Graphene oxide-based liquid crystals, binary liquid crystal systems, 3D sponge-like scaffold materials, liquid crystal gels, biological tissue engineering scaffolds

Abstract

The present invention provides graphene oxide-based liquid crystals, binary liquid crystal systems, 3D sponge-like scaffold materials, liquid crystal gels, and biological tissue engineering scaffolds, relating to the technical field of repair medical materials. The present invention grafts cholesterol-polyethylene glycol-amino groups onto graphene oxide to prepare a novel liquid crystal composite material, a graphene oxide-based liquid crystal composite. Its aqueous solution exhibits birefringence, and the liquid crystal gel has an anisotropic structure. It exhibits good osteogenic activity, printability, biocompatibility, degradability, mechanical properties highly similar to bone tissue, and biomimetic properties. It is a novel multifunctional integrated nanostructure platform. The graphene oxide-based liquid crystal composite can be compounded with biomedical materials, photoinitiators, growth factors, and stem cell chemotactic factors to obtain tissue engineering scaffolds through 3D printing-photocrosslinking, showing promising application prospects in bone repair, cartilage repair, vascular repair, tendon repair, or myocardial repair.

Description

Technical Field

[0001] The present invention relates to the technical field of restorative medical materials, and specifically to graphene oxide-based liquid crystal composites and preparation methods and applications thereof, binary liquid crystal systems and applications thereof, 3D sponge-like scaffold materials and applications thereof, liquid crystal gels and applications thereof, and biological tissue engineering scaffolds and preparation methods and applications thereof. Background Art

[0002] The development of ideal bone repair materials is currently a hot topic in bone tissue engineering research. The demand for novel bone scaffold materials is becoming increasingly urgent in both clinical applications and scientific research. Therefore, developing osteoinductive bone repair materials remains a top priority in bone tissue engineering research. Simulating the structural composition of natural bone tissue to design and construct scaffold materials with nanostructures or nanosurface / interface structures, while simultaneously engineering the scaffold materials at the molecular level to impart their own osteoinductive activity, is a key development direction for comprehensively enhancing the bioactivity of traditional bone tissue repair materials. Nanoscale surface / interface regulation, as well as scaffold material structure control and functional assembly, are effective means of imparting specialized properties to tissue engineering scaffolds.

[0003] Liquid crystals are the fundamental form of life in the human body. Proteins, nucleic acids, polysaccharides, lipids, and other substances in living organisms can self-assemble into liquid crystals. Using liquid crystal biomaterials for modification or as direct scaffolds in tissue engineering can achieve excellent blood compatibility and cell affinity, reduce inflammatory responses, and promote tissue regeneration and repair. The development of liquid crystal biomaterials is gaining increasing attention.

[0004] Graphene oxide has been widely studied in the field of soft and hard tissue engineering such as bone, cartilage, muscle, tendon, skin, nerve, heart, brain, and blood vessels due to its special nanostructure, excellent physical and chemical properties, and biocompatibility. For example, the existing technology "Sun Kai. Construction of biomimetic liquid crystal graphene oxide / polysaccharide composite system and its effect on the biological behavior of hBMSCs [D]. Jinan University, 2020." discloses a liquid crystal structure that simulates the extracellular matrix (ECM). Using graphene oxide (GO) liquid crystals (LCs) as liquid crystal elements and hyaluronic acid (HA) / alginate (SA) as matrices, two liquid crystal systems, GO / SA and GO / HA, were constructed and freeze-dried and Ca-treated. 2+ 、Mn 2+ 、Sr 2+ The cross-linking modification technology constructed a 3D scaffold of the binary liquid crystal system, which not only maintained the long-range orientation and porous interconnected structure of the liquid crystal state, but also significantly enhanced the mechanical strength of the scaffold material (the compressive strength increased by about 5 times); the results of in vitro and in vivo experiments showed that: Ca 2+ or Sr 2+Both cross-linked liquid crystal scaffolds and self-assembled liquid crystal fiber membranes facilitate the adhesion, spreading, proliferation, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), demonstrating significant activity in promoting skull defect repair. However, this prior art liquid crystal system serves only as a matrix material for stem cell growth and lacks activity in promoting bone defect repair. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide graphene oxide-based liquid crystals, binary liquid crystal systems, 3D sponge-like scaffold materials, liquid crystal gels, and biological tissue engineering scaffolds. The graphene oxide-based liquid crystal composites provided by the present invention themselves have the activity of promoting bone defect repair.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a graphene oxide-based liquid crystal composite, comprising graphene oxide liquid crystal and cholesterol-polyethylene glycol-amino groups grafted into the graphene oxide liquid crystal.

[0008] Preferably, the mass ratio of the graphite oxide liquid crystal to the cholesterol-polyethylene glycol-amino group is 1:0.05-5;

[0009] The weight average molecular weight of the cholesterol-polyethylene glycol-amino group is 200-2000.

[0010] The present invention provides a method for preparing the graphene oxide-based liquid crystal composite described in the above technical solution, comprising the following steps:

[0011] Cholesterol-polyethylene glycol-amino, graphite oxide liquid crystal, MES buffer solution, a dehydrating agent and a condensation accelerator are mixed and subjected to an amidation reaction to obtain a graphene oxide-based liquid crystal composite.

[0012] The present invention provides a graphene oxide-based binary liquid crystal system, comprising a liquid crystal unit and a biomedical material dispersion; the liquid crystal unit is the graphene oxide-based liquid crystal composite described in the above technical solution or the graphene oxide-based liquid crystal composite prepared by the preparation method described in the above technical solution; the biomedical material dispersion is an aqueous solution of the biomedical material or a sol of the biomedical material.

[0013] Preferably, the mass ratio of the liquid crystal unit to the biomedical material is 0.005 to 1:1;

[0014] The biomedical material comprises one or more of hyaluronic acid, methacryloyl hyaluronic acid, alginic acid, methacryloyl hyaluronic acid, silk protein and methacryloyl hyaluronic acid;

[0015] The concentration of the biomedical material in the graphene oxide-based binary liquid crystal system is 0.1 to 100 mg / mL.

[0016] The present invention provides a liquid crystal gel, which is obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with a photoinitiator and then photocrosslinking.

[0017] The present invention provides a 3D sponge-like scaffold material, which is obtained by freeze-drying the graphene oxide-based binary liquid crystal system described in the above technical solution or the liquid crystal gel described in the above technical solution.

[0018] The present invention provides a biological tissue engineering scaffold, the chemical composition of which includes the graphene oxide-based binary liquid crystal system described in the above technical solution, growth factors and chemokines.

[0019] The present invention provides a method for preparing the biological tissue engineering scaffold described in the above technical solution, comprising the following steps:

[0020] A graphene oxide-based binary liquid crystal system, growth factors and chemokines are mixed and freeze-dried or 3D printed-photocrosslinked to obtain a biological tissue engineering scaffold.

[0021] The present invention provides the graphene oxide-based liquid crystal composite described in the above technical solution, the graphene oxide-based liquid crystal composite prepared by the preparation method described in the above technical solution, the graphene oxide-based binary liquid crystal system described in the above technical solution, the liquid crystal gel described in the above technical solution, the 3D sponge-like scaffold material described in the above technical solution, the biological tissue engineering scaffold described in the above technical solution, or the biological tissue engineering scaffold prepared by the preparation method described in the above technical solution, and their use as biorepair materials.

[0022] The present invention provides a graphene oxide-based liquid crystal composite (abbreviated as CLS-PEGn-NH2-g-GO), comprising a graphene oxide liquid crystal and a cholesterol-polyethylene glycol-amino group grafted into the graphene oxide liquid crystal. In the graphene oxide-based liquid crystal composite provided by the present invention, the anisotropic structure of the liquid crystal material has a positive regulatory effect on osteogenic differentiation, and cholesterol, as a bioactive molecule, has a regulatory effect on the osteogenic differentiation of bone marrow stromal stem cells, so that the graphene oxide-based liquid crystal composite itself has the activity of promoting bone defect repair, without the need for the additional addition of Ca 2+ 、Sr 2+ The present invention uses cholesterol-polyethylene glycol-amino (CLS-PEG n-NH2) as the active molecule and graphene oxide as the regulatory element of the nanoscale surface / interface structure, the liquid crystal microenvironment of the bioactive molecule cholesterol in the organism, especially the microenvironment of the mosaic liquid crystal membrane, was biomimetically simulated. Cholesterol-polyethylene glycol-amino molecules were grafted onto the edges of GO nanosheets in graphene oxide liquid crystals, effectively simulating the characteristics of cholesterol molecules exerting bioactivity through lateral movement in the liquid crystal membrane. Compared with pure cholesterol, the graphene oxide-based liquid crystal composite has excellent bone-promoting activity. The graphene oxide-based liquid crystal composite provided by the present invention has an anisotropic structure and good osteogenic activity, and is printable, biocompatible, degradable, and has mechanical properties highly similar to bone tissue and biomimetic properties. It is a new type of multifunctional integrated nanostructure platform and an ideal scaffold material for 3D printing. The graphene oxide-based liquid crystal composite is used as a long-range orientation structure construction model for bone tissue. The orderly flow and self-assembly properties of the graphene oxide-based liquid crystal composite are utilized to achieve effective biomimetic construction of a multifunctional assembled bone tissue engineering scaffold, comprehensively enhance the bone tissue repair activity of the scaffold material, and provide a new solution to the problem that the current bone tissue engineering scaffold cannot be effectively biomimetic and leads to inconsistent in vivo experimental results. Moreover, the present invention has confirmed through in vitro and in vivo experiments that the graphene oxide-based liquid crystal composite is beneficial to the adhesion, spreading, proliferation and osteogenic differentiation of human periodontal ligament stem cells. It is a new type of liquid crystal composite material with excellent biocompatibility and bone-promoting activity. The graphene oxide-based liquid crystal composite can not only be used for biomimetic construction of 3D scaffolds of extracellular matrix-like structures, but also the scaffold material itself has bone-promoting activity.

[0023] The present invention provides a liquid crystal gel, which is obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with a photoinitiator and then photocrosslinking. The present invention uses a graphene oxide-based liquid crystal composite as a liquid crystal unit, composites it with natural medical materials, and utilizes the orderly flow characteristics of liquid crystals to produce anisotropic liquid crystal gels. With the help of 3D printing technology or freeze-drying technology, the liquid crystal gel can be used to prepare a 3D sponge-like scaffold material with a long-range ordered layered structure, and can also be used to prepare highly oriented fiber materials and film materials. Compared with non-liquid crystal-based 3D materials and 2D materials, it not only has a long-range ordered structure but also significantly improves its mechanical properties. Moreover, the 3D sponge-like scaffold material has its own biological activity, structure and mechanical properties, can effectively mimic bone tissue, and is expected to become an ideal scaffold material in bone tissue engineering.

[0024] The present invention provides a liquid crystal gel obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with a photoinitiator and then photocrosslinking. The present invention uses a graphene oxide-based liquid crystal composite as a liquid crystal unit and natural medical materials as a matrix. By adding a photoinitiator, a liquid crystal gel (a multicomponent composite liquid crystal system) is obtained. Using the liquid crystal structure of the extracellular matrix as a model, extrusion-type 3D printing technology and photocuring technology are used to prepare a 3D-printed liquid crystal gel scaffold with controllable mechanical properties and a long-range oriented structure. This achieves precise biomimetic preparation of the extracellular matrix, solving the scientific problem that current bone tissue engineering scaffolds cannot combine their own osteogenic activity with the structural properties of the extracellular matrix liquid crystal gel state, resulting in the inability to precisely simulate biomimetic structures. This establishes a new model for the research of bone tissue engineering scaffolds.

[0025] The present invention provides a biological tissue engineering scaffold, which is obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with growth factors and chemokines and then freeze-drying or 3D printing-photocrosslinking. Based on the unique properties of cholesterol and graphene oxide, the present invention uses the liquid crystal state that is ubiquitous in organisms as a biomimetic model, integrates it into a nano-platform, and prepares a new type of graphene oxide-based liquid crystal material to exert a synergistic effect, which will open up a new research direction for bone defect repair. The present invention utilizes the orderly flow characteristics and self-assembly properties of the graphene oxide-based liquid crystal composite to load cell chemokines and increase the activity of cell chemokines, composite with natural medical materials, and through 3D printing technology, through 3D printing technology, personalized and precise construction of bone defect repair 3D tissue engineering scaffold products.

[0026] This composite material can be used to formulate a series of materials and develop new dental and orthopedic bone transplants to accelerate bone regeneration. It has broad application prospects and important scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a polarizing microscope image of lyotropic liquid crystal in a graphene oxide dispersion with a concentration of 5 mg / mL;

[0028] Figure 2 CLS-PEG at a concentration of 1.5 mg / mL n -Polarized microscopy image of lyotropic liquid crystal of NH2-g-GO dispersion;

[0029] Figure 3 CLS-PEG at a concentration of 5 mg / mL n -Polarized microscopy image of lyotropic liquid crystal of NH2-g-GO dispersion;

[0030] Figure 4 GO, CLS-PEG n -NH2 and CLS-PEG n-Infrared absorption spectrum of NH2-g-GO;

[0031] Figure 5 GO, CLS-PEG n -NH2 and CLS-PEG n -Raman spectrum of NH2-g-GO;

[0032] Figure 6 This is a polarizing microscope image of the graphene oxide-based binary liquid crystal system prepared in Example 4;

[0033] Figure 7 Polarized light microscope image of 1.5 mg / mL CLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2 w / v% LAP prepared in Example 6;

[0034] Figure 8 This is a digital photo of a 1.5 mg / mLCLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2 w / v% LAP liquid crystal gel scaffold obtained by 3D printing combined with photocrosslinking;

[0035] Figure 9 This is the SEM image of the cross-sectional structure of the 3D printed CLS-PEG-NH2-g-GO / FibMA liquid crystal gel scaffold after liquid nitrogen brittle fracture;

[0036] Figure 10 SEM and AFM images of CLS-PEG-NH2 crystals, GO liquid crystal base films, and CLS-PEG-NH2-g-GO composite liquid crystal base films;

[0037] Figure 11 The diagrams show the life and death of hPDLSCs on CLS-PEG-NH2 crystal, GO liquid crystal base film and CLS-PEG-NH2-g-GO composite liquid crystal base film;

[0038] Figure 12 The cell activity of hPDLSCs on CLS-PEG-NH2 crystal, GO liquid crystal base film and CLS-PEG-NH2-g-GO composite liquid crystal base film;

[0039] Figure 13 The cytoskeleton microfilament staining of hPDLSCs cultured on CLS-PEG-NH2 crystals, GO liquid crystal basement membranes, and CLS-PEG-NH2-g-GO composite liquid crystal basement membranes for 24 hours;

[0040] Figure 14The cytoskeleton microfilament staining of hPDLSCs cultured on CLS-PEG-NH2 crystals, GO liquid crystal basement membranes, and CLS-PEG-NH2-g-GO composite liquid crystal basement membranes after 7 days;

[0041] Figure 15 This is a qualitative test result diagram of alkaline phosphatase, an early osteoblastic marker;

[0042] Figure 16 This is a qualitative test result of calcium nodules, a late osteoblastic marker;

[0043] Figure 17 This is the result of mouse skull repair in animal experiments using CLS-PEG-NH2-g-GO composite liquid crystal. DETAILED DESCRIPTION

[0044] The present invention provides a graphene oxide-based liquid crystal composite, comprising a graphene oxide liquid crystal and a cholesterol-polyethylene glycol-amino group grafted into the graphene oxide liquid crystal. In the present invention, the mass ratio of the graphene oxide liquid crystal to the cholesterol-polyethylene glycol-amino group is 1:0.05 to 5, more preferably 1:0.5 to 3, and specifically preferably 1:0.05, 1:0.5, 1:1, 1:2, 1:3, 1:4 or 5:1. In the present invention, the graphene oxide liquid crystal preferably has a nematic phase, a lamellar phase or a chiral liquid crystal phase structure. In the present invention, the cholesterol-polyethylene glycol-amino group (CLS-PEG n -NH2) preferably has a weight average molecular weight of 200 to 2000, more preferably 500 to 1500. In the present invention, the graphene oxide-based liquid crystal composite preferably has a nematic phase, a lamellar phase or a chiral liquid crystal phase structure.

[0045] The present invention provides a method for preparing the graphene oxide-based liquid crystal composite described in the above technical solution, comprising the following steps: mixing cholesterol-polyethylene glycol-amino, graphite oxide liquid crystal, MES buffer solution, a dehydrating agent and a condensation accelerator, and performing an amidation reaction to obtain the graphene oxide-based liquid crystal composite.

[0046] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0047] In the present invention, the preparation method of the graphite oxide liquid crystal preferably includes the following steps: flake graphite is acidified, expanded, pre-oxidized and oxidized in sequence to obtain graphite oxide liquid crystal. Specifically, the method preferably includes the following steps: flake graphite, concentrated sulfuric acid and fuming nitric acid are mixed and acidified to obtain acidified intercalated graphite; the acidified intercalated graphite is expanded to obtain expanded graphite; the expanded graphite, potassium persulfate, phosphorus pentoxide and concentrated sulfuric acid are mixed and pre-oxidized to obtain pre-oxidized graphite; the pre-oxidized graphite, concentrated sulfuric acid and potassium permanganate are mixed and oxidized to obtain graphite oxide liquid crystal.

[0048] The present invention mixes flake graphite, concentrated sulfuric acid, and fuming nitric acid, and acidifies them to obtain acidified intercalated graphite. In the present invention, the particle size of the flake graphite is preferably 100-1000 μm, more preferably 200-800 μm. In the present invention, the ratio of the mass of the flake graphite to the volume of the concentrated sulfuric acid is preferably 1-10 g:50-300 mL, more preferably 3-8 g:100-250 mL, and even more preferably 5-7 g:150-200 mL; the concentration of the concentrated sulfuric acid is preferably 98 wt%. In the present invention, the ratio of the mass of the flake graphite to the volume of the fuming nitric acid is preferably 1-10 g:20-100 mL, more preferably 3-8 g:30-80 mL, and even more preferably 5-7 g:40-60 mL; the concentration of the fuming nitric acid is preferably greater than 86 wt%, more preferably 86-98 wt%, and even more preferably 90-98 wt%. In the present invention, the mixing is preferably performed by mixing concentrated sulfuric acid and fuming nitric acid to obtain a mixed acid; flake graphite is mixed with the mixed acid; and the mixing is preferably performed by stirring. In the present invention, the acidification temperature is preferably room temperature, the acidification time is preferably 12 to 36 hours, more preferably 24 hours; and the acidification is preferably performed under stirring.

[0049] After the acidification, the present invention preferably further comprises washing the obtained acidified product with water and then drying it to obtain acidified intercalated graphite. In the present invention, the number of water washings is preferably 3 to 5 times, more preferably 4 times; the ratio of the mass of the flake graphite to the volume of water used for a single water washing is preferably 1 to 10 g: 500 to 4000 mL, more preferably 3 to 8 g: 1000 to 3000 mL. In the present invention, the drying temperature is preferably 45 to 65°C, more preferably 50 to 60°C; the drying time is preferably 24 to 72 hours, more preferably 30 to 50 hours; the drying is preferably vacuum drying, more preferably drying in a vacuum drying oven.

[0050] After obtaining the acidified intercalated graphite, the present invention expands the acidified intercalated graphite to obtain expanded graphite. In the present invention, the expansion temperature is preferably 800-1200°C, more preferably 900-1000°C; the expansion time is preferably 5-15 seconds, more preferably 10-12 seconds; and the expansion is preferably carried out in a muffle furnace.

[0051] After obtaining the expanded graphite, the present invention mixes the expanded graphite, potassium persulfate, phosphorus pentoxide, and concentrated sulfuric acid for pre-oxidation to obtain pre-oxidized graphite. In the present invention, the mass ratio of the expanded graphite, potassium persulfate, and phosphorus pentoxide is preferably 1-10:0.84-8.4:1.2-12, more preferably 3-8:2-6:3-10. In the present invention, the concentration of the concentrated sulfuric acid is preferably 98 wt%; the mass ratio of the expanded graphite to the volume of the concentrated sulfuric acid is preferably 1-10 g:50-500 mL, more preferably 3-8 g:100-300 mL. In the present invention, the mixing is preferably performed by mixing potassium persulfate, phosphorus pentoxide, and concentrated sulfuric acid to obtain a mixed solution; then mixing the expanded graphite with the mixed solution; and the mixing is preferably performed by stirring. In the present invention, the pre-oxidation temperature is preferably 65 to 85° C., more preferably 70 to 80° C., and the pre-oxidation time is preferably 3.5 to 7.5 h, more preferably 4 to 6 h; and the pre-oxidation is preferably carried out under stirring conditions.

[0052] After the pre-oxidation, the present invention preferably further comprises sequentially cooling the resulting pre-oxidation product liquid to room temperature, washing with water, and drying to obtain pre-oxidized graphite. The present invention does not specifically limit the cooling method; any cooling method familiar to those skilled in the art may be employed, such as natural cooling. In the present invention, the ratio of the mass of the expanded graphite to the volume of the dilution water is preferably 1-10 g:500-5000 mL, more preferably 3-8 g:1000-3000 mL. The washing is preferably performed by filtration, and the filter membrane used for the filtration is preferably a 0.22 μm acid- and alkali-resistant microporous filter membrane. The acid- and alkali-resistant microporous filter membrane is preferably made of polytetrafluoroethylene or polypropylene. In the present invention, the drying temperature is preferably room temperature, and the drying time is preferably 2-4 days, more preferably 3 days.

[0053] After obtaining the pre-oxidized graphite, the present invention mixes the pre-oxidized graphite, concentrated sulfuric acid and potassium permanganate, and oxidizes the mixture to obtain graphite oxide liquid crystal. In the present invention, the concentration of the concentrated sulfuric acid is preferably 98wt%. In the present invention, the ratio of the mass of the pre-oxidized graphite to the volume of the concentrated sulfuric acid is preferably 1-10g:40-400mL, more preferably 3-8g:50-300mL. In the present invention, the mass ratio of the pre-oxidized graphite to potassium permanganate is preferably 1-10:5-50, more preferably 3-8g:15-40. In the present invention, the temperature of the mixing is preferably ≤8°C; the mixing is preferably performed by adding the pre-oxidized graphite to the concentrated sulfuric acid in an ice bath with stirring, cooling to below 5°C, and then adding potassium permanganate for mixing. In the present invention, the oxidation preferably includes a first oxidation, mixing the obtained first oxidation liquid with water, and performing a second oxidation to obtain an oxidation liquid; the temperature of the first oxidation is preferably 35-55°C, more preferably 40-50°C, and the time of the first oxidation is preferably 1-4h, more preferably 2-3h; the ratio of the mass of the pre-oxidized graphite to the volume of water is preferably 1-10g:50-500mL, more preferably 3-8g:100-400mL; the temperature of the second oxidation is preferably 65-95°C, more preferably 70-80°C, and the time of the second oxidation is preferably 0.5-3.5h, more preferably 1-3h; the oxidation is preferably carried out under stirring conditions.

[0054] After the oxidation, the present invention preferably further comprises mixing the obtained oxidation liquid with water and then adding hydrogen peroxide to terminate the oxidation, separating the solid and liquid, and acid-washing the obtained solid product and then washing it with water to obtain oxidized graphite liquid crystal. In the present invention, the ratio of the mass of the pre-oxidized graphite to the volume of water is preferably 1-10 g: 20-2000 mL, more preferably 3-8 g: 500-100 mL. In the present invention, the concentration of the hydrogen peroxide is preferably 20-60 wt%, more preferably 25-30 wt%; the ratio of the mass of the pre-oxidized graphite to the volume of the hydrogen peroxide is preferably 1-10 g: 10-100 mL, more preferably 3-8 g: 30-80 mL. In the present invention, the solid-liquid separation is preferably allowed to stand and then discarding the supernatant; the temperature of the standing is preferably room temperature, and the standing time is preferably 12-36 h, more preferably 24 h. In the present invention, the pickling acid preferably comprises a hydrochloric acid solution, the concentration of the hydrochloric acid solution is preferably 1 to 5 mol / L, more preferably 1 mol / L, and the pickling is preferably centrifugal pickling. In the present invention, the water washing is preferably centrifugal water washing. The present invention has no particular limitation on the water washing, as long as the pH value of the last water washing solution is 5.5 to 6.5 (more preferably 6).

[0055] After obtaining the graphite oxide liquid crystal, the present invention mixes cholesterol-polyethylene glycol-amino, graphite oxide liquid crystal, MES buffer solution, dehydrating agent and condensation accelerator, and performs amidation reaction to obtain a graphene oxide base liquid complex.

[0056] In the present invention, the cholesterol-polyethylene glycol-amino (CLS-PEG n The mass ratio of -NH2) to graphite oxide liquid crystal is preferably 0.05 to 5:1, more preferably 0.5 to 3:1.

[0057] In the present invention, the pH value of the MES buffer solution is preferably 5, and the concentration of the MES buffer solution is preferably 0.1 mol / L; the ratio of the mass of the graphite oxide liquid crystal to the volume of the MES buffer solution is preferably 1 g: 200-1000 mL, more preferably 1 g: 500-800 mL.

[0058] In the present invention, the dehydrating agent is preferably a carbodiimide dehydrating agent, more preferably including one or more of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC); the mass ratio of the graphite oxide liquid crystal to the dehydrating agent is preferably 1:5-8, more preferably 1:6-7, and further preferably 1:6.5.

[0059] In the present invention, the condensation accelerator preferably includes one or more of condensed N-hydroxysuccinimide (NHS), sodium salt of N-hydroxysuccinimide sulfonate (Sulfo-NHS), Carter's condensation agent (BOP), 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU); the mass ratio of the graphite oxide liquid crystal to the condensation accelerator is preferably 1:6-9, more preferably 1:7-8, and further preferably 1:7.8.

[0060] In the present invention, the mixing is preferably performed by premixing cholesterol-polyethylene glycol-amino, graphite oxide liquid crystal, and MES buffer solution, and then mixing the resulting dispersion with a dehydrating agent and a condensation accelerator under a protective atmosphere; the premixing time is preferably 1 to 2 hours, more preferably 1.5 hours; the mixing time is preferably 4 to 10 hours, more preferably 5 to 6 hours; the premixing and mixing are preferably performed by ultrasonic mixing; the ultrasonic power of the ultrasonic mixing is not particularly limited in the present invention, and any ultrasonic power familiar to those skilled in the art can be used. In the present invention, the protective atmosphere preferably includes nitrogen, argon, or helium.

[0061] In the present invention, the temperature of the amidation reaction is preferably 20 to 40° C., more preferably 25° C., and the time of the amidation reaction is preferably 12 to 24 h, more preferably 16 to 20 h.

[0062] After the amidation reaction, the present invention preferably further comprises: subjecting the resulting amidation reaction liquid to solid-liquid separation, washing the resulting solid product with water and then dispersing it in water, dialyzing the resulting aqueous dispersion and then drying it to obtain a graphene oxide-based liquid crystal composite. In the present invention, the solid-liquid separation method is preferably filtration. In the present invention, the concentration of the aqueous dispersion is preferably 2-6 g / mL, more preferably 3-4 g / mL. In the present invention, the dialysis temperature is preferably 4-25°C, more preferably 4-10°C, and the dialysis time is preferably 3-5 days, more preferably 3.5-4 days; the dialysis bag used for the dialysis preferably has a molecular weight cutoff of 6-8 kDa. In the present invention, the drying is preferably freeze-drying, the freeze-drying temperature is preferably -80--45°C, more preferably -60--80°C, and the freeze-drying time is preferably 24-72 hours, more preferably 40-48 hours.

[0063] The present invention provides a graphene oxide-based binary liquid crystal system, comprising a mesogen and a dispersion; the mesogen is the graphene oxide liquid crystal described in the above technical solution or the graphene oxide-based liquid crystal composite prepared by the preparation method described in the above technical solution; and the dispersion is an aqueous solution of a biomedical material or a sol of a biomedical material. In the present invention, the biomedical material preferably includes one or more of hyaluronic acid (HA), methacrylated hyaluronic acid (HAMA), alginic acid (SA), methacrylated alginic acid (AlgMA), silk protein (SF), and methacrylated silk protein (SilMA), and more preferably includes hyaluronic acid, methacrylated hyaluronic acid, alginic acid, methacrylated alginic acid, silk protein, or methacrylated silk protein. In the present invention, the molecular weight of the biomedical material is preferably 20 to 3000 kDa, and more preferably 500 to 2500 kDa. In the present invention, the concentration of the biomedical material in the graphene oxide liquid crystal gel is preferably 0.1 to 100 mg / mL, more preferably 10 to 50 mg / mL. In the present invention, the concentration of the aqueous solution of the biomedical material is preferably 1 to 20 mg / mL, more preferably 5 to 15 mg / mL; the concentration of the sol (solvent is water) of the biomedical material is preferably 20 to 100 mg / mL, more preferably 30 to 50 mg / mL.

[0064] In the present invention, the concentration of the mesogen in the graphene oxide liquid crystal gel is preferably 0.02 to 10 mg / mL, more preferably 0.1 to 5 mg / mL. In the present invention, the mass ratio of the mesogen to the biomedical material is preferably 0.005 to 1:1, more preferably 0.01 to 0.5:1.

[0065] In the present invention, the method for preparing the graphene oxide-based binary liquid crystal system preferably comprises the following steps: mixing a mesogen, a biomedical material, and water to obtain the graphene oxide-based binary liquid crystal system. In the present invention, the mixing preferably comprises sequentially performing ultrasonic mixing and stirring mixing, wherein the ultrasonic mixing time is preferably 10 to 30 minutes, more preferably 20 minutes; the stirring mixing time is preferably 1 to 3 hours, more preferably 2 hours; and the mixing temperature is preferably room temperature.

[0066] The present invention provides a liquid crystal gel, which is obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with a photoinitiator and then photocrosslinking. In the present invention, the photoinitiator preferably includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), and the ratio of the mass of the photoinitiator to the volume of the graphene oxide-based binary liquid crystal system is preferably 0.05-2.5g:500mL, more preferably 0.5-2g:500mL, and further preferably 1g:500mL. The present invention has no special restrictions on the mixing, as long as the raw materials can be mixed evenly. In the present invention, after the mixing, the obtained mixed system is further 3D printed, and the 3D printing is preferably performed using an extrusion-type 3D printer. In the present invention, the photocrosslinking is preferably carried out under light conditions, the wavelength of the light is preferably 350-450nm, more preferably 405nm, the power of the portable point light source curing device is preferably 1-5W, more preferably 3W, and the time of the photocrosslinking is preferably 10-30s. In the present invention, the liquid crystal gel is an anisotropic liquid crystal gel.

[0067] The present invention provides a liquid crystal gel, which is obtained by mixing the graphene oxide-based binary liquid crystal system described in the above technical solution with a photoinitiator and then photocrosslinking. In the present invention, the photoinitiator preferably includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), and the ratio of the mass of the photoinitiator to the volume of the graphene oxide-based binary liquid crystal system is preferably 0.05-2.5g:500mL, more preferably 0.5-2g:500mL, and further preferably 1g:500mL. The present invention has no special restrictions on the mixing, and the raw materials can be mixed evenly, such as ultrasonic mixing. In the present invention, after the mixing, the obtained mixed system is further 3D printed, and the 3D printing is preferably performed using an extrusion-type 3D printer. In the present invention, the photocrosslinking is preferably performed under light conditions, the wavelength of the light is preferably 350-450nm, more preferably 405nm, and the light intensity is preferably 10-2000mw / cm 2 , more preferably 100 to 1200 mw / cm 2 ; The photocrosslinking time is preferably 10 to 30 seconds. In the present invention, the liquid crystal gel is an anisotropic liquid crystal gel, and the liquid crystal gel is freeze-dried to obtain a 3D sponge-like scaffold material with a long-range ordered layered structure. In the present invention, the biomaterial in the graphene oxide-based binary liquid crystal system preferably includes one or more of methacryloylated hyaluronic acid (HAMA), methacryloylated alginic acid (AlgMA) and methacryloylated silk protein (SilMA).

[0068] The present invention provides a 3D sponge-like scaffold material, which is obtained by freeze-drying the graphene oxide-based binary liquid crystal system described in the above technical solution or the liquid crystal gel described in the above technical solution. In the present invention, the freeze-drying temperature is preferably -80 to -45°C, more preferably -80 to -60°C; the freeze-drying time is preferably 24 to 72 hours, more preferably 48 hours. In the present invention, the 3D sponge-like scaffold material obtained by freeze-drying is preferably a 3D sponge-like scaffold material with a long-range ordered layered structure ("onion-shaped").

[0069] The present invention provides a biological tissue engineering scaffold, the chemical composition of which includes the graphene oxide-based binary liquid crystal system described in the above technical solution, growth factors, and chemokines. In the present invention, the growth factors preferably include growth factors related to osteogenesis, angiogenesis, or chondrogenesis, and more preferably include one or more of BMP-2, VEGF, TGF-β1, and bFGF. In the present invention, the chemokines preferably include stem cell chemokines, and more preferably include one or more of cell chemokines, monocyte chemoattractant protein-1 (MCP-1), and KGN; the cell chemokines preferably include SDF1α and / or interleukin-8 (IL-8).

[0070] The present invention provides a method for preparing a biological tissue engineering scaffold according to the above technical solution, comprising the following steps: mixing a graphene oxide-based binary liquid crystal system, a growth factor, and a chemokine, and performing freeze-drying or 3D printing-photocrosslinking to obtain a biological tissue engineering scaffold. Specifically, the method for preparing the biological tissue engineering scaffold preferably comprises the following steps: (1) mixing the graphene oxide-based liquid crystal composite with the growth factor to obtain a growth factor-loaded graphene oxide-based liquid crystal composite; (2) mixing a biomedical material dispersion with the chemokine to obtain a biomedical material mixed dispersion; (3) mixing the growth factor-loaded graphene oxide-based liquid crystal composite with the biomedical material mixed dispersion, and performing freeze-drying or 3D-photocrosslinking to obtain a biological tissue engineering scaffold; steps (1) and (2) are not performed in any chronological order.

[0071] The present invention mixes a graphene oxide-based liquid crystal composite with a growth factor to obtain a graphene oxide-based liquid crystal composite loaded with a growth factor. In the present invention, the mixing is preferably stirred mixing, and during the mixing process, the graphene oxide-based liquid crystal composite (CLS-PEG-NH2-g-GO) is loaded with a growth factor through π-π stacking. The present invention has no special restrictions on the mixing, and the raw materials can be mixed evenly, such as stirring and mixing; the mixing temperature is preferably 4 to 25°C, more preferably 14 to 10°C; the mixing time is preferably 0.5 to 6 hours, more preferably 3 to 4 hours.

[0072] The present invention mixes the biomedical material dispersion with the chemokine to obtain a biomedical material mixed dispersion. In the present invention, the mixing is preferably stirring.

[0073] After obtaining a growth factor-loaded graphene oxide-based liquid crystal composite and a biomedical material mixed dispersion, the present invention mixes the growth factor-loaded graphene oxide-based liquid crystal composite with the biomedical material mixed dispersion, and performs freeze drying or 3D printing-photo cross-linking to obtain a biological tissue engineering scaffold. In the present invention, the mixing is preferably ultrasonic mixing or stirring mixing. In the present invention, the freeze drying conditions are preferably the same as the freeze drying conditions in the preparation process of the aforementioned 3D sponge-like scaffold material, which will not be repeated here; the freeze drying can obtain the scaffold shape. In the present invention, when 3D printing-photo cross-linking is adopted, a photoinitiator is preferably added in step (2) for mixing. In the present invention, when 3D printing-photo cross-linking is adopted, the biomaterial preferably includes one or more of methacryloylated hyaluronic acid (HAMA), methacryloylated alginic acid (AlgMA) and methacryloylated silk protein (SilMA). In the present invention, the 3D printing-photocrosslinking preferably includes sequentially performing 3D printing and photocrosslinking, and the conditions of the 3D printing and photocrosslinking are preferably the same as the 3D printing and photocrosslinking conditions in the preparation process of the aforementioned 3D sponge-like scaffold material, which will not be repeated here. In the present invention, after the 3D printing-photocrosslinking, it is preferred to further include: freeze-drying the 3D printed liquid crystal gel scaffold obtained by the 3D printing-photocrosslinking to obtain a biological tissue engineering scaffold; the freeze-drying conditions are preferably the same as the freeze-drying conditions in the preparation process of the aforementioned 3D sponge-like scaffold material, which will not be repeated here; the present invention uses 3D printing-photocrosslinking to load chemokines inside the 3D printed liquid crystal gel scaffold, thereby achieving sequential sustained release of chemokines and growth factors, and giving full play to the synergistic effect of multiple factors; the present invention can achieve precise design and preparation of more ideal biological tissue engineering scaffolds through 3D printing.

[0074] In the present invention, the tissue engineering scaffold is preferably a tissue engineering scaffold material having a long-range ordered layered structure ("onion-like"). In the present invention, the sheet structure of the tissue engineering scaffold material is CLS-PEG-NH2-g-GO nanosheets, which are loaded with growth factors; and the spaces between the CLS-PEG-NH2-g-GO nanosheets are filled with a biomedical material, which is loaded with a chemokine.

[0075] The preparation method provided by the present invention is simple to operate, low in cost, and environmentally friendly. It can be used not only for the construction of bone tissue engineering scaffold materials, but also for the biomimetic construction of other tissues with highly oriented structures, and has important biomimetic value.

[0076] The present invention provides the use of the graphene oxide liquid crystal described in the above technical solution, the graphene oxide-based liquid crystal composite prepared by the preparation method described in the above technical solution, the graphene oxide-based binary liquid crystal system described in the above technical solution, the liquid crystal gel described in the above technical solution, the 3D sponge-like scaffold material described in the above technical solution, or the biological tissue engineering scaffold described in the above technical solution in bone repair, cartilage repair, vascular repair, tendon repair, or myocardial repair. In the present invention, the bone repair preferably includes in situ regeneration and repair of periodontal bone. Based on the unique properties of cholesterol and graphene oxide, the present invention uses the liquid crystal state that is ubiquitous in the body as a biomimetic model and integrates it into a nanoplatform to prepare a novel graphene oxide biomimetic liquid crystal system, which exerts a synergistic effect and will open up new research directions for bone defect repair. By analyzing the anisotropic structure, composition, and biomechanical properties of periodontal bone tissue, an innovative method is proposed to use CLS-PEGn-NH2 grafted graphene oxide to prepare NH2-PEGn-CLS-g-GO liquid crystal composite material. Utilizing the ordered flow and self-assembly properties of NH2-PEGn-CLS-g-GO, chemokines (such as SDF1α, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1)) can be loaded onto the scaffold and their activity enhanced. Furthermore, by combining it with natural biomedical materials like chitosan and alginate, 3D printing technology can be used to precisely construct personalized 3D tissue engineering scaffolds for periodontal bone defect repair. This enriches endogenous periodontal ligament stem cells and induces in situ periodontal bone regeneration. This development of a new liquid crystal molecular repair model for periodontal bone defect repair has broad application prospects and important scientific significance.

[0077] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0078] The flake graphite used in the following examples was purchased from Qingdao Henglide Graphite Co., Ltd., cholesterol-polyethanol-amino (CLS-PEG-NH2) was purchased from Shanghai Pengsheng Industrial Co., Ltd., and concentrated sulfuric acid, fuming nitric acid, potassium permanganate, hydrochloric acid, hydrogen peroxide and nitrogen were all purchased from Sinopharm Chemical Reagents.

[0079] Example 1

[0080] (1) Preparation of graphene oxide liquid crystal (GO)

[0081] 300 mL of 98 wt% concentrated sulfuric acid and 100 mL of 90 wt% fuming nitric acid were pre-added in a three-necked flask, 10 g of flake graphite with a particle size of 100 to 1000 μm was added under stirring at room temperature, acidified for 24 h under stirring at room temperature, washed with deionized water and filtered 3 to 5 times, and dried in a vacuum drying oven at 60°C for 48 h to obtain acidified intercalated graphite; the water consumption for a single washing was 4000 mL;

[0082] placing the acidified intercalated graphite in a muffle furnace at 1000° C. for 10 seconds to obtain expanded graphite;

[0083] First, 8.4g K2S2O8 and 12g P2O5 are dispersed in 500mL of 98wt% concentrated sulfuric acid, and then 10g of the expanded graphite is slowly added under stirring. The mixture is pre-oxidized at 80°C under stirring for 6h. After cooling to room temperature, 500-5000mL of deionized water is added for dilution and the mixture is filtered and washed with an acid- and alkali-resistant 0.22μm microporous filter membrane. The dilution-filtration-washing process is repeated 5 times, and the mixture is dried at room temperature for 2-4 days to obtain pre-oxidized graphite.

[0084] 400 mL of 98 wt % concentrated sulfuric acid pre-cooled in a refrigerator was injected into a three-necked flask, and 5 g of the pre-oxidized graphite was slowly added in an ice-water bath with stirring, and the temperature was controlled below 5° C. Subsequently, 50 g of KMnO4 was slowly added and the temperature was controlled below 8° C. The temperature was raised to 45° C. and the reaction was carried out under stirring for 3 h. 500 mL of deionized water was added, the temperature was raised to 80° C. and the reaction was carried out under stirring for 2 h. 1000 mL of deionized water was added, and 100 mL of 30 wt % H2O2 was added to terminate the reaction; the mixture was allowed to stand for 24 h, the supernatant was discarded, and 1 M HCl solution was added to the bottom precipitate for centrifugal washing once, and deionized water was centrifuged and washed multiple times to a pH value of 6 to obtain graphite oxide liquid crystal.

[0085] (2) Preparation of graphene oxide-based liquid crystal composites (CLS-PEG n -NH2-g-GO)

[0086] Cholesterol-polyethylene glycol-amino (CLS-PEG n -NH2)(M W=200) and the graphite oxide liquid crystal were mixed in a MES buffer solution (0.1 mol / L, pH=5) at a mass ratio of 0.05:1, and ultrasonicated in a water bath for 2 h to obtain a uniform dispersion; under nitrogen protection, dehydrating agent EDC and condensation accelerator NHS were added, ultrasonicated in a water bath for 6 h, stirred for reaction for 16 h, filtered and washed with water 5 times, and the obtained solid product was dispersed in deionized water. The obtained aqueous dispersion was dialyzed at 4 ° C (MWCO=6-8 kDa) for 5 days and freeze-dried at -70 ° C for 48 h to obtain graphene oxide-based liquid crystal (CLS-PEG-NH2-g-GO), wherein GO and CLS-PEG n The mass ratio of GO to MES buffer solution was 1:0.05, the mass ratio of GO to EDC was 1:6.5, and the mass ratio of GO to NHS was 1:7.8.

[0087] Example 2

[0088] The graphene oxide-based liquid crystal composite was prepared according to the method of Example 1. The only difference from Example 1 was that GO and CLS-PEG n -NH2 mass ratio 1:0.1.

[0089] Example 3

[0090] The graphene oxide-based liquid crystal composite was prepared according to the method of Example 1. The only difference from Example 1 was that GO and CLS-PEG n -NH2 mass ratio 1:0.5.

[0091] GO prepared in Example 1 and CLS-PEG prepared in Examples 1 to 3 were respectively n -NH2-g-GO was added to deionized water and dispersed by ultrasonication and stirring to obtain GO solution and CLS-PEG solution with a concentration of 10 mg / mL, respectively. n -NH2-g-GO solution. GO dispersion and CLS-PEG were mixed with deionized water. nThe -NH2-g-GO dispersion was diluted to 8 mg / mL, 6 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.08 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.03 mg / mL and 0.02 mg / mL in sequence, and ultrasonically dispersed for 15 min, continuously stirred magnetically for 1.5 h, and allowed to stand for 12 h. 150 μL of the above dispersion was pipetted into the liquid crystal cell or the droplets were directly placed on a glass slide, and the GO solutions of different concentrations and CLS-PEG were observed under a polarizing microscope. n Typical Schlieren structure when -NH2-g-GO is in liquid crystal state.

[0092] Figure 1 This is a polarizing microscope image of lyotropic liquid crystal in a graphene oxide dispersion with a concentration of 5 mg / mL. From the image, it can be seen that the dispersion has strong anisotropy, presenting colorful schlieren structures and filamentous structures, which have the characteristics of nematic liquid crystal.

[0093] Figure 2 CLS-PEG at a concentration of 1.5 mg / mL n Polarizing microscope image of the lyotropic liquid crystal of -NH2-g-GO dispersion. From the image, we can see the radially ordered fluidity and strong optical anisotropy of the cholesteric grafted graphene oxide dispersion, showing an ordered striped texture.

[0094] Figure 3 CLS-PEG at a concentration of 5 mg / mL n Polarized microscopy of lyotropic liquid crystal of -NH2-g-GO dispersion, from which we can see that with the increase of concentration, CLS-PEG n The fluidity of the -NH2-g-GO solution deteriorated and its viscosity increased. Its structure under a polarizing microscope changed from a radial stripe texture to a concentric band texture, resulting in a stable cholesteric grafted graphite oxide liquid crystal solution.

[0095] Figure 4 GO, CLS-PEG n -NH2 and CLS-PEG n -NH2-g-GO infrared absorption spectrum, from which we can see that GO and GO-g-NH2-PEG n -CLS has a very similar chemical structure and has GO at 3398 cm -1 、2933cm -1 、1655cm -1 、1254cm -1Absorption peaks appear at the same places, corresponding to the characteristic peaks of -OH, -CH3, C=O, COC and other groups, indicating that graphite has been successfully oxidized. n GO-g-NH2-PEG with mass ratios of -NH2 of 1:0.05, 1:0.1, and 1:0.5, respectively n -CLS at 1654cm -1 C=O and 1530cm -1 NH has a characteristic absorption peak, indicating that CLS-PEG n -NH2 combined with GO to form an O=C-NH amide bond, indicating that CLS-PEG n -NH2 was successfully grafted onto graphene oxide.

[0096] Figure 5 GO, CLS-PEG n -NH2 and CLS-PEG n -NH2-g-GO Raman spectrum, the Raman spectrum shows that GO has two characteristic peaks: D band and G band, however, different grafting rates (ie GO and CLS-PEG n GO-g-NH2-PEG with a mass ratio of -NH2 of 1:0.05, 1:0.1, and 1:0.5, respectively n -The D and G bands of CLS are the same as those of GO, both at 1587.9 cm -1 and 1339.38cm -1 This shows that the grafting of cholesterol onto graphene oxide has no effect on its structure and does not change the physical and chemical properties of graphene oxide.

[0097] Example 4

[0098] The CLS-PEG-NH2-g-GO aqueous dispersion with a concentration of 0.2 mg / mL and the hyaluronic acid aqueous solution with a concentration of 20 mg / mL were ultrasonically mixed at a volume ratio of 1:1 for 30 min and stirred for 2 h to obtain a stable graphene oxide-based binary liquid crystal system.

[0099] Figure 6 This is a polarizing microscope image of the graphene oxide-based binary liquid crystal system prepared in Example 4. As can be seen from the image, the colloidal liquid crystal has a stable, dense, layered, ordered structure. This is due to the displacement effect (or volume effect) of the hyaluronic acid molecules, which regulates the alignment of the CLS-PEG-NH2-g-GO mesogens, achieving "attraction without aggregation," thereby significantly reducing the critical concentration of the mesogens.

[0100] Example 5

[0101] Graphene oxide-based binary liquid crystal systems were prepared according to the method of Example 4. The only difference from Example 4 was that the concentrations of the CLS-PEG-NH2-g-GO aqueous dispersions were 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and 6.0 mg / mL, respectively; the hyaluronic acid was replaced with alginate or silk protein, and the concentrations of the biomedical material aqueous solutions were 1.0 mg / mL, 5.0 mg / mL, 10 mg / mL and 20 mg / mL, respectively, to obtain a series of stable graphene oxide-based binary liquid crystal systems.

[0102] Example 6

[0103] A 1.5 mg / mL CLS-PEG-NH2-g-GO aqueous dispersion, a 20 mg / mL methacryloylation silk protein (FibMA) aqueous solution, and LAP were ultrasonically mixed for 30 minutes and stirred for 2 hours to produce a liquid crystal gel (denoted as 1.5 mg / mL CLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2% LAP (w / v)). This liquid crystal gel was then used as a 3D printing ink for 3D printing using an extrusion-type 3D printer. The resulting scaffold was then exposed to UV light for 15 seconds and freeze-dried to produce a 3D sponge-like scaffold (denoted as 3D-printed CLS-PEG-NH2-g-GO / FibMA liquid crystal gel scaffold). The volume ratio of the CLS-PEG-NH2-g-GO aqueous dispersion to the FibMA aqueous solution was 1:1, and the LAP concentration in the liquid crystal gel was 0.2% (w / v, i.e., 2 g / L).

[0104] Figure 7 This is a polarizing microscope image of 1.5 mg / mL CLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2 w / v% LAP prepared in Example 6. It can be seen from the image that the liquid crystal gel has an ordered ring-layer structure.

[0105] Figure 8 This is a digital photo of the 1.5 mg / mLCLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2 w / v% LAP liquid crystal gel scaffold obtained by 3D printing and photocrosslinking of 1.5 mg / mLCLS-PEG-NH2-g-GO / 20 mg / mL FibMA / 0.2 w / v% LAP.

[0106] Figure 9This is the SEM image of the cross-sectional structure of the 3D printed CLS-PEG-NH2-g-GO / FibMA liquid crystal gel scaffold after liquid nitrogen brittle fracture. It can be seen from the figure that the biological tissue engineering scaffold has a layered and ordered fiber network structure.

[0107] Example 7

[0108] 3D sponge-like scaffold materials were prepared according to the method of Example 6. The only difference from Example 6 was that the concentrations of the CLS-PEG-NH2-g-GO aqueous dispersions were 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and 6.0 mg / mL, respectively; the methacryloylated silk protein was replaced with methacryloylated hyaluronic acid or methacryloylated alginate, and the concentrations of the aqueous solutions of the biomedical materials were 1.0 mg / mL, 5.0 mg / mL, 10 mg / mL and 20 mg / mL, respectively, to obtain a series of 3D sponge-like scaffold materials.

[0109] Test Example 1

[0110] The cell compatibility of CLS-PEG-NH2-g-GO composite liquid crystal was evaluated through three aspects: dead cell staining experiment, cell proliferation experiment and cell adhesion and spreading detection.

[0111] First, CLS-PEG-NH2 aqueous solution (concentration 5 mg / mL), GO liquid crystal or CLS-PEG-NH2-g-GO composite liquid crystal was cast into a 6-well cell culture plate, dried in air to form a liquid crystal base film, and sterilized by Co 60 irradiation. Human periodontal ligament stem cells (hPDLSCs) were cultured at 2.0×10 4 / cm 2 hPDLSCs were seeded at a density of 100 nm in a 6-well plate coated with a liquid crystal basement membrane. Cells were collected on day 1, day 4, and day 10. The hPDLSCs were stained using a live / dead staining kit according to the instructions. Calcein (AM) can penetrate the membrane of living cells and stain living cells, exhibiting strong green fluorescence. Propidium iodide (PI) only stains dead cells and produces red fluorescence. The live / dead status of hPDLSCs can be visualized by fluorescence microscopy.

[0112] Figure 10 These are the SEM and AFM images of CLS-PEG-NH2 crystals, GO liquid crystal base membrane and CLS-PEG-NH2-g-GO composite liquid crystal base membrane. It can be seen from the figures that CLS-PEG-NH2 is strip-shaped crystals and locally arranged in parallel and orderly. The surface of the GO liquid crystal base membrane has a staggered network-like structure. The CLS-PEG-NH2-g-GO composite liquid crystal base membrane has a ridge-like wrinkled structure, and its order is stronger than that of the GO liquid crystal base membrane.

[0113] Figure 11 This is a diagram of the life and death of hPDLSCs on CLS-PEG-NH2 crystal, GO liquid crystal basement membrane and CLS-PEG-NH2-g-GO composite liquid crystal basement membrane. It can be seen from the figure that some dead cells appear on CLS-PEG-NH2 crystal and GO liquid crystal basement membrane (red), while no dead cells are seen on the CLS-PEG-NH2-g-GO composite liquid crystal basement membrane. At the same time, the cell density on the composite liquid crystal basement membrane is higher and the cell proliferation is faster. The cholesterol grafted graphene oxide composite liquid crystal basement membrane has better cell compatibility.

[0114] CLS-PEG-NH2 solution, GO liquid crystal or CLS-PEG-NH2-g-GO composite liquid crystal was cast into a 96-well cell culture plate, dried in air to form a liquid crystal base film, and sterilized by Cobalt 60 irradiation. Human periodontal ligament stem cells (hPDLSCs) were cultured at 2.0×10 4 / cm 2 The cells were seeded at a density of 100 μg / mL in a 96-well plate coated with a liquid crystal base film. The cells were collected on days 1, 3, 5, 7, and 10. 10 μL MTT solution (5.0 mg / mL) was added to each well. After incubation in an incubator for 4 h, the mixed solution was discarded and 150 μL DMSO was added. The cells were shaken at low speed on a shaker for 10 min to fully dissolve the blue-purple crystalline formazan in the living cells. The absorbance at 490 nm was measured with a microplate reader. The OD value indirectly reflects the proliferation activity of hPDLSCs.

[0115] Figure 12 Figure 3 shows the cell activity of hPDLSCs on CLS-PEG-NH2 crystal, GO liquid crystal base film and CLS-PEG-NH2-g-GO complex liquid crystal base film. It can be seen that there is no significant difference in the cell proliferation activity of hPDLSCs on GO liquid crystal base film and CLS-PEG-NH2-g-GO complex liquid crystal base film, but both are better than the cell proliferation activity on the CLS-PEG-NH2 crystal surface.

[0116] CLS-PEG-NH2 solution, GO liquid crystal or CLS-PEG-NH2-g-GO composite liquid crystal was poured into a laser confocal microplate (35 mm outer diameter, 10 mm inner hole sealed with a glass slide), dried in air to form a liquid crystal base film, and sterilized by cobalt 60 irradiation. Human periodontal ligament stem cells (hPDLSCs) were cultured at 2.0×10 4 / cm 2The cells were seeded at a density of 100 nm in a small culture dish specially used for laser confocal microscopy with a liquid crystal base film, and rhodamine-labeled phalloidin was used to stain the microfilaments in the cytoskeleton of periodontal ligament stem cells after 24 hours. Specific steps: First, wash three times with PBS, fix with 4% paraformaldehyde for 15 minutes, remove the fixative, add 0.1% TritonX-100, let stand for 3 minutes, wash three times with PBS, add 100nM rhodamine phalloidin working solution (containing 1% BSA blocking solution) to cover the cells, incubate in the dark for 60 minutes, wash the cells three times with PBS, add 200ng / mL DAPI staining solution and incubate in the dark for 20 minutes, wash three times with PBS, add mounting solution to mount the slices, and finally observe the microfilaments in the cytoskeleton by laser confocal microscopy to evaluate the effect of cholesterol-grafted graphene oxide on the adhesion and spreading of periodontal ligament stem cells.

[0117] Figure 13 The cytoskeleton microfilament staining of hPDLSCs cultured on CLS-PEG-NH2 crystal, GO liquid crystal base film and CLS-PEG-NH2-g-GO composite liquid crystal base film for 24 hours. Figure 14 The cytoskeleton microfilament staining of hPDLSCs cultured on CLS-PEG-NH2 crystal, GO liquid crystal base film and CLS-PEG-NH2-g-GO composite liquid crystal base film after 7 days of culture. Figures 13-14 All three materials facilitated cell adhesion and spreading, resulting in a polygonal cell pattern. The CLS-PEG-NH2-g-GO composite exhibited the highest fluorescence intensity, indirectly indicating its beneficial effect on actin expression. By day 7 of culture, cells converged in a nematic orientation, forming spindle-shaped growth patterns.

[0118] Test Example 2

[0119] First, CLS-PEG-NH2 solution, GO liquid crystal or CLS-PEG-NH2-g-GO composite liquid crystal was cast into a 24-well cell culture plate, dried in air to form a liquid crystal base film, and sterilized by Cobalt 60 irradiation. Human periodontal ligament stem cells (hPDLSCs) were cultured at 2.0×10 4 / cm 2 hPDLSCs were seeded at a density of 100 μg / mL and plated in a 24-well plate coated with a liquid crystal basement membrane. Cells were harvested on days 7 and 14 and stained for alkaline phosphatase using the calcium-cobalt method according to the manufacturer's instructions (KeyGen Biotech KGA353, Osteogenesis Identification Kit - Alkaline Phosphatase Calcium-Cobalt Staining). The formation of gray-black precipitates in the cytoplasm, i.e., alkaline phosphatase (ALP), was observed microscopically to identify early osteogenic differentiation of hPDLSCs. Cells were harvested on days 14 and 21 and stained for calcium nodules according to the manufacturer's instructions (KeyGen Biotech KGA363-1, Alizarin Red Staining Solution). The formation of bright red calcium nodules stained with Alizarin Red was observed microscopically to identify late osteogenic differentiation of hPDLSCs.

[0120] Figure 15 This is the qualitative detection result of alkaline phosphatase, an early osteogenic marker. The more gray-black precipitates in the hPDLSCs cytoplasm on CLS-PEG-NH2 crystals, GO liquid crystal basement membranes, and CLS-PEG-NH2-g-GO composite liquid crystal basement membranes, the more alkaline phosphatase is secreted. It can be seen that the CLS-PEG-NH2-g-GO composite liquid crystal basement membrane is more conducive to the secretion of alkaline phosphatase in hPDLSCs, suggesting that it is beneficial to the osteogenic differentiation of periodontal ligament stem cells.

[0121] Figure 16 This figure shows the qualitative detection results of calcium nodules, a marker of late-stage osteogenesis. More bright red precipitates on the CLS-PEG-NH2 crystals, GO liquid crystal basement membranes, and CLS-PEG-NH2-g-GO composite liquid crystal basement membranes indicate more calcium nodules and better osteogenesis. A comparison revealed that the CLS-PEG-NH2-g-GO composite liquid crystal basement membrane favored the formation of calcium nodules, regardless of whether or not an osteogenic induction solution was added. The CLS-PEG-NH2-g-GO liquid crystal basement membrane itself possessed a certain degree of osteogenic induction activity, suggesting that it facilitated the osteogenic differentiation of periodontal ligament stem cells.

[0122] Test Example 3

[0123] High concentrations (5 mg / mL) of graphene oxide (GO) liquid crystals or graphene oxide-based liquid crystal composites (CLS-PEG-NH2-g-GO) were injected into 48-well plates, freeze-dried, and then cut into cylindrical scaffolds with a diameter of 5.8 mm and a height of 2.5 mm. These GO LC scaffolds and CLS-PEG-NH2-g-GO scaffolds, respectively, were sterilized by Cobalt-60 irradiation and used as scaffolds for skull healing in a mouse skull injury model. In all mouse skull injury models, a 6.0 mm diameter and 2.7 mm deep skull defect was constructed. The sterilized GO LC scaffolds and CLS-PEG-NH2-g-GO scaffolds were aseptically implanted into the skull injury wounds of mice. A control group without material implantation served as a control. The mice were housed under the same conditions for 4 to 12 weeks. Because the material swells after being implanted into the wound as it absorbs surrounding tissue fluid and blood, the implant size is slightly smaller than the wound itself. Mice were euthanized at 4 and 12 weeks, and skull specimens were obtained from the experimental and negative control groups. Gross morphological evaluation of skull defect repair was performed using the naked eye and a microscope, and new bone formation was observed using micro-CT.

[0124] Figure 17This is the result of mouse skull repair in animal experiments using graphene oxide-based liquid crystal composite (CLS-PEG-NH2-g-GO) scaffolds, verifying the bone repair-promoting effect of graphene oxide-based liquid crystal composites. Micro CT images show that compared with graphene oxide (GO) liquid crystal scaffolds and cholesterol-polyethanol-amino (CLS-PEG-NH2) scaffolds, the bone defects in the graphene oxide-based liquid crystal composite (CLS-PEG-NH2-g-GO) scaffold group healed faster, with more new bone mass, and had a better bone repair-promoting effect.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A graphene oxide-based liquid crystal composite, characterized in that: The invention comprises graphene oxide liquid crystal and cholesterol-polyethylene glycol-amino groups grafted onto the graphene oxide liquid crystal; the mass ratio of the graphene oxide liquid crystal to the cholesterol-polyethylene glycol-amino groups is 1:0.05-1; and the weight average molecular weight of the cholesterol-polyethylene glycol-amino groups is 200-2000.

2. The method for preparing the graphene oxide-based liquid crystal composite according to claim 1, wherein: The following steps are involved: Cholesterol-polyethylene glycol-amino, graphite oxide liquid crystal, MES buffer solution, a dehydrating agent and a condensation accelerator are mixed and subjected to an amidation reaction to obtain a graphene oxide-based liquid crystal composite.

3. A graphene oxide-based binary liquid crystal system, characterized in that: It comprises a liquid crystal unit and a biomedical material dispersion; the liquid crystal unit is the graphene oxide-based liquid crystal composite described in claim 1 or the graphene oxide-based liquid crystal composite prepared by the preparation method described in claim 2; the biomedical material dispersion is an aqueous solution of the biomedical material or a sol of the biomedical material.

4. The graphene oxide-based binary liquid crystal system according to claim 3, characterized in that: The mass ratio of the liquid crystal unit to the biomedical material is 0.005 to 1:1; The biomedical material comprises one or more of hyaluronic acid, methacryloyl hyaluronic acid, alginic acid, methacryloyl hyaluronic acid, silk protein and methacryloyl hyaluronic acid; The concentration of the biomedical material in the graphene oxide-based binary liquid crystal system is 0.1 to 100 mg / mL.

5. A liquid crystal gel, characterized in that: The graphene oxide-based binary liquid crystal system according to claim 3 or 4 is mixed with a photoinitiator and then photocrosslinked.

6. A 3D sponge-like scaffold material, characterized in that: It is obtained by freeze-drying the graphene oxide-based binary liquid crystal system according to claim 3 or 4 or the liquid crystal gel according to claim 5.

7. A biological tissue engineering scaffold, characterized in that: The chemical composition comprises the graphene oxide-based binary liquid crystal system according to claim 3 or 4, a growth factor and a chemotactic factor.

8. The method for preparing the biological tissue engineering scaffold according to claim 7, characterized in that: The following steps are involved: A graphene oxide-based binary liquid crystal system, growth factors and chemokines are mixed and freeze-dried or 3D printed-photocrosslinked to obtain a biological tissue engineering scaffold.

9. Use of the graphene oxide-based liquid crystal composite according to claim 1, the graphene oxide-based liquid crystal composite prepared by the preparation method according to claim 2, the graphene oxide-based binary liquid crystal system according to claim 3 or 4, the liquid crystal gel according to claim 5, the 3D sponge-like scaffold material according to claim 6, the biological tissue engineering scaffold according to claim 7, or the biological tissue engineering scaffold prepared by the preparation method according to claim 8 as a biorepair material.

Images