A three-dimensional scaffold for cell-selective adhesion based on chirality and peptides, its preparation method and application

Abstract

This invention relates to a three-dimensional scaffold based on chirality and peptides for selective cell adhesion, its preparation method, and its applications, relating to the field of medical products. The three-dimensional scaffold is a stacked, three-dimensional ordered structure, with each layer comprising several ordered polyelectrolyte multilayer membrane soft carriers arranged at an angle between adjacent layers. Each layer of the polyelectrolyte multilayer membrane is formed by alternating deposition of positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes on the soft carriers, followed by modification with peptides. The two peptides in adjacent polyelectrolyte multilayer membranes are different and are used to promote the specific adhesion of different cells. The three-dimensional scaffold provided by this invention has specific adhesion properties, thereby avoiding excessive cell proliferation in incorrect locations and helping to prevent problems such as re-injury and stenosis of artificial blood vessels.

Description

Technical Field

[0001] This invention relates to the technical field of medical products, specifically to a three-dimensional scaffold based on chirality and polypeptides for selective cell adhesion, its preparation method, and its application. Background Technology

[0002] Tissue engineering scaffolds provide external support for cell growth and serve as a platform for the exchange of matter and energy. However, due to limitations in fabrication technology, biocompatibility, and surface-specific modifications, constructing chemically and biologically specific three-dimensional scaffolds within the scaffold and mimicking their in vivo growth environment has become a significant challenge in this field.

[0003] Previous studies have found that peptides with specific sequences or chiral molecules can specifically regulate cell adhesion. However, most studies have been limited to surface modification of two-dimensional planar or thin-film materials. Therefore, there is an urgent need to develop a strategy that is mild in experimental conditions, simple to fabricate, and uses universal materials to achieve selective cell adhesion, i.e., to achieve three-dimensional culture and selective cell adhesion by introducing active sites at specific locations on a scaffold.

[0004] Cardiovascular disease is a major killer threatening human health today. Artificial blood vessels with a diameter of less than 4mm often experience restenosis, embolism, and other damage after implantation, leading to implantation failure. These damages are usually caused by excessive proliferation of cells in the wrong location, with competition between endothelial cells and smooth muscle cells in the blood vessel wall being the main reason.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] Purpose of the invention

[0007] To address the problem of erroneous proliferation in existing technologies, the present invention aims to provide a three-dimensional scaffold for cell selective adhesion based on chirality and peptides, its preparation method, and its application.

[0008] This invention modifies two peptides (such as REDV and VAPG) and introduces chiral molecules (such as PDL). Through the synergistic effect of peptides promoting cell adhesion and chiral molecules inhibiting cell adhesion, it achieves the same three-dimensional scaffold, promoting endothelial cell growth while inhibiting smooth muscle cell growth, or promoting smooth muscle cell growth while inhibiting endothelial cell growth, preventing excessive cell proliferation in the wrong location. This is of great significance to the development of tissue engineering scaffolds.

[0009] Solution

[0010] To achieve the objectives of this invention, in a first aspect, this invention provides a three-dimensional scaffold for selective cell adhesion based on chirality and peptides. This scaffold is a stacked, three-dimensional ordered structure, with each layer comprising several ordered polyelectrolyte multilayer membrane soft carriers arranged at an angle between adjacent layers. Each layer of the polyelectrolyte multilayer membrane soft carrier is formed by alternating deposition of positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes on the soft carrier through multiple depositions followed by peptide modification.

[0011] The two polypeptides in adjacent polyelectrolyte multilayer membranes are different and each promotes the specific adhesion of different cells.

[0012] The two specific recognition groups in the adjacent polyelectrolyte multilayer membranes are different and are a pair of host-guest interaction groups that can achieve supramolecular self-assembly.

[0013] Furthermore, the polypeptide is grafted into the polyelectrolyte multilayer membrane using simple adsorption, click chemistry, or azide and thiol reaction.

[0014] Furthermore, the soft carrier includes a soft material doped with magnetic nanoparticles. Optionally, the soft material is selected from one or more of polydimethylsiloxane (PDMS), polyethylene glycol diacrylate (PEGDA), methacrylic anhydride gelatin (GelMA), and collagen.

[0015] Further, the polyelectrolyte is selected from one or more of hyaluronic acid modified with C=C double bond groups and carboxylated chitosan modified with C=C double bond groups; optionally, the raw material used for C=C double bond group modification is one or more of methacrylic anhydride, methacrylic acid, and acrylic anhydride.

[0016] Furthermore, it also includes a substrate for supporting the polyelectrolyte multilayer film. The substrate is a polyelectrolyte multilayer film in which positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes are deposited alternately on the substrate through multiple cycles. The substrate material is selected from one or more of quartz, silicon, and titanium.

[0017] Further, the specific recognition group includes a host-guest interaction group; optionally, the host-guest interaction group is selected from one or more of β-cyclodextrin, cucurbita, adamantane, azobenzene, cholesterol, and anthracene; optionally, β-cyclodextrin is the host group (optionally the raw material used is 6-NH2-β-CD), and the corresponding guest group is adamantane (optionally the raw material used is adamantaneacetic acid), azobenzene, or cholesterol; optionally, cucurbita is the host group, and the corresponding guest group is anthracene.

[0018] Further, the chiral molecule is polydextral lysine, and optionally, the molecular weight of the chiral molecule is 70,000 to 150,000.

[0019] Furthermore, the polyelectrolyte multilayer film is formed by alternating deposition of positively charged chiral molecules modified with double bond groups and specific recognition groups and negatively charged supramolecular polyelectrolytes on a soft carrier for n or n+0.5 cycles; wherein, one cycle is the deposition of positively charged chiral molecules and negatively charged supramolecular polyelectrolytes once each, and n is optionally an integer, optionally n≥35, optionally n≥40, and optionally n+0.5 is the deposition of another layer of positively charged chiral molecules based on the nth cycle; optionally, n in two adjacent polyelectrolyte multilayer films is independently selected from an integer n≥35;

[0020] Optionally, the outermost layers of two adjacent polyelectrolyte multilayer films are respectively a positively charged chiral molecule grafted with a specific recognition group and a negatively charged supramolecular polyelectrolyte, which are used to connect the two adjacent polyelectrolyte multilayer films through positive and negative charges.

[0021] And / or, the multilayer membrane soft carrier includes at least two types, namely a host polyelectrolyte with host-guest interaction groups deposited thereon and a guest polyelectrolyte, and the deposition periods of the two types of multilayer membrane soft carriers are n and n+0.5, respectively.

[0022] Optionally, the polyelectrolyte multilayer membrane is selected from (PDL / MA-HA-Ad) membranes further modified with peptides. 40 (PDL / MA-HA-CD) 40.5 One or more of the multilayer PDMS films;

[0023] Optionally, the polyelectrolyte multilayer membrane is selected from (PDL / REDV-HA-Ad). 40 (PDL / VAPG-HA-CD) 40.5 One or more of the multilayer PDMS films;

[0024] Optionally, it also includes a substrate, said substrate being modified with (PDL / CCS-CD). 40.5 Quartz plates.

[0025] Furthermore, the polypeptide includes one or more of the following: REDV polypeptide, VAPG polypeptide, GFOGER polypeptide, PAP polypeptide, KHI polypeptide, and IKVAV polypeptide.

[0026] Furthermore, the cells include one or more pairs of specific adhesions between human umbilical vein endothelial cells and rabbit aortic smooth muscle cells, human bone marrow mesenchymal stem cells and mouse mononuclear macrophage leukemia cells, or human neural progenitor cells and astrocytes.

[0027] Furthermore, the first layer is modified with (PDL / REDV-HA-Ad). 40 The second layer of the multilayer PDMS membrane is modified with (PDL / VAPG-HA-CD). 40.5 The PDMS multilayer film has a third layer modified with (PDL / REDV-HA-Ad). 40 PDMS in multilayer films.

[0028] Secondly, a method for preparing a three-dimensional scaffold based on chirality and peptides for selective cell adhesion is provided, comprising the following steps:

[0029] 1) Prepare negatively charged supramolecular polyelectrolytes with host-guest interaction groups and C=C double bonds, wherein the host and guest of the host-guest interaction groups are modified on different polyelectrolytes respectively;

[0030] 2) The soft carrier is sequentially deposited into a film in a positively charged chiral molecular solution and then in a negatively charged supramolecular polyelectrolyte solution from step 1), and the process is repeated for n or n+0.5 cycles to prepare (chiral molecules / polyelectrolytes with host-guest interaction groups and C=C double bonds). n A multilayer membrane soft carrier, wherein a cycle consists of the sequential deposition of positively charged chiral molecules and negatively charged supramolecular polyelectrolytes, where n is optionally an integer, optionally n≥35, optionally n≥40, and optionally n+0.5 is the deposition of another layer of positively charged chiral molecules on the basis of the nth cycle;

[0031] Among them, the multilayer membrane soft carrier includes at least two types, and the supramolecular polyelectrolytes of the two types of multilayer membrane soft carriers are the host group modified with host-guest interaction groups and the guest group modified with supramolecular polyelectrolytes, respectively, and the deposition periods of the two types of multilayer membrane soft carriers are n and n+0.5, respectively.

[0032] 3) The multilayer membrane soft carrier of step 2) is grafted with peptides through simple adsorption, azide and thiol reaction or click chemical reaction to obtain polyelectrolyte multilayer membranes.

[0033] 4) A three-dimensional scaffold is constructed using multilayer membrane soft carriers that can interact with each other through host-guest interactions. The peptides and polyelectrolytes in adjacent multilayer membrane soft carriers have different host-guest interaction groups.

[0034] Further, in step 1), the polyelectrolyte is selected from one or more of hyaluronic acid modified with C=C double bond groups and carboxylated chitosan modified with C=C double bond groups; optionally, the raw material used for C=C double bond group modification is one or more of methacrylic anhydride, methacrylic acid, and acrylic anhydride; optionally, the concentration of the polyelectrolyte is 0.5-2 g / L.

[0035] And / or, in step 1), the specific recognition group includes a host-guest interaction group; optionally, the host-guest interaction group is selected from one or more of β-cyclodextrin, cucurbita, adamantane, azobenzene, cholesterol, and anthracene; optionally, β-cyclodextrin is the host group (optionally the raw material used is 6-NH2-β-CD), and the corresponding guest group is adamantane (optionally the raw material used is adamantaneacetic acid), azobenzene, or cholesterol; optionally, cucurbita is the host group, and the corresponding guest group is anthracene.

[0036] And / or, in step 2), the chiral molecule is polydextral lysine; optionally, the concentration of the chiral molecule is 0.1 to 1 g / L, optionally 0.5 to 1 g / L.

[0037] Optionally, in step 2), the multilayer membrane soft carrier is (PDL / MA-HA-Ad). 40 (PDL / MA-HA-CD) 40.5 One or more of the multilayer PDMS films.

[0038] Optionally, in step 3), the polypeptide includes one or more of the following: REDV polypeptide, VAPG polypeptide, GFOGER polypeptide, PAP polypeptide, KHI polypeptide, and IKVAV polypeptide; optionally, the concentration of the polypeptide is 0.5–2 g / L, or optionally 1–2 g / L.

[0039] Optionally, in step 3), the polyelectrolyte multilayer membrane is selected from (PDL / REDV-HA-Ad). 40 (PDL / VAPG-HA-CD) 40.5 Multilayer PDMS.

[0040] Furthermore, it also includes a substrate for supporting the polyelectrolyte multilayer film. The substrate is a polyelectrolyte multilayer film in which positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes are alternately deposited on the substrate through multiple cycles. The substrate material is selected from one or more of quartz, silicon, and titanium. Optionally, the substrate is modified with (PDL / CCS-CD). 40.5 Quartz plates

[0041] Thirdly, the application of the chiral and polypeptide-based cell-selective adhesion three-dimensional scaffold described in the first aspect or prepared by the method described in the second aspect in the preparation of soft tissue repair products, bone regeneration devices and / or nerve repair products, wherein the soft tissue repair products optionally include artificial vascular scaffolds.

[0042] In the first, second, or third aspect described above, the polypeptides in adjacent polyelectrolyte multilayer membranes are REDV polypeptide and VAPG polypeptide, respectively; optionally, the REDV polypeptide is CREDV polypeptide, and the cells used to promote specific adhesion are endothelial cells; optionally, the VAPG polypeptide is CVAPG polypeptide, and the cells used to promote specific adhesion are smooth muscle cells.

[0043] Beneficial effects

[0044] (1) This invention achieves three-dimensional selective adhesion of various cells through the synergistic effect of PDL chiral molecules and peptides. Positively charged chiral molecules (such as PDL) and negatively charged polyelectrolytes (such as MA-HA-Ad / MA-HA-CD) in a polyelectrolyte multilayer membrane are adsorbed layer by layer through electrostatic interaction to form a multilayer membrane. Then, peptides are grafted onto the double bonds on the polyelectrolytes through click chemistry (other grafting methods can also be used). In this way, chiral molecules (such as PDL), host-guest interaction molecules (such as CD, Ad) and peptides (such as REDV and VAPG) are modified on the surface of the three-dimensional scaffold soft carrier.

[0045] (2) The present invention modifies two or more polypeptides (REDV and VAPG, etc.) and introduces PDL chiral molecules. Through the synergistic effect of polypeptide promoting cell adhesion and chiral molecules inhibiting cell adhesion, the invention achieves the same three-dimensional scaffold, which promotes endothelial cells and inhibits smooth muscle cell growth, or promotes smooth muscle cells and inhibits endothelial cell growth.

[0046] (3) The three-dimensional scaffold provided by this invention comprises a soft carrier, chiral molecules coated on the surface of the soft carrier, a polyelectrolyte, biocompatible host and guest molecules disposed on the surface of the soft carrier, and REDV and VAPG polypeptides grafted onto the surface of the soft carrier via click chemistry. This enables the three-dimensional scaffold provided by this invention to have specific adhesion to human umbilical vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (CCC-SMC-1), thereby avoiding excessive cell proliferation in incorrect locations and helping to prevent problems such as re-injury and stenosis of artificial blood vessels. Furthermore, this scaffold is simple to prepare, low in cost, and has mild usage conditions, possessing significant clinical value and economic and social benefits. The preparation method of the above-mentioned three-dimensional scaffold provided by this invention is simple in process, convenient in operation, and suitable for widespread application. Attached Figure Description

[0047] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0048] Figure 1 This is a structural simulation diagram of the three-dimensional tissue engineering scaffold of Embodiment 8 of the present invention, wherein 1 represents the first layer (PDL / REDV-HA-Ad). 40 Multilayer PDMS membrane, 2 is the second layer (PDL / VAPG-HA-CD) 40.5 PDMS in multilayer membranes, where 3 is the third layer (PDL / REDV-HA-Ad). 40 PDMS in multilayer films.

[0049] Figure 2 This is a simplified diagram of the construction process of the three-dimensional tissue engineering scaffold in Embodiment 8 of the present invention.

[0050] Figure 3 These are the biocompatibility test results of MA-HA-CD and MA-HA-Ad in Experimental Example 1 of this invention.

[0051] Figure 4 These are the experimental results of cell adhesion in Experimental Example 2 of the present invention, where a represents the adhesion of endothelial cells to different polyelectrolyte multilayer membranes within 48 hours; and b represents the adhesion of smooth muscle cells to different polyelectrolyte multilayer membranes within 48 hours.

[0052] Figure 5 This is the experimental result of cell viability in Experimental Example 2 of the present invention.

[0053] Figure 6 These are the experimental results of cell viability in the three-dimensional scaffold of Experimental Example 3 of the present invention. a is a schematic diagram and fluorescence image of endothelial cell adhesion after 48 hours of culture on the three-dimensional scaffold; b is a schematic diagram and fluorescence image of smooth muscle cell adhesion after 48 hours of culture on the three-dimensional scaffold; c is a schematic diagram and fluorescence image of endothelial cell and smooth muscle cell adhesion after 48 hours of co-culture on the three-dimensional scaffold. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0055] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0056] Example 1: Fabrication of three-dimensional scaffold soft substrates using magnetic PDMS, PEGDA, or GelMA

[0057] In this embodiment, PDMS is used as the soft substrate of the three-dimensional scaffold: 10g of PDMS prepolymer liquid and 1g of curing agent (SYLGARD) are weighed. TM 184Silicone Elastomer Curing Agent (commercially available, for use with PDMS) and 100mg Fe3O4 magnetic nanoparticles were mixed and stirred for 20 minutes, then vacuumed for 30 minutes. After that, the mixture was sandwiched between two glass sheets consisting of two 200μm thick cover glass sheets at each end, and heated at 65℃ for 4 hours to obtain a 200μm thick PDMS film.

[0058] The PDMS film described above was cut into 2mm×2mm×200μm pieces, embedded in resin and frozen. The pieces were then cut into 2mm×200μm×100μm three-dimensional scaffold soft carriers (hereinafter referred to as soft carriers) using a cryostat for the construction of three-dimensional scaffolds.

[0059] Example 2: Synthesis of methacrylated hyaluronic acid (MA-HA):

[0060] Prepare a mixed solvent by mixing N,N-dimethylformamide (DMF) and deionized water at a volume ratio of 1:2. Then, dissolve 1g of hyaluronic acid (HA) in 100mL of the mixed solvent and place the solution in an ice-water bath at 4℃. Adjust the pH of the solution to between 8 and 9 with 5M sodium hydroxide to obtain the HA mixed solution.

[0061] 7.4 mL of methacrylic anhydride (MA) was slowly added dropwise to the HA mixture, and the mixture was stirred in an ice-water bath at 4 °C for 12 h, followed by stirring at room temperature for another 12 h.

[0062] After the reaction was completed, the cells were dialyzed for 5 days using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0063] The solution after dialysis was freeze-dried to obtain a white flocculent product, MA-HA.

[0064] Example 3: Synthesis of carboxylated chitosan grafted with cyclodextrin (CCS-CD):

[0065] Dissolve 500 mg of carboxylated chitosan (CCS) in 100 mL of phosphate buffer solution (PB, pH = 7.2).

[0066] Dissolve 289.15 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 172.95 mg of N-hydroxysuccinimide (NHS) in 25 mL of PB buffer solution (pH = 7.2), and then rapidly add the solution dropwise to the above CCS solution. Stir the mixture at room temperature for 30 min.

[0067] Finally, 1136.35 mg of 6-amino-β-cyclodextrin (6-NH2-β-CD) was dissolved in 100 mL of PB buffer solution (pH=7.2), and the solution was added dropwise to the above mixture. The mixture was stirred at room temperature and the reaction was continued for 12 h.

[0068] After the reaction was completed, the cells were dialyzed for 5 days using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0069] The solution after dialysis was freeze-dried to obtain a white flocculent product CCS-CD.

[0070] Example 4: Synthesis of methacrylated hyaluronic acid (MA-HA-CD) grafted with cyclodextrin:

[0071] Dissolve 500 mg of the synthesized MA-HA in 100 mL of PB buffer solution (pH = 7.4) to obtain a mixed MA-HA solution.

[0072] Dissolve 838.5 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 501.5 mg of NHS (N-hydroxysuccinimide) in 25 mL of PB buffer solution (pH=7.4), and then rapidly add the solution dropwise to the above MA-HA mixed solution. Stir at room temperature for 30 min.

[0073] Finally, 2.5 g of 6-NH2-β-CD was dissolved in 50 mL of PB buffer solution (pH = 7.4) and slowly added dropwise to the above solution. The mixture was stirred at room temperature for 24 h.

[0074] After the reaction was completed, the cells were dialyzed for 5 days using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0075] The solution after dialysis was freeze-dried to obtain a white flocculent product, MA-HA-CD.

[0076] Example 5: Synthesis of methacrylated hyaluronic acid grafted with adamantane (MA-HA-Ad):

[0077] Dissolve 3.0g of MA-HA completely in 150mL of deionized water to obtain a MA-HA solution.

[0078] Add 9.0g of Dowex 50w×8 cation exchange resin to the above MA-HA solution, stir for 30min, filter to remove resin, adjust the pH of the filtrate to between 7.02 and 7.05 with tetrabutylammonium hydroxide (TBA-OH) aqueous solution, and freeze-dry the filtrate to obtain MA-HA-TBA.

[0079] Weigh 2.50g of the synthesized MA-HA-TBA, 2.04g of adamantaneacetic acid and 0.32g of 4-dimethylaminopyridine, add 125mL of anhydrous DMSO and stir until completely dissolved, then purge with N2 for 30min.

[0080] After thoroughly removing air from the bottle, 350 μL of di-tert-butyl dicarbonate (BOC2O) was added dropwise to the above solution, and the reaction was carried out at 45 °C for 20 h. The reaction was then terminated by adding water.

[0081] After the reaction was completed, the dialysis was performed for 3 days using a dialysis bag with a molecular weight cutoff of 3500 Da. The precipitate in the dialysis bag was then filtered, and the clear filtrate obtained by filtration was further dialyzed for 5 days.

[0082] The solution after dialysis was freeze-dried to obtain a white flocculent product, MA-HA-Ad.

[0083] Example 6: Preparation of polyelectrolyte multilayer films:

[0084] Prepare a piranha solution (H2SO4:H2O2 = 7:3 v / v), and soak the quartz plates one by one in the piranha solution for 2 hours.

[0085] The treated quartz sheets were cleaned, dried with nitrogen, and then immersed in a 0.5 g / L poly(D-lysine) solution (PDL, molecular weight 70,000–150,000, Sigma-Aldrich, commercially available) for 12 hours. After immersion, they were rinsed with deionized water to remove excess moisture.

[0086] The above-treated quartz plates were then subjected to the following treatments:

[0087] 1) Immerse a quartz sheet in a 0.5 g / L PDL solution for 5 minutes, rinse with deionized water to remove excess water, then immerse the quartz sheet in a 1 g / L MA-HA-Ad solution for 5 minutes, rinse with deionized water to remove excess water, and repeat this process for the first cycle. Repeat this cycle 40 times to obtain a surface-modified (PDL / MA-HA-Ad) solution.40 The quartz plate 40 indicates that the polyelectrolytes have been deposited alternately for 40 cycles, with the outermost layer being MA-HA-Ad.

[0088] 2) Preparation of surface-modified (PDL / MA-HA-CD) 40 Quartz plates: The difference from 1) above is that the 1 g / L MA-HA-Ad solution is replaced with a 1 g / L MA-HA-CD solution, and the surface-modified (PDL / MA-HA-CD) is obtained by referring to the cyclic operation method of 1) above. 40 The quartz sheet, 40 indicates that the polyelectrolytes are deposited alternately for 40 cycles, and the outermost layer is MA-HA-CD.

[0089] 3) Preparation of surface-modified (PDL / CCS-CD) 40.5 Quartz sheet preparation: The quartz sheet was immersed in a 0.5 g / L PDL solution for 5 minutes, rinsed in deionized water to remove excess water, and then immersed in a 1 g / L CCS-CD solution for 5 minutes, marking the first cycle. This process was repeated 40.5 times to obtain the final surface (PDL / CCS-CD). 40.5 The quartz sheet, 40.5 indicates that the polyelectrolyte is deposited alternately for 40.5 cycles, with the outermost layer being PDL.

[0090] The PDMS soft carrier prepared in Example 1 was then used to prepare (PDL / MA-HA-Ad) modified versions according to the above method. 40 (Outermost layer is MA-HA-Ad), (PDL / MA-HA-CD) 40.5 PDMS (with PDL as the outermost layer).

[0091] Example 7: Grafting REDV peptide with VAPG peptide, GFOGER peptide with PAP peptide, or KHI peptide with IKVAVA peptide onto a multilayer membrane, taking REDV peptide with VAPG peptide as an example:

[0092] Using PB buffer solution with pH=9 as solvent, 1 g / L solutions of photoinitiator 2959 (Ig 2959), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), CREDV (purchased from Hefei Guotai Biotechnology), and CVAPG (purchased from Hefei Guotai Biotechnology) were prepared respectively.

[0093] The surface has been modified with (PDL / MA-HA-Ad). 40The quartz sheet of the multilayer film was placed in a 24-well plate, and 1 mL of CREDV peptide solution, 150 μL of TCEP solution, and 30 μL of Ig 2959 solution were added to each well.

[0094] The surface has been modified with (PDL / MA-HA-CD). 40 The quartz sheet of the multilayer film was placed in a 24-well plate, and 1 mL of CVAPG peptide solution, 150 μL of TCEP solution, and 30 μL of Ig 2959 solution were added to each well.

[0095] Quartz plates immersed in the above reaction solution were irradiated under a 365nm wavelength ultraviolet lamp for 10 minutes to allow the click chemical reaction between the double bonds and thiol groups to occur fully. After the reaction was completed, (PDL / REDV-HA-Ad) grafted with peptides was obtained. 40 Or (PDL / VAPG-HA-CD) 40 Multilayer quartz sheet (in which the double bond of MA in MA-HA-CD reacts with the thiol group of VAPG polypeptide through a click chemical reaction, so that VAPG is grafted onto MA-HA-CD to obtain VAPG-HA-CD).

[0096] The modified (PDL / MA-HA-Ad) prepared in Example 6 40 (PDL / MA-HA-CD) 40.5 The PDMS soft carrier was used to obtain (PDL / REDV-HA-Ad) grafted with REDV peptide using the same method. 40 Multilayer PDMS membrane with VAPG-grafted peptides (PDL / VAPG-HA-CD) 40.5 Multilayer PDMS.

[0097] Clean the quartz plate or PDMS with deionized water, remove excess moisture, and then dry it with N2.

[0098] Example 8: Fabrication of three-dimensional tissue engineering scaffolds using macroscopic supramolecular assembly (MSA) and magnetic field-assisted localization strategies.

[0099] (PDL / REDV-HA-Ad) will be modified. 40 The PDMS building blocks of the multilayer membrane were placed in water using magnetic field micromanipulation and placed in the (PDL / CCS-CD) of Example 6. 40.5 On a quartz substrate.

[0100] Example 7 was modified with (PDL / VAPG-HA-CD). 40.5 The PDMS multilayer film is used as the second layer and is placed perpendicularly aligned with the first layer.

[0101] Then, the (PDL / REDV-HA-Ad) modified in Example 7 is used. 40 PDMS, as the third layer of the multilayer membrane, is placed at a 45° angle to the second layer. During construction, it is crucial to ensure that the building blocks of each layer are arranged in parallel and evenly distributed. Figure 1 , 2 As shown.

[0102] The scaffolds modified with different multilayer membranes were sterilized using 75% medical alcohol and ultraviolet light.

[0103] Experiment Example 1: Biocompatibility Experiment

[0104] Biocompatibility of host-guest polyelectrolytes MA-HA-CD and MA-HA-Ad

[0105] First, human umbilical vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (CCC-SMC-1) with a spread area of ​​more than 80% were removed from the incubator and sterilized at high temperature. Then, the metabolic waste and impurities generated on the cell surface in the culture flask were rinsed with phosphate buffered saline (PBS) at pH 7.4 for cell culture.

[0106] Adherent cells were resuspended by trypsin treatment, and digestion was terminated by adding a special culture medium. The cells were centrifuged, the supernatant was removed, and the cell culture was prepared to a density of 2.5 × 10⁶ cells / mL. 4 A suspension of cells / mL.

[0107] Add 200 μL to each well of a 96-well plate. In addition, add another row of cell-free wells at the same ratio. Place the plate in an incubator and incubate for 20 h.

[0108] Next, the drugs MA-HA-CD and MA-HA-Ad synthesized in Examples 4 and 5 were sterilized by ultraviolet light for 30 min. After sterilization, MA-HA-CD was diluted with freshly prepared HUVEC-specific growth medium (purchased from MeisenCTCC) and MA-HA-Ad was diluted with CCC-SMC-1-specific growth medium (purchased from Gibco) to prepare solutions with concentrations of 0.025, 0.05, 0.1, and 0.2 g / L, respectively.

[0109] Remove the old culture medium from the well plate and add 200 μL of the drug solution. The wells without cells are the zeroing group. Another control group with a drug concentration of 0 is set up. The amount of culture medium added to the control group is also 200 μL. After the addition is complete, put the 96-well plate back into the incubator and continue to incubate for 24 h.

[0110] A 5 g / L thiazolyl blue (MTT) solution was prepared using PBS as the solvent. After the MTT was completely dissolved, it was filtered through a 0.22 μm sterile filter membrane.

[0111] After culturing the cells for 24 hours, add 20 μL of MTT solution to each well and continue culturing in the incubator for another 5 hours.

[0112] After the culture was completed, all the solution in the wells was removed, and anhydrous dimethyl sulfoxide (DMSO) was added to each well at a ratio of 150 μL / well. After the formazan formed by the reduction of MTT was completely dissolved, the absorbance at 570 nm was measured.

[0113] The results are as follows Figure 3 As shown, the cell viability of both HUVEC and CCC-SMC-1 cells was greater than or equal to 80%, which indicates that the two synthesized host-guest polyelectrolytes have excellent biocompatibility and can be used to construct multilayer membranes and for cell culture.

[0114] Experiment Example 2: Cell Adhesion and Activity Experiment

[0115] Polypeptide-modified polyelectrolytes enhance cell adhesion and activity on quartz sheets

[0116] First, the modified (PDL / MA-HA-CD) prepared in Example 6 was used. 40 Quartz sheets with multilayer films and quartz sheets grafted with REDV prepared in Example 7 (PDL / REDV-HA-Ad). 40 Or quartz plates grafted with VAPG peptides (PDL / VAPG-HA-CD) 40 Place the quartz plates in 24-well plates and add 1 mL of 75% medical alcohol to the wells containing the quartz plates to sterilize the surface of the materials, while turning on the UV lamp.

[0117] After sterilization, remove the alcohol and wash three times with sterilized PBS. Finally, add DMEM high-glucose basal medium to incubate the quartz slices for 12 hours.

[0118] Take HUVEC or CCC-SMC-1 cells that have reached 80% coverage and are in good growth condition, and digest them with trypsin to resuspend them. Then, add HUVEC or CCC-SMC-1 specific growth medium to terminate the digestion, centrifuge to remove the supernatant, add fresh medium, and dilute the solution containing cells to 5 × 10⁻⁶. 4 Concentration of cells / mL.

[0119] Remove the basal culture medium from the well plate and add 1 mL of the cell suspension at the above concentration to each well, and add 1 mL of PBS solution to each well around the perimeter.

[0120] The well plates were placed in an incubator for cell culture, which lasted for 12, 24, and 48 hours, respectively.

[0121] A calcein-AM stock solution with a concentration of 1 g / L was prepared using anhydrous DMSO as the solvent; a propidium iodide (PI) stock solution with a concentration of 1 g / L was prepared using sterilized deionized water at high temperature as the solvent.

[0122] Take 10 μL of Calcein-AM stock solution and 15 μL of PI stock solution, and dilute them together in 4 mL of sterilized PBS.

[0123] Remove the culture medium from the well plate and wash the quartz slide with PBS to remove any remaining liquid and waste. Then add 1 mL of solution to each well and continue culturing for 15 min.

[0124] Finally, the quartz slide was removed and the cell staining was observed under a microscope.

[0125] Cell viability was then tested using a CCK-8 assay kit.

[0126] Prepare a working solution by mixing HUVEC or CCC-SMC-1 growth medium with the CCK-8 kit at a volume ratio of 100:1, and shake on a shaker for 3 minutes to mix thoroughly.

[0127] Add 1 mL of working solution to each well of the new 24-well plate and carefully transfer the quartz disc into the working solution. Simultaneously, set up a zero-adjustment group with only working solution and no quartz disc to eliminate the influence of the culture medium on the experimental results, and continue incubation for 2 hours.

[0128] The solution was transferred to a 96-well plate at a ratio of 150 μL / well, and the absorbance of the solution at 450 nm was measured.

[0129] The results are as follows Figure 4 As shown, a quartz plate grafted with REDV peptide (PDL / REDV-HA-Ad) 40 The adhesion of HUVECs was significantly increased in the quartz sheet grafted with VAPG peptides (PDL / VAPG-HA-CD). 40 The adhesion of CCC-SMC-1 was significantly increased. Endothelial cells and smooth muscle cells were cultured on a quartz matrix modified with REDV and VAPG peptides (two-dimensional), and the adhesion behavior of endothelial cells on different multilayer membranes was observed over 48 hours. Multilayer membranes without modified peptides served as a control group. Figure 4As shown in Figure a, the number of attached cells was low, and cell proliferation did not occur, indicating that the right-handedness of PDL inhibited cell adhesion, and the VAPG peptide had no effect on endothelial cell growth. In contrast, we can clearly see an increase in the number of attached endothelial cells and cell proliferation on the modified polypeptide multilayer membrane, indicating favorable conditions. This demonstrates that the combined action of PDL and REDV peptide can promote endothelial cell adhesion (REDV's role in promoting endothelial cell adhesion is the main one). Furthermore, these multilayer membranes showed almost no dead cells and good biocompatibility. Similarly, we investigated the adhesion behavior of smooth muscle cells on different multilayers, such as... Figure 4 As shown in b, unlike endothelial cells, REDV peptides failed to promote smooth muscle cell adhesion under right-handed chiral antiadhesion. However, after 48 hours of culture with VAPG peptides, smooth muscle cells showed good growth and significant proliferation. These results indicate that VAPG peptides, in synergy with the antiadhesion substrate PDL, can promote specific adhesion of smooth muscle cells.

[0130] All modified multilayer membranes contained PDL and polyelectrolytes. PDL alone is anti-cell adhesion. When PDL acts in conjunction with peptides, the peptides play a dominant role in promoting cell adhesion. Therefore, cell adhesion on multilayer membranes depends on the type of modified peptide; for example, REDV-modified sites promote endothelial cell adhesion, while VAPG-modified sites promote smooth muscle cell adhesion. We were able to promote selective adhesion of various cell types in different regions of the scaffold by using PDL and peptides modified at different locations.

[0131] Regarding cell viability, the results are as follows: Figure 5 As shown, the two cell types were modified with (PDL / REDV-HA-Ad). 40 Multilayer PDMS or (PDL / VAPG-HA-CD) 40.5 Cell activity on the multilayered PDMS membrane was similar to, or even higher than, that on the blank quartz matrix. This demonstrates that the synergistic effect of chiral molecules and peptides can achieve selective adhesion to specific cells.

[0132] Experiment Example 3: Cell Selective Adhesion Experiment

[0133] Example 8: Selective cell adhesion on a three-dimensional tissue engineering scaffold

[0134] Using an anti-adhesion multilayer film (PDL / CCS-CD) 40.5 Modified quartz sheets are used as substrates for scaffolds to prevent cells from growing in non-target locations.

[0135] (PDL / REDV-HA-Ad) will be modified. 40 The PDMS building blocks of the multilayer membrane serve as the first scaffold; then, (PDL / VAPG-HA-CD) modified... 40.5The PDMS multilayer film was used as the second layer and placed perpendicularly to the first layer; finally, (PDL / REDV-HA-Ad) was added. 40 The PDMS multilayer film is used as the third layer and is placed at a 45° angle to the second layer.

[0136] A 2 mM stock solution of 3,3'-bis(octadecyloxacarbonyl)chlorate (DiO) and a 3 mM stock solution of 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindole carbonyl cyanine perchlorate (DiL) were prepared using anhydrous DMSO as the solvent.

[0137] Using PBS as a solvent, the above DiO and DiL stock solutions were diluted to 25 μM and 15 μM working solutions, respectively.

[0138] Select HUVECs or CCC-SMC-1 with a spreading area of ​​80% and good growth condition, wash, digest, centrifuge, and then stain with 1 mL of working solution at a concentration of 1×10⁻⁶. 6 Cells were stained according to the proportion of cells. CCC-SMC-1 cells were stained with DiO for 25 min, and HUVEC cells were stained with DiL for 5 min.

[0139] After staining, the staining solution was removed by centrifugation, and the two cell types were prepared separately with special growth medium at a concentration of 5 × 10⁻⁶ m² / g. 4 Cell suspension at a concentration of cells / mL.

[0140] Two types of cells were seeded onto a three-dimensional scaffold and allowed to grow freely. 500 μL of each cell suspension was added to each well, and the two types of cells were cultured together in an incubator for 12, 24, and 48 hours.

[0141] After the culture time is reached, surface impurities are cleaned with PBS, and the culture results are observed under a fluorescence microscope.

[0142] The results are as follows Figure 6 As shown, HUVECs adhered extensively to the first and third layers modified with the REDV peptide, and the endothelial cell density was significantly higher than that in the second layer. On the second layer modified with the VAPG peptide, CCC-SMC-1 cells adhered extensively, and the smooth muscle cell density was significantly higher than that in the first and third layers. Furthermore, the number of dead cells was low in both cell types, demonstrating the scaffold's good biocompatibility.

[0143] Therefore, by simply combining peptides and chiral molecules with a precise magnetic manipulation-assisted method, we achieved selective adhesion of cells to desired locations in three-dimensional ordered structures based on the MSA strategy.

[0144] This invention can further extend the application of REDV peptides and VAPG peptides as artificial vascular scaffolds, and utilize GFOGER peptides and PAP peptides or KHI peptides and IKVAVA peptides, etc., for applications in bone regeneration engineering and nerve repair devices. This invention can further extend the use of hydrogel materials such as PDMS and PEGDA as three-dimensional scaffold materials, and utilize rigid materials such as Si and Ti. Multiple scaffold materials can be applied to the repair of soft tissues, such as fat, skin, or blood vessels; and hard tissues, such as cartilage or jawbone. This invention enables selective cell adhesion within composite three-dimensional scaffolds, applicable to the repair and regeneration of complex craniofacial tissues. The scaffolds proposed in this invention can serve as a platform for studying cell-substance interactions and are applicable to multiple fields such as artificial blood vessels, bone regeneration, and nerve repair devices.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A three-dimensional scaffold for cell-selective adhesion based on chirality and peptides, characterized in that, It has a layered, three-dimensional ordered structure, with each layer consisting of several polyelectrolyte multilayer membrane soft carriers arranged in an ordered manner, and the polyelectrolyte multilayer membrane soft carriers of adjacent layers forming an angle; each layer of polyelectrolyte multilayer membrane is formed by alternating deposition of positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes on the soft carrier through multiple depositions and then modification with peptides; the soft carrier includes soft materials doped with magnetic nanoparticles; The two polypeptides in adjacent polyelectrolyte multilayer membranes are different and each promotes the specific adhesion of different cells. The two adjacent polyelectrolyte multilayer membranes contain two different specific recognition groups, which are a pair of host-guest interaction groups that can achieve supramolecular self-assembly. The outermost membranes of the two adjacent polyelectrolyte multilayer membranes are respectively a positively charged chiral molecule grafted with specific recognition groups and a negatively charged supramolecular polyelectrolyte, which are used to connect the two adjacent polyelectrolyte multilayer membranes through positive and negative charges. The chiral molecule is poly-dextral lysine. The basic structural elements of each layer are arranged in parallel.

2. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The polypeptide is grafted into a polyelectrolyte multilayer membrane using one or more of the following methods: simple adsorption, click chemistry, or azide and thiol reaction.

3. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The soft material is selected from one or more of polydimethylsiloxane, polyethylene glycol diacrylate, methacrylic anhydride gelatin, and collagen.

4. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, It also includes a substrate for supporting the polyelectrolyte multilayer film. The substrate is a polyelectrolyte multilayer film in which positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes are deposited alternately on the substrate through multiple cycles. The substrate material is selected from one or more of quartz, silicon, and titanium.

5. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The polyelectrolyte is selected from one or more of hyaluronic acid modified with C=C double bond groups and carboxylated chitosan modified with C=C double bond groups.

6. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The raw materials used for C=C double bond modification are one or more of methacrylic anhydride, methacrylic acid, and acrylic anhydride.

7. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The specific recognition group includes a host-guest interaction group.

8. The cell-selective adhesion three-dimensional scaffold according to claim 7, characterized in that, The host-guest interaction groups are selected from one or more of β-cyclodextrin, cucurbituril, adamantane, azobenzene, cholesterol, and anthracene.

9. The cell-selective adhesion three-dimensional scaffold according to claim 8, characterized in that, When β-cyclodextrin is the host group, the corresponding guest group is adamantane, azobenzene, or cholesterol.

10. The cell-selective adhesion three-dimensional scaffold according to claim 8, characterized in that, When cucurbituril is the host group, the corresponding guest group is anthracene.

11. The cell-selective adhesion three-dimensional scaffold according to claim 8, characterized in that, The molecular weight of the chiral molecule is 70,000 to 150,000.

12. The cell-selective adhesion three-dimensional scaffold according to claim 1, characterized in that, The polyelectrolyte multilayer film is formed by alternating deposition of positively charged chiral molecules modified with double bond groups and specific recognition groups and negatively charged supramolecular polyelectrolytes on a soft carrier for n or n+0.5 cycles. In one cycle, positively charged chiral molecules and negatively charged supramolecular polyelectrolytes are deposited once each, and n is an integer. n+0.5 means that another layer of positively charged chiral molecules is deposited on the basis of the nth cycle.

13. The cell-selective adhesion three-dimensional scaffold according to claim 12, characterized in that, n≥35。 14. The cell-selective adhesion three-dimensional scaffold according to claim 12, characterized in that, n≥40。 15. The cell-selective adhesion three-dimensional scaffold according to claim 12, characterized in that, The n values ​​of the polyelectrolyte multilayer membranes in two adjacent layers are independently selected from integers n≥35.

16. The cell-selective adhesion three-dimensional scaffold according to claim 12, characterized in that, The multilayer membrane soft carrier includes at least two types: a host polyelectrolyte with host-guest interaction groups deposited on it, and the deposition period of the two types of multilayer membrane soft carriers is n or n+0.5, respectively.

17. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, The polyelectrolyte multilayer membrane is selected from (PDL / MA-HA-Ad) membranes further modified with peptides. 40 (PDL / MA-HA-CD) 40.5 One or more of the multilayer PDMS films.

18. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, The polyelectrolyte multilayer membrane is selected from (PDL / REDV-HA-Ad). 40 (PDL / VAPG-HA-CD) 40.5 One or more of the multilayer PDMS films.

19. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, It also includes a substrate, which is modified with (PDL / CCS-CD). 40.5 Quartz plates.

20. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, The polypeptide includes one or more of the following: REDV polypeptide, VAPG polypeptide, GFOGER polypeptide, PAP polypeptide, KHI polypeptide, and IKVAV polypeptide.

21. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, The cells include one or more pairs of specific adhesions between human umbilical vein endothelial cells and rabbit aortic smooth muscle cells, human bone marrow mesenchymal stem cells and mouse mononuclear macrophage leukemia cells, or human neural progenitor cells and astrocytes.

22. The cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 16, characterized in that, The first layer is modified with (PDL / REDV-HA-Ad). 40 The second layer of the multilayer PDMS membrane is modified with (PDL / VAPG-HA-CD). 40.5 The PDMS multilayer film has a third layer modified with (PDL / REDV-HA-Ad). 40 PDMS in multilayer films.

23. A method for preparing a three-dimensional scaffold based on chirality and peptides for selective cell adhesion, characterized in that, Includes the following steps: 1) Prepare negatively charged supramolecular polyelectrolytes with host-guest interaction groups and C=C double bonds, wherein the host and guest of the host-guest interaction groups are modified on different polyelectrolytes respectively; 2) The soft carrier is deposited sequentially into the positively charged chiral molecule solution and the negatively charged supramolecular polyelectrolyte solution in step 1) to form a film, and the process is repeated for n or n+0.5 cycles to prepare (chiral molecules / polyelectrolytes with host-guest interaction groups and C=C double bonds). n A multilayer film soft carrier, comprising a soft material doped with magnetic nanoparticles; wherein a positively charged chiral molecule and a negatively charged supramolecular polyelectrolyte are sequentially deposited once, where n is an integer, and n+0.5 represents the deposition of another layer of positively charged chiral molecules based on the nth period; the chiral molecule is poly-D-lysine; The outermost layers of two adjacent polyelectrolyte multilayer films are respectively a positively charged chiral molecule grafted with a specific recognition group and a negatively charged supramolecular polyelectrolyte, which are used to connect the two adjacent polyelectrolyte multilayer films through positive and negative charges; the chiral molecule is poly-dextral lysine; 3) At least two types of multilayer membrane soft carriers from step 2) are grafted with peptides via simple adsorption, azide and thiol reaction or click chemistry reaction to obtain polyelectrolyte multilayer membranes; the building blocks of each layer are arranged in parallel. 4) A three-dimensional scaffold is constructed using multilayer membrane soft carriers that can interact with each other through host-guest interactions. The peptides and polyelectrolytes in adjacent multilayer membrane soft carriers have different host-guest interaction groups.

24. The preparation method according to claim 23, characterized in that, In step 2), n ≥ 35.

25. The preparation method according to claim 23, characterized in that, In step 2), n ≥ 40.

26. The preparation method according to claim 23, characterized in that, The multilayer membrane soft carrier includes at least two types, wherein the supramolecular polyelectrolytes of the two multilayer membrane soft carriers are a host group modified with host-guest interaction groups and a guest group modified with supramolecular polyelectrolytes, and the deposition periods of the two multilayer membrane soft carriers are n and n+0.5, respectively.

27. The preparation method according to any one of claims 23 to 26, characterized in that, In step 1), the polyelectrolyte is selected from one or more of hyaluronic acid modified with C=C double bond groups and carboxylated chitosan modified with C=C double bond groups.

28. The preparation method according to any one of claims 23 to 26, characterized in that, In step 1), the raw material used for C=C double bond group modification is one or more of methacrylic anhydride, methacrylic acid, and acrylic anhydride.

29. The preparation method according to any one of claims 23 to 26, characterized in that, In step 1), the concentration of the polyelectrolyte is 0.5~2g / L.

30. The preparation method according to any one of claims 23 to 26, characterized in that, In step 1), the concentration of the polyelectrolyte is 0.5~2g / L.

31. The preparation method according to any one of claims 23 to 26, characterized in that, In step 1), the specific recognition group includes host and guest interaction groups.

32. The preparation method according to claim 31, characterized in that, The host interacting groups are selected from one or more of β-cyclodextrin, cucurbituril, adamantane, azobenzene, cholesterol, and anthracene.

33. The preparation method according to claim 32, characterized in that, β-Cyclodextrin is the host group, and the corresponding guest group is adamantane, azobenzene or cholesterol.

34. The preparation method according to claim 32, characterized in that, Cucurbitaurea is the host group, and the corresponding guest group is anthracene.

35. The preparation method according to any one of claims 23 to 26, characterized in that, In step 2), the concentration of chiral molecules is 0.1~1 g / L.

36. The preparation method according to any one of claims 23 to 26, characterized in that, In step 2), the concentration of chiral molecules is 0.5~1g / L.

37. The preparation method according to any one of claims 23 to 26, characterized in that, In step 2), the multilayer membrane soft carrier is (PDL / MA-HA-Ad). 40 (PDL / MA-HA-CD) 40.5 One or more of the multilayer PDMS films.

38. The preparation method according to any one of claims 23 to 26, characterized in that, In step 3), the polypeptide includes one or more of the following: REDV polypeptide, VAPG polypeptide, GFOGER polypeptide, PAP polypeptide, KHI polypeptide, and IKVAV polypeptide.

39. The preparation method according to claim 38, characterized in that, The concentration of the polypeptide is 0.5~2g / L.

40. The preparation method according to claim 38, characterized in that, The concentration of the polypeptide is 1~2 g / L.

41. The preparation method according to any one of claims 23 to 26, characterized in that, In step 3), the polyelectrolyte multilayer membrane is selected from (PDL / REDV-HA-Ad). 40 (PDL / VAPG-HA-CD) 40.5 Multilayer PDMS.

42. The preparation method according to claim 41, characterized in that, It also includes a substrate for supporting the polyelectrolyte multilayer film. The substrate is a polyelectrolyte multilayer film in which positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes are deposited alternately on the substrate through multiple cycles. The substrate material is selected from one or more of quartz, silicon, and titanium.

43. The preparation method according to claim 42, characterized in that, The substrate is modified with (PDL / CCS-CD). 40.5 Quartz plates.

44. The use of a chiral and polypeptide-based cell-selective adhesion three-dimensional scaffold according to any one of claims 1 to 22, or a chiral and polypeptide-based cell-selective adhesion three-dimensional scaffold prepared by any one of claims 23 to 43, in the preparation of soft tissue repair products, bone regeneration devices, and / or nerve repair products.

45. The application according to claim 44, characterized in that, Soft tissue repair products include artificial vascular stents.

46. ​​The application according to claim 44, characterized in that, The peptides in the adjacent polyelectrolyte multilayer membranes are REDV peptide and VAPG peptide, respectively.

47. The application according to claim 46, characterized in that, The REDV polypeptide is the CREDV polypeptide, and the cells used to promote specific adhesion are endothelial cells.

48. The application according to claim 46, characterized in that, The VAPG polypeptide is a CVAPG polypeptide, and the cells used to promote specific adhesion are smooth muscle cells.

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