A biological nanocomposite of decellularized porcine pericardium crosslinked with poss-peg-cho and a preparation method and application thereof

Abstract

The present application relates to the biomedical technology field, especially to a kind of decellularized pig pericardium and POSS-PEG-CHO cross-linked biological nanocomposite material and its preparation method and application.The present application is based on Schiff base reaction, and the star-shaped eight-arm eight-functionalized POSS-based benzaldehyde end-capped polyethylene glycol is used to cross-link the decellularized pig pericardium, and the POSS-PEG-PP biological nanocomposite material is successfully prepared.Compared with GLUT-PP, the POSS-PEG-PP provided by the present application presents more compact fiber arrangement, and the mechanical property is also better.In vitro experiment shows that POSS-PEG-PP has excellent cell compatibility, endothelialization capacity and blood compatibility.In vivo evaluation further confirms that the material has significant anti-inflammatory and anti-calcification properties.Through RNA sequencing analysis of subcutaneous implant, it is found that the changes of AMPK and IL-17 signal pathways play an important role in the process of POSS-PEG-PP regulating inflammation and immune response.Therefore, POSS-PEG-PP shows good application prospect in the field of biological heart valve, and is expected to become a very potential alternative material.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a biological nanocomposite material of decellularized porcine pericardium and POSS-PEG-CHO cross-linking and a preparation method and application thereof. BACKGROUND

[0002] Valvular heart disease (VHD) is a common cardiovascular disease, and its incidence rate is only next to coronary artery disease and hypertension. At present, heart valve replacement is the only cure for VHD, and mechanical heart valve (MHV) or biological heart valve (BHV) can be selected. In recent years, BHV is more favored due to the need for lifelong anticoagulant therapy of MHV, and the application advantage of BHV in transcatheter heart valve replacement (THVR) is obvious.

[0003] Commercially available BHV is mainly prepared by glutaraldehyde cross-linking porcine or bovine pericardium. In the field of biomedical materials, cross-linking technology has a significant impact on material performance, and glutaraldehyde cross-linking is a common method for preparing BHV at that time. Glutaraldehyde cross-linking can meet the basic performance requirements of BHV to some extent, so that BHV can be applied in heart valve replacement surgery, providing a treatment approach for VHD patients. However, this preparation method has significant drawbacks. Studies have shown that BHV often fails due to calcification after 10-15 years of use, and the calcification is mainly due to immune response, thrombosis and toxicity of glutaraldehyde. In order to solve these problems, researchers have explored various methods for replacing glutaraldehyde for cross-linking of BHV in the past few decades, such as using epoxy compounds, polyphenols, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS), oxidized hyaluronic acid and photo-crosslinking technology. These alternative solutions have potential advantages such as reducing cytotoxicity and improving anti-calcification ability, but due to problems such as poor biocompatibility, insufficient durability or complex cross-linking process, they have not been widely used in clinical practice. At present, BHV for heart valve replacement mainly faces two problems: one is that BHV cross-linked with glutaraldehyde is prone to calcification, which seriously affects its service life and treatment effect; the other is that although there are various cross-linking methods to replace glutaraldehyde, they cannot effectively solve the existing problems of BHV due to their own defects, and it is difficult to meet the clinical needs.

[0004] Therefore, it is urgent to develop a biological heart valve material and a preparation method which can not only solve the problems of calcification and failure of existing BHV, but also have good mechanical properties, cell compatibility, blood compatibility and anti-calcification properties. SUMMARY

[0005] Therefore, the present application provides a biological nanocomposite material of decellularized porcine pericardium and POSS-PEG-CHO cross-linking and a preparation method and application thereof.

[0006] The technical scheme of the present application is implemented as follows:

[0007] In a first aspect, the present application provides a preparation method of a biological nanocomposite, which comprises the following steps: subjecting decellularized porcine pericardium to Schiff base reaction with a POSS-PEG-CHO solution with a concentration of 30-50 mg / mL, so that the aldehyde groups in the POSS-PEG-CHO and the amino groups in the decellularized porcine pericardium are subjected to condensation reaction to generate carbon-nitrogen double bonds, thereby cross-linking the POSS-PEG-CHO and the decellularized porcine pericardium, and obtaining the biological nanocomposite.

[0008] In a second aspect, the present application provides an application of the preparation method in the preparation of a biomedical material.

[0009] In a third aspect, the present application provides a biological nanocomposite, which is obtained according to the preparation method of the biological nanocomposite.

[0010] In a fourth aspect, the present application provides an application of the biological nanocomposite in the preparation of a biomedical material.

[0011] The present application has at least the following beneficial effects:

[0012] The present application cross-links DPP with star-shaped eight-arm eight-functionalized POSS-based benzaldehyde-terminated polyethylene glycol, and obtains POSS-PEG-PP based on the Schiff base reaction between the amino groups in DPP and the aldehyde groups in POSS-PEG-CHO. Compared with the traditional glutaraldehyde cross-linked BHV, it exhibits outstanding performance in mechanical properties, endothelialization, blood compatibility, biocompatibility, anti-inflammatory and anti-calcification ability.

[0013] In addition, the present application performs RNAseq analysis to further study the mechanism, and finds that the changes in the AMPK and IL-17 signaling pathways may play an important role in the anti-inflammatory performance of POSS-PEG-PP, and therefore, POSS-PEG-PP is an excellent alternative material for BHV, and is expected to be transformed in the clinic.

[0014]

Term Explanation

[0015] In some embodiments of the present application, the meanings of the relevant terms include the following:

[0016]

[0017]

[0018] BRIEF DESCRIPTION OF DRAWINGS

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the preparation process of POSS-PEG-PP according to an embodiment of the present invention;

[0021] Figure 2 The relevant detection results for the embodiments of the present invention are as follows: (A) POSS-PEG-CHO 1 H NMR spectrum; (B) POSS-PEG-CHO 13 (C) C NMR spectra; (D) Relative amino content of DPP, GLUT-PP and POSS-PEG-PP (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL and 50 mg / mL); (E) FTIR spectra of POSS-PEG-CHO, DPP, POSS-PEG-PP and GLUT-PP; (E) HE, Masson and DAPI staining of DPP and PP; (F) Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of the surface morphology of DPP, GLUT-PP and POSS-PEG-PP; Data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0022] Figure 3 In this embodiment of the invention, the relevant test results of the physicochemical properties of DPP, GLUT-PP, and POSS-PEG-PP are as follows: (A) strain-stress curves of DPP, GLUT-PP, and POSS-PEG-PP; (B) water contact angle of DPP, GLUT-PP, and POSS-PEG-PP; (C) fluorescent yellow CH staining of DPP, GLUT-PP, and POSS-PEG-PP; (D) elastic modulus of DPP, GLUT-PP, and POSS-PEG-PP; (E) maximum tensile strength of DPP, GLUT-PP, and POSS-PEG-PP; (F) fluorescence intensity of DPP, GLUT-PP, and POSS-PEG-PP after incubation with fluorescent yellow CH for 12 h; (G) heat shrinkage temperature of DPP, GLUT-PP, and POSS-PEG-PP; data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0023] Figure 4For the related test results of DPP, GLUT-PP and POSS-PEG-PP in the embodiments of the present application, (A) Calcein-AM / PI staining of L929 cells cultured in DPP, GLUT-PP and POSS-PEG-PP extract solution for 3 days; (B) Calcein-AM / PI staining of L929 cells grown on the surface of DPP, GLUT-PP and POSS-PEG-PP; (C) CCK8 assay of L929 cell viability cultured in DPP, GLUT-PP and POSS-PEG-PP extract solution; (D) CCK8 assay of HUVEC viability grown on the surface of DPP, GLUT-PP and POSS-PEG-PP; data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0024] Figure 5 For the related test results of DPP, GLUT-PP and POSS-PEG-PP in the embodiments of the present application, (A) confocal fluorescence microscope images of BSA-FITC and FBG-FITC adhered to the surface of DPP, GLUT-PP and POSS-PEG-PP; (B) fluorescence intensity of DPP, GLUT-PP and POSS-PEG-PP after incubation with BSA-FITC for 2h; (C) fluorescence intensity of DPP, GLUT-PP and POSS-PEG-PP after incubation with FBG-FITC for 2h; (D) representative images of hemolysis experiment (E) absorbance at OD 490nm of supernatant of DPP, GLUT-PP and POSS-PEG-PP hemolysis experiment; (F) SEM images of recalcified whole blood coagulation of DPP, GLUT-PP and POSS-PEG-PP; (G) representative images and LDH quantitative detection of recalcified whole blood coagulation of DPP, GLUT-PP and POSS-PEG-PP; (H) SEM images of platelet adhesion of DPP, GLUT-PP and POSS-PEG-PP; (I) LDH quantitative detection of platelet adhesion of DPP, GLUT-PP and POSS-PEG-PP; data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0025] Figure 6In this embodiment of the invention, the relevant detection results of DPP, GLUT-PP, and POSS-PEG-PP are as follows: (A) HE staining, CD3 immunohistochemical staining, and CD68 immunohistochemical staining of DPP, GLUT-PP, and POSS-PEG-PP 1 week and 2 weeks after implantation; (B) the ratio of CD3 positive cells around DPP, GLUT-PP, and POSS-PEG-PP 1 week and 2 weeks after implantation; (C) the ratio of CD68 positive cells around DPP, GLUT-PP, and POSS-PEG-PP 1 week and 2 weeks after subcutaneous implantation; the data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0026] Figure 7 The relevant detection results in this embodiment of the invention are as follows: (A) HE, Alizarin Red S and Masson staining of GLUT-PP and POSS-PEG-PP 1 month, 2 months and 3 months after implantation; (B) ICP-OES quantitative analysis of calcium content of DPP, GLUT-PP and POSS-PEGPP 1 month, 2 months and 3 months after subcutaneous implantation; the data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0027] Figure 8 The relevant detection results in this embodiment of the invention are as follows: (A) Gene heatmaps and the top 20 differentially expressed genes of DPP, GLUT-PP and POSS-PEG-PP 2 weeks after implantation; (B) Trends of 9 gene clusters; (C) Venn diagrams of differentially expressed genes (DEGs) of 3 control groups (DPP vs GLUT-PP, DPP vs POSS-PEG-PP and GLUT-PP vs POSS-PEG-PP); (D) Volcano diagram of DEGs (DPP vs POSS-PEG-PP, with DPP as a control); (E) Gene Ontology Enrichment Analysis of DEGs of DPP and POSS-PEG-PP; (F) KEGG (Kyoto Encyclopedia of Genes and Genomes) Enrichment Analysis of DEGs of DPP and POSS-PEG-PP. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] (I) Preparation of POSS-PEG-PP

[0030] 1. Materials and Reagents

[0031]

[0032]

[0033] 2. Synthesis of star-shaped eight-armed eight-functionalized POSS-based benzaldehyde-terminated polyethylene glycol (POSS-PEG-CHO)

[0034] Based on the references [1] The method was used to synthesize POSS-PEG-CHO (wherein the number-average molecular weight of polyethylene glycol is 2 kDa; the patent applicant guarantees to release the biological material to the public within twenty years from the filing date). Then, using... 1 HNMR and 13 CNMR characterization of POSS-PEG-CHO.

[0035] 3. Preparation of decellularized porcine pericardium (DPP)

[0036] Take fresh porcine pericardium (PP) and wash it with PBS. Then, gently remove the adipose tissue from the PP. For methods of obtaining decellularized porcine pericardium (DPP), please refer to the literature. [2][3] According to the report, PP was immersed in a 0.5% (v / v) Triton X-100 solution for 12 hours, and then in a 0.5% (w / w) SDS solution for another 12 hours, both with continuous stirring at 37°C. Subsequently, the material was washed three times with deionized water for 10 minutes each time. DPP was then obtained.

[0037] 4. Preparation of glutaraldehyde-crosslinked DPP (GLUT-PP)

[0038] GLUT-PP was obtained by continuously shaking DPP in a 0.625% (w / w) glutaraldehyde solution at 37°C for 24 hours. Before testing, GLUT-PP was washed three times with deionized water for 10 minutes each time.

[0039] 5. Preparation of POSS-PEG-PP

[0040] DPP was immersed in POSS-PEG-CHO solutions of different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL) and continuously shaken at 37°C for 24 h. Five types of POSS-PEG-PP were then obtained. Before testing, the POSS-PEG-PP was washed three times with deionized water for 10 min each time.

[0041] [REFERENCES]

[0042] [1]Li C,JiangT,Zhou C.Injectable self-healingchitosan-basedPOSS-PEGhybridhydrogel aswounddressingtopromote diabeticwoundhealing[J].CarbohydratePolymers:Scientific andTechnologicalAspects ofIndustriallyImportantPolysaccharides,2023.DOI:10.1016 / j.carbpol.2022.120198

[0043] [2]FindeisenK,MorticelliL,Goecke T,etal.Towardacellularxenogeneicheartvalveprostheses:Histological and biomechanical characterization ofdecellularizedanddenzymaticallydeglycosylatedporcinepulmonaryheartvalve matrices[J].Xenotransplantation,2020,27(5).DOI:10.1111 / xen.12617.

[0044] [3] SnyderY, Jana S. Strategies for development of decellularized heart valve scaffolds fortissue engineering. Biomaterials. 2022 Sep; 288: 121675. doi: 10.1016 / j. biomaterials. 2022. 121675. Epub 2022 Jul 18. PMID: 35953330.

[0045] (II) Testing

[0046] 1. Characterization

[0047] DPP, GLUT-PP, and POSS-PEG-PP samples were precisely cut into squares (10mm x 10mm) and sandwiched between two glass slides to maintain their shape. The sandwiched samples were then freeze-dried. To further analyze the properties of these samples, several advanced characterization techniques were employed. Specifically, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used. Since the samples are non-conductive, they were sputter-coated with gold at 40mA for 60 seconds before SEM observation.

[0048] 2. Uniaxial tensile property test

[0049] DPP, GLUT-PP, and POSS-PEG-PP were cut into rectangles (30mm x 10mm) and immersed in PBS before testing. The thickness of the samples was measured at three different points using calipers. Each sample was then firmly clamped at both ends into the fixtures of a tensile testing machine. The initial length (L0) of the sample between the fixtures was recorded for later analysis. The PP was stretched at a constant speed of 20mm / min in the tensile testing machine until fracture. Force-displacement curves were recorded during this process for further analysis.

[0050] 3. Water Contact Angle (WCA) Test

[0051] DPP, GLUT-PP, and POSS-PEG-PP were cut into squares (10mm x 10mm) and rinsed three times with deionized water. The materials were then placed between two glass slides and freeze-dried. After drying, the samples were flattened onto a smooth glass slide, and the water contact angle was measured using a tensiometer.

[0052] 4. Heat shrinkage temperature test

[0053] The heat shrinkage temperature of the samples was determined using differential scanning calorimetry (DSC). DPP, GLUT-PP, and POSS-PEG-PP were cut into squares (10 mm x 10 mm) and rinsed three times with deionized water. The materials were then placed between two glass slides and freeze-dried. After drying, the samples were placed in a sealed crucible and heated from 30 °C to 120 °C at a rate of 5 °C / min under nitrogen atmosphere. The maximum endothermic peak value was recorded as the heat shrinkage temperature.

[0054] 5. Aldehyde content test

[0055] The aldehyde content of the samples was quantified using the fluorescein yellow CH assay. DPP, GLUT-PP, and POSS-PEG-PP samples were cut into circular patches. (n=6) The sample was washed three times with PBS to remove any residual contaminants. Subsequently, the sample was placed in the wells of a 96-well plate and incubated for 12 h with 500 μL of fluorescein yellow solution at a concentration of 10 μg / mL per well. After incubation, the sample was washed three times with PBS to remove unbound fluorescein yellow. The fluorescence of bound fluorescein yellow was then measured using a confocal laser scanning microscope with an excitation wavelength set to 405 nm and an emission wavelength set to 540 nm.

[0056] 6. Protein adhesion

[0057] DPP, GLUT-PP, and POSS-PEG-PP were cut into circular patches. (n=6) The samples were washed three times with PBS. The material was then placed in the wells of a 48-well plate and incubated at 37°C for 2 hours with either 1 mg / mL fluorescein isothiocyanate-conjugated bovine serum albumin (BSA-FITC) or 0.1 mg / mL fluorescein isothiocyanate-conjugated fibrinogen (FBG-FITC). After incubation, the samples were washed three times with PBS. The fluorescence of the samples was then measured using a confocal laser scanning microscope, with the excitation and absorption wavelengths set to 488 nm and 525 nm, respectively.

[0058] 7. In vitro cytotoxicity evaluation

[0059] In vitro cytotoxicity of the materials was assessed using L929 cells. DPP, GLUT-PP, and POSS-PEG-PP samples were washed three times with deionized water to remove any contaminants. The samples were then cut into small pieces, sterilized with 75% ethanol for 24 h, and then irradiated with UV for 2 h. The samples were then washed three times with PBS to remove residual ethanol. Complete culture medium was prepared using RPMI 1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. Then, 30 cm³ of cells were used to... 2The sample blocks were soaked in 10 mL of complete culture medium at 37°C for 3 days, with constant shaking to obtain the extract. L929 cells were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells / well. Once cells adhered, the medium was replaced with material extract (n=6), and complete medium was used as a negative control. Cell viability was assessed using the Cell Count Kit-8 (CCK-8) assay after incubation at 37°C and 5% CO2 atmosphere for 24 h and 72 h. For the assay, 100 μL of Dulbecco modified Eagle medium (DMEM) containing 10% (v / v) CCK-8 reagent was added to each well. After 1 h of incubation, the reaction mixture was transferred to a new 96-well plate, and absorbance was measured at 450 nm using a microplate reader. Additionally, L929 cells were seeded in confocal culture dishes and cultured as previously described. After 3 days of culture, cells were gently washed with PBS. Subsequently, cells were incubated with Calcein-AM / PI assay reagent at 37°C for 30 min. Cells were then washed again with PBS. Finally, cells were observed using a confocal laser scanning microscope to assess their viability and morphology.

[0060] 8. Growth of human umbilical vein endothelial cells

[0061] Cut DPP, GLUT-PP, and POSS-PEG-PP into circles. (n=6) and washed 3 times with PBS. Then, sterilized with 75% ethanol for 24 h, followed by UV irradiation for 2 h. Afterward, rinsed the samples 3 times with PBS to remove residual ethanol. Place the sterilized samples in the wells of a 48-well plate at a density of 1.5 × 10⁻⁶ μL per well. 4 Human umbilical vein endothelial cells (HUVECs) were seeded onto samples at a density of 10 cells / well. Cells were incubated at 37°C in a 5% CO2 atmosphere for 24 h, 72 h, and 120 h to allow adhesion and proliferation. The culture medium was changed every 24 h. After culture, cell viability was measured using the Cell Counting Kit 8 (CCK-8) assay. Each well was replaced with 300 μL of DMEM containing 10% CCK-8 reagent. After 1 h of incubation, 100 μL of the reaction mixture was transferred to a new 96-well plate, and absorbance was measured at 450 nm using a microplate reader. The remaining samples were washed with PBS, and each well was incubated with 300 μL of Calcein-AM / PI assay solution at 37°C for 30 min. After incubation, each well was rinsed with PBS, and the samples were observed under a confocal laser scanning microscope to assess cell viability and morphology.

[0062] 9. Hemolysis test

[0063] Cut DPP, GLUT-PP, and POSS-PEG-PP into circles. (n=6) and washed three times with PBS. The samples were then placed in the wells of a 48-well plate. Fresh rat arterial blood was collected using an anticoagulant vacuum tube. The blood was centrifuged at 1500 rpm for 15 min to separate the components, and the supernatant was discarded. 350 μL of the lower red blood cell suspension and 700 μL of PBS were added to each well, and the mixture was incubated together at 37°C for 2 h. Deionized water and PBS were used as positive and negative controls, respectively. After incubation, the samples were removed, and 1000 μL of the mixture was transferred to a 2 ml centrifuge tube and centrifuged at 3000 rpm for 10 min. Finally, the supernatant was transferred to a 96-well plate, and the absorbance at 541 nm was measured using a microplate reader.

[0064] 10. Recalcification of whole blood coagulation

[0065] Cut DPP, GLUT-PP, and POSS-PEG-PP into circles. (n=6) and washed three times with PBS. The samples were then placed in the wells of a 48-well plate. 500 μL of fresh rat blood containing 2% (v / v) 100 mM CaCl2 was added to the PP plate and incubated at 37°C for 1 h to allow thrombus formation. After incubation, the thrombus-containing samples were carefully transferred to new wells and washed with PBS to remove any unbound blood components. After photographing, the samples were analyzed using an LDH cytotoxicity assay kit, and absorbance was measured at 490 nm using a microplate reader.

[0066] 11. Platelet adhesion

[0067] Cut DPP, GLUT-PP, and POSS-PEG-PP into circles. (n=6) and washed three times with PBS. The samples were then placed in the wells of a 48-well plate. Fresh rat blood was collected and centrifuged at 1500 rpm for 15 min to obtain platelet-rich plasma (PRP). Subsequently, 300 μL PRP was added to the sample and incubated at 37°C for 1 h to allow platelet-sample interaction. After incubation, the samples were analyzed using an LDH cytotoxicity assay kit, and absorbance was measured at 490 nm using a microplate reader.

[0068] 12. Subcutaneous implantation trial

[0069] The animal experiments were approved by the Ethics Committee of Zhongnan Hospital, Wuhan University. Materials were cut into cubes (10mm*10mm) and rinsed three times with deionized water. The samples were then sterilized with 75% ethanol solution for 24 hours, followed by UV irradiation for 2 hours. Subsequently, the samples were rinsed five times with PBS, each rinse lasting 15 minutes in a biosafety cabinet to ensure a sterile environment. Subcutaneous implantation was performed on male Sprague Dawley rats (50±10g) housed in individual ventilated cages (IVCs). Under anesthesia with sodium pentobarbital (30mg / kg), incisions were made on the back of each rat, and samples were implanted on both sides of each incision. Six samples were implanted in three incisions per rat. After implantation, the wounds were sutured with 4-0 sutures. Samples, along with their capsules, were retrieved for further analysis at 7, 15, 30, 60, and 90 days post-implantation.

[0070] 13. Histological and immunofluorescence analysis

[0071] Samples were fixed using a 4% formaldehyde solution to preserve their structure. Subsequently, the samples were dehydrated, embedded in paraffin, and cut into 4 mm thick sections. These sections were then subjected to various staining protocols to reveal different components: hematoxylin-eosin (HE) staining for cell morphology, Masson staining for collagen fibers, 4',6-diamino-2-phenylindole (DAPI) staining for cell nuclei, and Alizarin Red S staining for calcium deposition. For specific labeling of immune cells, macrophages were labeled with CD68 antibody, and T cells were labeled with CD3 antibody. CD3 was quantified by analyzing images taken at 40x magnification. + Lymphocytes and CD68 + The proportion of macrophages.

[0072] 14. Calcium Analysis

[0073] The sample was carefully dissected and the membrane removed, then the sample was freeze-dried. The dried sample was weighed and digested at 95°C with 1 mL of 6M hydrochloric acid (HCl) for 12 h to dissolve the sample and release calcium ions. Subsequently, the supernatant was carefully filtered through a 40 μm filter, and the filtrate was diluted with deionized water for analysis. The calcium content of the sample was then quantified using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0074] 15. RNA-seq. Analysis

[0075] Two weeks after subcutaneous implantation, total RNA was collected from samples such as DPP, GLUT-PP, and POSS-PEG-PP. Total RNA was extracted using Trizol reagent. Subsequently, 3 mg of RNA was used as the starting amount for RNA sample preparation. To generate sequencing libraries, mRNA was isolated from total RNA using poly-T oligonucleotide-linked magnetic beads. Fragmentation was performed using high-temperature divalent cations in Illumina's proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and SuperScript II. Second-strand cDNA was then synthesized using DNA polymerase I and RNase H, with remaining protruding ends converted to blunt ends by exonuclease / polymerase activity and enzyme removal. After adenylation at the 3′ end of the DNA fragment, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. Library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA) to select a preferred cDNA fragment length of 400–500 bp. In 15 cycles of PCR, DNA fragments with linker molecules at both ends were selectively enriched using Illumina PCR Primer Cocktail. The products were purified (AMPure XP system) and quantified using Agilent High Sensitivity DNA Analyzer on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a NovaSeq 6000 platform (Illumina Shanghai Personal Biotechnology Co., Ltd.). For bioinformatics analysis, HTSeq was used to compare the ReadCount value of each gene as the original gene expression, and FPKM was used to normalize the expression. Differential gene expression was analyzed using DESeq2 (v1.38.3) with the following screening criteria: fold change |log2FoldChange| > 1, and a significance P-value < 0.05.

[0076] 16. Statistical Analysis

[0077] Calculate the data and display them as mean ± standard deviation. One-way ANOVA was used for statistical analysis of all data. P < 0.05 was considered statistically significant.

[0078] (III) Results

[0079] 1. Material preparation ( Figure 2 AE)

[0080] like Figure 2 As shown in A and B, the crosslinking agent POSS-PEG-CHO was synthesized according to the previously established method. 1 H NMR and 1312C NMR spectroscopy confirmed the structural integrity of POSS-PEG-CHO.

[0081] like Figure 2 As shown in Figure C, POSS-PEG-PP was prepared by reacting different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL) of POSS-PEG-CHO solution with DPP via a Schiff base reaction. The degree of crosslinking of POSS-PEG-PP at concentrations of 40 mg / mL and 50 mg / mL was not significantly different, both being lower than that of GLUT-PP. Therefore, this application selected 40 mg / mL POSS-PEG-PP for subsequent experiments.

[0082] like Figure 2 As shown in Figure D, the successful preparation of POSS-PEG-PP was verified by ATR-FTIR spectroscopy. The aldehyde absorption peak is at 1715 cm⁻¹. -1 The disappearance at 1035 cm⁻¹, and the Si-O-Si bond at 1035 cm⁻¹ -1 The CH bond is at 2882 cm⁻¹ -1 The presence of this substance confirms the reaction between POSS-PEG-CHO and DPP, and that POSS-PEG was successfully coupled with DPP.

[0083] like Figure 2 As shown in Figure E, H&E staining and DAPI staining indicate that cells were completely removed from PP, yielding DPP. Furthermore, Masson staining shows that DPP retains most of the collagen fibers found in PP. Compared to PP, the fiber arrangement in DPP exhibits a significant change, becoming looser and more disordered.

[0084] 2. Characterization ( Figure 2 F)

[0085] The surface morphology of the materials was examined using SEM. It was observed that after the crosslinking process, the fiber arrangement became more compact, and POSS-PEG-PP exhibited a denser fiber arrangement structure compared to GLUT-PP. Furthermore, both POSS-PEG-PP and GLUT-PP maintained their integrity, with almost no visible surface cracks or fiber breakage after the freeze-drying process. AFM further confirmed the surface properties of the materials.

[0086] Two-dimensional AFM images revealed denser fiber networks in POSS-PEG-PP and GLUT-PP. Three-dimensional AFM images provided a more detailed view of the surface morphology, with POSS-PEG-PP exhibiting the highest roughness (Ra = 69.1 nm), followed by GLUT-PP at 67.4 nm. In contrast, DPP showed the lowest roughness, with an Ra value of 24.3 nm. These findings indicate that the crosslinking process and the type of crosslinking agent significantly influence the surface properties of the resulting materials.

[0087] 3. Physicochemical properties ( Figure 3 )

[0088] (1) Mechanical properties:

[0089] Through the Figure 3 Analysis of A, D, and E shows that the maximum tensile strength of DPP is 7.95 ± 1.88 MPa. After crosslinking, its maximum tensile strength is significantly improved, with POSS-PEG-PP reaching 28.90 ± 2.26 MPa, significantly higher than GLUT-PP's 15.78 ± 1.30 MPa. Furthermore, the slope of the stress-strain curve (i.e., the elastic modulus) also shows an increasing trend for the crosslinked materials. Specifically, the elastic modulus of DPP is 19.45 MPa ± 3.23 MPa, while the elastic modulus of POSS-PEG-PP is as high as 97.44 MPa ± 10.87 MPa, significantly better than GLUT-PP's 28.29 MPa ± 2.55 MPa. This indicates that POSS-PEG-PP exhibits superior mechanical properties, especially in terms of maximum tensile strength and elastic modulus.

[0090] (2) Water contact angle (WCA):

[0091] like Figure 3 As shown in Figure B, WCA measurements of various materials revealed different surface properties. The WCA of DPP was 27.68° ± 0.77°. After crosslinking, the WCA of GLUT increased to 38.67° ± 2.00°, indicating enhanced hydrophobicity of its surface. Conversely, the WCA of POSS-PEG-PP decreased to 19.62° ± 0.53°, indicating that the POSS-PEG-PP surface exhibited stronger hydrophilicity.

[0092] (3) Aldehyde content:

[0093] The results are as follows Figure 3As shown in C and F, the relative fluorescence intensity of fluorescein yellow in the GLUT-PP group was significantly higher than that in the DPP group, which fully indicates that the content of residual aldehyde groups in GLUT-PP is higher. In contrast, the fluorescence intensity of the POSS-PEG-PP group was not significantly different from that of the DPP group, which suggests that the level of residual aldehyde groups in POSS-PEG-PP is lower. A lower residual aldehyde group content is beneficial to improving the cell compatibility and biocompatibility of the material.

[0094] (4) Thermal stability:

[0095] like Figure 3 As shown in Figure G, DPP exhibits relatively poor thermal stability, with a heat shrinkage temperature of 83.71℃ ± 1.47℃. Crosslinking with GLUT or POSS-PEG-PP significantly improves thermal stability, enabling it to maintain better performance and structural stability at high temperatures. The heat shrinkage temperatures of POSS-PEG-PP and GLUT-PP increased to 89.63℃ ± 3.02℃ and 93.55℃ ± 2.80℃, respectively, with no significant difference observed between the two.

[0096] 4. Cell compatibility ( Figure 4 )

[0097] To assess the cytotoxicity of the materials, L929 fibroblasts were exposed to extracts from different materials. Cell viability was assessed at two time points using a CCK8 assay: day 1 and day 3 post-exposure. Furthermore, cell survival status was assessed on day 3 using Calcein AM / PI. CCK8 results showed that ( Figure 4 C), compared with the negative control (complete culture medium), DPP and POSS-PEG-PP showed no significant difference in cell viability after 1 and 3 days of culture. In contrast, the GLUT-PP group showed significant cytotoxicity relative to the negative control, with a particularly pronounced effect observed on day 3. This indicates that GLUT-PP has significant cytotoxicity, while DPP and POSS-PEG-PP have no significant cytotoxicity. Calcein AM / PI staining images ( Figure 4 A) The results of the CCK8 assay were confirmed, further validating the cell safety of POSS-PEG-PP.

[0098] To assess the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) on materials, HUVECs were cultured on these materials, and their viability was assessed using CCK8 assay at 1, 3, and 5 days post-seeding. Furthermore, Calcein AM / PI staining was used to confirm cell viability at 3 and 5 days post-seeding. CCK8 assay results showed that ( Figure 4(D) Compared with the GLUT-PP group, both the POSS-PEG-PP group and the DPP group promoted the adhesion and proliferation of HUVECs, with the POSS-PEG-PP group showing the best performance. Calcein AM / PI staining images ( Figure 4 B) confirmed the CCK8 results, showing higher cell viability densities on both POSS-PEG-PP and DPP compared to GLUT-PP. Furthermore, images revealed endothelial cell confluence on POSS-PEG-PP at day 5. This observation is significant because it demonstrates that POSS-PEG-PP not only supports HUVEC adhesion and proliferation but also promotes monolayer cell fusion, a crucial step in functional endothelial formation.

[0099] 5. Blood compatibility ( Figure 5 )

[0100] Thrombosis is a complex process involving the nonspecific adsorption of proteins. To evaluate the protein adsorption properties of the material, adhesion experiments with bovine serum albumin (BSA) and fibrinogen (FBG) were conducted. The results showed that POSS-PEG-PP significantly resisted protein adsorption compared to other materials (see...). Figure 5 A, B, and C). Performance regarding platelet adhesion was evaluated using a lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Results showed that a large number of platelets adhered to the surfaces of DPP and GLUT-PP, while only a small number adhered to POSS-PEG-PP (see [reference needed]). Figure 5 H and I), indicating that POSS-PEG-PP has a low platelet adhesion tendency. For more comprehensive validation, a recalcified whole blood coagulation assay was performed. (See H and I). Figure 5 As shown in F and G, numerous thrombi were observed on the surfaces of DPP and GLUT-PP, while only a small number of blood cells adhered to the surface of POSS-PEG-PP, indicating that POSS-PEG-PP has superior antithrombotic properties. Furthermore, hemolysis rate (an indicator of erythrocyte compatibility) was also included in the assessment of the material's blood contact safety. The hemolysis rates of DPP, GLUT-PP, and POSS-PEG-PP all met the ISO 10993-1 standard (hemolysis rate <2%). (See...) Figure 5 (D and E).

[0101] 6. Evaluation of inflammation in the body ( Figure 6 )

[0102] Immunogenicity of biomaterials is a key factor in the failure of bio-based heart valves, with T lymphocytes and macrophages playing important roles in the immune response. To assess the in vivo immunoreactivity of the material, a subcutaneous implantation experiment was conducted in rats, with samples retrieved at 1 and 2 weeks for further analysis. The presence of T lymphocytes and macrophages was identified by positive staining for CD3 and CD68, respectively. Hematoxylin-eosin (HE) staining showed less inflammatory cell infiltration around POSS-PEG-PP compared to the other two groups. Immunohistochemical staining results indicated that, in all three groups, POSS-PEG-PP recruited more CD3-positive T cells (…). Figure 6 A and B) and CD68-positive macrophages ( Figure 6 The lowest numbers of A and C indicate lower immunogenicity. These findings suggest that POSS-PEG-PP can reduce immune rejection of implants, a desirable property for biomaterials intended for long-term in vivo implantation.

[0103] 7. Evaluation of in vivo calcification and degradation ( Figure 7 )

[0104] Calcification is one of the main factors affecting the lifespan of biological heart valves, and resistance to calcification is an important evaluation criterion for new materials used in the manufacture of artificial heart valves. Similar to in vivo inflammation assessment, in vivo calcification assessment also used a rat subcutaneous implantation model, with implants removed at 1, 2, and 3 months for further analysis. Hematoxylin-eosin (HE) staining was used to assess overall performance, alizarin red S staining and inductively coupled plasma mass spectrometry (ICP-OES) were used to detect calcium salt deposition, and Masson staining was used to assess collagen fiber degradation. Figure 7 As shown in Figure A, compared to GLUT-PP, POSS-PEG-PP exhibits good biocompatibility, with visible angiogenesis within the material. Furthermore, it demonstrates superior resistance to calcification. Figure 7 (B) Furthermore, three months after subcutaneous implantation, most of the collagen fibers remained intact. This finding is significant because it demonstrates that POSS-PEG-PP not only effectively resists calcification but also maintains the structural integrity of collagen fibers, which is crucial for the durability and functionality of bio-cardiac valves.

[0105] 8. RNA-seq analysis ( Figure 8 )

[0106] To investigate the mechanism by which POSS-PEG-PP regulates inflammation, we performed RNA-seq analysis on samples from subcutaneously implanted DPP, GLUT-PP, and POSS-PEG-PP two weeks prior. Gene heatmaps and the top 20 differentially expressed genes (DEGs) are shown below. Figure 8 As shown in A. Furthermore, the trends of the nine gene clusters are as follows:Figure 8 As shown in B, there are significant differences among the three groups. In the three group comparisons (DPP vs. GLUT-PP, DPP vs. POSS-PEG-PP, and GLUT-PP vs. POSS-PEG-PP), there are 40 common DEGs (…). Figure 8 C). Volcano map ( Figure 8 D) showed that, between DPP and POSS-PEG-PP, 196 genes were upregulated and 320 genes were downregulated in the POSS-PEG-PP group [log2(fold change)>1, P<0.05]. GO enrichment analysis indicated that DEGs between DPP and POSS-PEG-PP were significantly associated with immune system processing, immune responses, cell differentiation, complement activation, and signal receptor binding. Figure 8 E). KEGG enrichment analysis showed that DEGs between DPP and POSS-PEG-PP were significantly associated with the adenosine monophosphate-activated protein kinase (AMPK) and IL-17 signaling pathways. Figure 8 F).

[0107] (IV) Analysis and Conclusion

[0108] Because xenogeneic biomaterials are immunogenic, decellularization of porcine pericardium is crucial for the fabrication of bioprosthetic heart valves. However, this process often results in a loose fibrous structure and consequently reduced mechanical properties. Heart valves endure a complex hemodynamic environment and billions of opening and closing cycles in vivo, making it essential to enhance the mechanical properties of decellularized porcine pericardium (DPP). Traditionally, most commercially available bioprosthetic heart valves use glutaraldehyde crosslinking to enhance the mechanical strength of DPP. However, GLUT-PP exhibits poor performance in terms of cell compatibility, blood compatibility, and anti-calcification properties. Given these limitations, we chose to use POSS-PEG-CHO as the crosslinking agent for DPP to construct POSS-PEG-PP. The fibrous arrangement of POSS-PEG-PP is significantly denser than that of GLUT-PP, consistent with our observed superior mechanical properties. POSS-PEG-PP also exhibits higher maximum tensile strength and elastic modulus, enabling it to better resist blood flow stress. The superior mechanical properties of POSS-PEG-PP are likely closely related to the star-shaped eight-arm branched structure of POSS-PEG. Furthermore, POSS-PEG-PP exhibits good thermal stability, indicating its excellent structural stability.

[0109] Cell compatibility, particularly cell safety and endothelialization capacity, is crucial for bioprosthetic heart valves. If the implant material exhibits biotoxicity, such as GLUT-PP, it can lead to surrounding cell death and local inflammation. The toxicity of GLUT-PP is primarily attributed to residual aldehyde groups. In contrast, POSS-PEG-PP has been shown to have fewer residual aldehyde groups than GLUT-PP. This is due to the relatively large molecular weight of POSS-PEG-CHO, resulting in a limited number of aldehyde groups available for reaction with amino groups and insufficient cross-linking. L929 cell proliferation assays confirmed the cell safety of POSS-PEG-PP. Endothelialization of bioprosthetic heart valves is also very important, effectively preventing calcium ion accumulation and resisting fibrin and platelet adhesion. Therefore, enhancing the adhesion and proliferation of endothelial cells on the surface of cardiovascular materials is essential for reducing post-implantation complications. In our study, POSS-PEG-PP demonstrated superior performance in endothelialization. The hydrophilicity of PEG is known to promote endothelial cell adhesion and growth, supporting the enhanced endothelialization capacity observed in POSS-PEG-PP. This characteristic is particularly important because it can increase biocompatibility, reduce thrombosis and resist calcification, thereby potentially extending the lifespan of biological heart valves.

[0110] Because bio-cardiovascular valves come into direct contact with blood, blood compatibility is a crucial evaluation metric. Firstly, erythrocyte safety is an important aspect; in this study, the hemolysis rate of POSS-PEG-PP was less than 2%, indicating its safety in terms of erythrocyte interactions. Furthermore, resistance to thrombosis is essential for cardiovascular medical devices, as detached thrombi can be carried by the bloodstream and obstruct blood vessels within the body. Moreover, attached thrombi can lead to calcification and deterioration of bio-cardiovascular valves. Thrombosis is a complex process involving the activation, adhesion, and interaction of various serum proteins and blood cells. Adhesion of serum albumin (the most abundant protein in plasma) and fibrinogen (essential in thrombosis) is associated with thrombosis risk. Additionally, arterial thrombosis is primarily caused by platelet activation and adhesion. The POSS component in POSS-PEG-PP imparts unstable surface free energy to the material, thereby weakening the binding strength between proteins and platelets. Furthermore, the high surface fluidity of PEG helps resist the adsorption of plasma proteins and platelet adhesion. These properties collectively enable POSS-PEG-PP to exhibit superior antithrombotic activity compared to GLUT-PP and DPP. In summary, the combination of POSS and PEG in POSS-PEG-PP endows the material with excellent blood compatibility, making it a promising candidate material for biological heart valves.

[0111] The deterioration of bioprosthetic heart valves is closely related to immune responses, especially in xenogeneic bioprosthetic heart valves. Reducing the immunogenicity of bioprosthetic heart valves can decrease the deposition of calcifications, a key factor affecting valve lifespan. Macrophages and T lymphocytes play important roles in immune responses. Specifically, macrophages process antigens and present them to the HLA system, acting as a crucial bridge between innate and adaptive immunity. T lymphocytes are essential for adaptive immune responses and maintaining immune homeostasis. After POSS-PEG-PP implantation, macrophage and T lymphocyte infiltration was reduced, primarily due to the "stealth" effect of PEG and the low-inflammatory properties of POSS. To further investigate the mechanism by which POSS-PEG-PP modulates inflammation, we performed RNA-seq analysis on samples from DPP, GLUT-PP, and POSS-PEG-PP subcutaneously implanted two weeks later. GO enrichment analysis showed that DEGs were significantly associated with multiple aspects, including immune system processing, immune responses, cell differentiation, complement activation, and signal receptor binding. Furthermore, KEGG enrichment analysis revealed that the performance differences between DPP and POSS-PEG-PP were associated with alterations in the AMPK and IL-17 signaling pathways. The AMPK signaling pathway regulates cellular energy homeostasis and participates in many biological processes, particularly under conditions of malnutrition, hypoxia, and ischemia. On the one hand, activation of the AMPK signaling pathway can increase energy metabolism and reduce reactive oxygen species (ROS) levels, thereby reducing inflammatory factors. On the other hand, downregulation of the AMPK signaling pathway can weaken the activity of immune cells, thereby reducing the body's immune rejection response to implanted foreign bodies. The IL-17 signaling pathway is crucial in innate immunity and inflammatory responses, particularly in the recruitment and activation of neutrophils. Therefore, alterations in the AMPK and IL-17 signaling pathways play a significant role in the anti-inflammatory properties of POSS-PEG-PP.

[0112] Structural valve degeneration (SVD) is a common, unavoidable, and currently untreatable outcome after BHV implantation. It is characterized by irreversible pathological progression including granulation tissue growth, leaflet fibrosis, calcification, connective tissue delamination, and the occurrence of tears and perforations. Calcification is the most common manifestation of SVD, which can lead to BHV sclerosis, thereby affecting its opening and closing motion. The pathogenesis of BHV calcification is complex, primarily caused by inflammatory responses, immune responses, and subclinical leaflet thrombosis. In our study, POSS-PEG-PP exhibited excellent anti-calcification properties, attributed to its superior endothelialization and biocompatibility. Notably, reduced inflammation and decreased immunogenicity played a crucial role in its resistance to calcification, suggesting that POSS-PEG-PP holds promise for a longer lifespan.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an artificial bioprosthetic valve, characterized in that, Includes the following steps: S1 used phosphate buffer to remove the fatty tissue of porcine pericardium, and then successively immersed it in 0.5% v / v Triton X-100 solution and 0.5% w / w SDS solution, and shook and soaked at 37℃ for 10-15 h to obtain decellularized porcine pericardium; S2. Decellularized porcine pericardium was immersed in a 40 mg / mL POSS-PEG-CHO solution at 37°C with shaking for 24 h. The aldehyde groups in POSS-PEG-CHO and the amino groups in the decellularized porcine pericardium underwent a condensation reaction to generate carbon-nitrogen double bonds, thereby achieving cross-linking to obtain an artificial bio-valve. In the POSS-PEG-CHO, the number-average molecular weight of polyethylene glycol is 2 kDa.

2. An artificial bioprosthetic valve, characterized in that, The preparation method according to claim 1 is used to obtain it.

3. The application of the artificial bioprosthetic valve according to claim 2 in the preparation of artificial heart valves.

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