepared hydrogels (GM, GM / OD and GM / OD@PRP) and PBS (negative control) were added. Positive control) After incubating with bacterial suspension, S. aureus and E. coli in equal volume ratio at 37°C for 24 hours, the bacterial suspension was evenly spread on solid agar medium containing nutrient broth and cultured for another 18 hours for observation; the bacterial suspension was then diluted to 10 -6 Then take 20uL of bacterial suspension to coat the plate; after culturing in a 37℃ bacterial incubator for 18-24 hours, count the bacterial colonies (CFU) generated on the culture dish to calculate the antibacterial rate, and repeat three times for each group. The calculation formula of the antibacterial experiment is as follows:

[0053]

[0054] Among them, B is the CFU of the control sample, and C is the CFU of the experimental group.

[0055] 20uL S. aureus bacterial suspension was dropped onto the sterile hydrogel surface and incubated at 37°C for 24 hours. It was then treated with 2.5% glutaraldehyde for 12 hours and dehydrated using a gradient ethanol method (25%, 50%, 75%, 100%) for 15 minutes. The morphology of the bacteria on the hydrogel was then observed using SEM.

[0056] 3. Biocompatibility test

[0057] After the hydrogel extract and cells were co-cultured at 37°C for 24 hours, the in vitro biocompatibility of the hydrogel was evaluated by cell counting kit (CCK-8), hemolysis test, cell scratch test and live / dead staining test. First, in this embodiment, all biocompatibility experiments were completed using mouse fibroblasts (L929 cells). After the prepared hydrogel solution was irradiated with UV365 nm to generate a gel, it was irradiated with ultraviolet light for 30 minutes for sterilization. Then, the hydrogel was immersed in DMEM cell culture medium at a ratio of 1:2 (W / V) for 8 hours to obtain an extract. The hydrogel extract was co-cultured with cells. The CCK-8 experiment was carried out in a 96-well sterile plate. L929 cells (4×10 3 The cells were inoculated with complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, placed in a cell culture incubator at 5% CO2 and 37°C for 24 hours, and the DMEM cell culture medium of the hydrogel group was replaced with the hydrogel extract, and the complete culture medium group not treated with the hydrogel extract was the control group. The absorbance at 450 nm was then measured using an ELISA reader on days 1, 3, and 5.

[0058] About 5×10 5 After 24 hours of inoculation of L929 cells on a 6-well plate containing 2 mL of complete cell culture medium, the L929 cells showed a spindle morphology and adhered to the wall. The required culture medium solution was replaced according to the grouping, and then co-cultured for 1 and 3 days, respectively. The hydrogel GM / OD was detected by tetramethylrhodamine colorimetry, and the distribution of live and dead cells in GM / OD@PRP and the blank control group was observed under an inverted fluorescence microscope.

[0059] For cell scratching, after L929 cells completely cover each well plate, use a 200uL pipette tip to scratch along the midline, then add culture medium and culture medium for co-culture with hydrogel according to the group, place in a cell culture incubator and culture for 1 day and 2 days respectively, and observe the cell growth and migration under an electron microscope.

[0060] In the cell hemolysis experiment, 200uL of rat diluted blood (20%) was added to four 1.5mL EP tubes, and then 800uL of double distilled water, PBS, GM / OD extract and GM / OD@PRP extract were added respectively. After incubation at room temperature for 1 hour, the tubes were centrifuged at 1500g for 5 minutes, and the absorbance of the supernatant at 540nm was recorded with an ELISA reader. Then, the hemolysis rate was calculated according to the formula:

[0061]

[0062] (B) Establishment of anastomotic fistula model.

[0063] Thirty-three 8-week-old male SD rats were divided into three groups, with 11 samples in each group. The control group was subjected to normal surgical suture; the positive control group was subjected to the experimental group using Beixiu Glue and GM / OD@PRP. The rats were kept in an environment with suitable temperature and humidity for one week before animal modeling. They were fasted for 8-12 hours before surgery. After intraperitoneal injection of 1% sodium pentobarbital (45 mg / kg), the abdomen of the rats was prepared and disinfected, and then the abdomen was opened. Mesenteric vascular ligation and colon transection were performed at about 2-3 cm above the peritoneal fold, followed by interrupted suture of the broken ends. After the operation, the rats were kept for 2 weeks, during which the mental state, diet, and defecation of the rats were observed, and laparotomy was performed on the 5th, 10th, and 15th days after surgery to observe the healing of the anastomosis.

[0064] The anastomosis and the intestinal segments 2 cm above and below were taken for burst pressure testing and histological pathological analysis on the 10th day of the control group, the positive control group and the experimental group on the 15th day. The collected tissue samples were fixed with 4% paraformaldehyde solution and embedded in paraffin. The tissue sections were prepared into 3 μm thick sections and stained with hematoxylin-eosin (H&E) and Masson's trichrome to determine the tissue morphological changes of the samples at different healing stages and the collagen metabolism in the regenerated tissue. In addition, immunohistochemistry was used to detect the expression of VEGF, IL-6, TNF-α, CD31 and B-FGF at the anastomosis. All animal experiments in this study passed the animal welfare ethics requirements of Qingdao University (NO.20231125SD3320240205146).

[0065] (III) Results and Discussion

[0066] 1. Preparation and mechanical properties of GM and OD

[0067] The aldehyde functional group of OD was prepared by redox reaction with sodium periodate (NaIO4) (e.g. Figure 2 A), the substitution degree of OD calculated by hydroxylamine hydrochloride colorimetry is 69.3% ± 10%. GM is synthesized by introducing methacryloyl substituents to the active amines and hydroxyls on gelatin. The chemical structure is as follows Figure 2 As shown in B. Figure 2 As shown in C, in the FTIR results, the OD at 2130 cm-1 is higher than that of Dex. -1 The carbon-oxygen double bond stretching peak appeared at 3120 cm-1, which confirmed that a large number of alcohol hydroxyl groups in Dex were successfully oxidized to aldehyde groups by sodium periodate. -1The hydrogen-oxygen bond stretching peak appears at , indicating that the methacrylic acid group is successfully grafted onto the gelatin. Storage modulus (G'), also known as elastic modulus, is the modulus stored due to elastic reversible deformation when the material is deformed; loss modulus (G"), also known as viscous modulus, refers to the energy lost by the material due to the inability to deform viscously when it is deformed. Figure 2 As shown in D, when the shear frequency is in the range of 0.1-100 rad / s, the storage modulus of the hydrogel is higher than the loss modulus (G′>G″), indicating that the hydrogel has good stability and mechanical properties. Figure 2 As shown in Figure E, the swelling performance of GM in PBS is relatively stable, with a swelling rate of 42±2.6%. Thanks to the fact that the aldehyde functional group on OD can react with the amino group of GM to form a Schiff base reaction, it can also form a cross-linked network with the amino group of PRP. Therefore, with the addition of OD and PRP in the GM / OD@PRP group, the cross-linked network of the hydrogel in this group increased significantly, thereby reducing the swelling rate by 15.8±3.7% compared with the GM group. This is conducive to the adhesion strength of the hydrogel in this group when it adheres to the anastomosis, and will not be weakened by excessive swelling of the hydrogel. In the swelling experiment, the amount of liquid absorbed by the GM / OD@PRP group was slightly less than that of the GM / OD group, which also indirectly confirmed that the addition of PRR formed a third cross-linked network formed by OD and PRP in addition to the photo-cross-linked network formed by GM itself and the Schiff base network of GM / OD.

[0068] Degradable biomaterials can be absorbed by the human body or completely excreted from the body, and will not accumulate in the internal tissues or organs of the human body, so they have better tissue compatibility. Therefore, we tested the degradability of the three groups of hydrogels, GM, GM / OD and GM / OD@PRP, by simulating in vitro degradation experiments ( Figure 2 F). The hydrogel was developed to prevent the occurrence of AL after colorectal cancer surgery, so simulated intestinal fluid was selected as the digestive fluid. From the results, it can be seen that although the degradation rate of the simple GM hydrogel group was about 2 days faster, the corresponding durability of preventing AL would be reduced. With the addition of PRP, a component that promotes anastomotic healing, the degradation time was relatively prolonged on the basis of accelerating anastomotic healing, and the occurrence of AL was further prevented under the dual measures.

[0069] 2. Synthesis and self-healing properties of GM / OD@PRP hydrogel

[0070] like Figure 3 As shown in A, GM is placed under 365nm UV to irradiate CC bonds to undergo free radical polymerization to form the first cross-linked network. After GM is mixed with OD, a Schiff base reaction can occur to form the second cross-linked network. The addition of PRP, the amino group in it can also react with OD to form a third cross-linked network, thereby forming an interpenetrating triple network hydrogel. Figure 3As shown in Figure B, the average gelation time of GM hydrogel under 365nm UV irradiation is about 240±23s. Because the addition of OD can react with GM to form a Schiff base, its gelation time is significantly accelerated. However, the addition of PRP makes the hydrogel turn light pink, which affects the penetration of ultraviolet light, resulting in a slower gelation time for GM / OD@PRP hydrogel than the other two groups. The microscopic morphology of each group of hydrogels was observed by SEM. With the addition of OD and PRP, the pore size of the hydrogel became more and more dense ( Figure 3 C). This is because with the addition of OD and PRP, on the basis of the GM free radical polymerization network, the second cross-linking network of GM and OD and the third cross-linking network of OD and PRP are formed, which gradually increases the cross-linking density of GM / OD@PRP hydrogel and reduces the porosity and pore size of the hydrogel ( Figure 3 D), which is consistent with the previous swelling results, and the mechanical strength of the hydrogel is correspondingly strengthened. The results show that the addition of OD and PRP can enhance the crosslinking density of the hydrogel, making the GM / OD and GM / OD@PRP hydrogels more stable, which is also consistent with the degradation experiment results. In order to analyze the self-healing property of the hydrogel, the hydrogel was split and spliced ​​together for 60 minutes, and it can be observed that the formed hydrogel can maintain its integrity well under its own gravity ( Figure 3 E). The self-repairing ability of GM / OD@PRP hydrogel is due to the dynamic covalent bonds of Schiff bases formed by the amino groups on GM and PRP and the aldehyde groups on OD to rebuild the network. The prevention of anastomotic leakage by GM / OD@PRP hydrogel requires that it can adhere tightly to the serous membrane at the anastomosis and meet the requirements of being a blocking barrier under normal intestinal pressure of 12-15 mmHg. Therefore, we used shear adhesion to test whether the adhesion strength of each group of hydrogels can meet the requirements of the blocking barrier. From the results, it can be seen that ( Figure 3 F) With the addition of OD and PRP, the shear force of the hydrogel increased significantly, both greater than the normal intestinal pressure, and could adhere tightly to the anastomosis to prevent intestinal contents from overflowing, corroding surrounding organs, and causing abdominal infection.

[0071] 3. In vitro antibacterial effect of hydrogel

[0072] AL is the most life-threatening complication that is prone to occur after colorectal cancer surgery, because the host's immunity is reduced due to the impact of the surgery. If the intestinal microbial flora is imbalanced and displaced, abdominal infection is very likely to occur. In such a healing environment, it is necessary for the hydrogel to have a certain antibacterial property to resist bacterial invasion, which is a necessary condition for preventing AL hydrogel. During intestinal anastomosis surgery, trace amounts of bacteria in the intestine will migrate into the abdominal cavity through the anastomosis, causing infection, which in turn affects the healing of the anastomosis and the overall patient recovery process. Therefore, the barrier material covering the anastomosis should not only block the displacement of bacteria, but also kill the displaced bacteria, thereby avoiding the occurrence of abdominal infection. Therefore, the hydrogel used to prevent anastomotic fistula should have a certain antibacterial property. In this example, the two most common pathogens, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, were used to determine the antibacterial activity of the hydrogel. From Figure 4 A It can be seen that the commercial material Prandtl has excellent antibacterial properties in both Staphylococcus aureus and Escherichia coli, and all bacteria are killed. GM itself also has a certain antibacterial ability, although not as good as commercial materials, but after the introduction of OD and PRP, the antibacterial ability has been significantly improved, and the antibacterial effect is comparable to that of commercial materials. Figure 4 The quantitative data in Figure B show that the antibacterial activity of GM / OD@PRP hydrogel is similar to that of commercial materials. There is no difference, and the GM / OD@PRP composite hydrogel has excellent inhibitory ability against both S. aureus and E. coli. In order to better observe the antibacterial mechanism of GM / OD@PRP hydrogel, the morphology of S. aureus treated with GM / OD@PRP hydrogel was observed by SEM. It can be seen that the cell wall of S. aureus co-incubated with GM / OD@PRP was directly destroyed ( Figure 4 C), which is attributed to GM / OD@PRP hydrogel. The amino groups on GM can bind to the bacterial cell wall, causing dysfunction of the bacterial cell wall and achieving a certain antibacterial effect; OD can trigger the body's immune system and enhance the body's immune response to achieve an antibacterial effect; PRP is a good antibacterial agent, containing a variety of antimicrobial peptides, which can exert its natural antibacterial effect and has antibacterial effects on a variety of Gram-negative and Gram-positive bacteria. GM / OD@PRP hydrogel can not only act as a barrier to prevent intestinal flora from shifting into the abdominal cavity through the anastomosis, but also its excellent broad-spectrum antibacterial properties can effectively kill the already shifted flora, avoid the occurrence of abdominal cavity infection, and create a suitable microenvironment for anastomotic healing.

[0073] 4. Biocompatibility of hydrogels

[0074] Biocompatibility is crucial for any hydrogel that needs to be used in the biomedical field. Therefore, the biocompatibility of GM / OD@PRP hydrogel was tested by live / dead staining experiment, cell scratch test, cytotoxicity test and hemolysis test. The results of live / dead staining experiment are shown in Figure 2. Figure 5 As shown in A, L929 cells were almost completely stained green (live cells), and a few cells were stained red (dead cells). As the co-incubation time increased, it was observed that the number of live cells on the third day increased significantly compared with the first day. The cell survival rate of each group of cells in the live / dead staining was higher than 90% on the first and third days, and the live cell survival rate of GM / OD@PRP hydrogel was significantly higher than that of the blank control group and GM / OD group ( Figure 5 B), it can be seen that GM / OD@PRP hydrogel has good cell biocompatibility. Scratch test was used to evaluate the ability of each group of hydrogels to promote cell migration. Scratch test ( Figure 5 C) The cell migration ability of GM / OD@PRP hydrogel was significantly higher than that of the control group and GM / OD hydrogel group and control group. The migration rates of GM, GM / OD and GM / OD@PRP hydrogel on the first and third days were 8.84±1.05%, 11.58±1.71%, 35.27±1.85% and 21.56±2.43%, 20.65±1.67%, 45.99±0.53%, respectively. It can be seen that under the same conditions, the migration degree and proliferation ability of cells in GM / OD@PRP hydrogel are significantly superior to those in GM and GM / OD hydrogel groups, which is beneficial to promote anastomotic healing and treatment ( Figure 5 D). In the cell proliferation CCK-8 assay ( Figure 5 E), GM / OD hydrogel and GM / OD@PRP hydrogel had no obvious cytotoxicity to L929 cells on the first day; on the third day, the cell survival rate of GM / OD@PRP hydrogel was higher than that of the control group and GM / OD hydrogel group, which was attributed to the fact that PRP is rich in various growth factors, which is beneficial to promote cell proliferation; on the fifth day, it can be observed that there is no difference in cytotoxicity between the control group and the GM / OD@PRP hydrogel group. The hemolysis experiment can intuitively observe the direct effect of each group of hydrogels on cells. We used distilled water as the positive control group and PBS as the negative control group. The final results showed that GM / OD@PRP hydrogel would not damage the red blood cell wall and had excellent blood compatibility ( Figure 5 F).

[0075] 5. Animal experiments and pathological tissue section analysis

[0076] In order to further verify the preventive effect of GM / OD@PRP hydrogel on anastomotic fistula, a rat colorectal anastomotic fistula model was designed in this example. The rats were divided into a blank group (negative control), a GM / OD@PRP hydrogel group (experimental group) and There are three groups (positive control). Figure 6 As shown in A, on the 10th day, anastomotic fistula occurred at the surgical suture site of the rats in the control group, and feces leaked into the abdominal cavity, resulting in abdominal infection and death of the rats. The healing conditions of the GM / OD@PRP hydrogel group and the Bioseal hydrogel group were significantly better than those of the negative control group. The anastomosis healed well, and feces could pass through the suture smoothly without leakage. On the 15th day, there was no adhesion or damage at the anastomosis in the GM / OD@PRP hydrogel and Bioseal groups, and the healing was good. The mental state, diet, and bowel movements of the rats returned to the state before the operation. This shows that GM / OD@PRP hydrogel and Bioseal can effectively prevent the occurrence of AL. This example also evaluates the repair effect of GM / OD@PRP hydrogel through histological analysis of the anastomosis. As shown Figure 6 As shown in pathological sections B, the colon of the experimental site was taken for H&E staining and Masson staining on the 10th day after the death of the negative control group and the 15th day of the experiment of the Beixiu gel and GM / OD@PRP hydrogel groups. In the H&E staining, it can be observed that the GM / OD@PRP hydrogel and Beixiu gel groups have fewer neutrophils and the inflammation is better than that of the negative control group. Figure 6 In the quantitative data analysis of Masson staining of C, it was observed that the Masson staining collagen deposition rate of the Beixiu gel group and the GM / OD@PRP hydrogel group was significantly higher than that of the negative control group. The strength of the anastomosis depends on the production of fibroblasts and collagen networks. The GM / OD@PRP hydrogel group has a large number of collagen fiber networks, which can enhance the mechanical capacity of the anastomotic site and resist the increase in pressure around the anastomosis caused by defecation, playing an important role in preventing the occurrence of AL. Figure 6 As shown in D, the abdominal adhesion scores of the Beixiu gel group and the GM / OD@PRP hydrogel group were better than those of the negative control group. The higher the possibility of anastomotic leakage at the surgical site, the more serious the abdominal adhesion and abdominal infection will be. The occurrence of anastomotic leakage can cause severe abdominal adhesion, resulting in a large area of ​​intestinal tract with difficulty in intestinal peristalsis due to adhesion, which is consistent with the results of animal experiments. The rats in the negative control group had anastomotic leakage rupture on the 10th day, resulting in severe infection and death of the rats. Figure 6As shown in E and F, the intestinal tract at the anastomosis site was subjected to a burst test on the negative control group, Beixiu gel group and GM / OD@PRP hydrogel group 14 days after surgery. It can be observed that the GM / OD@PRP hydrogel group can withstand a higher burst pressure, which is consistent with the Masson score results of the pathological section. GM / OD@PRP hydrogel effectively enhances the mechanical strength of the anastomosis site.

[0077] 6. Immunohistochemistry

[0078] In order to further clarify the mechanism of GM / OD@PRP hydrogel in promoting anastomotic healing, immunohistochemical analysis was performed in this example ( Figure 7 A). Vascular endothelial growth factor (VEGF) is a factor that promotes angiogenesis and can enhance the generation of new blood vessels in existing vascular networks. It plays an important role in wound healing and platelet-endothelial cell adhesion molecule (CD31) is the most abundant membrane glycoprotein constitutively expressed on the vascular endothelium. Its participation in the stimulation site of active vascular endothelial cells is particularly important for maintaining homeostasis in the circulation. It can also remove aging neutrophils in the body and can be used together with VEGF to evaluate the formation of blood vessel shape. Figure 7 As shown in B and C, the VEGF expression in the Beixiu gel group and the GM / OD@PRP hydrogel group was higher than that in the negative control group. The experimental results show that the hydrogel helps the formation of blood vessels and provides basic nutritional conditions for promoting wound healing. The good expression of CD31 in the GM / OD@PRP hydrogel group is conducive to the healing and formation of blood vessels, which can promote the repair of tissue cells and wound healing. This experimental result is similar to the expression of VEGF. Basic fibroblast growth factor (B-FGF) is an angiogenesis inducer other than VEGF, and can also stimulate cell proliferation and migration ( Figure 7 D) IL-6 is a pleiotropic pro-inflammatory factor and a key factor in chronic inflammation and autoimmune diseases. It can secrete antibodies to participate in inflammatory responses, such as Figure 7 E. The GM / OD@PRP hydrogel group had lower IL-6 expression than the negative control group and Beixiu gel group. The occurrence of inflammation is not conducive to wound healing. The hydrogel can effectively reduce the expression of IL-6, providing a good environment for promoting wound healing and preventing the occurrence of AL. Tumor necrosis factor (TNF-α) is a member of the TNF / TNFR cytokine superfamily, involved in the maintenance and stability of the immune system, inflammation and host defense, and is an inflammatory cytokine produced by macrophages / monocytes during acute inflammation. Figure 7F, GM / OD2PRP hydrogel has the lowest expression of TNF-α, which plays a core role in initiating the inflammatory response of the innate immune system. It is essential for effective and controlled inflammatory response and is the pioneer of the inflammatory cytokine cascade. High expression of TNF-α can cause chronic inflammation in the body. In summary, the comprehensive evaluation score of GM / OD@PRP hydrogel is excellent.

[0079] GM contains arginine-glycine-aspartic acid (RGD) sequence and metalloproteinase (MMP) sensitive sequence, which is similar to the natural extracellular matrix (ECM) and has good biocompatibility. It can be used as a material for tissue regeneration and is conducive to cell adhesion. Dex is a biodegradable and biocompatible biomaterial that can promote tissue regeneration and angiogenesis. Dex is easily oxidized to OD, in which the hydroxyl group can be converted into an aldehyde group. The aldehyde group has strong chemical reactivity and can react with many functional groups on drugs and chemical reagents. OD and the amino group of GM can react with Schiff base to create an interpenetrating polymer network, which is used to enhance external energy dissipation and reduce the degradation rate of the hydrogel, which is conducive to prolonging the degradation time of the anastomotic protective barrier. GM is in a liquid state at body temperature and cross-linked into a gel under the action of Schiff base and ultraviolet light, which can better fill and close irregular anastomosis. PRP is rich in a variety of growth factors, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor, epidermal growth factor (EGF) and transforming growth factor-β (TGF-β), which can promote cell repair, angiogenesis and proliferation of fibroblasts, thereby promoting cell repair. PRP is also a good antibacterial agent that can release a variety of antimicrobial peptides and bioactive factors to exert its natural antibacterial effects in a variety of ways. It has antibacterial effects on a variety of Gram-negative and Gram-positive bacteria and can help treat / prevent tissue infections. At present, the application of PRP has become a very promising adjuvant therapy.

[0080] A three-crosslinked network hydrogel is formed by GM free radical polymerization, GM / OD Schiff base reaction and OD / PRP Schiff base reaction as a protective barrier to prevent AL. The three networks of GM / OD@PRP hydrogel not only increase the structural stability of the hydrogel, but also slow down its degradation rate and increase the durability of its barrier function. The excellent physical barrier function and antibacterial ability of GM / OD@PRP hydrogel can block the overflow of digestive fluid in the intestine, kill displaced bacteria, prevent corrosion of surrounding organs and abdominal infection, and provide a relatively sterile and suitable environment for anastomotic healing. Combined with the ability of GM / OD@PRP hydrogel to promote cell migration, control inflammation and promote angiogenesis, the healing of the anastomosis is accelerated, fundamentally solving the problem of anastomotic fistula. Finally, the ability to prevent anastomotic fistula was confirmed by the rat colorectal anastomotic fistula model. Surprisingly, GM / OD@PRP hydrogel reduced local inflammation, thereby preventing the occurrence of abdominal adhesion. In summary, the GM / OD@PRP hydrogel developed by the present invention is a new choice for preventing anastomotic fistula.

[0081] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP, characterized in that: The steps include: (1) preparing methacryloyl gelatin GM; (2) Preparation of oxidized dextran OD; (3) Preparation of platelet-rich plasma (PRP); (4) Preparation of GM / OD@PRP triple-crosslinked hydrogel.

2. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 1, characterized in that: Step (1) preparing methacrylated gelatin GM, specifically comprising: adding gelatin to phosphate buffered saline with a pH of 5, stirring in a 50° C. water bath until completely dissolved, to obtain a gelatin solution with a concentration of 10%; then slowly adding methacrylate to the gelatin solution, continuing to stir until the solution is completely mixed and turns milky white, diluting the resulting mixture with PBS, placing the diluted solution in a dialysis bag, and dialyzing in deionized water at 50° C. for 1 week, replacing the dialysis water every 6-8 hours to remove unreacted methacrylate; finally, freeze-drying the dialysis solution and storing it at -20° C.

3. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 2, characterized in that: Step (2) preparing oxidized dextran OD specifically comprises: adding dextran and NaIO4 together into deionized water, stirring continuously for 4 hours at room temperature in the dark, then adding ethylene glycol into the reaction solution and stirring continuously for 1 hour to quench unreacted NaIO4 in the solution; then, placing the reaction solution into a dialysis bag, and dialyzing the reaction solution thoroughly in deionized water for 5 days, replacing the dialysate every 4 hours; finally, freeze-drying the dialyzed solution and storing it at 4°C.

4. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 3, characterized in that: Step (3) preparing platelet-rich plasma PRP specifically comprises: anesthetizing SD rats by intraperitoneal injection of sodium pentobarbital, drawing whole blood from the rat's abdominal aorta after the rat is completely anesthetized, transferring the blood sample to an anticoagulant reagent tube, and then centrifuging the blood sample at 300 g for 7 minutes, transferring the supernatant to another tube, and then centrifuging at 1000 g for 5 minutes to obtain milky yellow platelet-rich plasma PRP from top to bottom, which is freeze-dried and stored at -80°C.

5. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 4, characterized in that: Whole blood was drawn from the abdominal aorta of rats and transferred into an anticoagulant reagent tube containing acid-citrate-dextrose solution formula A, with the volume ratio of anticoagulant to blood being 1:

4.

6. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 5, characterized in that: Step (4) preparing the GM / OD@PRP triple cross-linked hydrogel, specifically comprising: dissolving methacryloyl gelatin GM in PBS and heating to dissolve at 50°C, directly dissolving oxidized dextran OD and platelet-rich plasma PRP in PBS, respectively, to obtain 15% (w / v) methacryloyl gelatin GM solution, 9% (w / v) oxidized dextran OD solution and 25% (w / v) platelet-rich plasma PRP solution, respectively, mixing the three materials in an equal volume ratio, adding 0.5% (w / v) photoinitiator, and irradiating under 365nm ultraviolet light for 5-10 minutes to obtain the triple cross-linked hydrogel GM / OD@PRP.

7. The method for preparing a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP according to claim 6, characterized in that: The water-soluble photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

8. A photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. An application of a photosensitive multifunctional three-crosslinked hydrogel GM / OD@PRP, characterized in that: The hydrogel GM / OD@PRP described in claim 8 is used to prevent anastomotic fistula.

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