A polyurethane / small intestinal submucosa composite material and its preparation method and use

Abstract

The application provides a polyurethane / small intestinal submucosa composite material and a preparation method and application thereof, and belongs to the field of medicines. The polyurethane / small intestinal submucosa composite material with an asymmetric structure is prepared by using a freeze-drying and impregnation drying method and different structural forms of the same components. The composite material is porous sponge on one side and dense film on the other side, can effectively prevent leakage on the basis of guaranteeing mechanical properties, and performs full-layer repair. The composite material can adjust the component ratio according to different requirements, and then adjust the mechanical properties, so as to match the normal bladder mechanical behavior of different species. The preparation method of the composite material is simple and convenient, has high popularization, can be popularized to other polyurethane / extracellular matrix materials, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a polyurethane / small intestinal submucosa composite material, its preparation method, and its uses. Background Technology

[0002] To promote cell ingrowth and tissue regeneration, ideal soft tissue repair materials should possess biocompatibility, excellent mechanical properties (such as tensile strength and resilience), and a suitable three-dimensional porous structure. Currently, soft tissue repair materials are mainly divided into two categories: synthetic polymer materials and natural extracellular matrix. Synthetic materials have good physical and mechanical properties but lack biological activity; while natural materials, although possessing biocompatibility and biological activity, suffer from poor resilience and a tendency to collapse.

[0003] Among synthetic polymer materials, polyurethane (PU) is a widely used polymer in the biomedical field. Among natural materials, the submucosa of the small intestine (SIS) is a natural extracellular matrix material with good biocompatibility and biodegradability, and has been widely used in tissue engineering.

[0004] Patent CN104341608B reports a polyurethane / small intestinal submucosal composite material, the preparation method of which includes the following steps: a) mixing anionic aqueous polyurethane emulsion with small intestinal submucosal powder, and then freeze-drying under vacuum; b) cross-linking and freeze-drying under vacuum to obtain the final product. However, this material cannot meet the mechanical matching requirements of normal bladders of different species under different needs by adjusting the component ratio. Summary of the Invention

[0005] In order to address the problems existing in the prior art, the present invention aims to provide a polyurethane submucosal composite material with significantly improved mechanical properties, its preparation method, and its uses.

[0006] This invention provides a polyurethane / small intestinal submucosa composite material, which is obtained by reacting an asymmetric Janus membrane with a crosslinking agent solution, followed by washing and drying.

[0007] The asymmetric Janus membrane is obtained by immersing polyurethane / small intestinal submucosal slices in a polyurethane / small intestinal submucosal solution, followed by drying and sterilization.

[0008] The polyurethane / small intestinal submucosal solution is obtained by mixing polyurethane emulsion with small intestinal submucosal powder, wherein the mass ratio of polyurethane emulsion to small intestinal submucosal powder is 1 to 10:1.

[0009] The polyurethane / small intestinal submucosal section was obtained by freeze-drying the polyurethane / small intestinal submucosal solution, rehydrating it, freezing it, and then slicing it.

[0010] Further, the crosslinking agent solution is obtained by mixing a buffer solution and a crosslinking agent; the buffer solution is a 2-morpholine ethanesulfonic acid solution with a concentration of 20-40 mmol / L and a pH of 5-7, preferably with a concentration of 30 mmol / L and a pH of 6.5; the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

[0011] The soaking temperature is 2-6℃, and the time is 0.5-2h, preferably 4℃ and 1h; the drying temperature is 25-35℃, and the time is 20-30h, preferably 27℃ and 24h.

[0012] The mixing conditions are: stirring at a speed of 400-600 rpm at 10-40°C for 2-4 hours, preferably: stirring at a speed of 500 rpm at 15-35°C for 3 hours;

[0013] The mass ratio of the polyurethane emulsion to the submucosal powder of the small intestine is 2 to 8:1, preferably 6 to 8:1;

[0014] The freeze-drying conditions are as follows: first, store at 0 to 5°C for 10 hours or more, then pre-freeze at -30 to -10°C for at least 10 hours, and freeze-dry for 20 to 30 hours. Preferably, the conditions are: first, store at 4°C for 12 hours or more, then pre-freeze at -20°C for at least 12 hours, and freeze-dry for 24 hours.

[0015] The freezing conditions are: freezing at -90 to -70°C for at least 10 hours, preferably at -80°C for at least 12 hours.

[0016] Furthermore, the polyurethane emulsion is prepared by the following method:

[0017] (1) Prepolymerization: The hydroxyl donor, isocyanate and catalyst are reacted to obtain the prepolymer;

[0018] (2) Chain extension: Add chain extender and organic solvent to the prepolymer and heat to react;

[0019] (3) Neutralization and emulsification: Add alkali, stir well, and then add water, alkali and organic solvent to the reaction system and stir.

[0020] (4) Purification: Concentrate under reduced pressure and dialyze with deionized water to obtain the product.

[0021] Further, in step (1), the molar ratio of the hydroxyl donor to the isocyanate is 1:1 to 5;

[0022] The isocyanate is any one or more of isophorone diisocyanate, L-lysine diisocyanate, and diphenylmethane diisocyanate;

[0023] The hydroxyl donor is any one or more of polycaprolactone diol, polyethylene glycol, polytetrahydrofuran, propylene glycol, and 1,4-butanediol;

[0024] The catalyst is stannous octoate, triethanolamine, or dibutyltin dilaurate;

[0025] The reaction is carried out at a temperature of 70–80°C for 2–4 hours.

[0026] In step (2), the molar ratio of the chain extender to the hydroxyl donor in step (1) is 0.5 to 3:1;

[0027] The chain extender is any one or more of 2,2-dimethylolbutyric acid, 2,2-dimethylolpropionic acid, N-methyldiethanolamine, and diethanolamine; the organic solvent is acetone.

[0028] The reaction is carried out at a temperature of 50–60°C for 2–4 hours.

[0029] In step (3), the volume-to-mass ratio of alkali to chain extender in step (2) is 5-9:10-12; the stirring speed is 1200-1400 rpm, and the time is 1-3 hours.

[0030] The base is triethylamine or sodium hydroxide; the organic solvent is acetone.

[0031] Further, in step (1), the molar ratio of the hydroxyl donor to the isocyanate is 1:3; the isocyanate is isophorone diisocyanate; the hydroxyl donor is polycaprolactone diol; the catalyst is stannous octoate; the reaction temperature is 74°C and the reaction time is 3 hours.

[0032] In step (2), the molar ratio of the chain extender to the hydroxyl donor in step (1) is 1:1;

[0033] The chain extender is 2,2-dihydroxymethylbutyric acid;

[0034] The reaction was carried out at a temperature of 54°C for 3 hours.

[0035] In step (3), the volume-to-mass ratio of alkali to chain extender in step (2) is 7:11.11; the stirring speed is 1300 rpm and the time is 2 hours;

[0036] The base is triethylamine.

[0037] Furthermore, the submucosal powder of the small intestine is prepared by the following method:

[0038] 1) Cleaning and preparation: Take pig small intestines, clean them, and cut them into sections;

[0039] 2) Mechanical scraping: Scrape off the serous membrane, mucosa, and muscle layer tissue, soak in physiological saline, and clean.

[0040] 3) Degreasing: After washing and drying, place the product in a methanol / chloroform mixed solution for degreasing and washing;

[0041] 4) Digestion: Soak the cleaned pig small intestine in trypsin solution at 2-6℃ for 10-15 hours, then clean it.

[0042] 5) Decellularization: Soak the material in a protein denaturing detergent solution for 2–6 hours, then wash;

[0043] 6) Freeze-drying: Spread out flat and freeze-dry for later use;

[0044] 7) Ball milling: After cutting into small pieces, put them into a ball mill jar and mill them. Repeat 1 to 5 times to obtain the desired product.

[0045] Further, in step 4), the cleaned pig small intestine is soaked in trypsin solution and treated at 4°C for 12 hours, then cleaned.

[0046] In step 5), the material is soaked in a protein denaturing detergent solution for 4 hours;

[0047] In step 7), repeat 3 times.

[0048] The present invention also provides a method for preparing the above-mentioned polyurethane / small intestinal submucosa composite material, the method comprising the following steps: reacting an asymmetric Janus membrane with a crosslinking agent solution, washing, and drying to obtain the final product.

[0049] Furthermore, the reaction conditions are as follows: reaction at 30–45°C in the dark for 30–35 hours.

[0050] Furthermore, the reaction conditions are as follows: reaction at 37°C in the dark for 36 hours.

[0051] The present invention also provides the use of the above-mentioned polyurethane / small intestinal submucosa composite material in the preparation of soft tissue repair materials.

[0052] The present invention has achieved the following beneficial effects:

[0053] 1. This invention is the first to propose using a freeze-drying and impregnation-drying method to prepare polyurethane / small intestinal submucosa composite materials with different structural forms using the same components, resulting in asymmetric structures. The polyurethane / small intestinal submucosa composite material of this invention has an asymmetric structure, with one side being a porous sponge and the other a dense membrane, which can effectively prevent leakage and perform full-layer repair while ensuring mechanical properties.

[0054] 2. In the polyurethane / small intestinal submucosa composite material of the present invention, polycaprolactone diol 1000 is used as the soft segment for the synthesis of polyurethane;

[0055] 3. The polyurethane / small intestinal submucosa composite material of the present invention has relatively uniform surface pores, which avoids the influence of pore size on cell function and facilitates cell growth and enrichment;

[0056] 4. The polyurethane / small intestinal submucosa composite material of the present invention exhibits excellent biocompatibility;

[0057] 5. The polyurethane / small intestinal submucosa composite material of the present invention can achieve a strain ratio of 50%-200%. The component ratio of the composite material can be adjusted according to different needs, thereby adjusting its mechanical properties to match the normal bladder mechanical behavior of different species. It matches the bladder contraction and filling changes under normal physiological conditions in humans / pigs / rats. Among them, the PU / SIS 6:1 group and the PU / SIS 8:1 group are most matched with the mechanical behavior of normal human and rat bladders during physiological activities, and the mechanical properties are significantly improved.

[0058] 6. The polyurethane / small intestinal submucosa composite material of the present invention has excellent mechanical properties, with a tensile strength of over 1.5 MPa.

[0059] 7. The preparation method of the polyurethane / small intestinal submucosa composite material of the present invention is simple, convenient and highly scalable, and can be extended to other polyurethane / extracellular matrix materials, showing good application prospects.

[0060] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0061] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0062] Figure 1 The preparation of PU / SIS (A: Synthetic route of PU; B: 1H NMR spectrum of PU; C: Preparation process diagram of PU / SIS; D: FTIR spectra of SIS, PU before and after crosslinking, and PU / SIS).

[0063] Figure 2Morphological characterization of PU / SIS composite materials: (A) Scanning electron microscope images of the composite materials obtained in Examples 1-4, from top to bottom: PU / SIS slice, sponge surface of integrated Janus material, dense film surface of integrated Janus material, and cross-section of integrated Janus material; (B) Water contact angle test images of the dense film surface and sponge surface of integrated Janus material; Quantitative analysis of water contact angle of dense film surface (C) and sponge surface (D); (E) Swelling experiment.

[0064] Figure 3 Biocompatibility of PU / SIS composites with different ratios (smooth muscle cells were seeded into PU / SIS composites; A: live / dead staining results; B: cytoskeleton staining results; C: scanning electron microscopy (SEM) results; D: hemolysis; E: histogram of hemolysis rate).

[0065] Figure 4 Comparative data on the mechanics of different proportions of materials and pig bladders: (A) Cyclic tension; (B) Elastic modulus; (C) Ultimate strain; (D) Ultimate stress; (E) Stress-strain diagrams of pig and (F) rat bladders with different proportions of PU / SIS composite materials; (G) Nanoindentation.

[0066] Figure 5 Construction of finite element simulation models for repairing human and rat bladder defects using PU / SIS composite materials: under normal internal pressure, the maximum stress (A) and maximum strain (B) of human bladder repaired with PU / SIS composite materials; the maximum stress (C) and maximum strain (D) of rat bladder repaired with PU / SIS composite materials. Detailed Implementation

[0067] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0068] In this invention, "room temperature" refers to 25±10℃, and "overnight" refers to 12±5 hours.

[0069] Example 1: Preparation of PU / SIS composite material

[0070] 1. Synthesis of water-based PU

[0071] (1) Prepolymer Synthesis

[0072] A fixed support was erected, and after heating the oil bath to 74°C, polycaprolactone diol 1000 (PCL1000) (33.33 g, 33.33 mmol) and isophorone diisocyanate (IPDI) (21.48 g, 96.67 mmol) were added to a three-necked flask. The mixture was pre-stirred in the oil bath for 10 min, and then stannous octoate (approximately 4 drops, 0.02 mL) was added dropwise. The reaction was continued for 2 h 50 min to obtain the prepolymer. The viscosity of the system was observed during the reaction. If necessary, an appropriate amount of acetone was added to adjust the viscosity. The viscosity of the reaction system was considered acceptable if it did not accumulate on the stir bar during stirring, thus maintaining the reaction under stable conditions.

[0073] (2) Chain extension reaction

[0074] Cool the prepolymer (74℃→54℃). Weigh 4.94g (33.33mmol) of 2,2-dihydroxymethylbutyric acid (DMBA) into a small single-necked flask and add 25ml of acetone. Place the flask in a 54℃ oil bath to dissolve the prepolymer completely. Using a syringe (1ml / 5ml), slowly add the dissolved solution dropwise to the prepolymer and continue stirring for 3 hours. To ensure a smooth reaction, acetone can be used to adjust the viscosity of the system if necessary.

[0075] (3) Neutralization and emulsification

[0076] 1) Neutralization: Add triethylamine (TEA) (7 mL) to a three-necked flask and stir for 20 min at room temperature to neutralize.

[0077] 2) Emulsification: Take a 2L beaker, add 200ml of pure water, 14ml of triethylamine and 50ml of acetone, seal it with plastic wrap and tie it with a rubber band; place it under the stirring table, tear open a small opening and put in the stirring paddle, adjust the speed to 1300rpm, slowly add the reaction product from the three-necked flask to the solution in the large beaker through a syringe, and stir thoroughly and rapidly for 2 hours to obtain PU emulsion.

[0078] (4) Purification

[0079] Using a 1L single-necked flask, remove acetone by rotary evaporation (half a day), then change the water and dialyze for 2-3 days to remove small molecule organic residues, obtaining purified PU emulsion.

[0080] 2. Preparation of SIS powder

[0081] In this invention, SIS powder is prepared using the following method:

[0082] 1) Cleaning and preparation: Take fresh pig small intestines, clean them, remove the surface fat, turn the small intestines inside out, wash them clean and cut them into sections about 10cm-25cm long.

[0083] 2) Mechanical scraping: Scrape off the serosa, mucosa and muscle layer tissue, leaving only the submucosa of the pig small intestine (SIS), and repeatedly wash it in physiological saline (0.9% NaCl 9g / 1000ml diluted with pure water).

[0084] 3) Degreasing: After cleaning, the SIS membrane is blotted dry with paper towels and then placed in an equal volume of a methanol / chloroform mixture in a fume hood at room temperature overnight for degreasing. After thorough degreasing, it is repeatedly washed until there is no organic solvent odor, thus obtaining the SISI membrane.

[0085] 4) Digestion: Immerse the SIS membrane in a 0.25% trypsin solution (5L PBS + 125g trypsin) at 4℃ for 12h, and rinse repeatedly with deionized water.

[0086] 5) Decellularization: Prepare a 0.5% (5L pure water + 25g SDS) solution of SDS (sodium dodecyl sulfate), soak the SIS membrane at room temperature for 4 hours, and wash repeatedly with deionized water to remove residual reagents.

[0087] 6) Freeze-drying: Unfold and flatten the SIS membrane, and freeze-dry it in a vacuum freeze dryer for later use.

[0088] 7) Ball milling: Cut the SIS membrane into small pieces as much as possible and put it into a ball milling jar. Repeat three times: Place the ball milling jar in liquid nitrogen for 5 minutes to cool; ball milling speed 30 Hz, 5 minutes.

[0089] 8) Collect SIS powder and store it at 4℃ for later use.

[0090] 3. Preparation of PU / SIS composite materials

[0091] (1) Preparation of PU / SIS solution

[0092] The SIS powder was added to the purified PU emulsion at a mass ratio of 1:8, and stirred at 500 rpm at room temperature for 3 hours to obtain a slightly viscous and homogeneous PU / SIS solution (wherein the PU solid content was 24 wt% and the SIS solid content was 3 wt%).

[0093] (2) Preparation of PU / SIS slices (also known as sponge layer)

[0094] 1) The PU / SIS solution prepared in (1) is injected into each hole of a silicone mold (circular silicone mold with a diameter of 1 cm and 1 ml per hole) with 0.15 ml of solution. It is first stored at 4℃ for 12 h (the emulsion becomes jelly-like), then pre-frozen at -20℃ for 12 h (the jelly-like becomes ice cube-like), and then freeze-dried for 24 h for later use.

[0095] 2) After rehydrating the above-mentioned mixed material and freezing it at -80℃ for 12 hours (until the material reaches the state of ice cubes), the material was frozen and sectioned with a section thickness of 300 micrometers / section. The sections were then washed and freeze-dried to obtain PU / SIS sections.

[0096] (3) Assembly of composite materials

[0097] Frozen PU / SIS slices were placed above a PU / SIS solution and incubated at 4°C for 1 hour to ensure uniform wetting of the bottom pores. Then, they were placed in a 27°C oven for 24 hours to form an asymmetric Janus membrane. The PU / SIS slice side is referred to as the sponge side, and the other side as the dense membrane side.

[0098] (4) EDC / NHS chemical crosslinking

[0099] Each of the above-mentioned asymmetric Janus membranes was immersed in two volumes of a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS) crosslinking solution at pH 6.5 and reacted at 37°C in the dark for 36 hours. Afterwards, it was washed once with saturated saline solution for at least 30 minutes each time, and then washed at least five times with large amounts of deionized water for at least 30 minutes each time. After drying, it was sterilized with ethylene oxide to obtain the PU / SIS composite material (also known as integrated Janus material).

[0100] The crosslinking solution is prepared as follows:

[0101] 2-Morpholine ethanesulfonic acid (MES) was dissolved in pure water to prepare a 30 mmol / L MES solution. The pH of this buffer solution was adjusted to approximately 6.5 using Tris aqueous solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were weighed out at a mass ratio of 1:2.5 and added to the solution. The mixture was then added to obtain the crosslinking solution. The crosslinking solution was controlled to contain 1 g of EDC per 40 ml of solution.

[0102] Example 2: Preparation of PU / SIS composite material

[0103] Referring to the method of Example 1, the only difference is that when preparing the PU / SIS composite material, SIS powder is added to the purified PU emulsion at a mass ratio of 1:6 to obtain a PU / SIS solution with a PU solid content of 18wt% and a SIS solid content of 3wt%, and then the composite material is prepared.

[0104] Example 3: Preparation of PU / SIS composite material

[0105] Referring to the method of Example 1, the only difference is that when preparing the PU / SIS composite material, SIS powder is added to the purified PU emulsion at a mass ratio of 1:4 to obtain a PU / SIS solution with a PU solid content of 12wt% and a SIS solid content of 3wt%, and then the composite material is prepared.

[0106] Example 4: Preparation of PU / SIS composite material

[0107] Referring to the method of Example 1, the only difference is that when preparing the PU / SIS composite material, SIS powder is added to the purified PU emulsion at a mass ratio of 1:2 to obtain a PU / SIS solution with a PU solid content of 6wt% and a SIS solid content of 3wt%, and then the composite material is prepared.

[0108] The following experimental examples demonstrate the beneficial effects of the present invention.

[0109] Experimental Example 1: Structural Characterization of the PU / SIS Composite Material of the Present Invention

[0110] 1. Experimental Methods

[0111] Using 1H nuclear magnetic resonance spectroscopy, 1 The chemical structure of PU was characterized by 1H-NMR, and the structure of PU / SIS composite material was analyzed by Fourier transform infrared spectroscopy (FTIR). Attenuated total reflectance (ATR) was used to test SIS, PU before and after crosslinking, and PU / SIS, with a scan wavenumber range of 1000–3600 cm⁻¹. -1 .

[0112] Surface and cross-sectional morphology characterization of materials was performed using scanning electron microscopy (SEM): The surface and cross-sectional morphology of the PU / SIS composite material were characterized using SEM. After pretreatment, the samples underwent surface treatment using a metal coating process to improve conductivity and imaging quality. The pore size, distribution, and microstructure of the samples were observed using SEM.

[0113] Hydrophilicity of PU / SIS composite materials was assessed and quantitatively analyzed using a water contact angle test: A contact angle meter was used to test the hydrophilicity of the PU / SIS composite material. The hydrophilicity was evaluated by adding deionized water to the material surface and recording the contact angle between the water droplet and the material surface. Contact angles were measured at at least five different locations for each material group, and the average value was calculated for quantitative analysis. Materials with a contact angle less than 90° were considered hydrophilic; the smaller the contact angle, the stronger the hydrophilicity. Swelling was characterized using a swelling experiment: Material samples were immersed in deionized water for a certain period, removed, and dried with filter paper before being weighed to measure the swelling. The swelling ratio was calculated using the formula (Ws-Wd) / Wd×100, where Ws is the weight after immersion and Wd is the weight after drying. The swelling performance of the composite material was characterized by measuring the swelling rate over different time periods.

[0114] 2. Experimental Results

[0115] Figure 1 A and 1C represent the synthesis process route of PU and the preparation process diagram of PU / SIS composite materials, respectively, as shown by the 1H NMR spectrum. Figure 1 B) and FTIR plot ( Figure 1 D) The successful preparation of PU and PU / SIS were confirmed respectively.

[0116] 1H NMR spectrum Figure 1 B) Analysis showed that the signal peaks at 0.85–0.97 ppm corresponded to the chemical shifts of the methyl protons in DMBA and IPDI, confirming the presence of these monomers. The peaks at 3.99 ppm (-COOCH2-), 2.27 ppm (-CH2COO-), 1.55 ppm (-CH2CH2CH2-), and 1.29 ppm (-CH2CH2CH2-) were attributed to the methylene protons in the PCL segment, indicating the successful introduction of the PCL segment. In addition, the signal peaks at 4.00–4.21 ppm were attributed to the methylene protons (-NHCOO-CH2-) formed by the reaction of PCL and DMBA with isocyanate, further verifying the successful construction of the polyurethane structure.

[0117] FTIR spectra of SIS, PU before and after crosslinking, and PU / SIS. Figure 1 D) Display: 1651cm -1 The absorption peak at 1651 cm⁻¹ corresponds to the stretching vibration of the C=O group in the urethane group. The FTIR curve of the cross-linked PU / SIS shows a peak at 1651 cm⁻¹. -1 The absorption peak appearing at this point is a characteristic absorption peak of the amide bond formed by the crosslinking of the composite material via EDC / NHS, indicating that a chemical reaction has occurred in PU / SIS under the action of the crosslinking agent.

[0118] Scanning electron microscopy (SEM) results Figure 2 A) shows that the PU / SIS slices exhibit a trend of gradually decreasing pore size with increasing polyurethane (PU) solid content. However, the surface pore size of the composite materials with different proportions is relatively consistent, concentrated between 30 and 60 micrometers. This indicates that the preparation method of the present invention can effectively control the surface porosity of materials with different proportions, resulting in relatively uniform pore size. This consistency avoids the potential negative impact of pore size on cell function and contributes to uniform cell adhesion and proliferation. The porous structure provides an ideal microenvironment for cell adhesion and growth, promoting cell enrichment and proliferation on the material surface.

[0119] Water contact angle test results ( Figure 2 BD analysis showed that the water contact angle of all PU / SIS composites was less than 90 degrees, indicating good hydrophilicity. Hydrophilic materials are generally more conducive to cell adhesion and spread, which is particularly important for the application of biomedical materials, especially in promoting cell-material interface interactions.

[0120] In addition, the swelling rate test results ( Figure 2 E) shows that the composite materials in each group have low swelling rates, which has a positive effect on maintaining the mechanical properties of the materials. Lower swelling rates help prevent excessive expansion of the materials in body fluids, thereby maintaining their structural strength and stability.

[0121] These characteristics collectively indicate that the PU / SIS composite material of the present invention has a relatively uniform surface pore size, which helps the uniform attachment and proliferation of cells, promotes the enrichment and proliferation of cells on the material surface, and has good hydrophilicity and structural stability, making it an ideal tissue repair material.

[0122] Experimental Example 2: Biocompatibility Test of the PU / SIS Composite Material of the Present Invention

[0123] 1. Experimental Methods

[0124] The biocompatibility of the composite material was tested using cell liveness assays, cytoskeleton assays, and scanning electron microscopy.

[0125] (1) Cell live / dead experiment

[0126] To assess the cytotoxicity of the PU / SIS composite material, a Live / Dead staining method was used to detect cell viability. Sterilized material was placed at the bottom of 24-well plates, hydrated with SMC medium, and then inoculated with smooth muscle cells, with the medium being changed periodically. At 3 and 5 days, the material was removed and stained with propidium iodide (PI) and Calcein AM dyes for live and dead cell analysis. Cell morphology was observed using two-photon confocal microscopy; the FITC channel showed live cells (green fluorescence), and the TRITC channel showed dead cells (red fluorescence). By analyzing the live / dead cell ratio at different time points, the direct toxicity and cell viability of the material could be assessed.

[0127] (2) Cytoskeleton staining experiment

[0128] To further investigate the cellular state on PU / SIS materials, cytoskeleton staining experiments were performed. Sterile PU / SIS materials were seeded with smooth muscle cells, and staining was performed on days 3 and 5. After cell fixation, the cells were perforated and stained with CY5-labeled phalloidin to observe actin microfilaments in the cytoskeleton. The cytoskeleton structure (red fluorescence signal) was displayed using the CY5 channel of a laser confocal microscope (excitation wavelength 640 nm), allowing for analysis of cell adhesion, proliferation, and morphological changes on the material surface.

[0129] (3) Scanning electron microscopy experiment

[0130] To further investigate cell growth on the PU / SIS composite material, scanning electron microscopy (SEM) was used to characterize the cells on the material. After seeding the material with smooth muscle cells, fixation and freeze-drying were performed on days 3 and 5, respectively. Subsequently, the samples were sputter-coated with gold and observed using field emission scanning electron microscopy.

[0131] (4) Hemolysis test

[0132] In the hemolysis experiment, the test materials were first sterilized and pretreated in PBS at 37°C for 30 min. Anticoagulated rabbit whole blood was collected, centrifuged to remove the supernatant, and then repeatedly washed and centrifuged with PBS solution. The blood was then diluted to 5% rabbit diluted blood. The diluted blood was added to each sample; the positive control group received 1% Tritium-100, and the negative control group received PBS. The samples were incubated at 37°C for 1 hour. After incubation, the supernatant was separated by centrifugation, and the absorbance was measured at 545 nm using a spectrophotometer. The hemolysis rate was calculated by comparing the absorbance with the positive and negative control groups to assess the blood compatibility of the material.

[0133] 2. Experimental Results

[0134] Live / Dead staining results Figure 3A) shows that all components of the PU / SIS composite exhibit good biocompatibility. Live cells (green fluorescence) predominate, while dead cells (red fluorescence) are minimal, indicating low cytotoxicity of the material. The survival rate of smooth muscle cells on the material surface is high, and the number of live cells increases significantly with prolonged culture time, demonstrating the material's good support for cell proliferation and proving that the PU / SIS composite has strong cell compatibility.

[0135] Cytoskeleton staining results ( Figure 3 B) The biocompatibility of the PU / SIS composite material was further verified. Actin microfilaments (red fluorescence) in the cytoskeleton were clearly visible, and the cells exhibited good morphology, indicating good adhesion and spreading of cells on the material surface. With prolonged culture time, the distribution of the cytoskeleton became denser, indicating good cell proliferation on the PU / SIS material, further demonstrating the material's ability to promote cell adhesion and proliferation.

[0136] Scanning electron microscopy (SEM) results Figure 3 C) shows that a large number of smooth muscle cells grew on the surface of all PU / SIS composite materials. The cells were morphologically intact and had good extensibility, indicating that the porous structure of the material surface is conducive to cell growth and proliferation. The microstructure of the material provides a good adhesion environment for cells, further supporting its biocompatibility.

[0137] Hemolysis assays were used to assess whether the PU / SIS composite material would cause erythrocyte rupture, thereby determining the impact of the composite material on blood components, including erythrocytes, complement system, clotting proteins, and the activity of various enzymes. Experimental results ( Figure 3 (D, E) shows that the hemolysis rate of each PU / SIS ratio is less than 5%, indicating good blood compatibility.

[0138] In summary, the PU / SIS composite material can effectively promote cell adhesion and proliferation, demonstrating excellent biocompatibility.

[0139] Experimental Example 3: Mechanical Matching Test of the PU / SIS Composite Material of the Present Invention with a Normal Bladder

[0140] 1. Experimental Methods

[0141] Stress-strain curves, ultimate stress, and ultimate strain of composite materials, pig bladders, and rat bladders were obtained by in vitro dynamic testing using a universal tensile testing instrument.

[0142] Bladder dynamic mechanical properties testing: Normal pig and rat bladders and PU / SIS scaffold materials from each group were prepared into strip-shaped specimens of 75x4x2mm, 20x4x2mm, and 20x4x2mm, respectively. Three samples were taken from each group, and cyclic tensile and uniaxial tensile tests were performed using a universal mechanical testing system.

[0143] Cyclic tensile test: At room temperature, the tensile rate is 10 mm / min, the tensile strain is 50% of the specimen length, and the number of cycles is 10. Uniaxial tensile test: At room temperature, the tensile rate is 10 mm / min, the clamping length is 10 mm, and the tensile strength, tensile modulus, and elongation at break of each sample are tested.

[0144] The surface micro-stiffness of composite materials was obtained through nanoindentation experiments: The surface micro-stiffness of PU / SIS composite materials was measured using a nanoindenter. Mechanical parameters such as Young's modulus were calculated by analyzing the indentation data.

[0145] A finite element method was used to establish simulation models of human and mouse bladder repair: Based on MRI image data, a three-dimensional model of a healthy bladder was reconstructed using Mimics software. Combined with in vitro dynamic data, finite element simulation models of a healthy bladder and a bladder repaired with composite materials were established using ANSYS software to obtain the stress and strain distribution during the material repair process.

[0146] 2. Experimental Results

[0147] The mechanical properties of PU / SIS composite materials were evaluated through in vitro kinetic testing. Pig bladder, rat bladder, and the PU / SIS composite material were selected for the experiment. The experiment first subjected the bladder tissue and composite material to 10 cycles of cyclic tensile and uniaxial tensile testing to obtain their stress-strain curves. The stress-strain curves generated by cyclic tensile testing can reflect the viscoelastic properties of the material. The results (…) Figure 4 A) The curves of the PU / SIS 4:1 and PU / SIS 6:1 groups are closest to the mechanical behavior of normal pig bladders.

[0148] With increasing PU solid content, the mechanical properties of the material showed an upward trend. The elastic modulus of the PU / SIS 2:1 group was significantly lower than that of pig bladder, while the elastic modulus of the PU / SIS 8:1 group was significantly higher than that of pig bladder. In contrast, the elastic modulus of the PU / SIS 4:1 and PU / SIS 6:1 groups did not differ significantly from that of pig bladder, indicating that these two composite materials were well-matched with pig bladder in terms of elasticity. Figure 4 B). Furthermore, the ultimate stress and ultimate strain of the PU / SIS 8:1 group were not significantly different from those of the pig bladder. Figure 4 C, D).

[0149] The slope of the stress-strain curve increases with increasing PU solid content, indicating that both the ultimate stress and ultimate strain increase accordingly. This result shows that PU / SIS composites can achieve mechanical matching with pig and rat bladders by adjusting different ratios of PU and SIS. Figure 4 E, F). Nanoindentation test results ( Figure 4 G) Consistent with the above findings, this further verifies the mechanical properties of different PU / SIS composite materials.

[0150] Based on MRI image data and uniaxial tensile and cyclic tensile experimental data, finite element simulation models of healthy bladders and repaired bladders were established. Figure 5 This study used finite element analysis (FEM) to simulate and compare the deformation behavior of healthy and repaired bladders under physiological loads. The FEM effectively evaluated the biomechanical compatibility of different PU / SIS composite materials in the human body. The results showed that the simulation results of the PU / SIS 6:1 and PU / SIS 8:1 groups best matched the mechanical behavior of normal human and rat bladders during physiological activities, especially during bladder filling and contraction. These two material groups effectively simulated the deformation characteristics and load-bearing capacity of normal bladders.

[0151] The above experimental results show that the polyurethane / small intestinal submucosa composite material prepared by this invention can achieve a strain ratio of 50%-200%. The component ratio of the composite material can be adjusted according to different needs, thereby adjusting its mechanical properties to match the normal bladder mechanical behavior of different species. It matches the bladder contraction and filling changes under normal physiological conditions in humans, pigs, and rats. Among them, the PU / SIS 6:1 group and the PU / SIS 8:1 group best match the mechanical behavior of normal human and rat bladders during physiological activities, and the mechanical properties are significantly improved.

[0152] In summary, this invention provides a polyurethane / small intestinal submucosal composite material, its preparation method, and its applications. This invention utilizes a freeze-drying and impregnation-drying method to prepare a polyurethane / small intestinal submucosal composite material with an asymmetric structure using different structural forms of the same components. One side of this composite material is a porous sponge, and the other side is a dense membrane, which can effectively prevent leakage and perform full-layer repair while ensuring mechanical properties. The component ratios of this composite material can be adjusted according to different needs, thereby regulating its mechanical properties to match the normal bladder mechanical behavior of different species. The preparation method of this composite material is simple, convenient, and highly scalable, and can be extended to other polyurethane / extracellular matrix materials, showing promising application prospects.

Claims

1. A polyurethane / small intestinal submucosa composite material for bladder repair, characterized in that, It is an asymmetric structure obtained by reacting an asymmetric Janus membrane with a crosslinking agent solution, washing, and drying, with one side being a porous sponge and the other side being a dense membrane; The asymmetric Janus membrane is obtained by immersing polyurethane / small intestinal submucosal slices in a polyurethane / small intestinal submucosal solution, followed by drying and sterilization. The polyurethane / small intestinal submucosal solution is obtained by mixing a polyurethane emulsion with polycaprolactone diol 1000 as the soft segment with small intestinal submucosal powder, wherein the mass ratio of polyurethane emulsion to small intestinal submucosal powder is 6:

1. The polyurethane emulsion is prepared by the following method: (1) Prepolymerization: Polycaprolactone diol 1000, isophorone diisocyanate and catalyst are reacted at 70~80℃ to obtain the prepolymer; (2) Chain extension: Add chain extender and organic solvent to the prepolymer and heat to react; (3) Neutralization and emulsification: Add alkali, stir well, and then add water, alkali and organic solvent to the reaction system and stir. (4) Purification: Concentrate under reduced pressure, dialyze with deionized water, and obtain the product; The polyurethane / small intestinal submucosal section was obtained by freeze-drying the polyurethane / small intestinal submucosal solution, rehydrating it, freezing it, and then slicing it.

2. The polyurethane / small intestinal submucosa composite material according to claim 1, characterized in that, The crosslinking agent solution is obtained by mixing a buffer solution and a crosslinking agent; the buffer solution is a 2-morpholine ethanesulfonic acid solution with a concentration of 20-40 mmol / L and a pH of 5-7; the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The soaking temperature is 2~6℃ and the time is 0.5~2h; the drying temperature is 25~35℃ and the time is 20~30h. The mixing conditions are: stirring at 400-600 rpm at 10-40°C for 2-4 hours; The freeze-drying conditions are as follows: first, store at 0~5℃ for 10 hours or more, then pre-freeze at -30~-10℃ for at least 10 hours, and freeze-dry for 20~30 hours; The freezing conditions are: freeze at -90~-70℃ for at least 10 hours.

3. The polyurethane / small intestinal submucosa composite material according to claim 2, characterized in that, The buffer solution has a concentration of 30 mmol / L and a pH of 6.5; The soaking temperature is 4°C and the time is 1 hour; the drying temperature is 27°C and the time is 24 hours. The mixing conditions are: stirring at 500 rpm at 15~35℃ for 3 hours; The freeze-drying conditions are as follows: first, store at 4°C for 12 hours or more, then pre-freeze at -20°C for at least 12 hours, and freeze-dry for 24 hours; The freezing conditions are: freeze at -80°C for at least 12 hours.

4. The polyurethane / small intestinal submucosa composite material according to claim 1, characterized in that, In step (1), the molar ratio of polycaprolactone diol 1000 to isocyanate is 1:1~5; The catalyst is stannous octoate, triethanolamine, or dibutyltin dilaurate; The reaction time is 2-4 hours; In step (2), the molar ratio of the chain extender to polycaprolactone diol 1000 in step (1) is 0.5~3:1; The chain extender is any one or more of 2,2-dimethylolbutyric acid, 2,2-dimethylolpropionic acid, N-methyldiethanolamine, and diethanolamine; the organic solvent is acetone. The reaction is carried out at a temperature of 50-60°C for 2-4 hours. In step (3), the volume-to-mass ratio of alkali to chain extender in step (2) is 5~9:10~12; the stirring speed is 1200~1400 rpm, and the time is 1~3 hours; The base is triethylamine or sodium hydroxide; the organic solvent is acetone.

5. The polyurethane / small intestinal submucosa composite material according to claim 4, characterized in that, In step (1), the molar ratio of polycaprolactone diol 1000 to isocyanate is 1:3; the isocyanate is isophorone diisocyanate; the catalyst is stannous octoate; the reaction temperature is 74°C and the reaction time is 3 hours. In step (2), the molar ratio of the chain extender to polycaprolactone diol 1000 in step (1) is 1:1; The chain extender is 2,2-dihydroxymethylbutyric acid; The reaction was carried out at a temperature of 54°C for 3 hours. In step (3), the volume-to-mass ratio of alkali to chain extender in step (2) is 7:11.11; the stirring speed is 1300 rpm and the time is 2 hours; The base is triethylamine.

6. The polyurethane / small intestinal submucosa composite material according to claim 1, characterized in that, The submucosal powder of the small intestine is prepared by the following method: 1) Cleaning and preparation: Take pig small intestines, clean them, and cut them into sections; 2) Mechanical scraping: Scrape off the serous membrane, mucosa, and muscle layer tissue, soak in physiological saline, and clean. 3) Degreasing: After washing and drying, place the product in a methanol / chloroform mixed solution for degreasing and washing; 4) Digestion: Soak the cleaned pig small intestine in trypsin solution at 2-6℃ for 10-15 hours, then clean it. 5) Decellularization: Soak the material in a protein denaturing detergent solution for 2-6 hours, then wash; 6) Freeze-drying: Spread out flat and freeze-dry for later use; 7) Ball milling: After cutting into small pieces, put them into a ball mill jar and mill them. Repeat 1 to 5 times to obtain the desired product.

7. A method for preparing the polyurethane / small intestinal submucosa composite material according to any one of claims 1 to 6, characterized in that, The method includes the following steps: reacting an asymmetric Janus membrane with a crosslinking agent solution, washing, and drying to obtain the final product.

8. The method according to claim 7, characterized in that, The reaction conditions are: reacting at 30~45℃ in the dark for 30~35 hours.

9. The method according to claim 8, characterized in that, The reaction conditions were: reaction at 37°C in the dark for 36 hours.

10. Use of the polyurethane / small intestinal submucosa composite material according to any one of claims 1 to 6 in the preparation of bladder repair materials.

Images