Preparation and application of tea polyphenol cross-linked decellularized diaphragm tendon patch

Abstract

This invention provides a method for preparing and applying a tea polyphenol-crosslinked decellularized diaphragmatic tendon. The tendon is formed by decellularizing porcine diaphragmatic tendon with 1% TNBP and then crosslinking it with tea polyphenols. The tea polyphenol-crosslinked decellularized diaphragmatic tendon can downregulate the expression of apoptosis and inflammatory genes and upregulate the expression of antioxidant genes, exhibiting strong antioxidant properties, mechanical properties, anti-enzymatic properties, and low immunogenicity. It can enhance the antioxidant capacity of cardiomyocytes, promote myocardial tissue vascularization and inhibit fibrosis, inhibit ventricular dilation, improve fibrosis, and promote angiogenesis. It can be used in the preparation of patches for treating myocardial infarction. The tea polyphenol-crosslinked decellularized diaphragmatic tendon provided by this invention has promising applications in the treatment of myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials applications. It relates to a method for preparing a tea polyphenol cross-linked decellularized diaphragmatic tendon patch and its medicinal uses. Background Technology

[0002] Cardiovascular disease associated with myocardial infarction (MI) is one of the leading causes of death worldwide, partly due to the limited regenerative capacity of myocardial tissue. Following a myocardial infarction, the heart develops pathological conditions such as ischemia, inflammation, fibrosis, and left ventricular dysfunction. Current surgical methods are sufficient to enhance myocardial perfusion but cannot reverse pathological changes such as fibrosis. Decellularized biomaterials are widely used in research on myocardial infarction treatment. However, currently reported biomaterials, such as the submucosa of the small intestine, peritoneum, and pericardium, are relatively thin, with limited mechanical and enzymatic resistance. Multiple layers must be stacked to create a scaffold of appropriate thickness, but this stacking process easily leads to delamination in vivo, reducing the scaffold's strength and limiting its reparative effect. Porcine diaphragmatic tendon, primarily composed of collagen fibers, possesses excellent mechanical properties and a thickness between 0.4 and 1 mm, which is suitable for implantation in the infarct area. It also has the potential to promote angiogenesis, making it a promising candidate for a more ideal repair material. Animal biomaterials require decellularization to remove components that stimulate an immune response. However, decellularization significantly reduces the mechanical strength of materials, accelerates their degradation rate in vivo, and the α-galactosyl (α-Gal) antigen in the material can still trigger a strong biological immune response, all of which severely affect repair efficiency. Clinically, glutaraldehyde is widely used to cross-link decellularized extracellular matrix materials to improve these defects. Cross-linking can also shield antigens to some extent and reduce immune rejection, but glutaraldehyde has drawbacks such as high toxicity and the ability to cause calcification of biomaterials. Tea polyphenols are natural polyphenols whose phenolic hydroxyl groups can form numerous hydrogen bonds with the extracellular matrix (ECM), enhancing mechanical properties and resistance to enzymatic degradation. They also endow materials with strong antioxidant, antibacterial, and calcification-inhibiting abilities. More importantly, tea polyphenol cross-linking can shield surface antigens and reduce post-implantation inflammatory responses, making it a superior natural and highly efficient cross-linking agent compared to glutaraldehyde.

[0003] Therefore, the development of a decellularized porcine diaphragmatic tendon cross-linked with a natural cross-linking agent—tea polyphenols—is of great significance for the treatment of myocardial infarction. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method for preparing a tea polyphenol cross-linked decellularized diaphragmatic tendon patch, which is formed by cross-linking porcine diaphragmatic tendon with tea polyphenols after decellularization using 1% TNBP (tributyl phosphate). The specific method is as follows:

[0005] (1) Fresh porcine diaphragmatic tendons obtained from slaughterhouses were treated with pre-cooled PBS (phosphate-buffered saline). Under aseptic conditions, the diaphragmatic tendons were prepared into nearly circular slices of 8 mm using an 8 mm drill bit. 1% TNBP (tributyl phosphate) was prepared using PBS. The treated diaphragmatic tendon slices were added to a sterile 50 ml reagent tube, followed by the addition of 1% TNBP. The tube was placed in a shaker at a constant speed of 80 rpm / min and a temperature of 20°C for 72 h. After treatment, the samples were treated with sterile PBS in a shaker for 24 h. The obtained samples were then tested for vasoactive factors and other related components.

[0006] (2) Prepare 5 mg / ml and 1 mg / ml tea polyphenol solutions using PBS respectively, and prepare 0.625% glutaraldehyde solution. Add sufficient amounts of the above three reagents to the sample obtained in step (1), and treat in a shaker for 48 h (under the same conditions as step 1). Then treat with sterile PBS for 24 h to obtain the final tea polyphenol cross-linked decellularized diaphragmatic tendon sample. The final product was tested for in vitro mechanical properties, anti-enzymatic properties and other characteristics. The immunogenicity and toxicity to the body after cross-linking were explored through a mouse subcutaneous implantation model. Finally, the comprehensive therapeutic effect of tea polyphenol cross-linked decellularized diaphragmatic tendon patch on myocardial infarction was verified through a rat myocardial infarction model.

[0007] Another objective of the invention is to provide the application of the aforementioned tea polyphenol-crosslinked decellularized diaphragmatic tendon patch in the preparation of a patch for treating myocardial infarction. The tea polyphenol-crosslinked decellularized diaphragmatic tendon provided by this invention can downregulate the expression of apoptosis and inflammatory genes and upregulate the expression of antioxidant genes, thereby comprehensively treating myocardial infarction by inhibiting ventricular dilation, improving fibrosis, and promoting angiogenesis. Studies have shown that the tea polyphenol-crosslinked decellularized diaphragmatic tendon patch prepared by this invention has good antioxidant properties, low immunogenicity, and mechanical properties. In the short term (within 7 days) of treating myocardial infarction, it can improve the antioxidant capacity of cardiomyocytes, and in the long term (more than 30 days), it can inhibit ventricular dilation, improve fibrosis, and promote angiogenesis, thus exhibiting a good comprehensive therapeutic effect on myocardial infarction.

[0008] The tea polyphenol-crosslinked decellularized diaphragmatic tendon patch provided by this invention possesses excellent antioxidant capacity, mechanical properties, anti-enzymatic activity, and low immunogenicity. Furthermore, it is non-toxic to the body, has a simple preparation method, low cost, readily available materials, and can be mass-produced with significant effects. Pathological remodeling in rats treated with tea polyphenol-crosslinked decellularized diaphragmatic tendon was significantly alleviated. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and total RNA sequencing (RNA-seq) of myocardial tissue in the treatment area of ​​the tea polyphenol-crosslinked patch showed that the tea polyphenol-crosslinked decellularized porcine diaphragmatic tendon patch downregulated apoptosis and inflammatory genes and upregulated the expression of antioxidant genes.

[0009] In summary, the cross-linked decellularized diaphragmatic tendon patch with tea polyphenols has been verified to have strong antioxidant, mechanical, anti-enzymatic, and low immunogenic properties. It can improve the antioxidant capacity of cardiomyocytes, promote myocardial tissue vascularization, and inhibit fibrosis, showing promising application prospects in the treatment of myocardial infarction. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the cross-linking of tea polyphenols with decellularized diaphragmatic tendons and their application in myocardial infarction.

[0011] Figure 2 For quantitative analysis of DNA content.

[0012] Figure 3 HE and Masson staining.

[0013] Figure 4 Measurement of angiogenic factors VEGF and HGF.

[0014] Figure 5 Fourier transform infrared peaks were measured for the cross-linked patch.

[0015] Figure 6 Mechanical property testing of cross-linked patches.

[0016] Figure 7 This is a test of the resistance to enzymatic degradation of cross-linked patches.

[0017] Figure 8 The antioxidant capacity and free radical scavenging capacity of the cross-linked patch were tested.

[0018] Figure 9 The hemolysis rate of the cross-linked patch.

[0019] Figure 10 This study describes the expression of acute response proteins and cytokines in the body after subcutaneous implantation of a cross-linked patch.

[0020] Figure 11 This study describes the changes in spleen cells after subcutaneous implantation of a cross-linked patch.

[0021] Figure 12 This is a histopathological image of a cross-linked patch after subcutaneous implantation.

[0022] Figure 13 This study aims to assess liver and kidney function after subcutaneous implantation of a cross-linked patch.

[0023] Figure 14 These are histopathological images of various organs after subcutaneous implantation of the cross-linked patch.

[0024] Figure 15 Echocardiograms were performed on day 3 and day 30 during cross-linked patch therapy for myocardial infarction.

[0025] Figure 16 Analysis of NOX2 and SOD2 expression levels in myocardial tissue during cross-linked patch treatment for myocardial infarction.

[0026] Figure 17 Statistics on neovascularization during cross-linked patch treatment for myocardial infarction.

[0027] Figure 18 Statistics on the area of ​​fibrosis during cross-linked patch treatment for myocardial infarction. Detailed Implementation

[0028] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0029] Example 1. Decellularization treatment of porcine diaphragmatic tendon patch

[0030] (1) The diaphragmatic tendon of pigs was obtained from the pig slaughterhouse. After obtaining the whole diaphragm, the central tendon part was cut off with a scalpel, and the muscle part was discarded. The fresh central tendon was then immediately stored in chilled PBS and placed on ice for preservation.

[0031] (2) One piece was processed, and the rest were stored in a refrigerator at -80℃ for later use. The tendons were divided into two groups: the fresh group (FD group) and the decellularized group (DD group). Under sterile conditions, the diaphragmatic tendons were prepared into nearly circular slices of 8 mm using an 8 mm drill bit. Then, the fresh group was added with sufficient PBS, and the decellularized group was added with sufficient 1% TNBP. Both groups were treated on a shaker (at a constant speed of 80 rpm / min) for 24 h, and the shaker temperature was set to 37℃.

[0032] (3) The treated tendon was first rinsed three times with sterile PBS, then washed in a shaker with PBS for 72 hours before proceeding to the next step.

[0033] Example 2: Evaluation of the decellularization effect of porcine diaphragmatic tendon patch

[0034] After the decellularization process, the DNA content in the DD group decreased significantly, and the results of DNA quantification were as follows ( Figure 2 The results showed that the DNA content in the FD group was 603.28±80.37 ng / mg, while the DNA content in the DD group was 63.84±6.97 ng / mg, representing an 89.42% decrease in DNA content in the DD group compared to the FD group. This indicates that using 1% TNBP as a decellularization agent for porcine diaphragmatic tendon can remove most cellular components. Furthermore, in HE staining (… Figure 3 Before decellularization, numerous cell nuclei were visible in the tendon tissue, indicating that stromal cells were scattered among the collagen fibers. After decellularization, the blue-stained cell nuclei completely disappeared. In Masson staining ( Figure 3Collagen fibers appeared blue, while muscle fibers appeared red. Before decellularization, numerous red muscle fibers were present, but after decellularization, no obvious muscle fibers were observed. This indicates that the reagent effectively removed cells. More importantly, the tendon fibers in the fresh and decellularized groups were observed to be tightly connected, with no obvious breakage or twisting. This demonstrates that the decellularization method has high DNA removal efficiency without causing serious damage to the matrix.

[0035] The levels of two pro-angiogenic factors in decellularized tendons were analyzed using ELISA. Figure 4 The VEGF content was relatively high, while the HGF content was relatively low. Both VEGF and HGF levels decreased after decellularization, but the differences were not significant compared to pre-decellularization levels. The VEGF content before decellularization was 110.1±10.91 ng / L, and after decellularization it was 89.85±19.85 ng / L. The HGF content before decellularization was 20.65±9.2 ng / L, and after decellularization it was 11.61±1.25 ng / L. These results demonstrate that 1% TNBP is an effective decellularization agent for porcine diaphragmatic tendons. After 24 hours of treatment, it can remove most of the DNA without severely damaging the matrix, and can largely retain biologically active factors.

[0036] Example 3: Crosslinking and Fourier Transform Infrared Characterization of Decellularized Porcine Diaphragmatic Tendon Patch

[0037] 50 mg and 250 mg of tea polyphenol powder were weighed separately using an electronic analytical balance and dissolved in 50 mL of PBS. The mixture was then vigorously mixed using a high-speed Vortex mixer to ensure complete dissolution, preparing 5 mg / mL and 1 mg / mL tea polyphenol solutions. After soaking in PBS for 72 h, the DD group was dried with sterile filter paper and then crosslinked in the 5 mg / mL and 1 mg / mL tea polyphenol solutions for 48 h. Crosslinking was performed on a shaker at 37°C and a shaking speed of 100 times / min. After crosslinking, the group was treated with PBS for 24 h to obtain the 5 mg / mL crosslinked decellularized porcine diaphragm tendon group (GTP5 group) and the 1 mg / mL crosslinked decellularized porcine diaphragm tendon group (GTP1 group) (see...). Figure 1 After lyophilizing each group of cross-linked patches, the chemical composition of the tea polyphenol-cross-linked tendon membrane was analyzed by Fourier transform infrared spectroscopy (see [link to Fourier transform infrared spectroscopy]). Figure 5 All groups exhibited five characteristic absorption peaks: amide A, B, I, II, and III bands. The amide A band was located between 3200 and 3440 cm⁻¹. -1 Between these peaks, the absorption peaks are mainly caused by NH stretching vibrations. When a large number of hydrogen bonds are formed, the absorption peaks shift to lower wavelengths. The amide A band of GTP5 shifts more than that of GTP1 mainly because more hydrogen bonds are formed when the phenolic hydroxyl groups of high-concentration tea polyphenols crosslink with collagen fibers. The shift of the infrared peaks proves that the crosslinking of tea polyphenols is successful.

[0038] Example 4: Determination of the mechanical properties and enzymatic resistance of cross-linked patches

[0039] Mechanical properties of uncrosslinked and crosslinked patches were tested using a universal testing machine (see...). Figure 6 The elastic modulus of decellularized diaphragmatic tendon was 162.88±32.79 MPa. Crosslinking improved the elastic modulus to varying degrees, with the GTP1 group reaching 249.16±27.89 MPa and the GTP5 group reaching 392.64±96.79 MPa. Compared to decellularized diaphragmatic tendon, low-concentration tea polyphenol crosslinking did not improve the ultimate tensile strength, while high-concentration tea polyphenol crosslinking significantly improved it, with the GTP5 group reaching 67.75±18.83 MPa (P<0.05). These results indicate that crosslinking with 5 mg / mL tea polyphenols can significantly improve the mechanical properties of decellularized tendons.

[0040] Degradation rates before and after cross-linking were tested using type I collagenase (see...) Figure 7 The results showed that both the GTP1 and GTP5 groups exhibited varying degrees of resistance to enzymatic degradation compared to the DD group. The degradation rate of the GTP1 group was faster than that of the GTP5 group. By day 12, the DD group had completely degraded, while the GTP1 group had degraded by 92.38±0.83%, and the GTP5 group by 74.45±1.1%. This indicates that cross-linking between tea polyphenols and glutaraldehyde can mitigate the excessively rapid degradation of dECM, and that higher concentrations of tea polyphenols resulted in higher resistance to enzymatic degradation.

[0041] Example 5: Antioxidant and Free Radical Scavenging Capabilities of Crosslinked Patches

[0042] Total antioxidant capacity and free radical scavenging capacity were detected using the T-AOC kit and the DPPH kit (see [link to kit]). Figure 8 The total antioxidant capacity experiment showed that the DD group had almost no antioxidant capacity. Compared with the DD group, the total antioxidant capacity of GTP1 reached 52.59±0.69%, while that of GTP5 reached 77.78±0.84%. The DPPH experiment results were consistent with the total antioxidant capacity experiment; the DD group had almost no free radical scavenging ability, while the GTP1 group reached 73.78±0.74%, and the GTP5 group reached 82.56±1.03%. The experimental results indicate that tea polyphenols endow decellularized diaphragmatic tendons with strong antioxidant and free radical scavenging abilities, and the higher the cross-linking concentration, the better the antioxidant performance.

[0043] Example 6: Hemolysis test of cross-linked patch

[0044] The hemolysis test was performed using whole pig blood to verify the blood compatibility of the hydrogel (see...). Figure 9The patch was soaked in PBS solution for 24 hours to obtain the patch extract. To obtain red blood cells, whole pig blood was centrifuged at 1000 rpm for 10 minutes and washed three times with PBS. Red blood cells were diluted with PBS to a final concentration of 5% v / v (volume / volume). 100 μL of the red blood cell solution and 100 μL of the patch extract were added to a 96-well plate. The positive control was mixed with sterile water, and the negative control was mixed with PBS. The 96-well plate was incubated at 37°C with shaking at 180 rpm for 1 hour. The microplate was centrifuged at 1000 rpm for 10 minutes, and 100 μL of supernatant was added to the 96-well plate. The absorbance was measured at 540 nm using a microplate reader. The hemolysis rate was calculated as follows: Hemolysis rate = (sample OD - negative control OD) / (positive control OD - negative control OD) × 100%, where OD is the absorbance. Compared to the control group (sterile water), no excessive hemolysis was observed in any sample, indicating that no significant exudates from the patch interfered with the normal function of blood cells. The hemolysis rate in each group was within the acceptable range (5%).

[0045] Example 7: Inflammation Detection After In Vivo Implantation of Cross-linked Patch

[0046] Male BALB / c mice (weighing 18–22 g, 6 weeks old) were weighed after five days of acclimatization and randomly divided into four groups: sham-operated group (Sham), non-crosslinked group (DD), 5 mg / mL tea polyphenol crosslinked group (GTP5), and 1 mg / mL tea polyphenol crosslinked group (GTP1), with 5 mice in each group. Mice were anesthetized with acetaminophen (50 mg / kg, intramuscular injection), and a 1 cm incision was made on the back after shaving. Materials for each group were implanted subcutaneously into the muscle, while the sham-operated group was directly sutured. Mice were then fed as usual. On days 8 and 30, blood was collected by enucleating the eyes. After sufficient blood was collected, the cells were centrifuged at 3500 rpm for 10 min to obtain serum. TNF-α, IL-1β, IL-2, IL-5 cytokines, and C-reactive protein were detected according to the ELISA kit instructions (see [link to ELISA kit]). Figure 10On day 8 after implantation, C-reactive protein (CRP) levels increased in all groups, with the most significant increase in the DD group and the lowest in the GTP5 group. On day 30 after implantation, CRP levels remained high in the DD and GTP1 groups, while significantly decreased in the GTP5 group, showing no significant difference from the Sham group. The GA group also showed a decrease, but remained significantly different from the Sham group. Regarding TNF-α changes, on day 8 after implantation, the DD group showed the largest increase, followed by the GTP1 group. There was no significant difference between the GTP5 and Sham groups. On day 30, the DD and GTP1 groups remained at high levels, while there was no significant difference between the GTP5 and Sham groups. Regarding IL-1β changes, on day 8 after implantation, IL-1β levels were higher in all groups compared to the Sham group, with the highest in the DD group and the second highest in the GTP1 group. There was no significant change in the GTP5 group. On day 30 after implantation, the DD and GTP1 groups remained at high levels, while there was no significant difference in the GTP5 group. Regarding changes in IL-2, on day 8 after implantation, significant decreases were observed in the DD and GTP1 groups, while no significant difference was found between the GTP5 and Sham groups. On day 30 after implantation, further decreases were observed in the DD and GTP1 groups, while the GTP5 group remained significantly different from the Sham group. This result can be explained by the fact that the DD and GTP1 groups triggered abnormal activation and dysregulation of the body's immune system, while the GTP5 group did not significantly activate the body's immune response. Regarding changes in IL-5, on day 8 after implantation, significant increases were observed in the DD and GTP1 groups, while no significant difference was found in the GTP5 group. On day 30 after implantation, the DD and GTP1 groups remained at high levels, while the GTP5 group remained stable. These results collectively indicate that although decellularized tendons have removed a large amount of DNA, they still trigger an immune rejection response. Low-concentration tea polyphenol cross-linking did not reduce the immune rejection response, while high-concentration tea polyphenol (5 mg / mL) cross-linking significantly reduced the inflammatory response caused by immune rejection over a longer period of time.

[0047] Example 8: In vivo immunogenicity test of cross-linked patch

[0048] On days 8 and 30, spleen samples were aseptically removed from each group of mice, and spleen single-cell suspensions were prepared with the cell density adjusted to 10 × 10⁻⁶ cells / mL. 6 / mL, add 100μL of cell suspension to each flow cytometry tube. Dilute Zombie dye stock solution 1:100 with PBS, add 1μL of the diluted solution to each flow cytometry tube containing 100μL of cell suspension, incubate at room temperature in the dark for 30 min, and wash once with 2mL of flow cytometry staining buffer. Subsequently, mouse anti-CD45, anti-CD3, anti-CD19, anti-CD4, and anti-CD8 antibodies were added to each group of flow cytometry tubes according to the supplier's recommended dosage, and incubate on ice in the dark for 20 min. Add 2mL of flow cytometry staining buffer, then centrifuge at 3500rpm for 5 min, discard the supernatant, and repeat the washing twice. Finally, add 500μL of flow cytometry buffer to each tube for flow cytometry assay (see Figure 11 The experimental results showed that on day 8, the CD4+ T cell ratio in the implanted DD group and GTP1 group increased significantly, while no significant difference was observed in the implanted GTP5 group. This indicates that high-concentration tea polyphenol cross-linking can reduce the early immune rejection response of the implant. The significant increase in CD4+ T cells in the DD group may be due to α-Gal antigens and non-α-Gal antigens in the tendon. Meanwhile, low-concentration tea polyphenol cross-linking did not reduce the early immune rejection response of the implant. Thirty days after implantation, the CD4+ T cell level in the DD group remained high, indicating a sustained immunogenic response, while the GTP1 group showed a certain decrease. The CD4+ T cell ratio in the high-concentration tea polyphenol cross-linking group remained significantly different from that in the Sham group. These results indicate that 5 mg / mL tea polyphenol cross-linking can significantly inhibit the immune rejection response of the implant.

[0049] Example 9: In vivo toxicity test of cross-linked patch

[0050] The extent of local pathological tissue cell infiltration was detected 30 days after implantation (see...). Figure 12 Representative biochemical indicators of the liver and kidneys (see) Figure 13 ) and pathological tissue staining of the heart, liver, lungs and kidneys (see Figure 14 This demonstrates that the cross-linked patch containing tea polyphenols does not cause damage to the body. On day 30, the implant material was removed, and local pathological findings were observed. The DD group showed significant inflammatory cell infiltration near the material, but no tissue necrosis, fibrous encapsulation, or granulation tissue. Both the GTP1 and GTP5 groups showed a certain amount of inflammatory cell infiltration, but no granulomas or fibrosis were observed. ALT is a sensitive indicator of liver damage, with an upper limit of 35.36 U / L in Balb / c mice. CREA is a sensitive indicator of kidney damage, with an upper limit of 35.36 μmol / L in Balb / c mice. On day 30 after implantation, CREA and ALT in all groups did not exceed their upper limits, demonstrating that the implant did not cause liver or kidney damage. On day 30 after implantation, the heart, lungs, liver, and kidneys of the mice were collected for histopathological staining (see...). Figure 14No pathological changes such as inflammatory cell infiltration and damage were observed in any organs, indicating that neither non-crosslinked nor crosslinked materials would cause organ lesions.

[0051] Example 10: Treatment trial of cross-linked patches in rats with myocardial infarction

[0052] Male SD rats (weighing 230–250 g, 7 weeks old) were acclimatized for 5 days, then weighed and randomly divided into four groups: sham operation group (Sham group), myocardial infarction group (MI group), DD treatment group, and GTP5 treatment group. The treatment lasted for two time periods: 3 days and 30 days, with 5 rats in each group. Rats were anesthetized with butorphanol (2 mg / kg) intramuscularly and with salbutamol (50 mg / kg) intramuscularly. They were then intubated with a rat-specific endotracheal tube and connected to a ventilator with a tidal volume of 5 mL, a respiratory ratio of 1:2, and a respiratory rate of 100 breaths / min. After confirming anesthesia, the skin was prepared and disinfected. The skin was incised along the line from the axilla to the apical cartilage, and the subcutaneous fascia and muscles were dissected sequentially until the pleura was exposed. A notch was made in the intercostal space (third and fourth intercostal space) with curved forceps, and two ribs were transversely severed with scissors, followed by the third rib. The thoracic cavity was opened with an ophthalmic retractor to expose the heart, and the pericardium was torn open and hemostasis achieved. Using cotton swabs to help expose the ligation point, the coronary artery was ligated with a 6-0 needle. Immediate whitening of the left ventricular wall indicated successful obstruction. In the treatment group, a pre-fabricated patch was sutured to the left ventricular infarction site, the pleural cavity was sutured with a nodular suture, and the muscles and skin were sutured sequentially, ensuring no gaps. The sham surgery group underwent only open-chest and closed-chest procedures. Rats were housed individually post-surgery, with regular feed and water. Echocardiography was performed and data recorded on days 0, 3, and 30. On day 3, after euthanasia, qRT-PCR was used to detect the oxidase gene NOX2 and the antioxidant enzyme gene SOD2 in the myocardial tissue of the infarcted area. On day 30, after euthanasia, immunofluorescence was used to detect myocardial angiogenesis, and Masson's assay was used to detect myocardial fibrosis.

[0053] Echocardiogram results (see) Figure 15The results showed that on day 3, the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) decreased in the myocardial infarction group, while the left ventricular end-diastolic diameter (IVIDd) and left ventricular systolic diameter (IVIDs) increased significantly. The DD treatment group and the GTP5 treatment group showed the same trend. On day 30, EF and FS further decreased in the MI group, due to ventricular dilation following long-term pathological remodeling after myocardial infarction. The DD treatment group showed a trend of mitigation, but this was not significantly different from the MI group. The GTP5 treatment group showed a significantly lower decrease in EF and FS compared to the MI and DD groups, and a lower increase in IVIDd and IVIDs compared to the MI and DD groups. This may be because the infarcted area secretes a large amount of matrix metallocollagenase after myocardial infarction, leading to a faster degradation time in the DD group than in the tea polyphenol cross-linking group. This rapid degradation prevented the generation of mechanical stress that inhibited ventricular dilation. After cross-linking, the tea polyphenols significantly improved their resistance to enzymatic degradation, enabling them to generate effective stress to inhibit ventricular dilation and alleviate ventricular remodeling. This result demonstrates the importance of crosslinking agents in stabilizing dECM.

[0054] Results of quantitative real-time qPCR experiment (see) Figure 16 The results showed that three days after myocardial infarction, NOX2 gene expression was significantly upregulated in the MI and DD groups, while it was significantly downregulated in the GTP5 group compared to the MI and DD groups. SOD2 levels did not change significantly in the MI and DD groups, but were significantly upregulated in the GTP5 group. This demonstrates that GTP5 treatment can protect the myocardium by increasing cellular SOD2 levels to scavenge free radicals. Improvement of the early oxidative stress microenvironment after myocardial infarction is beneficial in reducing inflammation and cardiomyocyte death.

[0055] Msaaon staining results (see...) Figure 17 The results showed that GTP5 significantly inhibited ventricular fibrosis in myocardial infarction, with a lower degree of fibrosis compared to the DD and MI groups, and the DD group had a lower degree of fibrosis than the MI group. Immunofluorescence results at 30 days after myocardial infarction (see...) Figure 18 The results showed that the VEGF fluorescence intensity in the GTP5 group was significantly greater than that in the DD, MI, and Sham groups, with the DD group showing greater intensity than the MI and Sham groups. The stronger pro-angiogenic and anti-fibrotic effects of cross-linked tea polyphenols may stem from the stronger mechanical effects following cross-linking. Mechanical signals can influence transcription factor expression, protein synthesis, and cell function through transduction networks, and different mechanical signals can produce different pro-angiogenic effects.

[0056] In summary, the results indicate that the 5 mg / mL tea polyphenol-crosslinked decellularized diaphragmatic tendon patch has a good comprehensive therapeutic effect in myocardial infarction. In the early stage of myocardial infarction, it can clear free radicals generated after myocardial infarction, and in the later stage of myocardial infarction, it can inhibit ventricular dilation and fibrosis and promote vascularization. It is an excellent candidate material that can improve the pathological process of myocardial infarction.

Claims

1. Use of a tea polyphenol cross-linked decellularized diaphragmatic muscle tendon patch in the preparation of a treatment for myocardial infarction patch, characterized in that, The tea polyphenol cross-linked decellularized diaphragm tendon patch is obtained by the following preparation method: (1) The porcine diaphragm tendon is treated with a refrigerator-precooled phosphate buffered saline solution, and the diaphragm tendon is prepared into 8 mm thin slices in a sterile environment. The diaphragm tendon thin slices are added into a sterile reagent tube, 1% tributyl phosphate is added, and the mixture is placed in a shaker at a uniform speed of 80 rpm and a temperature of 20°C for 72 h. After the treatment is completed, the mixture is treated with sterile PBS in a shaker for 24 h. The obtained sample is subjected to a pro-angiogenic factor related component test; (2) A 5 mg / ml tea polyphenol solution is prepared using PBS, and the sample obtained in step (1) is added into the above reagent in a sufficient amount, and the mixture is treated in a shaker for 48 h. Then, the mixture is treated with sterile PBS for 24 h to obtain a final tea polyphenol cross-linked decellularized diaphragm tendon patch.

2. Use according to claim 1, characterized in that, In step (1), 1% tributyl phosphate is prepared using PBS.

3. Use according to claim 1, characterized in that, In step (2), the setting conditions of the shaker treatment are the same as those in step (1).

4. Use according to claim 1, characterized in that, The tea polyphenol cross-linked decellularized diaphragm tendon can down-regulate the expression of apoptosis genes and inflammatory genes and up-regulate the expression of antioxidant genes, thereby inhibiting ventricular dilation, improving fibrosis, and promoting angiogenesis.

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